False complementary peptide nucleic acid probe biochip and detection method based on SPR principle
A biochip, peptide nucleic acid technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of reducing the universality and selectivity of PNA, reduce the use cost and experimental conditions, simplify the operation steps, The effect of high sensitivity and specificity
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Embodiment 1
[0054] Example 1 The method of spreading a 50nm thick nano-gold film with surface plasmon resonance sensitivity on a glass slide, the steps are as follows:
[0055] (1). Prepare gold sol according to the trisodium citrate reduction method (Frens method): take 100ml of 0.01% chloroauric acid aqueous solution and boil, add accurately 0.75ml of 1% trisodium citrate aqueous solution under agitation, golden chloroauric acid The aqueous solution turns purple-red within 2 minutes, and continues to boil for 15 minutes. After cooling, it is restored to its original volume with distilled water.
[0056] (2) Gold film laying system:
[0057] A. After the glass slide is cleaned by pickling, put it in N-β-(aminoethyl)-γaminopropyl trivalent oxysilane (APTMS) / toluene solution and reflux for 12 hours, then wash it, take it out, and blow dry with nitrogen. , Placed in a clean and dry place for later use;
[0058] B. After the processed glass slide is soaked in the nano-gold solution for 12 hours,...
Embodiment 2
[0059] Embodiment 2 The method of dot-making pcPNAs probe arrays on the surface of the gold film using the surface self-assembly monolayer technology, the steps are as follows:
[0060] (1), the prepared nano gold film in piranha solution (30% H 2 O 2 : Thick H 2 SO 4 =1:3) Soak and clean, then repeatedly clean with double distilled water, and dry with nitrogen;
[0061] (2) Add 500nM SH-pcPNAs / PBS buffer to the self-assembly reaction on the nano-gold film, and react at 100% humidity for 24 hours to generate a self-assembled array of probes;
[0062] (3). Blot the buffer solution separately, block with 6-mercaptohexanol, rinse with double distilled water, and blow dry with nitrogen.
Embodiment 3
[0063] Example 3 Using the reagent Chelex-100 to quickly process the specimen and extract bacterial DNA by one-step method, the steps are as follows:
[0064] (1) Use a sterile inoculation loop to collect a loop of pure culture colonies and place it in a 1.5ml sterile Eppendorf tube;
[0065] (2) Add 50μl of 5% Chelex suspension, mix well, add 20mg / ml proteinase K, digest in a water bath at 56°C for more than 2h, then boil it in a water bath for 7.5min, then immediately put it in an ice bath for 3min, and centrifuge at 12000r / min for 5min , Take the supernatant as a DNA sample.
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