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Single molecule dynamic detection method and system based on external force regulation and control

A dynamic detection, single-molecule technology, applied in the field of processing and analysis, biomedical detection, can solve the problems of high cost, low throughput, low detection throughput, etc., to achieve low use threshold, low device cost, high throughput Effect

Pending Publication Date: 2022-06-07
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The disadvantages of existing single-molecule detection technologies are high cost, low throughput, time-consuming and not universal
SiMoA and SMC technologies rely on detection chips and laser light sources focused into Airy disks, which are expensive and unfavorable for clinical IVD applications
Secondly, SiMoA, SMC, and SiMREPS all have the problem of time-consuming detection. Although the detection efficiency of SiMREPS can be improved by selecting an appropriate affinity antibody, the complex antibody screening process makes this method not universal.
Finally, all current single-molecule detection technologies have the problem of low detection throughput, which limits their promotion in clinical practice

Method used

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  • Single molecule dynamic detection method and system based on external force regulation and control
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  • Single molecule dynamic detection method and system based on external force regulation and control

Examples

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Effect test

Embodiment 1

[0096] Surface plasmon resonance microscope SPRM was used for dynamic analysis and detection, and electrochemical workstation was used to provide electric field force for the detection system. The external force regulation dynamic analysis nucleic acid detection method of the target nucleic acid whose base complementary chain length of the target nucleic acid and the detection probe is 25base pair is as follows:

[0097] (1) Use a commercial total internal reflection microscope (Olympus IX-81) as the detection instrument, choose an objective lens with a magnification of 60 times and a numerical aperture of N.A = 1.49, the light source is an ultra-broadband light source SLED, and the intensity of the light source control current is 140mA. The field of view is 512×512pixels (full field of view: 33.28×33.28μm 2 ). The imaging signal of SPRM was recorded by using a micromanager to control a CCD camera (Photometrics). The Cellsens software is used to control the incident angle of...

Embodiment 2

[0103] In Example 1, a high-resolution surface plasmon resonance microscope (SPRM) was used as a single-molecule imaging platform to perform dynamic detection of single-molecule based on external force regulation, which has extremely high sensitivity to particle size, and can see and distinguish particles with a particle size of 30-160 nm. However, in the single-molecule dynamic detection based on the sandwich structure of the present invention, it is not necessary to use particles with very small particle diameters. Therefore, SPRM with a small field of view cannot well meet practical applications. Therefore, the inventors further optimized the detection system, using the surface plasmon resonance imaging platform SPRi with a large field of view for dynamic analysis and detection, combined with an electrochemical workstation, and used the electrochemical workstation to provide electric field force for the detection system. The target molecule (in this embodiment, the target mo...

Embodiment 3

[0112] The micronucleic acid miRNA has broad application prospects as a biomarker for early tumor screening. In this example, hsa-miR-29a (miR-29a) was selected as the target nucleic acid, and the base complementary chain length of the detection probe was 12bp. Its external force regulation dynamic analysis nucleic acid rapid detection method is as follows:

[0113] (1) The single-molecule dynamic detection platform under the control of external force was reconstructed from the surface plasmon resonance imaging SPRi based on the Kretschmann prism coupling structure on the optical platform as the detection instrument. The optical devices used in the optical path were purchased from Thorlabs and Daheng Optoelectronics , the air-floating optical platform is from Lubang Technology (LBTEK). A lens with a magnification of 60 times and a numerical aperture of N.A=1.49 is used. The light source is an ultra-broadband light source SLED (Qphotonics) from Superlum. The intensity of the li...

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Abstract

The invention discloses a single molecule dynamic detection method based on external force regulation and control. According to the single-molecule dynamic detection method, an external force regulation and control module is used for regulating combination and dissociation between a target molecule and a detection probe, a capture probe and nanoparticles, and a single-molecule imaging system is used for obtaining single-molecule imaging information and analyzing the dynamic state of the target molecule. The invention also discloses a system for detecting the dynamic state of the single molecule and application of the system. According to the invention, rapid, ultra-sensitive and high-throughput single-molecule immunodetection can be realized, and the detection limit can reach fM level or even lower; and the problem that the probe does not have universality is solved, the device cost is lower, the use threshold is lower, and the flux is larger.

Description

technical field [0001] The invention belongs to the field of biomedical detection, processing and analysis, and relates to a single-molecule dynamic detection method and system based on external force regulation, which is used for the detection of low-abundance proteins, nucleic acids and biological small molecules. Background technique [0002] In vitro diagnosis (IVD) plays an important role in the early diagnosis, prognostic detection and treatment evaluation of many diseases and cancers. At present, more than 80% of clinical disease diagnosis relies on in vitro diagnostic technology. The limit of detection (LoD) of traditional IVD techniques such as enzyme-linked immunosorbent assay (ELISA) is at the pM level (10 -12 M), which greatly limits the application field of IVD technology. According to reports, there are still a large number of low-abundance proteins, nucleic acids and small biological molecules in the human body that cannot be detected by traditional IVD tech...

Claims

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Application Information

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IPC IPC(8): G01N33/543C12Q1/6837G01N27/30
CPCG01N33/54346G01N33/54326C12Q1/6837G01N27/30C12Q2563/155C12Q2565/601C12Q2565/628C12Q2565/519C12Q2563/131C12Q2523/303
Inventor 余辉杨玉婷曾强
Owner SHANGHAI JIAO TONG UNIV
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