| Fluorescent assays combined with nanoparticles possess a series of advantages over traditional analysis methods, such as facile, sensitive and specific and so on. Recently, advances in nanoscience and nanotechnologies have provided a great number of luminescent nanomaterials, such as quantum dots (QDs), dyes-encapsulated silica nanoparticles, upconversion nanoparticles, single walled carbon nanotubes and nano-graphene oxide. These fluorescence materials could provide the most powerful tool for particularly fluorescent analysis methods. Nevertheless, most of them still have the disadvantages of the ineluctable covalent conjugation to allow the application in fluorescent methods. The multistep complex process is time-consuming and labor-intensive. Besides, combined with functionalized nanomaterials may decrease the affinity performance of the aptamers. Therefore, developing label-free fluorescent detection strategy will be more encouraging. Recently, oligonucleotide-templated AgNCs, typically possessing sizes below2nm and exhibiting outstanding spectra and biocompatibility, have attracted special attention in chemical-sensing and biomedical imaging. Additionly, DNA could specifically bind with lots of tagets because of the chemical groups, base-sequence or configuration of DNA, such as,"T-Hg2+-T""C-Ag+-C" complex structure and aptamer obtained from SELEX. Consequently, in order to realize a novel research thinking of label-free fluorescent analysis methods, this paper employed DNA as recognition ligand and DNA-templated silver nanoclusters as the fluorescence signal to design the label-free assay platforms for ions, micromolecules, tumor cells. The main research aspects are as follow:1. Highly sensitive label-free fluorescent detection of Hg2+ions by DNA molecular machine-based Ag nanoclustersWe presented here a highly selective and sensitive label-free method to detect Hg2+ions in aqueous solution by using DNA molecular machine-based fluorescent Ag nanoclusters (AgNCs). This mechanism is based on the Hg2+ions triggering machine-like operations of DNA and the "product" of the machine being used to stabilize fluorescent AgNCs. In this method, a tailored DNA, containing a sequence for Hg2+ions recognition, a sequence-specific nicking site for Nb BbvC I and a sequence complementary to the DNA as a template for the synthesis of fluorescent AgNCs, was firstly designed. In the presence of Hg2+ions, the machine’s function operations were triggered. A series of machine-like operations, including replication, scission, and displacement then occurred with the addition of polymerase/dNTPs/Nb BbvC I, which manufactured lots of "product" DNA. The "product" DNA could act as a template for the preparation of fluorescent AgNCs. Thus the fluorescence of the AgNCs could be used as a signal transduction of this DNA machine, which was related to the concentration of the Hg+ions. The repeated synthesis of the "product" and its template effect for AgNCs synthesis led to signal amplification in the assay of Hg2+ions. A linear response to the concentration of Hg2+ions was observed in the range from0.08nM to20nM and a detection limit of0.08nM was obtained. By contrast, the operation of the machine could not be executed in an Hg2+ion-free system. Moreover, the detection was not only label-free but also specific for Hg2+ions without being affected by other metal ions..2. DNA-templated Ag nanoclusters fluorescence probes for highly selective detection of Pb2+ions.Pb2+is one of severe environmental pollutant and cause acute damage to liver, kidneys and nervous system. Therefore, it is important to develop simple and rapid detection method for Pb2+ions. Herein, we have established a facile strategy for detecting Pb2+ions, which was upon the selective quenching effect of Pb2+ions to enhanced fluorescence of DNA-templated Ag nanoclusters (DNA-AgNCs) by approaching guanine-rich oligonucleotide. In our design, one cytosine-rich single-stranded oligonucleotide was used as template to synthesis dark fluorescence Ag nanoclusters. The dark species could be transformed to bright fluorescence species, when the other guanine-rich single-stranded oligonucleotide placed in proximity to DNA-AgNCs by partly base hybridization. In presence of Pb2+ions, the bright fluorescence DNA-AgNCs could be transformed to dark species. By monitoring the quenching effect of enhanced fluorescence of DNA-AgNCs, the content of Pb2+ions in aqueous could be quantitatively detected. With the optimized condition, our protocol could detect5nM Pb2+ions against other heavy metal ions in aqueous. This detection method exhibits many advantages, such as short response time, high selectivity. DNA-AgNCs, as stable and facile fluorophores, are promising to fluorescent sensing.3. Highly selective label-free detection of ATP by base sequence-dependent fluorescent DNA-templated Ag nanoclusters.This paper proposed a highly selective label-free detection method for ATP based on DNA ligation reaction and base sequence-dependent fluorescent DNA-AgNCs. Two DNA sequences were employed. One was used as template for preparing the Ag nanoclusters and the other one was the guanine-rich oligonucleotide and used for enhancing the fluorescence of AgNCs. These two DNA sequences were used as the building blocks for the DNA ligation reaction with cofactor ATP. In the presence of ATP, These two DNA sequences could be constructed to a single-stranded oligonucleotide through T4DNA ligase. The new oligonucleotide has been proved to obtain the highly bright fluorescence DNA-AgNCs. By monitoring the enhancing effect of DNA-AgNCs, the content of ATP could be quantitatively detected. The detection limit is5nM. Due to cofactor ATP dependented the DNA ligation reaction, this method could distinguish ATP from its analogues. Therefore, this novel label-free fluorescent assay for ATP was facile, sensitive and selective.4. One-step engineering of silver nanoclusters-aptamer assemblies as luminescent labels to target tumor cellsHerein, we developed a one-step engineering of intrinsically fluorescent AgNCs-aptamer assemblies that would allow developing facile and specific luminescent labels for target tumor cells recognition and analysis. The in situ synthesis protocol reported here is a simple and inexpensive one-step labeling process without covalent conjugation of bio-recognition molecules to fluorophores. This engineering relies on the design of one single oligonucleotide structure, which is competent to work both on the AgNCs synthesis and the target bio-recognition. A cancer-targeted DNA aptamer sequence (A-strand) and cytosinerich sequence for templated synthesis of fluorescent AgNCs (C-strand) were employed. Considering the conformational change in aptamers following cell binding and steric hindrance between aptamers and the synthesized AgNCs, we proposed to insert a linker sequence (L-strand), to associate the aptamer sequence with cytosinerich sequence. These structures were used to form the fluorescent AgNCs in the later stage and then to recognize the target tumor cells. To demonstrate this principle, the binding of AgNCs to two different tumor cells, CCRF-CEM cells and Ramos cells, was studied in this work.5. Label-free and Turn-on Aptamer Strategy for Cancer Cells Detection Based on a DNA-Silver Nanocluster Fluorescence upon Recognition-Induced HybridizationWe presented here a label-free and turn-on aptamer strategy for cancer cells detection based on the recognition-induced conformation alteration of aptamer and hybridization-induced fluorescence enhancement effect of DNA-silver nanoclusters (DNA-Ag NCs) in proximity of guanine-rich DNA sequences. In this strategy, two tailored DNA probes were involved. One is designed as a hairpin-shaped structure consisting of a central, target specific aptamer sequence at the3’-end, a guanine-rich DNA sequence and an arm segment at the5’-end. Here, we denote it as recognition probe. The other, serving as signal probe, contains a sequence for Ag NCs templated synthesis and a link sequence complementary to the arm segment of recognition probe. Recognizing and binding of the aptamer to the cancer cells enforces the recognition probe to undergo a conformational alteration and then initiates hybridization between the arm segment of recognition probe and the link sequence of the signal probe. The Ag NCs are then brought to close to the guanine-rich DNA sequences, leading to enhanced fluorescence readout. As proof of concept, the CCRF-CEM cancer cells fluorescence imaging and flow cytometry assay were performed by using the specific aptamer, sgc8c. It was demonstrated that the label-free aptamer strategy could specially and efficiently recognize and fluorescently image the CCRF-CEM cells. Determination by flow cytometry, this strategy allowed for detection of as low as150CCRF-CEM cells in200μL binding buffer. The general applicability of the strategy is also achieved in the successful imaging and special detection of Ramos cells. These results implied that this label-free and turn-on aptamer strategy holds considerable potential as a simple, rapid, sensitive, universal and specific cancer cell detection strategy with no required washing and separation steps. |
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