Three-dimensional DNA microsphere with fluorescence signal amplification function as well as preparation method and application of three-dimensional DNA microsphere
A microsphere, three-dimensional technology, applied in the field of analysis and detection, can solve the problems of unfavorable culture time for the detection and treatment of drug-resistant bacteria and related diseases, and achieve the effect of rapid response, high specificity and rapid detection
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Embodiment 1
[0055] Example 1 Synthesis of Ab-DNF that recognizes β-lactamase by means of closure and the technical route of single-molecule detection method
[0056] a. The preparation and characterization of Ab-DNF that specifically recognizes β-lactamase;
[0057] b. Construction of single-molecule detection method, testing the binding ability and selectivity of Ab-DNF and β-lactamase, analyzing and optimizing the key influencing factors of detection. After blocking with blocking buffer, add the target protein sample (β-lactamase standard protein or bacterial lysate), Ab-DNF and 1×SYBR-Gold fluorescent dye to the 384-well plate pre-coated with the capture antibody, and pass through the antigen-antibody The formation of a sandwich structure was identified, and Ab-DNF produced uniform fluorescent spots, which were observed and imaged under an inverted fluorescence microscope, and analyzed and counted using ImageJ software. Analyze and optimize the assay performance of the single molecule...
Embodiment 2
[0058] The preparation and characterization of embodiment 2 material
[0059] Specific steps for the preparation of specific recognition β-lactamase Ab-DNF ( figure 1 a), including the detailed process of DNF preparation, Ab-LD preparation, Ab-DNF preparation, etc. The procedure of this experiment is:
[0060] a. DNF was prepared by RCA reaction, 4 μM circular template precursor CDT-DNF (SEQ ID NO.1), 2 mM ATP, 5 μL 10×PNK buffer and 10 U PNK enzyme, incubated at 37° C. for 40 min. Add 3 μM template primer SEQ ID NO.2 (TP), heat at 90°C for 5 min to denature, cool to room temperature, add 10 μL of 10×T4 DNA ligase buffer and 5U T4 DNA ligase to the above mixture, and the resulting mixture (100 μL) is at room temperature Incubate for 1 hour and heat at 90°C for 5 minutes to inactivate the ligase. Concentrate and purify the circular template by standard ethanol precipitation and 10% dPAGE electrophoresis; 0.35 μM circular template, 0.7 μM template primer SEQ ID NO.2 (TP), 10 μ...
Embodiment 3
[0077] Example 3 Single-molecule detection method establishment and application
[0078] a. Counting ELISA procedure. Add 20 μL Ab (10 μg / mL capture antibody diluted in coating buffer) to a 384-well plate, incubate overnight at 4°C, discard excess Ab, wash 3 times with PBST, 3 min each time; add 30 μL blocking buffer containing 5% BSA solution, incubated at 37°C for 1 h, discarded excess blocking buffer, washed 3 times with PBST, 3 min each time; added samples containing target protein (β-lactamase standard protein or E. coli lysate), Ab-DNF and 1×SYBR -20 μL of Gold PBST, incubated at 37°C for 30 minutes, washed with PBST for 3 times, each time for 3 minutes; imaged with an inverted fluorescence microscope (excitation wavelength 490nm, emission wavelength 520nm), and then counted by ImagJ software;
[0079] b. Determine the detection limit of β-lactamase. First, verify the selectivity of the single-molecule detection method: add β-lactamase, bovine serum albumin (BSA), cyto...
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