CN217359914U - Electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device for amplifying DNA signals - Google Patents
Electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device for amplifying DNA signals Download PDFInfo
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- CN217359914U CN217359914U CN202122985010.3U CN202122985010U CN217359914U CN 217359914 U CN217359914 U CN 217359914U CN 202122985010 U CN202122985010 U CN 202122985010U CN 217359914 U CN217359914 U CN 217359914U
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Abstract
The utility model discloses a DNA signal amplified electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device, which comprises a reagent supply unit, a microfluidic channel and a waste liquid collecting device, wherein the reagent supply unit is connected with an inlet of the microfluidic channel, and the waste liquid collecting device is connected with an outlet of the microfluidic channel; the sample introduction process can be controlled by an injection pump and is completely finished on a micro-fluidic chip with micron grade, the repeatability of the whole process is good, and the diagnosis platform is suitable for mass production.
Description
Technical Field
The utility model relates to a protein detects the field, concretely relates to ultrasensitive micro-fluidic immunodetection method and detection device who uses based on big electrochemical detection of two replays of DNA signal.
Background
Point-of-care testing (POCT) is a potentially promising new testing technique in the field of modern medical testing. Compared with the traditional clinical examination, the method has the advantages of rapid detection, simple and convenient operation, low cost, small sample consumption and the like. With the wide application of various biosensing technologies such as screen printing electrodes, immunolabeling, enzyme-linked technology, biochip technology and the like in POCT, compared with the traditional biochemical analyzer, the analysis time is greatly shortened, the detection cost is reduced, and the convenience for monitoring the patient at any time is provided for the patient. At present, main development companies include Roche, Germany Bayer, Yapeh, Japan Kyoto, and the like. The main products with large market capacity are blood sugar test strips and uric acid test strips, but most of the commercially available test strips for detecting blood sugar, uric acid and the like are manufactured by using an enzyme biosensor and a screen printing technology. The enzyme has high cost, complex fixation and poor stability, and the activity of the enzyme is easily influenced by factors such as pH, temperature, humidity, toxic chemicals and the like, so that the commercially available test strip has high cost and short shelf life, and is usually stored in a refrigerator at 4 ℃, particularly the shelf life of the test strip after unsealing is only three months. In addition, such test strips require a patient to perform a needle blood sampling test and cannot achieve continuous dynamic monitoring, which limits the application of the test strips in daily life. Therefore, aiming at the problems and challenges existing in the practical application of the current enzyme electrochemical test strip, a new generation of bionic enzyme electrochemical sensing chip with high performance, low cost and non-needle blood sampling is urgently needed to be developed.
Disclosure of Invention
In view of this, an object of the present invention is to provide an electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device based on double amplification of DNA signals.
In order to achieve the above purpose, the utility model provides a following technical scheme:
the electrochemical ultrasensitive microfluidic enzyme-linked immunosorbent assay detection device based on DNA signal double-playback amplification is characterized in that the detection device generates electrochemical active substances based on a sandwich enzyme-linked immunosorbent assay, nucleic acid rolling circle amplification and an enzyme catalysis substrate and detects markers by detecting electrochemical signal intensity;
the detection device comprises a reagent supply unit, a micro-fluid channel and a waste liquid collecting device, wherein the reagent supply unit is connected with an inlet of the micro-fluid channel, and the waste liquid collecting device is connected with an outlet of the micro-fluid channel;
the reagent supply unit comprises a cleaning solution storage, an object storage to be detected, a primary antibody storage, a rolling circle amplification initiation segment modified gold nanoparticle-secondary antibody compound storage, a rolling circle amplification reaction liquid storage, an electrochemical enzyme modified DNA fragment storage and a substrate storage; the electrochemically enzyme-modified DNA fragments in the electrochemically enzyme-modified DNA fragment reservoir are capable of complementary pairing with the rolling circle amplification priming fragments;
the microfluidic channel is provided with at least one sensing electrode for detecting electrochemical signals, and the sensing electrode is modified with polypeptide for specifically identifying an object to be detected.
The utility model discloses it is preferred, microfluidic channel comprises one or more sampling channel, sampling channel is provided with at least one subchannel, and the sensing electrode who decorates the polypeptide sets up on the subchannel, sets up passageway more than 2 and subchannel formation array structure, and different sensing electrode decoration different polypeptide can detect multiple determinand simultaneously.
The utility model discloses it is preferred, still be connected with micro-fluidic cleaning unit between the export of waste liquid collection device and microfluid passageway, micro-fluidic cleaning unit one end and microfluid passageway exit linkage, one end is connected with waste liquid collection device, still is provided with the pipeline with substrate accumulator intercommunication for the substrate is retrieved and cyclic utilization.
Preferably, the cleaning solution is PBS; the rolling circle amplification reaction solution is prepared from deoxynucleotide triphosphate: bovine serum albumin: phi29 DNA polymerase: 10 × RCA reaction buffer: h 2 The volume ratio of O is 2: 1: 1: 10: 86; the electrochemical enzyme is alkaline phosphatase; the substrate is PBS containing aminophenyl phosphate.
Preferably, the gold nanoparticle-secondary antibody compound modified by the rolling circle amplification priming segment is prepared by the following method: adding gold nanoparticles into a mixed solution of T1 and T2 DNA at a concentration ratio of 100:1, shaking, dropwise adding a NaCl solution at a concentration of 1M to remove unreacted DNA, and dissolving the precipitate in PBS; then adding a CPP solution to form a T1-gold nanoparticle-T2 solution, further synthesizing a T1-gold nanoparticle-T2/antibody solution by EDC-sulfurized NHS cross-linked antibody to obtain a gold nanoparticle-secondary antibody compound modified by a rolling ring amplification initiation segment; the T1 and the T2 are DNA connecting fragments, and the T1 contains a complementary sequence of the DNA fragment modified by the electrochemical enzyme.
The beneficial effects of the utility model reside in that: the utility model discloses an electrochemical ultrasensitive micro-fluidic enzyme-linked immunosorbent assay detection device based on DNA signal double playback and application, firstly, a detection target object is combined with a cyclic amplification initiation fragment by utilizing a sandwich enzyme-linked immunosorbent assay and is fixed in a detection area of a chip substrate; subsequently, triggering a DNA amplification process to generate long single fragments containing the repeat units; finally, the long single fragment is complementary and matched with the protein modified DNA fragment, the catalytic substrate is converted into an electrochemical active substance, and then the electrochemical active substance can be detected by using an electrochemical workstation or a portable electrochemical device with low cost and high sensitivity. Due to the universality of the principle, the platform can also detect other protein or nucleic acid markers by changing corresponding antibodies and aptamers. The whole process does not need to use complex instruments, and is particularly suitable for countries and regions with underdeveloped economic level.
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In order to make the purpose, technical scheme and beneficial effect of the utility model clearer, the utility model provides a following figure explains:
FIG. 1 is a microfluidic immunodiagnostic chip;
FIG. 2 is a schematic diagram of an ultrasensitive microfluidic immunoassay;
FIG. 3 shows the RCA reaction process and its characterization (A: schematic diagram of RCA process; B: gel electrophoresis of RCA process; C: three-dimensional atomic force microscopy of RCA product; D: height of RCA product at 5).
FIG. 4 shows the synthesis process and characterization of DNA and antibody modified gold nanoparticles (A: synthesis process diagram; B: ultraviolet characterization; C: electron microscope result).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and can implement the present invention, but the embodiments are not to be construed as limiting the present invention.
Example 1 ultrasensitive microfluidic immunoassay chip based on DNA signal double amplification and electrochemical detection
The structure of the ultrasensitive microfluidic immunodiagnosis chip based on double amplification of DNA signals and electrochemical detection is shown in figure 1, and the ultrasensitive microfluidic immunodiagnosis chip comprises a reagent supply unit 1, a microfluidic channel and a waste liquid collecting device 3, wherein the reagent supply unit is connected with an inlet of the microfluidic channel, and the waste liquid collecting device is connected with an outlet of the microfluidic channel;
the microfluidic channel is provided with at least one sensing electrode 4 for detecting electrochemical signals, and the sensing electrode can modify polypeptide, such as antibody, for specifically recognizing the substance to be detected; preferably, the microfluidic channel consists of one or more sample feeding channels, the sample feeding channel is provided with at least one sub-channel, the sensing electrode capable of modifying the polypeptide is arranged on the sub-channel, and an array structure is formed by more than two sample feeding channels and more than 2 sub-channels.
The reagent supply unit comprises a cleaning solution storage 5, an object storage 7 to be detected, a primary anti-storage 6, a Rolling Circle Amplification (RCA) initiation fragment modified gold nanoparticle/secondary antibody complex storage 8, a rolling circle amplification reaction liquid storage 10, an electrochemical enzyme modified DNA fragment storage (the DNA fragment is complementary with the rolling circle amplification initiation fragment) 11 and a substrate storage 9;
and a micro-fluidic cleaning unit is connected between the waste liquid collecting device and the outlet of the micro-fluidic channel, one end of the micro-fluidic cleaning unit is connected with the outlet of the micro-fluidic channel, the other end of the micro-fluidic cleaning unit is connected with the waste liquid collecting device, a pipeline communicated with the substrate storage device is further arranged for recovering and recycling the substrate, and an oxidation reaction product PQI in the reaction liquid is reduced under the action of the electrode to generate an electroactive product PAP which is then conveyed back to the substrate storage device, so that the detection sensitivity is improved, and the using amount of a sample is reduced.
The reagent supply unit of the embodiment can be controlled by an injection pump and is completely finished on a micro-fluidic chip, the repeatability of the whole process is good, the micro-fluidic chip is suitable for mass production, and the diagnosis platform can be further manufactured into an ultra-sensitive micro-fluidic portable diagnosis device based on DNA signal double amplification and a portable electrochemical device. In addition, by changing specific antibodies or aptamers, the platform can be applied to the detection of other protein or nucleic acid markers, and clinical diagnosis application of point-of-care detection in families or urban areas is realized.
Example 2 method of ultrasensitive microfluidic immunoassay based on DNA signal dual amplification and electrochemical detection
The principle of the ultrasensitive microfluidic immunoassay method based on DNA signal double amplification and electrochemical detection is shown in figure 2, and the ultrasensitive microfluidic immunoassay method comprises the following steps:
(1) the sandwich enzyme-linked immunization method comprises the following steps: communicating an anti-reservoir with a primary antibody to modify an antibody (primary antibody) specifically recognizing an object to be detected in a microfluidic channel, then introducing the object to be detected into the microfluidic channel to capture the object to be detected by the antibody, then adding a cleaning solution PBS, introducing a gold nanoparticle/secondary antibody compound (T1/lock-hanging DNA-gold nanoparticle-T2/antibody) subjected to rolling circle amplification initiation fragment modification, staying for 30 minutes to complete the fixation of T1/lock-hanging DNA-gold nanoparticle-T2/antibody, and then introducing a cleaning solution PBS;
(2) nucleic acid rolling circle amplification: introducing RCA reaction solution into a microfluidic channel on which T1/locking DNA-gold nanoparticle-T2/antibody is fixed, staying for 30 minutes, amplifying by using DNA polymerase in the RCA reaction solution, and then flowing into a cleaning solution PBS;
(3) electrochemical active material generation and detection: introducing a DNA fragment which is complementary with the locked DNA and modified by electrochemical enzyme into the micro-fluid channel after the RCA reaction, fully reacting to make the locked DNA complementary with the DNA fragment, introducing a catalytic substrate to make the electrochemical enzyme convert the substrate into an electroactive substance, and detecting the reaction solution through an electrochemical workstation after the reaction solution flows out.
In this example, the synthesis steps of T1/lock DNA-gold nanoparticle-T2/antibody are as follows: 15nm gold nanoparticles were added to a mixed solution of T1 and T2 (concentration ratio 100:1), shaken for 20h, then a volume of NaCl (C ═ 1M) solution was added dropwise, centrifuged (12000rpm) for 15 minutes to remove unreacted DNA, and the precipitate was redissolved in PBS. And then adding a CPP solution with a certain concentration to form a T1/locked DNA-gold nanoparticle-T2 solution, and further synthesizing a T1/locked DNA-gold nanoparticle-T2/antibody solution by EDC-sulfurized NHS cross-linked antibody.
Wherein the nucleotide sequence of T1 is: 5'-tttttttttttttttaaggcgaaga caggtgttagtcga-3', respectively;
the nucleotide sequence of T2 is: 5'-tttttttttttttttttttt-3' are provided.
The volume ratio of the RCA reaction solution includes, but is not limited to, deoxynucleotide triphosphate, bovine serum albumin, phi29 DNA polymerase, 10 × RCA reaction buffer solution, H 2 O＝2:1:1:10:86。
The enzyme in the DNA fragment modified by the electrochemical enzyme is alkaline phosphatase, and the sequence of the DNA fragment is as follows: 5'-gtttcctttccttgaaacttcctttcga-3' are provided.
The substrate is aminophenyl phosphate, in the detection, alkaline phosphatase (ALP) p-aminophenyl phosphate (PAPP) is subjected to enzymatic conversion to generate p-aminophenol (PAP), PAP is an electrochemical active substance, and when voltage is applied to the working electrode by taking the reference electrode as a reference, PAP is oxidized into p-benzoquinone imine (PQI) to generate electrons, so that electrochemical detection is realized.
Example 3 ultrasensitive microfluidic immunoassay for human epidermal growth factor 2 based on DNA signal dual amplification and electrochemical detection
HER2 is a transmembrane tyrosine kinase receptor associated with breast cancer and indicates that a patient is at potential risk for breast cancer when HER2 levels in the blood exceed 15.0 ng/mL. In this example, a platform for ultrasensitive microfluidic immunoassay of human epidermal growth factor 2(HER2) based on DNA signal dual amplification and electrochemical detection is constructed, comprising the following steps:
the microfluidic immunodetection chip shown in figure 1 is used, HER2 specific recognition polypeptide is modified on ultrathin glass of a chip substrate, a HER2 sample, PBS, T1/locking DNA-gold nanoparticle-T2/antibody solution, PBS, RCA reaction solution, PBS, T3-enzyme complex, PBS and substrate solution are sequentially injected into a sample pool of the chip, and then detection is carried out by using an electrochemical workstation.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutes or changes made by the technical personnel in the technical field on the basis of the utility model are all within the protection scope of the utility model. The protection scope of the present invention is subject to the claims.
Claims (3)
- The electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device for amplifying DNA signals is characterized in that: the detection device generates an electrochemical active substance based on a sandwich enzyme-linked immunosorbent assay, nucleic acid rolling circle amplification signal amplification and an enzyme catalysis substrate, and detects a doubly amplified electrochemical signal to obtain the concentration of the protein to be detected;the detection device comprises a reagent supply unit, a micro-fluid channel and a waste liquid collection device, wherein the reagent supply unit is connected with an inlet of the micro-fluid channel, and the waste liquid collection device is connected with an outlet of the micro-fluid channel;the reagent supply unit comprises a cleaning solution storage, an object storage to be detected, a primary antibody storage, a rolling circle amplification initiation segment modified gold nanoparticle/secondary antibody compound storage, a rolling circle amplification reaction liquid storage, an electrochemical enzyme modified DNA fragment storage and a substrate storage;the electrochemically enzyme-modified DNA fragments in the electrochemically enzyme-modified DNA fragment reservoir are capable of reacting with rolling circle amplification priming fragment pairs;the microfluidic channel is provided with at least one sensing electrode for detecting electrochemical signals, and the sensing electrode can modify polypeptide for specifically identifying an object to be detected.
- 2. The electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device for DNA signal amplification according to claim 1, wherein: the microfluidic channel consists of one or more sample feeding channels, the sample feeding channel is provided with at least one sub-channel, and the sensing electrode for modifying the polypeptide is arranged on the sub-channel.
- 3. The electrochemical ultrasensitive microfluidic enzyme-linked immunoassay device for DNA signal amplification according to claim 1, wherein: and a microfluidic cleaning unit is also connected between the waste liquid collecting device and the outlet of the microfluidic channel, one end of the microfluidic cleaning unit is connected with the outlet of the microfluidic channel, the other end of the microfluidic cleaning unit is connected with the waste liquid collecting device, and a pipeline communicated with the substrate storage is further arranged and used for recovering and recycling the substrate.
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| CN115856284A (en) * | 2022-12-15 | 2023-03-28 | 西交利物浦大学 | Chromatographic test strip with bionic flow channel and application thereof |
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| CN115856284A (en) * | 2022-12-15 | 2023-03-28 | 西交利物浦大学 | Chromatographic test strip with bionic flow channel and application thereof |
| CN115856284B (en) * | 2022-12-15 | 2023-11-28 | 西交利物浦大学 | Chromatographic test strip with bionic flow channel and application thereof |
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