WO1990008313A1

WO1990008313A1 - Analytical work station

Info

Publication number: WO1990008313A1
Application number: PCT/US1990/000168
Authority: WO
Inventors: George Pontis; Michael Lacy; Vladimir Drbal; Calvin Chow; Thomas Smith; Rick Smethers; Debra A. Ross; Gregory Kirk; Jonathan Briggs; Lev Leytes; Suresh N. Mehta
Original assignee: Molecular Devices Corporation
Priority date: 1989-01-17
Filing date: 1990-01-17
Publication date: 1990-07-26
Also published as: CA2007923A1; US5529752A; US5252293A

Abstract

This invention encompasses a work station for conducting assays which comprises a filtering assembly (2), a reading assembly (40), and a filter element (4) with a porous filter membrane (5), wherein the filter element has a means (3) for aligning the filter element (4) within the filter assembly (2) to filter reagents on predetermined portions of the porous filter membrane (5) and also to position the filter element in the reading assembly (40) so that detection will occur in those same predetermined locations where analyte or analyte complexes have been filtered.

Description

ANALYTICAL WORK STATION

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of U.S. Patent Application Serial No. 07/297,767, filed January 17, 1989, now pending.

Field Of The Invention

This invention relates to the field of analytical devices where analytes are filtered and bound to a membrane and the membrane is then analyzed for the

10 presence of analyte.

Description Of The Prior Art

The prior art describes a wide variety of filtering devices ranging from filter paper to individual test units which involve binding specific binding

15 substances such as antibodies and antigens to porous filter membranes as described in U.S. Patent 4,642,285. U.S. Patents 4,703,353; 4,591,550; 4,758,786 and 4,704,353 and references therein disclose a photoresponsive electrode generally adaptable to measurements made in the

20 present invention. U.S. Serial Number 065,418 filed 6/18/87 assigned to the same assignee as this application discloses a Zero Volume Electrochemical Cell where analytes on porous filter membranes are measured by means of a photoresponsive electrode. U.S. Serial Number

25 258,894 assigned to the same assignee as this invention, is directed to Hapten Derivatized Capture Membranes useful as membranes for the slides of this invention.

References of interest include U.S. Patents Nos. 4,020,830 to Jense, et al.; 3,975,238 to Bean, et al.;

30 4,238,757 to Schenck; 4,486,272 to Fujihira; 4,293,310 to Weber; and 4,444,892 to Malmros; and International Patent Publications Nos. WO83/02669 and W085/04018. See also "Experimental Electrochemistry for Electrochemists. " Sawyer and Roberts, Wiley-Inter-science, pp. 350-353. U.S. patents of interest also include Nos. 4,168,146, which concerns a test strip for immunoassays, where the extent to which an analyte travels is related to the amount of analyte in the medium; 4,298,688, which involves a three-zone strip, where the extent of travel of an enzymatic product is determinative of the amount of glucose analyte; 4,299,916, which concerns an assay technique employing a support for detection of the analyte; 4,361,537, which employs strips in conjunction with RIAs; 4,366,241, which concerns employing a small test zone for concentrating a particular component of the assay medium in a small area; 4,435,504, which concerns an immunochromatograph employing channeling; 4,442,204, which concerns using homogeneous assay reagents on solid support where displacement of labeled conjugate-analyte complex by analyte provides the desired signal; 4,533,629, which employs a simultaneous calibration technique for heterogeneous immunoassays; 4,446,232 , which employs a solid support having a zone occupied by labeled conjugate, followed by receptor, where binding of analyte to the labeled conjugate allows the labeled conjugate to traverse the receptor zone to a detection zone; 4,447,526, which employs a homogeneous specific binding assay system in conjunction with carrier matrix; and 4.454,094, which involves displaced apart layers through which a medium traverses, where reagent from one layer diffuses to the other layer in relation to the amount of analyte in the medium.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Perspective view of the analytical work station.

Figure 2 Top view of filter assembly.

Figure 3 Cross sectional 3-1 view of Figure 2.

Figure 4 Disassembled filter assembly. Figure 4 Top view of the slide. Figure 5 Schematic of the vacuum system.

Figure 6 Top perspective view of the slide reading assembly.

Figure 6a Internal perspective view of the slide reading

5 assembly.

Figure 7 Cross-section of the filter assembly.

Figure 7a Holes in inner wall section of housing.

Figure 8 Schematic diagram of circuit.

Figure 9 Alternating current response curve.

10 Figure 9a First derivative of the curve of Figure 9.

Figure 10 Standard curve for DNA determination.

Figure 11 Continuous tape analytical work station

Figure 11a Section view of guide roller

Figure 12 Top plan view of tape filter element

15 SUMMARY OF THE INVENTION

This invention encompasses a work station for conducting assays which comprise a filtering assembly, a reading assembly, and a filter element with a porous filter membrane wherein the filter element has a means for

20 aligning the filter element within the filter assembly to filter an analyte or analyte complex at a plurality of predetermined locations on the porous filter membrane and also to position the filter element in the reading assembly so that detection will occur in those same

25 predetermined locations.

Those skilled in this art will recognize a wide variety of configurations of the filter assembly, reading assembly and filter element encompassed by this invention. Below are described two embodiments of this

30 invention. In one embodiment the filter element is a slide which is manually transported between the filter assembly and the reading assembly. In another embodiment described below the filter element is a long strip of plastic sheet material which defines openings covered

35 with a porous filter membrane. This strip or tape filter element runs through the filter assembly and then to the reading assembly. In each of these embodiments the filter assembly provides for filtration of analyte or analyte complexes at predetermined locations on the porous filter membrane. The reading assembly provides for detection of analyte or analyte complexes at those same predetermined locations.

Slide Filter Element Embodiment

This embodiment of the invention encompasses an analytical work station which has a filtering assembly, a slide with a porous filter membrane and a slide reading assembly.

The filtering assembly comprises a combination of: 1. a block with a top and bottom surface, the block having a plurality of channels with inlet ports on the top surface and exit ports on the bottom surface and a first alignment means;

2. a vacuum plate having an upper and lower surface wherein the lower surface has a vacuum fitting defining an area on the lower surface, with a plurality of vacuum ports from the upper to lower surface within the area defined by the vacuum fitting, the vacuum plate having a second alignment means;

3. a slide defining an opening which is covered by a porous filter membrane and further having a third alignment means.

The block and the vacuum plate fit together to form a space for removably inserting the slide between the bottom surface of the block and the upper surface of the vacuum plate and the first, second, and third alignment means align the block, vacuum plate, and slide such that the exit ports of the block, porous filter membrane, and vacuum ports are aligned so that when a vacuum is applied through the vacuum fitting, liquids in the block channels separately flow through the exit ports, through the porous 5 filter membrane, and through the vacuum ports so that liquids from one channel do not mix with liquids from other channels on the porous filter membrane. The analytes or analyte complexes in the filtered liquid are fixed on the porous filter membrane in a predetermined

10 location so as to substantially concentrate the analyte or complexes of the analyte.

The slide is a thin flat generally rectangular plastic sheet with a front and rear end. The front end of the slide has a notch as a means for aligning the slide

15 both in a filter assembly and a reading assembly. The rear end of the slide has an area for manually grasping the slide. The front, approximately a third of the slide, defines an opening which is covered with a porous filter membrane. A section of the front one third of the slide

20 is narrower than the rear section of the slide. The narrower section also serves as a means for aligning the slide in both a filtering assembly and the slide reading assembly. The alignment means provide for filtration of analyte onto predetermined locations on the porous filter

25 membrane in the filter assembly and for detection of analyte at these same predetermined locations in the slide reading assembly. The porous filter membrane has a specific binding substance such as a hapten, for example, biotin so that the porous filter membrane can capture a

30 specifically binding analyte complex at a specific location on the filter membrane. For example, an avidin or streptavidin complex is used in conjunction with a biotin labeled membrane.

The slide reading assembly has a housing with a

35 top, bottom, and inner and outer sidewall sections. The inner side wall section has at least one window or a plurality of holes as windows with a photoresponsive electrode mounted above the windows on the Inside of the inner wall section so that light from an external source can pass through the holes to the external surface photoresponsive electrode

The top section of the housing has an opening for receiving a slide to be analyzed. The slide has a porous filter membrane with analyte or analyte complexes to be determined at predetermined locations on the porous filter. The housing has a means for aligning the analyt -containing region of the porous filter membrane on the internal surface of the photoresponsive electrode opposite the holes in the housing which allow light to pass to the photoresponsive electrode. A resilient plunger is mounted in the outer sidewall section of the housing opposite the photoresponsive electrode. The plunger has a stem and pad which is pivotally mounted on the stem and located within the housing such that when the plunger stem is externally pushed into the housing by a stepping motor the pad compresses the porous membrane against the inner surface of the photoresponsive electrode. This reduces electrolyte volume and improves sensitivity.

There is a liquid chamber within the housing with exit and entrance ports for liquids located in the top section of the housing. The inner surface of the photoresponsive electrode is within the liquid chamber and exposed to the liquid. The porous filter membrane is aligned above the inner surface of the photoresponsive electrode also in the liquid chamber.

There is a light source comprising an array of light emitting diodes (LEDs) for independently irradiating portions of the photoresponsive electrodes with intensity modulated light through the holes in the housing. The area of the photoresponsive electrode exposed to intensity modulated light is opposite the area of the porous filter membrane having filtered analyte or analyte complex.

There is also an electrical means for measuring the rate of surface potential change of those portions of 5 the photoresponsive electrode which are exposed to the intensity-modulated beam of light through the holes in the housing. A chemical reaction which alters the surface potential is occurring between the analyte or analyte complex on the porous filter membrane and a reagent in the

10 liquid in the liquid chamber so as to change the surface potential of an exposed portion of the photoresponsive electrode. The electrical means includes a counter electrode or reference electrode and a control electrode, an operational amplifier, and a computer for processing

15 the signal data.

Thus, in operation a reaction between specific binding substances is conducted in a test tube, for example, the reaction between DNA, enzyme labeled anti- DNA, single strand DNA-binding protein bound to a biotin,

20 and avidin or streptavidin. This complex is filtered through a biotinylated porous filter membrane such that the (enzyme containing) analyte complex is captured by the porous filter membrane in small spots at a predetermined location on the membrane. The locations of these spots

25 are fixed by the alignment means in the block, slide, and vacuum plate. The slide is removed from the filter assembly and placed in the slide reading assembly which contains a substrate to the enzyme in the liquid in the liquid chamber. The plunger is pushed in by a stepping

30 motor to press the pad of the plunger against a photoresponsive electrode. This decrease in volume increases sensitivity by decreasing the amount of pH buffer in the measurement volume where the enzyme catalyses a pH change. The alignment means in the reading

35 assembly aligns the enzyme-containing spots of the porous filter membrane opposite the holes for light in the housing. The reaction of enzyme and enzyme substrate results in a change In surface potential in the local area of the photoresponsive electrode which is measured by the above electrical means in conjunction with the action of the intensity-modulated light on the photosensitive electrode.

Thus, the filter assembly provides for concentrating a complex containing the analyte to be determined in a small area of a porous filter membrane which is predetermined by the alignment means. This predetermined location of the spots on the filter membrane can then be aligned with a specific reading location in the slide reading assembly by complimentary alignment means. The pad which compresses the porous filter membrane against the photoresponsive electrode uniformly reduces volume and increases sensitivity. In this way picogram (pg) quantities of analyte such as DNA can readily be determined. The filter and slide reading assemblies provide for filtering and reading of standards, controls and unknowns under similar conditions as well as for the ease of identification of the sample, and for obtaining an analytical result from the same sample.

Tape Filter Element Embodiment

In this embodiment the porous filter membrane is located in openings in a long strip or tape of support material. Preferably, the tape is in the form of a thin plastic strip with sprocket holes on both sides that serve as an alignment means for aligning the porous filter membrane in the filter assembly and reading assembly so that analyte or analyte complexes are filtered onto

^• predetermined locations on the porous filter membrane and readings are performed by the reading assembly on those predetermined locations. In this embodiment the tape filter element moves automatically to the filter assembly where analytes or analyte complexes are filtered as described for the slide filter element onto predetermined locations of the porous filter membrane. After filtration the tape filter element containing porous filter membrane is automatically advanced to the reading assembly where 5 readings are taken at the predetermined locations containing the analyte much the same as described in the slide filter element. In this case, the sprocket holes in the tape serve as the alignment means. Thus, in this embodiment the porous filter membrane, the reagents and

10 assays, the method of filtering and the method of detection are essentially the same as the slide filter element embodiment. Both the tape and slide filter element embodiments rely on filtering analyte on predetermined locations and performing analytical

15 determinations on those predetermined locations.

DETAILED DESCRIPTION OF THE INVENTION The Slide

Figure 4a is a top view of the slide.

The slide 4 is made of a thin plastic sheet and

20 has an opening which is covered with a porous filter membrane 5. The notch 3 at the front end of the slide and the angle 25 defined by the narrow and wide portion of the slide serve as means for aligning the slide in the filter assembly and the slide reading assembly. Slits 3a permit

25 the notch to resiliently expand. The rear end of the slide has an area 7 for manually grasping the slide. A porous filter membrane suitable for practicing this invention is described in great detail in SN 258,894 assigned to the same assignee of this application. S.N.

30 258,894 is incorporated herein by reference.

The porous filter membrane is capable of filtering from a solution containing a specifically binding complex having an anti-hapten bound to a binding member of the specifically binding complex. The material of the porous filter membrane is selected from material to which protein or other macromolecule can be adhered. A variety of materials may be used. Those skilled in the art will appreciate that 5 porous membranes made of nylon, cellulose acetate, polyolefln, polyacrylamide, nitrocellulose or other porous materials may be employed in the present invention. Other synthetic or naturally occurring materials which will adhere a protein or other macromolecule may also be used. 10 A preferred membrane is made from nitrocellulose.

Physical characteristics of the porous filter membrane in the slide are:

(a) pore size 0.25 to 12.0_/jm

(b) thickness 50 to 180μm 15 (c) bubble point 75 to 6 psi

A membrane having 1-10 millimol (per liter of compressed membrane volume) buffering capacity is preferred.

Haptens are substances which do not elicit antibody formation unless complexed to macromolecules and

20 which may be employed as specific organic materials for which specific binding substances can be provided. Antibodies to haptens can be formed by binding the hapten to a protein so as to elicit an antibody response. A specific binding substance is any substance or group of

25 substances having a specific binding affinity for the hapten to the exclusion of other substances. The hapten must be able to bind to a protein or other macro-molecule directly or through an extended linking group. Examples of haptens which may be used include steroids such as

30 estrone, estradiol, testosterone, pregnanediol and progesterone; vitamins such as B.-, biotin and folic acid; triiodothyronine, thyroxine, hlstamine, serotonin, digoxin, pros aglandins , adrenalin, noradrenalin, morphine, vegetable hormones and antibiotics such as

35 penicillin. When the hapten is a substance having a naturally occurring receptor, the receptor can be utilized as the anti-hapten provided the receptor can be isolated in a form specific for the hapten. Illustrative haptens 5 which have naturally occurring receptors include thyroxine, many steroids, polypeptides, such as insulin, angiotensin, biotin and many others. Receptors for this class of haptens are usually proteins or nucleic acids.

Extended linking groups are groups that will

10 bind the hapten to the protein or macro-molecule in such a way that the hapten has better access to the anti-hapten. here an extended linking group is not needed, a hapten, such as biotin without an extended linking group, is bound to a functional group on a membrane or to a function group

15 on a protein which can be disbursed on the membrane.

Preferentially the extended linking group having an hapten bound to one end will be bound to the protein or macro-molecule with an amide bond; the amine of the amide bond arising from the protein and the carboxyl

20 of the amide bond arising from the carboxy terminus of the extender group. Free carboxyl or hydroxyl groups on proteins can likewise be used.

The proteins used as carriers include, but are not limited to, bovine serum albumin (BSA), bovine gamma

25 globulin, and fibrinogen. A preferred membrane for use with the slide has 5-20 molecules of biotin bound to bovine serum albumin (BSA) The BSA is further adhered to the surface of the membrane. The preferred linking group is the following:

Complexes typically removed from solution by the membrane include:

Porous Filter Membrane Complex membrane - hapten anti hapten - Ab Ag-Ab (enzyme) 5 membrane - (biotin) (streptavidin)-Ab' Ag'Ab (enzyme)

The antibodies employed may be either polyclonal or monoclonal antibodies and are produced in response to the target antigen of the assay. Methods for the production of antibodies to various biological substances

10 are well known in the art.

The antigens targeted by the assay include, but are not limited to antigens such as IgE, prostatic acid phosphatase, prostate specific antigen, alphafetoprotein, carcinoembryonic antigen, leutenizing hormone, creatlne

15 kinase MB, Human Chorionic Gonadotropin (HCG) and other antigens in serum, plasma, urine, or other liquid media.

Polydeoxyribonucleotides can be determined by reactions with single strand DNA binding protein (SSB) and anti-DNA antibodies. Thus, various combinations of

20 labeled SSB or anti-DNA and biotinylated SSB or anti-DNA are employed. In this embodiment streptavidin is bound to the biotinylated SSB or anti-DNA so that the complex can be bound to the capture membrane having biotin. The article in Biochemistry. £5:21 (1986) describes the large

25 scale over production of single-strand binding protein

(SSB) from E. coli. Monoclonal antibodies to DNA have been used to measure DNA in biological fluids, Journal of Immunological Methods. 88. (1986) 185-192. This complex and membrane, can be viewed as follows:

30 Porous Filter Membrane Complex membrane-biotin ^• streptavidin-biotin-anti-DNA/DNA-SSB-enzyme Examples of enzymes which may conveniently be employed are, malate dehydrogenase, lipase, delta-5- ketosteroid isomerase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase, alpha glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, and more preferably, urease. Normally, it is preferred that the enzyme be in a pure form, free of contaminating proteins.

10 The preparation of the enzyme-labelled biological substances can be accomplished in various ways known in the art. Examples of the coupling of biological substances to enzymes are described in, for example, L. A. Steinberger, Immunocytochemistry, Prentice Hall, New

15 Jersey (1974).

The enzyme label on porous filter membrane is made to react with an enzyme substrate and the extent of reaction is measured by the photoresponsive electrodes and methods described in U.S. Patents 4,591,550, 4,704,353,

20 and U.S. Patent Application 876,925, filed June 20, 1986 and assigned to the same assignee as this application. The slide reading assembly is an adaption and improvement of these devices to read the predetermined locations on the membrane where the enzyme has been filtered.

25 The photoresponsive electrode can be influenced by a wide variety of redox systems where the redox reaction occurs at the surface of the photoresponsive electrode where the light strikes the surface of the photoresponsive electrode.

30 The Filter Assembly

Figure 1 is a perspective view of the analytical work station 1. In this embodiment there are four filter assemblies 2 and a slide reading assembly 40. Figure 2 is a top view of a filter assembly which shows a

35 filter block 10, vacuum plate 11 and slide 4 and Figure 3 is a cross-sectional view of 3-3 of Figure 2. The filter block 10 has a plurality of channels 12 which narrow from the inlet port 13 to the exit port 14. The exit ports 14 are centrally located in a smooth surface area 14a at the bottom end of the block. The bottom surface of the block 5 has a hole 23 as an alignment means to align the slide with the vacuum plate 11. The block has small projections 15 at the lower end of the side walls which are gripped by the clamping mechanism spring clip 17 through opening 16 to fix and release the slide between the filter block 10

10 and the vacuum plate 11 by turning lever 18 as shown in

Figures 1 and 3.

The vacuum plate 11 has on its upper surface a plurality of radial cylinders 19 through which pass the vacuum ports 20 as shown in Figure 3 which is a cross-

15 section of 3-3 of Figure 2, also see Figure 4. The smooth lower surface of the filter block and the smooth upper surface of the raised cylinders of the vacuum plate compress the membrane 5. When the lever 18 is turned the rotation of the lever moves the spring block 29 up and

20 down as the cam 28 mounted on the lever shaft rotates 90 degrees. The spring clips 17 follow a cam surface 27a and spread in the upper position allowing an easy removal of the filter assembly from the vacuum. In the lower position the spring clips are closed firmly and without

25 any side forces hold the filter block locked against the vacuum plate. This compression prevents fluid from one filter pathway 21 crossing over to another filter pathway 21a . The vacuum plate has a peg 22 which interacts with the hole 23 of the block and the notch 3 of the slide

30 (shown in Figure 4a) to align the exit ports 14 in the block and the vacuum ports 20 in the vacuum plate. Slits 3a provide for expanding the notch 3 to resiliently clamp the peg 22. The vacuum plate further has a depression 24 which fits the shape of the slide 25 to rigidly fix the 35 slide in place as shown in Figure 2. Turning 18 in

Figures 3 and 4 releases the pressure exerted by block 10, and thus permits the slide 4 to be removed from the filtering assembly.

Figure 4 shows the filter assembly disassembled into its component parts and further illustrates the arrangement of the block 10, manifold bottom 8, slide 4, and vacuum plate 11 as they sit in the vacuum manifold 9. Further illustrated in Figure 4 are the liquid level sensor 26, manifold insert 27, and check valve 27b which engages vacuum fitting 27c on the vacuum plate 11. Figure

10 4 further illustrates how lever 18 interacts with cam 28 and spring block 29 to spring clip 17 which has hole 16 engaged with projection 15 of the filter block 10. Thus turning 18 lowers the block 10 and releases the porous filter membrane 5.

15 Figure 3 illustrates the interaction of clamping mechanism 18, 28, 29, 17, 16 and further shows the engagement of spring clip 17 with the manifold insert 27 at point 27a. The manifold insert 27 engages the vacuum plate 11 at the vacuum fitting 27c. The vacuum

20 ports 20 lie within the vacuum fitting 27c and the check valve 27b lies within the manifold insert 27. Projections 27d push against the check valve 27b to open the check valve.

The vacuum system is schematically illustrated

25 in Figure 5. The vacuum pump 30 is controlled by the vacuum pump control 31. The vacuum to the vacuum manifold 32 is controlled by the vacuum control 33 which operates a proportional valve 34. A pressure transducer 35 provides for the vacuum sensor output 36.

30 The vacuum manifold serves as an effluent reservoir and as a vacuum reservoir. The individual filter assembly receptacle 9 in the manifold has a manifold insert with a check valve 27b, which is activated with the insertion of the filter base to start the vacuum

35 flow. The liquid level sensor 26 determines when the manifold should be emptied of waste fluid. The vacuum system provides vacuum to pull solutions in the channels of the filter block through the porous filter membrane on the slide by way of the vacuum ports. 5 The Slide Reading Assembly

The slide reading assembly is illustrated in Figures 6, 6a, 7 and 7a. The housing 40 has a slot 41 in the top end 42 for inserting the slide 4. The housing has an area with a plurality of holes 43 or windows in the

10 inner wall section 44 (see Figure 7a) and a photoresponsive electrode 45 mounted over the holes. This photoresponsive electrode 45 has a transparent layer of silicon dioxide which defines areas of transparent circular spots 46 which are aligned with the holes 43 in

15 the housing. The windows 43 in the housing and transparent circular spots 46 permit light to pass from the array of LED's 47 from outside the housing to the surface of the photoresponsive electrode as shown in Figures 7 and 7a. Within the reading assembly housing is

20 a peg 48 which serves as a means for aligning the slide by engaging the notch 3 (see Figure 4a) in the front end of the slide. Also there is a depression 49 on a slide support member 50 which further aligns the slide so that the porous filter membrane 5 (see Figure 4a) is located

25 above the photoresponsive electrode 45 and also the predetermined areas in the porous filter membrane 51 where analytes or analyte complexes are located are aligned above the transparent circular spots 46 on the photoresponsive electrode. The housing also defines a

30 liquid chamber 52 and there is an exit 53 and an entrance port 54 for filling and emptying to liquid chamber 52. The liquid in the liquid chamber is in contact with the photoresponsive electrode and the porous filter membranae. The liquid serves as an electrolyte and contains a

35 reagent that reacts with the analyte or analyte complex on the porous filter membrane. For example, if the analyte complex contains an enzyme, the liquid in the liquid chamber will contain an enzyme substrate and this enzyme/enzyme substrate reaction causes a local change in pH on the surface of the photoresponsive electrode. As 5 shown in Figure 7 the slide reading assembly has a plunger

60 resiliently mounted by flexing member 55 in the top of the housing. The plunger 60 has a stem 61 and a pivotally mounted pad 62. The ball and socket mounting 65 gives flexibility to the mounted pad 62.

10 Figure 7 shows light from LED 47 passing through holes 43 in the inner wall 44 and through the transparent spots 46 on the photoresponsive electrode 45 which are aligned with the predetermined areas 51 in the porous filter membrane 5 where analytes or analyte

15 complexes are located.

This pivotally mounted pad 62 is pushed against the porous filter membrane 5 by a stepping motor 63 which is operatively associated with the stem 61 of the plunger 60. This pad 62 reduces the volume of liquid above the

20 porous filter membrane and greatly increases the sensitivity of the photoresponsive electrode. Figure 7a illustrates the holes 43 in the inner wall section 44.

U.S. Serial Number 065,418 filed 6/18/87 and assigned to the same assignee as this application

25 describes in great detail the photoresponsive electrode and the increase in sensitivity resulting from reducing the volume of liquid above the membrane. The specification of U.S. Serial Number 065,418 is incorporated herein by reference. The present invention

30 differs from the invention described in U.S. Serial Number

065,418 in that the slide is aligned so that spots on the membrane having analyte or analyte complex are aligned above the irradiated portion of the photoresponsive electrode and the pivotally mounted pad uniformly

35 compresses the areas of the porous filter membrane where the analyte is located to exclude excess volume of bufferlng electrolyte so as to greatly increase sensitivity of the photoresponsive electrode for measuring potential changes resulting from the reaction of enzyme substrate and enzyme bound to the porous filter membrane 5 due to the presence of analyte or analyte complex.

The operation of the photoresponsive electrode, including the reference electrode, controlling electrode, materials for the electrode, light source, nature of measurable chemical reactions, signal amplification and 10 measurement, types of measurable enzyme and redox reactions and the like are described in great detail In U.S. Patents 4,704,353; 4,758,786 and 4,591,550, which are incorporated herein by reference. A schematic of an electrical circuit usable in the present invention Is 15 shown in U.S. Patent 4,758,786.

Figure 8 shows a preferred circuit for use with this invention. Shown in Figure 8 is a schematic diagram of a computer-controlled electronic circuit which may be used to operate the potentiometric reading device in 20 accordance with the present invention. A photoresponsive electrode 45 (e.g. n-type 10-25 ohm-cm, 100 crystalline silicon) polished on one side is covered on the polished side with an insulator 82 which is in contact with an electrolyte 84 enclosed by a chamber wall 86 sealed to the 25 insulator surface by a silicon polymer 88. Operational amplifiers 90 and 92, together with resistors 94 and 96, reference electrode 98, controlling electrode 100, and digital-to-analog converter (D/A) 118 mounted on master circuit board 102, function to determine the potential of 30 the electrolyte with respect to the bulk of the photoresponsive electrode 45. Computer 112, having keyboard 114, varies the potential of electrolyte 84 via the output of D/A 118. The potential of the photoresponsive electrode 45 is held constant at virtual 35 ground by copper lead 106 which is attached to the underside of the photoresponsive electrode 45 through a brass spring and ohmic contact 104. Ohmic contact 104 is made by evaporation of 99% gold - 1% arsenic onto the non- insulated silicon surface followed by alloying above the gold-silicon eutectic temperature. One light-emitting 5 diode (LED) 47 of an array of nine similar LEDs (not shown) is powered by LED driver 110 so as to irradiate the photoresponsive electrode at the desired X-Y-coordinate with light of 50% duty cycle, on/off-modulated intensity. Any one of the nine similar LEDs may be selected for light

10 intensity modulation by computer 112 which acts on a switching circuit in LED driver 110. The frequency of intensity-modulation is determined by LED driver 110 to be about 10 KHz. Current-to voltage converter 107, connected through lead 106, measures the alternating photocurrent

15 generated in photoresponsive electrode 45. The voltage output of current-to-voltage converter 107, is filtered by bandpass filter 109 and rectified by rectifier 111 to give a dc voltage output that is proportional to the alternating current amplitude. This analog dc voltage

20 output is converted into digital form by analog-to digital converter (A/D) 116 and stored as data in the memory of computer 112. Experimentally-acquired data may be viewed on CRT display 120 and permanently displayed by printer 122.

25 In operation, modulated current is applied from

LED driver 110 to cause LED 47 to be modulated in intensity. The output of D/A 118 is ramped by a program in computer 112 so as to ramp the potential of electrolyte 84. The electrolyte potential, in turn, affects the

30 electrical field within the photoresponsive electrode which, in turn, affects the alternating current generated in the illuminated portion of photoresponsive electrode 45, flowing through lead 106, and measured by current-to voltage converter 107. Figure 9 shows a typical

35 alternating current response to changes in the electrolyte potential is determined. The values of current vs. electrolyte potential are stored for subsequent analysis of the rate of change in this relationship. In a preferred embodiment of this invention, the point of maximum slope in the curve of

5 Figure 9 is determined, i.e. that point at which the resulting alternating photocurrent finds its maximum change for a given change in electrolyte potential. A convenient way of determining this point of maximum slope of the photocurrent of Figure 9 is to take the second 10 derivative and determine where the second derivative is equal to zero. This is depicted in the graph of Figure 9a. To avoid consideration of the data points where the alternating photocurrent is varying only slightly vs. electrolyte potential, the data near the maximum or

15 minimum alternating photocurrents are not used in the analysis. For example, the data points associated with photocurrent less than 10% and more than 90% of the maximum alternating photocurrent are neglected. Alternatively, the same objective may be achieved by

20 considering only data points between the largest maximum and smallest minimum of the second derivative of curve in Figure 9. Alternative electronic circuits, methods of analysis, and variety of materials that may be employed In photoresponsive potentiometrie reading devices are

25 disclosed in U.S. Patent Application, "Photoresponsive

Detection and Discrimination," U.S.S.N. 231,091, filed August 11, 1988, assigned to the same assignee as this application and herein incorporated by reference.

The measured rate of change in the relationship

30 between the alternating photocurrent amplitude and the electrolyte potential is determined by the rate of change of the electrostatic surface potential present at the electrolyte-exposed surface of the photoresponsive electrode at the X-Y coordinate of Irradiation with

35 intensity-modulated light (Science 240. 1182-1185, May 27,

1988) . This surface potential is determined by the pH of the electrolyte exposed to the surface when a pH-sensitive surface, e.g. silicon nitride is employed and is redox potential sensitive when the surface is a noble metal, e.g. platinum or gold (see U.S.S.N. 231,981, 072,168, and 5 065,418).

A photoresponsive electrode can be influenced by the redox potential of the medium adjacent the electrode surface. Various redox systems can be employed, enzyme reactions, particularly oxldoreductases,

10 e.g., glucose oxldase, peroxidase, uricase, NAD or NADP dependent dehydrogenases, naturally occurring electron transfer agents, e.g., ferridoxin, ferritin, cytochrome C, and cytochrome b2, organic electron donors and acceptor agents, e.g., methylene blue, nitro tetrazolium, Meldola

15 blue, phenazine methosulfate , etallocenes, e.g., ferrocenium, naphthoquinone, N,N'-dimethyl 4,4'-dipyridyl, etc., and inorganic redox agents, e.g., ferri- and ferrocyanide, chloronlum ion, cuprous and cupric ammonium halide, etc.

20 According to the foregoing specification, the slide reading assembly has a photoresponsive electrode with a multiplicity of measurement sites, and includes both a reference and a controlling electrode, as shown in Figure 8. Alternatively, the reference electrode may be

25 made optional by shorting the output of operational amplifier 90 and the input of operational amplifier 92, shown in Figure 8, and making a common connection to a single counter electrode placed in the electrolyte 84. The counter electrode may be extremely simple, namely a

30 foil or wire of a metal such as platinum, gold, irridium, titatium, etc. which is inert to the electrolyte. In this alternative mode, the photoresponsive electrode has a multiplicity of measurement sites that includes at least one control site where no analyte is present and is

35 thereby able to, by difference, measure the rate of potential change generated by the chemical reaction due to presence of the analyte at other sites at the surface of the photoresponsive electrode.

In operation the stepper motor 63 in Figure 7 is engaged with the plunger stem 61 to push the pad 62 of the plunger against the porous filter membrane. A chemical reaction occurs at the surface of the photoresponsive electrode between reagent in the liquid such as an enzyme substrate with an enzyme which is part of an analyte complex. Light from an LED 47 goes through

10 the holes 43 in the housing and is absorbed within the photoresponsive electrode 45. The chemical reaction such as the reaction of urease with urea to release ammonia and carbon dioxide causes a change in pH or redox potential of the electrolyte. This in turn affects the surface

15 potential of the photoresponsive electrode, which in turn alters the effect of light on the photoresponsive electrode.

DNA Assay Standard Curve Membrane: biotln-BSA coated nitrocellulose

20 membrane (o.8μ pore side). DNA sample: 0, 5, 10, 25, 50, 100, 150, 200 pg of single-stranded Calf thymus DNA in 0.5ml of phosphate buffered saline buffer 50 mm aPθ , 150 mm NaCl, 1 mm

25 EDTA; 0.05% sodium azide pH 7.0. Reagent :0.5μg/ml Streptavidin, lng/ml SSB- biotln, 250ng/ml anti-DNA-urease, 0.1% BSA in lOmM tris-HCl buffer, lmM EDTA (pH 7.4) plus 0.25% triton X-100, 0.05%

30 sodium azide. Assay Protocol: 500μl of DNA sample was incubated with lOOOμl of reagent at 37° for 60 minutes. The mixture was filtered through the biotin-BSA coated membrane

35 at a flow rate of about lOOμl/min. The membrane was then washed with lcc of wash buffer (10 mm NaPθ , 100 mm NaCl, 0.05% Tween 20, 0.05% sodium azide, pH 6.5) at a maximum flow rate of about 6ml/min) . After washing, the slide was transferred to the slide reading assembly where the liquid contains 100 mM urea in the wash buffer as substrate.

10

15

The results are shown as a curve in Figure 10. Thus the 20 analytical work station permits the determination of the picogram (pg) qualities of DNA at quantities as low as 2pg over a dynamic range of 2-200 pg.

Continuous Tape Analytical Work Station

The tape analytical work station is illustrated

25 in Figure 11 and Figure 12. Figure 12 shows the tape 200 which defines openings 201 which are covered with a porous filter membrane 202. Preferably the porous filter membrane is 0.005 inch nitrocellulose and the tape is opaque polyester, 0.003 inch thick. Openings 203 are used

30 as alignment means to activate an optical interrupter switch 211 which serves to position the tape in the filter assembly and then in the reading assembly. Now referring to Figure 11, the tape is transported between a supply reel 205 and takeup reel 206. A fluid station 207 mixes sample and reagents and delivers the solution to be filtered to the filter assembly 208. The reading assembly 209 receives tape 200 from filter assembly 208. Guide

5 rollers 210 are located before the filter assembly 208 and after the reading assembly 209. These guide rollers 210 align the tape in the transverse direction for both the filter assembly and the reading assembly. An optical interrupter 211 is activated by openings 203 in the tape 10 200 and serve to align the tape filter membrane longitudinally in the filter assembly and reading assembly. The spacing between openings 203 on the tape is set to correspond with the longitudinal separation between the filter assembly and reading assembly. The sensor

15 electrode is shown as 212 and the plunger 213 is activated by overhead cam 214. Substrate for reacting with enzyme reagents on the porous filter membrane is delivered from chamber 215. 216 is a waste collection system which collects waste from both the filter asembly and the sensor

20 electrode.

In filtering, the porous filter membrane 202 is compressed between the filter 217 and the receiving manifold 218 by overhead cam 219 which are operated by stepper motors. Positive pressure in the filter channels

25 forces liquid to be filtered through the porous filter membrane at predetermined locations.

In operation, a porous filter membrane 202 is aligned between the filter channels 217 and receiving manifold 218 by the interaction of the optical interrupter

30 211 and guide rollers 210 with the tape 200. Overhead cam

219 compresses the porous filter membrane 202 between the filter channels 217 and receiving manifold 218. Solutions containing analytes or analyte complexes are forced through the porous filter membrane 202 to filter the

35 enzyme-containing analytes at predetermined locations.

Plunger 219 is released and the tape 200 is then advanced so that the porous filter membrane containing enzyme at predetermined locations is aligned by the optical interrupter 211 In the reading assembly 209. Substrate is added to the porous filter membrane and drive 214 pushes 5 plunger 213 so that the porous filter membrane 202 is compressed between the plunger 213 and the silicon sensor 212 so that measurements can be made at the predetermined locations or spots which contain enzyme as in the slide reading assembly described earlier. To allow the next

10 filter membrane 202 to be advanced to the reading assembly

209, plunger 213 is released and the tape is advanced to the takeup reel 206. Substrate is removed by vacuum evaluation of the reading assembly prior to tape transport.

15 The aforementioned embodiments illustrate the present invention and are not intended to limit the invention in spirit or scope.

Claims

What is Claimed is:

1. A filter element comprising a support member defining an opening which is covered with a porous filter membrane and wherein the support member has alignment means providing for aligning the filter element in a filter assembly and reading assembly whereby analyte or analyte complexes are filtered and read on the same plurality of predetermined locations on the porous filter membrane.

2. A slide filter element comprising a thin flat generally rectangular plastic sheet with a front and rear and wherein the slide has a means for aligning the slide both in a filter assembly and a slide reading assembly; the rear end of the slide having an area for manually grasping the slide; the slide filter element further defines an opening which is covered with a porous filter membrane and wherein the alignment means provide for deposition of analyte or analyte complex onto predetermined locations on the porous filter membrane in the filter assembly and for detection of analyte or analyte complex at these same predetermined locations in the reading assembly.

3. A slide filter element according to Claim 2 comprising a thin flat generally rectangular plastic sheet with a front and rear end wherein the front end of the slide has a notch as a means for aligning the slide both in a filter assembly and a reading assembly; the rear end of the slide having an area for manually grasping the slide; wherein the front, approximately a third of the slide, defines an opening which is covered with a porous filter membrane and a section of the front one third of

' the slide is narrower than the rear section of the slide, said narrower section also serving as a means for aligning the slide in both a filtering assembly and a reading assembly and wherein the alignment means provide for deposition of analyte complex onto predetermined locations on the porous filter membrane in the filter assembly and for detection of analyte complex at these same predetermined locations in a reading assembly.

4. A slide filter element according to Claim 2 5 wherein the porous filter membrane has fixed to it a hapten.

5. A slide filter element according to Claim 4 wherein the hapten is biotin.

6. A slide filter element according to Claim 5 10 which has a buffering capacity of 1-10 millimole per liter of compresed membrane volume.

7. A tape filter element according to Claim 1 comprising an elongated strip of sheet material which periodically defines openings in the central portion of

15 the strip which are covered with a porous filter membrane and with openings at the edges of the strip for aligning the porous filter membrane in a filter assembly and a reading assembly.

8. A tape filter element according to Claim 7 20 wherein the porous filter membrane has fixed to it a hapten.

9. A tape filter element according to Claim 8 wherein the hapten is biotin.

10. A method for determining analytes 25 comprising deposition of analyte or complexes of analyte onto a plurality of predetermined locations on a porous filter membrane, positioning the porous filter membrane on a photoresponsive electrode such that light strikes the photoresponsive electrode below the location of the

30 analyte or analyte complex on the porous filter membrane and reacting the analyte or analyte complex with a reagent in an electrolyte solution wherein the reaction between the analyte or analyte complex and reagent alters the potential of the surface of the photoresponsive electrode

35 and measuring the potential change with intensity modulated light.

11. An analytical work station comprising: (a) a filter element having a porous filter membrane and means for aligning the porous filter membrane; 5 (b) a means for filtering analyte or analyte complex at predetermined locations on the porous filter membrane; and

(c) means for analyzing the analyte or analyte complexes at the predetermined locations on the 10 porous filter membrane wherein the means for aligning the porous filter membrane aligns the predetermined locations where the analyte or analyte complex is filtered by the means for filtering and analyzed by the means for analyzing. 15

12. A filtering assembly comprising in combinatio : a block with a top and bottom surface, said block having a plurality of channels with inlet ports on the top surface and exit ports on the bottom surface and a 20 first alignment means; a vacuum plate having an inner and outer surface wherein the outer surface has a vacuum fitting defining an area on the outer surface, with a plurality of vacuum ports from the inner to outer surface within the area 25 defined by the vacuum fitting, said vacuum plate having a second alignment means; a slide defining an opening which is covered by a porous filter membrane and further having a third alignment means; 30 wherein the block and the vacuum plate have spacing means providing space for removably inserting the slide between the block and the vacuum plate and wherein the first, second, and third alignment means align the block, vacuum plate, and slide such that the exit ports of 35 the block, porous filter membrane, and vacuum ports are aligned so that when a vacuum is applied through the vacuum fitting, liquids in the block channels separately flow through the exit ports, through the porous filter membrane, and through the vacuum ports without liquids from one channel mixing with liquids from other channels 5 on the porous filter membrane and wherein analyte or analyte complex in the filtered liquid are fixed on the porous filter membrane in a predetermined location.

13. A filter assembly, according to Claim 12, wherein the first alignment means is a hole in the bottom

10 of the block; the second alignment means is a peg in the vacuum plate that is received by the hole; the third alignment means is a notch in the front end of the slide which engages the peg; and the top surface of the vacuum plate has a depression that conforms to the shape of the

15 sides of the slide.

14. A slide reading assembly comprising:

(a) a housing with a top, bottom, and inner and outer sidewall sections wherein the inner side wall section has a window with a photoresponsive electrode

20 mounted above the window on the inside of the inner side wall section so that light from an external source can pass into and be absorbed within the photoresponsive electrode; the top section of the housing having an

25 opening for receiving a slide to be analyzed wherein the slide has a porous filter membrane with analyte to be determined on predetermined locations on the porous filter membrane, said housing having means for aligning the predetermined locations on the porous filter membrane on

30 the inner surface of the photoresponsive electrode opposite the window in the housing which allow light to pass to a photoresponsive electrode; a resilient plunger mounted in the outer side wall surface of the housing opposite the

35 photoresponsive electrode, the plunger having a stem and pad such that when the plunger stem is pushed inwardly, by an operatively associated stepping motor, into the housing the pad compresses the porous membrane against the inner surface of the photoresponsive electrode; a liquid chamber within the housing with exit and entrance ports for liquids in the top section of the housing and wherein the inner surface of the photoresponsive electrode is within the liquid chamber and the porous filter membrane is aligned above the inner surface of the photoresponsive electrode in the liquid chamber;

(b) an intensity-modulated light source for independently irradiating the photoresponsive electrode through the window in the housing on each area of the photoresponsive electrode opposite the area of the porous filter having a predetermined location of filtered analyte or analyte complex; and

(c) an electrical means for measuring the potential change of the surface of the photoresponsive electrode when the photoresponsive electrode is exposed to an intensity-modulated beam of light through the window in the housing and a chemical reaction Is occurring between the filtered analyte or analyte complex on the porous filter membrane and a reagent in the liquid in the liquid chamber, the electrical means Including a counter electrode, and an operational amplifier.

15. A slide reading assembly according to Claim 14 wherein the inner surface of the photoresponsive electrode has a coating of insulation.

16. A slide reading assembly according to Claim 14 wherein the pad of the plunger is pivotally mounted on the stem of the plunger.

17. A slide reading assembly according to Claim 14 wherein the slide alignment means is a slide support member with a depression that fits the configuration of the slide and a peg that fits the slide notch.

18. A slide reading assembly according to Claim 14 wherein the alignment means is a peg at the bottom end of the slide reading assembly and a depression on a slide support member to fit the shape of the slide.

19. A slide reading assembly according to Claim 14 which has a reference electrode.