Homogeneous Electrochemical And Fluorescence Biosensing Strategies For Early Detection Of Cancer Biomarks

Cancer is one of the most serious threats to human beings. Cancer survival rates tend to be poor most likely due to the late stage diagnosis. The early detection of cancer is highly important for clinical diagnosis and ultimately for the successful treatment of cancers. Cancer biomarks are present in cancer cell, mainly including enzymes, metabolites and microRNA. The detection of cancer biomarks may pave the way for the early diagnosis of cancer. Thus it is highly desirable to develop rapid and efficient strategies for detection of cancer biomarks. Using biosensor to detection of cancer biomarks has become an important direction in the field of analytical chemistry. Among them, homogeneous DNA biosensors using nucleic acids as molecular recognition elements have attracted many researches attention. Since it has immobilization-free, simple and rapid, high sensitivity, strong stability, as well as good biocompatibility, it has been widely applied in gene analysis, disease diagnosis, etc. In this paper, we use nucleic acids to construct several homogeneous DNA biosensors to realize simple and rapid sensitive detection of DNA methyltransferase, human telomerase and microRNA.The chapter firstly introduces the biosensor and DNA biosensor including basic structure and working principle. Electrochemical DNA biosensors and optical DNA biosensors are further highlighted. Subsequently, nucleic acid amplification techniques are introduced and classified. Finally is the brief overview of DNA methyltransferase, human telomerase and microRNA.A simple and novel homogeneous electrochemical strategy for ultrasensitive DNA MTase activity assay has been successfully developed, which is based on methylation-triggered exonuclease (Exo) ?-assisted autonomous isothermal cycling signal amplification. A duplex DNA (P1-P2 hybrid) containing the methylation-responsive sequence is ingeniously designed. In the presence of DNA adenine methylation (Dam) methyltransferase (MTase), P1-P2 hybrid is methylated and subsequently recognized and cleaved by Dpn I endonuclease, which triggers the Exo ?-catalyzed autonomous cycling cleavage processes. Therefore, a large amount of methylene blue-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the Dam MTase activity assay. The directly measured detection limit down to 0.004 U/mL is obtained, which is one or two orders magnitude lower than that of the approaches reported in literature. Since this assay is carried out in homogeneous solution phase under isothermal condition and sophisticated probe immobilization processes are avoided, it is very simple and easy to implement. Due to its advantages of ultrahigh sensitivity, excellent selectivity and simple operation, the as-proposed strategy has great potential in the applications in DNA methylation related clinical practices and biochemical researches.A homogeneous electrochemical strategy based on T7 exonuclease-aided target recycling amplification is proposed for simple, rapid, and highly sensitive assay of human telomerase activity from crude cancer cell extracts. In this strategy, a 5'methylene blue (MB)-labeled hairpin (HP) probe is designed, which can hybridize with the telomerase reaction products to initiate the subsequent digestion by T7 exonuclease, and large amount of MB-labeled mononucleotides are released to result in the significantly amplified electrochemical signal. By taking advantage of the high amplification efficiency of T7-aided target recycling, the present assay enables the detection of telomerase activity at the single-cell level, which is superior or comparable to that of the reported literatures. Furthermore, the assay was carried out in homogeneous solution without complex modification or immobilization procedures, which has the merits of simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensors. With the ability of fast detection, outstanding sensitivity and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early-stage cancer diagnosis.For the first time, a simple label-free and enzyme-free homogenous electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains. Thus, with the electrochemical indicator methylene blue (MB) selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is depended on the concentration of the target miRNA. Thus, by this "signal-off" mode, a simple, label-free and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative method for simple, sensitive and selective miRNA detection.We developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green ? (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO has multiple functions of protecting miRNA from enzymatic digestion, selectively absorbing single-stranded DNA, and quenching the fluorescence of free and single-stranded DNA intercalated SG. In the presence of target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA related clinical practices and biochemical researches.