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Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form ‘electrical hot spots’ in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures. Field effect transistors based on graphene hold promise for sensing applications. Here, the authors report a millimeter-sized transistor based on deformed graphene as a biosensor that can detect nucleic acid molecules having detection limit of ~18 molecules of DNA in physiological buffer solution and ~600 molecules in human serum.

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand–receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

Despite its early promise as a diagnostic and prognostic tool, gene expression profiling remains cost-prohibitive and challenging to implement in a clinical setting. Here, we introduce a molecular computation strategy for analysing the information contained in complex gene expression signatures without the need for costly instrumentation. Our workflow begins by training a computational classifier on labelled gene expression data. This in silico classifier is then realized at the molecular level to enable expression analysis and classification of previously uncharacterized samples. Classification occurs through a series of molecular interactions between RNA inputs and engineered DNA probes designed to differentially weigh each input according to its importance. We validate our technology with two applications: a classifier for early cancer diagnostics and a classifier for differentiating viral and bacterial respiratory infections based on host gene expression. Together, our results demonstrate a general and modular framework for low-cost gene expression analysis.

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains. A DNA probe that translocates from one anchor site to another by toehold-mediated DNA strand displacement is used to monitor transient molecular encounter events on live cell membranes.

A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target’s reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered. Graphical abstract

There is intense interest in quantifying the levels of microRNA because of its importance as a blood-borne biomarker. The challenge has been to develop methods that can monitor microRNA expression both over broad concentration ranges and in ultralow amounts directly in a patient’s blood. Here, we show that, through electric-field-induced reconfiguration of a network of gold-coated magnetic nanoparticles modified by probe DNA (DNA–Au@MNPs), it is possible to create a highly sensitive sensor for direct analysis of nucleic acids in samples as complex as whole blood. The sensor is the first to be able to detect concentrations of microRNA from 10 aM to 1 nM in unprocessed blood samples. It can distinguish small variations in microRNA concentrations in blood samples of mice with growing tumours. The ultrasensitive and direct detection of microRNA using an electrically reconfigurable DNA–Au@MNPs network makes the reported device a promising tool for cancer diagnostics.

Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.

Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array. Monitoring DNA binding and single-base mismatches accurately in real time is difficult, especially for miniaturized devices. Here the authors report a graphene field-effect transistor array capable of reliably measuring DNA hybridization kinetics and affinity at the picomolar level.

Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed. A molecular probe has been designed that distinguishes double-stranded DNA with single base-pair specificity. In this approach, two destabilizing bubbles, in which the base pairs are mismatched, are generated for each point mutation in the target DNA.

Healthcare is in demand of technologies for real-time sensing in order to continuously guard the state of patients. Here we present biomarker-monitoring based on the sensing of particle mobility, a concept wherein particles are coupled to a substrate via a flexible molecular tether, with both the particles and substrate provided with affinity molecules for effectuating specific and reversible interactions. Single-molecular binding and unbinding events modulate the Brownian particle motion and the state changes are recorded using optical scattering microscopy. The technology is demonstrated with DNA and protein as model biomarkers, in buffer and in blood plasma, showing sensitivity to picomolar and nanomolar concentrations. The sensing principle is direct and self-contained, without consuming or producing any reactants. With its basis in reversible interactions and single-molecule resolution, we envisage that the presented technology will enable biosensors for continuous biomarker monitoring with high sensitivity, specificity, and accuracy. Biomarkers are natural indicators of some biological conditions, often used in diagnostics. Here, the authors developed a biosensor that continuously measures concentrations of DNA or protein biomarkers, and is based on particles that change mobility by directly interacting with individual molecules.