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A single-molecule protein detection chip and method based on ultramicroelectrode array electrochemiluminescence

A protein detection and single-molecule technology, which is applied in the direction of material analysis, measuring devices, and scientific instruments through electromagnetic means, can solve the problems of unstable reaction process, complex device preparation, and high difficulty of preparation, so as to improve the capture rate and uniformity , fast and sensitive response to signals, and the effect of improving detection sensitivity

Active Publication Date: 2022-05-10
SHENZHEN INST OF ADVANCED TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Among the single-molecule protein detection technologies that have been disclosed so far, there is no ultra-microelectrode array electrochemiluminescence-related technology. The main problems are: complex device preparation, unstable reaction process, and inconvenient effective control, which makes its detection consistency and poor repeatability
[0009] Among the currently published electrochemiluminescence immunoassay techniques, they mainly focus on the preparation and surface modification of three-electrode systems or bipolar electrodes, and the chip cannot detect trace proteins. The main reasons are: 1) the number of electrode arrays and The density is usually not high, usually less than 1,000, which is far less than the requirements of digital trace protein detection (the number of electrodes must be at least greater than 40,000)
2) The preparation of high-density electrodes cannot be achieved, and the preparation is difficult; 3) If the size of the microwell electrode is too large, the intensity of the electrochemiluminescence on the microbeads is very weak, so it cannot meet the requirements for collecting the electrochemiluminescence signals on the microbeads

Method used

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  • A single-molecule protein detection chip and method based on ultramicroelectrode array electrochemiluminescence
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  • A single-molecule protein detection chip and method based on ultramicroelectrode array electrochemiluminescence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Preparation of immune complex-modified microbeads and ruthenium-labeled functionalization

[0086] In order to ensure that each microbead binds at most one single-molecule protein and increase the intensity of electrochemiluminescence, an immune complex modified microbead and ruthenium-labeled functionalization method were designed. It mainly includes carboxylated microbeads activated by carbodiimide-N-hydroxysuccinimide (EDC-NHS) coupled with primary antibody, bovine serum albumin (BSA) blocks the immune active site, captures the target protein in the sample and Combined with ruthenium-labeled secondary antibody to form immune complex modified microbeads:

[0087] Specifically:

[0088] 1) 2.8 μm magnetic microbeads modified with carboxyl groups (10mL solution contains 6×10 8 ~7×10 8 microbeads) as the carrier to carry out sufficient carboxylation on the outside of the chip;

[0089] Prepare N-hydroxysuccinimide (NHS) with phosphate buffer (0.01M, pH 7.4)...

Embodiment 2

[0093] Example 2 Design and preparation of a single-molecule protein detection chip

[0094] Design the specific structure of the single-molecule protein detection chip (see image 3 ): The basic microwell structural unit used to accommodate 2.8 μm microbeads is a semi-elliptical inclined groove microwell. axis, the inclination angle of the chute, etc., to construct the standard elliptic equation; the semi-major axis of the semi-ellipse is a, the semi-minor axis is b, and the inclination angle of the chute is α. According to the calculation, its size requirements need to meet the standard Ellipse equation:

[0095]

[0096] x and y are the coordinates of any point on the semi-ellipse, and the length of the semi-major axis of the semi-ellipse is a, then we can know a according to the formula 2 =4R 2 / sin 2 α, the length of the semi-minor axis is b, then it can be known that b is calculated according to the formula 2 =4R 2 The tilt angle of the / 3 tilted microwell is α,...

Embodiment 3

[0099] Embodiment 3 Research on the Specific Dimensions of the Microwell Array Unit

[0100] Firstly, it is determined that the SU-8 inclined microwell array prepared on the ITO electrode is 4000 rows × 20 columns, and the potential difference at both ends of a single microwell depends on the ratio of the length of the microwell to the distance between the two copper wires. When the potential difference is large enough, an oxidation or reduction reaction occurs on the electrode surfaces at both ends of the microwell, causing electrochemiluminescence. The highest potential is obtained when the drop point of the microbead is close to the semi-short axis. Therefore, the specific dimensions of the designed microwell array unit are 10 μm in the semi-major axis and 1.6 μm in the semi-minor axis. When an external voltage is applied across the ITO electrodes and a solution exists in the microwell array, a potential gradient is generated due to resistance. Because the resistance of t...

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Abstract

The invention relates to a single-molecule protein detection chip and method based on electrochemiluminescence of an ultra-micro electrode array, and specifically discloses that the single-molecule protein detection chip has a substrate with an ITO electrode and a slanted microstructure with a three-dimensional concave on the ITO electrode. Well array; two ends in the lateral direction of the inclined micro well array are respectively provided with wires connected to the positive and negative electrodes of the power supply. The single-molecule protein detection chip of the invention is simple to prepare, the electrochemiluminescence detection system further amplifies the signal, and can realize the highly sensitive single-molecule detection of trace proteins.

Description

technical field [0001] The present invention mainly relates to the fields of electroanalytical chemistry and microfluidics, and proposes a method for arraying microbeads on a microchip and an electrochemiluminescent method for matching ultramicroelectrode arrays, and develops a set of integrated bionic microfluidics accordingly. chip for single-molecule protein detection. The microfluidic chip obtains a fast and sensitive response signal by designing an ultra-microelectrode array, and secondly uses electrochemiluminescence to improve detection performance. It has the advantages of high detection sensitivity, short detection time, and low cost, and is especially suitable for single-molecule protein detection. potential economic value. Background technique [0002] With the continuous improvement of protein detection sensitivity requirements in the modern medical field, the development of advanced protein detection technology is the only way to improve the diagnostic ability ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/416G01N33/68G01N33/552G01N33/543G01N33/532
Inventor 吴天准惠允
Owner SHENZHEN INST OF ADVANCED TECH
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