Biosensing Studies Based On Rolling Circle Amplification And Gold Nanoparticles

In recent years, a seris of new biology techniques have been developed as a powerful platform for sensitively detection of proteins and DNA. However, with the development of the scientific research, greater sensitivity and specificity techniques are required. Therefore, the development of the high sensitivity, selectivity and accuracy strategies, as well as proved a high performance platform for proteins and DNA assays are of paramount importance for biomedical research and clinical diagnosis. In this thesis, a series of novel biosensing strategies were developed for ultrasensitive detection of proteins and DNA. The proposed methods were implemented in the analysis of realistic biological samples and were in good agreement with the classical methods. These results primarily proved that the proposed technology was feasible, reliable and accurate. The detailed content was described as follows:(1) Aptamers are nucleic acids (DNA or RNA) that selectively bind to low-molecular-weight organic or inorganic substrates or to macromolecules such as proteins. In chapter 2, based on rolling circle amplification, a novel versatile electrochemical aptasensor was developed for the ultrasensitive detection of model analyte PDGF-BB. First, PDGF-BB antibody was immobilized on the electrode surface via the SAM of cysteamine and the bifunctional linker of glutaraldehyde and then used to capture the protein target. The surface-captured protein was then sandwiched by an aptamer-primer complex. The aptamer-primer sequence mediated an in situ RCA reaction that generated hundreds of copies of a circular DNA template. Detection of the amplified copies via enzymatic silver deposition then allowed enormous sensitivity enhancement in the assay of target protein. In conclusion, the presented method exhibited a linear correlation to target concentration through a 4-decade range of 10 fM~100 pM, and a detection limit as low as 10 fM. The wide dynamic range of the presented sensor was about 3 orders of magnitude larger than those shown previously.(2) We have reported an aptamer-based immuno-RCA assay for protein detection in chapter 2. However, aptamers for most important protein biomarkers are currently unavailable, and like the DNA-antibody conjugates, aptamers are also exposed to the risk of electrostatic adsorption on proteins as well as chemical or enzymatic degradation. In chapter 3, we reported for the first time a DNA encapsulating liposome based RCA immunoassay, liposome-RCA immunoassay, as an alternative strategy. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg/mL to 0.1 ng/mL with a limit of detection as low as 0.08 fg/mL and a high dose-response sensitivity.(3) In chapter 4, based on rolling circle amplification and terminal protection assay, a novel photochemical biosensing strategy was developed for the ultrasensitive detection of model analyte folate receptor (FR). This assay was based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule could be protected from the degradation by exonuclease I (Exo I) when the small molecule moiety was bound to its protein target. The small-molecule-linked protected DNA was served as a template of the ligation probe to trigger the rolling circle amplification. And then generated a long tandem repeated sequences that could be selectively and sensitively detected by an exonuclease III-aided oligonucleotide recycling assay. This strategy was demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor. And quasilinear correlation was obtained in the concentration range from 1 pM to 1 nM with a readily achieved detection limit of 1 pM.(4) In chapter 5, a novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNAbinding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. In the absence of the DNA-binding protein, Exo III will stepwisely and nonprocessively digested the double-stranded DNA. This caused the dissociation of the AuNP aggregates into dispersed AuNPs with a concomitant purple-to-red color change for the solution. In the presence of the DNA-binding protein, tight binding of the proteins to the consensus sequences induced steric hindrance to Exo III in approaching the 3′-termini, which protected the antisense strands from Exo III-mediated digestion. This retained stable AuNP aggregates in the solution with no color change obtained. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM.(5) In chapter 6, we described a novel colorimetric assay method for detecting of the activity and kinetics of T4 polynucleotide kinase (PNK) by using of molecular beacons-modified gold nanoparticles (AuNPs) coupled withλexonuclease (λexo) and recj exonuclease (recj exo) cleavage. The assay was performed at salt concentrations so that DNA-modified AuNPs were barely stabilized by the electrostatic and steric stabilization. The 5′-hydroxyl group of the molecular beacons modified AuNPs was first phosphorylated in the presence of T4 PNK, then initiatedλexo and recj exo cleavage. Enzymatic cleavage of DNA chains on the AuNP surface destabilized the AuNPs, resulting in a rapid aggregation and a red-to-purple color change. In conclusion, the presented method exhibited a linear correlation to target concentration with a linear range of 0~4 U/mL and a detection limit of 0.24 U/mL.(6) In chapter 6, a rapid, simple colorimetric sensor with unmodified AuNPs for highly sensitive and selective detection of silver ion was developed. It was based on the specific recognition property of Ag~+ with a cytosine-cytosine mismatched base pair. In the absence of Ag~+, single strand oligonucleotide adsorbed on the surface of AuNPs and protected them from aggregation with the addition of high concentration salt. In contrast, the presence of Ag~+ drived the formation of C-Ag~+-C duplex structure and enabled the oligonucleotide to be desorbed from the surface of the AuNPs, resulting in the aggregation of AuNPs. The results revealed that the method allowed a specific, simple, and rapid assay of Ag~+ with a linear response range from 0.5μM to 6μM and a detection limit of 0.1μM.