ABSTRACT
The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.
Subject(s)
Fluorometry/methods , Nucleic Acids/analysis , Benzothiazoles , DNA Probes/chemistry , DNA, Single-Stranded , Diamines , Fluorometry/standards , Indoles , Nanotubes/chemistry , Nucleic Acid Amplification Techniques/methods , Organic Chemicals , Polymerization , Polymers , QuinolinesABSTRACT
A label-free fluorescence assay has been developed for sensitive and selective detection of adenosine triphosphate (ATP) by using poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) as fluorescent indicator. In our design, ATP aptamer was split into two fragments, both of which were elongated with poly T strands that can be utilized as efficient template for the formation of copper nanoparticles through the reduction of copper ions by sodium ascorbate. In the presence of ATP, the two split aptamers could be dragged to form aptamer-ATP aptamer complex, which drew the poly T strands close to each other and induced a remarkable fluorescence enhancement of poly T-templated CuNPs. Thus, an elevated fluorescence enhancement of poly T-templated CuNPs was obtained with the increase in ATP concentration. Under optimized conditions, a good linear range for ATP detection was realized from 100 nM to 100 µM with a detection limit of 10.29 nM. In addition, the application of this biosensing system in complex biological matrix was demonstrated with satisfactory results. This assay provided a simple, label-free, cost-effective, and sensitive platform for the detection of ATP.
Subject(s)
Adenosine Triphosphate/analysis , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Copper/chemistry , Fluorescent Dyes/chemistry , Metal Nanoparticles/chemistry , Thymine/analogs & derivatives , A549 Cells , Humans , Limit of Detection , Spectrometry, Fluorescence/methodsABSTRACT
Glutathione (GSH) serves many cellular functions and plays crucial roles in human pathologies. Simple and sensitive sensors capable of detecting GSH would be useful tools to understand the mechanism of diseases. In this work, a rapid fluorescence "switch-on" assay was developed to detect trace amount of GSH based on carbon dots-MnO2 nanocomposites, which was fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. Due to the formation of carbon dots-MnO2 nanocomposites, fluorescence of carbon dots could be quenched efficiently by MnO2 nanosheeets through fluorescence resonance energy transfer (FRET). However, the presence of GSH would reduce MnO2 nanosheets to Mn(2+) ions and subsequently release carbon dots, which resulted in sufficient recovery of fluorescent signal. This proposed assay demonstrated highly selectivity toward GSH with a detection limit of 300nM. Moreover, this method has also shown sensitive responses to GSH in human serum samples, which indicated its great potential to be used in disease diagnosis. As no requirement of any further functionalization of these as-prepared nanomaterials, this sensing system shows remarkable advantages including very fast and simple, cost-effective as well as environmental-friendly, which suggest that this new strategy could serve as an efficient tool for analyzing GSH level in biosamples.
Subject(s)
Carbon/chemistry , Fluorescence Resonance Energy Transfer/methods , Glutathione/blood , Manganese Compounds/chemistry , Nanocomposites/chemistry , Oxides/chemistry , Biosensing Techniques/methods , Fluorescence , Humans , Limit of Detection , Nanocomposites/ultrastructureABSTRACT
A novel label-free nanosensor has been developed for detecting biothiols including cysteine, glutathione and homocysteine based on poly(thymine)-templated fluorescent copper nanoparticles (CuNPs), which were controlled through thymine-Hg(II)-thymine coordination. This assay provides a simple, cost-effective, and sensitive platform for the detection of biothiols. By employing this turn-on sensor, biological thiols, such as cysteine, glutathione and homocysteine, are successfully detected at concentrations as low as 12.5nM for cysteine, 15nM for glutathione, and 20nM for homocysteine. The sensing system also exhibits high selectivity against other amino acids, and the application of the sensor for biological fluids shows that the proposed method works well for real samples.
