Novel Turn-on Fluorescent Biosensing Methods Based On The DNA-Au Nanoclusters

The metal nanocluster of DNA template is composed of several to hundreds of metal atoms.Its size is between metal atoms and nanoparticles.In addition,it is a transition form between atoms and nanoparticles.The metal nanoclusters of DNA template mainly include silver nanoclusters(DNA-Ag NCs),copper nanoclusters(DNA-Cu NCs)and gold nanoclusters(DNA-AuNCs)of DNA template.DNA-AuNCs as a new type of fluorescent nanoprobe has great potential application value in the fields of biosensor and biological imaging because of its small size,low toxicity,high fluorescence intensity,strong photobleaching resistance and excellent optical properties.Taking A30 DNA as a template,high fluorescence intensity DNA-AuNCs was quickly synthesized.A general platform of unlabeled multiplex molecular logic gates based on DNA-AuNCs was designed,and a series of signal enhanced fluorescence sensing platforms were constructed for sensitive detection of L-histidine,protamine and trypsin activity.The main research contents were as follows:1.DNA-AuNCs were used as fluorescent probes,take advantage of Hg²⁺could interact with biothiols/melamine to form more stable Hg(II)-thiols/Hg(II)-melamine coordination complexes.Similarly,Cu²⁺also acted with pyrophosphate(PPi)to form a stable Cu(II)-PPi coordination complexes.We have designed and constructed a universal platform of label-free multiple molecular logic gates(including NOR,XNOR,IMPLY,OR,AND,INHIBIT).These greater coordination interactions could inhibit the Hg²⁺/Cu²⁺-mediated fluorescence quenching of DNA-AuNCs,leading to the enhancement of fluorescence intensity.These molecular logic gates utilize Hg²⁺,Cu²⁺,biothiol,melamine,and pyrophosphate(PPi)as signal inputs,and the signal output is defined according to the fluorescence intensity of DNA-AuNCs.Through the signal output results,the universal platform can sensitively detect Hg²⁺,melamine,Cu²⁺and PPi with the detection limits of 3 nmol/L,15 nmol/L,60 nmol/L and 350 nmol/L,respectively.In addition,these logic gates have the advantages of low cost,operation simply and flexible design.2.By the interaction of L-histidine with DNA template,the fluorescence signal of DNA-AuNCs was significantly improved,and a novel signal-enhanced fluorescence sensing strategy was constructed for sensitive detected of L-histidine and imidazolyl drugs.In the presence of L-histidine,DNA-AuNCs exhibited a strong fluorescence emission signal at 475 nm,and its fluorescence quantum yield(QY)increased from1.9%to 6.5%.The enhanced fluorescence of DNA-AuNCs was mainly attributed to the interaction between L-histidine and DNA,which results in the altered conformation of the A30 DNA template,thus providing a better microenvironment for the protection of AuNCs.The linear range was 1?50 nmol/L,and the detection limit was 0.3 nmol/L.The relative standard deviation(RSD)of the three repeated measurements was less than4.5%.In addition,the method can also be used for the determination of imidazoles in commercial drug samples.3.Based on the interaction between cationic polyelectrolyte and DNA template,a new type of fluorescent sensing platform was designed to detect protamine and trypsin sensitively.When the cationic polyelectrolyte is present,the negatively charged DNA backbone in DNA-AuNCs and the positively charged cationic polyelectrolyte will be electrostatically attracted,resulting in a change in the spatial conformation of the DNA template,thereby providing a better microenvironment for stability DNA-AuNCs.Therefore,the fluorescence intensity and stability of AuNCs are improved at the same time,and there is almost no change in the maximum excitation wavelength and emission wavelength.Similarly,when positively charged protamine is present,the fluorescence intensity of DNA-AuNCs increases,and trypsin can catalyze the hydrolysis of protamine to form polypeptide fragments,which leads to a decrease in the fluorescence intensity of AuNCs.This strategy realized the sensitive detection of protamine and trypsin activity.The linear equation of protamine is F/F₀=0.2419C+0.9784,and the detection limit is1.7?g/mL,The recovery was 98.4%?101.2%.The linear equation of trypsin activity was F₀-F=0.0152C+1.0012,the linear range was 3?18 m U/mL,the detection limit was 1 m U/mL,and the recovery was 98.6%?102.6%.