Novel Fluorometric Biosensors Based On DNA-templated Metal Nanoclusters

Metal nanoclusters (NCs) are smaller than2nm which contain from two to tenmetal atoms. In recent years, DNA templated silver nanoclusters (DNA-AgNCs) haveattracted extensive attention in biosensors and chemical sensors. DNA-AgNCs consistof a few silver atoms and possess many excellent fluorescent properties includingtunable fluorescence emission, good quantum yield, high photostability, low biologicaltoxicity and excellent biocompatibility. Based on all these advantages, DNA-AgNCshave been applied to detect biomolecules, metal ions, DNA, RNA and proteins, andfurthermore biolabeling and bioimaging. Compared to traditional organic fluorescnetdyes and fluorescnet nanoparticles, the synthesis and property of tunable DNA-AgNCsare simple and convenient. Therefore, DNA-AgNCs possess extensive potential in theapplication of biosensors and chemical sensors. Based on DNA-AgNCs andDNA-Cu/AgNCs, the following biosensor research was carried out:1．A novel acetylcholinesterase (AChE) activity sensor was constructed based onthe fluorescence quenching of DNA-Cu/AgNCs. In this assay, AChE catalyzes thehydrolysis of acetlythiocholie (ATCh) to form thiocholine which contains sulfhydryland induces the fluorescence quenching of DNA-Cu/AgNCs. Therefore, the activity ofAChE can be monitored through this indirect mode. The fluorescence quenchingmechanism of AChE activity detection is that sulfhydryl of thiocholine has strongaffinity with silver atom, and thus the produced thiocholine can associate with thesilver atoms of DNA-Cu/AgNCs leading to fluorescence quenching ofDNA-Cu/AgNCs. The fluorescence intensity change of DNA-Cu/AgNCs shows goodresponse to the activity of AChE. Compared to many previous methods, this methodhas a lower detection limit of0.05mU/mL, and the linearity range is from0.05mU/mL to2.0mU/mL. Two typical AChE inhibitors (tacrine and methamidophos)were detected using this AChE assay described above. The IC50value of tacrine wasestimated to be16.9nM which is close to those by other reported methods. Thecorresponding IC50value of methamidophos was obtained to be about0.075mg/L, andits low detection limit was0.0001mg/L, which is much lower than the safetymethamidophos content in food. Furthermore, this method was also applied to detectspiked methamidophos in agricultural products successfully and the results weresimilar with that obtained in pure water. In this assay, the synthesis of DNA-Cu/AgNCs is simple, and the DNA templates used do not require thelabor-intensive and expensive modification with any label. This sensor is simple,cost-effective, label-free and with high sensitivity for the detection of AChE activityand its inhibitor.2．A novel DNA-AgNCs synthesis strategy was developed based on terminaldeoxynucleotidyl transferase (TdT) which can catalyze the sequential addition ofdNTPs to the3’ hydroxyl terminus of DNA. This assay was successfully applied to thedetection of protein combined with protein aptamer. In this assay, TdT was employedto catalyze the sequential addition of natural deoxycytidine triphosphate (dCTP) at the3′-OH of a single-stranded DNA primer which cannot be used as template forDNA-AgNCs synthesis. Due to the fact that cytosine-rich DNA can be used astemplate for DNA-AgNCs synthesis, then the cytosine-rich sequential addition DNAcatalyzed by TdT can be employed as template for the flurescent DNA-AgNCssynthesis. Furthermore, to demonstrate that this DNA-AgNCs synthesis assay haspotential for protein detection, a novel approach for protein detection based on theassay described above and protein aptamer was developed. In this protein detectionapproach, thrombin was chosen as the model analyte of interest.