Kit for detecting circulating nucleic acid based on microfluidic chip and g-quadruplex-heme dnazyme and its preparation method and application
A technology of microfluidic chips and quadruplexes, applied in the field of micro-total analysis systems, can solve problems such as high throughput and high sensitivity, and achieve the effect of not relying on complex equipment and low cost
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[0072] The invention provides a preparation method of the kit, including a preparation method of a microfluidic detection chip coated with functionalized microspheres and a preparation method of gold nanoparticles whose surface is modified with a second probe and an initiating probe.
[0073] The preparation method of the microfluidic detection chip coated with functionalized microspheres described in the present invention comprises the following steps:
[0074] 1) Mix and incubate the avidin-modified microsphere solution with a mass concentration of 1% to 3% after affinity washing and 0.3 μmol / L biotin-labeled first probe solution, and wash to obtain functionalized microspheres;
[0075] 2) Put the functionalized microspheres in the step 1) into the microchambers in the chip through the microsphere loading channel, fix them, and peel off the microsphere loading channels, then attach the reagent delivery sheet and the microsphere fixed array sheet, A microfluidic detection chi...
Embodiment 1
[0113] Method for preparing a kit for mRNA detection of alpha-fetoprotein coding gene
[0114] 15 μm avidin-modified polystyrene microspheres were used as the solid-phase interface for the immobilization of the first probe. Take 100 μL of avidin-modified microspheres with a concentration of 2% in a centrifuge tube and wash with 100 μL affinity eluent (20 mM Tris (pH 7.5, 1M NaCl, 1mM EDTA, 0.0005% TritonX-100) was washed twice, centrifuged at 3500rpm for 5min, and the supernatant was removed; 44μL of affinity eluent and 3μL of 0.3μM biotin-modified first probe were added respectively. needles (see Table 1 for the sequence), incubate at room temperature for 10-15 min; remove unbound molecules by centrifugation and wash, and suspend the functionalized microspheres in 100 μL of affinity eluent. The first probe specifically binds to the avidin on the surface of the microsphere through the modified biotin and is fixed on the surface of the microsphere, thereby forming a functionali...
Embodiment 2
[0121] Preparation method of standard curve for detection of mRNA molecule of alpha-fetoprotein coding gene
[0122] Add the alpha-fetoprotein sequence (Seq ID No.10 in the sequence listing) at a concentration of 0.1 pM to 10 nM to the buffer system to construct a 15 μL hybridization solution, including: 10 mM Tris-HCl (pH 7.5), 750 mM NaCl, 0.025% Tween 20, in Driven by pressure, pass through the detection area of the microfluidic bead array chip containing the functionalized bead array, incubate at room temperature for 30 min, wash with 55°C TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) for 5 min; flow 15 μL Containing 2nM gold nanoparticles modified on the second probe and the priming probe, incubate at room temperature for 30min. Wash with 55°C TE buffer (10mM Tris-HCl, pH 8.0, 1mMEDTA) for 5min; pour 10uL of a mixed solution of 0.5uM first hairpin probe and second hairpin probe into the reaction well, and incubate for 6 hours. Wash with 10 nM HEPES buffer for 5 min to ...
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