Novel Fluorescent Biosensors Based On Supercharged Green Fluorescent Protein And Quantum Dots

Today, human hea lth issues attract more and more attention, and the disease isthe most important threat to the security of human life. It has been reported that thedisease are closely re lated to the abnorma l expression of b iolo gical enzymes and theconcentration level o f some important nutrients. DNA methylation is c loselyassociated with tumorige nesis and hereditary disease, and telomerase is invo lveme ntin the85-90%cancer inc idence. DNA MTase and telomerase have been regarded asa bio marker for early stage cancer diagnose as well as a potentia l target foranticancer drugs. In additio n, ascorbic acid (Vitamine C) is an essentia lmicronutrient for numerous phys io logical and bioche mical functions in human body.And the concentration leve l of ascorbic acid has been treated as a key indicator ofsome diseases and nutrition analys is. Therefore, the sens itive detection of DNAMTase, telo merase and ascorbic acid has great practical s ignificance. Based on thecons ideratio ns above, we developed three methods for label-free fluorescentdetection of DNA MTase, telomerase and ascorbic acid, respective ly, by takingadvantage of uniq ue optical properties of na no-materia ls (fluorescent protein andquantum dots). The specific works are summarized as fo llows:(1) A fluorescent analytical method for Dam MTase detection was constructed,which is based on the positive charge and fluorescence properties of superchargedgreen fluorescent protein (scGFP), and the fluorescence quenching capacity ofgrapheme oxide (GO). scGFP can be absorbed by GO through non-covale ntinteraction, fo llowed by the fluorescence quenching o f scGFP. The negative chargedDNA can bind with scGFP, resulting in the aggregation of scGFP. Such aggregatio ncan protect scGFP fro m quenching by GO and retain the fluorescence of scGFP. Onthese basis, DNA probe with rationa l design can canse effic ient dua l-enzyme (Da mMTase and Dpn I endonuc lease) coupling reactio n, whic h will induce template-freeDNA polymerization by terminal deoxynuc leotidyl trans ferase (TdT). The n theproduced long cha in DNA can bind with scGFP, leading to the aggregation of scGFP,and retain the fluorescence of scGFP. Thus, the solution fluorescence can respond tothe activity of Dam MTase. This proposed method exhib its a wide linear range(0.1-100U mL-1) and a low detection limit of0.1U mL-1, and has been successfullyapplied to the Dam MTase activity inhibition experiment by5-fluorouracil. This method has some merits, such as simple operation, high sens itivity, and no need ofcomp licated and expens ive DNA modificatio n. Furthermore, the present approachwould be potentia lly extended to TdT, endonuc lease and their suitable inhibitorsdetection.(2) Based on the positive charge and fluorescence properties of superchargedgreen fluorescent protein (scGFP), and the fluorescence quenching capacity ofG-quadruple x-hemin comp lex, a nove l fluorescent analytical method for telo merasedetection was deve lpoed. Telo merase is a ribonuc leopro tein comp lex which adds therepeats of TTAGGG on the telo meric ends of chromosomal DNA by using thetelomerase RNA component of the comple x as a temp late, which will initiateisothermal strand disp lacement to produce more sequence of TTAGGG. In thepresence of he min, this TTAGGG can form G-quadruplex-hemin complex, which canbind with scGFP through electrostatic interaction, accompanied by fluorescencequenching of scGFP. Thus, the solution fluorescence can respond to the activity o ftelomerase. This proposed method exhib its a wide linear range (100-1000cells) anda low detection limit of60cells.(3) We designed a novel fluorescence method for ascorbic acid detection, basedon the quantum dots and the reduction properties of ascorbic acid. Since ascorbicacid can reduce Pb2+, and damage the structure of PbS, fo llowed by fluorescencequenching of BSA-PbS QDs. This method enables sens itive detection of ascorbicacid concentration with the detection limit of0.83μM. Furthermore, this method hasbeen successfully applied for the detection of ascorbic acid in the practica l fruitsamp les, and satisfactory recoveries for the spiked fruit samples analys is wereobtained (between95.2%and97.9%). Compared with the previous methods, thismethod does not require comp licated process for quantum dot preparation andmodificatio n, and the detection is very fast (within2min).