| Recently, many different nucleic acid ampli?cation techniques are proved to be increasingly valuable for the quantitative detection of nucleic acids, proteins and small molecules. Enlightened by the advances of the isothermal circular ampli?cation technique, we have developed new ampli?ed quartz crystal microbalance and ?uorescence strategy for sensitive assay. In this article, a series of novel biosensing assay systems were developed for ultrasensitive detection of nucleic acids and small molecules based on aptamer-target recognition, bio-bar-code nanoprobe enhancement and enzyme assisted multiple cycle amplification strategy. Moreover, the proposed strategise exhibited an excellent speci?city and were successfully applied in real sample assay which demonstrates potential application in practical samples.The detailed content was described as follows:1. A simple and novel quartz crystal microbalance(QCM) assay is demonstrated to selectively and sensitively detect the adenosine triphosphate(ATP). The ampli?cation process consists of circular nucleic acid strand-displacement polymerization, aptamer recognition strategy and nanoparticle signal ampli?cation. With the involvement of an aptamer-based complex, two ampli?cation reaction templates and AuNP-functionalized probes, the whole circle ampli?cation process is triggered by the target recognition of ATP. As an ef?cient mass ampli?er, AuNP-functionalized probes are introduced to enhance the QCM signals. As a result of DNA multiple ampli?cation, a large number of AuNP-functionalized probes are released and hybridized with the capture probes on the gold electrode. Therefore the QCM signals are signi?cantly enhanced, reaching a detection limit of ATP as low as 1.3 nM. This strategy can be conveniently used for any aptamer-target binding events with other biological detection such as protein and small molecules. Moreover, the practical determination of ATP in cancer cells demonstrates the feasibility of this QCM approach and potential application in clinical diagnostics.2. A self-assembled DNA nanostructure as an effcient signal ampli?er was introduced to create a simple quartz crystal microbalance biosensing platform for highly sensitive and selective detection of nucleic acids. The ampli?cation process consists of hybridization chain reaction(HCR) and the biocatalytic precipitation(BCP). Upon the recognition of the molecular beacon(MB) to target DNA, the target DNA could propagate a chain reaction of hybridization events between the two hairpin probes, and where long nicked DNA polymers could be formed on the modi?ed electrode. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled horseradish peroxidase(HRP), which would further stimulate the biocatalytic precipitation(BCP) onto the electrode surface for signal amplification, producing insoluble products on the electrode surface that changed the frequency greatly. As the result of the multisignal amplification in this HRP catalyzed BCP-based aptasensor, the QCM signals were significantly enhanced, reaching a detection limit of DNA as low as 5×10-11 M.3. Isothermal circular strand-displacement polymerization ampli?cation assay is developed for highly speci?c and sensitive detection of adenosine triphosphate(ATP). The ampli?cation process consists of circular common target molecule-displacement polymerization(CCDP) and circular nucleic acid strand-displacement polymerization(CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced ?uorescence probes are obtained, achieving a detection limit of ATP as low as 2.6×10-10 M. Moreover, the proposed strategy exhibits an excellent speci?city and is successfully applied in real sample assay which demonstrates potential application in practical samples.4. A fluorescent platform was designed for ultrasensitive DNA detection based on the cascade signal amplification strategy. TEHP can produce a large number of primers and templates for RCA with the initiation of a few target DNA, which then act as the catalyst of the CHA to initiate hairpin recycling assembly for realizing the cascade signal amplification as reported previously. More importantly, the presence of target DNA can trigger the operation of the DNA machine and be easily read out through the fluorescencemeasurements. The high sensitivity and selectivity of this strategy might be attributed to three factors:(1) the TEHP offers the primer and template to RCA;(2) the RCA product with numerous catalysts can simultaneously induce the CHA and dramatically increase the sensitivity;(3) ‘‘Y’’ structure provides excellent selectivity toward sequence mismatch DNA, showing great potential for single nucleotide polymorphism analysis. This research opened new horizons for integrating different disciplines, which provides a versatile tool for detecting nucleic acids for bioanalytical and clinical bio-medicinal applications.5. We had developed a fluorescent method for site-specific methylation detection and MTase activity assay by a novel rolling circle amplification strategy to enhance the sensitivity. This method taked advantage of specific recognition of MTase and methyl-sensitive endonuclease, with high amplification effciency of RCA. Compared with the traditional methods, this assay did not require expensive instruments, achieving sensitive assay of Dam MTase activity with a low detection limit down to 0.6 U/m L. Moreover, the proposed assay was successfully applied in screening of inhibitors for Dam MTase, which held great promise for the discovery of anticancer drugs. The developed method had potential application in cancer risk assessment, molecular diagnostics and therapeutics through monitoring the DNA methylation level and change. |
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