Construction Of Fluorescent Biosensors Based On DNA-templated Copper Nanoparticles

Recently,fluorescence biosensors have attracted considerable interest because of their rapidity,sensitivity and simplification.In addition,due to the ultrafine size,low toxicity,good biocompatibility,outstanding photophysical properties,metal nanoparticles can observably enhance the analytical performances of fluorescence biosensors.Biomaterials including protein,peptide and DNA,can serve as templates to synthesize numerous metal nanomaterials.DNA-templated nanomaterials become a better choice with excellent biological sensing advantages,due to its inherent molecular recognition,designable sequence and simple conformation.Compared with DNA-templated silver,gold and platinum nanoparticles,the synthesis process of DNA-templated copper nanoparticles(CuNPs)is shorter,milder and easier.With the development of self-assembly technique and fluorescence methods,DNA manipulation can be studied deeply and construction of DNA-templated CuNPs biosensor could be further widely applied in various biomedical and environmental aspects.Using alkaline phosphatase(ALP),mercury ions(Hg2+)and microRNA as targets,respectively,based on the DNA-templated CuNPs as fluorescence indicators and induced enzyme catalysis reaction,these works design biosensors with high sensitivity and excellent specificity.1.The activity of ALP is a crucial index of blood routine examinations,since the concentration of ALP is highly associated with various human diseases.Recently,we find that fluorescence of poly(thymine)(poly(T))-templated CuNPs can be directly and effectively quenched by pyrophosphate ion(PPi).In addition,it has been confirmed that phosphate ion(Pi)does not affect the intense fluorescence of CuNPs.Since ALP can specifically hydrolyze PPi into Pi,fluorescence of CuNPs is thus regulated by an ALP-triggered reaction,and a novel ALP biosensor is successfully developed.As a results,ALP is sensitively and selectively quantified with a wide linear range of 0.06 to 600 U/L and a low detection limit of 0.035 U/L.Besides,two typical inhibitors of ALP are evaluated by this analytical method,and different inhibitory effects are indicated.More importantly,by challenging this biosensor with real human serums,the obtained results get a fine match with the data from clinical tests,and the serum sample from a patient with liver disease is clearly distinguished,suggesting promising applications of this biosensor in clinical diagnosis.2.Concentration of Hg2+ in the environment dramatically varies from picomolar to micromolar.Herein,reticular DNA is constructed by introducing thymine(T)-Hg2+-T nodes in poly(T)DNA,and CuNPs with aggregate morphology are prepared using this reticular DNA as a template.Intriguingly,the prepared CuNPs exhibit enhanced fluorescence.Meanwhile,the reticular DNA reveals evident resistance to enzyme digestion,further clarifying the fluorescence enhancement of CuNPs.Relying on the dual function of DNA manipulation,a high signal-to-noise ratio biosensor is designed.This analytical approach can quantify Hg2+ in a very wide range(50 pM to 500 ?M)with an ultralow detection limit(16 pM).Besides,depending on the specific interaction between Hg2+ and reduced L-glutathione(GSH),this biosensor is able to evaluate the inhibition of GSH toward Hg2+.In addition,pollution of Hg2+ in three lakes is tested using this method,and the obtained results are in accord with those from inductively coupled plasma mass spectrometry.3.This work reports the fact that 3'-phosphate group-linked poly(T)(30T-P)can effectively prevent the digestion of exonuclease ?(Exo ?).Considering the specific hydrolysis of ALP,this strategy is demonstrated by introducing alkaline phosphatase(ALP)as a model.In this assay,the phosphate group of 30T-P can be hydrolyzed in the presence of ALP,and then the DNA would be digested by Exo ?.Hence,the activity of ALP can be quantified based on the fluorescence intensity of CuNPs with a wide linear range from 0.067 to 665.64 U/L and a low detection limit of 0.03 U/L.In order to further research,taking 30T-antiRNA-P as a template to instead 30T-P,we combine the amplification strategy of duplex-specific nuclease(DSN)with the phosphate group terminal protection together,and construct a highly sensitive and selective biosensor for microRNA-21 detection.In the presence of target microRNA,3 0T-antiRNA-P entirely hybridizes with microRNA-21 and forms heteroduplexes.Then,DSN subsequently cleaves the heteroduplexes to recycle the microRNA-21,which lead to continually exposure of 3' terminal of DNA without phosphate group,and the fluorescence signal would decrease due to the digestion of Exo ?.The proposed method reveals an advantageous linear range from 25 aM to 250 nM for quantifing microRNA-21 with a low detection limit of 16 aM.Importantly,the strategy can significantly distinguish cancer which is correlated with microRNA-21 and which is non-correlated.Moreover,this method also can indicate the cure and restore situation of postoperative.On the basis of these research foundations,we believe this proposed study holds great promise of becoming a routine tool for early diagnosis of cancer and post-operative evaluation.In conclusion,combined the fluorescence regulation of CuNPs with manipulation of enzyme,this paper establishes several biosensors for different targets with excellent sensing performances.