Development Of Colloidal Gold Nucleic Acid Strip Biosensor For Visual Detection Of Heavy Metal And Biomedicine

The colloidal gold nucleic acid strip biosensor has the advantages of low cost,short detection time(5?15 min),user-friendly,visual and can be used for in-field detection,etc,which possesses great potential in multiple detection fields.Recently,heavy metal pollution incidents and cancers such as liver cancer are increasing.They all threaten human life and health seriously.The heavy metal Pb has the characteristics of strong toxicity,accumulation,persistence and irreversibility.Liver cancer is one of the malignant tumors that seriously endangers human health.Chronic hepatitis B virus(HBV-DNA)infection accounts for at least 80%of HCC cases.In addition,the occurrence of HCC is closely related to heredity and susceptible genes(or SNP).In summary,the detection of Pb²⁺in the environment,human HBV-DNA and SNP sites can effectively prevent the occurrence of Pb pollution and liver cancer.At present,traditional detection methods are highly accurate and sensitive.However,these methods require large instruments and professional operations,and the high detection cost makes it not suitable for in-field or immediate detection.Therefore,the development of low-cost,user-friendly,simple and fast Pb²⁺or SNP,HBV-DNA detection method is important for application.Therefore,the methods for visual detection of Pb²⁺,SNP and HBV-DNA are established based on the colloidal gold strip biosensors.The details are as follows:1)Based on GR-5 DNAzyme,Pb²⁺nucleic acid strip biosensor with the assistance of graphene oxide(GO)has been established.GO could reduce the false positive interference by removing unhybridized ss DNA during the annealing of GR-5 DNAzyme.Meanwhile,conjugate pad was sprayed with two kinds of Au NP-DNA probes,thereby,the sensitivity of Pb²⁺detection was extensively improved.The limit of detection of this strip biosensor was estimated to be 0.05 n M(S/N=3)and 1 n M(with naked eyes)with a linear range from0.01 to 100?M.For real soil samples,the obtained recoveries were in the range from 91.5%to 113.1%.2)PS2M aptamer was selected as sensing element to construct Pb²⁺nucleic acid strip biosensor.The formation of a G-quadruplex structure between Pb²⁺and PS2M resulted in the release of the single strand Anti-PS2M from the PS2M/Anti-PS2M duplex.The single strand fixed the Au NP-DNA probe G to TZ via sandwich structure.It was the first time that Au NP-DNA polymer was used as sensitivity enhancement probe(SEP)to improve sensitivity.The limit of detection of Pb²⁺strip biosensor was 2.5 n M by adding the SEP,which was 40 times better than that of strip without enhancement.The test strip obtained the recovery of 90?111%of Pb²⁺in the spiked detection of soil and lake samples.3)A method for visual detection SNP sites(A/A,G/G,A/G)in CYP1A1 gene related to cancer based on colloidal gold nucleic acid strip biosensor and primer-specific polymerase chain reaction(PCR)was established.This method could directly distinguish SNP sites on a pair of strips by introducing twice PCR.The designed strip biosensor could only combine with the ss DNA,thus visual detection of SNP could be achieved by color difference of a pair of strips.The detection results of 61 human blood samples were identical with those of sequencing by this method.4)SNP sites(T/T,C/C,T/C)on the CYP1A1 gene associated with cancer were detected based on strip biosensor.Au NP-DNA probes A and G on strip A and G were completely complementary to ss DNA containing T/T site(complementary base of A/A site)and ss DNA containing C/C site(complementary base of G/G site),respectively.In this case,only completely complementary ss DNA to the optimized strand length of Au NP-DNA probes(12A and 11G)could effectively develop color on the TZ of strip.21 human blood samples detected by this method were consistent with those of sequencing.5)Microplate and HCR amplification were used to improve the sensitivity for detection of HBV-DNA on strip biosensor.The microplate amplification-based strategy was to use HBV-DNA to fix the dual Au NP-DNA probe on the microplate.The amplification of the HBV-DNA detection signal was achieved by detecting SH-RS-DNA dissociated from the surface of dual Au NP-DNA probe by adding DTT.For the HCR-based strategy,HBV-DNA triggered the cascade of Au NP-H1 probe and H2-Biotin,which formed a large amount of HCR products.Thus,a large amount of Au NPs was fixed on the TZ of the strip.The detection limit of the two strategeies could reach 0.1 n M(10 times higher)and 5 p M(200 times higher),respectively.And the HBV-DNA recovery spiked in human serum was97.2?102%.In conclusion,this study was developed colloidal gold nucleic acid strips for detection of Pb²⁺,SNP and HBV-DNA,which provided new approach for prevention and treatment of the environmental Pb²⁺and low-cost and visual detection of early diagnosis of liver cancer.