WO2007124669A1

WO2007124669A1 - Apparatus for electrochemical screening and early diagnosis of malignant tumor

Info

Publication number: WO2007124669A1
Application number: PCT/CN2007/001237
Authority: WO
Inventors: Huangxian Ju
Original assignee: Nanjing University
Priority date: 2006-04-30
Filing date: 2007-04-16
Publication date: 2007-11-08
Also published as: CN1866018A; CN1866018B

Abstract

An apparatus for electrochemical screening and early diagnosis of malignant tumor comprises an immunoassay chip (1), a time resolution multi-channel constant potential device (2), and a data processing and displaying system (3). The immunoassay chip (1) is connected with the time resolution multi-channel constant potential device (2) via an nterface, and the time resolution multi-channel constant potential device (2) is connected with the data processing and display systeming (3) via an interface. The immunoassay chip (1) comprises eight work electrodes fixed respectively with a different antigen molecule functional film, Ag wires (a), an Ag/AgCl reference electrode (b), a carbon counter electrode (c), and an insulating film (d). Some of the enzyme labelled antibodies are connected to the surfaces of the electrodes to acquire catalyzing current and acquire indirectly the quantities of the eight antigens to be measured in the solution, by means of the antigen molecules fixed on the electrode surfaces competing with the antigen molecules for the enzyme labelled antibodies in the warm culture solution. The assay results are processed by the software and displayed in visual image colors with legibility.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medical diagnostic apparatus, and more particularly to an immunoassay for tumor markers and a rapid electrochemical screening and early diagnostic apparatus. 2. Background Art Immunoassay is a highly sensitive and highly selective method for detecting antibodies, antigens or related substances by using a highly specific reaction between antigens/antibodies, which is a clinical immunoassay for serum tumor markers. Provides a powerful means. In general, clinical immunoassay methods for serum tumor markers mostly use the "double antibody sandwich" method, such as radioimmunoassay, enzyme labeling, time-resolved fluorescence, chemiluminescence, and electrochemiluminescence. Due to the radiological hazards of radioimmunoassay and the limitations of reagent life, non-radiolabeled immunoassay methods have evolved. These methods have greatly facilitated the automation of clinical immunoassay projects and the automation, detection and networking of detection tools. However, these methods require the use of special coated plates (tubes). The detection process requires long incubation times, multiple plate washings, and special instrument testing. The detection cycle is long, the operation steps are cumbersome, and the cost is high. Although automatic immunoassay analyzers have emerged, the number of markers that can be detected simultaneously is limited, instruments and reagents are also quite expensive, and it is difficult to promote them. Therefore, applications in early diagnosis, prognosis monitoring, and large-scale screening of malignant tumors are limited. .

In recent years, with the development of gene chips and protein chips, immune chips have attracted people's attention. The company has introduced an instrument that combines chemiluminescence and CCD methods to simultaneously detect 13 tumor markers. However, the instrument is very expensive and has limitations in quantitative detection, which makes it difficult to promote. How to simplify the operation steps, shorten the analysis time, reduce the measurement cost, develop new immunoassay technology, improve the sensitivity and accuracy of detection methods, and develop a high-throughput multi-tumor marker detector to adapt to large-scale screening of malignant tumors. The need for early diagnosis and prognosis monitoring is an urgent problem to be solved.

Electrochemical analysis has many advantages, such as the miniaturization of the test probe, which is not affected by the turbidity and color of the system. The method has high sensitivity, high speed, low cost and low harm. It also has the characteristics of simple detection instrument and easy miniaturization, and the ampere detection signal intensity has a linear relationship with the concentration of the substance to be tested. The linear range is wide and the sensitivity is high, so it has potential for high-throughput multi-tumor marker detection. In 2005, there were reports of electrochemical immunoassay methods for simultaneous determination of two tumor markers. In 2006, these authors further proposed a method for simultaneous immunoelectrochemical analysis of multiple markers. These methods have some practicality, but they cannot be given intuitively. Concentration values also do not provide image results that are generally useful in chip analysis, and do not break through the defects associated with tumor-associated antigens, develop detection instruments, and promote their use.

Screen printing technology has been widely used in the preparation of electrochemical sensors due to its strong ink layer thickness, suitable for various types of inks, flexible printing methods, and no restrictions on substrate size and traits. The cost of the electrode is low, the consumption of samples and reagents is small, and the repeatability is good. The result is accurate and reliable. Under the dry condition of 25 °C, the sensor can obtain good stability. By using this technology and electrochemical analysis method, the present invention proposes a multi-channel electrochemical detection chip capable of simultaneously and rapidly measuring a plurality of tumor markers, and sets thresholds according to the needs of screening and early diagnosis of malignant tumors through experiments and clinical comparisons. Combine the image results with the joint diagnosis, and further develop the electrochemical screening and early diagnosis instrument for malignant tumors. The instrument converts amp detection signals into intuitive image results for use by non-professionals. At the same time, the concentration values of various tumor markers associated with the course of the disease can be visually reported for disease monitoring.

The invention comprises a multi-channel electrochemical detection chip and a multi-channel electrochemical detection instrument, and the report result is intuitive, the detection cost is low, the degree of automation is high, and combined with the combined diagnosis overcomes the defect of tumor-related antigen specificity, and can be used for the early stage of malignant tumor. Discovery, early diagnosis and disease monitoring, to achieve universalization and familyization of testing, in order to improve the utilization efficiency of cancer prevention and control resources, is also very meaningful for the development of cancer prevention and treatment in China. Third, the content of the invention

1. The object of the present invention is to provide a high-throughput immunoassay method, a measuring instrument and a malignant tumor electrochemical sieve capable of simultaneously measuring a plurality of tumor markers based on electrochemical analysis techniques. Check with early diagnostic equipment.

2. Technical solutions

1) Electrochemical screening and early diagnosis of malignant tumors:

The electrochemical screening and early diagnosis instrument for malignant tumors is shown in Figure 1. It consists of an eight-channel immunoassay chip (1), a time-resolved multichannel potentiostat (2), and a data processing and display system (3); The immunoassay chip (1) is connected to the time-resolved multi-channel potentiostat (2) through an interface, and the data processing and display system (3) is also connected to the time-resolved multi-channel potentiometer (2) through the port;

2) Eight-channel immunoassay chip, including functionalized membranes for immobilizing different tumor-associated antigens and eight working electrodes modified by these functionalized membranes (Ml, M2, M3, M4, M5, M6, M7, M8), Ag/ AgCl reference electrode (b) and carbon counter electrode (c), silver wire (a), insulating film (d), as shown in Figure 2. The chip (1) is directly connected to the time-resolved multi-channel potentiostat (2) for measurement. 3) Electrochemical screening of malignant tumors and electrochemical detection of early diagnostic instruments: Based on the competitive reaction mode, a mixture of sample (antigen) and a specific concentration of various enzyme-labeled antibody solutions is used as an incubation solution on the immunochip. The immobilized different antigens and sample antigens compete for the corresponding horseradish peroxidase-labeled enzyme-labeled antibody in the reaction incubation solution, thereby immobilizing horseradish peroxidase on the electrode surface, catalyzing H ₂ 0 ₂ and electron transport medium The reaction of the body, indirectly detecting the content of the antigen to be tested by the magnitude of the catalytic current.

4) This process is achieved by the development of a time-resolved multi-channel potentiostat (the second component in Figure 1). A typical potentiostat can only detect one sample in one operation, and a commercial multi-channel potentiostat includes multiple potentiostatic systems, which is costly, and the number of detected objects is directly dependent on the number of potentiostatic systems. Conducive to improving practicality. The time-resolved multi-channel potentiostat designed by the invention uses a single potentiostat system to change the order of the working electrodes on the detecting chip in a short time by designing a control system, and obtains multiple objects simultaneously (multiple tumors). The information of the content, and the peak shape curve image shows that the peak shape curve of the various tumor marker contents in the detected sample is compared with its corresponding threshold value, and finally the image is displayed as a qualitative and The quantitative results are shown in Figure 3.

The usual potentiostat shows the peak shape result in Fig. 3. By automatic processing, the peak current value can be given, and the working curve can be used to obtain the concentration of the test object.

5) Data processing and display system: According to the needs of malignant tumor screening and early diagnosis, the present invention sets a threshold value through experiments and clinical comparisons, and displays an electrochemical immune amperometric detection signal obtained by the multi-channel electrochemical detection chip. A sample with a peak signal exceeding the threshold of 15% is set to a black book, indicating positive; a peak signal near the threshold of 15% of the sample is set to a semi-black 0, indicating suspicious; a sample with a peak signal below the threshold of 15% is set to white 0, indicating Negative and display qualitative and quantitative results in the form of images (see Figure 3).

6) Further introduce the concept of joint diagnosis into the results, automatically analyze the detection results of a variety of tumor markers, and propose screening results for early diagnosis. At the same time, quantitative results were used for disease monitoring. Proposed malignant tumors Electrochemical screening and early diagnostics overcome the defects associated with tumor-associated antigens, and the detection cost is low, which is convenient for non-professionals.

3. Benefits

1) The present invention combines the immobilized antigen on the immunochip and the sample antigen to compete with the horseradish peroxidase-labeled enzyme-labeled antibody in the reaction incubation solution, thereby allowing the horseradish peroxidase to react to the electrode. The surface, thereby catalyzing the reaction of H ₂ O ₂ with the electron transporting medium, indirectly detecting the content of the antigen to be tested by the magnitude of the catalytic current. The method does not require special instrument detection, has a short detection cycle, simple operation steps, is easy to be automated, and is intelligent, and is convenient for non-professionals. 2) The invention combines chemical modification, micro-machining and screen printing technologies to develop an immunochip which can prepare a batch of multi-tumor markers at the same time and rapidly measure, not only reduces the consumption of samples and reagents to microliters, but also shortens the temperature. Time is spent, sample consumption is reduced, and multiple tumor markers can be measured at one time, reducing the cost of the assay, increasing the speed and efficiency of the assay.

3) The invention proposes the concept of time detection of electrochemical detection, and develops a multi-channel potentiostat with time-resolved multi-channel potentiostat. By sequential detection, the signals of the samples on different detection electrodes on the chip are obtained, and the "simultaneous" is obtained. Information on the content of multiple subjects (multiple tumor markers).

4) Image display of electrochemical results. Through clinical comparison, the corresponding threshold is obtained, and the qualitative and quantitative results are displayed in the form of images using the comparison of the threshold and the signal.

5) The invention introduces the combined diagnosis concept and displays the detection results obtained by the multi-channel electrochemical detection chip, and provides screening results for early diagnosis and breakthrough of tumor-related antigen-specific defects.

The diagnostic instrument is low in cost, miniaturized and intelligent, and promotes the popularization and outdoorization of immunological detection technology and early diagnosis of malignant tumors, and has broad application prospects in large-scale screening and prognosis monitoring. IV. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic diagram of electrochemical screening and early diagnosis of malignant tumors

(1) Eight-channel immunoassay chip, (2) Time-resolved multi-channel potentiostat, (3) - Data processing and display system

Figure 2. Schematic diagram of an eight-channel immunoassay chip

(a) — Ag wire, (b) an Ag/AgCl reference electrode, (c) carbon counter electrode, (d) an insulating film, Ml, M2, M3, M4, M5, M6, M7, M8 respectively Eight working electrodes with different antigenic functionalized membranes.

Figure 3. Schematic diagram of electrochemical screening and early diagnosis of malignant tumors

One positive (warning), 0 - suspicious, 0 negative (excluded). Cl, C2, C3, C4, C5, C6, C7, and C8 represent the measured concentrations of the eight tumor markers, respectively. V. Specific implementation methods

1. Preparation of an eight-channel immunoassay chip

A substrate integrated with an Ag/AgCl reference electrode, a carbon counter electrode and a plurality of (8) carbon working electrodes is printed on a PVC film by screen printing technology, and the electrode is printed by first printing a silver conductive layer on the PVC film. And then printing an Ag/AgCl reference electrode (b) on the reference electrode to form an Ag/AgCl reference electrode (b) at the working electrode The carbon paste is printed on the carbon counter electrode to form 8 carbon working electrodes M1, M2, M3, M4, M5, M6, M7, M8 and carbon counter electrode (c), and finally printed with green oil insulating film (d) . The green oil insulating film (d) protects the silver conductive layer (a) as well as the volume of the reaction.

2. Preparation of functionalized membrane of antigen molecule

The chitosan powder was sonicated in a 1% acetic acid solution to prepare a 1% chitosan solution, and a certain concentration of the standard tumor marker (antigen) solution to be tested was proportional to the 1% chitosan solution. The mixture was uniformly mixed and placed at 4 ° C for 12 hours, and the mixed solution was pipetted onto the surface of the working electrode to obtain eight working electrodes containing functionalized membrane modifications immobilized with different tumor-associated antigen molecules.

3. Determination of tumor markers

3. 1 measurement principle

The malignant tumor electrochemical screening and early diagnosis instrument utilizes amperometric analysis method, combined with competitive immunoassay method, to rapidly and freely detect tumor markers. When only a proper amount of horseradish peroxidase-labeled tumor marker antibody (enzyme-labeled antibody) is present in the incubation solution, the enzyme-labeled antibody immunoreacts with the antigen molecule on the functionalized membrane of the antigen molecule immobilized on the electrode surface after incubation. Connect to the electrode surface. At this time, the immobilized HRP can catalyze the reaction of _{02 in the} solution with the electron transporting medium, so that a catalytic current for catalyzing the oxidation of 0 ₂ can be obtained on the working electrode, and the catalytic current is inversely proportional to the content of the antigen to be tested in the solution. The content of the antigen to be tested in the solution can be obtained indirectly by the response value of the catalytic current.

3. 2 measurement process

(1) Cross-interference reaction experiments. An eight-channel immunoassay chip was used in the electrochemical screening and early diagnosis of this automatic malignant tumor. In order to prevent cross-interference reactions that may occur between the electrodes, a series of immunodetection chips with different distances between the working electrodes are designed to select the minimum electrode spacing that does not cause cross-interference during the detection.

(2) Optimization of immunoassay conditions, including incubation time, incubation temperature, pH, determination of the concentration of ₀₂ in the solution and the concentration of the electron transporting medium, and the amount of the enzyme-labeled antibody in the reaction solution.

(3) Preparing a series of incubation solutions containing different concentrations of the standard solution of the antigen to be tested and a fixed amount of the enzyme-labeled antibody, and determining the reaction of the labeled enzyme catalyzed Η ₂ _{2 2} with the electron transporting medium under the optimal conditions. The catalytic current is obtained to obtain a measurement standard curve of the antigen to be tested.

(4) incubating the antigen to be tested and a fixed amount of the enzyme-labeled antibody under the optimal measurement conditions, and measuring the catalytic current of the labeling enzyme catalyzing the reaction of Η ₂ ₂ with the electron transporting medium, and checking the standard curve of the antigen determination The corresponding concentration is given. (5) Compare the concentration of the tumor marker in the measured actual sample with its corresponding threshold, and design a processing software to set the sample with a peak signal exceeding the threshold of 15% to be red, indicating positive; the peak signal is 15% near the threshold. The sample is set to orange, indicating suspicious; the sample with a peak signal below the threshold of 15% is set to blue, indicating negative. Qualitative and quantitative results are displayed in the form of images.

(6) Compare the catalytic current signal with the threshold signal to display the qualitative detection result in the image color.

(7) The results of the joint diagnosis concept are introduced, and the detection results obtained by the multi-channel electrochemical detection chip are analyzed and processed, and the screening results are presented.

3. 3 specific examples:

Carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), ovarian cancer-associated antigen (CA125), breast cancer-associated antigen (CA153), pancreatic gastrointestinal cancer marker (CA199), cancer marker (CA50), digestive system Cancer markers (CA242) and Yulie adenocarcinoma specific antigen (PSA) are examples of the application of electrochemical screening and early diagnosis of malignant tumors.

1) Preparation of detection chip for electrochemical screening of malignant tumors and early diagnostic equipment

The chitosan powder was ultrasonically dissolved in a 1% acetic acid solution to prepare a 1% chitosan solution. The standard solutions of CEA, AFP, CA125, CA153, CA199, CA50, CA242, and PSA were uniformly mixed with the 1% chitosan solution at a ratio of 1:1 (V:V) and allowed to stand at 4 °C for 12 hours. The above mixed solutions were each applied dropwise to the surface of eight working electrodes and dried at room temperature for 5-6 hours. 4 X Store in a dry condition for use.

2) Determination of CEA, AFP, CA125, CA153, CA199, CA50, CA242. PSA

(1) Optimization of measurement conditions: change the incubation time, incubation temperature, pH value, the concentration of H ₂ 0 ₂ and thiopurine in the solution, and the amount of various enzyme-labeled antibodies in the reaction solution, and select the maximum current response. The corresponding amount is taken as the optimum measurement condition.

(2) Determination of CEA, AFP, CA125, CA153, CA199, CA50, CA242, PSA: Under optimized experimental conditions, a series of standard CEA, AFP, CA125, CA153, CA199\CA50, CA242, PSA antigens and The incubation solution of the fixed amount of the enzyme-labeled antibody is added to the immunoassay chip of the automatic malignant tumor and the immunoassay chip of the early diagnostic apparatus, and the catalytic current of the labeled HRP catalytic oxidation of H ₂ 0 ₂ is determined after the incubation, respectively The standard curves of CEA, AFP, CA125, CA153, CA199, CA50, CA242, and PSA were obtained to determine the concentrations of various antigens of CEA, AFP, CA125, CA153, CA199, CA50, CA242, and PSA in the samples.

(3) Comparing the concentrations of CEA, AFP, CA125, CA153, CA199, CA50, CA242, and PSA antigens in the measured samples with their respective thresholds, and directly converting the detection signals into rich by software processing. The value and screening results are displayed.

(4) Analyze the concentration of different tumor markers and use the combined diagnosis to present the screening results.

Claims

Rights request

1. An electrochemical screening and early diagnosis instrument for malignant tumors, characterized in that it consists of an eight-channel immunoassay chip (1), a time-resolved multi-channel potentiostat (2), and a data processing and display system (3). The eight-channel immunoassay chip (1) is connected to the time-resolved multi-channel potentiostat (2) through an interface, and the time-resolved multi-channel potentiostat (2) is connected to the data processing and display system (3) through an interface; Channel immunoassay chip (1), including eight working electrodes (Ml, M2, M3, M4, M5, M6, M7, 8) and Ag/AgCl reference electrode modified by functionalized membranes respectively immobilizing different tumor-associated antigens b) and carbon counter electrode (c), silver wire

(a), insulating film '(d), the chip (1) is directly inserted into the time-resolved multi-channel potentiostat (2) through the interface, and is measured by the data processing and display system (3) in image color result.

2. The electrochemical screening and early diagnostic apparatus for malignant tumor according to claim 1, characterized in that the time-resolved multi-channel potentiostat uses a single potentiostat system, by designing a control system, in a shorter time The order of the working electrodes on the detection chip is changed internally, and information on the content of a plurality of subjects (multiple tumor markers) is obtained "simultaneously".

3. The electrochemical screening and early diagnostic apparatus for malignant tumor according to claim 1, wherein the eight-channel immunoassay chip (1) prints a silver conductive layer on a PVC film and then at a reference electrode portion. The Ag/AgCl reference electrode (b) is printed on the Ag/AgCl paste, and the carbon paste-shaped 3⁄8 carbon working electrodes M1, M2, M3, M4, M5, M6 are printed on the working electrode and the carbon counter electrode. , M7, M8 and carbon counter electrode (c), and finally printed with green oil insulating film (d).

4. The electrochemical screening and early diagnosis apparatus for malignant tumor according to claim 1, characterized in that the electrochemical color qualitative detection result is displayed by using an image color, and the processing software is designed to compare the peak signal by a threshold value by 15% by comparing with a threshold value. The sample is set to red (Qin), indicating positive; the peak signal is set to orange (0) near the threshold, and the sample is set to orange (0), indicating suspicious; the sample with peak signal below the threshold of 15% is set to blue (0), indicating negative. . And quantitative results can be obtained.

5. The electrochemical screening and early diagnostic apparatus for malignant tumor according to claim 1, wherein the functional membrane-modified working electrode is prepared by ultrasonically dissolving chitosan powder in a 1% acetic acid solution. 1% chitosan solution; mix the standard tumor marker to be tested, ie, the antigen solution, with 1% chitosan solution at 1: KV/V) and place it under 4 12 for 12 hours to absorb 1 μί of the mixed droplet. Applying to the surface of the working electrode, eight working electrodes containing functionalized membrane modifications immobilizing different tumor-associated antigen molecules were obtained.