Signal-enhanced test strip biosensor for detecting histone methylation
A histone methylation and biosensor technology, applied in biological testing, material inspection products, etc., can solve the problems of high toxicity of reagents, long operation time, cumbersome steps, etc., and achieve improved sensitivity, high specificity, and avoid operating steps complex effects
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Embodiment 1
[0026] Embodiment 1: Preparation of the signal-enhanced test strip biosensor for detecting histone methylation according to the present invention.
[0027] The present invention will take the detection of H3K9 in HeLa cells as an example, and construct a sensor that utilizes the signal enhancement probe of AuNP-DNA to amplify the signal of the detection result of the test strip (see figure 1 ).
[0028] The preparation method of the test strip biosensor for detecting the signal enhancement of histone methylation according to the present invention comprises the following steps:
[0029] 1) Design a pair of complementary oligonucleotide sequences, denoted as DNA and c-DNA respectively.
[0030] The 5' end of DNA is marked with a sulfhydryl group whose sequence is:
[0031] 5'-SH-atcataagctcatacaatcactaa-3' (SEQ ID NO. 1).
[0032] The 5' end of c-DNA is labeled with biotin, and its sequence is:
[0033] 5'-biotin-ttagtgattgtatgagcttatgat-3'.
[0034] 2) The monoclonal a...
specific Embodiment
[0043] 1. Preparation of gold nanometer (colloidal gold) solution:
[0044] Add 100 ml of 0.01% HAuCl to a 500 ml round bottom flask 4 solution, magnetic stirring and heating to boiling; then add 4 ml 1% sodium citrate to the above solution, after the solution turns wine red, continue to boil for 10 minutes, stop heating and continue stirring for 15 minutes to obtain gold nano (colloidal gold) solution .
[0045] The colloidal gold solution was stored in the dark at 4°C, and the gold nanoparticles were identified by the maximum absorbance value at 520 nm.
[0046] 2. Preparation of AuNP-DNA signal enhancement probe:
[0047] Dissolve 1OD DNA in 100 μl deionized water, add to 5 times volume concentrated colloidal gold (15nm) solution, shake at low speed, 4°C overnight; after blocking with 1% bovine serum albumin for 30 minutes, add Na 3 PO 4and 1% SDS, respectively, to a final concentration of 0.01M and 0.01%, and gradually increase the NaCl concentration to 0.15 M in t...
Embodiment 2
[0058] Embodiment 2: The detection method of the test strip biosensor for detecting the signal enhancement of histone methylation according to the present invention.
[0059] In this example, the test strip biosensor prepared in Example 1 was used to detect the trimethylation of histone H3K9 in HeLa cells.
[0060] Taking the detection of H3K9me3 as an example, the detection method is as follows:
[0061] 1) HeLa cells were lysed, and histone crude extract was obtained by acid extraction.
[0062] 2) Add histone extract solution dropwise to the sample pad, after 10 minutes, add AuNP-DNA dropwise, and observe the experimental results after 5 minutes.
[0063] The following is a specific example:
[0064] 1. Obtaining crude histone extracts from HeLa cells:
[0065] a) HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum.
[0066] b) When the cells grow to a confluence of 90% in a 6cm culture dish, aspirate the culture medium, wash it twice wit...
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