Purely electrical SARS-CoV-2 sensing based on single-molecule counting.

The majority of RNA based COVID-19 diagnostics employ enzymatic amplification to achieve high sensitivity, but this relies on arbitrary thresholding, which complicates the comparison of test results and may lead to false outcomes. Here we introduce solid-state nanopore sensing for label-free quantification of SARS-CoV-2 RNA in clinical nasal swab samples. This PCR-free method involves reverse transcribing a target gene on the viral RNA before enzymatically digesting all but the resulting dsDNA. Ratiometric quantification of RNA abundance is achieved by single-molecule counting and length-based nanopore identification of dsDNA from a SARS-CoV-2 gene and a human reference gene. We graded nasal swab samples from >15 subjects and find that the SARS-CoV-2 ratiometric nanopore index correlates well with the reported RT-qPCR threshold cycle for positive classified samples. Remarkably, nanopore analysis also reports quantitative positive outcomes for clinical samples classified as negative by RT-qPCR, suggesting that the method may be used to diagnose COVID-19 in samples that may evade detection. We show that the sample preparation workflow can be implemented using a compact microfluidic device with integrated thermal control for semi-automated processing of extremely small sample volumes, offering a viable route towards automated, fast and affordable RNA quantification in a small and portable device.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows.

In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers.

Quantification of mRNA Expression Using Single-Molecule Nanopore Sensing.

RNA quantification methods are broadly used in life science research and in clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for RNA quantification. However, in cases of rare transcripts or inhibiting contaminants in the sample, an extensive amplification could bias the copy number estimation, leading to quantification errors and false diagnosis. Single-molecule techniques may bypass amplification but commonly rely on fluorescence detection and probe hybridization, which introduces noise and limits multiplexing. Here, we introduce reverse transcription quantitative nanopore sensing (RT-qNP), an RNA quantification method that involves synthesis and single-molecule detection of gene-specific cDNAs without the need for purification or amplification. RT-qNP allows us to accurately quantify the relative expression of metastasis-associated genes MACC1 and S100A4 in nonmetastasizing and metastasizing human cell lines, even at levels for which RT-qPCR quantification produces uncertain results. We further demonstrate the versatility of the method by adapting it to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA against a human reference gene. This internal reference circumvents the need for producing a calibration curve for each measurement, an imminent requirement in RT-qPCR experiments. In summary, we describe a general method to process complicated biological samples with minimal losses, adequate for direct nanopore sensing. Thus, harnessing the sensitivity of label-free single-molecule counting, RT-qNP can potentially detect minute expression levels of RNA biomarkers or viral infection in the early stages of disease and provide accurate amplification-free quantification.

Microfluidic device for coupling isotachophoretic sample focusing with nanopore single-molecule sensing.

Solid-state nanopores (NPs) are label-free single-molecule sensors, capable of performing highly sensitive assays from a small number of biomolecule translocation events. However, single-molecule sensing is challenging at extremely low analyte concentrations due to the limited flux of analytes to the sensing volume. This leads to a low event rate and increases the overall assay time. In this work, we present a method to enhance the event rate at low analyte concentrations by using isotachophoresis (ITP) to focus and deliver analytes to a nanopore sensor. Central to this method is a device capable of performing ITP focusing directly on a solid-state NP chip, while preventing the focusing electric field from damaging the nanopore membrane. We discuss considerations and trade-offs related to the design of the focusing channel, the ITP electrolyte system and electrical decoupling between the focusing and sensing modes. Finally, we demonstrate an integrated device wherein the concentration enhancement due to ITP focusing leads to an increase in event rate of >300-fold in the ITP-NP device as compared to the NP-only case.

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification.

We have developed a method for spatially resolved genetic analysis of formalin-fixed paraffin-embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 µm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF-7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Plasmonic-Nanopore Biosensors for Superior Single-Molecule Detection.

Plasmonic and nanopore sensors have separately received much attention for achieving single-molecule precision. A plasmonic "hotspot" confines and enhances optical excitation at the nanometer length scale sufficient to optically detect surface-analyte interactions. A nanopore biosensor actively funnels and threads analytes through a molecular-scale aperture, wherein they are interrogated by electrical or optical means. Recently, solid-state plasmonic and nanopore structures have been integrated within monolithic devices that address fundamental challenges in each of the individual sensing methods and offer complimentary improvements in overall single-molecule sensitivity, detection rates, dwell time and scalability. Here, the physical phenomena and sensing principles of plasmonic and nanopore sensing are summarized to highlight the novel complementarity in dovetailing these techniques for vastly improved single-molecule sensing. A literature review of recent plasmonic nanopore devices is then presented to delineate methods for solid-state fabrication of a range of hybrid device formats, evaluate the progress and challenges in the detection of unlabeled and labeled analyte, and assess the impact and utility of localized plasmonic heating. Finally, future directions and applications inspired by the present state of the art are discussed.

Extraction of electrokinetically separated analytes with on-demand encapsulation.

Microchip electrokinetic methods are capable of increasing the sensitivity of molecular assays by enriching and purifying target analytes. However, their use is currently limited to assays that can be performed under a high external electric field, as spatial separation and focusing is lost when the electric field is removed. We present a novel method that uses two-phase encapsulation to overcome this limitation. The method uses passive filling and pinning of an oil phase in hydrophobic channels to encapsulate electrokinetically separated and focused analytes with a brief pressure pulse. The resulting encapsulated sample droplet maintains its concentration over long periods of time without requiring an electric field and can be manipulated for further analysis, either on- or off-chip. We demonstrate the method by encapsulating DNA oligonucleotides in a 240 pL aqueous segment after isotachophoresis (ITP) focusing, and show that the concentration remains at 60% of the initial value for tens of minutes, a 22-fold increase over free diffusion after 20 minutes. Furthermore, we demonstrate manipulation of a single droplet by selectively encapsulating amplicon after ITP purification from a polymerase chain reaction (PCR) mix, and performing parallel off-chip detection reactions using the droplet. We provide geometrical design guidelines for devices implementing the encapsulation method, and show how the method can be scaled to multiple analyte zones.

Focusing analytes from 50 µL into 500 pL: On-chip focusing from large sample volumes using isotachophoresis.

The use of on-chip isotachophoresis assays for diagnostic applications is often limited by the small volumes of standard microfluidic channels. Overcoming this limitation is particularly important for detection of 'discrete' biological targets (such as bacteria) at low concentrations, where the volume of processed liquid in a standard microchannel might not contain any targets. We present a novel microfluidic chip that enables ITP focusing of target analytes from initial sample volumes of 50 µL into a concentrated zone with a volume of 500 pL, corresponding to a 100,000-fold increase in mean concentration, and a 300,000-fold increase in peak concentration. We present design considerations for limiting sample dispersion in such large-volume focusing (LVF) chips and discuss the trade-off between assay time and Joule heating, which ultimately governs the scalability of LVF designs. Finally, we demonstrate a 100-fold improvement of ITP focusing performance in the LVF chip as compared to conventional microchannels, and apply this enhancement to achieve highly sensitive detection of both molecular targets (DNA, down to 10 fM) and whole bacteria (down to 100 cfu/mL).