Novel Biosensing Technology Based On Metal Nanomaterials

The development of biosensors has greatly promoted the research for biologicalproblems due to their high sensitivity, quick response, high selectivity, no need ofpretreatment, easiness in real-time online monitoring. Recently, the new emergenceand development of nanomaterials have attracted more and more attention because oftheir special physical and chemical properties. Combination of the advantages ofnanomaterials such as gold nanoparticles, silver nanoclusters, etc and electrochemicalor fluorescent biosensors, several electrochemical or fluorescent detection methodshave been developed for the detection of several life-related materials such aspolynucleotide kinase, micrococcal nuclease, cholesterol, etc. Compared withtraditional methods, these developed methods described in the present dissertation aremore sensitive and convenient, with enhanced selectivity. The detailed contents aredescribed as follows:(1) Based on the phosphorylation-induced DNA digestion by λ exonuclease and adual signal amplification using streptavidin–gold nanoparticle biocomplexes andalkaline phosphatase, an electrochemical method for the determination ofpolynucleotide kinase (PNK) activity has been proposed (Chapter2). In the presenceof PNK, the5’-terminus of the immobilized PNK substrate probe was phosphorylated,and the substrate probe would be cleaved by λ exonuclease after being hybridized withthe complementary detection probe biotinylated in the3’-hydroxyl terminus. As aresult, the detection probe would be released, with an electrochemical signal decreased,due to less biotinylated alkaline phosphatase loading on the electrode surface. Theelectrochemical signal exhibited a linear correlation to the logarithm of PNKconcentration ranging from0.01U/mL to5U/mL with the detection limit of0.01U/mL.The inhibiting effect of (NH4)2SO4on the activity of PNK was also evaluated.（2）Based on the digestion of phosphorylated terminus by λ exonuclease and thecycling cleavage of a molecular beacon by the autocatalytic DNAzyme, a fluorescentmethod for PNK is established (Chapter3). In the presence of PNK, the5’-terminusof C-DNA would be phosphorylated, which would be digested by λ exonuclease afterbeing hybridized with E-DNA. And then E-DNA would be released and catalyzed thecycling cleavage of the molecular beacon in the presence of Mg2+, resulting in thestrong fluorescence. Under the optimal experimental conditions, the fluoresence intensity exhibited a linear correlation to the PNK concentration ranging from0.5U/mLto100U/mL with the detection limit of0.16U/mL.（3）Based on the fluoresence quenching by H2O2, a new fluorescent method forcholesterol has been developed (Chapter4). At first, the DNA scaffold for silvernanoclusters (Ag-NCs) was optimized based on the reported one. The experimentalresults showed that when the DNA scaffold was modified with one or two guaninebases at the3’-end, strong fluorescence intensity was obtained at605nm. We alsofirstly found that H2O2could quenched the fluorescence of Ag-NCs. According to thisfinding, we synthesized Ag-NCs based on the optimized scaffold, which was used as afluorescent indicator for the determination of cholesterol. The fluorescence intensityexhibited a linear correlation to the logarithm of cholesterol concentration rangingfrom0.2μM to200μM with the detection limit of0.15μM and the linear correlationcoefficient is0.9943. This method can be extended to the determination of othermolecular species.（4） Based on the digestion of single-stranded DNA scaffold of AgNCs bymicrococcal nuclease (MNase), a novel, sensitive label-free fluorescent method hasbeen presented(Chapter5). The ssDNA was introduced as the substrate for MNaseand also as the scaffold for the synthesis of the AgNCs. In the absence of MNase, thessDNA was not digested. As a result, the fluorescent AgNCs were formed andexhibited strong fluorescence. In the presence of MNase, the DNA was digested, whichprohibited the formation of the AgNCs due to the lack of the DNA scaffold, resultingin weak fluorescence. The fluorescence intensity exhibits a linear correlation to MNaseconcentration in the range of1×10-5U/mL to2×10-4U/mL with a detection limit of8×10-6U/mL. Given its simplicity, easy operation, sensitivity and cost-effectiveness,this method can be extended to other nuclease assays.（5）An electrochemical method for microRNA (miRNA) has been proposed with adual signal amplification strategy relied on polymerase extension and two-step signalamplification using streptavidin-gold nanoparticle biocomplexes and alkalinephosphatase(Chapter6). The target miRNA can hybridize with the capture DNAtemplate, which can act as a primer and be extended along the template in the presenceof DNA polymerase and dNTPs. Biotin group was introduced into the duplex due tothe incorporated biotin-11-dUTP. Thus, biotinylated alkaline phosphatase (biotin-ALP)would bind to the duplex using SA-AuNPs as linkers, which resulted in an amplifiedelectrochemical signal. The electrochemical signal exhibited a linear correlation to thelogarithm of miRNA concentration ranging from1pM to10nM, with the detection limit of0.9pM. The specificity of the method allowed single-nucleotide differencebetween miRNA family members to be discriminated. The established biosensordisplayed excellent analytical performance toward miRNA detection and might beused as a convenient tool for biomedical research and clinic diagnostic application.（6） Based the excellent separation–enrichment of magnetic nanoparticles andDNA–SG (SYBR Green I)-based signal amplification, a new fluorescent method forbiotin has been established(Chapter7). Biotin in the sample and the biotinylateddsDNA would compete for the binding sites of streptavidin coated on magnetic bead(SA-MB) surfaces. Followed by magnetic separation, the unbounded dsDNA would bewashed away from the magnetic nanoparticles and SYBR Green I would intercalateinto the bound DNA, resulting in the strong fluorescence emission, which is inverselyrelated to biotin concentrations. Under the optimal experimental conditions, thefluorescence intensity at532nm decreases with the increase of biotin concentration,and the linear regression equation is F=-16.62C+466.8with the linear correctioncoefficient of0.9971and the detection limit of1.19ng/mL, which is comparable to ormore sensitive than traditional methods. Moreover, we also used the proposed methodto measure the biotin level in actual samples, for example flour and peanuts, for whichsatisfactory results were obtained.