| Optical biosensing technique has the advantages of short analysis time,good selectivity,simple operation and high sensitivity.Until now,optical biosensor has been widely applied for detection of nucleic acid,protein,bioactive small molecules and viruses,etc.which indicates a broad application prospect of optical biosensing technique in the field of biochemical analysis.Nanomaterials have special optical and electrical properties,which opened up a new situation for analytical chemistry.Using nanomaterials to construct the optical biosensor can enlarge the way of building biological sensing system.Microchip electrophoresis was increasingly recognized as one of the most important analytical separation technique.It has been widely used in field of biological chemistry,environmental sciences and medicine sciences characterized with its small sample volumes,short analysis times,high separation efficiency and easy to achieve high throughput analysis.Laser induced fluorescence detection technique is a very sensitive optical detection technique,and has great application potential in the microchip technology.The nucleid acid signal amplification technology can significantly improve the sensitivity of analysis.The nucleid acid amplification technology is applied to microchip technology,which could significantly improve the sensitivity of microfludic chip electrophoresis methods and improve the analysis performance of system.Based on optical biosensing technology,the fluorescence quenching effect of nanomaterials,microchip electrophoresis-laser induced fluorescence detection technique and nucleid acid signal amplification,this thesis has developed four new methods for detecting antitumor drugs and protein,and applied for bleomycin(BLM),S1 nuclease and interferon gamma(IFN-y)analysis.The main contents of this work can be concluded as follows:Part one:Based on different affinity of WS2 nanosheet toward different length ssDNA,and fluorescence quenching ability toward fluorescence molecule,a novel fluorescent sensing platform was developed for simple,fast and sensitive detection of BLM and nuclease.The nuclease activity was monitored by using S1 nuclease(ssDNA-specific nuclease)as a model enzyme analyte.A fluorescein(FAM)-labeled ssDNA could be adsorbed on the surface of WS2 nanosheet and the fluorescence of FAM-labeled ssDNA was therefore quenched.In the presence of BLM-Fe(?)or S1 nuclease,an irreversible scission of long ssDNA was underwent through the BLM-induced oxidation cleavage or S1 nuclease-induced enzymatic hydrolysis.Short FAM-linked oligonucleotide fragments which could not be adsorbed on the nanosheet surface.Thus,the fluorescence of FAM was restored.The proposed fluorescence sensor displays a wide linear range and a high sensitivity with a detection limit of 3.0×10-10mol/L for BLM and 0.01 U/mL for S1 nuclease.Moreover,it also exhibits a good performance in complex biological samples.This method not only provides a strategy for BLM or S1 nuclease assay but also offers a potential application in biomedical and clinical study.Part two:A homogeneous label free colorimetric sensing strategy for BLM assay was developed on the basis of BLM enhanced Fe(?)-H2O2-ABTS reaction.Fe(?)exhibits a catalytic effect on H2O2-mediated oxidation of ABTS by fenton chemistry.In the presence of oxygen,BLM and Fe(?)form a complex BLMˇFe(?).BLM Fe(?)possessed a much higher catalyticability than Fe(?).Therefore,bying the color change of Fe(?)-H2O2-ABTS reaction system,we can detect BLM by the naked eye and UV-vis spectroscopy.The peak absorbance exhibits a linear relationship with BLM concentrations from 2.5×10-8 mol/L to 1.0x10-6 mol/L and a detection limit(3?,?=S0/S)was estimated to be 1.6×10-8 mol/L.The present method was successfully applied to the determination of BLM in serum samples with satisfactory results.Part three:A novel strategy for highly sensitive detection of S1 nuclease activity and its inhibitor was developed base on microchip electrophoresis-laser induced fluorescence platform.S1 nuclease which is known to be a single-strand-specific nuclease and hydrolyzes primarily fluorescein(FAM)-labeled long ssDNA(FAM-DNA)to yield 5 '-FAM-nucleoside monophophates tes and a very small amount of dinucleotides.Monitor of S1 nuclease activity and inhibitor can be achieved by microchip electrophoresis for separating FAM-DNA and 5'-FAM-nucleoside monophophates and laser induced fluorescence for detecting the fluorescence signal.The calibration curve showed a good linearity between the peak height of 5'-FAM-nucleoside monophophates and the concentrations of S1 nuclease from 0.002 U/mL to 0.2 U/mL.Based on a S/N of 3,the limit of detection was estimated to be 0.0012 U/mL.In addition,by taking pyrophosphate as an example,we use the assay to evaluate the prohibition effect on S1 nuclease.This present method was applied to the recovery experiments in serum samples with satisfactory results.Part four:A new method for simple,sensitive,highly specific detection of IFN-? based on T7 exonuclease(T7 Exo)assisted signal amplification using microchip electrophoresis-laser induced fluorescence detection.In this method combined with T7 Exo assisted signal amplification,separation of microchip electrophoresis and detection of laser induced fluorescence achieve highly sensitve detection IFN-y.The results showed a good linearity between the peak height of FAM-nucleoside monophophates and the concentrations of IFN-?from 1.5×10-11 mol/L to 2.5×10-9 mol/L.The limit of detection(S/N=3)was estimated to be 6.5×10-1mol/L.The present method was successfully applied to the determination of IFN-? in human plasma with satisfactory results.Furthermore,this detection system appears to be a universal approach for the detection of other target molecules by simply changing the aptamer sequence of the hairpin probe.And based on principle of this method,bying design different length of the fluorescence label probe can achieve multiple detection of target under single excitation.Thus it could be greatly expanded the scope of application of the method. |
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