A plug-and-play aptamer diagnostic platform based on linear dichroism spectroscopy.

A plug-and-play sandwich assay platform for the aptamer-based detection of molecular targets using linear dichroism (LD) spectroscopy as a read-out method has been demonstrated. A 21-mer DNA strand comprising the plug-and-play linker was bioconjugated onto the backbone of the filamentous bacteriophage M13, which gives a strong LD signal due to its ready alignment in linear flow. Extended DNA strands containing aptamer sequences that bind the protein thrombin, TBA and HD22, were then bound to the plug-and-play linker strand via complementary base pairing to generate aptamer-functionalised M13 bacteriophages. The secondary structure of the extended aptameric sequences required to bind to thrombin was checked using circular dichroism spectroscopy, with the binding confirmed using fluorescence anisotropy measurements. LD studies revealed that this sandwich sensor design is very effective at detecting thrombin down to pM levels, indicating the potential of this plug-and-play assay system as a new label-free homogenous detection system based on aptamer recognition.

Linear Dichroism Activity of Chiral Poly(p-Aryltriazole) Foldamers.

Controllable higher-order assembly is a central aim of macromolecular chemistry. An essential challenge to developing these molecules is improving our understanding of the structures they adopt under different conditions. Here, we demonstrate how flow linear dichroism (LD) spectroscopy is used to provide insights into the solution structure of a chiral, self-assembled fibrillar foldamer. Poly(para-aryltriazole)s fold into different structures depending on the monomer geometry and variables such as solvent and ionic strength. LD spectroscopy provides a simple route to determine chromophore alignment in solution and is generally used on natural molecules or molecular assemblies such as DNA and M13 bacteriophage. In this contribution, we show that LD spectroscopy is a powerful tool in the observation of self-assembly processes of synthetic foldamers when complemented by circular dichroism, absorbance spectroscopy, and microscopy. To that end, poly(para-aryltriazole)s were aligned in a flow field under different solvent conditions. The extended aromatic structures in the foldamer give rise to a strong LD signal that changes in sign and in intensity with varying solvent conditions. A key advantage of LD is that it only detects the large assemblies, thus removing background due to monomers and small oligomers.

Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR.

A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.

Combining bacteriophage engineering and linear dichroism spectroscopy to produce a DNA hybridisation assay.

Nucleic acid detection is an important part of our bio-detection arsenal, with the COVID-19 pandemic clearly demonstrating the importance to healthcare of rapid and efficient detection of specific pathogenic sequences. As part of the drive to establish new DNA detection methodologies and signal read-outs, here we show how linear dichroism (LD) spectroscopy can be used to produce a rapid and modular detection system for detecting quantities of DNA from both bacterial and viral pathogens. The LD sensing method exploits changes in fluid alignment of bionanoparticles (bacteriophage M13) engineered with DNA stands covalently attached to their surfaces, with the read-out signal induced by the formation of complementary duplexes between DNA targets and two M13 bionanoparticles. This new sandwich assay can detect pathogenic material down to picomolar levels in under 1 minute without amplification, as demonstrated by the successful sensing of DNA sequences from a plant virus (Potato virus Y) and an ampicillin resistance gene, ampR.