Application Of Silver Nano Mate Rials In Cell Apoptosis And Enzymatic Biosensor

In recent years,biomacromolecules(DNA and peptides)have been widely used in the synthesis of metallic nano materials due to the interactions with group IB metals(gold,silver and copper),and they have attracted much attention in the field of analytical chemistry.Silver ions has a strong binding force on N3 of the cytosine,so that it can selectively bind to the heterocyclic base in the DNA oligonucleotides.Under reducing conditions,Ag atoms bind to cytosines of oligonucleotide very tightly,obtaining the DN A-stabilized silver nanoclusters.As a new class of fluorophores,DN A-templated silver nanoclusters have excellent properties,such as large Stokes shifts,photostability,tunable fluorescence emission and easy of synthesis,attracting a large amount of research interests.Combining the molecular recognition function of DNA sequences with the characteristics of nanocluster templates,DNA/AgNCs can be widely used for biosensing and imaging.In addition,cysteine and other thiols can be used as a thiol ligand to coordinate with Ag,forming the Ag-thiol coordination polymer.At present,some work has been done by using Ag(I)-S(R)coordination polymers for biosensing such as ions,small molecules and bioactive molecules,but the research is still relatively limited.In this article,based on the affinity of silver with cytosine bases and the coordination of silver with thiol group in thiols,we synthesized DNA/AgNCs and Ag(I)-S(R)coordination polymers,respectively.Furthermore,the metal nano material was developed for analytical system construction in detecting human health-related disease-apoptosis or enzyme testing.The concrete examples are as follows:1.Based on enzyme-polymerized polyadenylic acid(poly-dA)DNA chain and toehold strand displacement reaction,we constructed a novel DNA/AgNC probe for DNA.Terminal deoxynucleotidyl transferase(TdT)is a template-free DNA polymerase that catalyzes the addition of dNTP molecules to the 3' hydroxyl terminus of DNA.The extended poly-dA DNA chains can reach to 400-base produced by TdT-activated polymerization.The strand displacement initiated by the target poly-dA DNA chain releases the quencher labeled-DNA from DNA/AgNCs probe,leading to a significant fluorescence lighting-up of DNA/AgNCs for sensitive detection of single strain DNA with a high signal-to-background ratio(S/B = 58),and a detection limit of 0.2 nM.2.Considering many DNA fragmentations producing during apoptosis,we further used DNA/AgNC probe for in situ apoptosis detection and imaging.In the section,dATP was used as a substrate.and the genomic DNA fragment,a biochemical hallmark of apoptosis,was tagged with a polyadenylation fragment(poly-dA)by TdT mediate polymerization.The extended poly-dA can specifically activate the DNA/AgNC probe,as low as 20 apoptotic cells can be detected in vitro.We further applied this method to in situ imaging of HepG2 cell apoptosis,and the quantitative results were consistent with those of the commercial kit method.Compared with the traditional TUN EL analysis,this assay is a "Turn-on" detection mode and does not require marking of dNTP monomers,thus avoiding cumbersome washing and separation steps.3.Based on the coordination of Ag(1)with thiol peptide and the aggregation quenching properties of rhodamine dyes,we developed a simple and rapid assay for the thrombin activity.The peptide containing an N-terminal tag TAMRA and a C-terminal cysteine residue was used as thrombin substrate.The silver ions are coordinated with the cysteine in the peptides,forming Ag-peptide coordination polymers.And adjacent TAMRA in coordination polymer forms J-dimer,resulting in the quenching of TARMA fluorescence.After the thrombin cleavaging,TAMRA was released,destroying the J-dimer of TAMRA,with the recovery of TAMRA fluorescence.The method for thrombin activity has a good linear range of 20-300 pM and a detection limit of 5 pM.Compared with traditional dual label peptide fluorescent probes,this method only needs to mark a single fluorescent group,which greatly reduces the detection cost.