Formation Of Fluorescent CuNPs Research Articles

A label-free and enzyme-free ultra-sensitive transcription factors biosensor using DNA-templated copper nanoparticles as fluorescent indicator and hairpin DNA cascade reaction as signal amplifier

Detection and quantification of specific protein with ultralow concentration play a crucial role in biotechnological applications and biomedical diagnostics. In this paper, a label-free and enzyme-free amplified fluorescent biosensor has been developed for transcription factors detection based on AT-rich double-stranded DNA-templated copper nanoparticles (ds DNA/Cu NPs) and hairpin DNA cascade reaction. This strategy was demonstrated by using nuclear factor-kappa B p50 (NF-κB p50) and specific recognition sequences as a model case. In this assay, a triplex consists of double-stranded DNA containing NF-κB p50 specifically binding sequences and a special design single-stranded DNA (Trigger) which is able to activate the hairpin DNA cascade amplifier (HDCA). In the presence of NF-κB p50, the triplex became unstable since the target bound to the recognition sequences with strong affinity. The selective binding event confirmed that the Trigger was successfully released from the triplex and initiated HDCA to yield the product which could effectively template the formation of fluorescent Cu NPs. The experimental results revealed that the advanced strategy was ultra-sensitive for detecting NF-κB p50 in the concentration range from 0.1 to 1000 pM with a detection limit of 0.096 pM. In addition, the relative standard deviation was 4.08% in 3 repetitive assays of 500 pM NF-κB p50, which indicated that the reproducibility of this strategy was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, cost-effective, and simplified operations. In addition, the proposed biosensing platform is versatile. By conjugating with various specific recognition units, it could hold considerable potential to sensitive and selective detect various DNA-binding proteins and might find wide applications in biomedical fields.

A fluorescent biosensor for protein detection based on poly(thymine)-templated copper nanoparticles and terminal protection of small molecule-linked DNA.

In this paper, a fluorescent biosensor has been developed for protein detection based on poly(thymine) (poly T)-templated copper nanoparticles (Cu NPs) and terminal protection of small molecule linked-DNA. This strategy was demonstrated by using small molecule biotin and its binding protein streptavidin (SA) as a model case. In this assay, biotin-linked poly T (biotin-T30) probe was specifically bound to the target protein SA with strong affinity in the presence of SA. The selective binding events confirmed that biotin-T30 probe was protected against the hydrolysis by exonuclease I (Exo I), which could effectively template the formation of fluorescent Cu NPs. The results revealed that the developed strategy was highly sensitive for detecting SA in the concentration range from 0.5 to 1000 nM with a detection limit of 0.1 nM. In addition, the relative standard deviation was 3.6% in 5 repetitive assays of 50 nM SA, which indicated that the reproducibility of the method was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, low cost, and simplified operations. Thus, it could hold considerable potential to construct a simple, selective and sensitive fluorescent platform for detection of small molecule-protein interactions in molecular diagnostics and genomic research.

Highly thymine-dependent formation of fluorescent copper nanoparticles templated by ss-DNA

Double-stranded DNAs (ds-DNAs) have been identified as efficient templates favoring the formation of fluorescent copper nanoparticles (Cu NPs). Herein, we have tried to synthesize fluorescent Cu NPs using single-stranded DNAs (ss-DNAs) as templates and to identify the critical DNA sequences. By comparing the results using homopolymer DNAs, hairpin DNAs, and pristine ss-DNAs as templates, we found that DNA thymine base plays a dominant role in producing red-emissive fluorescent Cu NPs on ss-DNA templates. The thymine-dependent growth of the fluorescent Cu NPs is confirmed by Hg2+ mediated T–T base pair in comparison with the other non-specific metal ions, which could be developed into a practical sensor for turn-on fluorescence detection of Hg2+ with a high selectivity. The mechanism is briefly discussed according the DNA sequence-dependent formation of fluorescent Cu NPs. This work demonstrates the sequence role in producing fluorescent Cu NPs that could serve as promising fluorescent nanoprobes in biosensing and DNA-hosted Cu nanomaterials.