CA2688795A1 - Systems and methods for monitoring health and delivering drugs transdermally - Google Patents

Abstract

The present invention pertains to a system and method for transdermal sampling, comprising: at least one sampler for retrieving and transferring at least one analyte obtained transdermally from the skin of a subject; at least one detector system for identifying and quantifying said at least one analyte; and at least one logic module for: (i) receiving and storing input data from said at least one detector, (ii) relating the input data to other data obtained from the subject, (iii) displaying output information, (iv) transmitting the output information to another system, and (v) controlling the operation of said at least one sampler and at least one detector.

Description

SYSTEMS AND METSODS FOR MONiTORING HFYI[,=
.AND DELIV~',MG DRUGS TRANSD-ERMAUY

s (:R SMBRfi1+1IQ M~,4T.. A"PLLC TIOM
'1lris vptication claims priority to provisional appiicatiou seriat no.
60/208,327, filed June 1, 2000 which has issued as U.S. Patent No.
6,887,202 entitled "TR.ANSDERMAL HEALTH MONITORING AND

DRUG DELIVERY SYSTEM".

BACKGROUND OF THE INVffiVTIOl~i Field of the Invention:

The prasent invention relates gonarally to portable biomedical 1s monitoring. Mme spa:iScaily, Ws inveartton rdates to na-invasive and minimally invasive molwuler aLonitoring, and optionally the implemeutation of protecdve feedbuk messnres aud remote monitoring through telemetry.
Oeeaipgon of~R,elated Art Non-iavesive tansdarmel sempling of body fluids bas long been a goal in modicel nesearcb. Tbe notion that valuable diagnostie inforuation eomprising the concen~tions of key anelybes witbin the bloodstreatn could be obtained without bmwhng the sida has apurred mmy linas of reseeroh. With, such technology, long-term convenient health monitoring and screening without needtes or outpabW care would become a nality: diabetics could monitor blood gtucoee without drawtng blood; mearkers for microbisl, fangal or virel iafec:tions could be monitored; and environmental mciwsura to toxins could be essessed non-invesively.

Biomarloera hanre been ntilized ef&civeiy to deexR; m~at+R Oaa~essg.~
etposure levela to mviromaenid c.haYnicals daemed bazardoos and toxic to himoaon life. Tla sensitivity of biomarkezs allows them to aact as early warning 1tUdlCatotB to Sqbde dECCad01lS $n the CDvfroment. Their 3peCi$City can be used S to estabiish tbe nafim oftba imposing chmicat agmt debermiae acposiae level and de5ne a suitable conree ofactiom 8nvironwuaataUy indaod dmues affect everyona to one degrroe or aoatlw, howr.wmr iadividual swceptibilities can prtdispox the degree of toxic reaction of one group over anotbar: It is worthwhile notiag that in 1996, there vrere 86,912 aases of pesticide exposures reported to American Assoaiation of Poison Ceoters, of whiah 26 were fatalitie,s. In patticulari individnale in thanr developmmtiai atages, raaging from the embryonic phase to ablesomux, ffie ,patticuIarly susceptible to such environmental strasaes aince key body fun~~ctions hm not nnozed to a level where thay can toletate, praceea and huadle such mtpomvs. The uw of 1s biomarkera for ddmminatian of cMldren'e eavIeonmental health wili allow for the early deteetion of #axina, pmvenlian of impsirnoont in tbeir physieai condition, and detamine a oourse of treamnat for duldrem wbo have; been exposed to a taxic anviramment.

Especially impvrw in du fietd of pediatrica ia the use of health evaluation toola duat are minimaUy infinidve.

Many trausimmal seuapUng teobaiques have been reporbed. but all to date suffer from one or more serious drawbaelcs. Conventionat techniques have disadvantages of beimg gt+Usly invasive (and potentially mjurious) and swat or intarstitial fluid dapendent, except for the: pessive, non-swe.at depanda#

2s aansdr,rma! analybe collection and dettcfion bechniqnea.

One appmuh to ttansdarIDal s9mplin$ bes ed*yed dle co7teac8Od o"P
swest. Fuc eocample, M. Phdip and M.H.1NicAlooa Alcohol Clia. Exp. R,es. 4 391 (1980) diaclose an adsorbent pach which is a salt impregoated, cellulose pad under an occlnsive, ac8:eadve covet. However, such a method of t~aasdemnai sampling is dependemt upon tha swrst rate, requires sareat extraction by contrifittgation, and calls for externai chemical analysis. S.
Balabonova and E. Sch4aider. Beitr. Cierichd. Mcd 48,45 (1990) disclose Pflocapine-indnced swaat seaeti,on, but ttw system requires Iontophoreais-induced 'mfiWon of plocarpine and enatybe dihudon. U.S. Pabcnt No. 5,203,327, iesuW to Sohoendofor at at., disclosea en absorbaat pad under a watw vapor-permeable, occlusive, adhesive cover, but the system is svweat rate dependent and reqtdra chemical extrecdon and eutemal chemical an$lysia. F.P. Smith and D.A. IKidvhell, Forensk Sd. Iat. 83,179 (1996) discloses a cotton sweat wipe, but this systcm is sweat volume,dependenE and requires extrackion and extarnal ts chemical aaalysis. G.L. Henderson and B.IC. Wilson,. Res. Comman. Chem.
PatiioL PlmacoL 5,1(1973) dWoses the colkdion of liquid svweat following exeroise, but tha system reqnixea vigorons acercise, is sweat volume=dependcnt, and reqniYva extwtiom and uternsl chamical anelysia C.C. Peclc, D.P. Conner, et al. Skin PlumuCol 1,14 (1988) discloses a gat with an analybo bindiag rtservoir under an ooclusive adheeiva cover.
However, this reBKeme requirea exiracdon and external cbamical analpeis.

U.S. Pakat No. 4,909,2Sb, igsuad to Peck dixtoses a dry binding reservoir nndar an occlusive adhesive cover. Howe.wer, this re;fa+ence requires extraction and 23 extertw chemical analysis.

U.S. Patmt No. 4,821,733, issaed to Peck diaclbses a coIItction and detextion system under an ooclusive adhesive cover. However, this referewe raquirea highly mddve deteation components.

U.S. Patent No. 4,775,361, iswed to Jacques disclosea enhanwd s migration of aaalyte to a skin snrface. However, this referaeoe requires introducoion of Iight avecgy inft the body.

U.S. Patant No. 5,362,307, issued to (3uy discloses iontophoretic enhanced analyte collection across skin. Hovuevar, this refweme requires the introduction of electrical energy into the body.

U.S. Patd No. 5.=97, isaued to Bppstein discdoses attrmund enbaaced analyte collection across ekia. However, this ref+erance requires the introdwation of sonie ema and chemieals into the body.

U.S. Patient No. 5,885,211, issued to Eppstein, discloses micropore formatiou using heated wxxr vapor, pbysicai lancat, sonic energy, high pusure jet of fluid, or eloehricity. l:Iowever, this rr,fa+cnce sequires punchu+c of Yhe skin usbng heM, soalq or daati3cd eoarBY, Ahydcal or hydraulic ibrtix.

There is, thcrdore, a need within the transdermal sampliag field for a minimally invasive saqling tehmiqna amd apperatus suitable for rapid, inexpeusive, anobttusive, and pain-frte monitoring of important biomedioal markars and eaviroamental toarin exposure. 'The.se propeaties and advant:tges of ttlE pvnd 3nvEtdGII W121 bECOm WwOIIt to tlYoaC Of WMWBk opon reading the followimg discloswre.

BnIEF 3UlNIlMARY 0 g 1'HB 1Z3Vl~TON

1'Ire pzaaW iavaition pextaina to a tiraosdeml sampiing syst n, comprisiW at leaat oaa aaanpler for rairieving and transfwing at laa$t one analybe obtainad tranedeamaliy from the skin of a subject; at least one detoctor system for idmtifying emd quantifying said at loaat one analyte; and at least one logic modula for CI) r+ecaivieg and atocing iogut clata from said at least ow dewor, (ii") relating ft input data to Aer data obtained from to snbjoct, (iii) displaying ouVA bfm"oa, (iv) transmitbing the outptrt informatloa to anotlm syswn, and (v) cong+olliag *e operaiicrn of said at least one sampler and at least one debactox TLe preseat invention also partaWto a micmfabcicated dovica for ts allowing ranote monitoing of a enbjaM cwmpisiag: at lesst one sampler unit body for rett ieviag and =dwiag at Wst oae analyte obtained hansdemuUy fram th+e aldn of a subject; at lesat one deteabor system contectxd to said at least one sampler mnit body :6or idenffy3ng and qwaatifyimg at least one analyte obtained it+o a subject; and a tra~na,itter/rectiver foir mansmitting data ralating to at leaA one analyte dctected by said deuction syatm to a logic modnie for processing thac+cby, aod for allowiag control of tlu tnnerofabrieated deviee by a togic ayodule The praaeot inveation also pmloine to a miarofabdcat~ed devlce for sampling eaalytes from tM akin of a subjeet, comp:iainF a detection cbembar for rwaviag analytes retriaved fmm the akin of a subjec% a photonic detection system, comprising a photonics source located attached to said microfabricated device in association with said detection chamber, and detectors associated with said detection chamber for detecting analytes received in said detection chamber.

The present invention also pertains to a microfabricated device for sampling analytes from the skin of a subject, comprising: a detection chamber for receiving analytes retrieved from the skin of a subject; a patch which changes color when contacted by predetermined analytes, located attached to said microfabricated device in association with said detection chamber; and detectors associated with said detection chamber, for detecting a change of color of the patch indicating the presence of a predetermined analyte.

The present invention also pertains to a microfabricated device for sampling and detecting analytes retrieved from the skin of a subject, comprising: at least one conduit for retrieving and transmitting an analyte from the skin of a subject to a detector, and means for enhancing permeability of the skin ofa aubjed fas retrieving said at least one analyte therefrom.

It is an aspect of the present invention to provide a transdermal sampling SysteoL
It 's anotber aspect of the present invention to provide an integrated deectlcut sysbem using patch type detector.

It is still another aspect of the present invention to provide an integrated detecti(m spsbam using integrated photonics.

It is a farther aspect of the present invention to provide a microfluidic perfusion system for enhancing transdermal transfer of biological molecales.

It is ]td apOdw aspect of thC pmW1t lllVpttion to pG'VidC a dwmal abia;icei modmnign by noistvre heating for removal of the shatnm corncum.
It is sO anotaac aspeat of the preeW inventim to provide a laser abladon moch9niam f+or, removal of strahim corncnm.

s It is a fartfiar aapoct of the present invention to provide a microfluidic ~ df~t~ eepillary a~ion.

T't is anotbw aspect of tbe present invention to provide an adhesive for hokding WwM~nW is*pn8 syatem on sldn It is amodur stpect of die present imntion to provide a chiaaical modi8cationi of channel surfaces with satz'bodies containing fluaresceutly labeled aatigms that are expelled from the surraea and dcbected down stream by competitive bindbg;

A greader understanding of the present invention and its concomitant advantagaa will be obtamad by rafaning to the following figures and datailed d+escription provided 1s below.

BRIEF DESCRtP'T ION OF THE FIGFiJRES

Figare I is a schamettc illnstradon of the overall architecture of the microsystem of the prasW inventiion.

Figure 2 illastratts in am"mdm a single reservoir capillary pait.

Figure 3 shows test results obtained for a back of the band colorimetric test for blood alcohol.

Figun 4 shows the 9eei stsucta+e as viewed (a) from the bottom, and (b) in cross-section.

F'igava 5 is a aross-sectioa of a davioa of ds pis" invmfion illtouzting the non-iwuive samplite acqueax=

Fignrt 6 is a cross-seAiooml view iIlushatiag the sequwm of operation of the Bio-Fhadic Indegmble Trmdmi (B-F1T) microsystmn.

s Figure 7 scbemticaliy illnstraft the badc febdadon sWps for tlre thsee mAin components of &e sysoma, shoaa in aou-section Figure 8 is awbougc ilhstrafan of adetectioo4 scheme using fluoreecently labeled proteins or mwMbolitm Figure 9 illusuma in traosverse aection an atternative waveguide and sample to chamber contiguration.

Figure 10 iUusfttes a&F!T mi,ccoeqebem.

Figm 11 illustrabes a eeoae=seWanal viaw of type C bed iUusdrating the detoction scheme.

Figtme 12 Muataees an mrerview of the BLI3A microsyatem informational is connponent Figtms 13 illnaeaft a croea-secdonsl view of type CI svowing the microfluidic inimomect, ww1in8 tba extanwl tabin with the ailicoa capillary.

Figure 14 Modrdo a aoae-sectionsi view of an altienutiv+e which uses a siiicon stem acoimd tbe DRffi cgillat,F hole, 6owiog t6e siliooa sleave micaofluidie 2o ivwc~. coupling tttia eactarnal ttibitg with the SilieCa Capillary-fabTlCated as wafer tlu+otgh,hola, aud ex0enai Q~b1ag c0olrECied to siliCOri txitillarl-.

Figure 15 illndtaaa a a+osasecttoa- view of a tlubt+d bed savdm iacorporeting a collectio- ciuamber fs the smeb% wbich 1u flovred up ftougb DRtB cepillary througlk-wafer hole by capillary ac.tioa a Figure 16 illustrates a crosa-sectional view of a fourth bed, deaigmte8 type CIC, incorporating eollecdon chamber amd fluidic interconnect Figure 17 illiistratea the gaoarat Wcation process for tlve type Cl srray, showin5. (a) Pl-Oboresist (PR) pattamiug for siIic,on sleeveõ (b) oxide pffitarning of s slecve, (c) re-applicstion of PR, (d) pattern for DRIE of bore hole, (a) remove PR and DRtS slee.,vq and (f) remove oxide.

Figure 18 illn*dw a cross-szction showing the double sided grocessiog necessary to fsbricate de type CC (and type CIC) ckvice.

Figure 19 illushates amagiufied view of anchored spiropyraria in a silicon capillary.

Figare 20 illuserates a single nsservoir cqdlary pair.
Ftgnra 21 illu.4trms a siagle reservoir calrilluy pait:
Figure 22 illusttates fabrication steps for wafer #2.

Figure 23 illwtrates reQention volumes at varying concentrations of [3H]=EB.
1 s Figure 24 illustrates retention volumes at varying pH and ionic stcangt6s.
DETAII..ED DE9CRZTION QF THS PREEgtRCm RMBQDtAM

The pa+cseat inveotion provida an eubaneod systam and method for mo~it~o~ing t}m liealth of an individual and deliveft divgs to an Wividual transdeamaily. spacifically, tlte present inveWon provides an integrated, cost-dfoctive, rapid and unobtrusive assemnat of a subjeet' modical condition.
The invention tiuther providea meana for transdamal de2ivery of drugs in rGsponse to the -- oned aseLumnt of asubject's modioal condition.
Embodbmob include, for atampk, monitioring a subjed for pesticide exposure, monitoring the shess stabis of a war-fightrx; phenotyping using the enzyme N-ecetyl traasfacasa to indicm an infecGed or distseed stata; monitotiag extunal expomu+e and inwrrnal contamiaadon of a pecaoon with eithtr orgaaopl~osPhate ne~re ag~C (tabun, saria, so~~n) a or~C-gbsp~a~e inaecticidas (perrathion and metabolitea tbee+aofl; monkoring inflammatory sequeli ia rasponae to micmbial iafectiom (inlerlcvkin 1, iaRerleukin-6, tumar nec:+osis ficxm); monitoring microbial toxina (antbrax, bodvlinnm, endotioxin);
moaitoriag spm motabolites ariaing fmm hmnan catabolism via lymphatic or hepatic pAways; monitoi3ng shmulatrts srr,h as caffeine, aatihistaminee (deatomAmPhan- aaMirm); moutooring sbress fto* ahasiioos in blood glucoae concwfttm cr altmed metabolism of iasulia/glocase.

An ovecell architectxn of a pa~eferred embodimw of tha pream invention ie shown in Figure 1. The disposable B-FIT 100 is adaptQd to detect analytas of inoreat and Is moutxed in a neceptacle 101 to provide meclanical support and electrical conneetiona, including eteetical connectiona to the is thermal hestexs of the B-PIT. Tlu cwaecflon receptacle 101 also acottately aligns the B-FiT with respect to a switchable photonic baclcplaa.e. The connectioa recqtacIe aLso parefrably comaina a power source 102; logic control 103; and elatomic circuits for power managannant, electtoaic storage of results, elecwnic cireuits for procesaing bioc~l analysis data, elecianic circuits for timing events, and mem for communicating resulb 104 either dimctly or via tQhmetry. Optica! componauts ara providcd, paefarably located within H-FTT 100 or MEibLS physiochip 106. In one embodimaat, fluovmme meaaurennents are made sequentiatly upon each of a plmaiity of easlysis chambas eoatained in the B-FIT. Drug delivery chips 105 are siso optionally 2s provided by the present iavention aad at+e used to deliver potent druga ~Cat~Bder~ly, for exYffiple, alllp 1380d to OOUQtomet nom pS li>q be deiivea+ed. In adddOO, aphysiocb* 106 is optiomUq pc+ovided t1o sdbm coramuous biWc vital inormullao, ioclnding blood poressuro and pulae rate.
Tba toadanoauat mb"om localed wttbin the B-F1T aod dcag detivary s chip, fuocdons to contact tha akin with a phyaiologically compatible aolntlon or a physiologically compatible eolntion containing a drug. IU B-F1T is organiud iata a dose acmy of 5emavvhd mdepeodat a~ngle reservoir eaplllatl- pedrs. The capillacl+ pairs each eoeapaige a rexn-oir capiUatq 211 for retaining a Physiok4ocally wmpablle aoludoo, the nseavoir haviog a brr,alcable aeat 215 (itlushited in a suphzed atate), and an adjaeent traospat capillary 212 for ftupordn physiologically ca opatille aolndon6 wbio8 has contacted skin. to an aaalyta nmsming aite. An adbeaoive *er 216 is peovided apon the lower swface of the B-FIT. In use, tbe adhesive layer is totaposed between the lower smrfam of tle B-F1T and the siva, and a#aohes the &FiT to the sldm is In a praferred ambodiment, a themnal pwfotation aubaystan fianctions to ablate a mianecopia paaton of the slrahms oomeum, the topmost layer of skin, so thffi the iatasdtiwa cm be eqmed. IU tawmai perfaeation subsystem is preferably oompdsed of a micraheaDet In close ptoximity to the skin mrfaee, together with eleotticml eocmponenRa tbat control current to tho micro-hoatmcs.

A eapdlary arM a*qstem is pcehrably p:avided micxr-fabrlcded into ailicm weSets that eomgase the B-FIT. IU imentwu pmaibrably providoa a Phnrality of capiliasy=y snbsyomk eaah of wbich compnm a ftuid cklivery ahamber or remvoir chambm 201 to deliver a fluid to the skin surfw^ a capillary chemael 202 to recovae flnid from the sldn aiufaca, and at least one traaaverse aapdlry cheead in whtch the emlyu or aaalyiea an dcbecDed. IU
II

B-FiT 200 is pareferably co priged of e multilayetad essembly of miprmacbned silicon wafas: a Srst wafer 204, a seooad wafca 206, and a doEeatioa layrt 203. Tw ddeem layar pmvhrably comptism a PhOtonics syabem for viable ar fluorpcoe meaanamaNa, or a layec that comprises s oolorimatric reWmb that davelop a aolaar cbenp f n the prewm of an analyte, or otlw rneans for delerdoa of an andyta. T3te capiUwry aubayatem thus m fm mlY OMPiM capiittaiea for atone, pMage aad toalyaia of pbysiolosiCal $uids. The ftmetim' aad maPeoe aoadap of the capitlarfes are P4'elew,abil' oFtimsxed fnr controllln,g flow of t1Le fluid and im prevent to non specl8c adKrbdon of fluid compownts onto the cVillay wall9.

An opdmat iabegrated pbotomica syatam is povided by tbx Preaen-t invantiom to ddannine, eithet quaiitattvely or quaatilativelyy, tbo preaeoce of one or more analyteL Tba inawaed pboton3ea syabRm oompesm waveguides, lenses, wittMrs,liSht 3oucr40, and light datectM. Preferably, the integtated is p[wtonica sysm is housed witUia conaeatioa raxptac2e 101, which is attachod to a surfaoe of B-PIT 100 tlut feca away finn ft aidn. In some ambadbnents, the iaten*d photonlcs subMtam is replsced by a colmiaetric analyte seaaitiva region. whacin a c,olor change, pervadved directly by an obsecva, indicatu the pvsme oian matytL

20 F.uit of ihese aabsyalame aod the intemcam betwoen de sabastems is described in gmft detail below The B-1+[T praferabla+ aonidwns an ansy of somewha icdera-dew malyle seaamg davicm tmmod "3ngle rasarvo3r capillary pairs" 200. As used herein, the term "physiological flnid" rapxesub a fluid that is biologicially 25 compatr'ble with liv;ng eiaaut6 and ia, axrdore, isocrnnicauy and o&erwise physiologicatly (ft example, pM suitable as a modiam for centacft for exampK viable epidetmat ceUs or cella of tbe shibum mneucn. An example of a physiologicd solntm within the crnrart meaning is physiological satina solution. Each single reamvw capfllary pair preferably contains a reservoir s capillary 201 ibat atam and releam a physiological fluid to irrigate the skin suiface at a smalt region of the stratim4 comaam and recover anatytes. A
breakabk sea1205 is prefaably provided to control the timing of the release of the fluid to iaigo the sloin. The fluid is prefasbly recovered into a capillary chsane1202 that carries t1w fluid to an analysis location, for erample a detection patch 203. The ftmudwrnW sulsystem ptefdably utilizes single naervoir capillary pairs to ensm that the analytc of interest, if pa+a=t, is accessible to tiae fluid.

As the tearm is used in the present application, "ttaaadecmal dosifnetry"
refers to dw colkctioa and detecdon of tfaoa quanti#tes of analytes that reach the i s surfwce of the sldn by peasive dffusion from intarstitial fluid underlyiag the outeamoat layer of skin, tha stratum corneum. It will be appredaud that, in one ombodiment of the yar,seat invention, the iutestitiai fluid is sampled for the pmsemce of aflalybos of in" It will fintW be apprcciated ihat, in another embodiment of thc present iavention, ablation of a microscopic portion of stratum coraeum ensbles &e physiologic solution from the reservoir to come into contact wifh @ie upper regiea of the uaderlying viable qpidemnisr embling aaelyteat in inwWtial flnid ta migrnte into the physiologic solutian via passive diffa9ion for analysis.

"Noll--invalive han9dannal detection," as the term is wed in the presant applicadon, means ddecticm of snbstarices below the skin that is achieved witlwut phyaicai or cbemsW wodfficdim of tba namsi s1~m bearr*. SMU
moleatl~r ~t maty~es t~apt eo~abit bolh waoeaand l~?id solubilibiea caa be am3pW by aon kvaai" Uobiqnea.

For aaomaple, saad can be sampled fm tlre surface ofte sida and s analyiied ft aicoboi contmt by a colamwbic tat iadiative of blood alcohol concantcatiM ae illuutrakd in FWurre 3.1a thie acample of oon-invm3vt dadeotionq ak;olwl ia deteded in meat obtainod from tiw bab of the bands of sem mate aubjecta wbo have iosoed 0-4 alcoholic driab prior to the test.
Aloobol c,aamsiaed ia ft saroai rarm wft rageutb oontaloed within areactiv+e 1o 1ay+er, rwdfing ia aquesdindve mmre of alooW cawed of ttre blood.
IYoro-em, nao-invadve techniqteas aae M pu" wha+e the aaetyte la a high moleaular wbl* (for ecaba* Pvtoin), is highly pola' (hr cxampl0.
ohrooeo), or is poody solubla T!m outwrud ft of auch molocntes mcas the sfdn caa be grotly anbanaed by ablation of the aoratum commm Ablation is 1s pe~f'armod to a typicai depA of 30-60 m, expoaiag the undarIyieg viable apid=i4 from whoirb 8uid can be collectai md atiatyr,ed for analyt+ea that only poaaly pmOtata umblaeed most= corneam. This toclmique is b-erein teomed "muumally iavWva" bacwae only the akstom r.orna-nn is ablated v--bala the wdakjM VW1Q CpWmg813 Dd bTEaOloI. In one pd=ed emWdhug of 20 ttw presed invend0 , minimally ial/asiva bansdet =d ddoction is ac:idOl+ed by IIlicros~p~C l>ad 8bla&v of tlb *wum Qonm= 1ayet: In auAer pr4&SlIOd ambo1-mamt of $w pelft iaNaafioat, rnsa>mwly invadVC tz'aoedemal detaCdon is ecbiave8 by laser abk*n of the"atwawm commm tayea:

An adhadva layer prahrably provides tm inartm batween t2te clevice of 25 the peuant Iaventtco snd t!e aidn. Thc ahulva layv is affixed to tha lowat sarfice of the 13-FIT assembly aod funchhons to attaoh the B-FIT assembly to a suitable pordon of skin anrface, t3wreby minimizing motion of the 13-FIT
ameatbly rolative to tlhe sidn for effciem sampling. Gaps ia the adheaive laydr ace provided over each cqpllary pair to permit the physiological soludm to s contaot dw skm. The adhesive lsyer prevas leahw of fluid ktmlly, and is p~efmal~y coaoprlsed of a BendAid-type adhasive that is relstively aaber impermeabk.

It will be appccciated t1wt thet portion of the B-PIT that Wahm with the dermia pn+fecably fimatione to finly and occlueively pleux the B-FIT

system in direct contad the actatnat suuhce of slcin (strehmn onnncum) or nppormost region of the viable epidami9. Occlusive contect are preferably such that prevent letasl or vertioal movemant of the B-FIT from its initial position on the sldn, that limit release of B-FTT matecials acternslly, and pArechude entry of euternat mataials. Movemarrt pteventive properties iaclade prtfaably an adhesive element located peripherally on the lowermast surface of tiu B-F1T mu1/or covaring the entire 13-F1T and a4ecect akin swtsce.
AdditIonally, the lovennoat atnrhee of the B-FIT can be adbered tio the dermis to pmevmt sbeer ftm thet would diaplaoe the B-F1T from fts inftial poaitm The ocxdnsive mtura of t>re euwhmed of 8ye 8-FIT to the skin saves to confim all substances migceting fi+ont dw body or skin witLin the B-F1T, inaludiag water vapor. TTbis eapdu+od water vspor hatahaa tumdennsl pemmadon by hydrefmg the stcatum c.oraaum, r+exdaring it more pamewble to a wide variety of anelytea or tberVeutie dtnga, In one pra8erred anbodiment of the preeeat invention, minimslly fnrresive heat ablat3od of the stratum corneum is employed to achiwe significant is anhancernat of the eBlm of caain aoalytes. In pvfared embodiments, thenmal ablation is used to rmaove the stratum cornaum over a microscopic regicm of tha 9ioin tlsroogh a meclamism of resistive heating. A miao-allation unit eontaobv a micro-heater is pr+eFbarably fabaricated npon the mfsae of the B-FiT 4acent to each aepiltaey pair, and provides a cmduotive heat path to the stratnm comma. The mia+o-heater prefambly coaprisea a pair of elecErodes conaecW by a conductive pathway that is arramged, either by the use of a rasistive material or by a serpentine conductive pathway, to provlde sufficieat regstence to the flow of elect<Icitp snoh that an effwdve amount of luat is produced so as to locally ablate an appaapriate pardm of tba stratum corneum.
Electrical counections au also provided to each of the two eleotrodes to connect the micro-hwating unit to a controller that controls te application of an electrical ourrent souroe to the dectrodes. In prefecred embodianents, i# is advantagoous that the miGro-heatet protrude from the surface of the silicon ts sabsttate of tire B-F1T to provide improved heat tiansfer to the stratum conpaum anA rediux the power cons ption of the mioro-heamr. In one embodiment, a heat-sink maftIai is inowporated on top of the micro-heattr to direct the theanal Bow towards the sicin batnta rethec than through the bnilc silieoa nuaeaial. In notha embodimoat, the mscro-heatec is faboticxted onto a silicoa men that protrudes from the main silicon anbatrate of the B-FIT. Such an embodimmt may pcvferabty require non-plsnair hbrteation of eieetcical conneCtiona to pcovide conducting pathways from the silicon mesa to &e coadiguous bnllc sffiecro snbstrate. Such noo-plamr fabnica#ion ter.haiques are known to tizOSe of skill in the ark as illustrated in Paraqjape et aL, Technical Digest,1997 Internadonal Confeaence on 9olid-State Sensors and Actoators, Chicago, lilinois, Vol.1, pp. 397 (1997).

The thgomal ablation micro-hoater is pulsed with a suitable alteaaating or direct murent to provide local ablation. Control of the duration aad intmsity of s the heati* pulae is prefeaably eeuried out to effect ablation of the coneet area and depth. Tha micro-ablation preferably ocaus in a confined volume of the stratum corneum of appcoximately 50 pm x 50 pm x 30 pm.

Figum 4(a) ead 4(b) iIlustrate the seai strochue as viewed (a) from the bottom and (b) in cmn-seation.

A physiologicai compatlbk mMai that may or may not oonWn one or more dcngs is ramined vAin the rewvoir oapillaty 401 by a breakable sea1405 prior to use. Tlm seel prefatably peovide,s an electronically addressable means for opening the resesvoiar eapillary and contactir-g the aidn stufaoe or exposed stratum corneum to the physiological solution. The seai comprises a closure at t s the bottom end of the reseraoir espillary and a means for opening the reservoir capillary- In a prefeued embodiment, the seal comprises a thin meanbrane 400 that is preferably a dielectric bitayer that nTdues at elevated tempcratures and a metal conducting path. Preferably, any thin, noa toxic, membraneous material that is suffieiently tough not to teer prior to iatended use, is not electrically conducbn& and nWbm at elavded tannpmratwea is a soitable watmYal for use as the ar,al closure. Apa(mrod mgmial is low streas nitride. Control of the film stmn of the manbrme is reqniral daring fabrica#ion. Kinard et a[:, IEBB Trans. on 1nst. Meaa., 46(2), 347 (1998).

To fabricate the seal, a metal conducting paxh 402 is surface deposited upon a low-stress s0icon dielectrlc 400. Preferred metals for miCCohoWng eentEllts include e'YanobIIl. SinCC tbe bW uSd to rupture the wW

is optiomlly dw uaed to ablate dw skin in certain embodimcntaõ a carafiil balance of film stesus, thiclmess and rea3atance is prcferably achieved so as to provide both the deaired heatiag and rupture pmperties. Deposition of the metal upon dw f l:a dw requires deposition of inetat upon an in+egular topogcaphy.
Such techniqm are lmavim to tlwae of sicill in the ad. (iast et a1.,MT 7ournal of Resunah 95(6), 631(1990).

The conductive path prefetably taminates at two electrical contact pads 403, 404, to facilitate passage of electricity through conduetive pathway 402. In a preferred mode of operation, an electrical current passing through the thin conductive pathway hesta the metai of conductive pathway 402 and causes dw ropturing of the uoderlying dielectric layer, thus, opeft the reseavoir capiUary. It ahould be noted *st an advantaga of tbis pm'and embodiment of the presend inveation and tlds preferr~ed seai, in partieular, is that mechanical movmg pares are absent, thereby enhancing reliability.

In certain prefwW anbodWmnts, the seal seals both the reservoir aapiIlary and the capillay channel, and both are thereby opened simultanaonsty.
Figure 5 illvabam a B-FIT device cross-section, showing details on the non-invasive/minimaliy invasive sampling sequence. An exeatplary unused capillary pair S02 has an intact seal vvherain the physiological solution is re'taiined withia the resGrvoir capillarq; upon application of a suitable electric cnrrent, ttu seel 501 is nVurod and the physiological compatible solution firat contacb the sldn and is then t+ecovared into the transport capilisry; and finaliy a used capillary with a rtspture,d seat 500 is illustrated. In this prefetred is embodiment, each capillary pair fitrtctiona as a singie-vaa unit so as to utilim do seal snd physiological solydion.

gimilarly, Figure 6 illosfttw the sequence of opaations of a minimally fnvssive embodiment of a H-F1T system to detamine blood glucose concentratlon A micro-hesier 603 prefetabiy operates to ablm a portion of ttw stcatum comeom located below a gap in the adhesive layer 604 at thhe same time, or imaaediately prior to, ropture of the seal 601. Such a device ln+ovides an "on-desnsnd" analysis. A pbyaiological solution is preferably expelled onto the exposed viable epidermis and recovered into the transport capillary. Tlo to tansport capltlary psaferably conduts the aolntion to a detection pdch wlun the ghyoose is detactad in a eolorimetric rcaction that produces a blue reacion.
Note that in this pdmred embodimeat, the curreirt pulsea deliva+ed to the micro heater and the saal may be the same or difibrent; the heater and seal may, tlierefore, be electricaily connected either in series or paraIIel.

CapiUariea within tthe silicon body of the B-FIT device can preferably be fabrica~ed by se,weral techaiquea, for example by miero-machining, or by aMm8 m Pbm using deep mstive ion etohing (DRIE) toabniqwes. Rekrria8 now to Figtu+e 7, conshvction of a pmferred embodiment of the B-FTT is illustrated. The device compdrisea three main parts: the main body 700 which is preferably made of silicon 702, and contains aeverol serpentine cagillery ahannals 706, each with its own reservoir channe1707; a bottom capping seatan 701 that fmms the lower part of the aarpmtine atrxbm and coadaiae the micro-heating elemeats 703; and a top oapping setdon 704, whieb forms the upper part of the supmtine cbamdei 706, and which optionally contaim electrodes for assising the $ow of physiological fluids using electm)-osmotic pumping throagh ft harizontal segaunts of ft s pentine channel. The top-capping section 704 is, in some embodimenta, bonded to the main body: aII
advanbp of such an arrangement is good coupling of light into ft cqiltary that is tlmraby achieved. Tbe main body is praf'erably made of silican. TU m-ain body 700 and ft bottom capping sec,tion 701 are preferably pmandy affixed to each odher to compise a sensor 705, that can, in certain ambodiments, be detached from ft top capping aeetim 704 aflar nse and replaced with a fi=h array The re,tervoir and aapillary chmmels are prafecably fabrioatod within a standard silicon wafar. The dimensions oÃthe aapillaries ara selected to facilitate the tianspost of swed, interstitlal fluid, or other physaological flund, out of the open end of the reserwir mnder the fome of geavity, and into a capillary cheanet throagh capillary actioa. In a preferred embodiment, the capiUary channets are 25 m in diameter awd are appzoximately 500 m in is tength, and the reservoir cbatmels are 50 m in diameter but are etched slightly shorw t6an 500 pm in kngth to provide a back wall. A lateral portioa of tba sezpmtiae capillmy chamie1708 is provided, which provides for a region of fluid flow that is parallel and adjacent to the upper sinficx of the mWn body of the device for optical detection of anaiyte. Ths lower inside surfic.a of the lataal portion Is optionalty provided with a reflective suuface, such as a reSedive metal coating, to facilitate optical deteotion. Tle lataral portron of the serpartine capillary is, in a pi+afenmd embodimtnt, oomplebed by amrfwe of the top capping sadon. In nae, the transport of physiological fluids and the recovery of analyte is erhanced by rinAiag tlhe skin with $ufd pmevionsly maintained wlthin the remmir cbmml and t}xn reooveriag ttu sacg hto the cmEqOfld1Dg CaOUNy Cb8M0l.

The surfaee of tho capillary am system is pa+aferably fuochonalixod to iaipova &e propartias of ttm smfwk for axxampla to pmwart adsaption of pratein, aadlor to aftch bimolaw?res Wh aa antibodies m dW Mtface.
Molaculea ffiat bod specific amdybes ate uaed to inamobiliza udyto for ~ ddecdon and. quaoduive anelysia. 3uitabda biomoiaeulos Mude, but are not limitel t% anfi'bodias, anti'body fixgoen% actiEiciat aalfbodica, lectina, hykidizabla nuoleic aada, nuckic scid bindin8 Protemsi potem ffid bind nackac acids, pooins that birid otbec proiains, proteina #laet bind cofadoia, cc&ctors (fOr OMMPIO. &vins, PWin% thigMiN P)-ridoaal'!. qiaane), and othar reagaafi that VaciScally biad biologicat analytes.

Capiliuy tabes mre preferably modified by edher chemical or pdasma ~ant. This step alds siufaca cleaning of organic contaminaata aad i1lkoducE9 811fAce hydt+o7[yl groups on d0 tqnllaly StRf8C0, Wilich am pmftably r+eactied with a si1 e soh as aminoga+oPyl trimdhoxysilane (APTS) to pmvlde a fi+ee amine giovp aa an aod= for coupling raagenta such aa antibodim Iu a prefarad ambodimant, Polyaftfkno glyaol {PTO') silane derivatives are used to provide a snrFwe coating that preveub adsoqtion of protein.

In am enibodimezz, a sdudon containiag anti'bo8ies directad to an aflatyce of isftlest is expoaed to mMy oxidizing conditions kaawn to #hose of skill in the att wltich provides aldehyde gmups om the surface of the antibodies. Tht aidehyde ftctionatity is tbm caVled to a frce amim on the capillary tube surfem via a Schiffbasa reactiM thns immoWIzWg uto eatMM
to t~.' cqma[y t4om sIC1y1Ce.

In aprefernd ambodimatit, &ftction of the analyte of intaest is by done by means of fluoresctnce. A substance thai is capable of specifically bindin$
an s analybe (faf eocaonple, an antibody) 802 is coveleatly attacied to the mufam of the cepilmy, as deactibed previonsly. The binding sites of the immobilizal substanoe 802 am filled with fluorvsceMly labeled analybe 801, prior to use of the invention. When anaiyte, 800, is puresent, it oompetea for the specific binding sites, displacin fl a portion of tiw labeled analyte molexuiea into the solution. The degree of dispbeemat of labeled analybe depends upon the corceatcation of analyte in the aolutiM T'herefoae, mumumynt of the amount of fluorescence displaced into the sohrtion, wben suitably calibtated, puovides a qaantitative measure of th,c concentudw of analybe 800.

By preferably immobilizing a plurality of antibodies of difftrent binding is speoificity, the binding sitea of which are separately felled with their respective analytes tagged with fluotophaes with distinct emiasion and axckation spectra, multipie anatybe daermiaations can pr+eferably be made within a single capillary pair. The use of spectral fiftecs and/or aiternative ligbi souraes is used in a prafen-ed embodiment to photoaxcite and detect florescence from the different f lnorophomea, and tbeaeby; dcDamine the contrib+rtion of each tluompluun to the total fluamescant ptopaties of the sampk.

Praferrod flooroplores for the present invwtiom include drodaminea, fluoresaeiaa, Texas red, OreBon 8areen, Bidipy dyes, and aminonaphthalenes.

In one embodiment, N-acetyl transferase, isozyme 2, (NAT-2) activity is 2s measured as a marlcer of adverse drug effa:ta, toxicity and predisposition to disease. Tha NAT-2 phenotype can detected, for example, by detecting the rAHo of two metabolitea of caffeine produoed by NAT-2, S-acetylamiao-6-formylamina3-methyl uracil (ARMU) and 1-mCthylxanthine (IX). Utiliziung the ratio of AFMU to 1X, the aetivity of NAT-2 can be s _etenmiued. Poly+cloiwl aatibodies can be raised to tlm tvw metabolites and thea purified. Tbae autibodies can etad be used to detcct AFMU and 1X in urine sample~ by ELISA.

In a prdw+ed enbodimapit of the presw iaveatioq the reservoir aapillary is provided with a micso-heaiing elemat located at the opposite end of die capillary to the scal. The micxo-beateac is activated to provide local heating of the physiological 8uid so as to produce a bubble, tiureby Sorcibiy expelling the physiological solutm from tlue capillary once the aeal is ruptiu+od. Note that the micro-letw Ametions aa a pump measis, but that the pumping is achieved without mecbanically movin8 Puts, 6mby =urin8 Wmused reliability.

i s The micro-heefmg elements are prefarably comprisal of a resistive Gmducting pathway depaaited by convmtional deposition methods upon the mufte of the sllicon. UnMs &e brearable aeal, tlw heating elements are desiSnad to withstand ekvated tmnpasdm without destruction of the eanductive pathway. The conductive gathvvay is, in one pr+eferred ambodiment, aBerpcntine pathway, in which a high-resiatance pathway and localizad beat pnoteon are achieved ftougb the use of a ecrpentine pathway c.anm9ed of dia conductiva pathways drwely aaanged upon within a small snrt~ce area.

Amt>w pmfenW aspect of the present inventioa is an iAtegrated photoa,ica analysis suaayatem. The inftgration of photopics components into the &FTT system permita incceand density of assays, roduced size, lower power constanption, and demeased cosk rn a p+eferred enbodiment, the photanics com~anenb as+e liouaed wiwn a plastic housiag that ccm;rises the top cappM
section of the davfcx. Note *at other detwtioa methods a[e em-isnged in the pcesant invemtion snd an discussed beloa.

In snah a an intepatad phhota ios analyais mlbsystem, photanics sources, for eakmple LED's or lasacs, aire combined with detectos% waveguddes, couplers, and mmms, io provide a fiilly-inWded optical dataction system for detecting aaalytes in the pcasant invention. The photonics componeno are p7r.ferably located npm and attached to, the top surfix of the main body of the B-FIT device in a tap capping xction.

Polyma waveguides with conplers for source and deteaor arrays are fabricated as inftgrated "tlac ciranits" for mouoting. Fully integrated waveguide struchrres me falxieated by means known tu thoae of sldll in the art, sach as monolithic febrication of the waveguide by dry resist procasses. -Low rl wavegnide material (ij <qw =1.33) is preferre&

Figure 9 illnstratss a pceferxed embodiment of a waveguide and sample chamber. Ia ap+efetred embodinnen% capillety fluorescence is used to fleterx the analybe wdthin the capillaiy. LED sources emitdng green, blue, yeJtow, or red light, can be used to excite flumphrnres. The choice of exciting wavelength is dietated pdmoAy by the excitation spectrum of each fluorophore. In otha embodimamts, laser sources can be used to provide specific excrtadon wavelengtba, aithough the cost, size, and powex consnmptxon of lasexs is generaily higta than for LBD's.

In a preferrod embodiment, tlu upper innar surface of the la#eral portion of the sarpentiae cepillary is completed by a sudace of the top capping section 9M. QpttCal dl.hCtktt is pda" pwfoumd 1A1i1hUi ft jaftd p0lboa. T.&

is conducted to arrd $om ft ]atmel portion by aa inagial wavegaide febricated witbin the, pref=bty plastic, top tappiag section. The orierWon of the waveguidie rmos pardlei to tlm sl&on 3mhce.

In-mwtbes embod{tnent, tha treasverae aapillary iaterrupta tlo path of the wavegmide 903, so thet the light condocted by the waveguide pasxs directly througb-a pocdon of ft eolntion contaWd in traasvarse eVWmny 900. Tbis embodimenthes the advantage of simpluity: lenses end mirrors are not:+equired to diveat aad collat4 *a ligbtbam Fluorescence or 6wrbance measuremeats are pr+afierably made within tlo palion of the treasvero conduit tbd mwWts tio wavegadde. A ltt+eoeding oondcric partim 902 prdmbly wnftm the bindiag reagents that give r3se to the diaplacemaat iuto tlW soltuion of fluorophore when analyte ia puant. Subaeqeront comdtmt 901 prdaZably conductrs the sohrt&m out of the iigfit path.

is In an a>teraative embodimM of the preseat invention, light measure-enb are made within a cepMary tbet is constuuctod of a material haing an Wac of reftection lower thatt dmt of vvater. TNs embodimeat, aLio eliminaLea the need for leasas aad miimrs and offers superior signal to noise properties.

Figute 10 illustrates a pr+efirred embodbnart of the &Fit sysbem platform, To facilidbe the coupliq of light from tlm waveguide 1008 iato the laterai pordoq, and fmsn the lderal pmtion into the war+eguide, a mioro-miimr 100S is prehaWy provided.lU mirrct is bterded as a premd component in the tOP caPPiaB sudak or is a seporate compaoft placed witisin the plastic housing by injectioa molding, or is fabdcated by any other appropriate means.

Preferably the micro-miri+or 1005 is orieated at appwximadely 45 ralative to the silicon smfacro of the latral portion of the capillary, aad is positioned directly above the lateral secti.en. A highly raflective surfsce coatin8, such as a metal coatang, is preferably deposibed upon the nuface of the mirror to reflect light from te harizozdsl waveguide downwards into the laterd partion of the capillary. A lens is preferably pmovided to collate the Snoreacence exeitatton and emission light beama.lNticro-lem 1012 is, in one embodiment, convex to provide diverrce of tha light beam entering the lattral seation from the wavega{de, and canveWnt with reqPect to liglrt leaving the lateral portion and enteting the waveguide 1008. In embod'mments in wbich 8uavscex detection is used,liglrt firnn a}echral region capable of acciting the flnorophm is conducted along the wavegaide, stt&es the divergant mirmr and artera liquid contaimd vrithin the kteral oonftt. A fluo:opbare within the lateral cAndnit is preferably excited and emits light of a longer wavelength. The emitted light ts strikes t~e miuror, which converge.s the light, and re-enters the wnveguide.
Bandpass or notch filters may prefarably be interpascd in the light path to optimiu the signalto-noise ratio of the detected fluoresceacx, depending on the bandwidth eensitivity of photodetedor embodiment.

Light sources for the integcated photonics analysis subsystem include so LEIYa, wbich have recxntly bawnne available in light-emissio~a colars &om blue to green, thus essentiatly covaWg at least a portion of the excitation spectra of most crommonly used fluotvsow probes. See, for exxmaple, Fluorexmt and Lwmnocent Probes for Biologioal Acdvity. A Pracdcal Gnide to Teclmology for Qmntitative Reat Tune Analysis, 3econd Ed. W.T. Mason, ed. Amdemic 25 Pres9 (1999). Alternatively, mfcroelactroaic lasers can preferably be used whera speo& wavelmgths an required. Any Hght dCtection means can be used to ddCd thC emitted fl11o1EdaM W PbOtDdiodE!S, pbobotlansiBtOTa, Da[Hngtn jall' p~DLotiau68tffiS, or pbOti0reS13toTs Qan be fabTiCatOd onto d1C

silicon surfa<x of *e main body, or cen be provided as separate componeats.
Standacd low power CMOS fabcication is preferably used to powar the microsystmn, to provide sequmtial logic control, and to permit atoragc of data in mamory and its manipulatjon.

It should be noted tlat, dsapite the foreQoing diaciosnre of fltwrescencc datection of anatybea, the greuat invention is not rastricted to fluoresaence mesauremants. Othw dttaetion methods that ara advantageously used in the present invention inclnde, but are not limited to, Raman, UV-ViS, and FTIR
spectroscopy, iaoluding two-dimansional techniqwes, and fluorescence con+elation spectrmcopy. Ftndbamore, nuliation scnsars and magnatic field sensors are also usefW as the besis of clcaxtion in certain embodiments. For ts monitoring radiation wodcas and the like, a p¾efrrred sensor embodimeart is an optical random access memory (ORAM} matGrial. These mataiaLq are coiriposed of a phoWchromic cnolecule such as spirobenzapyram embedded in a poly(methyl metbseylate) matrix. Tlm measurament apprnach is based upon measmmiw of radiation-indnced tracks in optical momory media An optioal deflaton magic field amm is pmhrabiy utiliroed whera magnetie field monittozing is desiz+ai. Tln microaenm campaisa an ahuminum beam that is suapended above a miaomachinai silkon sabatrate using four aivminum soppport arme. These arms }mld the beam at its nodal gointay which ara points of zero displacanaxt when the beam vibrates at the faadmmentei resonant frequenay. A siewsoidal cnn+ent is forced to flow thtough one support aim, thrwagh ft kqh af tba bemn, aad ont ftough te othar auppozt am. The frequency of the sirntsoidal cuaKat is eeamaaity idw" to chat of the mechanical mood 5+equeaq of the bam. In ffie aboeace of a mopofc fidd, the beam is unaffected. Howevor, in the pmoom of a magaatic Seld mianted s parpmdianlac to dya beem, a magaetic fares =Lses defloction of eemes, wbich in thun cuaes tba beam tD vibrate at its resonw Frequwoy. The amplitude of the vi'bodoa ia directly proportional to the magmeHc fultl stran^ wbich can be measmtid nsiag a laae:.

Figure 10 ilualraw the opmation of an embodian+enl of the B-FIT

systrra with msped to amly#e dataaioa. The physiological solndon pefmbiy camtacts the exgosed viable apidarmis following operadon of microheaters 1006 to ablaa~ a poctio of tbe nhamn oornann, n4hu+e the seal, and expel the physiologicel solution from the reearvoir chamel 1002. Solution cmtaiaiag amayle rooovaea from the nftWd.t flW batbing tlra viable epiaezmia ts prefarably amera the cwillaty cbenne11004. Withia the capiilary cbaanel, anulyte displacea flm+e=atly ]abeled amlybe fmn aoatybe biumdiq molecuka affixed to the capilluy waile. Diaplaced flutoreacwtly labeled analyDe is pa'oftably cwried to the laberal pation whot+a it is excited by light con&xbed by the waveguida 1008, miec+a-mi= 1903, and micrm-lens 1012. Light of a longa wavelength that is emitEed by the fltamophora is, in one preferred embodimcant, con&ietzd back into the waveguide 1006 by the reversed optiaal pethway, and Ttt'oPagWas to a deftetor.

TU inbgmdm sepect of tie preunt kvmtioas also pmfaably inclnaes ft aspect t6et reat-time moaitmiag of a anbja:t pmmits the use of adaptive CCnW algoddom to OQmGi7E tb0 omddm (Aw examb, haoftg pltl8e C?lBiaC~[!$ttC~ smpZ>>g 18tei auCIIg others~ od drug davC1y regimen, in ICSpo178C Wdta o1twAe& jA ft pref=M embodimea of dLC lnvEndon, dft machine-teaining tecbaiquea are pcefaably employed to derive or learrn some function that relatos oae measure of the heaft of a subject to analyte s measuce~neats, thertby possibly acquiring the ability to predict tisr heaith maasuce from sibseqaed anatyte measoraaenta. Adaptive eontrol atgorithms utilizod in the present invention embody the stoxs of learning, adapWtim fieedbaclc, and daciaion maldn& 3im tle body is a dynamic sysbenn, dese steps oceur simultaneously and coatinuously throughout the Iife of the device of the to present invention.

Figure 12 illv.ftes an ovarview of the BI.ISA microsystem iaformadonal component A profeaei espect of the praseat invention is the large mimba of individual measmancots that are possible over an extended time period, With exLmded pGriods of ineasurement, baseline drift must be 15 accounted for so that significaat deviations are acciagtely detected. The present invention pseferably provides camputedonal meana for accounting for baselim drift, and for thereby detecting deviations from a current baseline. Tltia meems is illuscrated for an embodiment directed to monitoring health in a sabjeCk With improved monitoring techaiqnee, day-to-day variations in metabo}isn are 20 prefaably established in the healthy individoai, and limits set to deW
early stages of infecdon, disease progression, and expostue to toxins.

1U melabolism of exogenous compounds such as draga is mediatied by a series of enzyme.a. The type and amount of these enzymes in each individual is reflected ia the persan's genotype and, based upon the genetic iaformatioa, 23 individuals can be clamifial as more effici tt metabotizexs (FA3T) and others as less efficie,at metabollms (3LOW').1n lualth,y individuglL% tha relationship bdINl:EU gEnEt{c ntakbllp (gwx)t)p) and 11s G8pC3S1Ca1 {pbEDO") is conserved, i.e. FAST genotypes prodmce FA3T pbmotypaa, while SLOW
gemtypes pcoduce SLOW phenotypes. However, a diaease state of ttr, individual em alter tbis relati+cs,ship, aa em diet, smoldn& alcohol, environmantal cliemicals, and biolu;icat or chemical warfara ageub, among ot>srr faetors. The determination of a person's NAT-2 ganotype and the monitacing of that individual's NAT-2 phenatype can be und as a direci and sonsitive probe of luath and clinical statns.

In this apprnaeh, polyelonsl anti'bodie9 sre prafarably developed a8ainat the caffaine metabolites AFMU and 1X, and arc used to datemmiae NAT-2 phawtypes in an embodimart of the present invention. Blood glucose levels, cytokine levels, and d~n~Orphan metabolite levels, can also be monitored.

Macbine-leanoiag algarithms are prefembly used to acquite a metabolic baseline and to indicata whm an indfviduai's body begina to antcr a state of distress or diaease. The Wivaow and Weiglrted-Moority Algorithms (Lattlestone & Wannnth, Infosmtion and Computations 108, 212, (1994) can preftrably be used. Tlxse algoriftms, with well-understood format properties, are capable of learning and perfomiing in non-atationary aivironments (i.e., in the presence of baselise drift).

Tlm reedings of the two caffeine metabolites, AFMU aad 1X, am TrefamMy provided as inputs for compuatiwn, and the computation preferably proceeds in two altemating and cooperative modes: a lesming mode and a performam mode. In the leaming mode, the device prefacably coatinoally calibrates itself to the vearees body chemifty umnB ~~ipntbM

Wh1Ch atljuIb asa of VUCigbffiõ Wft ft ag of hodbwk LT9a bftwdoA IS
neCwmy o* lf ft b* is sftnt1awd in StlCtl B VYay- dw ft 1QNa),a of f'1d metab0lftes atE wt hmfiatii/!i of normid b* ft>iw10i1 (i.G., &0usG[ WlU
p20YtdQ fCEdback only ft h1SO-l1DgmiVCt). In dw pdollnatlCC moiC, tC device S prakably talm dw re&&W ofthe caffebe moabolite aa<l, uft the ctIIrint concaapt amipdona C~.a, waiorts~ makaa a decision abaut the bWs stdo of 2seatdt, wbich is tteeR coan~mmi~e~od to tlo wsar. Simcx tlo body is a dynamic sysoun, ft process of teenaing.. a&qWdon, f+cadbeck, od de:cision-moking paeferably ocaura cauiauovely md dmmobout ffie lifb of the davica The devioe slao pcrfrably acqni= a nmodal of the wearm'a be"y stgce, aad naas thia ntodd to ptediat swes of bealth in the fimura. Formally, maaltiwieaining ae6ods derive adr [m some ftmcdan fa from a act of x, y p" such ttzast y=ftx~ Nadnilty, fo is an approximation to the trua fimtion, which isunimown;

t~ &wova, wlben levels o$ saY, h'oP+mia I begim to increase (sngpspng an imm#aea~ imt a#ac3r), t6ar the devne wltl pctf~ably need to sample more fiequeady, aa dw rate of change fiam ona meesutommt to ft =ct wilt be incraasing. In this aitualion, adaptive conrol algodthms are praEecabip employed, powarfuT cougb to pmpearly contvl the sampliiag rate, bnt simpla enough to be realiztd in micro-lwdwat+e.

'I'!m in a prafatnd ftubodtm-mrt, a&ptivre conrol algeaWms m be nsed eo tasic tha aandoma3 componout tu aazvie its vweana far dw ww subatancm aod macl~ineie~autng algoritim can be uead to acqaira a mockl, wbicb may aloaA of the aeeacWa heatthy staft ~t ReOaTmO brdc, Plgura 10 illtstratee a pmCenvd embvdiaeut of a H-FTT
IIdCloq3aL lUt101dmodt118t sy*mpmhnMy ]11d1dES: (l) ta$Wd transpmt system iaahdiag nsarvoir dmmne1100% and cepillary cbamel 1004;
(2) micro-ba~ects)1t~06y (~} tbe ~ syelesa ~t wnv~udda 1008.

3 micro-mim 1005, and micro-laos 1012, sod (4) the chemistry for anaalysis of sabxEod anslyaes. The iu~1 fluid ooataining mmoleouies indictive of biaamadcers ara prdbnbly obtsiaed mdag andWangy iavaeive.ftchnkla onployng comtcollod tiewd mica+o-ablstion of the sEratttrn comaum, rw macao4mier(s)1006 neal for tins sre pmhtably incoapmvted dieoatlY iuo 8ra ailican-bmd snbaystemIhst,is paeof the B-F1T mia+osysbm For optimal tcmnspad of iakotitiat $uids or mabft through the snalpais capillmy. a second remr,oirevitlalr. codwnbag aCOMPIMe Suid, iepewi fix"O! 14^1y uiei to dtiis all fluiceftwada the "er swf,aoe of 11ss modula. The driving.
force is pctfQrablY Pmvided by micavheattr(s)1006 that produtce bubbles to ~s forrx ibe liqumd to $orov out of the mswMr capiliary and over the thermally, ablated region of the alria. Once the intekdal and physlologicd Vpopiate liquurds cam WWoS Ngpd end nnta0~a~d molaulee reach the toP hoMWS-cutY, maiysis caa begiu. The top of Ns total ttaosdermal drrtootian platEorm can PM& b1Y be bugmted with optlal waveguidea, comprLlns mioz+o-Wrror(s)l005 snd miom-lenses 1012 for pnopedy dhoeft the light within the lmlaino-chambat TU lightthat stiiloes t}w aualysiai rregion of the hotding-cavliy is asod to aaoaibe tlw fluososoady bned molecules. 'Tlm ittleoslty of this 8uaMeaee is preBeeaWy pickad up Onou,gb the rem peth by tlae seme ooeW wavegnida The modaba nstm of the miaroaystan providea an excelient plaiform that can be eoaity adapted for many itmovative gpphcattams by applying new chemistrias for tia detection of selecLed analytea. For example, one eaialyte or biomancm tbai is especially impottant tq c.bildhen expased to pesticidea is s aaeylcholioa. Aottyicboline is located throughout ttie body and whm it is released, it acts as en rxatatory ~urokaasanitta to pnnpagak nava conduation in &e peripireral aad ceatrai rervoua syatenas, or to initisto muscle eontraction.
Exposure to or~optmapboive peaticides causes iabibition of acetylrholiaesterage activity resuft in an sccuanilatiton of acetylcholina.
This iaane in acetylaholine oonceubiWon will act as a biontarkcr, maaured using the device by fat eetablisbing a baseline in aa unexposed cbild. The MEMS-based patch is smatl and unobtruaive, permitft a ehild to live his/her daily life while being coatiaatiowly monitored for exposm to peaticide contamination and ixovid'mg early vvming diagnostics.

t s Thus, one embodiment of tho portable biomedical monitoring device of the present iuveWon is as a pediatric micm pateh system (PpP). In providing sach a P P device there are three taslcs. Task i is the fabrication of silicon bed stefiur.~ tbat lLnctioa miato oua to the B-FtT Microsystem. As described above, the bed functions to deliver fluids to the intarior of the capillarien and to the collecticm cbffinber. In adclition, the bed mi.nora the &FIT mierosystem with regard to the integration of the chemistry. Task 2 is the chemistry to detect ac.etyleholine. Thia tak includes chemicaity modifying a 8k sample of silieon, wbich enablea a fimctioniag method for inugm*g chomiatrjr to the bed. Task 3 involvee the testiag and validation phassi whera the chanistry pxotocol is adoptcd for the cqfiIary bed Detedion limita o#Uetytctx-tme ara estabtistiaa md ssmaple bodily intiastitial Snid is tested.

with regen+d to the B-F1T system, the fabrication of the capiUary bed stru+churep relies, in one embodiment, on bulk micr~mnachiaing of silicon, accompiished through oitla deep reaaive ion etching (DRIE) or wet chemical etching. The DRIE procxsa prefiarably enablea the fabrication of high aspect ratio through-wafer holee that form narraw micro-capillariea of varying diameters. Wafess wrth a nomiaal thickness of 500 pm ara used; lrowever, a prefmre~d thiclaam can be established tbrouglt suface modification testing.

io For tltia eacemplary bed st<vcda~a shown in Figure 10, (type C), an arrzy of calrillariea with varying diam+eDars arie preferebly fomaed using lithographic patterniag and DR1E. This " of st<ucdme aaables selection for the optimal dimenaions reqnired for cqillary action to allow liquids to be dtawa up and inside the channel, becansa wet chemistries are involved in both capillary-wall t s surface modi8cations and during fluorescence vatida6on using a test solution cantainiog acetylcholine. Oace the capillaries have been chemically modified, testing for fluorescence is pteferably accomplished using a laser sonrce at the top-side entrance port of the silicon capillary, and a detector located at the exit port on the bottoam-side.

20 R,ehnning to Figare 11 illnstrating this paefened detection scheme, the spot size of Oe laea Iigtrt path 1102 am prefiably be adjusted to match the diameDer of salicon cgillary hole 1104 etclud in silicon sabstrate 1106, while its exatatioa w-avelength is preEerably held at 430 run, tD match the frequancy reqWred to excite the fluorophore causiAg it to emit fluonscot liglrt 1108.
The 25 detector preferably inclndea photomultiplier 1110, and monochromator 1112 is pteftably 11sal1 to tm 1h0 ddOCbOI' to *6 flUGraCCd00 W8VEl0II gl11 of 567 DN.

In ad8idon, notch fllw 1114 is preistably used to gra* Mmub the ~ law H& fiq11lmy from lwhia$di6 p~10~it~~af.

A second acampWy bed *a=a, tUMOd type CI, is sinails~ to the haaio s capnllM arM with amodgicWm to ft entraave pM ilO is fittad with a microfluudic iat +connoat. This deda is prderably used as an atberaefive to type C, in a eitvation whese capitlary aadon pethepa does not Aactitcm app~opr;abe~y. In soch awamftncak type Cl providea an iaterooimecc cnAchanism allowing ft extmMal tubing or syntige pMU to be dfractly oouglad to the siliam capitlsry for siwfece modification aad besttng purposeL

Figoie 13 illnsadas a croes-sactiomal view of a type CI bed structure show3ng tha nAkroflnidic wmmuect cowlimg &e a:tbmtnal Uibmg with the siHeon capdlwy. TYpicaUy, a irole produced by DRIE can preferably be made so that its innm and outer diwutws match did of the intarcanacet tubing, which is 1s insa6ed into die opming and Lieid in plm with adha9ive 1302.1"MA DRIB
microcapillades fabucaad as wafa throngh boles 1304 ara in dlicon subetrata 1306. The holee ara p'eharabIy prodaadd m6 dtat the iam and oabes diameters match 0lat of extamel tubing coamected to ailion capfllarp 1308, wbacredn the tubiwg is held in plarx with adhesive 1310. Floarever, cate must be takan such that the adlesiv+e used to hold flte tnbing does not seap iutio the capillaries blocking the flow.

Figm 14 iltusUdes a aoas-sedonal view of an allbenndiva embodiment which uaes a silieon sleCVe aroimd the DRIB cepjllary lyole, showing the silicon ,leeve micrmfluidic iatarcomreet, coupling the exwal tubing with tbe silicon 23 capillaq-. The sleen pmhta* pravides eahaaced meebukat in"* for ft oftvAt fluldic coqonml, bnt W0 pevmb adhooiva 14tr1 lYOm soepina mad Ptut~in~ tIN aopfllaq hole. Om tbs aokmt thing Is atbahed to tbe sUcon au6M, duniods ad unlytas adt pohroW be h&wd uft eftet Pmmra gndiaa or syfluga-1tie eocbermd tab" is tbm rmnovied, having 3 hMMM 3b putpm of inu+OdOCin# &ft i1o ft ImiCo01 ap11Wy CwmW a0d ft verMcWoat peOCOdurb to ddet3t fiammcenoa can soK as for the typa C
davZm ftm 14 that slaws adhosive 1402. gicat anbdOe 1444, DRIB
miaoc+qull~tier febcisoed:r wdhc thtough-bals 1406, and cacbewt tubft d to alioon ap0lary 140.

ta FiQun+e 13 iUumstu i. aw*4eaaomd vjeov afa ftd bM annwbzs i~~oorpavoting a oollec,tian atumabet for ft acAl* vr1dd-1as 8oweid up ftauah DRIB cqflLuy *=Sb-aft hok 1 S02 by cqUlary ediOn. For dds miaaohactoa, alkd type CC, tlw slloOa csp3ll" ie pr~sbdy l~brk~abed usini DRIB LoIIowed by sn anbow* weR a&Oa etch to c*te t1o collactton t s chssmber on #1re fiod4de of dUoon Meme 1504. With 116 bed, it is aafficiart to ahemdaally moft only ft aa0,1 1 of ft a~eoaber. Tlte snatyte pnforably avm up the csAaq abm" and reachr witlt the inumMized ahaminr3r ia ft chambar t4 paroduoe $uocmoenoe Ii& p~h 1S06.

pi~u~+e 13 alao ~ p~rted mccit~ton tasac 1308 end fluoteacao~ce li~t padt 1510 ddta~a~ aet~ The atat~ou and c~ecti~
nm~wd p[~ly mabe~ qse of aodt~don I~ 1 SOS, pho~omult~liet 1512, and manocl:roaneier 1514, respectlvaly, a be&% hoaswer, aaw ft map is P~eSea~jr a~- a~ tbs fraa~mlde oithe bad sOrootnse. T3e ~on~letpNat 1512 and mo~rocIroma~er 1314 +ae pe+a~ntbly set dirady above the coIlemou resacvoir to act n 6e 8uosesc oe damctoer. In thie slhudaa, tbe i laaer ligM an preforoW be diraKed Wwaeds the calloctiem chembw at an angle snch tlut ib tdWaa doa nat aaaubm to p6oto-u1Hp11er 1512 dabec"
Navedwlak notdi-fiitat 1516 can p+e6rably be used betwaen de datectiaa acit and the cbw6er to elimina0e mpr ahay liSU 6raom acciuga Isw 1508.

s F'*uea 16 ilhd*w a cross-aeatoiW view of a fm& poreibcred bed, dmvxbd tppa CIC, iaompm*bg colla~ta cfiamba 1602 and 8uidia it , ~ aamneot. In edditioa to the aMcan enollacy cltaad and colleadan clnoiber of tyge CC, tLfia bed pcct'enbiy alsa tnchuda a Snldic kmeonnect meclwaism a t6a bacic-aide of the wSer. As bdore, this des,tp cem pc+e8aebly aave aa a ta tltillbeelc modanim if t8e capillmy aoiio does not provide eao* cVWary ftm to draw de fidd up to *e collaction ctumbe:: Tu Saidia kft=med ptefiembly maioes nn c-f tlre ailicon slem 1604, as described in tbs type CI
taw"cd airuohnn DRlB pplllary thcojo-wdw lrole 1606 and silim aubs4rata 1606 are almo diown.

t s Foe wch variatlon ia bed *Udm% Mcic.dom of *e ttnrough-wafac cspiltay arrp is made on AnStco-aided poliabed, <100>-type 4 inch sili=
wafara. T6e mdty of capilltrl- hoka prdwsbly aandh of four diometw values (u W4 501ttm, 75 pm, and 100 pm) w&h a noffiimd Iasgth of s00 m, which oaaspoada to t6e wafrx tldc. For the typa C deWSn, the pattao for the 20 holm aro pea6em* fatmod in a ploor+eele tayM wbiOh acft aa an ideal m"o6Wror to the DRiB pocm A sm& (staadm'd1 hm OiC atep pc~efevobly produom Palta=aodke koat-"of the polished aitimsmbm Altttou6h the DRM pram mWers an tmilaoropically dcbed can*, some imderoutdng ot 8re mmlc ta1m plffix. This, dre pdoem cf *e naalc Woaa into 25 atu,onot this nnavaidebie laftnt etch to eclueve the desired dIameEers for the capilleries. DepmWing on ft type of DR1E system used, ft ratio of venipd..ta.labecei etch is betw thmt 50.to-1. That is, far every SO pm of et+ch depth, tluxe is qpwdmoaly 1 pm of under-etch bmeath the maaldng layer.
Tba maldag dbmmm m tharofm depmdmt on ffiis etch pffundw, which em be detarminod tMongh Pm testiA& DRtE services cm be obtainA for epwmple,Ajough om of tbe Nationml Nano- aa Faailitiea, or drongh the MEM3 Excheage pwon.

Figures 17(a) to (t) iIIustra6e a prafeaed gmeral fabcIcdion pi+ncesa for the tyPe Cl wY~ "win5. (a) Pbotwe" (PR) PftMill8 for silicon sleave, (b) oxide gatnning of steeve, (c) re-applicxtion of PR, (d) pmmn for DRIE of bore hole, (e) rrneaaove PR and DRtE sleeve, md (t) remove oxide. The initiel atep, prior to lithography, is to grow a thin layra of ftrnaal silicon dioxide over the enAim silim stnrbee. Pbotocesist is qpliod, and dre oxide ia patftud and etahed to deliaeate the locations of dye silieon sleevea that ate located atotmd t s each cpillary lmle praviding for mia+oSuidic ' Thia abCp is followed by aaothec application of pbotoresist, Ntd tltie capillary locstiona are patt~aed inta both phomocaeiat a d oxide. TJgou*wafier holes ace again formed using DRIE, as with the type C device. The pbotoreaist is subsequently retnovad, leavIng the pe-panmied layear of t>umat ocide on tbe Erot-aide of the silicon surfix. A much etwrtea DRtE step is perfow-,d, with the oxide layer actinQ as tba maskiog tayar, to cxese tlw amcon sleeve.

For both type CC and CIC the Wcsiion pzocess preferahly requires the wafers to be double-sided poliahed eiaoa baclc to fl,oa ali~t is reqWred. For the type CC device, the colleetion cbember is preferably bulk miao~aclri~
into the fiaot-siCb of tbe wa&ar using an anisotropie wet demiql ethamt.

Snbsoquantly, a thin ihetmal oxida is grown only on thehsradt"&w passivedon layer, while the back-side ia coabd widt pb,ctoroist. 'TU QRIB
pt I J jie for the capillaries is peaformed firom tbe baclc side so the cepillary hole aligns with otre side of the collection channbar.

Figure 18 illustrates a c~-section sbowing the donbte sided procaing necxssery to falnicaft to tM CC (and type CIC) device. Tha wa*r is then inverted so that subaoquent procasaing of the capillarias end siliccro sleevo intcrconwts on the back-aide follows the same procedura as that for the type CI davica.

With tc8ard to a psefntd staface modification aspect of the present invoation, the txAnicd qpoaob to providin8 a siufaoe bowd Bwmwat probe speci$c for the biomasler acatylcholine is preferably accomplished by modifsnag dw method developed for liquid pBase detection sat forth ia lnouye, M. at al., Napdatuciive Detection of Acetyl Cbolina in Protic Media:
Artificiai ts Sioaling Acxtylclmline R,cceptAta, J. Am. ChenL Soc.,116,5517 (1994). In a prafefvm d ambodinuk, dw method nd'lixea spiropyrans, wbich are inexpcnsive and n,edily availabla from comme+cial sourm. They aca lcnown for their spxtral popeaties amd ate very robuat. especially compared with molecules used for standard BLISA dataction methods. Thc spiropyrans are synthetically aurfice immobilized on dw silicon bed, in eidw a collection chamber or in a capiAarl+, uain8 sitane chumiatry and dandacd coupling cheaaoistry.

Figme 19 illustWes a magai$ed view of anchored spixopycaos in a silicon capillsry. A spfropyren (for enamplai C-methyicaliC(4kesorcinarene) is prefaably modified to incrntporeta a csrboxylic acid cc+asa-linking group that can be coupled to a frae amino-silane modified ailicon surfgce.
Stoichioineoric addition of base to the spfropyrmn allows for the rftctton of att m-bromocatboxylia acad (for exampla, S hromopentemic acid). '1U leugtb of this molewe is cdabd to its sotab4iityr and teeetion dfimewy. Lonm catbon chaia-leagft en mora solubie, but hacder to couple to the surface, wfiile low s carbon chain-lengths iurie kss soluble and mat+e Moely Lv couple to the smfaca.
The syn&esia can be followed by NMR speatroseopy as needed to eacamine and chawoeemresction poducM.

The sphmpyran or resorcinol/aeeWdehyde tatrmec prefaably fama a tetroplmolate in akaiioe nnedis that attanges ia a bowl shaped caviiy and can complax ~um cations. When complexed with a pytew modified N-aryl pyridiaiam cation (PPC), no flooreeomce is obmvecL Tha PPC may be pvrchmed ot syn&esized depaoding aa the selected method. One pnfarned method of incorporation of PPC is by solution complexafion with the spiropyran. After anchoring the spisopyran, PPC is inorodaced'and the complex is is fomsed. Tha mupedtiva biading of acetyio6oline kkcs off PPC and Modaoes a fluorowrt eompiau. This complex ia dexactiod osiag the laseddebecoor scbme described above in the microEab~ic~tioa approach. PPC can also be incorporated by forming mixed moswlaqera of the apiropyran and the PPC. Ia this way.

complaxes ara fmmed at the solution surface iaderhm Anodwr petfaTr,od way of malciag PPC complex with ffie 3pkopyrn is by synf>e3ticatly attaCbitAg PPC
to the spiwpyrm as deecdbe by Ynotrye et al., supas. This mthod allows for iatramoleailar queuching of the fluortsceace as opposed to intermoleaular qneaching as described in the fisat two metlada.

Upon compleSm of ayadmia, ahom anbabatea are pareibsably derivatized with si1a= sach aa 3-amiaopropylhimethoxysilaae. The reaction pwvidea a hee miao gtwp oa t6e s&on sur*ce do can ie oo*W nsing a anler,solubIc earboft'ida, such aa EDC, to the catoacylirc acid of do madi8exl VimP7+rm. 7C-nY Fbmdeuroa spectroscopy (XP3) +md tontm aqla aneasmuman can be employed to an*ze the progen of varyLq surFace s atdahmaat reaodouL 1 s prsIkaqd embodimaM the klg*at suilm covaop is acbiavad. Ia addition ta smrhCe covarop. the flmsacame efSciocia is aatamined nft s flnaamoenoe mia+mope. Tus " tt qualiiy* the activity of do atfa" spsimryeot. A aimple axperlmW monitoring tbe qnaiitadve fluoeaoeaos i~mbm~tiy be~e a~on of aaetykbo~lae aal ~bec t)~ ad~tiOn c~

to aoetyLaholias provides a 6uetiaa. At thtr poiut. do mmetbad is tremaitioned 9ato bed davkas hCtaft Dutiag do microfibtIcatiaa taalr, DRIE and wet ccmaW ets~- rataa m 91tib bly ddermhaed mftt* amapiar in aadac to prodoce the poper bed adrnctum In adcttl3m p[~e~aabt7r eampka are claved and viewed bouo a 1S sm mini eladron mim+ompe to datermim whadw de paper crOea-seraional ~eoase4y of dye Arough-wdbe ~ll~m haa been acldavvad. For tbs ahemistry maic, vx&ve madi8Wadon and csbm". ayndwWa is p+ehrably used to validate the iam~obil~tan ptmoco! oa a 8d anpte of aisoso. Z% ddmmiwdaa requires tba ddeattbn of 8namoam aRec oaritafio- on a nedativdy laW

20 nowle; t1wo, a flnovxammimveoape ia und during dds iesdagPeocedue.
Onoe ffs ckmical *z&wIs and strfts modiFoation tmb ace 6000 n m 1~ ~togfu6. do dwmidry is lhar teibd on the amail-acule capillarp bad structim For this pheik a,phOtondcaMued tw at-up is pmhra* anwloyed Woed on the ccaftda and acoiaa;aa of 25 flwrophomL BxclwOn is thnonA direct abeowpdm bom an exbaew laaer sotlYOe. SCf-0181 pmfeled soriTm are available for vaCloUs ftstn$ stCatliem, inclnding tunsble contianons wm (CW) acgon ion pumped dye laser, an aimooled arg,on ion lasar, a Nd:YAa nanosecond pnlsed laser which pumps an optical parametric osciIlator, and several smalle~ HeNe lasers with both red (632.8 nm) and gm (543 mn) wavel+ength outputs. T6e Ar ion pumped dye laser hes outputs from the pump lasex at wavelengtha of 488 nm and 514 nm, with a maximum powa of 9W. T6e maximum power from the CW dye laser is 3W, and is tunable in the ranges of 590 nm. to 600 nm and 610 nm to 630 nm, with an addiiional output at 577 mn. The air-eooled Ar ion laser has a single output at 514 nm and provides appwdmwly 70 mW of power. The Nd:YAa laxr has a flutdamantal wavelength of 1064 nm, along with the doubled (532 nm) and tripled (3.55 am) outputs achieved with inte:nal batmonic garargtors (KDP crystsls). The puise width is S to 7 nsnosewnds, with peak 1ula pmws of over 200 mJ. However, since the Suorophore being immobilized on the silicon surfice needa to be excited at 430 nm, the power from the tripled output Nd:YACI lasec can be used to pump an optical parameMic oseillator (OPO), based on a beta Barium borate crystal (BBO). T'his is essentially a resonant opticai cavity containing the nontinear BBO crystal. The pump beam is converted to the o-catled signal and idler beams, whene t}m wavelengtbs following the relation:

fvmv ' baw+ adpd which arisea finm the photon energy conservation requirament. 'The ratio betwm the two ouiput waveleugdw is govecned by the aagle of the BBO
crystal wiih respect ta the incident beam. Using this, the output wavelength can 2s be hmed by changing the crystal angle. Either the signal or idler outpat can be eliminated nalos a h* or low pssr op" ffitet d the ooxpnt pat of tha OPO.
T7a ouot is mwbb jn diffiawd rum &a sbcnt 400 nm to 2?AQ mn. The ronM ars sdt by the reeoeat csvity miaor peoparde md 6a ouqstit Sltera.
Pealc puLa meeget iia &wreftaa mon tbe ordar of I O mJ.

s Tbe oudpat fluoresoenos is pcehably deoecbed either !n the &rvvs[d dit+ecdazs at at aaode m& depeaft on t6e microattti~tura geooft In eider casq, the incident lesac li& is pcefaebly htooked uwq botb sholopphic aa" fiboc md s ma~oc~+nm~mor.1'Le notoh Stter, =mo* wed in Remsn speatromduk caq ed li& at ft wavalagth oftbe naMon li^ whs&w comias aectly fromn ft lma or $+ow ltayleigb scsftNin& Ths monoc~romatof pmvidm fitt&a rajeotion of nnwvmtied H^ botL =*mw ad to lsxr.
Ths manoVnromx dso allom tmft to tLe mdmima of ft eanisdos specaum, foe opGMkon of ft sipst-to-noise ratlio. Pimlly, detecdon is A1ato~itipl~a~ md a baxoat~inagrdoc P~~y ~~ n~ut a 13 aaoMAon schme, witti Pftframn We lawdwboaicLAftndsty, the signaf, is dftcW with a silTcoa- svslaocbe photoalode, pa+avidiag l*hae deteation aftift".

With ft type C bed, c*Ulay addon ie Pmhmbly tesed in oadar to, fuK modify the miermd affiaw mbo wtp of the cagillary boto; and tl!%
second, to draw up amtyEe, wbkh resCb with the im obi&ed aidmaii chomistty. If ft c*Uy faxces m nwt offaing to arsw up idpdkxa aa4amb of thM for dre c*Uwy dimmioos ba3ng testA than tbe type Cl teat-bed is pnimbh- naed. Thtt altowrr direet phy" inswdon of Suids witbia d,s cgillay udog a peoca patiw oe a ayriW pmp comecW to tbe mica+o$uidk MWCOaaoecta,ln eitlw ces0. the photonic datection ayatem is oft- illy USOdtob*d1Cfeasibft ofpa*caft bKapffWy $110rescom D~wuraIIUala. In calmrad, ft type CC oeOed l+tqtrites chmncu mdification 0* within ft A&M smrfaae of ft collection ctmbar Tha+eorr, ft device caa Wo be uacd to to capillmy acdon as vwell as t6e *tioaia ddection system s situated on ft frwl-" of tho wefer. Agmdn, the device cm pmfrably be fted pWallp by iaating ft eaa,lyoe 3nto ft eapillary srrey, us3q bed tM
CIC.

Once a partlcnla bed Is selaatedõ Rathw tesfia8 podmbly relatea to deberminin-tio seleelivitsr and aonsittvity of the bioovatec to tLe immobwzed demisky witbin the cxpitlBy mta. Thie type of teaft Is pmferaw omdmted, ft c=* by bft&mg= aotutlm containiag acetyicholiaa at varylns concentraation levels. By dakmlwna the amam of Suoavecence vwiadoq usingbe cv~ut ftm tlw wi& pwaspoiMng Chang" in acaqtcholine oonceatWon lftvU, a qumdtafve ia&aticn of the t s lower limit of detection of acxtyloholiaa, aad therafora davia saWvity, is obMined To Aetamiae selectiirily, anodW aariea of tOsts are prefmxbly perFrnmed to inaadrioe otber namiotrM.4 mitEo~ in variata aotuotraHone within the andyte sotuthm md to Mataine their relativm Quovscmce r+n& respeet to ft obbuned f bc eeetylc&line. The m&e eommoa n~uottaa4miitara tmt caa be used ia tbia teating ,ptaaa otbar tbaa acery+lcholinq iuol,* &&md*
dovamina. s=oooin, trypUmiM bdapuQdae, and gtyana. Far conoprabariadve teeft of selaadvity, tHe daviCe is pre92ably teod for other naaoitemt~e~s such e6 nomdraoatiae, "aaaime; glubm& aad, aspardc aGid, Wulaa, aad proline ft coiild intr+oduoe an imwamoed crab-sms;ttvity flnozmence 23 responee. Finally, in vitro tesft of the davice Ptef"lY using traftmlly C7Lt1!-Cted hmwsutW $iltd from h1maII donors is conducted. Samples aTC t8lCen from nndividuale acpoeod to high levds of pestiadea, and a COnti+ol sat is taken fiorill'11o8C WM WCiiC IIot C7Lpo9ed to toX,1C e2lviTOiliIIeIItS.

A sample result of this fabcication and tasting gncess is preferably a siliCoo-based capWlaty attay bod (PNP} wi& a clumicai inmo4obili~tioa paUtoool for the detcction of acaiyic>mline, which is a biomarket for oganophosphate type ppea4icide arcposuco. In addition, a sensitivity profile is established.
The P P
miaroatructure design allows it to be readily intatficed and fategcated as a module to the B-FIT transdermal sampling platform. Tltie transdetnial sampling prooess is prderably initiatM using a minimally invasive micro-thermal ablation iioatw to reach the stxtum cornemmb/viable epidemnis interface, allowing for mctiraction of iatetatitial fluid. The B-FTf miorosystem makes use of silicoa fabricated capillmry anays to allow for interstitial flvid transport to a gtuoosasensing patch situated on top of t6e acray. The tuie capillary array is structue of the PpP microdevice can be incorporated into the B-FiT. The debection mechanism for the biomarlcer acetylcholina in PpP prafaably consists of the synthesis of a flum+eseed spyropymn that is surface immobilized. With the chmaistry identififed, sudwe modiScaion of the bed is cairied ont, allowing for eaae of manufaetace of low cost, minimalty-intrusive ehip scale detoction.

The chanistry developed relies upon an alternate przfeared embodiment of the B-F1T miarosystem device, 'tttcorpasating a photomiCs compoaew instead of a gtnoose patch. Thia system is prafaa* fitted with waveguide technoiop-on the top of the am, wbich is used to tcansmit and dekd wtcitation and flworaacenoe lig6t, respectively. Apin, the P P microdevice is ideally suited as a module to the H-F1T, in view of the fluoresoencn datxtion of acelyichaline.

Tesft of simulded bodjr fluld and humun swm cae prefersbly be dnna on the test bed to detecmine ft xuitivity ad tLe Veai8city of tbe chemistiy.

Tbe praent inv"on aUom for tbe hbdeafiaa- aod chmmical imnamo 'b~tia~oa of mrir numbmr of biomadaassõ th=8y amft a xt of maduleu s to be "ptngqed itmo" the B-FIT pimtorm. Nummna exsmplee, possibilitiee, and appplicatom exK rm*g S+om a vast rnaaW of moleculer bomakrm for Wtb monitoft ~ mzyme ad mutabolite datoatim to hounonm For pesticide deoecdon soma of ft oOar kay biomakars would bs acetylclwl'm~ratie, aceft add, ad c.hotiaa. lt is also impartwd to deteat other aadytm eAW fi+a afgump~a~ made poaei* ftouo tbe use of the P P mi~x+odevica Thmie mcluda ~micticl~nesoense i~soctiddas (plwsphotothioaatasl opu,wdioriire hmx*ida (DDT, nioldrto, Lia&m).
PYredn'oid Wmdcidu {Pftmetluh4 Feavaie:ahib haMcides (fCDD. Paaqust), nd todenWiles (i1Vahninõ DipbackwiM sodiwn 8uoroaeetata, sdrydmina).

ts Utbec lcay biomadoers to trm rvoulcl be the aatidotas sarh as atropine and Par~lidooatn~e=

Pr+occoWns stap aod t6e respecdve equ~pmee in ft fabdogon of the P P pmfm* iaclnde cha l6llowtng: (1) tithogrephq: a fl+onNs;de mask alignar capabla of 1 m lioa reaadntion with W and cteap-UV photolfthopOy; a fncfiue capable of iwo sdded alignaxm a photo-nW spinnar, pro. and post bab ovens and asoeLoed ptncesdag aloudealt (2) depositioa4: a magne"+oa spt4tedng ryaEem cvble of depositiaQ meteJa (A1, W, Ni, Ti, P'k eoc.), and mag,ottron reaative VftinQ of oxsdea which can be provlded in the fabriaaioo; aa o-beam mparatar with three heartbs for low eanergy dqosifiion of inetas; and deposition %Vazu fcr PECYD oxides and nitiid+es for ooatiing mfices to adjust fa *mea aod adhadon; (3) frlm ftasWmtd. to adjust the dtassm and strengths of fihas aad mmbmw using rapid thmmal aaunling capability; (4) P6oto-maRtc dadga and fftcation; (5) eWing: deep t+ea+ctive ion ete,hear (DRIE), RIB eV*nent and wet TMAH eW=V (6) diffmon and heat s tcea:~ bigh oempaatta+e Rzrnaces capable of wet and dry oxide growth and fiamacx soak annealing which can be required far heateas comprised of dolled sdicon; and (7) measuteanent: a thin fdm streas tasDec and a Leitz thin film anatyzer or a Nanometrics Autamatic Film Tliiulaiesa meaguring apparatus for ~asuring film thielanm.lVlicroscopic examination is available arit8 a high to quatity Leitz micx+ascaFe and a Zeiae SEM with EDS.

The traasdermal tram~afes system (TTS) ia Prefmabty mainuacturod using various sdandaed processing and fabcicatfon fthaologiea. The TTS micmdevice fabrication also relies on sCveral micrnmaahining stepe, fiom simple bullc mia+omachining to deap reaetive ion etching (DRIE) procedures.

ts Tba fabtication procx,ys steps of the TTS microdevice pzefeiabiy involve silicon procming of two wafe,raõ as iadicahd in Figure 20. Wafer #1 prefssrably COII1j!!lSeB the YeSeCVoir C,haffiel, capillary ch90IIe1, n1iCr0-8bhfi0II
11nit, eIId b~+eakable seal. The mkio-oblation umt contsins ft micr+o-hester along with a heat sink to pvvide a 3rigbly oondnative tharmat path towards the stcntum 20 oomeum. By incorporating a heat-sink on the micaro-heatex, the ]est traasfer is more favorably direcEed bavards the stratum conaeum. Wafac #2 p+efersbly contains the rrsemir micao-heder, which is prefexably aligned to mAa with the top of t6e reservoir cliamei.

Figaee 21(a-e) provides crosrsoational fabrication diagraras of the wafar 25 processing sbeps for waEec A. Double-sided polished, 300 m thick silicon VVdn aY0 jlmfwft 11SOti in d13 pmoosft lbp bOCWeO VUo11dq VYIu be dot10 on bok dw $+nnt and bwk ades. InMaUy, in one ambodbnmt, the micaoabladoa hamor is fam~ed by depodft and pmnmabg a maal lryv ondo apammed eUcon di*cda tqr to hm a hatig ekanant "i sh w1dd s cunaa is pessed. Tlm dieleca3 c b pmhm*p~0ecned as squue rq* vahm the beafts elam" rea{des. A pr - A od lemar mrtaW can be saladed ftough tham-, Amalatba. ta additioa to the boadal etamed a tompadure aenoar can p~mabty ba iatqgded alcmgs.ide to mo~itoe the locat temparaWm pmaated by tha c~reat @n,+on& dw boiidng ooL Thia ie iadiaded in Ptiura 2Is, vdae to proae~ ccoun on tha top46 of f8s waft, bnt wt!! evantWly be invaormd to beoo s d6e bottom ?a dds vaMm offt desiM alt bonft peds and traoes m located in dye pluu of dw beadog elemat The meWHe tram and hoWas elamoulr are pt+ehrably iasulated iurd pmftaoed by dqmiftg a layet of lovv-a~rea. alica nitride scrou the wa&x Alffia-ugh sEfeat-fi+oe nittide is not ~s requhed fbr dds pedvation pzposa. It osa find an application in the subeequent Stp.

Renias Lacic to Fkne 4(arb)r tie aeemd oy b pe+e6eee* tn fabcicata the bcnk" saif. The nal b pdan* composed of a bilayer formed by dse toar-stzees 'alim nitdde 1a^ depoaitea in dw earlier sto-p, and a 20 aretal that m vvaan at devmd owfawwm Tha m+et whea+p the matsl is dapoaited data:mbm the location of ths ras&veit capillary-. Since the capillmy dimodon is prahmUy an dw order of 7S pmõ the baawlmble a,et must be sidMod ia ttb 75 m ration ia oWt to opan dw Marv* caANy. FWa+a 4 shom the bottom view deap of ths beadcabls sed. It eonsisb oftvao u low-em sikon niaiae tlap bddpd by the 9ea1 metaL By pmng targe =uSt cmzgnb ftcu# ft mmW ab* ft bed it dMMM to vreatoa me ' metal sW t3tmbry mess4 tba aitride Asp& AJft* the WW& Iayat is low in *Cas, ~'hy tana iS some coautta* tongdik or ampraaahv skain. By 4USIbM dW 4eVOSWM COMMIM C1f ft WICM Wk!tei it CM bC MA& IYl S.1&

s campflacra sv *a 4+bm dw aeal rVtmts, t1e aitride flap middy ad as uakkiitwiamd r+eim The ptrnfeanarl thud mjor procuoiag si+cp is to form ttm he:9-shA on top atdw mkr*4bW= heWng elemeaat As 4L%u=d abaM abmt-ikic pref=aWy dinca ft heat owarda ft *at= *ra*= rn*o1 of witWa ft buiic S&M sudW4 *Wrdh*Lt &a h*4h*, ft mgoft of do Not ftvds thmugh tt* sHoon, tmmm its &=W cmdcutivity is hagW tu ariar, By d~pWi #I a *1=3inm but-m'io3c an sa bodw, 60 ra*~* W* ftw is.
Vpuftapcly 4v3dad +a sre* bwnn aWcatt md al=MIML M is baatuse ft tbcmg condudvita d4o* sll= and aIumia~n ara oompuabln, bvt by 1s seltaing amew wJ& ah4bw tbem-1 axducdvfty *w d* dt'mlWm sad aiumiftum, a+tffCieat Im uusft m be acsevva& In a ~
~ftdWunt, aluaismma is oed os the hest-aink m*miai, haw4a~or ad&tionM
matzrWl can be ap-2i"bta. In aSffltift to *=W #ttw towards the *atm conwum. #ba pkcemmt of a heal-snk psabably redum s3w ovm12 distam betwoco the mm oE'hcat and ft s1dn bmia, taeby toducinj power =m=pi'i= 'in aiumimaaai Is pcefi,rbly gaftna .,.usmga gftmaffpwceduirv~
Howevtr, sime a*kk m+Cisl tria may be swdCd, Neftap eit2gà a t~i~ck *t=" is usd or ft nt=imn fs dqwft1 ovet the entire waft. 3'Ws may csm prablaas wft #he moW wat, thaefiamathinprvoWve 3so1a6g layer is 23 Frafaa* depwiWd prWr to- the aIwninwmr, T1wAminuzn is ttm litbographically pattaned atba' the fourth step, to rtmain oaly on tba micro-ablation haster. TU paitaaing is done at a latat staga in otda to keep the surfaee plamr f(it subsequGmt pcoceesing atepa.

'I'he fourth pndoned prooessing step involves imrerting the donble-sided s poliahed wafar tu reveat tbe as yet unprocessed sida A photoresist masking la,yer is preferably deposited in order to patbern tbe opaainga whare both reaeavoir and thin capillaries ara foimed aimultaneously. The.se capilIaries are both fabdcated using deep reacive ion etching in order to obtain nsrnw, high aspect mtio tturough-wafer hoIes. The thin capillary is pnferably deWgned to be 25 m in c3iametier while the rr,seavoir oapillary is about 75 m in diameter, both with nominal lengths of 300 um. During the DRIE process, the silicon is anisotropically etchea until holes eae made through the wafar. However, for tha reservofr capillffiy, the atGhing process temninatea an the silicon-nitride that is alr+eady present on the wafear backmde. Tbis is becsuse the nitride acb as an Is etch-stop fw the DIt1E etch process. As all pa+ocessiag of wafer #1 is now complete, the almninum luat sinic can be defimed and the isolating layer can be removed.

'Ttve pefwed piocessing steps far wafef #2 can also be ontiined by a cross-sectional fabdfcation diagiam, as shown in Figure 22. The sequence of steps is far less laborioua, however son-e atigament issuea still exist. The first rtep in tlib pcefarsed pnocen is to fabricate tbe rcaavoir hoatiag elemeent on tlw &ont side of the ailicon wafrx: Thia is paeferably done, as befora, by dapositing a heater materia! ontio a siliton dielectric mzbx. The material is pzeferably patterned in the form of a 6eating coil, and is subsequently covered by a protective siliocm nitride paasivatlon tqa. The prefernd next step is to deposit so on tlLe muBm an e~ch-stop dielecWo lqa. Next; the wafar Is inverbed md PdomW uftDR1B to Scrnn dw coamooft caFiWc! opaeJa& Onoe coambft tbe ftd a~eP Pcofmabir involvea da)oift a layw of aUca dio)dde cato ft Aie caaWnia, tbe nser+rok hatar. This aqom far dw Std arop, aoodiuily bonft wst6or # I wi& wafer #'I. Caro and attentio pWeaably ia taken to enm+e tta dw cesecvi* ladee ma0es vv3th tbe resacvok cgillary opeob& and alsa to mmnau+e 6at dw cepil-tzy bom wda # 1 coeaaects pcopady witle ttee capollaty formed ia w+tfa #Z.

F.ech capolsry and raeevoix peic ie parefan* ad~aod iadivikatly aa es Vo expoee mly m auch peir ie tle ei3n atfim m ordw to pert6cm a sWe 8idd andyeia. Oaoe emptayed, dw opea ad of tbe cwillwy oomdinw to remain mcposed to tba alda, bat b not sWc+ased far eny hr&w um A pee#ba+ed =bodimad boWo ad3hioad cowWwafim rapnftg the timing for signaL to open aeats and oo*al bedwt.lU total enuotart of is mergy bnpuEed to the 1atm that affeat the ablato of the skibm coaraiam and the dw over wLich tbat meW b imptzled we aleo consideradioiu. 1hm syatemv is dad~at aud tested foc min3md abutiw aereegp. TMt is, the miaimeum mmfae ibe miahmm dwwmia a aigi8c+mt parmww&n opmatioa- resuftgin mjnime! dae4pof the underlyingviable epideraiia, and 6wmSoce mininoizin~ the imrm4v*mdors dte pc+ooeee.

Otba timinS iam ara aLo oooidaatioiuõ Inaiuft the *mg of dw =
ablWan po ooees (!wln) widk rairiiaa to the apmdtg of a cspiUary aed. Dnniog the time dw 9est far a givem capilleh- ie being vVbnd, de miaoablatioa heater is prderabiy pvlW whb an appopda akernatiag ccoarmt to tiwm=lly is raaave mmuive layw of tba stratum camuem st Anotha timing considen#iott is the heater pulses amciated with the reservoir emptlring prooesa. 'Tht tiwymng of heatcr pulsmg is a censidendoII
to keap ft reseivoie flowiag. and not tahin up fluid from the stcatum corneum.
The heater at the top of tha fluid resavoir preferably farces out the liquid contents. Cax*ol of this ls3atar pecmits eomrol of the flow of iiquid during the reservoir/capillaty anetysia lifctim Prefmably tests of the varions aftystems an done to establish the detmatoxicoiogical and clinical pharnnacological advantsges. The test saquence is prefecably sequential, staraag with simple tests on varfous mataeials.

progreasing to in vitro usta on hmwa cadavw skin or animal skin, thGn to complete ammai te,ting end Snatly clinical p-henmacotogigal testing with human subjecta. Om sla valves covering the capfllary and reservoir are prefarably tested od opttcally acuaned for smusM deployment. Tha liquid reservoir is prcferably initially tested to prove tbat it can be emptied of liquid eontarts.

1s Initial tesb are prefarably atso done on adsorbeut sarfacea. Furtber testing is preferably done on a nonabsazbeut sofam to prove flow of liquid up the capillacy. Deoumination of the opamal flow rate for the resarvoir ad cqillary combiaation is preferably detmiaed besed on glucase conomhvfion at the peRch detector.

The glocoee deteeW patch materiai is prefeably tested for smdtivity using sMadard in vitro vwet chemistry methoda, to sasure tbat its cellutose platfuam-glmae debedc material is capable of reflectance densitometric detection of at lea,t 10 fg of glncose per pm3.

In vitro (using cadaver skin) and in viyo animat and hwnan biomeclamicd beata of FDA approved liiacompetibte adhesives and adbesive membranes obtnined from 3Mõ ?ne and Adtaaivea Resesach, Ina, are prefmbly conducted to detesmine optimal adhdfva componenb and skin preparamton conditions for occlusim fluid dght adhesion requiremeats of the B-F!T dmce.
Upon comrpletion of initial tesb of the B-FIT system, paroeliaical denmaLoxicological tevwg begins. Tlm te~s pedmibly consiat of a demonstration of the bioplryaies of tlbe device, done in vitro using human cadaver sldn or animal akin, aad evaluation of local der=-tological effects, done on live animais.

~logiaal testing fs paeferably uadertaican to aemonsbate the biophysical propasties of the B-FIT device. Biophysioat testing is preferably conducted on animal or human oadaver skin. Fnll thiclomeas hnman abdominal skin spacmx= can be obtained commec+cially fi+om vitron, Ino. (PLoenix, AZ) and ot1ur vendors. Ammal tim sampla can preferably be used to establish a baseline and mitigate cosb. Tbe sidn ssmples preferably serve as a platform to iavestigate and optimize the thermal abtation mea6anism. Tha B-F1T system has several diffmw vusya to ablate sidbn. Tbe goal ofthe heat/ablation step is ta remove the stratnm cxnneum witfi no dasaage to the viable egicermis. The first set of eacperimab prafecably debesmim the optimal ablation conditions, for example, tempanttue peak, puise dumtion, number of pulses, among others.

Testa to determim the opttmwn ablation condidone are preferably accomplislud using optical and elaNran mloroscopy and surfiwe profilomwy using an atomic force microscope in order to view and measni+e (a) depth and volwne of ablation hole, and (b) the epidermal cell suucthuat ,integrity so as to provide sufficient ahletion of the shatum corneum without peaetrating the 23 viable apidernnis.

To obtain a proclinicol evahuuiofl of seW in vivo animel testing of the B-FlT systmn is pratggabiy undwbb% utilizing for example, a luidm rat, guinea pig, or fuzay rae spooia Clinicd obsarvattons for gcoaa evidence of sicin initatioq ulcar fomoatian, and inflamoamtory reaotians ara prekably made.

S 3kin biopsiesõ axamiaed wdng Hght and aleatrarn miccosoopy provide a alosm arenaioatm of tire devioa biophysical affeda. Seial clinioal and micosoopic observations following zemoval of the device eaeble aasmmeat of the hailing time for the tharmal ablation lesions.

cHnkalP- mmoolo8ical testing m pdambly waeWm to aatamirne dw malyticai pt+eciaiau mnd acxuracy of mdiiode to detomine ghmse levels via tiranadamal swmpling relative to previously validaticd plasma assaya. A
prefarned aseay teobnalogq ia based on Ouco e oxidase imm~ob~ttion in micromachiaad capillkies. A validated plasma aasay fot glocoae arlth aaceptable limits of detwtion and qumaeation and with acceptabla infrs. and t3 intanday coeficimts of vsaafion is used to compare with thm assya in the clinicel sattindeecrUbod in the twa trials outtitted in datait below. The disclooed ttials am of idmticai dosign: the first in oormal volunteant, and the seeond in pai" wldt lype II(Adult Oasct) "ebes mollitua.

In ordet to vaUdW the analytical xmtivity of tran' damai sampliag to meam glume, tzn henithy men and aoa prepW women who have siped an infonmed conaaat, fa:bed ovaaoig6R, and bave bean scroened to sat fy the itrclaaion and awlmim cxiteria of the atndy aca mtolled in a aliniwl triat to moasure glucose eonexntrations in their plaema or intmdsl $uid before and during aglucooae tolmaRx tast. Tha B-NT systam 9s atmebed to the dorsal smrfam of the right hend using adhesive ispe. An 18-gauge iatravenoas catheter sa ia ineecbed im tft~ao veia in tbt !aft am. Veou bloo*lmhgw (aq.o' daWel,p S cc, ml not leea 8w 4 cc) m takm at qpoprimme iorvda for u debmimdm of pLams gluawe concentmtfom. C,omc~ons of Simon in the prluat ere ddeemined by a voidatad mapr, routiaely need in clinicai s xtdnp. TLees canoemwtiomt m caqu+ed to 8rae daoanmind in ktomittal flntd ~ d~e B~T gys~em. P1a~r aoa~iaw ~+e n~a~s~ed on 8 occAei+m at 1S minub iamrwle over two bom% wbile iataretitW Svid caacmtrtelom emnoeaocad fac O.S. l, 2. 5.10 aad 13 miauta periodsovar the aam 2 laun be5ors ft adtaWmadon of 7s omti of glucore by mouh These deft aa nsed to opdmin the smswft t3me ft tha &FCP ay3ani. Afbw dw WminlsMdcn of Ouco34 pLwmt aoomuadm md S-F!T syftm-aHmtted co~xxIratiome ere meerurad a 30 miaute intetvala fbt a fiatbat two hataa. ln 6alibqr vobmeem tto stuoore Plaou co~mu abovld rsnp $nan 80 to 140 mW/dl mdat flraaa cooMow (WnbWOn Mma ofMe"

is ThwqmWcs, 28tb edidoa.,1995.) Inclwian critaris fm tht clinW trail ma, n fcilows: Clsong 1: mm aod .
wamom who ~ wac iLe e0e of 21 ye~s emd tidaer tbe a~e of 75 yan. (lmnp 2: maie and f+bmtie voGmlema who m ava ths e,e ot2l and nerder the asa of 7S, and who cony dw dimpods of adah-owd dfab" by a bond-cer4tfied endoarivo1o21st. Group 1: Wft no pc+eacciption mecliatiom, or ndml pwducb. t3wop 1: Aorow&'Wg- clincaily memW labormoy vL%nes for ccmplebe blood coum% .ernm clembbiat tNt,1C. C1. Ht(6BUIK Stncoae md =atia=) and clinicatlyr mmalliva enzyme pro8let Sa0'!', St3pT, alkaline phosphebn aod bc'lirubin; ability to nadeeatand and caay out a sigaed infarmed oonod ckmlWng the plabocoL
ss Tho foIlowbag suNectb an exchuded from tbe trial: snbjocts wbe, in tha opinion of the inveshgokw, is nottcompliant rvitb the protocol requirecmeats;
and wommi who ara pa+egaant.

Once a subjed has wmwbd to pMfiicipata in the eendy, tbe fnllowing s prooedm+a~ are conducted. Sareaoaag ptooedures ere conducted within 21 days of atqdy initiatian and inciude: medical hiabory and pbysical exaominatioak reviaw of incinsion and exclusion ctit%ia; and blood and uuine specimm collection. Subaequant to inehdon in the study, subjects tmdergo the followiag 11 cm (1) snbjects aarive at tlx location of tlre clinuw tciat at appaoximately 9a.m. in the maming af ber an avaaight fast. Vital siens (heait tate, resphntory rate, blood praaa<me and tennperatin) are racordel.

The H-F1T system is placed on tlu dorsal smfaw of the right haad and attached aqcurely with tape. Rccotding ocovts via a 50 micron cauterizod lesion In the skin made by a small needle on the undGrside of the monitor that is not ts visible. The monitor is choCtced t4 ensure that it is recording. Vital sigoa (heart rate, respiratory sate, blood pcmn and temperahme) are reoorded once the device has bem attached owx more. Samples of venous blood (S cc or one teaspoon) ae drawn fi+om a catheter inserted in tlie a left foreacm vein for the measuremeat of glnooae acoording to the above schedala wbile dw subject is supin& Additional blood sampiea are drawn four hours and eight hours after the first. Tlm B-PIT syatGm monitor tape and device ia removed. Patiem am discharged and allowed to retmn lome.

With regatd to tte3 blood sampling schedule, five mL venou,t blood sainples m collected in vabaim in the mamm described above. The total 'õ .
number of blood draws duriug the conrse of the study inciuding the sareening samples ia 14,(12 study draws an+d 2 saceeaina dravre foar haaostolog3r, cheenietry, and liver enzymes, iespectively.) Tbe toal volume of blood drawn should not exceed 100 mIN Vital sigrn (leart rate, ngmAmry rale, hlood pesm and tempawriure) are Wen before and aftear pbcanxnt of tlx device and cathetar and after the last blaod draw baa boem taken and the davice and csthew has beea removed. Patiaau are encoucaged to report any wtable icritation on the am1 whm tbe davviee is placed. A pl~yaician is consanfly avaUsble to sabjaft enro}kd in die study ix ooncecna related to bsuisiag or ia"oa in the akia dae to the igavetm c~hetcr ear mnlt* blood drawa. In adcl'itim sympmms of polym3a aod polydypma are caref'u* noW and ludd attention to dutiag the study with diabebic patimta, and inanlin is available for immediate injection by plq+siclsm and ntusea ehould the need a,riae.
Statistical aaalyais include plearna glneose consxamhaCiaw deftmin ad using the clinicai plasma assay as compared with the valaes obtained neing the B-FTT system. if 1s the correlation coemcient is> 0.8 wi@t a significance p< 0.05, the measuramnb we deemed vaHd.

Tlre second clinial hiel with diabetic patieats is conducted u9ing an ideatical study desiga. Patiea#s are aIIovuw~eid to tats md hypogtyoemic medications on tha day bel'oTe, bnt not on the monning of the study, md are askcd not to itect insulia dutimg the stady period: oce the study pedod is over, patienta aro allovaed to eat and to resume ttj* ronfte diabetic regune.
In addition to the safety considaatiiona described above, carefut ciinicat monitoring md the availability of iasalia is paid great atftdon to wliile tlm subjects are mder study.

lU H-1!ff SysMM pVf0M* 11lliZl3nlny diffa=AIDiC1Ali'lcattOn bDChLOk&Aridftdeg[Kno&ghmsm&bL111[ ln1CNmachiningto ft miore com,plicmd dM rftdive iat dcft (DRiL). Refoaing beck to Figure S, a aros-so6m of a ps+ehrred one chrmnet dyet+em rmirrodevioe *ahows, s pnaferabty complsing of tlme maia ocmyonecft (1) the me3a body coniaioing wvecal ampmtitre cgglery chmmeta each with its own roeanroir chumd to unPls and anolym pkuotOpcdty cromptalb flu3d: (2) a bottm cWitog aection to farm @re loaror pot of t>se sapaaiint owtm aad to coniain micoo-iteatial elemeata m*=lly posate tba todumel layar for aubmamtial p}qr~olo~lly compe'bla Sttid mtftefim mad (3) a top cqping aecttoai which fofine the nPpw Pwt ofthe mpeadw eboud " if mesmy, to cmain electroder for asdstiaR the flow of Pl-ysiobttically caupmNe fl" using elactm- osmotic pumping ft+oagh harizontni awunts of tlw serpendne o>sumel. IU Srat and second componnb topthex &um the diaposabla modules t s of the ayetdao. Theaie iabetohwgeabla B-PTT elomwb ere inseroed into the main comect~n receptacle aft all amlarsis cepiltem bave bm used.

Tht reservoir aod eapillwy clmnob m+e pacoftblq hb9desbd ia a sundwd silicon waft uft dup reacNtre ien etcltiag in order to obtala nwmw, high aspct ratia ft+au*vwafat hoLes. The cspfliary a~etnelu m pzdoaably dWpcd to be 23 Ema in din*w with s n4winei lmgth of 500 m, while the reaarvoit ebsnael: m+e SO m in diaeaatar, but dchad aHghtly lesa thm S00 Wa.
T!m leteral portion of 6e sUpeRtIOa capitlacy damet is pmfaSbly formed by reewins the ailicon ntfaae by 25 }Wn. Thie r+gim ai'or m*rably has a highly reflective odei dqw" on the s3thce to MitO the mecfwnism ft op"

2S defectiOn of the awlyW AfEer bonft the silicon wId- a top cappieg sccttM
S~

ft sarpeaM stenctmca becoaus coMplebe. With tlM ditncasioae pDq~iolo~ic~' oonVatibk S" such ai papiraft or intecMal flnid% can be cUawn inroe tba opm als of the cbaeamb ftouo cepill:U acdan.
Ptiuftmare, ft fiuid within the rr,wioir is pmhably used in c*unctlon , w4* cegleay acoon, ad weew ow ft de:mil re#= 6eig tded. ftft udaftg in tt toopm of tlw Ph**"Y compaiible fluida thro* tbe 3maUer dmmnel. By aefivaiq the mutwl capifty claoei sudkm !o viftin a apeci8c au'&ndy 11hatloo, ft Wds cn be pee%ably snalyLed by aak"oody-anftm ComphagioL A swim of such ampffinift esch with its own to reearti-oir c,heoned. is caaWaed within asintle device ekmnmt. Facb soatqais a~ttui- an~d resecw~r peir ie ~ea~ai~ add~eaeed i~vi~ll~r ~o expoes anty one sueds peir tio tbe sian ra8ce in owee bo pearfoM s st*e fln&1 amtysi.t, Owe empb*ed, dN open end of tha capiiiq conthmn to remWn opo ed to the atan.

ts The botcom cappinQ unit is also pdm* made nsin sZtiooa and ewm two odier mejot flmcdam, eeida fiosn tlo mle of fOrMigg thelowler strochre of tbe eacpentine chomW. Miara acbined iogting elrmoanb mcorprded within tbds meeion aee preferabtynaedto dwmayporvfttbe owa s~rlloq akn~
Srodw awitaMlity of Waratttiat and PhYWalo"3' conipaul& hids witbin 20 tbe cbomeL. 3im~almaeoualyoaMming "02 the ~IDCIIIMGM Pxation peooedure; the miao-syabe= a+a pdaably ueed to iadivfiduatiy addrara esebt of the cepilLt"aer"k pdm Initiellj, an OPat eads of tlre chounal I in contact witb tbe akiia era covm+ed by a sat t1at ean be "btown" to reveel a siagle amaiyoie cplbry. This aeci3ng pcooodm cen be effeatWy cownollad naiM
21 large thermat gradients ia oloesproxnnity to the seeL such as thoae afforded by so 8mtbn In1C[D =IC~lE. lU m1Clo-boaftd we pTOfCtaMy InbCgCBbOd in tbe SffiCOII

region aurrommding each of 1Le aapillarry-=eawvoir ahaoneia. The coamGctiag micro-capillaries in this section am pnfarably formed usidg DRIB, eech iieing aligned with *s vatical mioro-cepilis" fi+om tbe maia body.

s Unlloe the two pev4oua compaments, the top cWin8 layer is pdmably made ont of plastie od used to aaomplish 9ev" tsalcs. F'ustly, it caanpleces ft uppar stivcdne, or laterd pmtion, of t3o aecpodae oimomL Thia area, preferahly, is ft deoection ragion of our mica+oaqabem. 3econdly, by uaing plastic, an imbedc{ed waveguide can pt+derably be fabricded wittinn tbe maoatiai, with its odwtdion ttmning peraliel to t8a silioon surfaos and forming the basis of tho intepated photomics analysis systemn. In addition, to couple the li& 8rm the we,vaguide into ft detectioa regIen, amicro-mbror is prderably inaegrateed within the plaatic. In additiao, for more effative light coupling and better effidancy, a micro-leos is preferably integrded arith3n the plastic located directiy above &e ddecun region. Tla mian-min+or is preferably intagratod as a pcesaed componeat within tbis top seatioa by using a tdangular form to indent the plaatia. Tlo resdting iadeftdm pse8aaably ha a 45 angle with raspoct to the sarface. Deposition of a higbly refleadve material is made on the ratiilftg beveled aagle to remder a anioro-minor that reflects the imrimontally-ditectAd light fivn tla; veaqpude clownwacd. Tha iaueamted miaolmm can also be stmnped dim* inbo the plaatie or cam be incorporated as sepamte unita placed witLin tbt p}Astic by ming q*d= nsvlding. In effiw caae, the lm dAes not naed to be of high quality, bnt ehoutd simply be " to diverge ligbt originatiag fim the waveguida TU illunination poduced by the light from the waveguide will eanse the taWd anaiytes to 8 oaeoe.lLe li& pmdueed is tben reflected from the bottom snrfsoe of tlre detecion region b cic through the now convarpng Iens.

R"ning to Figure 6, the dnrae poeeible (controllable) stataa of the individual microcnpolary systems sre shown. The laftmost mic=o-cgpillary s system (# i) sliows an achausted csqillmy pek *at has aiready been used for an anatysis pmcedare. This first pair showa the comgtdod thamat ablation, miccofluid flow awd captuee of ghwose ftom cxpoaed mtmstitiat Snid, encoimter with lbe gluaose detocdon petck and bluish color reaetion avidant at the upper sucface of the chip. TIo middle micro-c,apallary syscem (#2) is pwfoming an on-demsnd aaalysis. Tha riAhnost capillary sysem is ready for a firtura, on-demand analysis.

A pwrpose of the miaodeviee is to fecilitda the firaasfec of molecules of glucose or otber poorly peeneable analyte(s) from intentitial flund in the viable epidermis, located just baneath the inner sarface of tha shatm cornamn, to the 1 s dataetion patch situated on top of the microdevice. The microdevicx enables conted of the microfluidic sampting fluid directly with intastitial fluid by &amal micro-ablation of the shaum corneom, By dir+ect inDerfaca with indersdtiai fluid, tlo micradtvice mables aampling o& not only the normally-inaconsible polar moleaaias, but also impameabla larger molecutas snch aa grafieim The first in a ps+efeaod pragcammed seqneaca of events is the flow of alectiicai cxarm-t tbmngh the resavoir heabng alenient to create a mimrte $ydrautic prmm in the seeled reservoir eontainirtg phys.iologicaity competible fluid. The second and tiud steps occur almpst in uaison, and comprise two scpenaa caurents ftoatgh both the braalcable seal and the micro-ablstion buft.

lU sqil pL+kifl,lably is ameW-MOctric bllayei t'11At 11ij}tim at elevaLEd maq 1110 gd. nB l11eW SCaI is jrftfxft surflCe depo8ltCd on a IoVi- 3ti'G9S
siliCG31 dieleCti'ic elettlt'!at to TedtiCe te c11anD!`S of CC'lIIpl+0mi8itlg ft Seal 1IIteV* pft LD itS opet8do11. QQCe ft 6E81 is bmkeA dlt[ii]g an 80a1y51s s procedwre, tbe phydologicaily cemapatible fluid pmferably flowa down from the reservoir an+d aaross the negion ft haa boeai thermatly ablated by the micro-heatex. During t1u time tbe seal is beiag rupUured, ft micro-ablation heatrx is pAtably beiag pulaed with altornating cun+ent to thermaily remove suceeasive Iayers of the stratum comeum, which is typically about 30-60 }-m in tlrickness. The micro-ablation prefarably owirs in a highly confined volume of the *aIrm. cornaum, appt+oximateip 50 pm x 50 pm x 30 Wm. ThQ
Physiolo*cally compatible fluid fim the now opaa reservoir intefwes with the iatwstitial fluid and, due to the dnal actions of da resea^voir heater and cepillary fove~ ttia mixda+e is ttansported towerds the detxtioa patch. The bulk ts of the physiolagical sampliag fluid is p7refeiably fteed out of the reservoir, emptying over the skia suBice region wd into the aborbw detection patch. In addition, a,stmn& Band-Aid-like adhesive film preferably lceeps the mi<xodavice ia Suid-tight contact with the slcin, preventiag aeeape of intecshtLal and physiologically compatible fluids Erom the analysis region. The fluids are pzeferably faaced up the soalyaia eapillary to tbe dete.ctioa patch, duectly above the capillades on de miorodevice and, foz exempla in we enibodimemt, gmorate a color cbange to ind,icate the presem of giwose.

TU mia+osystims component of the pcesW iavartion is pmeferabiy towd oa molccular aca(e mmipulaHon using enhanced tansdermal ftnfa of 23 matabolites from intasdtial8uida, and resultent deuction with enzyme imawbiliud ctmiatrieL 9ompla am prefienbly oolkcbed uft a mioimaliy nou-6avadws teusdemd nbodevi s md tnos qamWn af wiy0e4 ahdch fftch the sioa swfkco by pueive Msion Erom Wm*st flndd uadendft ft ,..
on oecanooz bqmrcfdia tft sttatm catommm) cm be aatxtee. sinoe t1m anatpm 'on&m bn othar para of t!r 1wd3-, tmparW to tio iobmdU
floid via blood dmvlx&4 tLsy redeat a vedety af plgreioloScd peocesees Inclnft body cqpsm to asvircmmrtd cbutdcab oe miarabm as wait a iatosad me4boliam. Mao-lqeai of tto ~comm m go* rammvoed ~ uphtae of WmdW 8nid fum ths viabb apWecmio, wmclt 1ia juet to bemeo& tbe dtdm coorArr.

A pc~efted datedla echam OOe deftmadon of heddi and oHw impcet,~eot 6dd,*cal noedcros utitizoz aiaalar stwilCe and bdochmishy fa each aaasy. Rdmiol to Figae 8, a Fmhrred proce&m for the detbctian soboare i!.
Aown. A pdwad ptoaedsuta Is to covdantly aftch mb"oo" of the pmEein or ts metab" of inmecast tq a capilLry waU 69 inmcpamto a ftaaawentljr twd aadm. The oWd autiten Is re&ced by empeOva bin&q with the protein or nmarabolite ot9onat$aa the kftrMd fluid so"iod. TU$uoroscart ~ Is kidced off ift aolatioo to be deoected dawn straam in the eollecft chsmber by ft phoooWo ecmpowmL Me Snoropbow aodoatan mxl musion 20 c1eraatorisdce are mddwd to ft pbotoWa aaid. viso va[so for idepaLing ft ,i& "vedamm aouM de~eoboe aad Ahn le peopaely aoft ond ddermdna aWaim in tbs *Ww ma"

As mmmok de Eabwint tln" pe+owiw of vuious motewVr weigbb ean be osod for monking ha& PmPada: h+opooin 4 Cracdve 25 pc~otei% md predbnmmia. Avliaroponin I ia oovdamyy attached to t}re capiilecy 1Nau 13uoWiTlg! SIlg= m]Lftb ucauftwoY MWOPOMM, (ppM TmPonin I is &wKesoe1y taWd using asdbr fluoruceis ac rhodemim ad botmd to tba atdbody aftched in ths capillm. Usin compedtivre bindin of Iftm swvft- tDe fltwMcw* bWd tropcmdn I is iepLmd inoo sotWOn md &etected downoem. The 4bovro pcocedm cea alw und for both C-r+esctiw protain Od pmaogouaW, al6eit modi8ad to talce into eocant dWbreoCes betwoen &m pmteins.

'1ha aurfeoe cb+emiaay is dowGaized atopwin to easm+e ad%chft surAce wabog. T!u mount of brnmd en8body od cauopetittvre bdnft s"a is uted naiq s vn* of diffmed hetnmamb such m XP3, Ammem plate raadw, flnorescw micaosoape, ct aepiuradon %cbaiquae.

In moffiac fta ple, polyotooai wtibodies raised egaiW tba ed'aiae noatabolites S-Mcdylamino-b-fm,yl 3-meft+i urea (AMU} aad 1 raethybamt~im M m immobili~ed on the micxomachinod cqi&zimThe ts aapilta:y tnbo are modiScd by cdunicat traftand in mder to iatrodnce hy+dti'oxyl moieda on the cepiUWy snfta. The surfiwce hydeOxyl gi+ottpe ene &m reacted with AP'I'9. prodnctaa t molecular utr wieh $+ea emina moieties at &e eod of the ftvi.bon ch"

The sW red&ws in the Fe r4on of tho amtibodie'raiaed apinat AFMU and IX an mcidized uaiq Padodate to genonft aldahydae. Tln aotibod~ea aze anchorred to sutbee of tbe mir+amsc6i:red ca)Waia tlu+oq*
Scbiffbese &anadou. betwoea 6e stde6yda on the autibodiw od tbs emina on *A moleilw tether. In ti3o mamar, tire antibody Wmft r4ams is dk%-ted away firomm the auf ux of the miaromshiaed cheaneJn TbC amotmt of agibody 3A11nObl~izatloll on thCowim Pf*

microclamcl is preftzbly de#eamioed by analysis of the protedn contmt of tba biading solution before aud aft aq)Oua to the microcbaancla The binding sctivity of tttie immobil'rmd antibodiee is deGanninad using displar,emmt of ft s fluorescent-labaled AFMU and 1X probes and ge obsaved aarvity is compared to the aativity of eqtrivaleot caorLCantrations of non=immobll'rzod antibodies to yield binding affinity per mg of immobilizad aatibody iadices (BAl}.

For #he caftu metaboliites, in vivo and in vitro testing is conduobed to assm dw speaificitq of the aolibodies AFMU and 1X. The abilfty to measure the ratio of t6ese metabolites nsing the device of the pnse,nt invmdon is asussed by compaiag tbe ratio obtained using tLe portable biomedical monitor3ag system with the ratio obtained employin,g conventionai HPLC
mediods. Daviaes modified with this assay are tested both in vivo and in vitro for provide a prealnical evaluauon. These data are ntilized as a baselin+e and Freliminary dats for testiaa ft algoritimn.

Aatibodies for prreelbumin, CRP, troponin I are ciutmtly available on the malaet and sre w ed to aeaees the specificity of these eatibodies to tbeir protein complimaat. As some commaciet aatibodies are not active or specific, this pnscseening test is pabind to determiae wtivity and specificity for axich protein of i~t. 3peci8city is prefably tesbed for eacA antibody by adding other submm similar in aowtura, wh{ch sltould not arowreact. For uamplq in assesaiag proelbmmm the protesana such as albumia and gtobnlim, among others, ane added. In as9essimg caffeim metabolities, xamtbiam and xanth3ne metabolites are added. Assaya using tho aaribody, the ]igand and t~e ~r L~bded u~od ttte P~e~b1y ~v~oped nd~ ~ech~qae~ a~ us ft.r i*Womt Eaoh camnpleted amqr IS soomed fat acau=y. ~epeoduc~itF. litmearitY, amd rawtat tuoa ooooqud wbhthome of msft pcaoedna amu* naed in ft A" bbaaioy. A poii" enht" fa ia viro md in vitro taft ie done eAd ffis dqa b uu=d k #ie a1sorltimn devektpod.

11te,pawle btame" monitodno eyarban nttbe praant invmbaa ia pt 1 ieeOP ly issed on molseWw sca16 numtpnLdioa mft eaimmcod awatdomW
traow6e oimedrbolite~ rrd odtat body mddyler uft todemd dosImetry to immbibilind aadbaft, In mktoabowli, oapMo =iau tbr 8ni& mobflitY, and mbgrbd phatxdw ~ior doWodoiL Tlnu, the aoicro-6ddiC cdp iaredce tacbnalo~ier of ft pssft iavido peo" coftelied swgle a&cdm fi+om lW fluids. (cdaoWjory md noadralawy) ned ft ft catroIIed Mvay of ttaids (Mov, abemicstls) nd tanet prob" (paHbodiat, "einer aSnea is molecniU). In addittaa. sampW collectiau pWfwms of ths pasW invantfan cta siaoutt~meoueiy pmp~oy a~ "owtvva~d Sc~" da~viae croaapom~a~ fa ~mpling mr a: iiqaid vome mu:vhuwn@uW terpt su*w ood an "inwmd flciug" compoaeart ft detecdca oit>tr" =*" amwmft ftat ft " 0ur~x dc aooeaft bWyH"

20 Tbue appanftmd prooei~ atpact"bdome"moafto& disalosed haeia is adpb& tia a wW wft of &wft%& Far examplq ihe pormble biomedat monift* dwfce catn iuethde ct3ps w&ch =dw loft (C*
"A") and illaeaa cc ia"an (t* "B"}_ Ch*A cm mam maleaulea l&a ghmee to estsbliah a b"ae ottht subjecb heeUh ate& in both madat aind 23 1>;I* stm dtusdam CMqea hm thm bueliAe lircib wi11 signal a nad for Chip H. Chip B is deeigned to determiae the eusct caute of illnds. For example, Chip B can coutain antibody ornopugatm for pamthion and ita metabolites that emulste a chanioai w+ahrce agent. Tha saucture of the miaosystem is adaptable for manW other types of ohemiatries based qpon drag meiaboJism and/or "probe S draga".

Drug metabolisn is the praxes by which druga are converted, by enzymo-catalyzed reactioas, to praduote or mefabolitea which we roadily excreted in the urine aad bile. One pathway of drng metabolism are phase I
reactiimm which involve the creebon or modification of a fumtioaet group in the suba4ste moleeule. The cytocbrome P450 -depodeant (CYP) micrmmal mixed ftac*on oxidase system ia a very importaat enzyme syateau for these reactioae. A second ma,{+ot pathway involves phaae lI reactions, in which the drug or a pbase I matabolite is cbnjugaed with a water soluble endogwouw sulsrate. Pheso II nactions iavolve a divem gmup of enzymea knawa is oollectively as tudmim. This gtoup inoludes UDP-glncuronyltransfcraae, UDPSIYcoqftMnshm-e, 61"bionee-S-tranatbrase, sulpbotcaa~ese, mechyl>zaa~a0. atrd N-autylti"aashrese.

Drug metaboliam is affected by dietsay aad emmonmwntal faatora. For exaMplq akOhol, caataia food oonstiamts atud compounds in cigacette smoke have been obsecved tu affat t18 biotraaafurmation of mamy dnugs, as have iurdustrial pollutaats md peetioides. Gematia factora also play an importGent part in the canlsvl of drug medtboliun and it has been observed that there is much variabiou ia dr% affeota betweea iu&vidtuta, For some enzymea, discnte gemetic subgranps are present in the human popniation. These gaxtic polymorpbiams ere pnaded by mutationa in the genes coding for theae ailZynOe WN& otlilQ d0a=4 LmvuW ot 8bw anzym aQlmWm Or aCdVlty. Qenotic p0bn0l*sm oFwVCAI GWs j1aV0 bm Wooddd nd du*

sctivity fails im im ctemly cieSned atrd 9~m"lY diffmat Pwabdoor.
individuab w&ore rau al mctent otnetabolimm is pm (poor meWmli=ra.
P1Na) ad tlase vrlw liawe 5kft or more aocomodva med)oliam (aftdve matabolizors, BW Omddc potymonphisms of warrs phase II anzyimes also exiiL For euample. Nmcyltrmdwue.x (NAT-2) is dbted In ft way and th3s ecatyladon poIymaYpinam reLdea- fla te metabolimt of a vuriely o(drup aad aada*m& Nuuwm SHOa m+e aascaided with diecroeed limctioaof to this agyirie and a bimodat d3fskbutton fiobsbrveds 50409f- of iadividuals are ganofiypicaY slow acafyLroora aad tbe reet of tto popjktiao sta $ad:

In healthy Wividad* tlw mdabolic vwtyPe roM* PMdiM ft ,met~bolic pbamotypa T6at foc a pard¾nk mzym4 Vnctypdaatly- uftdve mebboiitias ae observod to officim1y metebolize &W t}m are sabstratas for ~ s that enzyme, and gmotypically poor rnetabolizera ara de8cieot ia tlm process.
Howem, ¾irm interactiom, b*cdm dimm pmpow aion awd muLnaWtton maY
prodm channa in the ralativi levale md aadvittes of naatabolizing mzzymee.
Thuk in hedttiy Mvidaai'the relatioaft betwem genotype aod its acprP A is on (phenah-pe) Is cmavad; i.e. PAST gmrotypes produce FA3T

20 phawypes, while SL4w gasotypes p.rodm* 3LOW pbmotppes. Howeirw. a dimm sho of the iadivi" caaaioec t1A ida doop, as cm diatõ smolcia alcobor. emrua-M-LUM-1-u-ml chomiade, ad bialoskat or chaanisW vvaefare aratL
For ft raason, the detumhwdn of inebbobc pluxotype (the maaauv of acdW aonma activity) ia of gtae impocdm aod m be uaed as a direct ad 25 sensitiva probs of hWffi od ciimcal stns. Ia a piePared an6odimeit, 6s IdaWfiabon and quutiSeatiiami of spocift matabolia paanrns pcoduad by iaaocvxm tw compo<mds or probe dtugs ca be uWizod to detdnnint the metabolie phemutype of s snbject. For axam~e, caSine is metebol'umd by sevarai rontee including one involvM NAT-2. T5w Bre muury ratio of 2 s autabotitee, S-a~c~fo~mylffinirio-3mmthylu~cil(AFMU) to 1 mcihytycaudidve (DI) is an indn of NAT-2 adivity.

Bxsmplr,e of numerous mibodimenta follow. F.ach mbodimmi can be praoKced alm or in uoqjmncAioa- with othec embodmunts of t6e inveatioaL

For axampic, ea ma4tioned above, diawsit'wnal or matabwiic marlGers of "stress" can be nonitoned. includiag bat not hmited to, ciLemistries for the detection of c4'fl'a1 art clreniorl pcobaa of human health, such as glucw, cafficine, eihenol, and deoctromatborphen. "8tr+ew" can manifest itself via detectable sitamtioms of many intamal membolic pedhways, such as in ahered iasutin-gtucm patterns or abaranc >apedie ca.mbofiam of ue, commonly used ls stimuI~ta (eatefdae) or eatibistamiaes (dacftnmett,orphaa).

7he wzym N-acetql U=dnn (NAT 2) metaboliaes caffiim Tbis enzyme is higbly polymaphie. T6e adivity of NAT-2 is lnmawn to be assoeiabed with advarge divg dbcs, divecse toxmdiea and predispoeition to dmnae. Two majar metabolia phwotypae bave boea WentiSed: tat atd slow N-acCtylators..

1le expremed activity of NAT 2(plmotype) bes beea shown to be affected by acoe and ctmaic diawn Aamee. Fot eamrqde, in HTV+ aod AIDS pstieuts, tbe pmsecx of an acft illnm reduces the exprea,sad activity of NAT-2, changiaQ a patient with a faat NAT-2 glenatypo ido one with a slow NAT-2 phenotype.
When the illnew is reswhrod aad ft patient Is returned to the iaitiat etiteicai atste, the ptbw apin opum a fast NAT-2 pllmotypa. TMus, the a d~-nniaattan of an indlvidwCa NAT 2 plwotype and do mooitming of ahsga ia " ptumotype am be a dkCt aod xnadtive probe of dw imdi~a haslth and ciiaical steM=. M doNrmiaadian allam prediction of vvhetber patieats ai^a FA9T or 31AW metabolizms pdcc to iomsfing d% tagiamm 'L'!ris approach do allows ft tha ecsreemag of dl pdY" bafOr+s drug tmtuuat is iaitieDed so do approprista doeap regimm ara givea at do onbet of beabnw aad chvg ovaboaftned ac nodmtreabaeat is avoided.

The NAT-2 pbenot,yps cag be dooermiW by a aambw of pobes. In the pahnad embodimeat, caffaine is used becwse of iis wide dlaEdbottan, and relaiive sa8ety. In saxliw uft ceffeine ea do pQoba, tbe phenotype of the anzyrone is detecmiaed by the rAdo of tm caSaiame metebolifes: AFMU to I X.
Baud on the ratio of theae mahbolitea, the activity of t6e enzyme can be detenminod. Polyrdond antibodies ane gi+own ago{net the metaboliu AFMU
and 1X and tlw puri$+ed. T6eee etbodiu ara mucoeeaMy used to deoamina is NAT 2 phano#ypae.

Tha prdared datectioa acheme consift of t'ha mdioring of mtibodiae of a patiealar metaboft or cbm" anftn to the acuface of do capillary. The atar`body is bound to a apea3al sotWen attachied to a fluonescot tag swh as rhodamine. As the aadgart fl+aws into the ch=el it will release the fluomwAa tag which is deceetad dorovnso+am.

T'hae, in en ahanatrve mnbod'immt *w0ing usteg NAT-2 is coa&ated to indicase an infbeted or diaea:ed staos. Tha enzym NAT Z is higbly pobuaoia= Tha activhy ofNAT 2bse ban associated with advane drug eflbcts, diverse tioxicities and pedpoeition of dixase. The monftming of Qhe*n in thir plntyp0 if a direat md smsitiv0 po0lie Of W&W! MM
etd h"OQ SUOL

In a ftdw eanbofted, acgwopbosplute demicala (mm) aaenb ue IDOnitOtEd wft dd idOCd" stiQog* mOdEl CmpOmld psCstdOa SiIICO

s '4wve p" typa chwtaii weepmme T*^ Stdo, end Somtm id by 'Wbftin otMxt~-lchol~aaae, tbe arcgi mopb~ophrme ineect-cide, Parathim (or its mwadofts) ptovidee m aaocelaot "amaraW onlye to atlect wcpmm A
paa~ moa~itoAr, t~ lua ~d i~due~ri~l m~d aiviliim applic,e~ta~s.

In alWthac anbodbasK bdonmwy seqwg to mimob3d tcaim are la m~d, inctnung, !br mcampta, i~n-1(1L 1): intierleaida6 (g, ftmd toqmor mUO& lbctor {'m,amoulcdmw amdMft II.1,U and TNP
prVSW c=didm ndym dut cen be oabcted and ddeccood asi* permeahon mtlurwed trnrdamat wabdqne~. md advmad dateotian aystm dedpe ia acoordance wiih 6611 P, etd invan"

ts In a fbcther embodimant, miurobiei toxins are monitored, jnclndinL for botAHn!um toxn, aadoooxi4 mnoqoibor. Al&aao miarobisi toxiu ace typicsdly twmolewlek &* mctramiy biob bdialogicai po0eac1-, caupied vridi mtoaood amwmd migatim uidng m&xoso*c pk"
ber:ier moailicabon tacbmfqna (for mcenWK ftmet microablsioa) can pamit 20 tir~t do~atry emobybl de"au ayabane LOrcapwidol toacin rcop OSWve cOMponmta. b eddttiM anOody tap an be used ft idwAficarion ofWbcbg Mb mf ddeemhndm otbrWW and vinl loals. Mo+eovdr, the debecmiatian of D- amiat- ecide (fl+om bwoecisi sounms) can e,able the mouitmiug of tLa raqmse to smiblatio thwW.

~~

In a further ambodiment, apora metabolities can be monitored.
Circnlating biochmmical metabolites arising from humaa catabolism via lymphatc and hepatic pathways of microbisl spores ere coilactod and detected nsing te techaiqoas and opRimi2ed de0sctian systam designs of the pmsent invmrtion.

In a fiather embodimat, spoaffcc proteias are monitored, such as those reftrred to in the table below.

Proteiu Concentratloa Mol. Wt.
Preatbumin 70-390 54 C-reaettve Pretean 0.06-8.2 115-140 T ' I < 0.0001 76 io FreaUwmin (MW 54,000) is known as being an impcntant marker for nutritional status. Tha referaice range for 0-1 month old is 70-390 mg/L. Uses of this enibodfinent intclude but are not limitad to screeaing inner city pediatric populations for nutritional status, as well as scrowiiag all patients for autritional stata4 particulacIy prior to surgrry.

is C-reactive proteia (MW 115,000-140,000) is an acute phese raadant and as such is elevated in many disaase ptocessea. Tb,a refeaence range in adults is 68-8200 g/L. The mearzoment of this proteia provides a good indication of heatth vs. diaeaee, Greac4ve protein is also an impartaat prognosticawr of hesrt diseasa and impending myocarW infmfton. Thus, this assay could also be 20 used to ser+ean for aardiovaaeular health.

Troponin t is racognind as a tiscRil and specific markar for acute myocerdial infarction. The refwence renge in adults is < 0.1 }tg/L,. In myocar3ial fnfantion pationts it is > 0.8 g/L This assay provides a real time evaluateon of troponin I in tbe emuvnay moms of hospitals and provide the eerliest recogio{tion tbat a pWent needs to be admitted to intensive cam units.
In addition to monitoring, the biomedical monitorias system of ffie present iavention can provide drug delivery with feodbaalc coah+ol in bursts to maintain concxntrations of a specifie ageg witbin the body at specific levels tbronghout the day, levels whieb ean vary on a day to day basis and during the day. Examples of such agnta inalnde the iwmonea estrogen and testoderone.
The decrease that ocxnra in eatrogm with aga is wgmately related to the incramed risk of osoeoporosia and eardiovascular disease in women. Moreover, the raplaceanGnt with pdmnaeologie estrogen may iMrove mortality frean cardiovascular disease, redwce the risic of osteopomtie frachm and may play an impa~tant role in protecting woramt apinst Alzbeimel's diseaae. Theae diaeaaea have immensa societal impaat nd financiat cost, bat their tmftw wi#6 repWment este+ogan is associated with a host of side effects inaluding, not least t s the development of bneast cancer and uterine cancer, but also a host of ottiar effecb inchuiing skin cbanges, weight changes and depressioa. Altbough no medicine lAts bcen shown to be as effective as estrogen itsoA a lmge effort has been expended to devalop modified esdtogens that have seleative actions on bone, bnmt or othrx tissues (the development of speciflc estrogen receptor modulators or SERMs). The appcoacb of admiw~ effective esbrogen in a p-ysiologic, controlled and monitored manaar is attractive in tha# it remains tlve most effctive medication and innovative tberapentic regimens ntilizing it may prove of g:+eat beaefit.

The dalivery of beataebetm in a controlled and monitored manner can also be uaeful. Savm conoenMons of testosterone also dacline with age, as ttu.y do in a numba of pathological conditiona, includft'HIWU*olftuo#K
replaatmend strategies far the "aument of FIIV and caam wasting, male osteoporosis and cbmc obatewctive pnlmonary disease ere anecgi% and can a)SO be uaetiil for short term controiled aftministration post-apmtively after major surgery to entom tix rate and the likelihood of mceaslhl recovery.
Feasibility of tlese embodiments is investigated tht+ough twodemW detection of estradiol (E2) in Rhem mmkeys and human femalea. A prototype solid phase E2 de'kabton aystan (TED) can be incorporated in a transdetmal patch that immobilizes atrtibody against 82 in the TED, aW analyzed ex-situ using a radioinuntmoassay proce&u+e. The E2 detection system is capable of dewbon less than 0.125 picosmo. TEDs aTe 8rst ftsW by emplamnft for 24 honra on the ahests of partially or fiilly castrated fanale Rhesus monkaye (n=3), treated with placebo or 20 ugJkg estradiol benzoate. The TED meastirameats distingnish bet.ween monkeys that have high circulating E2 concxntraxions and 1s those who have none. TE)s can also be affixed on the forearms of four repmoductive age lnmm females who exhibit a 1ar e ranp of circulating E2 concentcations (48-382 pB/nol). E2 collected in TEDs range from 0.06 to 0.5 pg.
and correlate rnnghly a-ith ciranlating E2 eaeemtrationa. These data are consisteirt with an in vivo penneability coefficient of 4.3 +/- 0.5 x 1015 cm/hr.

In a fiutlus ambodimwit, the portable biomedical monitoring device of the pesmt invontion coa be usad for pain maoageanent, detemining how best to delivet codeina and moThineõ umg others, to minimiu cytotoxicity, while achieving pain controL

In a furdw embodiment of the preseat invention, a MEMS-based physiocldp can be used to non-invasively monitor fundameatal physiological aspects refated to hnman f=tion unft typical and etypicat mYirommantal cwnditions. By csraf+illy ma itoring of relevant physiological data mh as body wmpereture, pulse ratie, blood preseure, and heffit activlty (eleauoeardiogi=) sa infinitesimal change or anomalone beiavior can provide an early indic,ator of st[esa to tlo human system.

tn a fucthw embodimeat, passive or nan-invaaive transdennat dosimetry is used without pbysW or chamioal modification of the nozmad sldn bacrier.
This embodianeat is prectical for small molecular weight analytea that exhibit both lipid and wata solubilitiee.

The following description of expatiments and clinical trlala is provided so as to demonstrate how variona embodiments of the prescnt iavmrtion puform. Suftable analybes for demonstirating the operation of these mubodimenb of tbe presant invention are provided below. However, it is to be recognized that the eystems aad methods of the preseut invention contemplate analysis of a much Iarger set of analytes in the vatious embodimeats of the present inveniion.

1ase&HPI,C ~. *a nd dre aoolicadon oftbese nheses to ft on-line $nparattoa of nAC,b&deteraeni sohrtion. Rat whole forebraia or transfected cetls are suspended in 50 mM Tris-HCI, pH 7.4, (bnffer A), homoganiud far 30 aeoonda with Brinlctaaun Polyaron, and cantrifuged at 40,000 x g for 10 min at 4' e. The pellet is resaspended in b ml of 2%

deoxycholate or 2% cholate in buffer A and stirnd for 2 hours. The mixture is centrifi~ged at 35,000 x g for 30 miautes, and the suparnatant centaining nAChR-deaxycholate solution is collected.

Dried IAM parli~icles ere suspended in 4 ml of tha obteined detwgmnt solutions containing nAChR sabumits or subtypes. For the immobilizstion of one nAChR
subtype, the mixdurc of IAM-detagft-receptor is stirred for 1 hour at room tianpaawm. The suspmsion is dialyzed againat 2 x 1 L buffer A for 24 hours at 4 C. T!u LAM LC snpport with 9mmobilized nACbRs is tha- washod with buffer A. centrlfugod and the solid colleoted.

A dried lipid mixture of 60 mg L-aLr,citin (20% phosphatidylcboline), mg L-a-phosphatidpiserine, and 20 mg clLolestarol is solubilized with 4 ml of 10 obtained nACblt4dwpd soluttoa. The nAthR lipid-cbolate solution is mixed wtth 50 mg dry Supmdmc 200 beads. The suspension is dialyzed agaiasi buffer A for 24 honrs at 4 C. Non-immobilixed liposomes are removed by eentritbgal washing with baft A at 2,000 x g.

([4]-cp9batidine ([3Hj-EB) binding amyg for the suspmsions of nACbR IAMperlicdes and nAChR-Superdex 200 beada: The nAChR IAM
particles, IAiVt perticlea, nAChR-Superda 200 gel beads and Supardac 200 gol beatis, coQespandiag to 30 mg dry nnataial, are each saspended in 1.25 ml buffer A. A 250 gl aliquot of each suspension is inoubated with 250 gi of ['Kl-EB [1.5 dAlfor 4h at ?A C in a final volume of 2.5 ml.

Expaiments are carried ont with arai without added 100 t of 300 pM
(-}nicotine. Hotmd and fitie lipads ere aepastd by vaautmQ filtration throngh Whitrm GF/C filte,ra treated with 0.5% potyettrytimimine. Tle filtear-retained radioactivity is determined by liquid scintillation counting. Specific binding is defined as the differmee betweea total binmding and nanapxifie binding. 1-m SII2oUtlt of pt+OtGitl is deftrmined Umv BCA 1agM (p1e!'ce,1ZoCICfoTd,11., USA) measured at 570 DmL

Cbuomatography based on nACbR LAMolmmn or nAM-lipogome=Superdeac 200 cohmon: The nACbR IAM pertiolea or s aAChR-Supmxiax ge1 beads are packed in a HRSI' glasar column and aonnectiod to a HPLC puaop. [3H]4M is used a's a marlcer and an on-lime flow scistMadon detector (525 TR,) nwnitars the elution pe+oSle. AU eltromatogrepbic acpaimec~a are pecformed ac flow raoe o.a mVmin at room tampaature.

In zoaal abnomatomphta e;Kpaimwts, a 100 l-loop is used to apply lo ft sample. The chramatographie data is summed up in 0.5 mia intervals and smoothed using the A~'i+aomft Eaccel program with a 5 point moving average.

In frontal chromatogram 50-mi sample superloop are used to apply a series of [3A]-EB conemtration through the nAChR-colmmn to obtain elution profil+ee showing a fi+wtt and plateau regions. The chromatogiaphic data is is summed up in 1-min inbervais and smoothed using the Mieroso$ Excel program with a 10 point moving avecage.

Remb: Immobilisation of nAC'hRmbmitsor s About 63 mg potein isolated fi+am the membraoa of tauafected cells and 14 mg of protein pceparcd fmm t?he brain tiwus ase respectively immobilixed on the per gram of 20 IAM perticlea or Superden 200 gl beads. Rocepvor binding assays using [3Hj-EB slwwed tbat the nAChR banding activitiea are retained atEea the immobiluxtion prac,educre as shown in the table below. In parallel eacperimenta, no specific bWing of [3H]-EB is ddocW on 1Ar+t particlea and Sup=dex 200 gel beads.
=

3smpb 9peaiM HWft nAChR Daasity M (MMY$ P"tdn) w{/P2 aAOlt-ddurd solution 62 0.14 at4i02 up,MR TAM 49 0.81 a3/04 nAClR4Aacgeat solution? 100 8.57 a31P4 nAChR IAM'Z 97.$ 5.09 a3/04 nACblt liposoma Supetdex 2W 29.4 1.45 'ptepend from rt forebmtt with damVat deoxytholate.
to 2 prepared from tanhaed cells with ddftVW cholate.

Frontal clMmabomMdrn withg3j@4 nACb$-IAM sgio= vhase= The rztention volumes of [3H] BB sne 23 ml at ilye concwbation of 60 pM. This ratardatiort is pdmarily due to tlle specific biadinQ to s-ttugble sites of the recoptora as indicated by a dacreaee in rebantion volume to 8 ml when the conaentration of [3H]-8B is incrtased to 4S0 pM (Figure X, profile B). The bindng of [sH]-ES to the a3/04 nAehR-IAIuI statioteery phase oonld be rednced in compeWve displecamed acperiments uamg ]movhn a3104 nAChR ligands in the mobita pheae. For example, the ratentioo volme of 60 pM [3HJ-EB

decreased fiom 23 ml to 18 ml whhem a 60 nM cowmaudon of the nAChR 1iSaad (}nicotine is added tD the mobile phaase and felt to 0.9 ml when the ()-nicotine concentradon is ina+eaeed to 1000 nM. The decreases in retention volvme,s of [%-F33 t+elstive to mobile plm concewWons of a displacxr reflect tttia binding affinity of the displacer for the receptor.
Usipg this teclmique, the relative affinities of nicotiaic drugs for the a3/04 nAChR are readily classised by aeUnmiaiag the conomtmtions required to a xease the retention volumes of [3Ii] BB to apzuWmmined leval.

To daarrsee the ratendoa volumes of 60 pM [3Hj-EB fivm 9.S ml to 6 ml on an 63/P4 nACbR column (0.5 x 125 cm), requirm mobile pbase aonceatratioaa of 0.12 nM of (f)-E9,1.7 nM of A8S380, 45 nM of (-}nicotiae, 1,200 nM of carbarLol or 21,000 nM of atopina, reapectively. Tlwrelative affinities of theae drup for the a3/(34 nAChR dabermined by this method arro ~e (f)-BB > A85380 > (-}nicotine > caatdcho! > atrapine which is conaisbem with cesults $+om ligend lrind'mg assays using mamnbmae lyomoge.natss. The relative affiaities m be classiSed by the assoc9mtion constetrts calculated &om the resulting dats in the table below.

Llgsnd K't' (n11'Y) T4s (Um (1)-Epi"batidina 0.27 t 0.05 0.38 t 0.07 A85380 17.2*0.5 73.6&6.3 (-)-Nfcodne 88 t 33 475 f 52 Carbachol 1,280 +t 30 3,839 f 276 Atropi= 14,570 f 2600 -1 Front:i chroxpatograpby with a3/(341AM stattionaty phese (0.5 x 1.3 cm).
'Binding assw using oell membrane homogansfies.

These dissociation conatanis (Kd) vahua show the sanu rank order as t6Ose of the values meamu+od with binding assays nsiag membuae homogenates. Tlu low affinity of atvpina (Kd: 17,200 aM) is also consigtent with litezattn+e valnes.

____~vi 'eAolimntubilized nAChRs subtvoea: Binding of [3H]-EH is also measured in zonal format on the columns containing a3 subunits only, 04 submits only, a mixtiue of th+e two cell types, or a3/04 nAChRs. The retention of [3Hj-EB on a3 nAOit tAM (peah 1, Figure 23a), 04 nAChR 1AM (peak 2, Figura 23A) aad a3/p4 nACLR IAM (peak 3, Figure 23A) is low, and no sigaif-aatrt change in the reteation volumes is observed v+hen a displacer, (-)-nicotina, is iachulyd in the m,obile Phw, [41-SB is muiwd on the IAM

column containing the immobilized a3/p4 nAChR IAM (peak 4, Ftgare 23A).

The nKention volume is demtoW when the comanftxtion of ['H'j-EB is hweand or whau (-}nicotime is included in the IDobile phase, peak 4(dash line) Figarie 23H.

lie rC8111t8 s of bindWg to immobil~' ed neceptors eltiowed that [3H]-EB and (-)-nicotine have higb,er binding affinities at nAChR a4/02 sublype tban at a3/p4 -subtype and ttusa resutts are consistmt with the resulta dd4miaed fim ]igand binding essqs osing mmibrana hamogeaates as shown in ffie table below. The Kd values obtdned from aW nAChR lipoaome=Supadex 200 colmm are similar as those dcDermined using W02 nACbR IAM column.

Formats of nACBRs Kd of (+)-epibatidine (nM) Kd of (-)-nicotiue W
a3/04 - nAChR 1AM 0.27 f 0.05 88 f 33 13 a3/p4 - nAChR mambrana 0.3810.07 475 f 52 a4/O2 -nACbR IAMB 0.044 f 0.00S 1.0 t 2.3 a4/02 -nAChR aaembraue 0.053 f 0.002 7.2 f 1.3 aa/02-nAChlt iiposomo-superdec 200 0.020 f 0.08 7.4 +2 Bffwta of ionic stt+enath and pH of the mo ' nhase on the b'gft of [3H1-EB: T6e effeat of mobile phase ionic stremgth aad pH on tiba binding affinities of [3H] BB are demdned with a a3/04 nAChR-colmmn. The ratention volumes inereasea when the pH of mobile pbase is increesed from pH 4.0 to pH
7.0 and ramainod constant bdwea pH 7.0 to 9.5. T'be retution volnmes of [3H]-EB are higlur at low ionic strongth (5-mM ammoniwn aoetate) and ' decmse as the ionic concentation of the mobile phase increases.
Stabilih- and renroftibf litv of nAChlt colmnns= One a3/04 aAChR-IAM oolmnn is uscd crontimiously over a tm day period and tfm stored for 40 daya at 4 C. The retmtion volumes for 60 pM (3 H]-EB are 9.5 + 0.05 ml so (from day 1 to day 10) and 9.7 f 0.08 ml (day 50). The ralative affinities of EB
and (-) nicotine obtsin,ed on tbree a3/04 nAChR IAM columns pmpared from diffiwmt batch of cell Hnes ate reproducible as shown in the table below, alt}iough the retadon volumes of EB at the seme concenretion diffaad from column to column.

Colamia sise ICa al EB YCd of (.}Nkotier Bisdia8 dtoo (M) (am (aM) (Paioi/W bed) 0.5x1.8 0.34*0.04 52110 7.5+0.2 0.5xl.3 0.274.05 88133 13.5+Ø3 0.5 x 1.7 0.21 4.06 130:L45 15.0f0.4 Preaaration Qfjmmobilized GABAA and ni,ccdnig_acetvlftliae receptore on an1AM saenort $+om rg whole brain: Rat whole brain (4 brains) is is homogenized in 30 nml of TRIS-HCI bnffer [50 mM, pH 7.4] containing 5 mM
EDTA, 3 mM bamamictine and 0.2 mM PMSF (Phenpl meW salfonyl chloride) for 3 x 20 seconds using a Brinlmsn Polptron at setting 6. The mixture is kepA in an ice bath for 20 soeonds betweoa each homogenintion step to prevest excessive beatig of the tissue. Homogenized brain tissue is centifugod fbr 10 min/4 C at 21,000 rpm. Supenatamt is removed using a Pasteur pipertte and discarded. The pellets are suspended in 10 ml of Soluhilization Buffer contaiaing 100 mM N*C1, 2 mM MgCl2, 3 mm CaC12, 5 mM KCI, 2 % Narclxiiate and 10 glmll.evpeptin in TRIS-HC1 buffer [50 mM, pH 7.4]. Tio rasWtiog mixture is atirred for 12h/4 C and centrifuged at 21,000 rpm.

Supowatimt (receptor-cholate snsmsion) is mixed with 200 mg of dried IAM PC pacldng mataial and stirred gentty for l W25 C, ftnsfared into dialysia tubing and dialyzod for 48 h/4 C against 3 x 600 ml of Dialysis Buffer containing 5 mM EDTA,100 mM NaCI00.1 mM CaCl2 and 0.1 mM PMSF in TRIS-HCI buffer [50 mM, pH 7.41.

The raxptor4AM-PC ia ceatrifnged for 3 miaJ4 C at 2,000 rpn.
Supanatant is digaarded. PeHets are washed with TRiS-HCI baffiar [50 mM, pH
7.41 and emtrifaged until thse supmatnt is cte:rc. The reuiting pelleb are used to pack the column.

Tkdiemineti= of bigdn A4L-i$wa tn tHw jmp 'li QARA A~pr (=,~,¾ frontat chro oatoacaahv: Tbe G1R-L4MM patticlea ata pecbed in a HR5/2 glaaa column and conmccEed to a HPI.C pnmp. [3ffl-Ftumtrazapam.

([3Hj-FTZ), a(IABAA receptor lipnd, is o$ed as a marloer and an on-line flow sci,ntMetion detector (525 TR) monitored tto elntlon p:oSle. All chromatograp}ric eacperima4ts are parformed at flow rate 0.4 mUmin at room u~nper~me. Ia frontal cd~aar~aatography, a 50-mi sample superloop is used to apply a seriea of 0 In-FTZ eonoentrations through the aR-column to abtain i5 elution profiles showing a front and plateau regions. The chromatogra~ic data is summed up in 1-mia intavals and smoothed using the MicrosoR Excel program with a 10 point moving averagL

When the OABAA receptor ligand diazepem (DAZ) is added to ft mobile phase, the natentIM vohune of [3ffl-FfZ is reduced in proportion to the cancentration of DAZ in the mobile phase. Tbese multa indicate that the retantion of FTZ on tlte OR-LAM is due to specifie iauracoioaa with tY-e immobilized GABAA recqrtor. Tbe dimoraation constenta (Ka) of FTZ and DAZ are determined on ft t3R-1AM. T]za calculated I{d of FTZ and DAZ
obtaiaed by frontal chmnaatogcaphy ara consisttat with those determined by claasM binding aaseya, as shawn in the table below.
~

L1gmd Firodd Q-oaoato-py BindftAsup Flumitra~san 1.3 1.7 DLmep~a 1.0 1.3 s 'I~e B~e~ RecaOmr (ffi) is prtt of dr Nhclae Recept{ot 3operbody. It ia made up of flve diffemm m&m A. D. C, D smd H. The Brq6m, ebo luwvrm as, ft li0sad 6iftft io dmatn "D) b whm tbie arWb md aotegooiab biad. Tbe BR I.ED 6o beea aopoeed ia yodt aal also in boamia via albaiaa podaet between pmtin A ao8 ttre LBD. Tbe Produdioai of ncornbWW Fftpa Rocapor ProteLa it daacdbod: T5a DNA ~eqi~eqao oodta0 fct tha Iipnd bin doamsin of da buma adroRan racxptor s p+oob (miw Wds 302-395) Is obWnad by is PmiaAs tlra fia11 ta90 oDNA astlue immpWL ?Le pmduat of tie PCR
rrwtton ia suhclooed into dro pR38T plasmid ia ftaro wit4 a b hiatidiaa tag on the N-umdnd a d of ft pe+otdo. TU His tag is "ed foc ffie pvdficadoa of die protain ,6ront tba bacteeial pcobems. The plsmid is tandomed inbo dra HI.21 codcar+ beaonda T!ro beccer3a an grorvn in smnd+ad LB &o!h to an opticel 20 dmeit,y id X= 600 of- I.S.

Tbs boctedat are hasvated by oeontiifugatio aad ffraun at -8M mrtit fiatbarpudficodo . TLe bacb&palla m lyeed in amosM&41yds bift by sonlaWm aad clariied 6gr %=-Uzfllodm and ffitsdmTbe lynt is laded oofia a S ml N!-NTA ni~Od a~ cod~mmot tl~t ia preequilibea~ed vri&flr 25 mwHE'F~41y* buft: Tha 1WWfA coban sateoKvaky binds pcatm widt le 6-ft mg. T'3re oomtawd p:oW= un aa" off ft colmn wi6 6*
=JME9 buffec. Tlos eettoQen raxpft is refolded on ft colmn by gmdwlty cbao&& t6a bnffir to a PBS (phoephete bufficed aafte) bnft.

Fntelly, tbc eauogn :aceptor pratoin ie eluted with a PBS btrtfer contatntng imidsa~k, whirh compews with tlu Hia tag fbr bindina to the Ni NTA calwam.
The fractYo s containiag the ew%m raoeptor protein are deundned by gel eixttoptwzeais an+d staing wfth C1elcode Hlue md by wrsdan Wt analysis s uafng a aadbody agtbW the himoan estrogen z+eoepW. The conoalIMon of ProtAm Pun$ad is debmnined via bicitrohoninfc aaid (BAC) protein assay.
Biadin¾wA& o the F.R-LW. A bind'mg assay is c.airied out to datennina the acdvity of the lUaion pcotcin Tlr cfmsical mcthod using deften-coakd cbaroast is initially used and givea the aativity of the protein.

1o However, the method is improved with the use of Niclael-NTA agarose beads to ieolata tlre Oadon potein. RougMy 200 pnioles of protdin is plaoed per tube.
For total bdndins vazylog omxmthtim ef (3'HI-nstradiol is added aad hr nonapeciflc bdnding a 200 fold excess of the cold estradiol is added prior to the addhion of the radiolabeled estradiol. The aohtions are ittcabated at mm is oennparha+e for 2 houa. FollowinS hoibdion, the Nickd NTA is added. After one weah, the protein ia dlsplaccd with imidezola The ICd is detemnined to be app~ximaddy 3.4 nM (an avara e IGd of wveral experfineuts~ Althoagb eattadiol hsd a stW* stcoogec affinity for the native ER (0.2 nM), this is sofftcient.

20 bogwinam Qfft EL-_LBD. The initial immobilizati,on of the isolabed fiudon pvtein is cacriod out mimg a ailica besed immobilizod arti8cial mambcana IANLPC. This membrane contaias a a'slfca coxa, wbich is wtacb d to a hyckrnphobie spacac with a polat }ad Smup. Tbe pa+ocedure for immob'sli~ion of tlo pvWn onto *m membmes is kncwa in the art.

ZS Varyiag concoomdoea of LAM m ued to deenmine the optimal conditions for imm~bili~ion. It is dttamined that 25 mg of 1AM is opMM'an*"3! 9e it1ColponfiOD.

Howam, upon testing for activity it becomes appaceat that [3 .i]-estradiol is not onty bWding to the proteia bm slao to the bydrophobic laya of the manbcane. Incaaaslrng ft afl=! conawhatioa ia solutim doas not signi5cantiy reduce tba Mnding to the membraae. Using a modified IAM
stationery phaae, the IAM MG, that is mnote hydraphific omly sligWy redncea de aomspeccific bindiag,.

The ER-LBD is tbat immobilized in a new coiumn fornnat containing a to silica bachbone and a hydrophobic spacer (C 10). The ER,-LBD is immobilixed and nWned its bindiog aativity but ft nonspecific bivodiag of [sHj-estradiol is still to great for effective use of the colunnn. The C10 spacer is replaced by a hydrophilic spew and ft nonspecific binding of [3H]-estadiol is alin4inabed and the ER-LBD-SP column is syntheaized.

15 TU K,d of tha estradiol marlcGr ligand is then detemdaed on-line using the ER I,BD-3P cohwQa. The ER-LBD-SP colnnm ia conneotod with oa-line flow saatillation asanitoring {kadiomdric FI.O-0NE Beta 500 TR ins~
Paalcwd Ins6roo69 Co., Merldien, C'I) arai rao at room tampmattn+c for 97.5 minutes at a flow rate of 02 mt/fmia. The system utup is as de~ibe~d by 20 Zhang, at al., Immabilized Nicotitic lteceptor 3tailonary Pha9e For Oa-I,iaa Liquid Cl~e+ommageapbie Ddamination of Drug-Receptor AfBmtias, Anal, Biochem. 264,22 (1998). 18 mL samples of 0.5 nM [3H'J-Estradiol ([3Hj EE2) suppkmenftd with a ranga of concentation of cold Eshadiol (0-7 nM) ara run by frontal ebmmatogcaphy. The elntion volurne data is used to calculate the 25 disaociation conataat of the ligand, The ICQ value of esbadiol is calculated by nonliam rpsmsion with Prism (CiiaphPad Sofs ware) using one site binding eqnatim-: Y=Bmax LB'Lltotav(Ka + Iffiltotal). The Kd values of estradiol is calcnlated as previoudy descn'bal to be (0.189 f 0.06) nM. Tha radioactive sigcial Is recorded emq 6 saoonds by an on-line flow scmtiltation deeeetar.

s ftaration of the MLBD: The ra:ombinant ER-LBD is obtained and parifled as described above.

' ImmohflizatiQn of the ER LBD: The EEL-LBD is then immobilized in the micromachined capillaries. Tha immabilizatton is acoamplishod through activation of the ailanol gmp on the silica chips uaing to dicyclol-exl+tcarbodiimide (DCC) and then oonpling of t1u C2 spacer with a free carboxyl Woup to the actlvated sisfecx. The ER-LBD is then bound to the derivatized aarface uft tBe procedures dzveloped in the previouB studies with the liquid clvromatographic stationary phaae composed of silioa pl beads. The amount of protan inmmobilired on the surfaae of the aaiaochanuels is 15 datecnnir~ed bq analysis of tha protein content of the biading solution before and after expostue to the rnitaracheanels.

If the initial ex,parinortal approach to the immobilization of BR LBD is not sucxessM the following procedtn+es ate investigated:l) if the problean exists at the darin the activation of the siiowl groups at the sitica surface, DCC is 20 replaced by diaQetMWlaminopyridiae (DMAP); 2) if the problem arises fibm the C2 spacor, C3 to C4 spacros are acamiaed; 3) if a psoblem exists with the immobitization to the new aufbae, an OPoxide activeted approaab is explored by a method sach as descn'bed in J.B. Wheatley et al.: Salt-induced immobilization of aftit3- ligands onto epoxide-activated supports, I. Cbromatogr. A, 849,1 25 (1999); D. 2hou, et al.: MMembum affinity clmomatography for analysis and purification of biopolymrs. Cbranns~graphia, 50, 27 (I999), or an approach ntiiizing streptavidin-biotin}-lation such as described by L.A. Paige, et al.:
F.droM recapbor (F1t) modulators each indtx;e distin.x c~fomnationsl changes in ERn and ERP. Pma. Nat. Aca+d. Sai., 96, 3999 (1999).

9,Mft gStivitv of & imcMind ER LBD: The binding activity of ft immabilized ER-LBD is deftminad using [3H]-estradiol (0.005 nM in phosphate buffer (0.1 M, pH 7.4) ([4-E2) supplemented with a range of concemmdon of cold estradiol to p~+odace a tsnge of from 0.001 to 0.050 nM in phospbato buffer [0.1 M, pH 7.4]). TU solutions conWnlag the ['F1jE'l ara appiied to tha mira+ocbannQls containing the immobilized ER LBD, microchaanels cmtaining the immobilimd sapport (without the ER-LBD, a positive comol) and bare nnicnacbsnaets (negativs contral). The solution conmidag microclonls Is incubatal at room tampaatine for 30 miuudo. The channels ara tbm weshed three timm with phosphate buft [0.1 M, pH 7.4], the washing is coIlected and aasaped for [3H]-E2 conLent using a scintillation detoctor. T7u Kd value of E2 is calculated by nonl'mear regression with Prism (GraphPad 3cdlvvare) using one site binffing equation: Y= Bma [E2]totaU(Ka +[E2]total). Tlm observed biading affiSities smd cdwt of binding is compared to the data Eroan parallel binding stndics canied out using an eqaivalent concortretion of non-immobilized ER LBD. These sindies wrill yield bindmg affinity/mg umnoblliwd ER LBD indicxs (BAIj which is used to charactariu the immobiti7ed receptw.

.ne innnobilizetion of the ERLBD is optimized through the invcstigation of the effect of ER-LBD concentration, reaetion time, tempaatm and ehemistry used in da immobilizs-rion. Bsch of the variables is independently investigatied in a step-wiae opttmizatioa approach. The outcom of each itaaration is assessed using te BAL Onoe an optimum immobilix~#on proceihm has been detecm,inai, dw Wka-day aad interday reprodncibifltp of the proceduce is detarmined. A variawx of no greater than 10% is deemed acc:eptable. If this cannot be acbieved under the initislly detenained "optimal" conditions, otber previously detmmined conditiom is iavestigated using the BIA as dw selecting vatiable.

Ddamgnstion of tye ]imits of quWbdm mid detecdaai of the to mmoWized EL-M clun= T'lue esaadiol ligand is dexivartzed witb flwmccin-5-meleianide to psodim the flumsont-ligand (E2-FM) wbich is used in the clinical petch. If this fluoreacxat tag does not paduce enongk sensitivity, other agents are utilizad. The immobilized ER L8D clip is suspended over and thmi baought into surface contact with solutions containing IS E2. The conccatrations of the E2 solutions are serially diluted from the initial conca-ttation of 0.050 nM until displacxment of E2-F1VI can no longer be obaerved. The measured opkcal daasity at 71ex = 488 mn and lam - 520 ntn is plotted againat the E2 conmutratiom of itie test solmions to construr,t standard curves. 3twndad iau+r-day and infis-day valiclaiion studiea are coaducted to 20 establish dw Mndumbility of the meesw+onents, the low limits of qnantitation and the lower limita of debection. Onae this has been establishad, the c,hfp is ready for ciinW taating.

Precmtion of tk Alt-LBD: Thx mxlrogen receptor ligand biading domiaia {AR-LBD} fusion protein is pmduced aW puzified foIlowing lamwn 25 procedum. Onee the protein is acprmcd and puiified, the binding affinity of tha AR L8D is dooamined by comrentioml methoda following tUa xc:e&u+e describEd for tlw 8R LBD.

pnomldmof tbe $:UD ft and velidati% of ita '~gy, t~,r' Tltie immobitization oft6e AR-LBD, ddmgwdon offt biiuding s activity of the immobilired BRLBD, deWnoaination of ft limits of quantifton and detection as well as the initial clinical validation is eaQied out besed apon tbe renilts obtained with ft ER LBD.

The p+otein is immobilized in a *d* feshion as the ER-LBD, and the Kd is deftniwd by fiontat ehmieoFWhy. The stabilfry of the calumn is also lo d:t m3ned.

The immobilizartion of ttm receptora sllows for rapid =eenkg and detennination of preaeace of biologicalty active/'niaotive compou'ds on the estrogen aud/or androgn recxpbora.

The rocombinant ER-LBD n obtained nnd pnrified as dembed above Is Iwmobiliution of the ER-LBD: The AR-LBD is tlm immobilized following the pcmeduwres described above for tbe ER-LBD.

The cliniical hiaie descn'bad baein dctannina the anmlytical precision and accuracy of inetWds to detamine estrogen ad testosterone via tiansdecmal samplimg in comparison with validsted plssma assays. Validated ptasms ELISA

20 assaya for estrogan and tastoaGexone widi scoeptable limits of deteation and quautification end widt a+cceptabla ints- and inter-day coefficiente ofvatistion are useci to compere aritfi ftee uMs in the elinieal sading,g described in the four trials onttfned in datail below.

Cli.+ ca! TrW I: A>'il~ Trid C__ -tntinaEUMM Concentrations in 2s pIs_M and IgteI +Ai Fluid in Pm and Pos~ MW+amad Women, In order to validate the anaiyticaf sansitivity of tranadermal samplinQ to measure eatrogm eight healft masWatn8 women and eight hattb,y post-menopausal women who ara taking no esftogen-contaiaing medications ara followed for two months to measm the esdc+agea coaoentrattons iu thair plastna or interstitial fluid.

.s Co~ons of eshnai in the plaama is determined by a validated clinical ELISA assay, routiaely used in climral sathngt These concentrstions are comparad to those detamined in interstitial fluid using estrogen raxptor-based assaya using the HW monituring davica. Plasma and interstitial fluid concentations are measurod daily during the period from the end of afber lo manstruation until 10 days atfimvvard in menstruating womm- and over a ten day period in post-menopausat woman.

Two groupa of women are reeauitcd: a gmW of eight women wlro are gre-mencyausal and who, by history, axpariem repila mensa ual cycles; and a group of women who have passed tmgb mewpmoe ls lwhudoB Cr'steria. t3mup 1: women who ara over the age of 21 years and under the age of 40 yms. C'mup 2: womnen who are over the age of 55 and under the age of 75. All women must conform with the followiag: (1) taking no Fued1ti(m medications or nauusl p+odneta intended to produce estrogen liice effects (for example, giaseag or blactc kolmsh), (2) with clinicatly normal 20 laboratory valuoa for eoanplft blood eoao.ta, saum ehanistriea (Na, K, Cl, HC03, BUN, gluaase and creatmm) and clinically nomial liver enzyme prof tea: S(3O T, SQPT, aikeiine Ompbst rw and bilirubin; (3) ability to imderstand aad carry out a signed informed coment desctibing this protocol.

Exalusion Criteria. The following individuals att excludod from the 25 study: (1)smokers; (2) impaicod liver or rmal RmcGiout as d=wns~ated by =

senun 3tiOT, SOP'T or bilirrnbsn AMve the ncrmsl rmye of labomaq valuas, or smm ctednine gmateer tbm 1.5 mg/dL; (3) positive nrine drug screen; (4) aubjecfs who test positive for IHuman rmmmodeficiency Virus or hepadtrs; (5) subjects wIa }vve talmn an kmeodpdonst dmg wMin 30 days of stady etait (6) subjecia taldag aaW enzyme in*ing oc iNiibiting medications (for eascnple, rifempin or phenytoin) for 30 days prior to doaing and (7) subjects who, in the opinion of the ?nvasugatw. is nmoomplimt with the protocol rcqunreneiNL

Restrlctmns mwhdo tbit aubjecta are insuvatod to ra6rejn froma tha to follorwinr. (1) talft mry pteeorpdon mediot'sons for two weehs priac to doalog; (Z) consummg cdMne and/or xantbt contalning products and aicohol fiom at kW 48 haurs pciac bo Study Day 1 mrtii after tiye last blood sampk has bceu oolleoted; (3) amidna, (4) skmnwns oweise during the entire study to avoid dWocation of t6e meaamiag device.

Once a subject has cooseated to partioipete In the study, the following procadem aoa oandnctad. ,Sereaing pmeeduur.t ffie cyonducod witbin 21 days of study iaitistion and inclade: mebsl history amd physical examiaation;
review of iachjsion and mcdmm aitm blood acd urine apeaimeu eollection;
anatyeis of blood umple for heaadology, aeram chemistry, lim aoaymes, HiV

and hepatitis B and C; and snelysis of ucioe sm-ple fot screaning of druga of abose.

3ub equet to inclusion in tba study, mbjeat will undecgoca the foltowinS pc+ocedun+es: sabjecb artive at the tee;fing facality at approximately 9a.m. in the mom* Wltffe tbe ptncise date is not importaut for 23 post manopmel women, menwuatiag worman ere ealoed to report aa &e last day of their regular period. At this tiaoe estrogmr leveis as+e low and the detection Umts of tha agays is appropriaGely tested. Vital signs (beart rate, rasptratory rate, blood pressure and ftmemm) are recoadetL The portabk biomedical monitoring devicx is placed on a formm and attached securely with tape.

s Recording ocaua via a 50 micron cmAulud lesion in thw skin made by a small noedle on the tnndmide of the monitor that is twt visibla The monitor is checlced to enmn that it is reooidin,g. Vital sigas (heayrt rate, raquatary rate, blood pressure and tanperatnze) are recorded.

A "e ssmple of venous blood (5 cc or one teaspoon) is drawn from a forearm vaiu for the messmumt of estrogen while the subject is sopine. The device is instrncted to measm eslrogen for pariods of 0.25, .5,1,1.5, 2, 3, 6, and 8 hours in orda to test the optimal time required. Additional blood sainples aae draw-n 4 hours smd eight hoius after the first. The tape and device is removed. Patim~ are tben diacbar8ed and allowed to return home before t s returaing at 9 am. the neact morniug. This procedua+e is repeated on the following 9 days fae a total of 10 days with each subject.

ivt mL vewus blood samples are colleooed in vacnWnm containiag EDTA on days I throngh 10 in the mannar deseribed above. The total nnmber of blood clnaws during the course of the study includiag the ecreening and axit semples, is 35, ('fhirty study clraws and itve' serxning drav--s for hatatology, chemistry, liver enzymes, HIV and Hepatitis and C
reqm*vely.) and the total vohma of blood drawn does not exceed 200 mL.

Vifal sigos (heart rate, respiratoary- rate, blood paessure aad taanparatute) are taken before and aftar placemmt of the device and before and aftet each blood dravv on days 1 through 10.

Pahero are encouraged to repm any notable iYrimfion on the arm where the device is placed. A phyeician is consCentty available to subjeM esmolted in the study for conmu related to biussing or infection in the skin due to mUli'xple blood dravvs.

s F.strogen aonoentmtiams dedetmintd using plamma ELISA is ooffipaed with the values obtsined uang the portable biomedicai mouitiar. If the correletion coeff ieient is > 0.8 with a signifeanae p < 0.05 the nneastaements are deemed valid.
A r" TrW QUjWW Tn== Cvwackado~Wesma and Ten heattby me,n, chosen to repreaent a range of ages between 21 anc170 years old and wJio are takin no rnedications have plsama and intastitial fluid teftstm+om conceWationa masaured daily for 5 consecutive days both by validatied plesnu asaay and by transdenmal sampling using the portable biomedical monitaring device.

Ioluaion Cr'~tRacia. Men w" $re over the ag,e of 21 and under the ago of 75. All men must confosm with the following: (1) taking no pre,x,ription Medications or natural P+oducts inteaded to paU&u7e testosterone like effects (far example, androatened~oae or DHBA); (2) with clinically normal laboratory values for complete blood couata, saraaa chamistriea (Na, K, Cl, HC03, BUN, glucose and creatinine) and clinically nowoal liver enzyme prafilea: S(IOTT, SC1PT, alkaline phaVbeoe and b ilirubiay end (3) ab>yity to mdmWd aad cerry out a sigaed iaforroied conuM docribing this protocoi.

Exctuaioi Criteria. TIu followiag individuals are exclnded flom the study: (1) smolcers; (2) impaired liver or renal fimation as demonstrated by serum SfiOT, SC3PT or b9lnnbin above ffie narmat range of lab+rnMory values, ct serum a+eadnin eP=tw tbaa 1.5 m01L; (3) paeffiva urine drug smeen; (4) sabjects who test positlve for Aumen ImmWOpclQficiwcy Virus ar hapatidis.
Rostcictiona includo satt}acb ace instrueted to rafcain ffom the followiag:

(1) snwWW ana {2) stumm aaancise dudog the amce study to avoid s dislocatioce of tle meaauciaS c3evice.

Onca a subjeet hat eonsented to parlicipate in the study, the followiag pracedurea aKa eondncted: Screening ptncedma am conducted arithin 21 days of study initiation and iaaltx}o; medCSt hiftry- andphysicat eamination;
reviov-- of mclu.gion and ewlusion criteria; blood and m4ne specimen eolleetion;

to anaiysia of blood sewoe fee hmwoloV, seum cbemisorq, $ver een.ymas, HIV
aod lepidtis B and C; aad snallysas of mine savnple faar ecreeming of dzup of abuse.

Sub oqumt to inelua;on in the awdl-, sabjeat will undergoes the followiaQ pu+noalue+es: Subj" aerive at the tstiag facalityr at aPPomzustelY 9 ~ s a.m. in 6e monring. Vital eigos (hestt rate, reepitataei- rate, blood preastroe and te~npd ) are reoorded. The pmtable biomedical mmkoring clcvico is pluxd on a fomtm and awled xcualy with tape. Recot+din owsra viaa So microan cautcriud lesion in t>ye skia made by a small nadle on the underside of the nwnitor mat is not visible. Tha monitor is cbecbod to aasare that it is recording.

20 V'rtal aians (hmt rate, mgatdorY raab% blood pmmn and mmpmem) aza recorcled. =

A siagte aample of vGnous blood (5 cc or one tea9poon) is drawa from a Soeeacm vein for tho mmemmunt of tdoswm abik t}te sabjat is ~.
'I'be device is ias~d to measure enrogen for pmioda of 0.25, .5,1,1.5, 2, 3, 25 6, and 8 houra in order to test tbe optimal time required. Additionat blood sannples ace drawn 4 b.ours ad eW Loura after the first. TFie ape and davica is neamed. patimta are tltien discharged ad allowed to rcdna hom befor' e rafiuft at 9 aim tha next morning. This pancedua is repoaaed on t8e follovving 4 daya for a total of 5 days with aach eubject.

Five mL vmous blood samples are coIIecbd in Vamminm containing EDTA on Days I tbn+ooQh S in the mmoaer dcscn'bed above. The total nunnber of blood draws during the conrse of tiw study iaclnding the smeeaing and adt samplas, is Z0, (F'ifiem study dtavvs snd $v+e screening drawa for hennatology, cheaniatrl-, liver enzymes, HIV and Hepatitis and C reslxxtivaly.) Hnd the total voluma of blood drawn does am accaad 200 mt..

Vital sigda (hesrt rate, respiratory rala, blood p=sure acl temperature) an talcace befor+e aad aftec plaxmM of the devica and before and after ach blood draw on days 1 tltrouglt 5.

Pasienta are encouraged to report any notabla irritation on ttm arm wbeae is the device is placed. A physiciaa is conetently availablc to sub,jeots anrolled ia the study for coneems ralatod to bruising or infec:tion in the sian due to multipla blood draws.

Testosterone concentmtiona datecmined uaing plasna SLI3A is compered witL *e valnes obAaiaod using tbe pocCable biooe" mcaft. ff the cornelation ooeffieiart ia > 0.8 with a sigaifiicance p < 0.05 the moasuremern.a , ane deemed vaiid.

The purpose of thit tbird clinical trial is to administer appropr3ate concad1-riona of em+ogen to poetnneaopausni wmea who ars Iredk volunteeta and to measdre the resulting c,oncentWons. P,strogm is admiaistered 23 in microgcam pnlgae over a perW of 3 days aAet a 3-day nm ia for baseline measurcm~t using tlte opdmet machine settings for the monitor as defined abov+e.

Tlm purpm of this fourth clinicst irial is to admiaister appropriate cvnc~tcattons of testosberooe to nm bdwftn the ages of SS and 75 years who are healthy volnateeas and to measm+e the resniting coacentrations.
Tcstosterone is adnninistmnd in microgcem pulses over a S day period ai3er a 3 day nu-in period that is used to deLermine b seluu testosterone eoncentrations in theae men bafore the adminis6ration of tesbosterone. The resulting camceatrations is detenmiuea using the snalydcal specificatiom as defined sbove.

lo It should be apparat to one of ordinary 90 in the art that other embodiments can be readily contemplated in view of the tear.iYings of the present specification. Such otiser embodiments, while not speci8cslly diaa[osed 13 nonetheless fall within the scope and spirit of the preseat invention.
Thus, the presant iavention should not be construed as bemg limited to the specifie embodiments de,scribed abovey and is solely deSned by the following ciaims.

~

Claims (13)

1. A microfabricated device for sampling analytes from the skin of a subject, comprising:
a detection chamber for receiving analytes retrieved from the skin of a subject;
a photonic detection system, comprising a photonics source located attached to said microfabricated device in association with said detection chamber, and detectors associated with said detection chamber for detecting analytes received in said detection chamber.

2. The device of claim 1, wherein said photonics source is at least one LED.

3. The device of claim 1, wherein said photonics source is at least one laser.

4. The device of claim 1, further comprising substances in said device which bind with selected analytes, and which fluoresce when said photonics source applies radiation thereto.

5. A microfabricated device for sampling analytes from the skin of a subject, comprising:
a detection chamber for receiving analytes retrieved from the skin of a subject;
a patch which changes color when contacted by predetermined analytes, located attached to said microfabricated device in association with said detection chamber; and detectors associated with said detection chamber, for detecting a change of color of the patch indicating the presence of a predetermined analyte.

6. A microfabricated device for sampling and detecting analytes retrieved from the skin of a subject, comprising:
at least one conduit for retrieving and transmitting an analyte from the skin of a subject to a detector; and means for enhancing permeability of the skin of a subject for retrieving said at least one analyte therefrom.

7. The device of claim 6, wherein said means for enhancing permeability comprises a microheater disposed to be in close proximity to a subject's skin surface, and configured for ablating a portion of the stratum corneum of the skin of a subject to allow collection of interstitial fluid from the subject's underlying viable epidermis.

8. The device of claim 6, wherein said means for enhancing permeability comprises a microheater disposed to be in close proximity to a subject's skin surface, and configured for ablating a portion of the stratum corneum of the skin of a subject to allow diffusion from interstitial fluid from the subject's underlying viable epidermis.

9. The device of claim 6, wherein said means for enhancing permeability of the skin comprises a radiation source positioned for generating and directing radiation to a subject's skin surface, effective to ablate a portion of the stratum corneum of the skin of a subject, to allow collection of interstitial fluid from the subject's underlying viable epidermis.

10. The device of claim 6, wherein said means for enhancing permeability of the skin comprises a radiation source positioned for generating and directing radiation to a subject's skin surface, effective to ablate a portion of the stratum corneum of the skin of a subject, to allow diffusion from interstitial fluid from the subject's underlying viable epidermis.

11. The device of claim 9, wherein said radiation source is a light source.

12. The device of claim 11, wherein said light source is a laser.

13. The device of claim 6, wherein said means for enhancing permeability of the skin is at least one of:
(i) means for chemically altering the surface of the skin;
(ii) means for puncturing the surface of the skin;
(iii) means for solubilizing the surface of the skin;
(iv) means for illuminating the surface of the skin sufficient to cause ablation thereof; and (v) means for irradiating the surface of the skin to cause ablation thereof.