Novel Method Development Of Optical Biosensors

Optical biosensors are powerful alternative to conventional analytical techniquesdue to its great potential for the particularly rapid, high specification, sensitivity, smallsize, and cost effectiveness. The research and technological development of opticalbiosensors have experienced an exponential growth during the last decade because thistechnology has a great potential for the direct, real-time, and rapid detection of manychemical and biological substances in clinic biomarkers detection, drug discovery,environmental, food monitoring, and public safety. Considering some key points in thebiomolecular detection especially enzymes, protein, a series of optical sensors weredeveloped with high sensitivity, high specificity, speediness and simpleness in thethesis. The details are described as follows:(1) According to the aggregation of gold nanoparticles (AuNPs) induce asignificant variation in the plasmon resonance absorption peak, in chapter2, a novelbiosensing strategy was developed for sensitivity and specificity screening ofpeptidase activity assay based on the proteolysis-mediated protection of substratepeptide-decorated AuNPs against the network-like assembly which can be triggered bythe biotin–streptavidin binding event. The substrate peptide is designed to have abiotinylated substrate sequence with a specific reacting site for peptide hydrolysisenzyme. In the absence of target peptidase, because the free terminal of the intactsubstrate peptide decorated on AuNPs is biotinylated, it can bind with the streptavidinthrough the biotin–streptavidin interaction. Thus, it can trigger a network-likeassembly of the peptide-decorated AuNPs, subsequently inducing a significantvariation in the plasmon resonance absorption peak with a visualized color change. Incontrast, in the presence of target peptidase, the peptidase catalyzes the hydrolysis ofthe specific reacting site in the substrate peptide. The enzyme-catalyzed hydrolysis,thus, releases a biotinylated peptide fragment from the substrate peptide-decoratedAuNP. As the added streptavidin can only bind with the released biotinylated fragment,AuNPs are well protected from the streptavidin-triggered network-like assembly.Therefore, the resulting absorption spectral response of AuNPs can be immediatelyused for quantifying the activity of the target peptidase. This strategy allowed ahomogeneous assay of the peptidase activity in a very simple format as well assimplified instrumentation owing to the visualized detection, which made the assays robust, easily automated, scalable for parallel assays of hundreds of samples. Usingpancreatic elastase as a model case, the developed strategy was demonstrated todisplay desirable selectivity and sensitivity in peptidase assay. Using pancreaticelastase as a model case, a wide linear response range from0.005to0.10U/mL with adetection limit of0.003U/mL was achieved. By designing proper substrate peptidesand decorating them on AuNPs, this strategy also can be used for sensitively andspecifically activity screening of other peptidases the developed peptidase biosensingstrategy was expected to provide an intrinsically robust, convenient, sensitive platformfor visualized peptidase activity analysis and related biochemical studies.(2) Based on the target-mediated consecutive endonuclease reactions, a simple,sensitive and selective homogeneous fluorescence assay strategy is developed for thedetection of O6-methylguanine-DNA methyltransferase (MGMT). The activity assay ofMGMT is firstly accomplished using a hairpin-structured DNA substrate to offer aspecific recognition site on the substrate DNA for restriction endonuclease PvuII, andthus to initiate the first endonuclease reaction. The product which activates the secondendonuclease reaction allows an efficient amplification approach to create anabundance of fluorescence signal reporters. The first endonuclease reaction offers themethod high specificity and the second one furnishes the assay improved sensitivity.The results reveal that the MGMT assay strategy shows dynamic responses in theconcentration range from1to120ng/mL with a detection limit of0.5ng/mL. Theresults indicated that this strategy offers a simple, cost-effective, highly sensitive andselective homogeneous detection platform for MGMT activity assay.(3) With the principle of active endonuclease PvuII reaction by MGMT that wasreported in chapter3, the more sensitive fluorescence biosensor has been developed fordetecting the O6-methylguanine-DNA methyltransferase. We proposed a novelbiosensor constituted by adsorbing the O6-methylguanine containing DNA that ishybrid with fluorophore-labeled probe onto the surface of graphene oxide (GO) as ahomogeneous assay platform for sensitive MGMT activity assay. Active MGMT couldremove the methyl group in the DNA probe, and further hydrolysis specific sites byrestriction endonuclease gave rise to high fluorescence. Thus, it provided a convenientapproach for MGMT activity quantification. Because of the unique ability of GO inuniversal fluorescence quenching, a low background fluorescence signal can beobtained for the efficient fluorescence resonant energy transfer from thefluorophore-labeled probe to GO sheet. A quite wide dynamic range from0.5to60ng/mL was achieved for MGMT assay and the detection limit was estimated to be0.2 ng/mL. The results indicated that this strategy offers a simple, non-radioactive,cost-effective, highly sensitive and selective homogeneous detection platform forMGMT activity assay.(4) According to the theory that plasmonic coupling between AuNPs can enhancethe electromagnetic fields in the interstitial spots of AuNPs by many orders ofmagnitude, which evokes dramatic enhancement of the Surface Enhanced RamanScattering (SERS) signal, a novel SERS strategy is developed based on assembly of the5-thio-nitrobenzoic acid (TNB) labeled hepatitis B surface antibody-AuNPs intonetwork structures mediated by hepatitis B surface antigent (HBsAg) for the HBsAgdetection in chapter5. The reaction between hepatitis B surface antibody (HBsAb) andHBsAg is detected via the strong SERS signal of TNB. A calibration curve wasobtained to detect the concentration of HBsAg in the range from10ng/mL to0.5μg/mL with the detection limit is5ng/mL. By the using only single step labeling of thehighly Raman-active reporter molecule TNB on gold nanoparticle, our detectionprocess is a lot more simplifed. This immunoassay can be applied to other analytes bysimply replacing with suitable antibodies.