WO1995020051A1

WO1995020051A1 - Optical chemical sensors based on enzyme inhibition

Info

Publication number: WO1995020051A1
Application number: PCT/US1995/000846
Authority: WO
Inventors: Otto S. Wolfbeis; Stanley M. Klainer
Original assignee: Fci-Fiberchem, Inc.
Priority date: 1994-01-21
Filing date: 1995-01-20
Publication date: 1995-07-27

Abstract

An optical sensor for detecting a first species, comprises an optical sensor which produces an optical signal which varies as a function of concentration of a second species, a chemical substrate, and an enzyme which acts on the chemical substrate to produce the second species and which is inhibited by the first species from acting on the substrate to produce the second species. Further, a method for detecting a first species, comprises exposing a chemical substrate to an agent which acts on the substrate to produce a second species and which is inhibited by the first species from acting on the substrate to produce the second species, optically detecting changes in concentration of the second species, and determining changes in the first species from detected changes in the second species.

Description

OPTICAL CHEMICAL SENSORS BASED ON ENZYME INHIBITION

BACKGROUND OF THE INVENTION

The invention relates generally to optical chemical sensors and more particularly to enzyme based optical chemical sensors.

A variety of optical chemical sensors (optrodes) are based on optical fibers and other waveguides. Many optical chemical sensors (OCS) are solid state; others are reservoir type. These sensors generally contain a chemistry which reacts with the target species and produces a measurable optical effect. However, many target species cannot be detected because a suitable reaction does not exist.

One approach to solving this problem of not being able to directly measure a target species is to convert the target species to a product which can be measured. For example, a number of biosensors have been produced by using a biological transducer, e.g., an enzyme, which converts the analyte into a species for which an optrode exists. Moreno- Bondi, et al., "Oxygen Optrode for Use in a Fiber-Optic Glucose Biosensor," Anal. Chem. 1990, 62, 2377-2380, describes an oxygen sensor based on luminescent quenching of a ruthenium complex. The complex is adsorbed onto silica gel and incorporated into a silicone matrix with high oxygen permeability placed on the tip of a fiber. The enzyme glucose oxidase is immobilized on the surface of the oxygen optrode. The sensor relates oxygen consumption as a result of enzymatic oxidation of glucose to glucose concentration. Similarly, an oxygen optrode with an oxygen sensitive indicator dye (decacyclene) and a C0₂ optrode with a pH sensitive indicator dye (HPTS) having the enzymes gluta ate oxidase and glutamate decarboxylase, respectively, immobilized thereon are used to detect L-glutamate, Dremel, et al., "Comparison of two fibre-optic L-glutamate biosensors based on the detection of oxygen or carbon dioxide, ...n, Analytica Chimica Acta, 248(1991)351-359. Microbial sensors, e.g. as described in U.S. Patent Applications Ser. No. 08/163,040 filed Dec. 6, 1993 and Ser. No. 08/101,977 filed Aug 4, 1993, contain micro¬ organisms, e.g., yeasts or bacteria, immobilized on an optical chemical sensor. The microorganisms act on the target species to produce a measurable change in a reaction product.

However, in some cases, a target species cannot be directly detected by a suitable reaction and cannot be converted to a detectable product. For example, heavy metals (HMs) are difficult to measure with a sensor chemistry and cannot be converted to a product which can be measured. Thus, an additional sensor mechanism is needed which does not involve direct measurement of the target species or one of its reaction products.

In accordance with the invention, it is desirable to find a mechanism by which the target species can affect another reaction which produces a detectable product which can then be related to the target species. Heavy Metals (HMs) are key water pollutants.

Optical detection and quantification of HMs is usually performed using spectrophotometric or fluorometric cuvette tests, or with commercially available test strips. While such tests are widely accepted as the state-of the art, they do not provide good sensitivity and selectivity and can, therefore, be used only as an indicator of the existence of a problem. The heavy metals are very similar in behavior and thus there is no single reagent or combination of reagents that allows them to be spectrally separated. Consequently, after selecting the best reagents, algorithms (Chemometrics) must be written to improve separation by the use of software. The excessive amount of spectral peak overlap becomes even more of a quandary if the objective is to detect not a single species of a HM, but rather the presence of any of the many HMs which may be present. Under this scenario, the procedure becomes tedious and costly, because a single test must be run for each of the 20 most important HMs, not to mention the less common HMs, and the data must be deconvolved.

E. Toren in Mikrochimica Acta. vol. 1968, pp. 1049-1056 has shown that urease is inhibited by the following HMs in decreasing order: Ag(I) > Hg(II) > Cu(II) > Cd(II) > Co(II) > Ni(II) > Mn(II) > Pb(II) . Based on this finding, L. Ogren in Analytical Chimica Acta 125. 45, (1981) has described an assay for heavy metals based on the inhibition reaction, by measuring the rate of the increase in pH as a function of inhibitor concentration. Detection limits as low as 10-200 ppb were achieved. Similar results were reported by G. Schwedt in Fresenius Journal of Anal. ChemistrY 346. 659, (1993).

The above methods suffer from two disadvantages: (1) They require tedious procedures, and are not easy to perform in the field (especially by unskilled personnel) , thus reducing the effectiveness of real-time, in-situ analysis and (2) The result depends strongly on the buffer capacity of the sample because the pH change caused by the enzyme may be overcompensated for by a strong buffering effect.

Therefore, one objective of the invention is to use the inhibition effect of a target species, e.g., enzyme inhibition by heavy metals, in an optical chemical sensor. Another objective of the invention is to provide the means for a test that can be performed in the field by unskilled personnel and to overcome the problem of varying buffer capacity.

This invention preferably addresses the need for: (1) General sensors which can be used for indicating the presence of general groups of analytes, (2) Individual sensors which can detect and quantify a particular species and (3) Group sensitive sensors which can identify and measure particular chemical sets, i.e. heavy metals. The general sensors are not intended to be species specific nor are they intended to be quantitative. They must, however, have part-per-billion (ppb) sensitivity to be useful. They are intended to be early warning devices to provide inputs as to when samples are to be collected and subsequently analyzed in the laboratory. Thus they provide information that a target or group of targets are present and furnish the mechanism for eliminating the analyses associated with negative samples. These sensors can also be used to assure that a target or the sum of several targets remain below a threshold level.

The analyte specific devices, on the other hand, are intended to be true monitoring devices. Their purpose is to look for a particular species; identify its presence and its concentration insitu and in real-time; assure that the amount present is within predetermined limits; and, if necessary, initiate the proper correction procedures or institute the appropriate warnings.

SUMMARY OF THE INVENTION

One aspect of the invention is an optical chemical sensor method and apparatus for detecting a target species by its effect in inhibiting the action of an enzyme on another species which produces a detectable product. An enzyme is selected which may act on a chemical substrate to produce a product which is detectable with an available optical chemical sensor, and whose action on the substrate is inhibited by the target species. A wide variety of chemical sensors can preferably be used including waveguide sensors and reservoir sensors. Both general sensors and species-specific sensors can be produced. In particular, a heavy metal sensor is based on inhibition of urease which hydrolyzes urea, producing ammonia or ammonium.

According to one aspect of the invention, there is provided an optical sensor for detecting a first species, comprising: optical sensing means which produces an optical signal which varies as a function of concentration of a second species; a chemical substrate; an enzyme which acts on the chemical substrate to produce the second species and which is inhibited by the first species from acting on the substrate to produce the second species; and a sensor comprising a buffer. According to another aspect of the invention, there is provided an optical method for detecting a first species, comprising: exposing a chemical substrate to an agent which acts on the substrate to produce a second species and which is inhibited by the first species from acting on the substrate to produce the second species; optically detecting changes in concentration of the second species; determining changes in the first species from detected changes in the second species.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a perspective view of a rectangular cuvette according to the invention.

Fig. 2a is a perspective view of a triangular cuvette according to the invention. Fig. 2b is a sectional view of a second cuvette containing substrate.

Figs. 3a,b illustrate an integrated waveguide capillary cuvette.

Fig. 4 is a sectional view of a waveguide sensor. Figs. 5a,b are sectional views of a reservoir sensor.

Figs. 6a-c show a multiple waveguide sensor. Fig. 7 is a response curve for an ammonia sensor at various concentrations. Fig. 8 is a response curve for an ammonia sensor as a function of pH.

Fig. 9 shows the Michaelis-Menten diagram and the Lineweaver-Burk plot of urease.

Fig. 10 shows the inhibition of urease by Ag'. Figs. 11a-j show the inhibition of urease by various heavy metals, when immobilized with and without covalent bonds.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is method and apparatus for measurement with an optical waveguide chemical sensor (OWCS) or other optical chemical sensor (OCS) of a target chemical 0051

species by enzyme inhibition. The OWCS or OCS includes a chemical substrate on which the enzyme acts to produce a detectable product. The target species inhibits the activity of the enzyme so that changes in the detectable product are related to the target species. The basic reaction is:

ENZYME A SUBSTRATE A - ■ PRODUCT A * OTHER PRODUCTS

A

INHIBITOR where Product A is detectable with the OWCS or OCS and the inhibitor is the target species.

If product A cannot be readily detected, e.g., no available optrode, a two enzyme system can be used where enzyme B converts Product A to Product B which can be detected. Enzyme A is still inhibited by the target species so a decrease in Product A leads to a decrease in Product B which is thus an indicator of the target species.

This invention involves the approach whereby an enzyme reacts with a particular analyte (substrate) to generate a species which can be measured by an optical chemical sensor (OCS) . This is the baseline reaction and represents no inhibitor present. The enzyme selected for the baseline reaction is picKed to be inhibited by the target molecule or ion of interest. When this inhibition takes place, the amount of species to be measured by the OCS is reduced and the amount of signal decrease can be related to the concentration of the molecule or ion of interest. There are two major considerations when employing this type of reaction: (1) The induced fit is a dynamic recognition process, i.e., the molecular conformation change is responsible for specificity and t2) The enzyme introduces an electronic strain in its substrate to expedite enhanced reaction rates. For these reasons, enzymes are often more suitable for initiating specific chemical reactions than reagents and dyes.

The use of enzyme inhibition represents a unique method of detecting and quantifying a variety of species, including the heavy metals and several anions, organics and gases. The approach has the general advantages of: (1) using a simple OWCS or OCS for making difficult chemical analyses, (2) doing complex analyses without sample preparation, (3) making real-time in-situ measurements, (4) selecting between reversible and non-reversible reactions, (5) choosing between analyzing for a single analyte or a group of analytes, (6) having good selectivity and sensitivity and (7) being amenable to solid-state sensor configurations. A particular benefit of this approach is that since enzymes can now be tailored to meet any reaction mechanism requirement, the potential for very broad analytical applications for this approach is enhanced.

Table 1 gives a limited list of analyses that can be done using the invention. In this table the first column gives the species to be detected and measured, the second column a selection of enzymes which are inhibited by the species of interest and the third column the OCS which will be used for the measurement. The number of targets that can be measured is only restricted by the available enzymes and OCS. Of particular importance is the limited number of OCS needed to measure a large number of target analytes.

θ

TABLE 1

ENZYME/INHIBITOR COMBINATIONS

1 0

FOOTNOTES

1) Galactosidase hydrolyzes esters of galactose to release galactose which, in a second step may be oxidized by galactosidase with the constmption of oxygen. If inactivated, galactosidase does not produce galactose and no oxidation can take place in Step 2. 2) Glycine is the product of Step 1 (dehydroxymethylation of serin). Glycine requires an additional sensor. If, however, glycine hydrogenase is added as a second enzyme

(not inhibited by silver ion) to convert glycine into a monia/arnnonium, detection can be accomplished using an ammonia OWCS.

3) The peptidase releases a mixture of free amino acids which are oxidized by the added oxidase with the consumption of oxygen.

^' ) The lyase forms oxaloacetate which in a second enzyme reaction is decarboxylated to form carbon dioxide.

5) The first reaction yields glutamate which, in a second enzyme-catalyzed reaction, is oxidized by glutamate oxidase/oxygen to form amnonia and hydrogen peroxide. 6) Arginase produces urea which is converted, in a second step, to carbon dioxide and amnonia by urease.

7) In this detection scheme, a competing enzyme system (creatinase and urease) is added to the enzyme (creatine kinase) which is inhibited, (a) If not inhibited, the kinase catalyzes the usual reaction (the formation of phosphocreatine). (b) j_ inhibited, the competing reaction takes place, which is the conversion of creatine into urea, followed by hydrolysis of urea to give ammonia and carbon dioxide. This scheme requires the kinase to have a higher activity than the creatinase. As to the appropriate substrates for the enzymes, there is an obvious substrate for each enzyme, e.g. listed in T.E. Barman, The Enzyme Handbook, which is herein incorporated by reference. Some enzymes are more general in their action and accept several substrates. Others are very specific; e.g. urease is specific for urea. The most appropriate substrate is usually the one given by the enzyme's name, e.g., glucose for glucose oxidase, acetylcholine for acetylcholine esterase, methane for -methane monooxygenase, creatine for creatine kinase, analine for analine carboxypeptidase and peptides for amino peptidase.

In accordance with the invention, the term optical waveguide covers optical waveguides per se; channeled optical waveguides; single and multimode fiber optics, with either side- or tipcoatings and optical "chips". There are a number of different embodiments of OWCS that can be used to implement the invention. The invention can also be carried out with other types of optical chemical sensors, e.g., reservoir type sensors, including simple cuvette systems. The reservoir sensors can include a fiber optic.

Another part of this invention is the need to maintain the enzymes in an active state. Enzymes are known to degrade as a function of time, temperature and other parameters which often cannot be controlled during a sensor's use or storage. To overcome these effects, with the exception of extreme temperatures, two steps have been taken: (1) The enzyme is immobilized onto a support, e.g., cell wall. (Preference would have been to covalently bond the enzyme, but this results in lack of participation in the reaction as will be shown later.) Thus, the enzyme, and also substrate and buffer, are deposited or immobilized on the walls of a cell before aqueous sample is added, but in a manner that they readily dissolve when the sample is added. (2) The stability of the enzyme is enhanced by the presence of water and, therefore, the use of a hydrophilic immobilization agent for the use of a hydrophilic overcoating or undercoating helps to preserve enzyme activity. Four types of sensors can be produced: (1) General sensors which use a single enzyme to measure a general class of compounds; (2) Analyte-specific sensors which use a single enzyme to measure a specific target molecule; (3) General sensors which use two or more enzymes to measure all, or some, of the species in a general compound class and (4) Target-specific sensors which use two or more enzymes to assure specificity to a single material. An example of a type (4) sensor is zinc. From Table 1 it can be seen that aminoacetyl histidine dipeptidase is the enzyme inhibited by zinc. Further examination of the Table, however, also shows that cadmium and copper also inhibit this enzyme. Separation of zinc from copper is accomplished by the use of sarcosine oxidase and separation of zinc from cadmium is accomplished by 4methoxybenzoate monooxygenase. Thus by using, for example, a three-channeled waveguide, each of which exploits a different enzyme, zinc, copper and cadmium can be specifically measured.

The enzyme inhibition reactions proceed in a liquid medium. Thus, it is an excellent technique for measuring dissolved species, i.e. heavy metals, organics, anions, cations, gases, etc. This leads to several cell configurations in which the sensing chemistry is solid state while the sample is a liquid. Figure 1 is a rectangular cuvette 10 with a sensor chemistry (paint) 12 deposited on one of the inner walls. On another wall substrate 13 and buffer 14 are deposited while on a third wall enzyme 16 is deposited. When the cell is filled with an aqueous sample, the sample will dissolve the enzyme, substrate and buffer 12 (but not the sensor chemistry) and the reaction will begin. This results in a liberation of a chemical species such as ammonia. This in turn, causes the color of the sensor chemistry (paint) 12 to change. In the presence of inhibitors such as listed in Table 1, the reaction is retarded or even suppressed. In this case there is a change in the absorption of the light in (I_w) which is shown in the intensity of the light out (Ϊ_OUT)" ^Tne ratio of I_N to I_ouτ is a direct measurement of the amount of reaction taking place and thus the analyte concentration. Fluorescence could also be measured, as in Figure 2a.

A second cell, like cuvette 10 in Figure 1, can be used as a reference cell. The reference cell is filled with plain water or buffer instead of sample and a difference measurement is performed with two beams. One beam measures non-inhibited activity in the reference cell while the second beam measures the inhibited activity in the sample cell.

Figure 2a is a triangular cuvette 22 configuration with the sensor chemistry (paint) 12 deposited on one wall. The substrate 13 and buffer 14 are deposited on the bottom of cuvette 22. The enzyme 16 is deposited on a second wall. When the cell is filled with an aqueous sample, the sample will dissolve all components (enzyme, substrate, buffer) and the reaction will begin. This results in a liberation of a chemical species such as oxygen or carbon dioxide. In the presence of inhibitors such as listed in Table l, the reaction is retarded or even suppressed. In this case, the excitation light 1^ causes fluorescence 1^ to occur. The fluorescence intensity 1^ is a measurement of the amount of reaction taking place and thus the analyte concentration.

Although a single cell configuration as shown in Fig. 2a can be utilized, a 2-σell configuration can be produced by the combination of Figures 2a and 2b. In this design a triangular cuvette 22 has sensor chemistry (paint) 12 deposited on one wall. A second wall is coated with enzyme 16 and buffer 14. The sample containing the inhibitor would be added to a second container, cuvette 28 of Fig. 2b, of known volume containing the solid substrate 30. The contents of cuvette 28 would then be poured into cuvette 22 and the reaction measured. When the cell 22 is filled with an aqueous sample, the sample will dissolve all components and the reaction will begin. This results in a liberation of a chemical species. If ammonia or carbon dioxide is to be assessed, the measurement will be made in absorption or fluorescence; while if oxygen is to be assessed, fluorescence is the detection method.

Figures 3a and 3b show two views, assembled and partly disassembled, of, an "integrated waveguide capillary cuvette" (IWCC) 32. IWCC 32 is formed of body 20 with cavity 24 formed therein and capillary inlet 26. The sensor chemistry (paint) 12 is on the inside surface of one side 21 of the IWCC and the immobilized enzyme 16 is on the inside surface of the other side 23. The sample containing the inhibitor would be added to a second container, cuvette 28 of Fig 2b, of known volume containing the solid substrate. The contents of cuvette 28 would then enter IWCC 32 by capillary action through inlet 26. If ammonia or carbon dioxide is to be assessed, the measurement will be made in absorption or fluorescence; while if oxygen is to be assessed, fluorescence is the detection method. Measurements may be performed through the wall(s) of IWCC 32 as in Figs. 1,2a or by coupling light into side 21 (evanescent waves) as in Fig. 4. Figure 4 shows a waveguide sensor 36. Opposed walls 33,38 of a cuvette (as in Fig. 1) are coated on the inside surfaces with sensor chemistry 12 and enzyme 16, respectively, and define therebetween a cell volume 34. In this design, absorption or fluorescence may be monitored using the evanescent wave mode- Wall 33 with sensor chemistry 12 can be utilized as a waveguide. A light signal

UN is input into waveguide 33 and changes in the light signal propagating through the waveguide 33 are detected.

Figure 5a shows a fiber optic reservoir cell 50 (Klainer, et al, U.S. Patent No. 5,059,790, U.S. Patent No. 5,107,133 and U.S. Patent No. 5,116,759) adapted to the present invention. In this configuration, a fiber optic consisting of core 42 and clad 44 is placed in a reservoir cell of known volume 46. Sensor chemistry 12 is placed on the tip of the fiber. One wall of the cell is coated with substrate 13 and buffer 14. A second wall is coated with enzyme 16. The sample to be measured enters through inlet 52 15 and exits through outlet 54. This approach is suitable for use with kinetic and static samples.

Figure 5b is a variation of Figure 5a wherein part of clad 44 is removed and replaced with sensor chemistry (paint) 12, i.e., the sensor chemistry is on the side (side-coated) rather than the tip.

Figures 6a-c show a sensor 58 with multiple waveguides on a chip 56. In the arrangement shown three different waveguides 60, 62 and 64 are used. This permits three different enzymes to be used at once and also provides for a reference channel 66. The system uses a single light source 68 and multiple detectors 70, 72 and 74 each of which is filtered to give a specific detection wavelength. A fourth detector 76 can be used to look at the reference channel 66. Light from source 68 in chip 56 is incident on sloped reflective end face 61 of waveguide 59 which reflects the light down the waveguide 59 to beam splitter 71. Beam splitter 71 reflects a portion of the light to reference detector 76. Beam splitters 73,75 divide the transmitted light into the three waveguides 60,62,64 which are coated with a sensing chemistry 12a,b,c, respectively. The light passing through waveguides 60,62,64, which is affected by chemistries 12a,b,c, then is reflected by sloped reflective end faces 63,65,67 into detectors 70,72,74, respectively. This arrangement can be used in two approaches: (1) Three different analytes can be analyzed simultaneously or (2) If there is a question of specificity, then coincidence or redundant analyses can be accomplished using two or more enzymes which respond to the target analyte. The number of waveguides that can be used is only restricted by source strength and geometric considerations.

Additional solid state and reservoir cell designs appear ih U.S. Patents 5,059,790, 5,107,133 and 5,116,759, filed herewith, all of which are herein incorporated by reference.

An example of the application of this invention is the detection of heavy metals (HMs) . In keeping with the objectives of the invention (to provide a means for detecting not only individual analytes, but also the presence of a certain level of groups of analytes) , the measurement of total HMs is shown as well as selected individual HMs. Clearly, this cannot be achieved with a single reagent or indicator dye since these would bind one or, at best, only a few of the common HMs. This is one of many instances where enzyme inhibition is key to success. HMs are known to inhibit species-specific enzymes (Table 1) or in other cases the activity of certain enzymes is reduced by many HMs. Phosphatase, glucose oxidase, pyruvate oxidase, alcohol dehydrogenase and lactate dehydrogenase are other enzymes inhibited by HMs. This propensity can be used to create many more versatile sensors.

Specifically, the inhibition of urease by HMs can be used in a sensor. Urease is an enzyme which hydrolyzes urea according to the following reactions:

H₂N'CO-NH₂ + OH^~ + H₂0 2 M, + HCO_^ (1)

H₂N'CO-NH₂ + H⁺ + 2 H₂0 - 2 NH^ + HC0₃- (2)

H₂N'CO-NH₂ + 2 H⁺ + H₂0 -> 2 NH_A + C0₂ (3)

Reaction (1) is predominant at pHs above 8, reaction (2) at pHs between 6 and 7, and reaction (3) at pHs below 7. In order to monitor the rate of the reaction, the consumption of hydrogen ion has been measured.

Rather than measuring pH changes, it is advantageous to measure changes in the concentration of ammonia (possible at pHs above 6) or ammonium ion (possible at pHs below 9) . The concentration of ammonia or ammonium formed from urea is independent of the buffer capacity of the sample.

From Table 1 it can be seen that the four key OWCS are oxygen (0₂) , carbon dioxide (C0₂) ammonia (NH₃) and ammonium (NH₄) . The oxygen and carbon dioxide sensors are covered in Patent Application 08/163,040 filed December 6,

1993 which is herein incorporated by reference.

To make an ammonia (NH₃) sensor, dissolve 10 mg of the lipophilic indicator bromothymol blue cetyltrimethyl-ammonium salt (BTB) [made by analogy to J. Reichert, et al.. Sensors & Actuators. 25A , 481, (1991)] and 1 g of silicone (Elastosil E4, Wacker, Germany) in 1 m£ chloroform. The use of chloroform as a solvent makes it possible to deposit the chemistry on a membrane, inside the wall of a sample cuvette, at the tip or side of an optical fiber, on a waveguide or on an optical chip. To make a disposable cuvette with an ammonia-sensitive chemistry (paint) on the inner wall (Figure 1) the solution was cast on one of the inner walls of a disposable 1 x 1 cm plastic cuvette and the solvent slowly evaporated at room temperature. The silicone cures on the wall and forms a thin yellow film. In order to complete polymerization and to remove the acetic acid released during polymerization, the cuvettes are then dried at 90° C for two days. The resulting membrane coating was estimated to be 10 /xm thick. Before actual measurements, the cuvettes were conditioned for 1 hour in a 100 mM phosphate buffer at pH 7.40. The membranes, when in contact with ammonia, assume the blue color of the BTB anion and this can be monitored in absorption photometrically at 605 nm. The time for the reaction to go to 95% completion (t₉₅) is » 100 minutes. Fortunately, it is not necessary to wait for completion and good results can be obtained by calibrating the system at a fixed time after the initiation of the reaction or by doing kinetic slope measurements.

If bromothymol blue is replaced by a fluorescent dye of a pKa similar to that of BTB (7.2), a fluorescent 18 sensor is obtained. One example for a fluorescent dye of similar pKa is l-hydroxypyrene-3,6,8-trisulfonate with a pKa of 7.3. It can be excited at 460 nm and fluoresces above 500 nm with a maximum at 512 nm. To make an ammonium (NH₄ ⁺) sensor, dissolve 2.40 mg polytvinyl chloride), 0.40 mg potassium tetrakis(chlorophenyl)borate K⁺ [B(C£-Ph)₄]", 0.50 mg N-octadecyl nile blue, 0.85 mg nonactin and 4.8 mg dioctyl sebacate in 1.5 ml of freshly distilled tetrahydrofuran (THF) (all from FLUKA, Switzerland) . One hundred μi of this solution were pipetted into a disposable cuvette located in a THF-saturated desiccator. This procedure is required in order to prevent the membrane from becoming turbid. After about 15 minutes, the cuvette with « 2 μm pvc coating on one of the inner walls was exposed to air for 15 minutes for further drying. Before measurement, the cuvettes were activated in 1 mM hydrochloric acid for 10 minutes and another 10 minutes in a 0.1 M solution of ammonium chloride. A fluorescent sensor for ammonium ion is described in U.S. Patent Application Serial No. 08/009,171 which is herein incorporated by reference.

The urease-catalyzed hydrolysis of urea has been found to be competitively inhibited by phosphate (X.M. Harmon, C. Niemann, 1948) . Maleate buffer (known to be appropriate to biological systems) was, therefore, chosen for absorbance measurements. Tris-HC£ buffer can also be used.

The standard solutions for calibrating the sensor were made up of various concentrations of NH₄C£ dissolved in maleate buffer at pH 7.2. The equilibrium concentration [NH₃] - [NH₄ ⁺] at pH 7.2 is related by the Henderson- Hasselbach equation:

log[.MB,] ^a log^[ θ - 2.1 ( Figure 7 is the response curve of the ammonia sensor at various NH₃ concentration as a function of time and absorbance when measured at 605 nm.

To identify the optimum pH, there must be a trade- off between optimum activity of the enzyme (pH 7 at 25°C) , the relative signal change of the ammonia sensor and possible precipitation of some heavy metals. Figure 8 shows the response of the optical sensor at various pH-values. Enzyme and urea concentrations were the same in all cases. Although the relative signal change increases with increasing pH. pH 7.2 was chosen to be the optimum pH. Nevertheless, some heavy metals, e.g., Hg²⁺, Cu²⁺, Ag⁺ precipitate at pHs above 7. If these are not target analytes, then the fact they precipitate out simplifies the total HM analysis. If they should be of interest, then the pH must be lowered to accommodate them.

To define 100% enzyme activity, the concentration of substrate (in this case urea) which leads to the highest rate (v_^ must be determined. Above this urea concentration, the velocity of the reaction is constant and the relative signal change of the optical sensor is the same as all concentrations. Figure 9 shows the Michaelis-Menten diagram and the Lineweaver-Burk plot of urease.

Urease is strongly inhibited by Hg²⁺, Ag⁺ and Cu²⁺ and also inactivated by other heavy metal ions. The inhibition of urease by Ag⁺ is shown in Figure 10. The relative responses of the urease inhibition to several key heavy metals are shown in Figures 11a through llj with the solid black dots. This data is obtained when the reactants are immobilized without covalent bonds. The open dots represent the behavious of the system with covalent immobilization. As can be seen, the covalently bonded material behaves very poorly when compared to simple immobilization. Since the behavior of the covalently bonded materials is predictable, as a function of analyte concentration, this can be used as an internal reference.

Other examples which are very amenable to the enzyme inhibition route include pesticides, cyanide, TNT, etc. Organometals (e.g. mercury-organic compounds) inhibit enzymes.

In a similar fashion all of the enzymes listed in Table 1, as well as many that are not listed, can be used to initiate reactions which can be measured using relatively simple OWCS or other OCS.

Although the invention has been described with reference to enzyme inhibition, the principles can also be applied using other materials, e.g., yeast or bacteria, whose activity can be inhibited by a target species.

Changes and modifications in the specifically described embodiments can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims.

Claims

1. An optical sensor for detecting a first species, comprising; optical sensing means which produces an optical signal which varies as a function of concentration of a second species; a chemical substrate; an enzyme which acts on the chemical substrate to produce the second species and which is inhibited by the first species from acting on the substrate to produce the second species.

2. The sensor of Claim 1 further comprising a buffer.

3. The sensor of Claim 1 wherein the optical sensing means comprises a cuvette and a sensing chemistry immobilized on a surface thereof, and wherein the substrate and enzyme are coated on other surfaces thereof.

4. The sensor of Claim 3 wherein the cuvette is a rectangular or triangular shaped cuvette.

5. The sensor of Claim 3 wherein the substrate and enzyme are dissolvable into a sample placed in the cuvette.

6. The sensor of Claim 3 wherein the surface df the cuvette with the sensing chemistry thereon forms a waveguide.

7. The sensor of Claim 1 wherein the optical sensing means comprises a first cuvette and a sensing chemistry immobilized on a surface thereof, and wherein the enzyme is coated on another surface thereof, and further comprising a second cuvette containing the substrate.

8. The sensor of Claim 1 wherein the optical sensing means is an integrated waveguide capillary cuvette.

9. The sensor of Claim 1 wherein the optical sensing means is a reservoir type chemical sensor.

10. The sensor of Claim 9 wherein the reservoir type chemical sensor comprises a cell having the substrate and enzyme coated on inner surfaces thereof, inlet means for introducing a sample into the cell, and optical fiber extending into the cell, and a sensing chemistry immobilized on the optical fiber in the cell.

11. The sensor of Claim 1 wherein the substrate is urea, the enzyme is urease, and the optical sensing means is an ammonia or ammonium sensor.

12. The sensor of Claim 1 wherein the optical sensing means is an oxygen sensor, a carbon dioxide sensor, an ammonia sensor, or an ammonium sensor.

13. The sensor of Claim 1 wherein the optical sensing means is an optical waveguide chemical sensor.

14. The sensor of Claim 1 wherein the optical sensing means comprises means for detecting a plurality of different species.

15. The sensor of Claim l further comprising hydrophilic means for stabilizing the enzyme.

16. An optical method for detecting a first species, comprising: exposing a chemical substrate to an agent which acts on the substrate to produce a second species and which is inhibited by the first species from acting on the substrate to produce the second species; optically detecting changes in concentration of the second species; determining changes in the first species from detected changes in the second species.

17. The method of Claim 16 wherein the agent is an enzyme.

18. The method of Claim 16 wherein the step of optically detecting changes in concentration of the second species is performed with an optical waveguide chemical sensor.

19. The method of Claim 17 wherein the substrate is urea and the enzyme is urease.

20. An optical method for detecting a first species, comprising: exposing a chemical substrate to a first enzyme which acts on the substrate to produce a second species and which is inhibited by the first species from acting on the substrate to produce the second species; exposing the second species to a second enzyme which acts on the second species to produce a third species; optically detecting changes in concentration of the third species; determining changes in the first species from detected changes in the third species.