DNA-templated silver nanoclusters (AgNC) are a class of subnanometer sized fluorophores with good photostability and brightness. It has been applied as a diagnostic tool mainly for deoxyribonucleic acid (DNA) detection. Integration of DNA oligomers to generate AgNCs is interesting as varying DNA sequences can result in different fluorescence spectra. This allows a simple fluorescence shifting effect to occur upon DNA hybridization with the hybridization efficiency being a pronominal factor for successful shifting. The ability to shift the fluorescence spectra as a result of hybridization overcomes the issue of background intensities in most fluorescent based assays. Here we describe an optimized method for the detection of single-stranded and double-stranded synthetic forkhead box P3 (FOXP3) target by hybridization with the DNA fluorescence shift sensor. The system forms a three-way junction by successful hybridization of AgNC, G-rich strand (G-rich) to the target DNA, which generated a shift in fluorescence spectra with a marked increase in fluorescence intensity. The DNA fluorescence shift sensor presents a rapid and specific alternative to conventional DNA detection.
Human papillomavirus (HPV) is one of the most common sexually transmitted disease, transmitted through intimate skin contact or mucosal membrane. The HPV virus consists of a double-stranded circular DNA and the role of HPV virus in cervical cancer has been studied extensively. Thus it is critical to develop rapid identification method for early detection of the virus. A portable biosensing device could give rapid and reliable results for the identification and quantitative determination of the virus. The fabrication of electrochemical biosensors is one of the current techniques utilized to achieve this aim. In such electrochemical biosensors, a single-strand DNA is immobilized onto an electrically conducting surface and the changes in electrical parameters due to the hybridization on the electrode surface are measured. This review covers the recent developments in electrochemical DNA biosensors for the detection of HPV virus. Due to the several advantages of electrochemical DNA biosensors, their applications have witnessed an increased interest and research focus nowadays.
Lactobacillus plantarum PA18, a strain originally isolated from the leaves of Pandanus amaryllifolius, contains a pR18 plasmid. The pR18 plasmid is a 3211bp circular molecule with a G+C content of 35.8%. Nucleotide sequence analysis revealed two putative open reading frames, ORF1 and ORF2, in which ORF2 was predicted (317 amino acids) to be a replication protein and shared 99% similarity with the Rep proteins of pLR1, pLD1, pC30il, and pLP2000, which belong to the RCR pC194/pUB110 family. Sequence analysis also indicated that ORF1 was predicted to encode linA, an enzyme that enzymatically inactivates lincomycin. The result of Southern hybridization and mung bean nuclease treatment confirmed that pR18 replicated via the RCR mechanism. Phylogenetic tree analysis of pR18 plasmid proteins suggested that horizontal transfer of antibiotic resistance determinants without genes encoding mobilization has not only occurred between Bacillus and Lactobacillus but also between unrelated bacteria. Understanding this type of transfer could possibly play a key role in facilitating the study of the origin and evolution of lactobacillus plasmids. Quantitative PCR showed that the relative copy number of pR18 was approximately 39 copies per chromosome equivalent.
Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH3)6,2Cl(-)] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10(-21) to 1×10(-9)M with a lower detection limit of 1.55×10(-21)M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish.