Preparation Of DNA-Templated Silver Nanoclusters And Their Application In Tumor Marker Detection

In the past few years,silver nanoclusters(Ag NCs)composed of a few to tens of atoms have emerged as novel fluorescent nanomaterials.When the particle size becomes comparable to the electron Fermi wavelength of silver metal(0.5 nm),the continuous band structure of the NPs breaks into discrete energy states;as a result,Ag NPs behave like molecules and display high fluorescence intensity.The size of Ag NCs was about-2 nm.Recently,DNA templated Ag NCs have become a novel label-free fluorescent molecular beacon for wide applications,such as biolabeling,biosensing,and chemical sensing,due to their high quantum yields.While the quantum yields of DNA templated Ag NCs over 0.40 is rare.In this article,in order to acquire superior DNA templates for forming Ag NCs with high quantum yields(over 0.40),various DNA strands with intermolecular i-motif structures were designed.Based on the obtained superior DNA templates and the recognition ability of aptamers to MUC1 protein,an aptamer-functionalized Ag NCs biosensor was prepared for MCF-7 breast cancer cell imaging.In order to reduce the dependence of Ag NCs on DNA sequence,a general DNA template was designed for forming Ag NCs with high quantum yields.The specific contents were as follows.1.Synthesis of Ag NCs using DNA templates with intermolecular i-motif structuresFive DNA libraries were designed through adjusting loop bases and tetraplex structure numbers of the intermolecular i-motif structure.Ag NCs with emision wavelength at 525-660 nm were formed using DNA libraries as templates.Some of the synthesized Ag NCs have high quantum yields,such as the quantum yields of red fluorescence Ag NCs stabilized by C4-ATAT-C4,C4-AAAT-C4,C4-AAAA-C3 and C4-AAAT-C3 were 0.78,0.48,0.56 and 0.75;the quantum yields of green fluorescence Ag NCs stabilized by C4-ATAT-C3,C3-TATA-C4 and C4-ATAA-C3 were 0.45,0.41 and 0.44,respectively.The fluorescence properties of the synthesized Ag NCs were affected by loop bases and tetraplex structure numbers of the intermolecular i-motif structures.The loop bases enhanced the stability of intermolecular i-motif structures and also promoted Ag NCs formation with high quantum yields.2.Synthesis of aptamer-functionalized Ag NCs for MCF-7 breast cancer cells imagingFour superior DNA(C4-AAAA-C3,C4-ATAT-C3,C4-AAAT-C3 and C4-ATAT-C4)templates were connected with MUC1 aptamer via poly A or T loop for obtaining aptamer-functionalized Ag NCs.Among four superior DNA templates,only C4-AAAA-C3 template was connected with MUC1 aptamer using-AAAAA-and could stabilize bright red fluorescence Ag NCs.This result indicated that MUC1 aptamer sequence could quench DNA templated Ag NCs fluorescence.Using C4A4C3-A5-MUC1 as template,the synthesized Ag NCs showed highest fluorescence intensity and could differentiate MCF-7 breast cancer cells(over expression of MUC1 protein)from MDA-MB-231 breast cancer cells and A549 human lung cancer cells.3.A general DNA template design strategy for synthesis of Ag NCs with high quantum yieldsA general DNA template was designed for forming red fluorescence Ag NCs with high quantum yields.The general DNA template was composed of nucleating sequence,linker,and G-rich sequence.The nucleating sequences which consist of C-rich sequence were used for forming Ag NCs.The linker was composed of adenine or thymine.The role of the G-rich sequence was activating Ag NCs fluorescence.Under the rule of design strategy,the obtained DNA template could stabilize red fluorescence Ag NCs with high quantum yields(over 0.60).The Ag NCs with highest quantum yields was stabilized by the C4-ATAT-G15 template(quantum yield was 0.91).4.A label-free fluorescent probe for tumors gene detection based on general DNA template stabilized Ag NCsDue to the fact that nucleating sequence of general DNA tenplate colud not form Ag NCs with bright fluorescence after hybridizing with its complement DNA,a DNA probe was obtained through adjusting the nucleating sequence to hybrid with G-quadruplex(an important tumor marker).The obtained DNA probe templated Ag NCs could detect c-kit gene(a marker of gastroenteric tumor),and the limit of detection was 0.6 nM.Based on that the activation ability of G-rich sequence will disappear due to the formation of double strand structure,the G-rich sequence could be designed to recognize i-motif structure whose single nucleotide mutation could lead to cancer.The BCL mutation gene(cause gastric cancer)was selected as the target,and the limit of detection was 0.8 nM.