Research And Application Of Functional DNA Biosensor

Functional DNA is broadly applied by good selectivity and simple synthesization.In the thesis,some biosensors were designed and developed based on functional DNA and microfluidic technology,using for the detection of disease biomarker and environmental pollutants.The thesis is divided into following six chapters.In chapter 1,the definition and characteristics of functional DNA biosensor was introduced in detail,and different kinds of biosensors were illustrated in brief.The concept and technology of microfluidics was described briefly.Meanwhile,the research and application of functional DNA biosensor was introduced for the point-of-care testing.Finally,we summarized the purpose and signification of the thesis.In chapter 2,based on the adsorption of a metal-organic framework(MOF)to DNA,a fluorescent biosensor for H5N1 antibodies has been developed.In this system,a short oligonucleotide was modified with the fluorescent dye 5'6-FAM(5'6.-carboxyfluorescein)as the fluorescent DNA probe.With the introduction of a MOF aqueous solution,the DNA probe can be adsorbed by the MOF and the fluorescence of the dye will be quenched by the MOF.Subsequently,exonuclease I(Exo 1)is employed to specifically hydrolyze the DNA probe at the 3'-terminus and the fluorescent dye FAM is released from the MOF,which results in recovery of the fluorescence.If the 3'-ends of the DNA probe are linked to H5N1 antigens,which can specifically recognize the H5N1 antibody,the hydrolysis of Exo I would be inhibited,so the fluorescence of the system would not recover.The fluorescence is related to the logarithm of the H5N1 antibody concentration in the range 1.0 × 10-6 mol/L?5.0 ×10-9 mol/L with a detection limit of 1.6 × 10-9 mol/L(S/N = 3).The proposed method has been applied to detect the H5N1 antibody in serum samples with satisfactory results.In chapter 3,on the basis of the different diffusivity and existence of electrostatic repulsion between long and short DNA on the negatively changed indium tin oxide(ITO)microelectrode,a simple but sensitive immobilization free solution-phase electrochemical method for DNA methylation detection and inhibitor screening has been developed.Electroactive substance(methylene blue)tagged at the penultimate base T close to the 3'-terminal first,in the absence of DNA methylation,methylene blue-labeled electroactive fragments cannot be generated by Dpn I and results in a weak electrochemical response being detected on the ITO electrode.On the contrary,a remarkable electrochemical response can be achieved by the cleavage in the presence of DNA methylation since methylene blue-labeled electroactive fragments can be generated and aggregate on the ITO electrode.The proposed system does not need complex operation procedures such as bisulfitetreatment,PCR amplification,and electrode immobilization.Six ITO microelectrodes had been assembled on the same microchip,which can achieve the parallel detection of the same sample and improve the experimental efficiency of drug screening.The system was used to conveniently and specifically monitor the change of the DNA methylation level with high sensitivity and selectivity.The proposed system has the potential application to screen the drugs as inhibitors on the activity of methyltransferase in the clinic.In chapter 4,a versatile point-of-care assay platform was developed for simultaneous detection of multiple targets based on a microfluidic p.aper-based analytic device(?PAD)using a target-responsive hydrogel to mediate fluidic flow and signal readout.An aptamer-cross-linked hydrogel was used as a target responsive flow regulator in the ?PAD.In the absence of a target,the hydrogel is formed in the flow channel,stopping the flow in the pPAD and preventing the colored indicator from traveling to the final observation spot,thus yielding a "signal off' readout.In contrast,in the presence of a target,no hydrogel is formed because of the preferential interaction of target and aptamer.This allows free fluidic flow in the pPAD,carrying the indicator to the observation spot and producing a "signal on" readout.The device is inexpensive to fabricate,easy to use,and disposable after detection.Testing results can be obtained within 6 min by the naked eye via a simple loading operation without the need for any auxiliary equipment.Multiple targets,including cocaine,adenosine,and Pb2+,can be detected simultaneously,even in complex biological matrices such as urine.The reported method offers simple,low cost,rapid,user-friendly,point-of-care testing,which will be useful in many applications.In chapter 5,a novel method that integrates glucoamylase-trapped aptamer cross-linked hydrogel for molecular recognition with cascaded enzymatic reactions for signal amplification and a pPAD for portable readout.Upon target introduction,the hydrogel decomposes to release glucoamylase,which catalyzes the hydrolysis of amylose to produce a large amount of glucose.With a simple folding of the ?PAD,the sample solution containing glucose product wicks and diffuses in parallel to each test-zone to carry out homogeneous assays,where glucose is used to produceI2for brown color visualization through multiple enzymatic and chemical cascade reactions.Through color gradient changes based on different concentrations of the target,a semiquantitative assay is achieved by the naked eye,and quantitation can be obtained by handheld devices.Detection of cocaine in buffer and urine was performed to demonstrate the utility of the hydrogel-?PAD system.More importantly,the hydrogel-?PAD system can be extended to the detection of various targets by incorporating the corresponding aptamer into the hydrogel.The hydrogel-?PAD system reported here provides a new platform for portable,disposable and visual detection of a wide range of targets.In chapter 6,A disposable,equipment-free,versatile point-of-care testing platform,microfluidic distance readout sweet hydrogel integrated paper-based analytical device(?DiSH-PAD),was developed for portable quantitative detection of different types of targets.The platform relies on a target-responsive aptamer cross-linked hydrogel for target recognition,cascade enzymatic reactions for signal amplification,and microfluidic paper-based analytic devices(?PADs)for visual distance-based quantitative readout.A "sweet" hydrogel with trapped glucoamylase(GA)was synthesized using an aptamer as a cross-linker.When target is present in the sample,the "sweet" hydrogel collapses and releases enzyme GA into the sample,generating glucose by amylolysis.A hydrophilic channel on the ?PADs is modified with glucose oxidase(GOx)and colorless 3,3'-diaminobenzidine(DAB)as the substrate.When glucose travels along the channel by capillary action,it is converted to H2O2 by GOx.In addition,DAB is converted into brown insoluble poly-3,3'-diaminobenzidine[poly(DAB)]by horseradish peroxidase,producing a visible brown bar,whose length is positively correlated to the concentration of targets.The distance-based visual quantitative platform can detect cocaine in urine with high selectivity,sensitivity,and accuracy.Because the target-induced cascade reaction is triggered by aptamer/target recognition,this method is widely suitable for different kinds of targets.With the advantages of low cost,ease of operation,general applicability,and disposability with quantitative readout,the ?DiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring,security inspection,personalized healthcare,and clinical diagnostics.