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Volatile organic compounds (VOCs) are common constituents of fruits, vegetables, and crops, and are closely associated with their quality attributes, such as firmness, sugar level, ripeness, translucency, and pungency levels. While VOCs are vital for assessing vegetable quality and phenotypic classification, traditional detection methods, such as Gas Chromatography-Mass Spectrometry (GC-MS) and Proton Transfer Reaction Mass Spectrometry (PTR-MS) are limited by expensive equipment, complex sample reparation, and slow turnaround time. Additionally, the transient nature of VOCs complicates their detection using these methods. Here, we developed a paper-based colorimetric sensor array combined with needles that could: 1) induce vegetable VOC release in a minimally invasive fashion, and 2) analyze VOCs in situ with a smartphone reader device. The needle sampling device helped release specific VOCs from the studied vegetables that usually require mechanic stimulation, while maintaining the vegetable viability. On the other hand, the colorimetric sensor array was optimized for sulfur compound-based VOCs with a limit of detection (LOD) in the 1–25 ppm range, and classified fourteen different vegetable VOCs, including sulfoxides, sulfides, mercaptans, thiophenes, and aldehydes. By combining principal components analysis (PCA) nalysis, the integrated sensor platform proficiently discriminated between four vegetable subtypes originating from two major categories within 2 min of testing time. Additionally, the sensor demomstrated the capability to distinguish between different types of tested fruits and vegetables, including garlic, green pepper, and nectarine. This rapid and minimally invasive sensing technology holds great promise for conducting field-based vegetable quality monitoring. 

Abstract Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent approach quantifies analyte concentration by sizing λ DNA reporters via gel electrophoresis, providing a simple solution for label-free detection. Here, we report an alternative strategy for label-free CRISPR-Cas12a, which relies on Cas12atrans-nicking induced supercoil relaxation of dsDNA plasmid reporters to generate a robust and ratiometric readout. The ratiometric CRISPR (rCRISPR) measures the relative percentage of supercoiled plasmid DNA to the relaxed circular DNA by gel electrophoresis for more accurate target concentration quantification. This simple method is two orders of magnitude more sensitive than the typical fluorescent reporter. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of rCRISPR for detecting various model DNA targets such as HPV 16 and real AAV samples, highlighting its feasibility for point-of-care CRISPR-Dx applications. 

Abstract CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn’t cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3′ overhang dsDNA substrates at least 3 times faster than 5′ overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule. 

Abstract CRISPR‐based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR‐Cas12a‐based sensing platform to detect ssDNA targets by sizing double‐stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massivetrans‐nuclease activity of Cas12a toward λ DNA is due to the presence of single‐stranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes thetrans‐cleavage activity and helps achieve sub‐picomolar detection sensitivity, ≈100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters. 

Abstract Biosensors are analytical tools for monitoring various parameters related to living organisms, such as humans and plants. Liquid metals (LMs) have emerged as a promising new material for biosensing applications in recent years. LMs have attractive physical and chemical properties such as deformability, high thermal and electrical conductivity, low volatility, and low viscosity. LM‐based biosensors represent a new strategy in biosensing particularly for wearable and real‐time sensing. While early demonstrations of LM biosensors focus on monitoring physical parameters such as strain, motion, and temperature, recent examples show LM can be an excellent sensing material for biochemical and biomolecular detection as well. In this review, the recent progress of LM‐based biosensors for personalized healthcare and disease monitoring via both physical and biochemical signaling is survey. It is started with a brief introduction of the fundamentals of biosensors and LMs, followed by a discussion of different mechanisms by which LM can transduce biological or physiological signals. Next, it is reviewed example LM‐based biosensors that have been used in real biological systems, ranging from real‐time on‐skin physiological monitoring to target‐specific biochemical detection. Finally, the challenges and future directions of LM‐integrated biosensor platforms is discussed.