Detection reagent, device and detection method for electrochemical immunoassay

Abstract

The invention relates to the technical field of biology, in particular to a detection reagent, a device and a detection method for electrochemical immunoassay. The interference of blood cells on magnetic beads is fully considered and dealt with, and the volume of the blood cells is reduced by increasing the salt concentration to enable the cells to shrink; by inverting the electrode, the content of blood cells around the electrode is reduced by using the density difference between the blood cells and plasma; the microspheres marked by the anti-erythrocyte antibodies are added into the system, so that blood cells are gathered around the microspheres to quickly settle, and meanwhile, the number of free erythrocytes is reduced, so that the purposes of reducing pollution of blood to the surface of the electrode and improving the passing rate of the magnetic beads are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a detection reagent, a device and a detection method for electrochemical immunoassay. BACKGROUND

[0002] As one of the important roles of biological fluids, whole blood carries a wealth of information related to the body's health status, and plays a crucial role in health management and disease monitoring. Most patients with acute onset, severe symptoms and complex conditions often first visit the emergency department, making the emergency department a department where acute and critical conditions are concentrated. Emergency testing is an important part of emergency medical diagnosis, and the types of emergency testing items, the accuracy and timeliness of the test results can provide important protection for the effective treatment of emergency patients within the golden time window. If whole blood can be directly detected without additional centrifugal separation, the time from sample to result can be effectively shortened, which will greatly improve the possibility of obtaining immediate diagnosis and subsequent treatment for patients in the golden treatment period.

[0003] The current mainstream immunodetection method mainly uses magnetic beads to enrich capture antibodies, and then labels signal molecules such as luminophore or peroxidase (HRP) on the detection antibodies, and captures and detects antigens by sandwich method. However, in whole blood detection, a large number of blood cells will hinder the magnetic adsorption of magnetic beads to the vicinity of the magnet, which will cause the decrease of the detection signal. More importantly, in the HRP-TMB system, red blood cells in the blood also contain peroxidase, and when red blood cells adhere to the surface of magnetic beads or electrodes, if not cleaned properly, the background signal will be increased due to blood contamination, resulting in a significant decrease in sensitivity in whole blood. SUMMARY

[0004] Therefore, the present application provides a detection reagent, a device and a detection method for electrochemical immunoassay, which can reduce the interference of blood cells on magnetic beads when determining analytes, especially blood samples, and is particularly suitable for solving the problem of insufficient sensitivity caused by high background signal and low detection signal of the magnetic bead-peroxidase (HRP)-3,3',5,5'-tetramethylbenzidine (TMB) system in whole blood detection.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a detection reagent for electrochemical immunoassay of a sample containing cells, the electrochemical immunoassay including qualitative analysis and / or quantitative analysis of target substances, and the total concentration of the compounds contained in the detection reagent causes the cells to shrink due to dehydration;

[0007] The compounds include organic compounds and inorganic compounds;

[0008] The inorganic compound includes a salt;

[0009] The sample to be tested includes a blood sample.

[0010] In some embodiments of the present application, in the detection reagent, the amount of substance of the compound is 0.01 mmol, 0.05 mmol, 0.1 mmol, 0.15 mmol, 0.2 mmol or 0.25 mmol, based on 50 μL of the sample to be tested.

[0011] In some embodiments of the present application, in the detection reagent, the salt includes one or more of a potassium salt, a calcium salt, a sodium salt, a magnesium salt, an iron salt, a zinc salt or a chloride salt.

[0012] In some embodiments of the present application, in the detection reagent, the salt is sodium chloride.

[0013] In some embodiments of the present application, in the detection reagent, further includes 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg or 50 μg of microspheres, based on 50 μL of the sample to be tested.

[0014] The microspheres are labeled with a protein capable of binding to the cells;

[0015] The protein includes an antibody.

[0016] In some embodiments of the present application, in the detection reagent, the microspheres include carboxyl latex microspheres with a diameter of 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm or 2 μm.

[0017] In some embodiments of the present application, in the detection reagent, further includes, based on 50 μL of the sample to be tested:

[0018] (i) 2-5 μg of an enzyme-labeled protein capable of binding to the target substance; and

[0019] (ii) 10-50 μg of magnetic beads coupled with a protein capable of binding to the target substance;

[0020] The enzyme-labeled protein includes an enzyme-labeled antibody.

[0021] The protein includes an antibody.

[0022] In some embodiments of the present application, the enzyme-labeled antibody in the above detection reagent is a horseradish peroxidase-labeled antibody.

[0023] In some embodiments of the present application, the protein in the above detection reagent includes streptavidin.

[0024] In some embodiments of the present application, the antibody or other biological molecule in the above detection reagent is one or more of an anti-human red blood cell membrane antibody, a mouse, rabbit, goat, etc. non-human antibody, mouse, rabbit, goat, etc. non-human serum, an anti-human white blood cell antibody.

[0025] In some embodiments of the present application, the above detection reagent further includes a substrate of the enzyme in the enzyme-labeled protein.

[0026] In some embodiments of the present application, the substrate in the above detection reagent is TMB.

[0027] In some embodiments of the present application, the magnetic beads in the above detection reagent are carboxyl-modified magnetic microspheres with a diameter of 0.1 µm, 0.15 µm, 0.2 µm, 0.25 µm, or 3 µm.

[0028] The present application also provides an electrochemical immunoassay device, which includes the above detection reagent and acceptable adjuvants or aids.

[0029] In some embodiments of the present application, the electrochemical immunoassay device includes a liquid bag (2), an air bag (3), an electrode (4), an upper substrate (5), a laminated layer (6), and a lower substrate (7).

[0030] The upper substrate (5) is provided with a sample inlet (8), airway holes, electrode holes, air bag holes, liquid bag holes, and a seal (10) connected to the airway (9) and the liquid flow channel (12) and attached with double-sided tape.

[0031] The laminated layer (6) is provided with a waste liquid bin (15), airway holes, air bag holes, liquid bag holes, a sample detection channel (13), and an incubation bin (11).

[0032] The lower substrate is provided with an airway (9), a liquid flow channel (12), air holes (16), a waste liquid flow channel, and a spike (17).

[0033] The air bag, the airway, the airway holes, and the sample detection channel are sequentially connected.

[0034] The liquid flow channel is provided with the spike.

[0035] The spike can contact the air bag through the air bag hole.

[0036] The sample inlet, the incubation chamber, the liquid flow channel, the sample detection channel are sequentially communicated.

[0037] The sample detection channel, the waste liquid flow channel, the waste liquid chamber, the air hole are sequentially communicated.

[0038] In some embodiments of the present application, the electrode in the electrochemical immunoassay device comprises an insulating substrate (18), a metal contact point (19) penetrating through both sides and used for connecting an instrument, and an array of metal electrodes (20).

[0039] In some embodiments of the present application, the electrode in the electrochemical immunoassay device comprises a working electrode.

[0040] In some embodiments of the present application, the electrode in the electrochemical immunoassay device further comprises a reference electrode.

[0041] In some embodiments of the present application, the liquid pocket in the electrochemical immunoassay device comprises a substrate.

[0042] The substrate comprises TMB.

[0043] The present application also provides a method for electrochemical immunoassay of a sample containing cells, which comprises qualitative analysis and / or quantitative analysis of a target substance, and is based on the electrochemical immunoassay device described above to obtain results.

[0044] In some embodiments of the present application, the method comprises the following steps:

[0045] Step (1): mixing the sample to be tested, the salt, the microspheres, the enzyme-labeled protein and the magnetic beads, so that the osmotic pressure of the sample to be tested is increased, and incubating to obtain a mixture;

[0046] Step (2): contacting the electrode with the mixture, and the relative position relationship between the electrode and the mixture is that the electrode is above and the mixture is below in the vertical direction.

[0047] Step (3): applying a magnetic field above the electrode in the vertical direction to attract the magnetic beads so that the magnetic beads are in contact with the electrode.

[0048] Step (4): contacting the substrate with the magnetic beads, and analyzing the signal obtained by the electrochemical immunoassay device.

[0049] In some embodiments of the present application, the electrode in the method is arranged in the electrochemical immunoassay device.

[0050] In some embodiments of the present application, the method comprises the following steps:

[0051] mixing the sample to be tested, the salt, the microspheres, the enzyme-labeled protein and the magnetic beads so that the osmotic pressure of the sample to be tested is increased, injecting the mixture into the incubation chamber through the sample inlet, incubating, and obtaining a mixture;

[0052] folding the seal to connect the gas channel and the liquid flow channel;

[0053] pressing the air bag to push the mixture into the sample detection channel through the gas channel, the gas channel hole, the incubation chamber, the electrode being in contact with the mixture, and the relative position of the electrode and the mixture being that the electrode is above the mixture in the vertical direction;

[0054] applying a magnetic field above the electrode in the vertical direction to attract the magnetic beads to make the magnetic beads contact the electrode;

[0055] Optionally, pressing the air bag to push the liquid out of the sample detection channel to the waste liquid chamber through the gas channel, the gas channel hole, the incubation chamber and the sample detection channel;

[0056] pressing the liquid bag to push the substrate into the sample detection channel through the liquid flow channel, the substrate being in contact with the magnetic beads;

[0057] connecting the electrode to an electrochemical workstation and applying a potential to the electrode;

[0058] the substrate generating a signal through electrochemical reaction on the electrode, and analyzing the signal.

[0059] In some embodiments of the present application, the sample to be tested in the method is a blood sample.

[0060] The reagent, device and detection method of the present application have the following advantages:

[0061] (1) increasing the salt concentration to make the cells shrink and reduce the volume of the blood cells, thereby reducing the physical barrier of the blood cells to the magnetic beads;

[0062] (2) by inverting the electrode, the density difference between the blood cells and the plasma is utilized, and during the incubation process, the blood cells will naturally sink away from the electrode, thereby increasing the content of the plasma around the electrode and reducing the content of the blood cells around the electrode;

[0063] (3) adding anti-red blood cell antibody (RBC) labeled microspheres to the system, utilizing the specific recognition between the red blood cells and the RBCs to make the red blood cells gather around the microspheres to quickly sink, and greatly reducing the number of free blood cells. BRIEF DESCRIPTION OF DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0065] Figure 1 Schematic diagram of principle;

[0066] Figure 2 Schematic diagram of electrochemical comparison after whole blood improvement;

[0067] Figure 3 Schematic diagram of the test card involved in the present application, wherein 1 represents a test card main body part, 2 represents a TMB liquid bag, 3 represents an air bag, and 4 represents an electrode;

[0068] Figure 4 Schematic diagram of the exploded view of the test card involved in the present application; wherein 5 represents an upper substrate, 6 represents a laminated layer, 7 represents a lower substrate, 8 represents a sample inlet, 9 represents an air channel, 10 represents a sealing port, 11 represents an incubation bin, 12 represents a liquid flow channel, 13 represents a sample detection channel, 15 represents a waste liquid bin, 16 represents an air hole, and 17 represents a spike;

[0069] Figure 5 Schematic diagram of the front and back structures of the electrode, wherein 18 represents an insulating substrate, 19 represents a metal contact point, and 20 represents an array metal electrode;

[0070] Figure 6 Schematic diagram Figure 5 Schematic diagram of the cross-sectional view of the circle area, wherein 7 represents a lower substrate, and 21 represents a reference electrode;

[0071] Figure 7 Schematic diagram of signal intensity of different treatment groups;

[0072] Figure 8 Schematic diagram of the recovery of magnetic beads in whole blood samples with different salt concentrations;

[0073] Figure 9 Schematic diagram of the aggregation of blood cells before and after the addition of microspheres. DETAILED DESCRIPTION

[0074] The present application discloses an electrochemical immunological test card and a test method thereof, and those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0075] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0076] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0077] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0078] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0079] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0080] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0081] like Figure 1 As shown in the figure, in a plasma environment, the magnetic beads labeled with antibodies will be enriched around the electrode due to magnetic adsorption. However, in a whole blood environment, due to the density difference between blood cells and plasma, blood cells will naturally settle near the electrode during the incubation process. At this time, the magnetic beads labeled with antibodies are difficult to reach the electrode due to the physical barrier of blood cells under the action of magnetic adsorption. The few magnetic beads that reach the electrode are also easily adhered by blood cells, thereby increasing the background signal.

[0082] The present invention combines three strategies: increasing the salt concentration, inverting the electrode, and adding anti-red blood cell (RBC) antibody-labeled microspheres into the system. This not only reduces the number and volume of free blood cells, but also increases the plasma content around the electrode, thereby reducing blood cell adhesion to the magnetic beads and physical barrier, ultimately achieving the purpose of reducing blood contamination of the electrode surface and increasing the rate of magnetic beads passing through the blood, restoring the background signal and electrochemical response in whole blood to plasma levels ( Figure 2 ).

[0083] The test card of the present invention is as follows Figure 3 、 Figure 4 As shown, 1 shows the device part, which mainly includes TMB liquid bag 2, air bag 3, electrode 4, upper substrate 5, interlayer 6, lower substrate 7, and other structures and components include injection port 8, air channel 9, seal 10 connecting the air channel and liquid flow channel and affixed with double-sided tape, incubation chamber 11, liquid flow channel 12, sample detection channel 13, waste liquid chamber 15, pore 16 and thorn 17. The electrode is attached to the sample detection channel through the interlayer. The front and back structures of the electrode are shown in FIG. Figure 5 As shown, it includes an insulating substrate 18, metal contact points 19 that penetrate through both sides and are used to connect to instruments, and an array of metal electrodes 20. The gold electrode array includes a working electrode, a reference electrode, a counter electrode, a control electrode, an electrode for judging the hematocrit, and a position electrode for judging whether the liquid flow is in place. Figure 6 for Figure 5 From the cross-sectional view of the circled area, it can be seen that when the test card is placed horizontally, the metal electrode is facing downward and is located above the flow channel, forming an inverted position. A reference electrode 21 is also provided in the electrode array. There is a magnetic field above the metal electrode to gather the magnetic beads in the liquid at a specified position.

[0084] The present invention provides an electrode test card that utilizes the above-mentioned strategy to reduce the interference of blood cells on magnetic beads. Taking the HRP-TMB system as an example, the test card structure includes: an air bag, a TMB liquid bag, magnetic beads, electrodes, and a waste liquid tank. One specific test operation step is as follows:

[0085] 1. Blood flows through the incubation chamber, washing away the magnetic beads fixed to the card;

[0086] 2. The blood mixed with magnetic beads flows through the flow channel into the reaction zone inside the electrode. Under the action of the magnetic field, the magnetic beads are concentrated near the working electrode.

[0087] 3. After capturing the antigen and labeled antibody using the sandwich method, the reaction solution is discharged;

[0088] 4. Press the TMB solution bag for the first time to fully flush and drain the electrode reaction area;

[0089] 5. Press the TMB liquid bag again to fill the electrode reaction area with TMB, and then apply a certain potential to perform subsequent electrochemical detection after incubation for a certain period of time;

[0090] The material of the test card substrate includes one or more of aluminum alloy, PET, PC, glass, and acrylic, PDMS;

[0091] The test card processing method includes one or more of CNC machining, laser processing, injection molding, 3D printing, reverse molding, die cutting, and soft etching;

[0092] The electrode metal layer is attached to the insulating substrate by screen printing or electroplating or magnetron sputtering or laser engraving;

[0093] The metal layer is made of a metal material, and the metal includes at least one of Cu, Ti, Ag, Pt, Pd, Gu, and Au;

[0094] The reference electrode is covered on the surface of the metal electrode by screen printing or electroplating or dispensing;

[0095] The magnetic field forming method uses one or more of an electromagnet, a neodymium iron boron magnet, an aluminum nickel cobalt magnet, and a samarium cobalt magnet, and the magnetic field area size is 0.5-2 times the diameter of the metal electrode;

[0096] The TMB liquid bag contains a horseradish peroxidase (HRP) substrate developing solution (single-component TMB solution), and the main components are 3,3',5,5'-tetramethylbenzidine and a peroxide mixture;

[0097] The single-component TMB solution is a commercial reagent, which can be obtained from one or more of Biyun Tian, Sigma-Aldrich, Merck, ThermoFisher Scientific, and Neogen;

[0098] The reagents used for antibody-labeled magnetic beads include carboxyl-modified magnetic beads, activation buffer, activation reagent, antibody or other biological molecules, quenching solution, and storage buffer containing blocking molecules;

[0099] The magnetic beads are carboxyl-modified magnetic microspheres with a diameter of 0.1-3 µm;

[0100] The activation buffer is MES (2-Morpholinoethanesulphonic acid) with a pH of 4.5-7.5;

[0101] The activation reagent is 5-20 mM carbodiimide (EDC, CMC, etc.) or mixed with 5-20 mM N-hydroxysuccinimide (NHS);

[0102] The antibody or other biological molecule is one or more of a related immunoglobulin, a related antigen, DNA, RNA, enzyme, aptamer, streptavidin;

[0103] The quenching solution is a mixed solution of 10-30 mM primary amine source and 0.01-0.1% (w / v) blocking molecule;

[0104] The blocking molecule is one or more of bovine serum albumin, casein, non-ionic surfactant, polyethylene glycol, commercial blocking agent;

[0105] The storage buffer is a storage buffer (pH 7.0-7.5) with 0.01-0.1% (w / v) blocking molecule.

[0106] The specific preparation steps of the antibody-labeled magnetic beads are taken as an example: 1 mg of magnetic beads is fully suspended in an activation buffer containing 20 mg / mL EDC, mixed and incubated in the dark for 50 minutes. Then the activated magnetic beads are mixed and incubated with 5 μg of cardiac troponin I antibody at room temperature in the dark for 8-16 hours. After the reaction is completed, the magnetic beads are washed repeatedly with the quenching solution for 3-8 times, incubated in the quenching solution for more than 2 hours, then washed and resuspended in the storage buffer to the required storage concentration (usually 10 mg / mL), and stored at 4°C until use;

[0107] The reagent method of antibody-labeled microspheres is the same as the other reagents and steps except for the microspheres and the antibody or other biological molecule; the microspheres are carboxyl latex microspheres with a diameter of 0.2-2 μm; the antibody or other biological molecule is one or more of an anti-human red blood cell membrane antibody, a mouse, rabbit, sheep, etc. non-human antibody, mouse, rabbit, sheep, etc. non-human serum, an anti-human white blood cell antibody;

[0108] The antibody-HRP complex is coupled by the sodium periodate method or the glutaraldehyde labeling method or the avidin antibody-biotinized peroxidase complex method or the biotinized antibody-avidin peroxidase complex method. The coupled antibody-HRP complex is stored in a commercial stabilizer for long-term storage.

[0109] One use scheme of the present application is as follows:

[0110] 1) Mix 50 μL of blood sample containing antigens with 10-50 μg of antibody-labeled magnetic bead complex, 2-5 μg of antibody-HRP complex, 0.01-0.025 mmol of salt, 10-50 μg of anti-RBC antibody-labeled microspheres, etc., then inject them into the incubation chamber through the sample inlet by a syringe or pipette, fold the seal to adhere to the substrate on the test card through the adhesive layer, and form a passage between the air channel and the liquid flow channel;

[0111] 2) Press the air bag to drive the blood sample, and push the blood sample from the incubation chamber to the sample detection flow channel. The sample is kept in the sample detection area by the in-place electrode, and incubated for 3-5 minutes.

[0112] 3) After the incubation is completed, the magnet is close to the working electrode and stops at a distance of 0-2 mm from the working electrode. The magnetic beads are gradually separated from the blood and adsorbed on the electrode surface by magnetic field attraction. This step takes 1-3 minutes.

[0113] 4) The sample detection flow channel is filled with TMB solution by alternately pressing the liquid bag and the air bag to continuously inject TMB solution and air into the sample detection flow channel. After 2-4 cycles, the sample detection flow channel is filled with TMB solution. After the TMB solution is incubated for one minute, the sensing device is connected to the electrochemical workstation, and a voltage of -0.2 V is applied to the electrode for 60 seconds. The current value at the 60th second is recorded.

[0114] When the electrode surface forms an antibody-labeled magnetic bead-antigen-antibody-HRP complex sandwich complex, the effective components in the TMB solution are oxidized under the catalysis of the enzyme. When a negative voltage is applied, the oxidized product is reduced and generates an electric current. The absolute value of the generated current is proportional to the amount of the formed sandwich complex.

[0115] The above-mentioned salt includes one or more of K + , Ca 2+ , Na + , Mg + , Fe 3+ , Zn 2+ , Cl - .

[0116] The application is further described below in combination with examples:

[0117] Example

[0118] 1) Mix 50 μL of blood sample containing antigen (Huadeng recombinant human cardiac troponin complex, product number 60005, containing 0 ng / mL, 0.1 ng / mL, 1 ng / mL or 10 ng / mL) with 2.5 μL of detection reagent, and then inject the mixture from the sample inlet into the incubation chamber by a syringe or a pipette. Fold the seal to adhere to the substrate on the test card through the adhesive layer to form a passage between the air channel and the liquid flow channel.

[0119] 2) Press the air bag to drive the blood sample, and push the blood sample from the incubation chamber to the sample detection flow channel. The sample is kept in the sample detection area by the in-place electrode, and incubated for 3-5 minutes.

[0120] 3) After the incubation is completed, the magnet is close to the working electrode and stops at a distance of 0-2 mm from the working electrode. The magnetic beads are gradually separated from the blood and adsorbed on the electrode surface by magnetic field attraction. This step takes 1-3 minutes.

[0121] 4) By alternating the pressing of the liquid bag and the air bag, the TMB solution and air are continuously injected into the sample detection flow channel. After 2-4 cycles, the sample detection flow channel is filled with TMB solution. After incubation for 1 minute, the electrode is connected to the electrochemical workstation, and a voltage of -0.2 V is applied to the electrode for 60 seconds, and the current signal at 60 seconds is read.

[0122] The above detection reagent includes detection reagent 1, detection reagent 2, detection reagent 3 or detection reagent 4, and the components and proportions are shown in Table 1:

[0123] Table 1

[0124]

[0125] Four different processing methods are set up, respectively:

[0126] (1): Control: 50 μL of blood sample mixed with detection reagent 1, and the electrode of the test card is upright;

[0127] (2): Upright: 50 μL of blood sample mixed with detection reagent 1, and the electrode of the test card is upright;

[0128] (3): Upright + salt: 50 μL of blood sample mixed with detection reagent 2, and the electrode of the test card is upright;

[0129] (4): Inverted + salt + microspheres: 50 μL of blood sample mixed with detection reagent 3, and the electrode of the test card is inverted.

[0130] Results: 1. Table 2 and Figure 7 It is shown that, compared with the upright whole blood signal, the signal in the upright salt whole blood sample is greatly improved, indicating that the magnetic beads can better respond to the magnetic field in the whole blood with more salt, and form more sandwich structures on the metal electrode surface, so that the analysis performance is improved, but there is still a certain gap with the control group (whole blood plasma). When salt, microspheres are added to the blood sample and the electrode is inverted, the signal response is further improved. Compared with the signal generated by the upright whole blood sample and the inverted sample at 10 ng / mL, the signal is improved by 243%. On the other hand, as shown in Table 3, by adding salt, microspheres, inversion and other measures, the background signal of the sensor is gradually reduced from 104.4 (-nA) to 57.7 (-nA), and the CV (%) is reduced from 9.1 to 5.7, improving the analysis performance and reducing the probability of false positives and false negatives.

[0131] Table 2

[0132]

[0133] Table 3

[0134]

[0135] 2. Based on the test conditions of the above (4), by comparing the recovery of magnetic beads in whole blood samples with different salt concentrations, it can be seen that the addition of 100-250 mM salt in whole blood can make the recovery of magnetic beads comparable to that in whole blood plasma (Figure 3) Figure 8 According to the result analysis, the main reason is that the increased salt concentration can make the blood cells shrink, so that the magnetic beads are more easily transferred in the blood sample under the action of the magnetic field.

[0136] By adding microspheres in whole blood, the antibodies and other biomolecules on the microspheres can make the blood cells agglomerate in a very short time, as shown in Figure 9 The agglomerated blood cells can quickly settle down under the action of gravity, which further improves the passing rate of magnetic beads and avoids the adhesion of blood cells on the electrode surface, thereby further reducing the background signal.

[0137] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Test reagent for electrochemical immunoassay of a sample to be tested containing cells, the electrochemical immunoassay comprising a qualitative and / or quantitative analysis of a target substance, characterized in that, The total concentration of the compound contained in the detection reagent causes the cells to lose water and shrink; The compound includes a salt; The sample to be tested includes a blood sample.

2. The detection reagent of claim 1, wherein The amount of substance of the salt is 0.01-0.25 millimoles, based on 50 μL of the sample to be tested.

3. The test reagent according to claim 1 or 2, wherein Further including 10-50 μg of microspheres, based on 50 μL of the sample to be tested; The microspheres are labeled with a protein capable of binding to the cells.

4. The detection reagent of claim 3, wherein Further including, based on 50 μL of the sample to be tested: (i) 2-5 μg of an enzyme-labeled protein capable of binding to the target substance; and (ii) 10-50 μg of magnetic beads coupled with a protein capable of binding to the target substance; The enzyme-labeled protein includes an enzyme-labeled antibody; The protein includes an antibody.

5. The detection reagent of claim 4, wherein the antibody is a monoclonal antibody. Further including a substrate of the enzyme in the enzyme-labeled protein.

6. An electrochemical immunoassay device characterized by Including the detection reagent as claimed in any one of claims 1-5, and acceptable adjuvants, auxiliaries and / or components.

7. A method of electrochemical immunoassay, which comprises qualitative analysis and / or quantitative analysis of a target substance, characterized by, Obtaining results based on the electrochemical immunoassay device as claimed in claim 6.

8. The method of claim 7, wherein, Including the following steps: Step (1): mixing the sample to be tested, the compound, the microspheres, the enzyme-labeled protein and the magnetic beads, so that the osmotic pressure of the sample to be tested is increased, incubating, and obtaining a mixture; Step (2): contacting an electrode with the mixture, the relative position relationship between the electrode and the mixture being that the electrode is above and the mixture is below in the vertical direction; Step (3): applying a magnetic field above the electrode in the vertical direction, attracting the magnetic beads so that the magnetic beads are in contact with the electrode; Step (4): contacting the substrate with the magnetic beads, and analyzing the signal obtained by the electrochemical immunoassay device.

9. The method of claim 8, wherein, The sample to be tested is a blood sample.

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