▎ 摘　　要

NOVELTY - Amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase comprises modifying gold nanoparticle (AuNPs)-zinc selenide nanosheets (ZnSeNSs) to graphene (GE), then fixing FP on AuNPs-ZnSeNSs/GE through gold-sulfur bond, adding the sample containing kallikrein related peptidase 2 (KLK2), where the FP on the electrode surface and the RP in the solution undergo RPA amplification in the presence of recombinase polymerase, amplifying the target on the electrode and capturing RP on the electrode surface, washing, where St-ALP is connected to the electrode through the affinity between biotin and streptavidin, ALP catalyzes the hydrolysis of p-NPP to p-NP, due to the high catalytic activity of ALP, producing many p-NPs, generating strong photoelectric signal under the action of MB and pNP, determining the KLK2 content according to the strength of the PEC signal. USE - The method is useful for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase. ADVANTAGE - The method: has high sensitivity for determining KLK2, and detection limit of 30 aM; and is simple. DETAILED DESCRIPTION - Amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase comprises modifying gold nanoparticle (AuNPs)-zinc selenide nanosheets (ZnSeNSs) to graphene (GE), then fixing FP on AuNPs-ZnSeNSs/GE through gold-sulfur bond, adding the sample containing kallikrein related peptidase 2 (KLK2), where the FP on the electrode surface and the RP in the solution undergo RPA amplification in the presence of recombinase polymerase, amplifying the target on the electrode and capturing RP on the electrode surface, washing, where St-ALP is connected to the electrode through the affinity between biotin and streptavidin, ALP catalyzes the hydrolysis of p-NPP to p-NP, due to the high catalytic activity of ALP, producing many p-NPs, generating strong photoelectric signal under the action of MB and pNP, determining the KLK2 content according to the strength of the PEC signal, achieving high-sensitivity detection of the target due to the co-regulation of MB and p-NP, the circulation of MB, and the amplification of recombinase polymerase, AuNPs-ZnSeNSs/GE generates strong photocurrent at 0V in the presence of MB and p-NP, has self-supply characteristics, combined with in situ recombinase polymerase amplification strategy, establishing method for liquid biopsy of circulating tumor DNA KLK2 for prostate cancer, the specific steps are: the preparation of zinc selenide nanosheets comprises obtaining zinc selenide nanosheets by peeling off by ultrasonic peeling method, adding 0.1-450 mg zinc selenide to 1-150 ml dimethylformamide solution, then dispersing the mixture by ultrasonic for 0.1-100 hours to obtain a yellow dispersion, centrifuging at 14,000 revolutions per minute for 20 minutes to separate the stripped zinc selenide from the dispersion and washing using ethanol for 3 times, using the ZnSeNSs suspension in the next experiment, the synthesis of AuNPs-ZnSeNSs comprises preparing AuNPs-ZnSeNSs by in-situ growth method, adding 0.1-30 ml ZnSeNSs and 0.1-10 ml chloroauric acid tetrahydrate into the flask, heating to 115 degrees C in an oil bath with stirring, the solution continues to reflux and stirring for 3 minutes, dropping 0.1-10 ml 1.66 mg/ml sodium citrate solution into the flask, refluxing the mixture at 115 degrees C for 3 minutes to obtain black AuNPs-ZnSeNSs, centrifuging for 10 minutes at 14,000 revolutions per minute to collect the final black precipitate, in the synthesis method of AuNPs-ZnSeNSs, chloroauric acid of different quality will affect the PEC signal, the mass ratios of ZnSeNSs and chloroauric acid are 1:0.2, 1:0.33, 1:0.66, 1:1, 1:1.5, respectively, the KLK2 detection comprises dropping 1-60 mu l AuNPs-ZnSeNSs onto the surface of the gold electrode and naturally drying in air, adding 1-60 mu l 10 mM TCEP to 1-60 mu l 10 mu M FP solution, incubating the mixture solution at 37 degrees C, after 60 minutes, adding 1-60 mu l of the mixture to the surface of AuNPs-ZnSeNSs/GE, incubating for 2 hours at 37 degrees C to obtain FP/AuNPs-ZnSeNSs/GE, soaking FP/AuNPs-ZnSeNSs/GE in 1 mM MCH solution for 1 hour to eliminate non-specific adsorption, preparing RPA solution for surface amplification of KLK2 gene, the RPA reaction solution is made of 2.4 mu l 10 mu M RP, 13.2 ml water without DNase, 31.9 ml recombinase polymerase buffer solution and 2.5 ml 280 mM magnesium acetate solution, then adding 1-50 mu l of the RPA solution and 1-50 mu l of the sample containing KLK2 to the surface of FP/AuNPs-ZnSeNSs/GE, after 30 minutes of amplification at 37 degrees C, washing the electrode with 0.1M phosphate buffered saline, soaking the electrode in 2-60 mu l 1 mu M St-ALP solution and incubating at 37 degrees C for 30 minutes, washing the electrode using 0.1M phosphate buffered saline, inserting the electrode into a solution of p-NPP and MB, where ALP catalyzes the hydrolysis of p-NPP into p-NP, generating strong photoelectric signal under the action of MB and p-NP, and realizing the determination of KLK2 content according to the intensity of PEC signal, the sequence of forward primer, reverse primer and KLK2 gene is as follows: KLK2 forward primer (FP) comprises fully defined nucleotide sequence of 5'-SH-gggggtccacttgtctgtaa-3' (SEQ ID NO: 1), KLK2 reverse primer (RP) comprises fully defined nucleotide sequence of 5'-ggtgagttccaagcttcagg-biotin-3' (SEQ ID NO: 2), and KLK2 gene comprises fully defined sequence of 250 nucleotides (SEQ ID NO: 3) as give in the specification.