Qingshan Wei’s research while affiliated with North Carolina State University and other places

This paper describes laser exposure to tune the infrared (IR) emissivity of a film of eutectic gallium indium (EGaIn) particles. EGaIn – a liquid metal at room temperature – forms a native oxide that keeps particles of the metal from spontaneously percolating. Photothermal energy from a CO2 laser percolates the particles into a conductive network. Here, it also causes a decrease in the IR emissivity of the film of particles from 0.4 to 0.24 over the range of 7.5–13 µm wavelength (measured by an IR camera) with the increase of laser fluence from 1.4 to 1.9 J cm⁻². The particles percolate most prominently at the bottom of the film, and thus, the apparent surface roughness does not change with laser exposure. This finding suggests the decrease in emissivity is not due to changes in the film's topography. Instead, the change in IR emissivity is attributed to a loss of the surface plasmonic resonance effect of EGaIn particles in the IR range after the sintering, which is confirmed by optical simulations. As a demonstration, it is shown that the ability to change the emissivity makes it possible to encrypt messages and camouflage laser‐processed patterns.

Crop breeding plays an essential role in addressing food security by enhancing crop yield, disease resistance, and nutritional value. However, the current crop breeding process faces multiple challenges and limitations, especially in genotypic evaluations. Traditional methods for seed genotyping remain labor-intensive, time-consuming, and cost-prohibitive outside of large-scale breeding programs. Here, we present a handheld microneedle (MN)-based seed DNA extraction platform for rapid, nondestructive, and in-field DNA isolation from crop seeds for instant marker analysis. Using soybean seeds as a case study, we demonstrated the use of polyvinyl alcohol (PVA) MN patches for the successful extraction of DNA from softened soybean seeds. This extraction technology maintained high seed viability, showing germination rates of 82% and 79%, respectively, before and after MN sampling. The quality of MN-extracted DNA was sufficient for various genomic analyses, including PCR, LAMP, and whole genome sequencing. Importantly, this MN patch method also allowed for the identification of specific genetic differences between soybean varieties. Additionally, we designed a 3D-printed extraction device, which enabled multiplexed seed DNA extraction in a microplate format. In the future, this method could be applied at scale and in-field for crop seed DNA extraction and genotyping analysis.

Liquid metals (LM) are emerging plasmonic nanomaterials with transformable surface plasmon resonances (SPR) due to their liquid-like deformability. This study delves into the plasmonic properties of LM nanoparticles, with a focus on EGaIn (eutectic gallium-indium)-based materials. Leveraging Finite-Difference Time-Domain (FDTD) simulations, we explored the localized SPR (LSPR) effects of EGaIn nanoparticles with various shapes, including nanospheres, dimers, nanorods, nanodisks, nanoellipses, nanocubes, and nanocuboids, in the broad range of ultraviolet (UV)-visible-near infrared (NIR) spectrum. EGaIn, known for its unique properties such as low toxicity, negligible vapor pressure, and excellent electrical and thermal conductivity, is appealing in broad wavelength plasmonic applications. In particular, this study reveals uncovered LSPR effects in the visible and NIR wavelength ranges, providing a comprehensive map of LSPR peaks and cross-sections for different shapes of EGaIn nanoparticles. The findings offer insights into correlating EGaIn nanoparticle geometry with their optical properties for diverse applications, ranging from biosensing, nanoelectronics, to optomechanical systems.

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 Cas12a trans-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.

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.

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

Use of tomato cultivars with the Sw-5 resistance gene cluster has led to the occurrence of resistance-breaking (RB) tomato spotted wilt virus (TSWV) strains globally, including California and, recently, North Carolina and Texas. We documented disease on tomato infected with either an RB strain from California (CA-RB) or a wild type (CA-WT) strain of TSWV on tomato with (cultivar Mountain Merit) or without (cultivar Mountain Fresh Plus) the Sw-5b resistance gene and detected virus incidence over time using microneedle RNA extractions and LAMP. We developed a LAMP/Cas12a assay for detection of the CA-C118Y mutation in a CA-RB strain and tested the assay with field samples. Disease in the susceptible cultivar was less severe with CA-RB than with the CA-WT strain. In contrast, the resistant cultivar had little disease when inoculated with the CA-WT strain but exhibited stunting of greater than 50% when inoculated with the CA-RB strain. In the susceptible tomatoes, the detection rates over time by LAMP reaction were higher in CA-WT than in CA-RB-inoculated plants. In resistant tomato, CA-RB remained detectable by TSWV LAMP over 14 days, whereas the WT strain was undetectable. A two-step LAMP/Cas12a assay differentiated the two strains in 1 h. Our methods were validated with samples from TSWV-infected North Carolina fields. A phylogeny of NSm gene sequences that included North Carolina field samples revealed two independent origins of the North Carolina RB isolates. The LAMP/Cas12 assay showed excellent detection of the CA-C118Y mutation. The TSWV LAMP/Cas12a assay is adaptable for in-field applications on either a smart phone platform or heat block. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent alternative approach quantifies analyte concentration by sizing native λ DNA reporters, providing a simple and label-free solution for ultrasensitive detection. However, this method faces challenges in accurately quantifying size reduction of long DNA reporters via gel electrophoresis due to the sensitivity of DNA band shift to other interferences such as gel distortion. To overcome these limitations, here we developed a simple and robust ratiometric signaling strategy using CRISPR-Cas12a-induced supercoil relaxation of dsDNA plasmid reporters. In the presence of target, we observed that the fraction of supercoiled plasmid DNA decreased, and the amount of relaxed conformation (circular) increased over time. The relative percentage of supercoiled DNA to the relaxed circular DNA was analyzed by gel electrophoresis to generate an intensity-based ratiometric signal for more accurate target concentration quantification. This simple and inexpensive method is ∼100 times more sensitive when compared with the typical fluorescent reporter system. 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 ratiometric sensing strategy using model DNA targets such as AAV and HPV 16, highlighting its feasibility for point-of-use CRISPR-Dx applications. Table of Contents (TOC)

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 minutes of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT). Graphical Abstract