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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. 

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles (MN) and combined these with LAMP assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated and disease severity was measured. Extractions were performed using MN and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, while P. infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid MN extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens. 

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 Molecular diagnostics for crop diseases can guide the precise application of pesticides, thereby reducing pesticide usage while improving crop yield, but tools are lacking. Here, we report an in-field molecular diagnostic tool that uses a cheap colorimetric paper and a smartphone, allowing multiplexed, low-cost, rapid detection of crop pathogens. Rapid nucleic acid amplification-free detection of pathogenic RNA is achieved by combining toehold-mediated strand displacement with a metal ion-mediated urease catalysis reaction. We demonstrate multiplexed detection of six wheat pathogenic fungi and an early detection of wheat stripe rust. When coupled with a microneedle for rapid nucleic acid extraction and a smartphone app for results analysis, the sample-to-result test can be completed in ~10 min in the field. Importantly, by detecting fungal RNA and mutations, the approach allows to distinguish viable and dead pathogens and to sensitively identify mutation-carrying fungicide-resistant isolates, providing fundamental information for precision crop disease management. 

A leaf-attachable multimodal plant wearable sensor was developed for monitoring biotic and abiotic stresses in real time.