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Homogeneous immunoassay method and equipment based on proximity strike effect and graphene oxide quenching acridinium ester chemiluminescence

A technique for chemical and immunological analysis of cyanidinium esters, which is applied in the fields of chemiluminescence/bioluminescence, analysis of materials through chemical reactions, and analysis of materials. There are problems such as stability problems, to achieve the effect of shortened turnaround time, lower requirements, and fewer modules

Active Publication Date: 2021-05-11
南京浦光生物科技有限公司
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Problems solved by technology

Wu Jie of Nanjing University et al proposed a no-clean homogeneous analysis technique for the detection of CEA carcinoembryonic antigen by using the proximity reaction to regulate chemiluminescence resonance energy transfer, in which graphene oxide was used as an energy switch for TCPO-H2O2-Cy5 adjustment, however this switch adjustment cannot be 100% effective
In addition, the above article also mentioned the use of a restriction endonuclease loop amplification system. Our research found that the signal amplification function of this system is limited, and due to the presence of enzymes, not only the cost is increased, but also the stability is problematic. It is speculated that the reason may be Because the TCPO-H2O2-Cy5 luminescent system needs to cut off luciferin Cy5 through enzyme digestion to be able to emit light effectively

Method used

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  • Homogeneous immunoassay method and equipment based on proximity strike effect and graphene oxide quenching acridinium ester chemiluminescence
  • Homogeneous immunoassay method and equipment based on proximity strike effect and graphene oxide quenching acridinium ester chemiluminescence
  • Homogeneous immunoassay method and equipment based on proximity strike effect and graphene oxide quenching acridinium ester chemiluminescence

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Experimental program
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Embodiment 1

[0073] Embodiment 1: in combination with figure 1 , illustrating the detection of troponin I in whole blood by a homogeneous immunoassay based on graphene oxide-antioxidant quenched acridinium ester chemiluminescence

[0074] 1. Configure detection reagents: mix DNA1-antibody 1 conjugate, DNA2-antibody 2 conjugate, AE-modified DNA3, GO-AOD (AOD is vitamin C), so that their final concentrations are 1-20nM, 1-20 nM, 0.05-0.2 μM and 20 μg / ml (optimal conditions are 10 nM, 10 nM, 0.15 μM and 20 μg / ml).

[0075] 2. Mix 50 μL of calibration solutions of different concentrations or whole blood samples containing troponin I with 200 μL of detection solution, place them in the detection well of the rotor, start the incubation, incubate at 37°C for 5-10 minutes, and the incubation is complete. The detection hole is turned to the detection position, facing the light source module.

[0076] 3. Through the control of the circuit board module and the host computer software, start the step...

Embodiment 2

[0083] Embodiment 2: in combination with figure 2 , illustrating the detection of hepatitis B surface antibody (HBsAb) in whole blood by a homogeneous immunoassay based on graphene oxide-antioxidant quenched acridinium ester chemiluminescence

[0084] 1. Configure detection reagents: mix DNA1-scaffold protein 1 conjugate (ZJ-Ag1), DNA2-antibody protein 2 conjugate (ZJ-Ag2), AE-modified DNA3, GO-AOD (AOD is vitamin E) mixed , so that their final concentrations were 1-20nM, 1-20nM, 0.05-0.2μM and 20μg / ml (the optimal conditions were 10nM, 10nM, 0.15μM and 20μg / ml). Scaffolds are project-independent immunoglobulins IgG.

[0085] 2. Mix 50 μL of calibration solutions of different concentrations or whole blood samples containing HBsAb with 200 μL of detection solution, place them in the detection well of the rotor, start incubation, and incubate at 37°C for 5 minutes. After the incubation is completed, the detection well is turned to detection The position is directly opposite t...

Embodiment 3

[0091] Embodiment 3: in combination with image 3 , illustrating the detection of free thyroxine (fT4) in whole blood by a homogeneous immunoassay based on graphene oxide-antioxidant quenched acridinium ester chemiluminescence

[0092] 1. Configure the detection reagent: mix DNA1-antibody 1 conjugate, DNA2-scaffold small molecule conjugate (ZJ-XFZ), AE-modified DNA3, GO-AOD (AOD is cannabidiol) to make their final The concentrations are 1-5nM, 5-20nM, 0.05-0.2μM and 20μg / ml (optimum conditions are 1nM, 10nM, 0.1μM and 20μg / ml). Scaffolds are project-independent immunoglobulins IgG.

[0093] 2. Mix 50 μL of calibration solutions of different concentrations or whole blood samples containing fT4 with 200 μL of detection solution, place them in the detection well of the rotor, start incubation, and incubate at 37°C for 5 minutes. After the incubation is completed, the detection well is turned to detection The position is directly opposite to the light source module.

[0094] 3....

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Abstract

The present invention relates to the technical field of chemiluminescence immunoassay, in particular to a homogeneous immunoassay method and equipment based on the proximity shock effect and graphene oxide quenching acridinium ester chemiluminescence; the detection method used in the analysis method The solution includes DNA1-conjugates, DNA2-conjugates, DNA3 labeled with acridinium ester (AE) and graphene oxide (GO) combined with antioxidants (AOD), etc.; this method is a homogeneous chemiluminescent immunoassay method without The separation and cleaning steps are easy to operate, and at the same time, the turnover time (TAT) of clinical test specimens is greatly shortened. Blood samples do not need centrifugation, and whole blood samples can be loaded, and the test report can be produced within 5‑10 minutes; acridinium ester derivatives ( AE) Different from fluorescein (Cy5, etc.) substances, it can realize anti-interference chemiluminescence detection; in addition to the detection of macromolecular proteins and antibodies, this method can also realize the immunological detection of chemical small molecules.

Description

technical field [0001] The invention relates to the technical field of chemiluminescence immunoassay, in particular to a homogeneous immunoassay method and equipment based on the proximity impact effect and graphene oxide quenching acridinium ester chemiluminescence. Background technique [0002] Chemiluminescence immunoassay (CLIA) is an analytical technique for various antigens, haptens, small molecules, antibodies and drugs, which combines high-sensitivity chemiluminescence assay technology with high-specificity immune reactions. . According to whether there is a separation and cleaning step, it can be divided into heterogeneous chemiluminescence method and homogeneous chemiluminescence method. At present, in the field of in vitro diagnostic testing, domestic and foreign testing products basically use heterogeneous chemiluminescence. Foreign manufacturers include Roche, Abbott, Beckman, Siemens, Sorin, and Hismicom, and domestic manufacturers include New Industry, Antu,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N21/76
CPCG01N21/76G01N33/5302
Inventor 曹丹
Owner 南京浦光生物科技有限公司
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