JPH10500212A - Once-through electrochemical biosensor - Google Patents

Abstract

(57) Abstract: Electrochemistry characterized in that the solid phase is intimately connected to the porous working electrode (5, 21) and that a suitable counter electrode (6, 22) is present in the system. Flow-through solid-phase immunoassay system or device with specific detectability. This new device provides very fast and sensitive diagnostic capabilities.

Description

DETAILED DESCRIPTION OF THE INVENTION                      Once-through electrochemical biosensor   Background of the Invention   The present invention relates to a flow-through solid-phase immunoassay system or device capable of electrochemical detection. It relates to a Muno assay device.   Immunoassay diagnostics are commonly used for a wide range of substances in serum, plasma or other fluids. Well-established as diagnostic methods for clinical use, and the principles of these assays are environmental monitoring or Can be extended to other fields such as DNA analysis.   A typical method for an immunoassay is (I) Biological capture antibody on solid phase, eg plastic microtiter plate On the target sample to bind to the antigen in question, washing and then a second This is a method of acting on a labeled antibody. The label on the antibody can be, for example, a radiotracer or It may be an enzyme. Further, detection of the label is performed following washing (so that The amount of antigen in the sample is detected). This method is a sandwich assay or Known as a two-site assay, or (Ii) a biological sample containing an antigen and an added amount of labeled antigen as the capture antibody on the solid phase; Is to act on . Labeled and unlabeled antigens compete for antibody sites on the solid phase. Of the label shown after washing The amount is inversely proportional to the amount of the true antigen in the biological sample. This method is a competitive asset It is known as a.   Indication of the sign can be achieved in various ways. For enzyme labeling, chromogen detection Choice of luminescence, luminescence or electrochemical detection methods . A chromogenic immunoassay that produces a colored product upon addition of a substrate for which an enzyme label is suitable. B) is well established under the general term "ELISA" and uses the principle of photometry. To detect discoloration in the substrate solution. This method is relatively easy to implement However, it must accept the difficulty of achieving long incubation times and low detection limits. Must.   Luminescence and fluoresence generated from substrate solutions are enzyme labeled Can be achieved by a sandwich assay compared to the chromogenic method. And a low detection limit with a factor of 10 (Portsmann, T. and Kiessig, S.T. , "Enzyme Immunoassey Techniques", J. Immuno. Meth., 150, 5-21. Pp. 1992), and the required sample volume is clearly reduced. But city Luminescence / fluorescence in stabilizing sales substrates and equipment costs Difficulties in the expenditure of the sense detector. Electrochemical immunoassays are In the field of And an electrochemically active species on the electrode immediately adjacent to the solid phase. The reaction product) is oxidized or reduced to detect this species. Mainly pursuing the combination. There are many such biosensors in the literature (See, for example, Heineman, W.R. and Haisall, H.B., "Strategies for elec trochemical immunoassay ", Anal. Chem. 57, 2, 1321-1331, 1985), most of which achieve the required sensitivity and detection limits. Have failed completely and cannot be used to produce viable equipment on the market. No.   Another aspect of a practical immunoassay is that it reduces the incubation time for the assay. Instrumentation with various high surface area solid phases to shrink. Big table The solid phase having an area includes a polymer bed, magnetic particles and a polymer film.   Different forms of flow-through technology select solid phase to reduce assay time and enhance sensitivity Has been implemented in the context of U.S. Pat. No. 5,006,309 describes an optical system A device having a membrane as a solid phase for an immunoassay for detection is described. JP -A-60188839 protrudes into the sample stream for detection It describes the use of a selection electrode.   EP-B1-0033188, EP-B1-0060082 And EP-A-046983 All of No. 0 improve electrochemical detection in analytical systems such as HPLC A structure of a porous electrode that can be used to perform EP-B1 No. 0122009 discloses that a sample is filtered by chromatography before the experiment. Detection of some electrochemically active biological molecules, provided that The electrodes for are described. The electrode described in this patent is a chromatographic system. Intended for use as a permanent and reusable detector in systems .   EP-A2-0352138 and EP-A2-0525723 Both documents incorporate membranes as solid phases and biosensors as electrochemical detectors. Describe special equipment that uses the standard solid electrode format normally applied during You.   Summary of the Invention   The present invention relates to the detection of porous solid-state immunoassay systems or devices. It can be clearly increased by having a solid phase that is tightly coupled to the poles. It is the foundation that we found. This effect is due to the fact that the electrode in contact with the electroactive species It appears that increased surface area and enhanced diffusion motion at the electrode / species interface can be achieved It is. The large surface area achieved by the porous electrode can be achieved under favorable flow and voltage conditions. Obviously leads to significantly higher oxidation and reduction efficiency of the electroactive species. Liquid passing through electrodes The effect of the flow of gas is to detect oxidation or return at the diffusion layer at the electrode / liquid interface. It is likely that the original seed will be replenished immediately. Oxidized or reduced species from two phases Leaving and refilling is usually done by stirring the solution on the solid detection electrode or This interface must be controlled by the flow of liquid, Is a limiting factor in thermochemical immunosensors and other biosensors . Therefore, the application of this electrode in the present invention may be an electrochemical immunosensor or other Two aspects that are very important for electrodes to be used in biosensors, The need for high efficiency in oxidation and reduction (surface area) and the two-phase Covers the need for rapid replenishment (flow-through measurement). This electrode is optional Any suitable porous conductive material, such as preformed graphite sheet, molten copper Printed on carbon powder, metal-based porous plugs or suitable porous substrates It may be a conductive ink.   Thus, the present invention provides that the solid phase is intimately connected to the porous working electrode and that Electrochemical detection capability, characterized by the presence of a counter electrode in the system. Flow-through solid layer immunoassay system or device.   Other objects will be apparent from the claims.   The sensitivity of the electrode according to the invention to a particular species controls the flow rate and the voltage at the electrode Can be operated by it can. Voltage control is achieved by a conventional electrostatic device, which controls the species in the solution flowing through the electrodes. Provides a current or charge reading in proportion to the quantity. Electrostatic potential control is optional electrification In a normal voltage-current or current measurement format for biological analysis. But preferably the application of a suitable dc potential for the oxidation or reduction of the species And the current at the electrodes is monitored by use of a suitable device.   Electrochemically active species (also known as electroactive species) are easily oxidized, Or any suitable enzyme / substrate pair that produces the species to be reduced (the enzyme is usually Sandwich assay when using an labelle (or forming part of an assay) Or in the course of a competition assay or other immunoassay type Can be. This assay is advantageously performed with the enzymes alkaline phosphatase and phosphate. Uses a substrate, but the substrate produces an electrochemically detectable species upon contact with the enzyme. Any other suitable enzyme / substrate pair that forms can also be used. For example, Arca Naphthylphosphorus in solution as a substrate during a rephosphatase-labeled immunoassay The use of a salt allows the electroactive species naphthol to be obtained under suitable buffer and substrate conditions. It is. It is easily oxidizable on a porous electrode under electrostatic potential control and produced One electron is formed for each molecule of naphthol. Similarly, aminophenylphospho Alkaline phosphatas It can be used as a substrate by using a protease. Enzyme substrates that can be used Another example of a pair is β-galactosidase and p-aminophenyl-β-D-galactosidase. Production of the electroactive species aminophenol from horseradish, horseradish peroxidase and Production of electroactive species hydrogen peroxide from lactose, lactate dehydrogenase and lactate This is the generation of the electroactive species NADH in the presence of NAD-.   The electrodes use a three-electrode system consisting of a working (detection) electrode, a counter and a reference electrode. A suitable flow cell or device to be used which allows suitable electrostatic potential control of the electrode / liquid environment For proper function at the applied potential between the electrodes. Includes devices that require only the presence of the poles, ie, the working and reference electrodes. Electrochemical Detection can be performed using peak current or total charge measurements, which Each special parameter required for an electrochemical immunoassay on the system Can be calibrated for the analyte.   A second aspect of the invention is that the immunoassay is performed in a sequential flow process or in a continuous flow. The porous electrode and the solid phase for carrying out the sealing are included in the finished device. This is due to the high surface area and antibody / antigen interaction on the solid phase Fluorescence or luminescence with enhanced diffusional motion due to the active detection layer A device that requires less sophisticated electrical control than detection and is practical and inexpensive to manufacture Manufacturing a device and providing very rapid and sensitive diagnostic capabilities. This All suitable protein binders exhibit high surface area to volume ratio and good porosity The material may be, for example, a polymer membrane, a polymer bed or a particle suspension. The solid phase is advantageously located close to the porous electrode and encapsulated in a complete immunoassay device. As included, the preferred solid phase is a polymer membrane.   A full description of the possible structures of the present invention is provided by showing a schematic diagram.   List of drawings   Figure 1: Porous membrane acting as solid phase for immunoassay and electrochemical detection Schematic cross section of a flow-through cell structure including a porous operation and a counter electrode for The figure is shown.   FIG. 2: a schematic longitudinal section through a flow-through cell with a wicking action.   FIG. 3: Graph showing the results of the assay described in Example 3.   FIG. 4: Graph showing the results of the assay described in Example 4.   Description of the device   FIG. 1 shows a sample in a preferred process order via a cell in a final electrochemical detection process. The passage of reagents and washing solutions According to the invention for connecting to a pump system for liquid flow control enabling 1 shows a longitudinal section of one possible structure for a flow device or cell. Cell body 1 can be any It may be a suitable preform or a mechanically inert material by machine, but is advantageously Molded from rimer material, here formed from two splittable halves And the case where the components of the device can be replaced as necessary . All reagents are introduced into the cell via the annular aperture 2 and in a suitable tube 15 It is sucked in the direction of arrow 3 by the pump 14 installed below the cell. In this case The cell consists of a three-electrode arrangement for complete electrostatic control, The electrode 5 is a working electrode, the porous electrode 6 is a counter electrode, and a solid Ag The / AgCl reference electrode 7 is exposed to contact with the reagent stream on the inner wall of the cell. Porous electrode Gaskets 10, 11 and 12 and "O" -ring 1 are fixed in place. 3, as well as a porous membrane 4 that provides a solid phase for the immunoassay reaction, Sealed to the cell body. This membrane 4 is any membrane suitable for immobilizing any protein. But may advantageously consist of one of the following materials: nitrocellulose, poly Vinylidene difluoride (PVDF), nylon or glass fiber. Membrane holes Diameter and thickness are tailored to the requirements of the individual immunoassay and required liquid behavior Can be The membrane 4 is a capture antibody or antigen in the field. Manufactured according to the usual aspects of immunoassays known to the expert and inserted into the cell It is. The cell inner diameter, d, can be varied, but the preferred dimensions are 2-10 m m and controls the sample volume required for favorable detection of the product. Limit. The thickness of the working electrode 5 is high in the concentration range of the product to be detected. Or reduction effect and thus a high sensitivity or, if necessary, a rapid fouling of the electrodes. Dyeing should be avoided. Pollution refers to the failure of oxidation or reduction of electroactive species. Produces a substance that is soluble and gradually deposits on the electrode, blocks the surface, and Describes a phenomenon that reduces the effect. Electrical connections are made to the electrodes through the cell. And is connected to an external electrostatic potential or other electroanalytical device at contacts 7, 8 and 9.   The cell was then subjected to competition or two-site electrical as shown in Examples 3 and 4. Creates a complete immunoassay environment for chemical immunoassays. In FIG. The conceptualized device is a suitable liquid for plasma, serum or other analysis in immunoassays. Can be analyzed.   Flow electrochemical immunoassay device concept pumps liquid through device Perform with a device adapted for wicking liquid by capillary action to eliminate the need You can also. A conceptual diagram of this type of device derived from the basic principle of the present invention is shown in FIG. FIG. 2 is a longitudinal sectional view of the apparatus. Depending on the parameter to be tested Samples and reagents required for a munoassay or competitive immunoassay It is introduced into the apparatus by the aperture 14, and the solid phase film 15, the spacer film 16a, Pass through the porous working electrode 21, the spacer film 16b, and the porous counter / reference electrode 22. To suck up into the absorbent material 19. Operation and contact with the counter electrode are electrostatic Or conductive materials 17 and 18 that can suitably contact other electroanalytical devices. Has been done more. The porous counter / reference electrode 22 is preferably made of a stable material. Ag / AgCl inks printed on a suitable porous support are advantageous. apparatus The final reagents that must pass through are immediately converted to electroactive products, in other words Substrate solution for enzyme labeling, detected at the pole by oxidation or reduction. this The device is placed in a container 20 for protection, and after one sample is measured, it is discarded. Plasma, serum or Other liquids that can be analyzed by immunoassay can be used with this device . In addition, the device may provide a blood / By attaching a plasma separation membrane, it can be applied to analysis of whole blood.   This device has been realized from the schematic diagrams of FIGS. 1 and 2 with different manufacturing and compounding processes can do. Porous electrodes are incorporated directly into the device as preform materials Or printed with a porous ink on a suitable porous surface. Wear. This device is Depending on the requirements of the analysis equipment, it may consist of a working electrode, a reference electrode and a counter electrode or Is simply configured as a two-electrode system. To reduce waste material generation, Equipment of the type conceptually described in the figures shall be constructed in two separable halves One containing a membrane or other antibody / antigen capture phase and the other a detection electrode system It contains.   The half containing the electrodes of the device is reusable for the expected number of tests and the solid phase Only the parts of the equipment that contain Should be replaced by   Example   Example 1   A commercially available disk made of porous graphite having a diameter of 5 mm and a thickness of 0.35 mm was used. (Toray Industries, Japan).   As a first test, only the liquid contact with the disc is on the upper surface of the graphite. In some conventional planar electrode cells, the usual solid-state electrodes for biosensors It was arranged as above. The cell contains an Ag / AgCl reference electrode and titanium for measurement. Stir to include counter electrode and ensure diffusion layer control in conventional manner I was able to. This can be achieved by using a conventional solid-phase electrode electrochemical immunoassay. A model for measurement of gas-active species was provided. + 300mV voltage to the reference electrode With the working electrode equilibrated with pressure, a diethanolamine DEA buffer (DEA50 naphthol solution in 100 mmol / l) 100 mmol / NaCl, pH 9.6) Measurement of 10 μmol / l gave a response average current peak of 100 nA.   In a second test, another disk electrode was cut from the graphite sheet. And placed in a flow cell of the type shown in FIG. Direct contact between electrode and liquid The diameter was 3 mm. See samples of various concentrations of naphthol in DEA buffer Using the electrode equilibrated at +300 mV with respect to the reference electrode Ag / AgCl, 30 The current obtained for each sample volume of 300 μl, flowing through the cell at 0 μl / min The peak was recorded. A current peak of about 100 nA has a naphthol concentration of 100 nmol. / L, the porous electrode is a conventional solid electrode at this voltage and flow conditions. The sensitivity was 100 times higher. This sensitivity was adapted to the flow rate at which the cell configuration was suitable By being able to total oxidize or reduce the electroactive species when passing through the electrode at potential Can be reached. This response current peak increases as the velocity through the cell increases. sell.   Background generated in a carrier solution (biological buffer) in this way The naphthol concentration to at least 0.5 nmol / l relative to the It is possible to This limit is not Caused by the flow of the system. Especially high enough to create bubbles in open systems Flow rates should be avoided. Example 2   Assay for measuring the amount of thyroid stimulating hormone (TSH) in a serum standard solution A comparison of an electrochemical flow detection system with a standard chromogen assay A conventional ELISA sandwich assay was used for this.   The assay was performed using a 96-well microtiter plate coated with a capture antibody for TSH. Was carried out at the site. Different concentrations of TSH standard solution, 100 μl / well on plate Incubate for 1 hour, then wash the wells and add alkaline phosphatase (AK P) labeled anti-TSH secondary antibody solution, 100 μl / well, 1 μl on plate The temperature was kept constant for hours. The plate is washed again with buffer to remove any unbound secondary antibody. Removed.   Half of the wells in the microplate are filled with a chromogenic substrate for ALP, p-nitro- Exposure to a 2.8 mmol / l solution of phenyl phosphate, 300 μl / well. The other half is the electrochemical substrate for ALP, one of naphthyl phosphate, the concentration Exposure to 1 mmol / l, 300 μl / well. After maintaining the temperature at 37 ° C for 20 minutes, Measure the color development of the solution in this well in a plate reader (chromogenic substrate), or Extracted from the hole and measured by amperometry with the flow method described in Example 1 ( Electric Chemochemical substrate). A typical conventional chromogen assay has a volume of less than 1 mU / l TSH. Reliable measurements are not possible, while the electrochemical flow detection method is 0.01. Sustain measurements up to mU / l.   Example 3   Setting up a competitive immunoassay for thyroxine (T4) in a flow-through system Was.   The T4-alkaline phosphatase (T4-ALP) linkage is converted to L-thyroxine sodium. Starting from the sodium salt and using alkaline phosphine with disuccinimidyl substrate (DSS) Manufactured by conjugation to tatase (bioenzyme).   The anti-T4 antibody was carbonated using an ELISA membrane assay system (Pierce). Nitrocellulose membrane (Schl) at 2 µg / ml in buffer, pH 9.6 to 0.45 µm. eicher and Schuell). The membrane was shielded with a shielding solution.   A disk (diameter 6 mm) of the membrane was cut from the prepared membrane and the disk shown in FIG. Loaded in the Ip flow cell. A new membrane was used for each test sample. antigen T4-ALP complex in Tris buffered saline, pH 8.6, for isothermal step 1 μg / ml of the body was mixed with the T4 standard solution at a ratio of 1: 5, and the total sample volume was 200 μl provided the competition assay. Updating in a cell performed continuously without interruption The Say process is as follows Was: (I) Passage of 1 ml buffer through the cell. (Ii) 200 μl passage of the sample / complex mixture. (Iii) 1 ml of buffer passed through the cell as a washing step. (Iv) 300 μl of substrate solution (1 mmol / l naphthyl phosphate in buffer) Passing.   This constant temperature electroactive product, naphthol, is applied to the Ag / AgCl reference electrode. And equilibrated at +300 mV, with the electrodes in the cell (Toray, 3 mm diameter liquid Contact with body) directly detected. Results consistent with those obtained from a typical competition assay The results are shown in FIG. The total assay time was 27 minutes at room temperature.   Example 4   Sandwich (or two-site) immunoassay for TSH in continuous flow Conducted by law:   Nitrocellulose membrane (Millipore) using ELIFA system (Pierce) Was loaded at a concentration of 10 μg / ml in primary buffer against TSH, carbonate buffer. The membrane was blocked using a blocking solution.   A 6 mm diameter disk was cut from this membrane and a flow cell of the form shown in FIG. Installed inside. A membrane of human standard serum containing TSH was prepared in a sample volume of 300 μl. Passed through. New membranes and electrodes were applied for each concentration. In this assay For this purpose, sample Flowing through the system and immediately in solution the second antibody-alkaline phosphatase complex, Then, 1.5 ml of the washing solution was poured. Finally, 1 mmol / l in DEA buffer 300 μl of 1 naphthyl phosphate solution was passed through the device. Serum sample Cleaning procedures to reduce the risk of electrode contamination with oxidation or reduction products from Only the cell was polarized.   Electrode with naphthol equilibrated at +300 mV against Ag / AgCl reference electrode Was directly detected at the porous electrode by the oxidation flow at All the time from sample addition to results The temperature retention time was 29 minutes at room temperature. The results are shown in FIG. Consistent with the expected response from a two-site immunoassay.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor William Henry Maren             United States 60030 Illinois Gre             Islake Deep Wood Court             833 (72) Inventor Nigel Kyle             United Kingdom Cambridgeshire Sea             B4 1Rs Cambridge             Lean End Road 79 (72) Inventor Fabio Lissandrero             Italy A-20135 Milan Via               Marco Greppi 2

Claims (1)

[Claims]   1. The solid phase is intimately connected to the porous working electrode (5, 21) and Electrochemical, characterized in that a counter electrode (6, 22) is present in the system Flow-through solid-phase immunoassay system or apparatus having an objective detection ability.   2. At least one multi-layer in which the solid phase is in intimate connection with the porous working electrode 2. The flow-through solid phase with electrochemical detection capability according to claim 1, which is a porous membrane (4, 15). Immunoassay system or device.   3. 2. The electrochemical detection capability of claim 1 wherein a flow control system is present. A once-through solid-phase immunoassay system or device.   4. 4. The electricity of claim 3, wherein the flow control system is a pump that creates a pressure differential. A flow-through solid-phase immunoassay system or device having chemical detection ability.   5. The electrochemical device according to claim 3, wherein the flow control system is a wicking material (19). A flow-through solid-phase immunoassay system or device with detectability.   6. 2. Electrochemical detection capability according to claim 1, wherein a reference electrode (7, 22) is present. Flow-through solid-phase immunoassay system or device.   7. The porous working electrode (5, 21) is a graphite or carbon paper electrode or a porous material Electrode printed on material 2. A flow-through solid-phase immunoassay system having electrochemical detection ability according to claim 1. System or device.