Cancer marker detection method combining DNA coding technology with nanopore technology
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A DNA molecule and antibody detection technology, applied in recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as inapplicability of antigens and coding errors
Active Publication Date: 2018-09-14
INST OF CHEM CHINESE ACAD OF SCI +1
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Because the pore size of glass pores is about 14nm, it is only suitable for detecting relatively large proteins such as antibodies, but not suitable for antigens
In addition, the distinction of these DNA-encoded molecules depends
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Embodiment 1
[0140] Embodiment 1, the synthesis of five kinds of probe molecules
[0141] 1. Artificially synthesize the five single-stranded DNA molecules listed in Table 1.
[0142] Table 1 Five coding DNA sequences and modification methods
[0143]
[0144] 2. Perform the following operations on the five single-stranded DNA molecules synthesized in step 1:
[0145](1) Take 3.3 μL DNA molecule solution (solvent is water, the concentration of single-stranded DNA molecule prepared in step 1 is 100 μM), 1.2 μL deionized water, 2 μL azide ferrocene solution (solvent is acetonitrile, azide The concentration of iron is 200mM), 1 μL sodium ascorbate solution (the solvent is water, the concentration of sodium ascorbate is 20mM), 0.5 μL copper nitrate solution (the solvent is water, the concentration of copper nitrate is 20mM), 2 μL HEPES buffer solution (200mM) mixed , a total of 10 μL of the mixed solution.
[0146] (2) The mixed solution obtained in step (1) was vortex reacted at room te...
Embodiment 2
[0160] Embodiment 2, establishment of detection method
[0161] The principle of simultaneous detection of five antigens by using the above five probes is shown in figure 2 . The antigen to be tested can be any antigen molecule.
[0162] The following experiments use five cancer markers PSA (prostate specific antigen), AFP (alpha-fetoprotein), CEA (carcinoembryonic antigen), NSE (neuron-specific enolase) and CA19-9 (cancer antigen CA19-9 ) as an example.
[0163] 1. Detection of antibody-modified gold nanoparticles
[0164] 1. Take 1ml colloidal gold solution (particle size 30nm, concentration 2×10 11 particles / mL), with 0.1M NaHCO 3 Adjust the pH of the colloidal gold solution to 9.2 with the aqueous solution, add a detection antibody corresponding to the antigen to be tested into the solution, and place it at 10°C and 90 r / min for 30 minutes to obtain a colloidal gold solution modified with the detection antibody.
[0165] The concentrations of different antibodies in...
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Abstract
The invention discloses a cancer marker detection method combining DNA coding technology and nanopore technology. The cancer marker detection method comprises the following steps: firstly, synthesizing a DNA coding molecule, and modifying the group and the length of DNA by virtue of chemical modification changing the DNA base, so as to generate a characteristic current signal with higher discrimination when the DNA coding molecule passes through nanopores; in combination with immunoassay technology, adding cancer markers to form a sandwich structure with magnetic beads and nano gold; and thenreleasing the encoded DNA on the surface of nanogold, so as to detect the cancer markers separately and simultaneously. A high-throughput multi-component detection platform based on nanopore single molecule technology outputs corresponding characteristic current signals with different DNA coding molecules, so as to eliminate background interference as well as signal interference between the DNA coding molecules, and overcome the defects of fluorescent bleaching, short fluorescence lifetime, background interference, fluorescence interference between encoded microspheres and the like of fluorescence encoding of a traditional method.
Description
technical field [0001] The invention relates to a method for detecting cancer markers by combining DNA encoding technology and nanopore technology. Background technique [0002] In recent years, the rapid and sensitive detection of tumor markers is of great significance in the early clinical diagnosis of tumors. The real-time detection of tumor markers can provide assistance for clinical tumor occurrence, monitoring, response to drugs in treatment, and prediction of tumor recurrence probability. However, the detection of a single marker is not enough to determine the occurrence of a certain cancer. The occurrence of cancer is often accompanied by the increase of the content of multiple cancer markers. The simultaneous detection of cancer markers can improve the efficiency of clinical cancer diagnosis. Therefore, it is becoming more and more urgent to develop high-throughput nucleic acid and protein detection technologies. High-throughput detection technology refers to the ...
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