Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.
The increased release of harmful dyes in water, along with the continuous reduction of the world's freshwater supplies has placed the textile industry under greater pressure to safely and effectively treat wastewater effluents. Resistance of reactive dyes to breakdown naturally has highlighted the need for specialised removal methods. The growing need for low-cost, efficient sorbents has led to the exploration of bioinspired silica (BIS) due to their green synthesis, proven scalability, and versatility for chemical functionalisation required for dye scavenging. Through a systematic approach, the removal of Reactive Blue 19 from water was studied using a range of BIS, and was compared to removal using a commercial sorbent.While 0% removal was denoted for the commercial sorbent, BIS showed up to 94% removal.The results obtained from a kinetic study suggested a pseudo-second-order reaction, indicating a chemisorption process via electrostatic interactions. Examination of the effects of various adsorption conditions (temperature, pH, sorbent and dye concentrations) using isotherm models (Langmuir and Freundlich) indicated that adsorption was of both chemical and physical nature. Examination of the adsorption mechanism suggest that dye adsorption on BIS was spontaneous. BIS showed higher adsorption capacity (334 mg g -1 ) compared to literature examples, with rapid adsorption under acidic conditions, excellent thermal stability and a good reuse potential. These findings highlight the potential of BIS as a sustainable, efficient and low-cost sorbent that could be brought forward for future implementation.
The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe−biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.
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