Here we describe the design of a bioluminescent stem-loop probe for the sensitive detection of HIV-1 spliced RNA. In this study, we employed Gaussia luciferase (GLuc), a bioluminescent protein that has several advantages over other bioluminescent proteins, including smaller size, higher bioluminescent intensity, and chemical and thermal stability. GLuc was chemically conjugated to the DABCYL-modified stem-loop probe (SLP) and was purified with a 2-step process to remove unconjugated GLuc and SLP. The binding of the target RNA to the loop region of the SLP results in the open conformation separating the stem part of SLP. GLuc conjugated to the stem acts as a reporter that produces light by a chemical reaction upon adding its substrate, coelenterazine in the presence of the target, while DABCYL serves as a quencher of bioluminescence in the closed conformation of SLP in the absence of the target. The optimized GLuc based-SLP assay resulted in a signal-to-background ratio of 47, which is the highest reported with bioluminescent SLPs and is significantly higher compared to traditional fluorescence-based SLPs that yield low signal to background ratio. Moreover, the assay showed an excellent selectivity against a single and double mismatched nucleic acid target, low detection limit, and ability to detect spiked HIV-1 RNA in human serum matrix.
In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported.Finally, the performances of the developed typing platform were validated by the analysis of real patient 2 samples. The HLA typing results gave excellent correlation when compared with those obtained using hospital based typing technologies.
Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.
Although bioluminescent
molecular beacons designed around resonance
quenchers have shown higher signal-to-noise ratios and increased sensitivity
compared with fluorescent beacon systems, bioluminescence quenching
is still comparatively inefficient. A more elegant solution to inefficient
quenching can be realized by designing a competitive inhibitor that
is structurally very similar to the native substrate, resulting in
essentially complete substrate exclusion. In this work, we designed
a conjugated anti-interferon-γ (IFN-γ) molecular aptamer
beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure
to the native substrate coelenterazine. To prove that a MAB can be
more sensitive and have a better signal-to-noise ratio, a bioluminescence-based
assay was developed against IFN-γ and provided an optimized,
physiologically relevant detection limit of 1.0 nM. We believe that
this inhibitor approach may provide a simple alternative strategy
to standard resonance quenching in the development of high-performance
molecular beacon-based biosensing systems.
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