CA1249025A

CA1249025A - Chemical sensor for n-acetyl primary aromatic amines

Info

Publication number: CA1249025A
Application number: CA000492807A
Authority: CA
Inventors: Anthony E.G. Cass; Helena Bramwell
Original assignee: Medisense Inc
Current assignee: Medisense Inc
Priority date: 1984-10-12
Filing date: 1985-10-11
Publication date: 1989-01-17
Also published as: EP0184895B1; EP0184895A1; AU4853385A; NZ213816A; US4948727A; JPH0684952B2; DE3563064D1; JPS61274250A; AU581690B2

Abstract

A B S T R A C T

The present invention is concerned with a chemical sensor, more specifically with a chemical sensor which is capable of detecting the presence of, measuring a quantity of or monitoring the level of N-acetyl primary aromatic amines, such as paracetamol, in whole blood.

The specification relates to a method of assay of the type in which an electrode poised at a suitable potential is contacted with a system comprising:
a) a sample suspected to contain paracetamol or a derivative thereof, and b) an enzyme capable of catalysing the hydrolysis of an M-acylated primary aromatic amine or a derivative thereof, and, wherein the current flowing in the electrode is a measure of the quantity of hydrolysis products formed and thereby of the concentration of N-acylated primary aromatic amine or derivatives thereof in the sample.

Description

-The present invention is concerned with a chemical sen-sor, more specifically with a chemical sensor which is capable of detecting the presence of, measuring a quantity of or monitoring the level of N-acetyl primary aromatic amines, such as paraceta-mol, in whole blood.

Paracetamol (N-acetyl p-aminophenol) is a widely used analgesic with a rapid action and a general lack of side effects.
Although the drug has an effective action and favourable interac-tions with other drugs~ its availability has led to an increasinguse of the drug in suicide attempts. Fatal dosage causes death as a result of extensive hepatic necrosis.

Paracetamol is believed to function by inhibiting the synthesis of certain classes of prostaglandins and lipopolysac-charides which are believed to sensitize pain receptors.

Excretion of the substance from the body is believed to occur after conjugation in the liver. Current theory holds that at low concentrations, paracetamol is conjugated with sulphate or glucuronide, while at I

higher concentrations an oxidative metabolic path is initiated which forms cysteine and mercapturic acid conjugates following the reaction o~ an activated intermediate with reduced glutathione. At very high paracetamol concen~rations it is bel.leved that ~he hepatic reduced glutathione pool become6 depleted and the subsequent high levels of the activated intermediate cause hepatic cell necrosis. It is further believed that the reactive chemical groups of the intermediate which cause the cellular damage are sulphydryl groups.

The clinical onset of hepatic necrosis is the only overt symptom of paracetamol poisoning and does not occur until some 10-12 hour after ingestion. Furthermore, the poisoning must be treated before necrosis occurs and therefore before the first clinical symptoms occur.
Treatment normally comprises ~astric lavage or ingestion of an adsorbant such as activa~ed charcoal (if medical aid is available shortly after ingestion) followed by administration of a sulphydryl group(s) containing ~ubstance which competitlvely inhibits the effects of the activated intermediate.

However, the administration of the sulphydryl containing antidote has been found to ba dangerous if la~er than ten hours af~er ingestion, or if not correctly matched to the serum parace~amol concentration.

It is therefore desirous to provide a method by which a rapid and accurate measure of paracetamol levels can be made, whereby early aiagnosis and effective levels of treatment may be ensured .

Previous attempts to provide such a~ method have been based on chromatographic procedures requirin~ time, skill and expensive equipment. ~lternatively more specific colorimetric techniques based on the detection of p-aminophenol (PAP) have been used. p-aminophenol (MP 186C) is produced by the enzymic degradation of paracetamol, and once again expensive equipment and skilled staff are required. A particular restriction of the colorimetric method is ln the use of serum or plasma, rather than whole blood, due to the interference of red blood cells.

It is therefore further desired to provide a method of assay for paracetamol which can be performed by relatively unskilled persons without bulky and expensive equipment. Prefer-ably such a method should be usable in vivo, or in vitro with whole blood.
Our Canadian Patent No. 1,212,146 issued September 30, 1986 describes and claims a sensor electrode composed of electri-cally conductive material and comprising at least at an external surface thereof the combination o an enzyme and a mediator compound which transfers electrons to the electrode when the enzyme is catalytically active.

The purpose of such an electrode is to detect the presence of, measure the amount of and/or monitor the level of one or more selected components caeable of undertaking a reaction catalysed by the said enzyme.

The present invention is based on the realisation that a particular general reaction scheme has a particular utillity as the basis for an assay for paracetamol and related compounds.

According to a first aspect of ~he present inven~ion there is provided a method of assay of the type in which an elect~ode poised at a sui~able potential is contacted with a system comprising;

a~ a sample suspected to contain paracetamo~ or a derivative thereof, and ~b) an enzyme capable of catalysing the hydrolysi~ of an N-acylated primary aromatic amine or a derivative thereof, and, wherein the current flowing in the e~lectrode i6 a ';, measure of the ~uantity of hydrolysis products formed and thereby of the concentration of N-acylated primary aromatic amine or derivatives thereof in the sample.

It should be noted that the presen1 syst0m does not employ a mediator, and in this respect differs from our earli~r applications.

Our Canadian Patent No. 1,~26,036 :Lssued August 25, l987 entitled "Analytical Equipment and Sensor Electrodes there-for" describe the nature and manufacture of sensor electrodes.
Such electrodes are preferred in the practice of the present invention in which preferably the electrode is provided at its surface wi-th the enzyme but the mediator compound is omitted.

Accordingly, a second aspect of the invention comprises a sensor electrode having at a surface thereof, an enzyme capable of catalysing the hydrolysis of paracetamol or a derivative of paracetamol to a product, wherein the current flowing in the ~0 electrode is a measure of the reaction taking place and thereby of the concentration of paracetamol or derivatives thereof at the said surface.

Conveniently, the enzyme is of the type defined as EC
3.5.l.l3 and named as aryl acylamidase ~International Union of Biochemistry] (otherwise known as aryl-acylamide amidohydrolase).

Preferably, the enzyme is obtained from a bacterium.

A number of bacterial enzymes have been studied which convert paracetamol directly into p-aminophenol, for assay at the electrode surface. The following reaction scheme has been postu-lated for the processes occurring in assay systems employing these aryl acylamidases.

Under the action of the bacterial aryl acylamidase, .

parace-tamol is hydrolysed into p-aminophenol, and acetic acid.
Electrons are accepted by the conduction band of the electrode when the potential difference between the p-aminophenol in solu-tion and tha electrode surface is such tha$ the electrons find a lower energy band in the electrode. An electro-oxidation occurs therefore when a potential difference is applied to the elec~
trode, with respect to a reference electrode~, In a particular embodiment of the invention, the enzyme is produced from a species o Fusarium.
In a further particular embodiment of the invention, the enzyme is produced from a species of Pseudomonas.

In order that the nature of the invention is better ~2~

understood it will be further explained by way of example and with reference to the accompanying figures wherein;

Figure l; Shows the postulated reaction scheme for the hydroly~is of paracetamol by aryl acylamidase.

Figure Z: Shows the standard three-electrode system Vo /~ r~'c!
used in all the ~Y}~*e~ measurments, ~, ,; ~

Figure 3; Shows a cyclic voltammogram of p-aminophenol at a lmM final concentration, Figure 4: Shows the effect of the addition of aryl acylamidase to a cyclic voltammogram of parace~amol at a lmM concentration, Figure 5: Shows a calibration curve for paracetamol at pH 7.0, FiguLe 6; Shows a calib~ation curve for paracetamol at p~ 7.5, Figure 7; Shows a calibration curve for paracetamol at pH 8~0, Figure 8; Shows a calibration curve for paracetamol ~2~

at pH 7.0 in 100% Control Serum.

Figure 9: Shows a comparison of an a~say according to the present invention with a standard paracetamol assay (Cambridge Life SCiencQ), and , Figure 10; Shows the results of using an immobilised enzyme electrode to detect a fixed amount of paracetamol, Working electrodes of different materials were used:
gold, platinum , glassy carbon and nickel (solid) electrodes we~e made ~rom an electrode disc and brass connecting rod housed in a teflon coating. Graphite paste elactrodes were constructed of a bronze connector housed in araldite (from Radio Spares) and were ~ade in the laboratory. The araldite coating (held in a glass collar) had a cup bored in one end to expose the connector. The graphi~e paste material was packed into the cup.

Graphi~e paste was made using graphite powder mixed with Nujol or graphite powder mixed with araldite which was allowed to harden in the cue. In every case the counter electrode was held close ~o the working elec~rode material to facilitate ea~y passage of electrons.

The following examples illustrate the use of technique&

:,.

~` ~.2~

comprising the present lnvention, EXAMPLE l;

~vclic Voltamme ry of p-aminophenol A buffer solution was prepared from potassium di-hydro~
gen phosphate ~5.31 g; Analar from ~3ritish Drug Houses (BDH~ and di-potassium hydrogen phosphate (13.92 g; Analar from BDH), which were dissolved in Milli-Q water, adjusted -to pH 7 and made up to a final volume of 1 litre.

p-amlnophenol solution was prepared by dissolving 54.56 mg of p-aminophenol ~BDH) in approximately 80 ml Milli-Q water and adjusted to pH 11 with 0.1 M NaOH. The solution was then acidified to pH ~ with 0.1 M ~Cl and made up to a final volume of 100 ml with Milli-Q water. The p-aminophenol solution was pro-tected from light and used immediately after preparation.

The working electrodes were made from a range of dif-ferent materials, e.g. gold, glassy carbon and most especially pyrolytic graphite. The electrodes were routinely cleaned between runs using a slurry of 0.3~ alumina (BDH) in water in order to remove impurities and oxidation products from the elec-trode surface. The

2~

alumina was subsequently removed from the electrode surface by ultra sonication.

Cyclic voltammogram~ were ~roduced from a range of solutions by ~weeping ~he potential from zero to + ~00 mV and back down to - 20Q mV vs a saturated calomel electrode (SCE). The potential applied was controlled by a potentios~a~ (from Jaytron I~s. A.S. Scientific, ~bingdon) using a scan rate of 50 m~/s.

The oxidation current produced was recorded on a Gould Series 60000 Chart Recorder in which the X-axis recorded the applied potential and the Y-axis recorded the current produced. A cyclic voltammogram of p-ami~ophenol (at lmM final concentration) is shown in Figure ~.

ExamDle 2 Sensor IncorPora~inq Aryl Acylamidase Paracetamol ~N-acetyl p-aminophenol; Sigma Chemical Co.) was di~solved in the potassium phsophate buffer to give A final concentration of 25 mM. Aryl acylamidase, extracted from a Pseudomonas species, was supplied freeze-dried in 10 ml glass vials by P.H.L.S. Centre for Applied ~icrobiology ~ Research, Porton Down, Salisbury. The enzyme was stored at - 20C and reconstitu~ed with 1 ml Milli-Q water per vial as required.

Aryl acylamidase, extracted from a Fusarium species, was supplied by Sigma Chemical Co.

The enzyme solution was routinely assayed using the method of Atkinson, A ., Hammond, P.M., Price, C.P. and Scawan, MoD~ (U~ Patent 2,089,978 B). In this system 1 ml of 1% (w/v) aqueous o-cresol and 0.1 ml of ammoniacal copper sulphate, com-prising 25 mls of a 0.2~ Iw/v~ aqueous solution of anhydrous cop-per sulphate mlxed with 0.4 ml of 0.880 ammonia, are added to 1.4 mls of water in a disposable cuvette. The solution is mixed thoroughly and 0.5 mls of a standard p-aminophenol solution are added. The solution is then mixed again and allowed to stand for five minutes after which the absorbance of the solution at 614 nm is measured. One unit of enzyme is defined as converting 1 mole of paracetamol to p-aminophenol per minute at pH 7 and 37C.

The electrodes used were identical to those described above with reference to Example 1.
In -the cyclic voltammograms the cells contained 24,~1 of paracetamol solution (25 mM as above) and 576,~1 of buffer solution.

Cyclic voltammograms were recorded both in the absence and presence of 0.9 U aryl acylamidase (120 ~1 of enzyme solu-tion above). In order to ensure that the reaction had been ini-tiated each sample was incuhated at 37C for 2 minutes before starting the scan.
A cyclic voltammogram is shown in Figure 4 which shows that on addition of the aryl acylamidase solution prior to initi-ation of the scan, a substantial change in the profile of the voltammogram is observed. This is ascribed as being due to the catalytic conversion of paracetamol to p-aminophenol by the en~yme.

Example 3. ~7 SteadY-State Measurements in Buffered solutions In steady-state measurements the current produced upon application of a fixed potential to a stirred solution was mea-sured on the Y-axis of the chart recorder using the X-axis as a time base. The potential was poised at +250 mV va SCE at 37C
after allowing 2 minutes for the system to come -to equilibrium.
Stirring of the solutions ensures that the layer of material close to the electrode and which is available for oxidation is replenished and thus the current produced at the electrode does not decay due to exhaustion of reagents.

...
. ..

The stirrqd solutions comprised of 200 ~1 of aryl acylamidase solution (1.5 Units) and 800 ~1 of buffer solution ~pM 7). Steady-state electrochemical measurements were made in the presence of increasing amounts of paracetamol solution to produce a linear calibration curve for paracetamol and is shown in Figure 5.

A similar linear calibration curve was produced in buffer solution at pH 7.5 (made from 2.18 g of po~assium dihydrogen phosphate (Analar from BDH) and 19.17 g di-potassium hydrogen phosphate (Analar from BDH) which was dissolved in Milli-Q water. adjusted to pH 1.5 and made up to a final volume of 1 litre) and is shown in Figure 6.

further increase in the pH of the incubation mixture to 8.0 (~ee prepared from 12.11 g Trizma base ~Sigma Chemical Co.) dissolved in Milli-Q water, adjusted to pH8 with 1 M HCl and made up to a final volume of 1 litre) showed a m~ch poorer response to paracetamol and yielded~ a calibration curve~shown in Figure 7 which deviated from linearity at concentrations of paracetamol above 0.5mM.

Calibration curve6 for paracetamol which were obtained 2~i using buffers betwee~ pH 7.0 and 7.5 can be used in conjunction with direct readings of unknown samples in order to determine the paracetamol concentration.

xample 4. VSteady-State Measurements in 100% Control Serum __ Control serum (Monitrol IIE: Merz and Dade AG, Switzerland~ was resuspended in 3.5 ml Milli-Q water per vial (70% of the volume s~ated in the manufacturers' instructions) and mixed according ~o manufacturers' instructions to yield 143% (final concentration) control serum. Paracetamol (Sigma Chemical Co.) was added to this control serum to give a final concetration of 4.29 mM. Dilution of the solution with 143% control serum gave a range of paracetamol concentration i-n serum from zero to 4.29 mM.

The sti~red cell comprised of ?00 ~1 of aryl acylamidase solution (1.5 uni~s) 100~ ~1 of lM
po~assium phosphate buffer solution lpH 7.0) and 700 ~1 of paracetamol solution in 143% control serum.
(The final concentration of serum in the stirred cell wa~ eguivalent to 100%). Steady-state currents were measured in duplicate as described in Example 3 above.
The calibration curve for paracetamol in 100% control serum is shown in Figure 8.

~2~

In a parallel study, the parace~amol solutions in 143%
control serum were assayed using a standard paracetamol assay (Cambridge Life Sciences (CLS)).

A good correlation was found between the s~eady-s~ate current measured and the paracetamol concentration determined by the CLS method which is shown in Figure 4.

A particular advantage of the Pseudomonas enzyme over the Fusarium enzyme is that the ~ormer operates well at ambie~t temperature and neutral pH (the pH of whole blood is around 7.2)- Such oetimal conditions facilltate the production of a biosensor which can be used in any environment (such as a surgery) without the need for special laboratory conditions.

E~AMPLE 5: Enz~me Immobilisation into Membranes All enzymes immobilised were mixed in varying concentrations with a solution of cellulose acetate in a¢etone. The solution was then placed on the electrode surface and the acetone allowed to evaporate (either without ald or by the application of a drying air-6tream~ leaving a cellulose acetate membrane containin~ entrapped enzyme. This enzyme electrode was .
then u~ed to detect a fixed con~entration of paracetamol ,.:..

(see figure ~0).

The enzyme clearly retains its activity when immobilised in this way. It is likely that trapping the enzyme provides no serious constraints on its structure as it forms no covalent bonds with the immobi:Lising membrane and this allows it to retain much of its na~ive activity.

The slow response time encountered with the immobilised enzyme is probabl~ due to a limitation on both enzyme activity [due to the small constraints on movement imposed by entrappment] and eroduct transportation through the membrane.

The particular advantages of being able to immobilise the enzyme are as follows;

a3 it allows the manufacture of a one-component device to sense paracetamol, b) it renders the enzyme reusable, and, c) it is possible that the enzymes characteristic6 may be altered by the immobilisation p~ocess, to make the response time of the sensor horter.

Various modifications may be made within the scop~ of the present invention. For example. it will be apparent that while the invention has primary relevance to a sensor electrode, especially such an electrode specific Eo~ paracetamol, it also relates to the combination of such an electrode and temporary or permaner.t implantation means, e.g. a needle-like probe. Also, such an electrode, connected or connectable, with signal or control equipment,constitutes an as~ect o~ the inverltion. The electrodes accor~ing to the invention permit the manufacture of an improved macro-sensor for use in hospital analytical paracetamol, or paracetamol derivative sensing instruments. The electrodes of the invention, on the macro-scale can be incorporated into simple, cheap electronic digital read-out instruments for doctors surgeries.

Use of a small version of the macro-sensor would be ~ossible in a device which automatically takes a blood sample rom the finger, brings it into contact with the sensor, amplifies the signal and gives a digital readout. In such an application it is envisaged that the sensor electcode would comprise aryl acylamidase, stabilisers,. buffer and graphite in a dry focm.

It is envisaged that the present invention can be employed in combination with the screen-pcinted electcodess ,... . . .

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of assay of a liquid sample to determine presence or content of N-aceylated primary aromatic amine, comprising the successive or at least partially simultaneous features of (a) contact of said sample with an enzyme capable of catalysing the hydrolysis of the said N-acylated primary aromatic amine.

(b) contact of said enzyme/amine system with an electrode, (c) poising said electrode at a potential responsive to direct non-mediated transfer of charge from the system to the electrode and (d) detecting or measuring the current flow as an indication of pressure or amount of N acylated primary aromatic amine in the system.

2. A method as claimed in claim 1 in which an electrode is contacted with a liquid system comprising both paracetamol as the primary aromatic amine, and an enzyme capable of catalysing the hydrolysis thereof, whereby the detection or measurement of the paracetamol is effected.

3. A method as claimed in claim 1 in which the liquid sample contains paracetamol as the primary aromatic amine and in which an enzyme capable of catalysing the hydrolysis of paracetamol is located at least at the surface of the electrode.

4. A method as claimed in claim 3 in which the enzyme is the aryle acylamidase defined as EC 3.5.1.13.

5. The invention of claim 1, 2 or 3, wherein the enzyme is screen printed onto a suitable substrate.

6. A sensor electrode comprising a substrate material having located at least at its surface an enzyme capable of catalysing the hydrolysis of paracetamol, for use at a poised potential at which charge is directly transferred to the electrode material, without presence of a mediator compound, when the electrode at said potential is contacted with a liquid sample containing paracetamol.

7. A sensor electrode as claimed in claim 6 comprising an electrode substrate upon which a composition comprising said enzyme is screen-printed.

8. A sensor electrode as claimed in claim 6 comprising a laminer support substrate upon whicha mixture of electrode material and said enzyme is screen-printed.

9. A sensor electrode as claimed in claim 6 comprising a membrane at the electrode surface in which said enzyme is immobilised.

10. A sensor electrode as claimed inclaim 6, 7 or 8 in which the electrode comprises electrically conductive material chosen from gold, platinum, nickel and carbon.

11. A sensor electrode as claimed inclaim 6, 7 or 8 in which the said enzyme is the aryl acrylamidase defined