The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR-based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification-free nucleic acid detection via harnessing the trans-cleavage mechanism of Cas13a and ultrasensitive graphene field-effect transistors (gFETs). CRISPR Cas13a-gFET achieves the detection of SARS-CoV-2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS-CoV-2 samples, including those with low viral loads (Ct value > 30). Overall, these findings establish our CRISPR Cas13a-gFET among the most sensitive amplification-free nucleic acid diagnostic platforms to date.
Electrolytes play a pivotal role in regulating cardiovascular functions, hydration, and muscle activation. The current standards for monitoring electrolytes involve periodic sampling of blood and measurements using laboratory techniques, which are often uncomfortable/inconvenient to the subjects and add considerable expense to the management of their underlying disease conditions. The wide range of electrolytes in skin interstitial fluids (ISFs) and their correlations with those in plasma create exciting opportunities for applications such as electrolyte and circadian metabolism monitoring. However, it has been challenging to monitor these electrolytes in the skin ISFs. In this study, we report a minimally invasive microneedle-based potentiometric sensing system for multiplexed and continuous monitoring of Na+ and K+ in the skin ISFs. The potentiometric sensing system consists of a miniaturized stainless-steel hollow microneedle to prevent sensor delamination and a set of modified microneedle electrodes for multiplex monitoring. We demonstrate the measurement of Na+ and K+ in artificial ISFs with a fast response time, excellent reversibility and repeatability, adequate selectivity, and negligible potential interferences upon the addition of a physiologically relevant concentration of metabolites, dietary biomarkers, and nutrients. In addition, the sensor maintains the sensitivity after multiple insertions into the chicken skin model. Furthermore, the measurements in artificial ISFs using calibrated sensors confirm the accurate measurements of physiological electrolytes in artificial ISFs. Finally, the skin-mimicking phantom gel and chicken skin model experiments demonstrate the sensor’s potential for minimally invasive monitoring of electrolytes in skin ISFs. The developed sensor platform can be adapted for a wide range of other applications, including real-time monitoring of nutrients, metabolites, and proteins.
The real-time monitoring of neurochemical release in vivo plays a critical role in understanding the biochemical process of the complex nervous system. Current technologies for such applications, including microdialysis and fast-scan cyclic voltammetry, suffer from limited spatiotemporal resolution or poor selectivity. Here, we report a soft implantable aptamer-graphene microtransistor probe for real-time monitoring of neurochemical release. As a demonstration, we show the monitoring of dopamine with nearly cellular-scale spatial resolution, high selectivity (dopamine sensor >19-fold over norepinephrine), and picomolar sensitivity, simultaneously. Systematic benchtop evaluations, ex vivo experiments, and in vivo studies in mice models highlight the key features and demonstrate the capability of capturing the dopamine release dynamics evoked by pharmacological stimulation, suggesting the potential applications in basic neuroscience studies and studying neurological disease-related processes. The developed system can be easily adapted for monitoring other neurochemicals and drugs by simply replacing the aptamers functionalized on the graphene microtransistors.
Neurochemical corelease has received much attention in understanding brain activity and cognition. Despite many attempts, the multiplexed monitoring of coreleased neurochemicals with spatiotemporal precision and minimal crosstalk using existing methods remains challenging. Here, we report a soft neural probe for multiplexed neurochemical monitoring via the electrografting-assisted site-selective functionalization of aptamers on graphene field-effect transistors (G-FETs). The neural probes possess excellent flexibility, ultralight mass (28 mg), and a nearly cellular-scale dimension of 50 μm × 50 μm for each G-FET. As a demonstration, we show that G-FETs with electrochemically grafted molecular linkers (−COOH or −NH 2 ) and specific aptamers can be used to monitor serotonin and dopamine with high sensitivity (limit of detection: 10 pM) and selectivity (dopamine sensor >22-fold over norepinephrine; serotonin sensor >17-fold over dopamine). In addition, we demonstrate the feasibility of the simultaneous monitoring of dopamine and serotonin in a single neural probe with minimal crosstalk and interferences in phosphate-buffered saline, artificial cerebrospinal fluid, and harvested mouse brain tissues. The stability studies show that multiplexed neural probes maintain the capability for simultaneously monitoring dopamine and serotonin with minimal crosstalk after incubating in rat cerebrospinal fluid for 96 h, although a reduced sensor response at high concentrations is observed. Ex vivo studies in harvested mice brains suggest potential applications in monitoring the evoked release of dopamine and serotonin. The developed multiplexed detection methodology can also be adapted for monitoring other neurochemicals, such as metabolites and neuropeptides, by simply replacing the aptamers functionalized on the G-FETs.
Extensive studies in both animals and humans have demonstrated that high molecular weight neurochemicals, such as neuropeptides and other polypeptide neurochemicals, play critical roles in various neurological disorders. Despite many attempts, existing methods are constrained by detecting neuropeptide release in small animal models during behavior tasks, which leaves the molecular mechanisms underlying many neurological and psychological disorders unresolved. Here, we report a wireless, programmable push-pull microsystem for membrane-free neurochemical sampling with cellular spatial resolution in freely moving animals. In vitro studies demonstrate the sampling of various neurochemicals with high recovery (>80%). Open-field tests reveal that the device implantation does not affect the natural behavior of mice. The probe successfully captures the pharmacologically evoked release of neuropeptide Y in freely moving mice. This wireless push-pull microsystem creates opportunities for neuroscientists to understand where, when, and how the release of neuropeptides modulates diverse behavioral outputs of the brain.
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