DNA-stabilized Fluorescent Silver Nanoclusters For Drug Sensing Assays

Nanoclusters are a kind of nanomaterials with the size less than 2 nm in diameter.Ascribed to the closeness to the Fermi wavelength of the electron,they possess strong fluorescence emission.Fluorescent nanoclusters usually include silver nanoclusters(AgNCs),gold nanoclusters(Au NCs),copper nanoclusters(Cu NCs),etc.Among them,AgNCs have received a lot of attention due to their broad application prospects.However,without appropriate template or stabilizer,AgNCs have a short fluorescence lifetime and tend to aggregate to form large nanoparticles,resulting in the disappearance of fluorescence characteristics.Many biomolecules such as mercaptan,dendrimers,polymers and DNA,can be used as templates for the synthesis of AgNCs.AgNCs synthesized using DNA as templates(DNA-AgNCs)have the characteristics of good biocompatibility,non-toxicity,good photostability and adjustable fluorescence,etc.,which have received special attention in recent years.In particular,the fluorescence properties of AgNCs depend on the sequences of DNA templates.Based on this characteristic,the response pattern of DNA-AgNCs to specific analytes can be regulated by selecting some appropriate DNA templates.Penicillamine is a natural amino acid containing sulfur,which is a metabolite of penicillin.It has good complexation and is used for the treatment of heavy metal poisoning.It can form complex with copper,iron,mercury,lead,arsenic and other heavy metals in the body to discharge urine.In addition,penicillamine has been shown to treat liver nuclear damage and anti-rheumatoid arthritis,as well as skin and soft tissue collagen disorders.However,the overuse of penicillamine will produce a large number of side effects on human body,so the rapid detection of penicillamine is of great significance for clinical diagnosis and medical treatment.Bleomycin(BLM)is a glycopeptide antibiotic,which has the advantages of low bone marrow suppression and low immunosuppression.BLM plays an important role in the treatment of some cancers,and while it is less toxic,overdose can cause pulmonary fibrosis and pneumonia.Activated metal-bound BLM can selectively cut DNA at the 5'-GC/GT-3'site.Based on the splicing effect of BLM on DNA,a variety of single fluorescent signal detection methods for BLM have been established,but the ratio fluorescence detection methods for BLM are rarely reported.In this paper,three kinds of fluorescence sensors have been designed with the novel DNA-AgNCs to detect D-penicillamine(D-Pen)and BLM,respectively.The specific research contents are as follows:(1)A fluorescence sensing platform for D-Pen has been constructed with DNA-AgNCs as the probe.Using single-stranded DNA as template,we synthesized DNA-AgNCs with red fluorescence emission by one-pot hydrothermal reduction of Ag~+.D-Pen can quench the fluorescence of DNA-AgNCs through static quenching mechanism,and the decrease of fluorescence intensity is proportional to the concentration of D-Pen.This method is highly sensitive to D-Pen detection,and the detection limit is as low as 8 n M,which is 1-3 orders of magnitude lower than that of other methods using fluorescent nanoprobes.In addition,the preparation time of DNA-AgNCs is shorter than that of other nanoprobes,and the method does not use heavy metal ions,which not only improves the detection efficiency but also is more environmentally friendly.This method has been successfully used for the determination of D-Pen in human serum samples and tablets with satisfactory recovery results.(2)A ratiometric fluorescence sensing platform for BLM has been constructed with transformed DNA-AgNCs as the probe.The sensing platform is constructed based on the following two processes:first,the transformed DNA can convert the green fluorescence emission of G-DNA-AgNCs to red fluorescence emission through hybridization reaction;second,BLM-Fe(II)can cleave the transformed DNA inhibiting the fluorescence changes of DNA-AgNCs.Based on the optical properties of DNA-AgNCs and the specific cleavage of BLM,a highly sensitive ratiometric detection of BLM has been successfully achieved with the detection limit as low as 30p M.This unlabeled fluorescent probe has the advantages of convenient synthesis and low cost.In addition,this ratiometric assay has been applied to the detection of BLM in human serum samples with satisfactory recovery results,providing an effective tool for the analysis and detection of BLM during cancer treatment.(3)A ratiometric fluorescence sensing platform for BLM has been constructed with double-emissive DNA-AgNCs.The sensing principle is as follows:the two ends of the hairpin DNA probe are rich in different cytosine sequences(C-rich),which are used to synthesize DNA-AgNCs.Due to the similarity of the two ends of AgNCs,the DNA-AgNCs exhibit a strong fluorescence emission at 622 nm(?ex=570 nm)and a weak fluorescence emission at 572 nm(?ex=504 nm).At the same time,the ring of the probe contains the cleavage site of BLM(5'-GC-3').When the BLM-Fe(II)complex is present,the hairpin probe is cut into two fragments,resulting in the separation of DNA-AgNCs at both ends.Thus,the fluorescence at 572 nm is enhanced while the fluorescence at 622 nm is weakened.The presence of BLM-Fe(II)induced the fluorescence color transformation of DNA-AgNCs from orange to yellow.With this method BLM can be detected sensitively with the detection limit of 67 p M.This method has been successfully applied to the analysis of BLM in serum samples with satisfactory recovery results.