Alkaline phosphatase detection method based on single-particle collision electrochemistry and application

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to an alkaline phosphatase detection method based on single-particle collision electrochemistry and application. The invention designs a novel nano collision electrochemical sensing analysis strategy based on reduction of silver ions on the surfaces of gold nanoparticles catalyzed by alkaline phosphatase. The core of the method is that alkaline phosphatase catalyzes hydrolysis of a substrate ascorbic acid-2-trisodium phosphate (AA-P) to generate ascorbic acid (AA) with reducibility, silver ions are reduced on the surfaces of gold particles to generate Au-Ag NPs nano-particles, and random collision behaviors occur on the surface of a microelectrode. The nano collision electrochemical biosensor disclosed by the invention is simple to operate, and does not need a tedious biomarking procedure and strenuous cleaning and separation steps. And the technology has the analysis characteristics of zero background, high sensitivity, high specificity and quick response.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to an alkaline phosphatase detection method based on single particle collision electrochemistry and an application thereof. Background Art

[0002] Enzymatic reactions play an important role in maintaining the normal physiological processes of organisms. Enzyme inactivation or changes in levels may induce or accompany the occurrence and development of some serious diseases. Alkaline phosphatase (ALP, EC3.1.3.1) is an important hydrolase in phosphate metabolism, plays a key role in the signal transduction of cell cycle, growth, apoptosis and intracellular regulatory processes, and is regarded as an important biomarker for diseases such as hepatobiliary and bone diseases, breast cancer and prostate cancer, and diabetes in clinical diagnosis. In addition, due to the high catalytic activity, good stability, wide substrate specificity and easy conjugation with antibodies of alkaline phosphatase, alkaline phosphatase is one of the most widely used label tracers in bioanalysis fields such as enzyme-linked immunosorbent assay. Therefore, the establishment of an efficient and accurate ALP activity detection method is of great significance for the early diagnosis of related diseases and human health monitoring, as well as the development of bioanalysis methods based on alkaline phosphatase catalysis. At present, researchers have established a variety of methods for ALP activity detection, including colorimetry, fluorescence, surface-enhanced Raman scattering, chemiluminescence and electrochemical methods. Although these methods have their own unique advantages, they still have some limitations, such as relatively long analysis time, complex instruments and data collection, high cost, background interference, etc.

[0003] In recent years, nano-collision electrochemistry has become one of the most attractive analytical methods at the single entity level. Unlike macroscopic electrochemical methods, nano-collision electrochemistry uses the transient current caused by the random collision of nanoparticles diffused in the solution with the microelectrode surface as a signal output to record single entity events. Based on the transient current response characteristics (current intensity, collision frequency, collision response time, etc.), the concentration, size, aggregation state and electrocatalytic performance of nanoparticles can be quantitatively analyzed in real time, and then used as a powerful tool for accurate analysis of specific biomolecules. Nano-collision electrochemical methods have the advantages of homogeneous operation, fast response speed, high sensitivity and high throughput. In recent years, nano-collision electrochemistry has been widely used in the analysis of various biological analytes, including nucleic acids, proteins, cells, bacteria, viruses, etc., and has broad application prospects. However, some reported nano-collision sensing strategies require the modification of biorecognition elements on the surface of nanoparticles, and the preparation process is relatively complex and difficult to control. In addition, biological assemblies are prone to cause background interference and limit the collision behavior of nanoparticles, which poses a challenge to the accurate analysis of analytes.

[0004] The present invention constructs a gold core-silver shell nanoparticle collision electrochemical sensing analysis platform by alkaline phosphatase-catalyzed silver ion deposition on the surface of gold nanoparticles, and provides a simple, homogeneous alkaline phosphatase quantitative analysis detection method. The method has low detection background signal (zero background), fast response, good selectivity, low cost, and can quickly and sensitively detect the activity of alkaline phosphatase. Summary of the invention

[0005] In view of the above technical analysis and existing problems, the present invention constructs a gold core silver shell nanoparticle collision electrochemical sensing analysis platform, and provides a simple, homogeneous alkaline phosphatase (ALP) and immunoassay method. Alkaline phosphatase catalyzes the hydrolysis of ascorbic acid-2-phosphate trisodium salt (AA-P), removes the phosphate group, and produces ascorbic acid (AA), and ascorbic acid can convert Ag into + It is reduced to Ag and deposited on the surface of gold nanoparticles to form gold core and silver shell nanoparticles (Au@Ag NPs). When the nanoparticles collide randomly with the surface of the microelectrode, an electrochemical oxidation reaction occurs on the surface of the microelectrode, producing a spike-like transient electrochemical response. The frequency of this transient current response is related to the number of Au@AgNPs produced by alkaline phosphatase catalysis. According to the corresponding relationship between the collision frequency and the activity of alkaline phosphatase, the activity analysis and detection of alkaline phosphatase can be realized. On this basis, combined with the enzyme-linked immunosorbent assay method, the specific binding effect of alkaline phosphatase labeled antibody and antigen is utilized, and the collision electrochemical response of Au@Ag NPs produced under the catalysis of alkaline phosphatase is used to realize the detection of human immunoglobulin concentration.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In one aspect, the present invention provides a method for detecting alkaline phosphatase based on single particle collision electrochemistry, comprising the following steps:

[0008] The alkaline phosphatase solution to be tested and the ascorbic acid-2-phosphate trisodium salt solution are mixed in a DEA buffer solution to form a mixed solution;

[0009] The mixed solution is mixed with Au NPs and AgNO3 to react to obtain an Au@Ag NPs solution;

[0010] The Au@Ag NPs solution is added with KCl solution as an electrolyte solution;

[0011] An electrochemical collision test was performed, and the random collision signals between Au@Ag NPs and the working electrode were collected using the chronoamperometry method and the collision frequency was counted. The activity of alkaline phosphatase was detected by the collision frequency.

[0012] As a preferred technical solution of the present invention, the volume ratio of the alkaline phosphatase solution to be tested and the ascorbic acid-2-phosphate trisodium salt solution is 1:19-21, and the concentration of the ascorbic acid-2-phosphate trisodium salt solution is 2.5mM, and the concentration of the DEA buffer solution is 40mM, and the pH is 9.8.

[0013] As a preferred technical solution of the present invention, the preparation method of the Au NPs is: add hexadecyltrimethylammonium bromide, NaOH and formaldehyde solution into a double-necked flask in a constant temperature water bath at 80°C, mix them under the action of magnetic stirring and add HAuCl4, after the reaction liquid changes from light yellow to wine red, continue heating the reaction for 10 minutes until the reaction is completed, continue stirring until it cools to room temperature, and the prepared gold nanoparticles are transferred to a clean brown bottle and placed in a 4°C refrigerator for standby use.

[0014] As a preferred technical solution of the present invention, the method of the electrochemical collision test is: using the gold ultramicroelectrode as the working electrode, Ag / AgCl as the reference electrode, and the Pt wire electrode as the counter electrode, the random collision signal between Au@Ag NPs and the electrode is collected by using the chronoamperometry, wherein the voltage is 0.6V and the sampling time is 300s, and then the transient current spikes in the chronoamperograph are identified and processed using the Origin software, and the collision frequency is counted.

[0015] On the other hand, the present invention provides the application of the above-mentioned alkaline phosphatase detection method based on single particle collision electrochemistry in the detection of human immunoglobulin concentration.

[0016] As a preferred technical solution of the present invention, the method for detecting the concentration of human immunoglobulin comprises the following steps:

[0017] Add the human immunoglobulin solution to be tested to the ELISA plate, incubate at 4°C for 12 hours, and seal the wells with the standard protein solution at 37°C for 1 hour;

[0018] Adding rabbit anti-human thymocyte immunoglobulin solution to the ELISA plate, incubating at 37° C. for 0.5 hour, then adding ALP-labeled goat anti-rabbit immunoglobulin antibody solution to the ELISA plate, incubating at 37° C. for 0.5 hour;

[0019] Then, the ascorbic acid-2-phosphate trisodium salt solution in the ELISA plate is mixed with the DEA buffer solution to form a mixed solution;

[0020] The mixed solution is mixed with Au NPs and AgNO3 to react to obtain an Au@Ag NPs solution;

[0021] The Au@Ag NPs solution is added with KCl solution as an electrolyte solution;

[0022] An electrochemical collision test was performed, and the random collision signals between Au@Ag NPs and the working electrode were collected using the chronoamperometry method. The collision frequency was then counted, and the concentration of human immunoglobulin was detected by the collision frequency.

[0023] By adopting the above technical solution, the beneficial effects of the present invention are:

[0024] The present invention quantifies alkaline phosphatase by collecting collision frequency information. Under optimized conditions, the detection limit of the nano-collision electrochemical biosensor for alkaline phosphatase activity is 2mU / mL, the linear range is 2mU / mL to 80mU / mL, and it shows a high degree of specificity. At the same time, the nano-collision electrochemical strategy can be applied to immunoassays, with a detection limit of 5ng / mL for human immunoglobulin concentration and excellent selectivity. Compared with the limitations of the prior art, the nano-collision electrochemical biosensor described in the present invention is simple to operate, without the need for cumbersome biolabeling procedures and laborious cleaning and separation steps. Moreover, the technology has the analytical characteristics of zero background, high sensitivity, high specificity and rapid response, which can provide a new strategy for enzyme activity detection and immunoassay, and shows great potential in the analysis and detection of a variety of biological analytes including nucleic acids, proteins, liposomes, cells, bacteria, viruses, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 The schematic diagram of the alkaline phosphatase detection method based on single particle collision electrochemistry of the present invention;

[0027] Figure 2 The feasibility analysis of detecting ALP in Example 1 of the present invention is as follows: (A) current-time curve; (B) UV absorption spectrum;

[0028] Figure 3 The test results of ALP at different concentrations in Example 2 of the present invention are shown as follows: (A) current-time curve recorded at different ALP concentrations; (B) calibration curve of collision frequency and ALP concentration; (C) linear relationship between collision frequency and ALP concentration (2-80 mU / mL);

[0029] Figure 4 This is a diagram of the anti-interference results in Example 3 of the present invention;

[0030] Figure 5The test results of different concentrations of IgG in Example 4 of the present invention are shown in Figures: (A) Current-time curve recorded at different IgG concentrations; (B) Calibration curve of collision frequency and IgG concentration; (C) Linear relationship between collision frequency and IgG concentration (5-100 ng / mL). DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0033] The term "alkaline phosphatase" of the present invention is (ALP), which is an enzyme widely distributed in tissues such as the liver, bones, intestines, kidneys and placenta of the human body and is excreted from the liver to the bile.

[0034] The term "ascorbic acid-2-phosphate trisodium salt" (AA-P) in the present invention is a chemical substance, and its aliases include L-ascorbic acid-2-phosphate trisodium salt and vitamin C phosphate sodium.

[0035] The term "ascorbic acid" in the present invention is (AA), which can convert Ag into + It is reduced to Ag element and deposited on the surface of gold nanoparticles to form gold core-silver shell nanoparticles.

[0036] The term "DEA buffer solution" of the present invention refers to diethanolamine buffer, which is an activated buffer and can serve as an acceptor of acyl phosphate and facilitate enzymatic reactions.

[0037] The term "Au NPs" in the present invention, i.e., gold nanoparticles, refers to gold nanoparticles with a diameter between 1 and 100 nanometers, commonly referred to as nanogold or gold nanoparticles. These nanoparticles have unique physical and chemical properties, including surface plasmon resonance, electrical conductivity, magnetism, thermal properties, optical properties, and biocompatibility.

[0038] The term "Au@Ag NPs" in the present invention refers to silver-coated gold nanoparticles, which have a core-shell structure, wherein the inner core is gold nanoparticles and the outer layer is metallic silver, and the particle size is generally 10 to 100 nm.

[0039] The term "chronoamperometry" in the present invention is an electrochemical method that applies a single potential step or a double potential step to the working electrode and then measures the current response as a function of time. In an electrochemical workstation, the electrochemical reaction process is studied by measuring the change in current over time. Chronoamperometry is widely used in electrochemical research, especially in biosensors and electrochemical analysis. It can help researchers understand the kinetics of electrochemical reactions and measure the concentration and reaction rate of substances.

[0040] The present invention designs a novel nano-collision electrochemical sensing analysis strategy based on the reduction of silver ions on the surface of gold nanoparticles catalyzed by alkaline phosphatase. The core of the method is that alkaline phosphatase catalyzes the hydrolysis of the substrate ascorbic acid-2-phosphate trisodium salt (AA-P) to produce ascorbic acid (AA) with reducing properties, and silver ions are reduced on the surface of gold particles to generate Au@AgNPs nanoparticles and undergo random collision behavior on the surface of the microelectrode. The principle is as follows: Figure 1 shown.

[0041] In order to verify the benefits of the present invention, multiple groups of comparative experiments were used for verification:

[0042] Example 1

[0043] Feasibility analysis of ALP activity detection:

[0044] Prepare 4 groups of electrolyte solutions:

[0045] Group 1: 2 mL Au NPs, 1 mL 0.1 M KCl, and 2 mL deionized water;

[0046] Group 2: 2 mL Au NPs, 50 μL 30 mM AgNO3, 200 μL 2.5 mM ascorbic acid-2-phosphate trisodium salt solution, 0.1 M KCl was added and the volume was adjusted to 5 mL;

[0047] Group 3: 2 mL Au NPs, 50 μL 30 mM AgNO3, 10 μL 50 mU / mL alkaline phosphatase solution, 0.1 M KCl solution was added and the volume was adjusted to 5 mL;

[0048] Group 4: 2 mL Au NPs, 50 μL 30 mM AgNO3, 200 μL 2.5 mM ascorbic acid-2-phosphate trisodium salt solution, 0.1 M KCl was added and the volume was adjusted to 5 mL;

[0049] Then electrochemical tests were performed respectively. The results are as follows Figure 2 As shown, Figure 2Figure A shows that only in the reaction system containing Au NPs, AgNO3, AA-P and ALP, a large number of transient current peaks can be observed. Figure 2 Figure B shows the characteristic absorption peak of silver particles in the UV spectrum, which confirms that the process of alkaline phosphatase catalyzing silver deposition to generate Au@Ag NPs is the core of collision electrochemical analysis. There is no collision current response in the it curve of other control systems, indicating that this nano-collision electrochemical sensing platform can be used for the activity determination of ALP.

[0050] Example 2

[0051] Alkaline phosphatase detection:

[0052] (1) Eleven groups of alkaline phosphatase solutions with activity concentrations of 0 mU / mL, 2 mU / mL, 5 mU / mL, 10 mU / mL, 20 mU / mL, 40 mU / mL, 50 mU / mL, 80 mU / mL, 100 mU / mL, 150 mU / mL and 200 mU / mL were selected in sequence as test liquids with different activities. 10 μL of each solution was mixed with 200 μL of 2.5 mM ascorbic acid-2-phosphate trisodium salt solution in DEA buffer solution (40 mM, pH = 9.8) to form a mixed solution, and the mixture was reacted at 37°C for 30 minutes.

[0053] (2) Then, the mixed solutions after the above reactions were added to 2 mL of Au NPs solution, and then 50 μL of 30 mM AgNO 3 was added to each of them. The mixtures were reacted at 37° C. for 30 minutes to prepare Au@Ag NPs solution.

[0054] (3) Take 2 mL of the Au@Ag NPs solution of each group above, add 1 mL of 0.1M KCl and 2 mL of deionized water, and the total solution volume is 5 mL as the electrolyte solution.

[0055] (4) The electrochemical test was performed using a Shanghai Chenhua electrochemical workstation. The gold ultramicroelectrode was used as the working electrode, Ag / AgCl was used as the reference electrode, and the Pt wire electrode was used as the counter electrode. The random collision signal between Au@Ag NPs and the electrode was collected using a chronoamperometer, where the voltage was 0.6 V and the sampling time was 300 s.

[0056] (5) Use Origin software to identify and process transient current spikes in the chronoamperogram and count the collision frequency.

[0057] By using the linear relationship between collision frequency and alkaline phosphatase activity concentration, the detection performance of electrochemical collision biosensor for alkaline phosphatase was analyzed. Figure 3As shown, it can be seen that the frequency and intensity of collisions are related to the activity concentration of alkaline phosphatase. In the concentration range of 2mU / mL to 80mU / mL, the collision frequency shows a rapid increase trend. When the activity concentration is greater than 80mU / mL, the collision frequency decreases slightly. The collision frequency and ALP activity concentration have a good linear relationship between 2mU / mL and 80mU / mL. The linear equation is F(frequency) = 0.0121 + 0.0035C (ALP concentration, mU / mL), and the coefficient is R 2 =0.9920. The detection limit of ALP activity of this method can reach 2mU / mL, which has excellent sensitivity.

[0058] Example 3

[0059] Anti-interference test:

[0060] The ascorbic acid-2-phosphate trisodium salt solution was mixed with 150 mU / mL PKA, 1 μg / mL LZM, 1 μM / mL TB, 1.0 μg / mL GOx, 1 μg / mL HRP, and 50 mU / mL ALP solutions, respectively, and reacted at 37°C for 30 minutes. Then, the experiments were carried out according to steps (2) to (5) in Example 2. The results are shown in FIG. Figure 4 As shown, it can be seen that except for alkaline phosphatase, the collision frequencies of other interfering proteins are negligible and comparable to that of the blank sample, indicating that the nano-collision electrochemical biosensor has excellent selectivity and specificity.

[0061] Example 4

[0062] Detection of human immunoglobulin (IgG):

[0063] (1) 100 μL of IgG (0.1 M carbonate buffer, pH 9.6) of different concentrations were added to a 96-well ELISA plate. After incubation at 4°C for 12 hours, the plate was washed three times with 1× ELISA washing solution. The concentrations of the above IgG were 0 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL, respectively.

[0064] (2) Add 300 μL of blocking solution (1% BSA) to the ELISA plate used in step (1). After sealing at 37° C. for 1 hour, rinse the microplate three times with 1×ELISA washing solution.

[0065] (3) Add 100 μL of 20 μg / mL rabbit anti-human IgG to the ELISA plate used in step (2), incubate at 37°C for 30 minutes, and then rinse the microplate three times with 1× ELISA washing solution.

[0066] (4) Add 100 μL of 1 μg / mL ALP-labeled goat anti-rabbit IgG to the ELISA plate used in step (3), incubate at 37°C for 30 minutes, and then rinse the microplate three times with 1× ELISA washing solution.

[0067] (5) Add 200 μL of 2.5 mM AA-P (40 mM DEA buffer, pH 9.8) to the ELISA plate used in step (4) and react at 37°C for 30 minutes.

[0068] (6) The reaction solution in step (5) was taken out from the ELISA plate and added to 2 mL of Au NPs solution, and then 50 μL of AgNO3 (30 mM) was added and reacted at 37°C for 30 minutes to prepare Au@Ag NPs solution.

[0069] (7) Take 2 mL of the mixed solution after the reaction in step (6), add 1 mL of 0.1 M KCl and 2 mL of deionized water, and the total solution is 5 mL, as the electrolyte solution.

[0070] (8) The electrochemical test was carried out using Shanghai Chenhua electrochemical workstation. The gold ultramicroelectrode was used as the working electrode, Ag / AgCl was used as the reference electrode, and the Pt wire electrode was used as the counter electrode. The random collision signal between Au@AgNPs and the electrode was collected using a chronoamperometer, where the voltage was 0.6 V and the sampling time was 300 s.

[0071] (9) Use Origin software to identify and process transient current spikes in the chronoamperogram and count the collision frequency.

[0072] The linear relationship between collision frequency and human immunoglobulin concentration was used to analyze the detection performance of electrochemical collision biosensor for human immunoglobulin. Figure 5 As shown in the figure, the collision frequency gradually increases with the increase of HIgG concentration and reaches a stable state. In the concentration range of 5ng / mL to 100ng / mL, there is a linear relationship between the collision frequency and HIgG concentration, and its regression equation is F=0.00295+0.00242C(HIgG concentration, ng / mL), R 2 =0.9924. The quantitative detection limit of the nano-collision electrochemical biosensor for HIgG is 5 ng / mL.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting alkaline phosphatase based on single particle collision electrochemistry, characterized in that: The steps include: The alkaline phosphatase solution to be tested and the ascorbic acid-2-phosphate trisodium salt solution are mixed in a DEA buffer solution to form a mixed solution; The mixed solution is mixed with AuNPs and AgNO3 to react to obtain an Au@AgNPs solution; The Au@AgNPs solution is added with KCl solution as an electrolyte solution; An electrochemical collision test was performed, and the random collision signals between Au@AgNPs and the working electrode were collected using the chronoamperometry method and the collision frequency was counted. The activity of alkaline phosphatase was detected by the collision frequency.

2. The alkaline phosphatase detection method based on single particle collision electrochemistry according to claim 1, characterized in that: The volume ratio of the alkaline phosphatase solution to be tested to the ascorbic acid-2-phosphate trisodium salt solution is 1:19-21, the concentration of the ascorbic acid-2-phosphate trisodium salt solution is 2.5 mM, and the concentration of the DEA buffer solution is 40 mM, with a pH of 9.

8.

3. The alkaline phosphatase detection method based on single particle collision electrochemistry according to claim 1, characterized in that: The preparation method of the Au NPs is as follows: cetyltrimethylammonium bromide, NaOH and formaldehyde solution are added to a double-necked flask in a constant temperature water bath at 80°C, mixed under magnetic stirring and HAuCl4 is added, and after the reaction solution changes from light yellow to wine red, the heating reaction is continued for 10 minutes until the reaction is completed, and stirring is continued until it is cooled to room temperature. The prepared gold nanoparticles are transferred to a clean brown bottle and placed in a 4°C refrigerator for standby use.

4. The alkaline phosphatase detection method based on single particle collision electrochemistry according to claim 1, characterized in that: The electrochemical collision test method is as follows: using the gold ultramicroelectrode as the working electrode, Ag / AgCl as the reference electrode, and the Pt wire electrode as the counter electrode, using the chronoamperometry to collect the random collision signal between Au@AgNPs and the electrode, wherein the voltage is 0.6V and the sampling time is 300s, and then using the Origin software to identify and process the transient current spikes in the chronoamperograph and to count the collision frequency.

5. Use of the alkaline phosphatase detection method based on single particle collision electrochemistry according to any one of claims 1 to 5 in the detection of human immunoglobulin concentration.

6. The use according to claim 5, characterized in that: The method for detecting the concentration of human immunoglobulin comprises the following steps: Add the human immunoglobulin solution to be tested to the ELISA plate, incubate at 4°C for 12 hours, and seal the wells with the standard protein solution at 37°C for 1 hour; Adding rabbit anti-human thymocyte immunoglobulin solution to the ELISA plate, incubating at 37° C. for 0.5 hour, then adding ALP-labeled goat anti-rabbit immunoglobulin antibody solution to the ELISA plate, incubating at 37° C. for 0.5 hour; Then, ascorbic acid-2-phosphate trisodium salt solution is added to the ELISA plate, and mixed and reacted in the DEA buffer solution to form a mixed solution; The mixed solution is mixed with AuNPs and AgNO3 to react to obtain an Au@AgNPs solution; The Au@AgNPs solution is added with KCl solution as an electrolyte solution; An electrochemical collision test was performed, and the random collision signals between Au@AgNPs and the working electrode were collected using the chronoamperometry method and the collision frequency was counted. The concentration of human immunoglobulin was detected by the collision frequency.

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