Diversity and distribution of bamboo-feeding true bugs in China.

The Bambusoideae subfamily, originating in the late Cretaceous, has evolved to include over 1500 species globally. Notably, China hosts the richest diversity of Bambusoideae, with 728 species documented. After a long period of coevolution, plenty of animals could feed on these plants rich in cellulose and lignin. As an important group of pests and participants in the ecosystem, bamboo-feeding true bugs (BFTBs, or bamboo-feeding Heteropteran insects) have attracted the attention of researchers. However, the diversity and distribution of BFTBs still lack systematic and generalized research. In this study, we reviewed the BFTBs in China and simulated the diversity pattern and the driving forces of this pattern. A list of 36 genera with 69 species of BFTBs in China was obtained through paper review and field surveys. And their bamboo-feeding habit had multiple independent origins. The spatial diversity pattern showed that the biodiversity hotspots of BFTBs are located in and around the tropics of southern China. Environmental driving force analysis showed that the minimum temperature of coldest month and annual precipitation were the dominant environmental factors shaping the spatial diversity of BFTBs. Our work quantified the diversity and distribution of BFTBs in China, providing fundamental data support for pest control and evolutionary research.

Ensuring food safety: Microfluidic-based approaches for the detection of food contaminants.

Detecting foodborne contamination is a critical challenge in ensuring food safety and preventing human suffering and economic losses. Contaminated food, comprising biological agents (e.g. bacteria, viruses and fungi) and chemicals (e.g. toxins, allergens, antibiotics and heavy metals), poses significant risks to public health. Microfluidic technology has emerged as a transformative solution, revolutionizing the detection of contaminants with precise and efficient methodologies. By manipulating minute volumes of fluid on miniaturized systems, microfluidics enables the creation of portable chips for biosensing applications. Advancements from early glass and silicon devices to modern polymers and cellulose-based chips have significantly enhanced microfluidic technology, offering adaptability, flexibility, cost-effectiveness and biocompatibility. Microfluidic systems integrate seamlessly with various biosensing reactions, facilitating nucleic acid amplification, target analyte recognition and accurate signal readouts. As research progresses, microfluidic technology is poised to play a pivotal role in addressing evolving challenges in the detection of foodborne contaminants. In this short review, we delve into various manufacturing materials for state-of-the-art microfluidic devices, including inorganics, elastomers, thermoplastics and paper. Additionally, we examine several applications where microfluidic technology offers unique advantages in the detection of food contaminants, including bacteria, viruses, fungi, allergens and more. This review underscores the significant advancement of microfluidic technology and its pivotal role in advancing the detection and mitigation of foodborne contaminants.

Integrative taxonomy, phylogenetics and historical biogeography of subgenus Aeschyntelus Stål, 1872 (Hemiptera: Heteroptera: Rhopalidae).

The subgenus Aeschyntelus includes six species that show variations in body color and shape, thus making it difficult to identify them based on morphological identification alone. To date, no genetic study has evaluated species within this genus. Herein, we collected 171 individuals from 90 localities of Rhopalus and employed an integrative taxonomic approach that incorporated morphological data, mitochondrial genomic data (COI, whole mitochondrial data) and nuclear genomic data (18S + 28S rRNAs, nuclear genome-wide SNPs) to delineate species boundaries. Our analyses confirmed the status of nine described species of Rhopalus and proposed the recognition of one new species known as Rhopalus qinlinganus sp. nov., which is classified within the subgenus Aeschyntelus. Discrepancies arising from nuclear and mitochondrial data suggest the presence of mito-nuclear discordance. Specifically, mitochondrial data indicated admixture within Clade A, comprising R. kerzhneri and R. latus, whereas genome-wide SNPs unambiguously identified two separate species, aligning with morphological classification. Conversely, mitochondrial data clearly distinguished Clade B- consisting of R. sapporensis into two lineages, whereas genome-wide SNPs unequivocally identified a single species. Our study also provides insights into the evolutionary history of Aeschyntelus, thus indicating that it likely originated in East Asia during the middle Miocene. The development of Aeschyntelus biodiversity in the southwestern mountains of China occurred via an uplift-driven diversification process. Our findings highlight the necessity of integrating both morphological and multiple molecular datasets for precise species identification, particularly when delineating closely related species. Additionally, it reveals the important role of mountain orogenesis on speciation within the southwestern mountains of China.

Development of a self-powered digital LAMP microfluidic chip (SP-dChip) for the detection of emerging viruses.

Point-of-care (POC) diagnostics have emerged as a crucial technology for emerging pathogen detections to enable rapid and on-site detection of infectious diseases. However, current POC devices often suffer from limited sensitivity with poor reliability to provide quantitative readouts. In this paper, we present a self-powered digital loop-mediated isothermal amplification (dLAMP) microfluidic chip (SP-dChip) for the rapid and quantitative detection of nucleic acids. The SP-dChip utilizes a vacuum lung design to passively digitize samples into individual nanoliter wells for high-throughput analysis. The superior digitization scheme is further combined with reverse transcription loop-mediated isothermal amplification (RT-LAMP) to demonstrate dLAMP detection of Zika virus (ZIKV). Firstly, the LAMP assay is loaded into the chip and passively digitized into individual wells. Mineral oil is then pipetted through the chip to differentiate each well as an individual reactor. The chip did not require any external pumping or power input for rapid and reliable results to detect ZIKA RNA as low as 100 copies per µL within one hour. As such, this SP-dChip offers a new class of solutions for truly affordable, portable, and quantitative POC detections for emerging viruses.

The ncRNA-mediated regulatory networks of defensins and lysozymes in Riptortus pedestris: involvement in response to gut bacterial disturbances.

Insects depend on humoral immunity against intruders through the secretion of antimicrobial peptides (AMPs) and immune effectors via NF-κB transcription factors, and their fitness is improved by gut bacterial microbiota. Although there are growing numbers of reports on noncoding RNAs (ncRNAs) involving in immune responses against pathogens, comprehensive studies of ncRNA-AMP regulatory networks in Riptortus pedestris, which is one of the widely distributed pests in East Asia, are still not well understood under feeding environmental changes. The objective of this study employed the whole-transcriptome sequencing (WTS) to systematically identify the lncRNAs (long noncoding RNA) and circRNAs (circular RNA) and to obtain their differential expression from the R. pedestris gut under different feeding conditions. Functional annotation indicated that they were mainly enriched in various biological processes with the GO and KEGG databases, especially in immune signaling pathways. Five defensin (four novel members) and eleven lysozyme (nine novel members) family genes were identified and characterized from WTS data, and meanwhile, phylogenetic analysis confirmed their classification. Subsequently, the miRNA-mRNA interaction network of above two AMPs and lncRNA-involved ceRNA (competing endogenous RNA) regulatory network of one lysozyme were predicted and built based on bioinformatic prediction and calculation, and the expression patterns of differentially expressed (DE) defensins, and DE lysozymes and related DE ncRNAs were estimated and selected among all the comparison groups. Finally, to integrate the analyses of WTS and previous 16S rRNA amplicon sequencing, we conducted the Pearson correlation analysis to reveal the significantly positive or negative correlation between above DE AMPs and ncRNAs, as well as most changes in the gut bacterial microbiota at the genus level of R. pedestris. Taken together, the present observations provide great insights into the ncRNA regulatory networks of AMPs in response to rearing environmental changes in insects and uncover new potential strategies for pest control in the future.

CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus.

The highly contagious nature and 100% fatality rate contribute to the ongoing and expanding impact of the African swine fever virus (ASFV), causing significant economic losses worldwide. Herein, we developed a cascaded colorimetric detection using the combination of a CRISPR/Cas14a system, G-quadruplex DNAzyme, and microfluidic paper-based analytical device. This CRISPR/Cas14a-G4 biosensor could detect ASFV as low as 5 copies/µL and differentiate the wild-type and mutated ASFV DNA with 2-nt difference. Moreover, this approach was employed to detect ASFV in porcine plasma. A broad linear detection range was observed, and the limit of detection in spiked porcine plasma was calculated to be as low as 42-85 copies/µL. Our results indicate that the developed paper platform exhibits the advantages of high sensitivity, excellent specificity, and low cost, making it promising for clinical applications in the field of DNA disease detection and suitable for popularization in low-resourced areas.

Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA).

Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

Shared phylogeographic patterns and environmental responses of co-distributed soybean pests: Insights from comparative phylogeographic studies of Riptortus pedestris and Riptortus linearis in the subtropics of East Asia.

Comparative phylogeographic studies of closely related species sharing co-distribution areas can elucidate the role of shared historical factors and environmental changes in shaping their phylogeographic pattern. The bean bugs, Riptortus pedestris and Riptortus linearis, which both inhabit subtropical regions in East Asia, are recognized as highly destructive soybean pests. Many previous studies have investigated the biological characteristics, pheromones, chemicals and control mechanisms of these two pests, but few studies have explored their phylogeographic patterns and underlying factors. In this study, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) dataset to investigate phylogeographic patterns and construct ecological niche models (ENM) for both Riptortus species. Our findings revealed similar niche occupancies and population genetic structures between the two species, with each comprising two phylogeographic lineages (i.e., the mainland China and the Indochina Peninsula clades) that diverged approximately 0.1 and 0.3 million years ago, respectively. This divergence likely resulted from the combined effects of temperatures variation and geographical barriers in the mountainous regions of Southwest China. Further demographic history and ENM analyses suggested that both pests underwent rapid expansion prior to the Last Glacial Maximum (LGM). Furthermore, ENM predicts a northward shift of both pests into new soybean-producing regions due to global warming. Our study indicated that co-distribution soybean pests with overlapping ecological niches and similar life histories in subtropical regions of East Asia exhibit congruent phylogeographic and demographic patterns in response to shared historical biogeographic drivers.

Identifying a potentially invasive population in the native range of a species: The enlightenment from the phylogeography of the yellow spotted stink bug, Erthesina fullo (Hemiptera: Pentatomidae).

The yellow spotted stink bug (YSSB), Erthesina fullo (Thunberg, 1783) is an important Asian pest that has recently successfully invaded Europe and an excellent material for research on the initial stage of biological invasion. Here, we reported the native evolutionary history, recent invasion history, and potential invasion threats of YSSB for the first time based on population genetic methods [using double digest restriction-site associated DNA (ddRAD) data and mitochondrial COI and CYTB] and ecological niche modelling. The results showed that four lineages (east, west, southwest, and Hainan Island) were established in the native range with a strong east-west differentiation phylogeographical structure, and the violent climate fluctuation might cause population divergence during the Middle and Upper Pleistocene. In addition, land bridges and monsoon promote dispersal and directional genetic exchanging between island populations and neighboring continental populations. The east lineage (EA) was identified as the source of invasion in Albania. EA had the widest geographical distribution among all other lineages, with a star-like haplotype network with the main haplotype as the core. It also had a rapid population expansion history, indicating that the source lineage might have stronger diffusion ability and adaptability. Our findings provided a significant biological basis for fine tracking of invasive source at the lineage or population level and promote early invasion warning of potential invasive species on a much subtler lineage level.

On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System.

Sepsis is an extremely dangerous medical condition that emanates from the body's response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsis-inducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsis-inducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 103 CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resource-limited settings, expediting the response for patients at risk of sepsis.

A low-background and wash-free signal amplification F-CRISPR biosensor for sensitive quantitative and visible qualitative detection of Salmonella Typhimurium.

In traditional CRISPR-based biosensors, the cleavage-induced signal generation is insufficient because only a signals is generated at a CRISPR-induced cleavage. Herein, we developed an improved CRISPR/Cas12a-based biosensor with an enlarged signal generation which integrated the hybridization chain reaction (HCR) and low-background Förster Resonance Energy Transfer (FRET) signal output mode. The HCR with nucleic acid self-assembly capability was used as a signal carrier to load more signaling molecules. To get the best signal amplification, three different fluorescence signal output modes (fluorescence recovery, FRET and low-background FRET) generated by two fluoresceins, FAM and Cy5, were fully investigated and compared. The results indicated that the low-background FRET signal output mode with the strictest signal generation conditions yielded the highest signal-to-noise ratio (S/N) (19.17) and the most obvious fluorescence color change (from red to yellow). In optimal conditions, the proposed biosensor was successfully applied for Salmonella Typhimurium (S. Typhimurium) detection with 6 h (including 4 h for sample pre-treatment) from the initial target processing to the final detection result. The qualitative sensitivity, reliant on color changes, was 103 CFU/mL. The quantitative sensitivity, calculated by the fluorescence value, were 1.62 × 101 CFU/mL, 3.72 × 102 CFU/mL, and 8.71 × 102 CFU/mL in buffer solution, S. Typhimurium-spiked milk samples, and S.Typhimurium-spiked chicken samples, respectively. The excellent detection performance of the proposed biosensor endowed its great application potential in food and environment safety monitoring.

Integrated analysis of miRNA profiles and gut bacterial changes in Altica viridicyanea following antibiotic treatment.

The gut bacteria involves in insect homeostasis by playing essential roles in host physiology, metabolism, innate immunity, and so forth. microRNAs (miRNAs) are endogenous small noncoding RNAs that posttranscriptionally regulate gene expression to affect immune or metabolic processes in insects. For several non-model insects, the available knowledge on the relationship between changes in the gut bacteria and miRNA profiles is limited. In this study, we investigated the gut bacterial diversity, composition, and function from Altica viridicyanea feeding on normal- and antibiotic-treated host plants using 16S rRNA amplicon sequencing; antibiotics have been shown to affect the body weight and development time in A. viridicyanea, suggesting that the gut bacteria of the normal sample were more diverse and abundant than those of the antibiotic-fed group, and most of them were involved in various physical functions by enrichment analysis. Furthermore, we executed small RNA transcriptome sequencing using the two experimental groups to obtain numerous sRNAs, such as piRNAs, siRNAs, and known and novel miRNAs, by data mapping and quality control, and furthermore, a total of 224 miRNAs were identified as significantly differentially expressed miRNAs, of which some DEMs and their target genes participated in immune- and metabolism-related pathways based on GO and KEGG annotation. Besides, regarding the regulatory roles of miRNA and target genes, a interaction network of DEM-target gene pairs from eight immune- or metabolism-related signaling pathways were constructed. Finally, we discovered that DEMs from above pathways were significantly positively or negatively correlated with gut bacterial alterations following antibiotic treatment. Collectively, the observations of this study expand our understanding of how the disturbance of gut bacteria affects miRNA profiles in A. viridicyanea and provide new valuable resources from extreme ranges for future studies on the adaptive evolution in insects.

High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems.

Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 103 copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.

CRISPR-based Biosensors for Human Health: A Novel Strategy to Detect Emerging Infectious Diseases.

Infectious diseases (such as sepsis, influenza, and malaria), caused by various pathogenic bacteria and viruses, are widespread across the world. Early and rapid detection of disease-related pathogens is necessary to reduce their spread in the world and prevent their potential global pandemics. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, as the next-generation molecular diagnosis technique, holds immense promise in the detection of infectious diseases because of its remarkable advantages, including supreme flexibility, sensitivity, and specificity. While numerous CRISPR-based biosensors have been developed for application in environmental monitoring, food safety, and point-of-care diagnosis, there remains a critical need to summarize and explore their potential in human health. This review aims to address this gap by focusing on the latest advancements in CRISPR-based biosensors for infectious disease detection. We provide an overview of the current status, pre-amplification methods, the unique feature of each CRISPR system, and the design of CRISPR-based biosensing strategies to detect disease-associated nucleic acids. Last but not least, the review analyzes the current challenges and provides future perspectives, which will contribute to developing more effective CRISPR-based biosensors for human health.

Micro- and nanosystems for the detection of hemorrhagic fever viruses.

Hemorrhagic fever viruses (HFVs) are virulent pathogens that can cause severe and often fatal illnesses in humans. Timely and accurate detection of HFVs is critical for effective disease management and prevention. In recent years, micro- and nano-technologies have emerged as promising approaches for the detection of HFVs. This paper provides an overview of the current state-of-the-art systems for micro- and nano-scale approaches to detect HFVs. It covers various aspects of these technologies, including the principles behind their sensing assays, as well as the different types of diagnostic strategies that have been developed. This paper also explores future possibilities of employing micro- and nano-systems for the development of HFV diagnostic tools that meet the practical demands of clinical settings.

Changes in the gut bacterial community affect miRNA profiles in Riptortus pedestris under different rearing conditions.

Insects possess complex and dynamic gut microbial system, which contributes to host nutrient absorption, reproduction, energy metabolism, and protection against stress. However, there are limited data on interactions of host-gut bacterial microbiota through miRNA (microRNA) regulation in a significant pest, Riptortus pedestris. Here, we performed the 16S rRNA amplicon sequencing and small RNA sequencing from the R. pedestris gut under three environmental conditions and antibiotic treatment, suggesting that we obtained a large amount of reads by assembly, filtration and quality control. The 16S rRNA amplicon sequencing results showed that the abundance and diversity of gut bacterial microbiota were significantly changed between antibiotic treatment and other groups, and they are involved in metabolism and biosynthesis-related function based on functional prediction. Furthermore, we identified different numbers of differentially expressed unigenes (DEGs) and differentially expressed miRNAs (DEMs) based on high-quality mappable reads, which were enriched in various immune-related pathways, including Toll-like receptor, RIG-I-like receptor, NOD-like receptor, JAK/STAT, PI3K/Akt, NF-κB, MAPK signaling pathways, and so forth, using GO and KEGG enrichment analysis. Later on, the identified miRNAs and their target genes in the R. pedestris gut were predicted and randomly selected to construct an interaction network. Finally, our study indicated that alterations in the gut bacterial microbiota are significantly positively or negatively associated with DEMs of the Toll/Imd signaling pathway with Pearson correlation analysis. Taken together, the results of our study lay the foundation for further deeply understanding the interactions between the gut microbiota and immune responses in R. pedestris through miRNA regulation, and provide the new basis for pest management in hemipteran pests.

GlMPC activated by GCN4 regulates secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum.

IMPORTANCE: Mitochondrial pyruvate carrier (MPC) is a pyruvate transporter that plays a crucial role in regulating the carbon metabolic flow and is considered an essential mechanism for microorganisms to adapt to environmental changes. However, it remains unclear how MPC responds to environmental stress in organisms. General control non-derepressible 4 (GCN4), a key regulator of nitrogen metabolism, plays a pivotal role in the growth and development of fungi. In this study, we report that GCN4 can directly bind to the promoter region and activate the expression of GlMPC, thereby regulating the tricarboxylic acid cycle and secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum. These findings provide significant insights into the regulation of carbon and nitrogen metabolism in fungi, highlighting the critical role of GCN4 in coordinating metabolic adaptation to environmental stresses.

The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.

Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.

Development of Nanobody-Displayed Whole-Cell Biosensors for the Colorimetric Detection of SARS-CoV-2.

The accurate and effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to preventing the spread of infectious diseases and ensuring human health. Herein, a nanobody-displayed whole-cell biosensor was developed for colorimetric detection of SARS-CoV-2 spike proteins. Serving as bioreceptors, yeast surfaces were genetically engineered to display SARS-CoV-2 binding of llama-derived single-domain antibodies (nanobodies) with high capture efficiency, facilitating the concentration and purification of SARS-CoV-2. Gold nanoparticles (AuNPs) employed as signal transductions were functionalized with horseradish peroxidase (HRP) and anti-SARS monoclonal antibodies to enhance the detection sensitivity. In the presence of SARS-CoV-2 spike proteins, the sandwiched binding will be formed by linking engineered yeast, SARS-CoV-2 spike proteins, and reporter AuNPs. The colorimetric signal was generated by the enzymatic reaction of HRP and its corresponding colorimetric substrate/chromogen system. At the optimal conditions, the developed whole-cell biosensor enables the sensitive detection of SARS-CoV-2 spike proteins in a linear range from 0.01 to 1 µg/mL with a limit of detection (LOD) of 0.037 µg/mL (about 4 × 108 virion particles/mL). Furthermore, the whole-cell biosensor was demonstrated to detect the spike protein of different SARS-CoV-2 variants in human serum, providing new possibilities for the detection of future SARS-CoV-2 variants.

Superhydrophobic Rotation-Chip for Computer-Vision Identification of Drug-Resistant Bacteria.

The transport, distribution, and mixing of microfluidics often require additional instruments, such as pumps and valves, which are not feasible when operated in point-of-care (POC) settings. Here, we present a simple microfluidic pathogen detection system known as Rotation-Chip that transfers the reagents between wells by manually rotating two concentric layers without using external instruments. The Rotation-Chip is fabricated by a simple computer numerical control (CNC) machining process and is capable of carrying out 60 multiplexed reactions with a simple 30 or 60° rotation. Leveraging superhydrophobic coating, a high fluid transport efficiency of 92.78% is achieved without observable leaking. Integrated with an intracellular fluorescence assay, an on-chip detection limit of 1.8 × 106 CFU/mL is achieved for ampicillin-resistant Escherichia coli (E. coli), which is similar to our off-chip results. We also develop a computer vision method to automatically distinguish positive and negative samples on the chip, showing 100% accuracy. Our Rotation-Chip is simple, low-cost, high-throughput, and can display test results with a single chip image, making it ideal for various multiplexing POC applications in resource-limited settings.