A pico-HPLC-LIF system for the amplification-free determination of multiple miRNAs in cells

MicroRNAs are a class of important biomarkers,and the simultaneous detection of multiple miRNAs can provide valuable information about many diseases and biological processes.Amplification-free determination has been developed for the analysis of multiple miRNAs because of its characteristic low cost and high fidelity.Herein,a method for the amplification-free analysis and simultaneous detection of multiple miRNAs based on a so-called pico-HPLC-LIF system is described.In this process,a bare open capilla ry with an inner diameter of 680 nm is used as a sepa ration column for a sample volume of several hundreds of femtoliters(300 fL),followed by separation and detection.The technique has a zeptomolar limit of detection.The method was applied to detect cellular miRNA from adenocarcinomic human alveolar basal epithelial(A549)cell extracts,and the simultaneous detection of the mir-182,miR-155,and let-7 a was achieved.The results showed that the expression of mir-182 and miR-155 was up-regulated and that of let-7 a was down-regulated in A549 cells.This method for multiple miRNAs detection is expected to have broad applications in miRNA-based disease diagnosis,prognosis,treatment,and monitoring.

DNA-directed assembly of copper nanoblocks with inbuilt fluorescent and electrochemical properties： Application in simultaneous amplification-free analysis of multiple RNA species

The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of copper nanostructures within the nucleic acid helix structure in a sequence-dependent manner. The resultant nanostructures have interesting fluorescent and electrochemical properties, which are attractive for novel biosensing applications. However, the potential of using DNA-templated nano- structures for precision disease diagnosis remains unexplored. Particularly, DNA- templated nanostructures show high potential for the universal amplification-free detection of different RNA biomarker species. Because of their low cellular levels and differing species-dependent length and sequence features, simultaneous detection of different messenger RNAs, microRNAs, and long non-coding RNAs species with a single technique is challenging. Here, we report a contemporary technique for facile in situ assembly of DNA-templated copper nanoblocks （CuNBs） on various RNA species targets after hybridization-based magnetic isolation. Our approach circumvents the typical limitations associated with amplification and labeling procedures of current RNA assays. The synthesized CuNBs enabled amplification-free fM-level RNA detection with flexible fluorescence or electrochemical readouts. Furthermore, our nanosensing technique displays potential for clinical application, as demonstrated by non-invasive analysis of three diagnostic RNA biomarkers from a cohort of 10 prostate cancer patient urinary samples with 100%-concordance （quantitative reverse transcription- polymerase chain reaction （PCR） validation）. The good analytical performance and versatility of our method may be useful in both diagnostics and research fields.

Multiplexed micro RNA TG-FRET assay with isothermal and amplification-free single-step

Human genome encodes more than 2000 distinct micro RNAs(Micro RNA database:http://www.mirbase.org/),a class of single-strand noncoding RNAs with a short length of 19 to 25 nucleotides[1].They take part as regulatory factors in almost all biological processes from cell proliferation,differentiation to death.Since the alternation in the micro RNA expression level can be associated with a num-

Nanomechanical assay for ultrasensitive and rapid detection of SARS-CoV-2 based on peptide nucleic acid

The massive global spread of the COVID-19 pandemic makes the development of more effective and easily popularized assays critical.Here,we developed an ultrasensitive nanomechanical method based on microcantilever array and peptide nucleic acid(PNA)for the detection of severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2)RNA.The method has an extremely low detection limit of 0.1 fM(105 copies/mL)for N-gene specific sequence(20 bp).Interestingly,it was further found that the detection limit of N gene(pharyngeal swab sample)was even lower,reaching 50 copies/mL.The large size of the N gene dramatically enhances the sensitivity of the nanomechanical sensor by up to three orders of magnitude.The detection limit of this amplification-free assay method is an order of magnitude lower than RT-PCR(500 copies/mL)that requires amplification.The non-specific signal in the assay is eliminated by the in-situ comparison of the array,reducing the false-positive misdiagnosis rate.The method is amplification-free and label-free,allowing for accurate diagnosis within 1 h.The strong specificity and ultrasensitivity allow single base mutations in viruses to be distinguished even at very low concentrations.Also,the method remains sensitive to fM magnitude lung cancer marker(miRNA-155).Therefore,this ultrasensitive,amplification-free and inexpensive assay is expected to be used for the early diagnosis of COVID-19 patients and to be extended as a broad detection tool.

Effects of symmetric and asymmetric salt conditions on a selective solid-state nanopore assay

Conventional solid-state nanopore measurements sense all translocating entities,necessitating meticulous analysis to differentiate target biomolecules.To address this,we have established a selective assay with the platform that has shown utility in quantifying several nucleic acid biomarkers.However,limited detection efficiency and intrinsic noise have so far limited assay resolution to 10 nM.Improvements in this value require manipulation of translocation dynamics.Here,we report the effects of NaCl conditions on assay performance.We first investigate symmetric conditions,finding sensitivity increases with salt concentration but selectivity is maximized at 1.0 M NaCl.We then probe asymmetric conditions,showing a remarkable impact on assay sensitivity and selectivity when measurement buffer NaCl concentration in the reservoir with the translocating molecules is low and the opposite reservoir is increased.Using optimum conditions,we demonstrate detection of target biomolecules down to a concentration of 100 pM which is an improvement of 2 orders of magnitude over past results.