Development Of Label-free Novel Fluorescent Biosensing Technology For The Detection Of Alkaline Phosphatase And Small Biological Molecule

Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removingphosphate groups from many types of molecules, including nucleotides, proteins, andalkaloids. ALP plays crucial roles in many biological processes involving genetictransduction, cell metabolism and neuronal functions. Adenosine-5′-triphosphate (ATP)is an important substrate in living organisms. Being a universal energy currency inliving cells, ATP plays a critical role in the regulation of cellular metabolism andbiochemical pathways in cell physiology. Therefore, sensitive and selectivedeterminations of ALP and ATP are essential for biochemical study and clinicaldiagnosis.A label free detection strategy can provide a fast and cost-effective assay whichhas gained much interest. SYBR Green I (SG) is the most frequently used DNAinteracting dye in label free assays. It has been widely applied for molecular detectionbecause of its favorable photophysical properties, thermal stability, and highsensitivity. However, it still suffers from problems caused by high background signaldue to the nonspecific adsorption, which might decrease the signal to background (S/B)ratio and sensitivity.Nowadays, graphene oxide (GO) based sensing platforms are being widelyapplied for the detection of a range of analytes attributing to a unique ability ofabsorbing DNA species as well as its super quenching capacity for a wide range offluorophores. GO could act as an effcient S/B enhancer for the label free assay.According to the above considerations and the reported literatures, thisdissertation focuses on developing a series of novel label-free biosensors for alkalinephosphatase and small biological molecule detection. The detailed methods aredescribed as follows:In chapter2, we present a label-free, simple and signal-on architecture forfluorescent alkaline phosphatase (ALP) biosensor utilizing a SYBR Green I (SG)assisted fluorescence amplification method. The strategy relies on the fact that ALPprovides a significant barrier to lambda exonuclease (λ exo) activity by adephosphorylating DNA hairpin probe (HP), and SG shows a considerablefluorescence intensity enhancement upon binding to double-stranded DNA (dsDNA)than single-stranded DNA (ssDNA). Our method is simple, sensitive and selective, which can also successfully detect the activity of ALP in complex biological fluids.The results have revealed that the method allows a specific and quantitative assay ofthe target ALP with a wide linear response range from0.4to200U/mL and a detectionlimit of0.05U/mL.In chapter3, we have developed a label-free, simple and highly sensitive hairpinfuorescent biosensor for the assay of DNA3′-phosphatases and their inhibitorsutilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer(HP) with a3′-phosphoryl end that served as the substrate for DNA3′-phosphatases.Once the phosphorylated HP was hydrolyzed by DNA3′-phosphatases, the resultingHP with a3′-hydroxyl end was immediately elongated to form a long double-strandproduct by Klenow fragment polymerase (KF polymerase). With SYBR Green I (SG)selectively staining the double-helix DNA, a very high fuorescence enhancement wasachieved. Furthermore, GO was introduced to quench the fuorescence of the HPwithout polymerase elongation, thereby further increasing the signal-to-backgroundratio. The proposed method is simple and convenient, yet still exhibits high sensitivityand selectivity. This method has been successfully applied to detecting the activity oftwo typical3′-phosphatases, T4polynucleotide kinase phosphatase (PNKP) and shrimpalkaline phosphatase (SAP). The effect of their inhibitors has also been investigated.The results revealed that the method allowed a sensitive quantitative assay of T4PNKPand SAP, with detection limits of0.07U/mL and0.003U/mL, respectively. Theproposed method is anticipated to fnd applications in the study of DNA damage repairmechanisms.In chapter4, a label free exonuclease Ⅲ (Exo Ⅲ)-aided fluorescence assay foradenosine triphosphate (ATP) was developed based on the ATP-dependent enzymaticreaction and graphene oxide (GO). This strategy relies on the principle that Exo Ⅲshows different cleavage capacity for a DNA substrate in the absence and presence ofATP and the preferential binding of GO to single-stranded DNA over double-strandedone. By combining the unique properties of SYBR Green I and GO adsorption, thissensor displays an improved sensitivity and a wide linear range covering the ATPconcentration from1nM to200nM with a low detection limit of0.2nM. Theproposed method is simple, cost-effective and convenient, which might create a newapproach for developing a sensitive ATP biosensor.