WO1997045741A1

WO1997045741A1 - Flow-through immuno-electrode for fast amperometric immunoassays

Info

Publication number: WO1997045741A1
Application number: PCT/US1997/008587
Authority: WO
Inventors: Ebtisam S. Wilkins; Andrey L. Ghindilis; Plamen B. Atanasov
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-05-29
Filing date: 1997-05-20
Publication date: 1997-12-04
Anticipated expiration: 1998-11-29
Also published as: AU3136097A

Abstract

An electrode (15, 19) and measuring cell (17, 50) for fast flow immunoassay of a wide range of analytes. The high surface area electrode (15, 19) comprises an electroconductive material with immobilized immuno-species deposited on glass, paper or plastic filter (18, 62). The solution containing the target analyte and/or the enzyme labeled immunoconjugate flows through the electrode (15, 19). The assay procedure is based on a competitive or sandwich scheme. The immuno-interaction between the immobilized immuno-species target analyte and the enzyme labeled immunoconjugate results in binding of the enzyme label to the electrode surface. The assay of the enzyme label is performed by flow injection of the corresponding enzyme substrate, and amperometric measurement of the product of the enzymatic reaction.

Description

Flow-Through Immuno-Electrode for Fast Amperometric Immuno-Assavs

Field of the Invention

This invention relates to sensors for detecting and quantifying a target analyte via immunological based reactions, and more particularly to a flow through amperometric sensor.

Background of the Invention

Immunoassay techniques are based on the ability of antibodies to form complexes with the corresponding antigens. This property of highly specific molecular recognition of antigens by antibodies leads to high selectivity of assays based on immune principles. The high affinity of antigen-antibody interactions allows very small quantities of a target analyte to be determined. Contemporary immunological techniques allow production of antibodies against a great number of antigens, and enables immunoanalysis of many substances such as low molecular weight drugs, short peptides, toxins, as well as polypeptides, proteins, viruses and bacteria.

Immunoassay techniques are used mainly in clinical analyses and medical diagnostics. Immunoassay techniques could, in principle, be utilized in many non-clinical applications if assay systems that were better adapted for field conditions were available.

Conventional immunoassay techniques (such as ELISA, immunoblot, immunoagglutination) are optimized for clinical situations in which a great number of (often identical) analyses are constantly conducted on a routine basis. However, this technique can be used only in specially equipped laboratories and requires technically trained personnel. Factors limiting the application of conventional immunoassay techniques are: (i) the conventional immunoassay is a multistage process, resulting in a general complexity of analysis and in the requirement for a highly qualified technician; (ii) the equipment for conventional immunoassay cannot be easily miniaturized; (iii) automation of the multistage measurement procedure is very difficult and (iv) conventional immunoassays are difficult to conduct in the non-laboratory conditions typically encountered in field settings. It should also be noted that the time for analysis by conventional immunoassay is normally from one to several hours, which makes this technique unsuitable for emergency cases in which fast determination of several analytes is of great importance.

During the last few years, a significant number of publications have dealt with alternative immunoassay techniques. The development of alternative immunoassay techniques aims in most cases at improvements in performance of conventional immuno¬ analysis, by such means as decreasing analysis times, increasing assay sensitivity, and simplification and automation of assay procedures.

The basic principles of the alternative immunoassay methods are the same as for conventional immunoassay techniques in that these alternative assays are also based on the detection of antigen-antibody interaction. Frequently the term 'biosensor' or 'immunosensor' is used to label an immunoassay system that is an alternative to a conventional assay system, developed with automated and fast, possibly direct, data acquisition.

In general, an immuno-sensor (biosensor) consists of a signal transducer and a biochemically interactive system employing principles of biological molecular recognition. Based on the nature of the physical detection used in the transducer, immuno-sensing systems can be classified as optical, gravimetric and electrochemical. In optical transducers, detection is based on light-sensitive elements. The optical signal detection can be conducted by spectrophotometric, spectrofluorimetric, hemiluminometric, reflectometric or other related techniques.

Gravimetric transducers are based on sensitive detection of mass changes following antigen-antibody complex formation. Piezoelectric detectors are typically based on acoustical resonators having resonant frequencies that are altered by the change in mass of a layer which is in contact with the resonator. This layer typically includes one member of an antigen- antibody complex. When the other member attaches to the layer, the resonance frequency shifts. These transducers cannot distinguish between specific binding and non-specific binding. Electrochemical transducers are based on detection of changes in electro transfer caused by the immuno-interaction. In particular, this detection is brought about using amperometric, poteniometric, conductometric (at constant voltage) or impedimetric (at alternative voltage) devices.

In conventional solid-phase immunoassay, the ratio of the immuno-interactive surface to the volume of the liquid reaction medium is quite low. This causes the reaction rates to be limited by diffusion of the immuno-species from the reaction media to the immuno- interaction surface. As a result, conventional immunoassay usually requires several hours.

Flow-injection principles can be used to enhance the efficiency of the immuno- interaction. Prior art flow-injection immuno-sensing systems are based on a principle of displacement. In this case, the immunoassay system is arranged as a column containing immobilized antibodies. The column is saturated with a solution containing labeled antigen. After antigen-antibody interaction has occurred, the column contains a solid carrier with immobilized antibody-labeled antigen complexes. The affinity of antibodies for labeled antigens is usually significantly lower than their affinity for unlabeled (free) antigen due to stearic factors. Therefore, injection of free antigen into the column results in displacement of the labeled by the unlabeled antigen. Labeled antigen then is detected at the outlet of the column. A similar scheme can be realized based on the use of immobilized antigen. In this case, injection of the analyte leads to replacement of antibody conjugated complex.

Flow-injection immunoassay system based on displacement schemes for real time (two-three minutes) determination of a number haptens have been reported by Liegler, et al. in US Patent No. 5,183,740. However, these flow-injection schemes have substantially less sensitivity than the conventional assay systems.

Traditional immuno-analysis schemes (competitive binding and 'sandwich' schemes) are also employed in flow-injection immunoassay systems. In these cases, the problem of column regeneration is an important issue. The problem associated with the necessity to renew the immuno-sorbent in flow-injection systems can be solved by development of disposable immuno-columns. The concept of disposability of the sensing elements is a general trend in development of analytical devices for fast assay (especially for biomedical applications). An immuno-sensor for dioxin, combining the advantage of flow-injection and the disposable principle, has been reported (Kaneki et al., Analytica Chimica Ada, 287, pp253-258).

Existing flow immunoassays employ a transducer situated outside of the immuno- column. Therefore, enzymatic reaction catalyzed by enzyme-label occurs in the immuno- column volume and the measurements of the concentration of the reaction is conducted by an electrochemical detector connected to the outlet of the column. This arrangement increases the complexity of equipment, measuring procedure, and sensor preparation.

Broadly, it is the object of the present invention to provide an improved immunoassay apparatus.

It is a further object of the present invention to provide a flow-immunoassay apparatus that does not require the products measured to leave the column.

It is yet another object of the present invention to provide an assay system with shorter measurement times than prior art ELISA systems.

Another object of the invention is to provide a portable immuno-sensor capable to detect both (i) high molecular weight and (ii) low molecular weight target analytes under non- laboratory conditions.

These and other objects of the present invention will become apparent to those skilled in the art from the following detailed description of the invention and the accompanying drawings.

Summary of the Invention

The present invention is an electrode and measuring cell for fast flow immunoassay of a wide range of analytes. The high surface area electrode comprises an electroconductive material with immobilized immuno-species deposited on glass, paper or plastic filter. The solution containing the target analyte and/or the enzyme labeled immunoconjugate flows through the electrode. The assay procedure is based on a competitive or 'sandwich' scheme. The immuno-interaction between the immobilized immunospecies target analyte and the enzyme labeled immunoconjugate results in binding of the enzyme label to the electrode surface. The assay of the enzyme label is performed by flow injection of the corresponding enzyme substrate, and amperometric measurement of the product of the enzymatic reaction. The measuring cell is equipped with an inlet, outlet, a reference electrode, a counter electrode, and a current collector attached to a disposable flow immuno-electrode.

Brief Description of the Drawings

Figure 1 is a schematic drawing of a flow-through sensor system according to the present invention.

Figure 2 is a cross-sectional view of an assay apparatus according to the present invention.

Figure 3 is an exploded cross-sectional view of assay probe 50 shown in Figure 2.

Detailed Description of the Invention

The present invention comprises a flow-through system in which the immuno- electrode utilizes a highly dispersed electro-conductive immuno-sorbent which also provides the immuno-electrode function. That is, the immuno-column doubles as the electrochemical transducer. The highly dispersed material comprising the immuno-sorbent provides a high surface area-to- volume ratio which, in turn, provides a high rate of immuno-complex formation. The high conductivity of the immuno-sorbent material allows the electrochemical detection of the enzyme label directly in the column. Therefore, the present invention provides the advantages of both the flow-immunoassay techniques and techniques that utilize immuno-electrodes. The present invention may be more easily understood with reference to Figure 1 which is a schematic drawing of a flow-through sensor system 10 according to the present invention. System 10 utilizes a miniature immuno-column 17 which includes a highly dispersed immuno-sorbent 15 which acts as a working (measuring) electrode. The current generated by the electrodes discussed below is measured by measurement assembly 16 which also includes conventional control circuitry for operating the various other components of the system. To simplify the drawing, the connections between measurement assembly 16 and the various other components have been omitted from the drawing.

Immuno-column 17 serves as both an immuno-reactor and an electrochemical measuring cell. Immuno-column 17 includes an inert body which supports a porous filter membrane 18. The immuno-sorbent 15 is deposited upon the filter membrane 18. Filter membrane 18 is preferably constructed from glass, paper, or plastic.

The manner in which the system shown in Figure 1 is used may be more easily understood with reference to performing a conventional 'sandwich' immuno-analysis for a target analyte, X. The immuno-sorbent 15 is assumed to be loaded with antibodies to X which are immobilized on the particles of the immuno-sorbent at the start of the assay. The sample is injected into immuno-column 17 via sample port 13 and valve assembly 12 in a suitable carrier liquid. Valve Assembly 12 may include a pump for moving the various fluids through the column. If molecules of X are present in the sample, they are bound to the immuno-sorbent by the antibodies attached to the particles. A second solution containing antibody to X is then introduced into the immuno-column from reservoir 22. These antibodies are labeled with an enzyme that catalyzes a reaction involving a substrate Y. After a predetermined quantity of the labeled antibody has passed through immuno-column 17, the immuno-column 17 is washed with carrier liquid from reservoir 24 to remove any unbound labeled antibody. A solution containing substrate Y is then introduced into immuno-column 17 from reservoir 23. Simultaneously, the amperometric output from the electrodes is measured by assembly 16. The output is the current between the working and counter electrodes generated when the potential between the working and reference electrodes is maintained at a constant value. The amperometric output is proportional to the concentration of the product of enzymatic reaction and therefore, proportional to the amount of the enzyme label bound to the immuno-sorbent. The bound concentration of bound enzyme is proportional to the amount of X bound to the column.

The present invention may also be used to perform assays based on competitive inhibition of the antibody binding. In this case, the immuno-sorbent is loaded with X', a compound that binds antibody to X via a group that is similar in configuration to X. The presence of X will inhibit the binding of antibody to X'. In this case, X and antibody to X' are introduced into the column. The antibody to X' is labeled with the substrate. After the washing step described above, the amount of labeled antibody is measured as described above by introducing Y into the column and measuring the output from the electrodes.

A variant of the sensor arrangement involves a current collector 19 affixed above the immuno-sorbent layer 15 in intimate contact with the later. The current collector 19 is used to uptake the amperometric signal from the immuno-sorbent layer 15 which may be viewed as an immuno-electrode, polarized as working electrode in amperometric mode. The current collector 19 consists of a conductive probe made from electrochemically inert material (such as carbon, graphite, gold, etc.).

In one preferred embodiment of the present invention, the immuno-column is a disposable assembly which is connected to the various electrodes when the column is attached to a non-disposable portion of the apparatus. Such an embodiment is shown in Figures 2 and 3 at 50. Figure 2 is a cross-sectional view of an assay apparatus according to the present invention. Figure 3 is an exploded cross-sectional view of assay probe 50. Assay probe 50 is constructed from three sub-assemblies shown at 60. 70, and 80, respectively. The immuno-column sub-assembly 60 is a disposable unit which includes the immuno-sorbent 63 on a filter membrane 62 which is supported on an insulating plastic subort 61. The immuno- column assembly fits into a reference electrode assembly 70 which includes the reference electrode 65 and counter electrode 64. The working electrode assembly 80 fits into the immuno-electrode assembly and provides the working electrode 66 which is preferably constructed from carbon. A flow tube 67 provides the connection through which the sample is pumped through the immuno-column. The flow tube may be constructed from a glass capillary tube. In all variations of the assembly, the electrodes contributing to the amperometric signal transduction: the working (measuring) electrode represented by flow immuno-sorbent layer 15, the reference electrode 20 and (or) the counter electrode 21 are in contact with the liquid flowing through the sensor assembly. Both a three electrode circuit scheme (with working electrode 15. counter 21 and reference electrodes 20) and a two electrode circuit scheme (with working electrode 15 and reference electrodes 20 where the reference electrode serves as a counter electrode as well) for the amperometric signal transduction can be employed.

EXPERIMENTAL

Having described the invention, the following examples are given to illustrate specific applications of the invention including specific techniques which can be used to perform the invention. These specific examples are not intended to limit the scope of the invention described in this application.

Example 1 : Determination of rabbit IgG utilizing a 'sandwich' assay scheme.

Immuno-sorbent preparation. 10 mg of Ultra Low Temperature Isotropic carbon (product of Carbomedics, Inc.) was suspended in a solution of Woodward's reagent K (20 mg/mL) in water and was adjusted to pH 4.5 using diluted HC1. The suspension was incubated at room temperature for 2 hours with shaking. The suspension was than washed 5 times with distilled water by repeated centrifugation and removal of the supernatant. Carbon treated with Woodward's reagent K was suspended in 2 mL of a solution of anti rabbit antibodies (0.5 mg/mL) in 0.1 M Na-phosphate buffer pH 7.8, containing 0.15 M NaC 1. The suspension was then incubated with shaking at room temperature for 2 hours. After incubation, 5 mg of trypsin inhibitor was added to the same suspension as a blocking agent, and the suspension was incubated for additional 2 hours at room temperature with shaking. The suspension was finally washed 10 times with 0.1 M Na-phosphate buffer pH 7.8, containing 0.15 M NaC 1 by repeated centrifugation (5 minutes each) and removal of the supernatant. The immuno-sorbent was stored in the same buffer solution at 4°C. Immuno- electrode was prepared by deposition of the immuno-sorbent suspension in the immuno- column and centrifugation.

Immunoassay procedure. The assay system was assembled by connecting the immuno-column to the liquid stream. The current collector was attached to the immuno- electrode. Then a sample containing target analyte (rabbit IgG) is introduced into the liquid stream formed by 0.01 M Na-phosphate buffer (pH 7.4), containing 0.15 M NaCl and 0.05% Tween-20 (PBST) and flows (for 2 minutes) through immuno-column. PBST containing peroxidase labeled antibodies against rabbit IgG (1 :50 diluted) is then allowed to flow through the immuno-column for 5 minutes. Then washing solution (PBST alone) is then allowed to flow through the immuno-column in order to remove unbound labeled antibodies. After 2 minutes washing, the solution containing 1 mM sodium iodide in of 0.1 sodium acetate buffer containing 0.05% Tween-20 and 1 mM H₂0₂ flowed through immuno-column. The amperometric output is measured simultaneously with flowing of sodium iodide solution through the immuno-column. The sensor response is obtained as a maximal signal from the immuno-electrode output. The flow rate at all the stages of the assay was 0.09 ml/min. The dependence of immuno-electrode response on rabbit IgG concentration is presented in Table 1.

TABLE 1

DETECTION OF IMMUNOGLOBULIN G (IgG) USING THE AMPEROMETRIC FLOW IMMUNOELECTRODE

Immunoglobulin G Concentration I (IgG) Amperometric Response (pM) (μA)

0 - 50 (background signal)

30 1.48 85

100 2.00 100

6000 3.78 150

30000 4.48 250

100000 5.00 330 Example 2: Determination of rabbit IgG utilizing a competitive binding protocol.

Immuno-sorbent preparation. The immobilization of rabbit IgG on ULTI carbon was performed using Woodward's reagent by the method described above with reference to Example 1 to form the immuno-sorbent. Immuno-electrode was prepared by deposition of the immuno-sorbent suspension in the immuno-column and centrifugation.

Immunoassay procedure. The assay system was assembled by connecting the immuno-column to the liquid stream. The current collector was attached to the immuno- electrode. Than a sample containing mixture of a target analyte (rabbit IgG) and peroxidase labeled antibodies against rabbit IgG (1 :400 diluted) was introduced into the liquid stream formed by 0.01 M Na-phosphate buffer (pH 7.4), containing 0.15 M NaCl and 0.05% Tween- 20 (PBST) and was allowed to flow for 8 minutes through immuno-column. Than a washing solution (PBST alone) was allowed to flow through the immuno-column in order to remove unbound labeled antibodies. After 2 minutes washing, the solution containing 1 mM sodium iodide in of 0.1 M sodium acetate buffer containing 0.05% Tween-20 and 1 mM H₂0₂ was allowed to flow through immuno-column. The amperometric output was measured while the sodium iodide solution flowed through the immuno-column. The flow rate at all the stages of the assay was 0.09 ml/min. The sensor response is obtained by the method described above (see Example 1). The immuno-electrode response was found to be inversely proportional to the concentration of rabbit IgG as expected.

Example 3: Determination of antibodies against Hantavirus in human blood plasma.

Immunosorbent preparation. The recombinant nucleocapsid protein was formed from expression of plasmid constructs with the complete nucleocapsid gene in E. coli. The product is a fusion protein with matose binding protein that is purified by passage through amaltose affinity column.

The immobilization of recombinant protein of Hantavirus on ULTI carbon was performed using Woodward's reagent by the method described above with reference to Example 1. The immuo-electrode was prepared by deposition of the immuno-sorbent suspension in the immuno-column and centrifugation.

Immunoassay procedure. The assay system was assembled by connecting the immuno-column to the liquid stream. The current collector was attached to the immuno- electrode. Then a blood plasma sample (diluted by PBST 1 :2) was introduced into the liquid stream and allowed to flow for 2 minutes through the immuno-column. PBST containing peroxidase labeled antibodies against human IgG (1 :50 diluted) was then allowed to flow through the immuno-column for 5 minutes. The immuno-column was then washed (PBST alone) for 2 minutes. The sensor response was then measured while a solution containing 1 mM sodium iodide in of 0.1 M sodium acetate buffer containing 0.05% Tween-20 and 1 mM H₂0₂ flowed through the immuno-column. The flow rate of all the stages of the assay was 0.09 ml/min. The sensor response to negative blood sample is practically the same as the control background response obtained for buffer solution. However, the Hantavirus positive blood sample demonstrates an electrode response about 5 times higher than the background sensor response.

It will be appreciated from the above discussion that immuno-columns according to the present invention can be provided as disposable units. The cost of such columns is minimal, since the structural members and electrodes may be constructed from inexpensive materials and the amount of material is small. Systems that are partially disposable will also be apparent to those skilled in the art from the above discussion. For example, a disposable element comprising the immuno-electrode element comprising the immuno-sorbent coated particles on a filter membrane may be utilized. The actual connection to current collector 19 may be made by a contact in the wall of the immuno-column. A more complex disposable unit that includes electrodes 20 and/or 21 may also be provided.

Various modifications to the present invention will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Accordingly, the present invention is to be limited solely by the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for measuring the concentration of an analyte, said apparatus comprising: a flow-through cell[17, 50]; a porous immuno-sorbent layer[15, 63]disposed in said flow-through cell[l 7, 50] such that liquid flowing through said flow-through cell[17, 50] passes through said immuno-sorbent layer, said immuno-sorbent layer[15, 63]comprising an analyte binding material that binds said analyte attached to a support material that is electrically conductive and chemically inert with respect to said analyte; a first electrode[19, 67] in contact with said immuno-sorbent layer; and a second electrode[20, 65] disposed in said flow-through cell[l 7, 50] so as to be in contact with liquid flowing through said flow- through cellfl 7, 50].

2. The apparatus[10, 50] of Claim 1 wherein said support material comprises dispersed particles applied to a filter membrane[18, 62].

3. The apparatus[10, 50] of Claim 2 wherein said particles comprise carbon.

4. The apparatus[ 10, 50] of Claim 2 wherein said particles comprise graphite.

5. The apparatusf 10, 50] of Claim 2 wherein said particles comprise gold.

6. The apparatusf 10, 50] of Claim 1 further comprising a third electrode[21 , 64] disposed in said flow-through cell[17, 50] so as to be in contact with liquid flowing through said flow-through cell[17, 50], aid third electrode[21, 64] being on the opposite side of said immuno-absorbent layer from said second electrode[20, 65].

7. The apparatus[10, 50] of Claim 1 further comprising a pump[12] for causing liquid to flow through said flow-through cellfl 7, 50].

8. The apparatus[10, 50] of Claim 7 further comprising a reservoir[22, 23] operatively connected to said pump[12] for providing said liquid.

9. The apparatusflO, 50] of Claim 8 further comprising an injection port[13] for introducing a sample to be tested into said apparatus[10, 50].

10. A method for determining the concentration of an analyte in a solution, said method comprising the steps of: causing a first solution containing said analyte to flow through a flow-through cell[17, 50] containing a porous immuno-sorbent layer[15, 63]disposed in said flow-through cell[17, 50] such that liquid flowing through said flow- through cell[17, 50] passes through said immuno-sorbent layer, said immuno-sorbent layer[15, 63]comprising an analyte binding material that binds said analyte attached to a support material that is electrically conductive and chemically inert with respect to said analyte; causing a second solution to flow through said flow-through cellf 17, 50], said second solution containing a chemical species that binds to said analyte, said chemical species also catalyzing a chemical reaction of a substrate; and causing a third solution containing said substrate to flow through said flow-through cell[17, 50] and simultaneously measuring the electrical output of a first electrode[19, 67] in contact with said immuno-sorbent layer; and a second electrode[20, 65] disposed in said flow-through cell[17, 50] so as to be in contact with liquid flowing through said flow-through cellf 17, 50].

1 1. The method of Claim 10 wherein said support material comprises dispersed particles applied to a filter membranef 18, 62].

12. The method of Claim 1 1 wherein said particles comprise carbon.

13. The method of Claim 1 1 wherein said particles comprise graphite.

14. The method of Claim 1 1 wherein said particles comprise gold.

15. The method of Claim 10 wherein said flow-through cell[17, 50] further comprises a third electrodef21, 64] disposed in said flow-through cell[17, 50] so as to be in contact with liquid flowing through said flow-through cellf 17, 50], aid third electrode[21 , 64] being on the opposite side of said immuno-absorbent layer from said second electrodef20, 65], and said method further comprises the step of measuring the electrical output of said third electrodef21, 64].

16. A method for determining the concentration of an analyte in a solution, said method comprising the steps of: causing a first solution to flow through a flow-through cellf 17, 50] containing a porous immuno-sorbent layer[15, 63]disposed in said flow-through cellf 17, 50] such that liquid flowing through said flow-through cellf 17, 50] passes through said immuno-sorbent layer, said immuno-sorbent layer[15, 63]comprising a binding material that binds a chemical species attached to a support material that is electrically conductive and chemically inert with respect to said analyte, said first solution containing said analyte and said chemical species, said analyte interfering with said binding of said chemical species, said chemical species also catalyzing a chemical reaction of a substrate; and causing a second solution containing said substrate to flow through said flow-through cellf 17, 50] and simultaneously measuring the electrical output of a first electrodefl9, 67] in contact with said immuno-sorbent layer; and a second electrode[20, 65] disposed in said flow-through cell[17, 50] so as to be in contact with liquid flowing through said flow-through cellf 17, 50].

17. The method of Claim 16 wherein said support material comprises dispersed particles applied to a filter membrane[18, 62].

18. The method of Claim 17 wherein said particles comprise carbon.

19. The method of Claim 17 wherein said particles comprise graphite.

20. The method of Claim 17 wherein said particles comprise gold.

21. The method of Claim 16 wherein said flow-through cell [17, 50] further comprises a third electrode[21, 64] disposed in said flow-through cellf 17, 50] so as to be in contact with liquid flowing through said flow-through cell[17, 50], aid third electrode[21, 64] being on the opposite side of said immuno-absorbent layer from said second electrodef20, 65], and said method further comprises the step of measuring the electrical output of said third electrode[21, 64].