Fluorescent Double-Stranded DNA-Templated Copper Nanoprobes for Rapid Diagnosis of Tuberculosis

Abstract

In this work, we investigate highly sensitive fluorescent Cu nanoparticles for use as rapid and specific nucleic acid amplification nanoprobes (NPs) for the diagnosis of tuberculosis. After applying polymerase chain reaction (PCR) to a tuberculosis (TB) sample, we demonstrate that the presence of the targeted IS6110 DNA sequence of TB can be easily and directly detected through the in situ formation of DNA-templated fluorescent Cu NPs and subsequently quantified using only a smartphone. Compared to traditional DNA analysis, this sensing platform does not require purification steps and eliminates the need for electrophoresis to confirm the PCR results. After optimization, this dsDNA-Cu NP-PCR method has the ability to analyze clinical TB nucleic acid samples at a detection limit of 5 fg/μL, and the fluorescent signal can be distinguished in only ∼3 min after the DNA has been amplified. Moreover, with the combination of smartphone-assisted imaging analysis, we can further reduce the instrument size/cost and enhance the portability. In this manner, we are able to eliminate the need for a fluorescent spectrophotometer to measure the clinical sample. These results demonstrate this platform’s practical applicability, combining a smartphone and on-site analysis while retaining the detection performance, making it suitable for clinical DNA applications in resource-limited regions of the world.

Copper nanoclusters: designed synthesis, structural diversity, and multiplatform applications

“…[63][64][65][66][67][68][69][70][71][72][73][74][75][76][77] Double-stranded DNA (dsDNA) with adjustable sequences and lengths has been considered as a template for the synthesis of fluorescent copper nanoparticles/nanoclusters. [78][79][80][81][82] Moreover, it has been shown that at lower concentrations of metal precursor, Cu 2+ first binds to backbone phosphate groups of DNA via a nonspecific interaction. However, with increasing the concentration, the metallization of high affinity bases is favoured.…”

“…dsDNA-templated CuNCs have been applied for the diagnosis of tuberculosis (TB). 79 The in situ cluster formation in the presence of PCR product of the targeted IS6110 DNA sequence of TB is only successful if specifically designed AT-rich primers are used. With this, the formation of dimers and other unwanted hairpin structures and their contribution to PL were kept at a minimum level.…”

Development of General Methods for Detection of Virus by Engineering Fluorescent Silver Nanoclusters

“…Metal nanoclusters (NCs) are ultrasmall nanomaterials (normally < ca. 2 nm), which contain a few atoms of a precise number. − The metal NCs interpose between metal nanoparticles (NPs) and the atoms . Traditional metal NPs of large sizes show surface plasmon resonance (SPR) effects, but metal NCs do not support SPR.…”

Recent Advances in Bio‐Templated Metallic Nanomaterial Synthesis and Electrocatalytic Applications

“…After reduction by NaBH 4 , Ag NCs with 1–4 atoms grew on the template, as indicated by mass spectra [57] . Fluorescent Cu NPs have also been fabricated on DNA by sodium ascorbate reduction, and later served as nucleic acid amplification nanoprobes for tuberculosis diagnosis [126] . Silver‐DNA NPs can also be prepared by electrodeposition in which a potential is applied in the presence of DNA and silver precursor [56] .…”