Functionalized Fluorescence Nano-biosensors Based On Semiconductor Quantum Dots And Graphene Quantum Dots And Their Application In Biomedical And Analytical Field

Nanobiotechnology is one of the fastest growing and broadest-ranged interdisciplinary subfields of the nanosciences.Meanwhile,fluorescence is a rapidly growing research field in nanobiotechnology.Recent advances in nanotechnology and photonics have led to a new generation for fluorescent nanobiosensor using different kinds of nanomaterials.As a kind of popular fluorescent nanoparticles,semiconductor quantum dots?QDs?have been widely concerned since their emergence.The outstanding properties such as strong signal intensity,high quantum yield,tunable size-dependent photoluminescence and narrow emission peaks make QDs a superior tool in the applications of life science.Labeled as a new sort of fluorescent carbon-based material,graphene quantum dots?GQDs?have attracted increasing attention in recent years.As a novel nanomaterial for biosensors,GQDs has excellent biocompatibility,remarkable optical properties and convenient surface modification.Serving as the most representative nanomaterial for biosensor,they have been applied in numerous areas: water quality monitoring,biological sensing,clinical diagnosis,drug delivery,gene diagnosis,bioimaging,etc.Furthermore,by functionalizing diverse molecules or materials onto the surface of them,surface-functionalized QDs orGQDs may participate in the subsequent attachment of target entities by affinity interaction,making specific detections possible.The modification of QDs and GQDs could expand their application in biosensing and help to build biosensors towards a multitude of target analytes.Here,we developed several kinds of biosensor based on functionalized QDs orGQDs and applied them in biomedical fields for the determination of biological molecules and enzymes monitoring.Details are as follows:1.We briefly introduce two kinds of most popular nano materials?semiconductor quantum dots and graphene quantum dots?,which are currently used in fluorescent nano biological sensors.Their characteristics,synthesis methods,luminescent mechanism,surface functionalization and the applications in the field of biomedical analysis are discussed in detail.2.A simple and sensitive fluorescence probe for adenosine 5'-triphosphate?ATP?detection was designed.A series of L-cysteine capped CdTe quantum dots?QDs?with different sizes were synthesized in aqueous phase.The effects of Zn²⁺ on different sizes of L-cysteine capped CdTeQDs were investigated and the fluorescence of CdTe with small size QDs could be effectively quenched due to the binding of Zn²⁺ to the L-cysteine on the surface of the QDs and the electron transfer from the photoexcited QDs to Zn²⁺.The phosphate groups of ATP have a high affinity for zinc ions through Zn-O-P bonds,with the addition of ATP,Zn²⁺ could bind with ATP based on the metal-ligand coordinative effect and the modulation of Zn²⁺ to CdTeQDs would be inhibited.Hence the quenched fluorescence of the CdTeQDs can be recovered.The recovered fluorescence of QDs was proportional to the concentration of ATP in the range of 5-50 ?mol L^-1 and the detection limit for ATP was 2.07 ?mol L^-1.The proposed sensing assay showed a good selectivity for ATP over other biologically important phosphates.It was applied to the determination of ATP in human serum sample with satisfactory results.3.A simple,convenient and high sensitive “turn-on” fluorescence platform for trypsin assay based on N-acetyl-L-cysteine capped CdTe Quantum Dots?NAC-CdTeQDs?was designed.A series of NAC-CdTeQDs with different sizes were synthesized in aqueous via refluxing routes.The fluorescence of NAC-CdTeQDs could be effectively quenched by hemoglobin?Hb?via the electron transfer interactions from NAC-CdTeQDs to hemoglobin due to the formation of QDs/Hb complex.In the presence of trypsin,hemoglobin would be hydrolyzed to small peptides,releasing inactive hemin molecules,which has slight effect on the fluorescent intensity of NAC-CdTeQDs,so the combination of hemoglobin and NAC-CdTeQDs would be dissociated,and leading to the fluorescence recovery of NAC-CdTeQDs.Therefore,we could monitor the trypsin activity by utilizing the different responses of NAC-CdTeQDs to hemoglobin and hemin.The recovered fluorescence intensity of NAC-CdTeQDs was proportional to the logarithm of trypsin concentration in the range of 0.2⁴0 ng m L^-1 and the detection limit for trypsin was 0.144 ng m L^-1.A model for trypsin inhibition was further established to verify the feasibility of the system.The IC50 values are 3.06 ?g m L^-1 for the inhibitor from soybean.Thus,a label free real time fluorometric assay for trypsin and inhibitor screening has been developed.The established method showed a high selectivity and sensitiveness for trypsin over other biological relevant enzyme,and it was applied to the determination of trypsin in human urine sample with satisfactory results.4.We developed a dopamine functionalized CdTe quantum dots as a fluorescence probe for the determination of L-histidine.Firstly,CdTe were covalently linked to dopamine to form a kind of fluorescence sensor with pyrocatechol structure on the surface.The photoluminescence intensity of CdTe-dopamine?QDs-DA?could be quenched by Ni²⁺ due to the strong coordination interaction between the pyrocatechol structure of QDs-DA and Ni²⁺.In the presence of L-histidine,Ni²⁺ preferred to bind with L-histidine for the high affinity of Ni²⁺ to L-histidine and the photoluminescence intensity of QDs-DA is recovered.The recovered photoluminescence intensity of QDs-DA was proportional to the concentration of L-histidine in the ranges of 1.0×10-6¹.0×10-4 mol L^-1 and the detection limit was 5.0×10-7 mol L^-1,respectively.The established method showed a good selectivity for L-histidine among other common amino acids,and it was applied to the determination of L-histidine in human serum sample with satisfactory results.5.A facile and rapid fluorescent assay based on redox reaction for successively detecting ascorbic acid and acid phosphatase was developed via Cr?VI?modulated grapheme quantum dots?GQDs?.Graphene quantum dots with yellow-green emission have been firstly synthesized via one-pot hydrothermal method.Based on the electrostatic adsorption of Cr3+ to GQDs and the strong chelation between Cr3+ and the-COOH and-OH groups on the surface of GQDs,the fluorescence of GQDs could be greatly quenched by Cr3+ ions.By the introduction of ascorbic acid,Cr₂O₇₂-could be reduced into Cr3+,which resulted in a turn-off fluorescence signal of GQDs.The quenched fluorescence intensity of GQDs was proportional to the concentration of ascorbic acid.Dynamic detection range for ascorbic acid is from 0.5²50 ?mol L^-1 with the detection limit?LOD?of 0.28 ?mol L^-1.Moreover,this phenomenon is further exploited for sensitive and selective detection of acid phosphatase?ACP?.L-ascorbic acid-2-phosphate?AAP?,a more stable phosphatase substrate,could be hydrolyzed by ACP to give ascorbic acid.Ascorbic acid then reduced Cr₂O₇₂-to Cr3+,leading to the quenching of GQDs fluorescence.Thus,the amount of ACP could be indirectly detected in a range from 0.02 to 3 m U m L^-1 with the LOD of 8.9 ?U m L^-1.Thus,a Cr?VI?modulated GQDs “turn-off” fluorescence sensor for ascorbic acid and ACP was constructed.The present strategy showed high selectivity for ascorbic acid and ACP.The feasibility of the proposed sensing system in real samples assay was also studied and satisfactory results were obtained.6.In the current study,reduced graphene quantum dots?rGQDs?with blue emission have been prepared by chemically reducing GQDs with Na BH4.The integration of rGQDs and biopolymers via self-assembly/disassembly offers an excellent design of label-free biosensors for alkaline phosphatase.The disc-shaped GQDs act as both the fluorescent elements and the self-assembly building blocks.The charged biopolymer used in this work is chitosan,which exhibits both electrostatic attraction and structure transformation-induced fluorescence quenching of the r-GQDs at the same time.Meanwhile,sodium hexametaphosphate??Na PO3?6?,which is used as the disassembly agent of the hybrid in this work,also serves as the substrate of the phosphatase to provide its biological functions.In the presence of?Na PO3?6,it competes with r-GQDs for the binding sites on the chitosan,which results in the disassembly of the combination between r-GQDs and chitosan.Consequently,the r-GQDs exhibit a turn-on fluorescence signal.By introducing alkaline phosphatase?ALP?into the system,?Na PO3?6 could be hydrolyzed to give phosphate anions,leading to the self-assembly of r-GQDs and chitosan for the second time,then the fluorescence signal of GQDs is decreased again.The quenched fluorescence intensity of GQDs was proportional to the concentration of ALP.Dynamic detection range for ALP is from 20⁵00 m U m L^-1 with the detection limit?LOD?of 7.8 m U m L^-1.Therefore,a?Na PO3?6 modulated “turn-off” fluorescence biosensor based on self-assembly/disassembly mode of r-GQDs/chitosan system for ALP was constructed.The present strategy showed high selectivity for ALP.When applied to real samples analysis,it exhibited satisfactory results.The developed approach displays merits such as good selectivity,low cost,free of toxins and a broad detection range.It also provides a new prospect on the application of the hybrid system of GQDs and biopolymers.