Gold nanoparticles-mediated fluorescent chemical nose sensor for pathogenic diagnosis and phenotype.

Pathogens are one of the important factors affecting national economic construction. An ideal detection system for pathogen control with excellent sensitivity, high specificity, and time-saving is needed. Here, we reported a method for bacterial detection using gold nanoparticles-mediated fluorescent "chemical nose" sensors (GFCEs). The technique consists of gold nanoparticles-coated magnetic particle using benzaldehyde, octyl aldehyde, and pyrimidine-4-formaldehyde modified, respectively. And these positively charged nanocompound interacting with three different fluorescent proteins (FPs) to form three kinds of GFCEs, respectively, named GFCE1, GFCE2, and GFCE3. Upon binding with pathogenic cells, functionalized gold nanoparticles could identify patches on hydrophobic/functional surfaces of microorganisms, and self-assemble with living bacteria by complementary electrostatic interactions. The binding ability between GFCEs and bacteria determines the change of fluorescence response of three FPs from GFCEs. These feature fluorescent level are pathogen-specific, highly repeatable, and can be analyzed by Linear Discriminant Analysis (LDA). The combination of GFCE1 and GFCE2 has the best performance when detecting pathogens with concentrations of 106 cfu mL-1 . The first discriminant within 15 minutes is 93.8%, which could be used for subsequent identification of unknown samples. The commonly applicable system provides a simple way for the rapid bacterial detection without preprocessing procedures.

Trypsin-Amplified Aerolysin Nanopore Amplified Sandwich Assay for Attomolar Nucleic Acid and Single Bacteria Detection.

Nanopore technology is promising for the next-generation of nucleic acid-based diagnosis. However, sequence reservation could still be hardly achieved in low-concentration. Herein, we propose a trypsin-activated catalysis reaction for amplified detection, which substantially improves the sensitivity of nanopore technique. The proposed trypsin-amplified nanopore amplified sandwich assay (tNASA) could contribute to a sensitivity approximately 100 000 times higher based on nucleic acid probe design. Remarkably, tNASA is capable of attomolar nucleic acid and single cell detection by using a miniaturized current amplifier without alignment algorithm. Also it allows 10 pathogenic species in serum to be accurately and robustly profiled, thus be utilized for the diagnosis of infectious diseases. tNASA may evolve the construction of nanopore techniques for nucleic acid detection and would facilitate its translation for pocket diagnosis and precision medicine.

Electrical Signal Reporter, Pore-Forming Protein, for Rapid, Miniaturized, and Universal Identification of Microorganisms.

Despite recent advances in signal reporter-based assays for bacteria detection and profiling, the low-cost, ultrasensitive, accurate, and fast diagnosis remains a challenge for better patient care. Herein, we present a novel bacteria identification method based on α-hemolysin-labeled sandwich assay (HLSA). A pore-forming protein, α-hemolysin, is used as an electrical signal reporter. The assay takes advantage of the specific binding of target nucleic acid with two hybridization probes: capture probe decorated magnetic microparticles and oligonucleotides detecting probe and α-hemolysin modified gold nanoparticles. α-Hemolysin was then released by competitive gold binding peptide incubation into an electric cell with a lipid bilayer between the electrodes. The nanopores formed by α-hemolysin on the lipid layer allowed target nucleic acid concentration-dependent currents for quantification. Sandwich probes against 16S rRNAs of 10 common bacteria pathogens were designed and single cell level nucleic acid concentration detection was achieved. Compared with nanopore technique-based DNA sequencing, HLSA gives a quantitive and straightforward readout that is not dependent on an ultrasensitive and expensive instrument (Axopatch 200B amplifier), thus, is faster and requires no large-scale instruments. Also, since α-hemolysin-modified nanoparticles will be washed out before the α-hemolysin releasing step without the target nucleic acid, no current will be detected, and thus, the assay is more specific. The current strategy based on the electrical signal reporter offers a new insight for pathogen and virus diagnostics.

CRISPR/Cas9-mediated deletion of EcMIH shortens metamorphosis time from mysis larva to postlarva of Exopalaemon carinicauda.

The recently emerged CRISPR/Cas9 technology is the most flexible means to produce targeted mutations at the genomic loci in a variety of organisms. In Crustaceans, molt-inhibiting hormone (MIH) is an important negative-regulatory factor and plays a key role in suppressing the molting process. However, whether precise disruption of MIH in crustacean can be achieved and successfully used to improve the development and growth has not been proved. In this research, the complementary DNA (cDNA) and genomic DNA, including flanking regions of the MIH gene (EcMIH) of ridgetail white prawn Exopalaemon carinicauda, were cloned and sequenced. Sequence analysis revealed that EcMIH was composed of three exons and two introns. Analysis by RT-PCR showed that EcMIH mainly expressed in eyestalks. During different development periods, EcMIH was highest in juvenile stage and extremely low in others but adult prawns eyestalks. In addition, we applied CRISPR/Cas9 technology to generate EcMIH knock-out (KO) prawns and then analyzed the changes in their phenotypes. We efficiently generated 12 EcMIH-KO prawns out of 250 injected one-cell stage embryos and the mutant rate reached 4.8% after embryo injection with one sgRNA targeting the second exon of EcMIH. The EcMIH-KO prawns exhibited increased the body length and shortened the metamorphosis time of larvae from mysis larva to postlarva. Meanwhile, EcMIH-KO did not cause the health problems such as early stage death or deformity. In conclusion, we successfully obtained EcMIH gene and generated EcMIH-KO prawns using CRISPR/Cas9 technology. This study will certainly lead to a wide application prospect of MIH gene in prawns breeding.

Purification and characterization of chitinases from ridgetail white prawn Exopalaemon carinicauda.

In this paper, we purified two native chitinases from the hepatopancreas of the ridgetail white prawn Exopalaemon carinicauda by using ion-exchange resin chromatography (IEC) and gel filtration. These two chitinases, named EcChi1 and EcChi2, were identified by chitinolytic activity assay and LC-ESI-MS/MS. Their apparent molecular weights were 44 kDa and 65 kDa as determined by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of EcChi1 and EcChi2 was 1305.97 U·mg-1 and 28.69 U·mg-1. The optimal temperature and pH of EcChi1 were 37 °C and pH 4.0, respectively. Co2+, Fe3+, Zn2+, Cd2+, and Cu2+ had an obvious promoting effect upon chitinase activity of EcChi1. For colloidal chitin, the Km and Vmax values of EcChi1 were 2.09 mg·mL-1 and 31.15 U·mL-1·h-1.

Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.

Ligation-dependent Cas14a1-Activated biosensor for one-pot pathogen diagnostic.

Pathogen identification requires nucleic acid diagnosis with simple equipment and fast manipulation. Our work established an all-in-one strategy assay with excellent sensitivity and high specificity, Transcription-Amplified Cas14a1-Activated Signal Biosensor (TACAS), for the fluorescence-based bacterial RNA detection. The DNA as a promoter probe and a reporter probe directly ligated via SplintR ligase once specifically hybridized to the single-stranded target RNA sequence, with the ligation product transcribed into Cas14a1 RNA activators by T7 RNA polymerase. This forming sustained isothermal one-pot ligation-transcription cascade produced RNA activators constantly and enabled Cas14a1/sgRNA complex to generate fluorescence signal, thus leading to a sensitive detection limit of 1.52 CFU mL-1E. coli within 2 h of incubation time. TACAS was applied in contrived E. coli infected fish and milk samples, and a significant signal differentiation between positive (infected) and negative (uninfected) samples was reached. Meanwhile, E. coli colonization and transmit time in vivo were explored and the TACAS assay promoted the understanding of the infection mechanisms of the E. coli infection, demonstrating an excellent detection capability.

Z-scheme heterojunction based on NiWO4/WO3 microspheres with enhanced photocatalytic performance under visible light.

The green treatment of dye wastewater has always been a research hotspot in the environmental field. The photocatalytic technology is considered to be a simple and effective strategy to remove dyes in wastewater. A new type of NiWO4/WO3 Z-scheme heterojunction microspheres were synthesized by a simple hydrothermal method and impregnation-calcination process. The crystal structure, microscopic morphology, optical and electrochemical properties of the samples were systematically characterized. The photocatalytic activity of methylene blue (MB) was studied by visible light irradiation. The results show that the direct Z-scheme heterojunction formed by NiWO4/WO3 effectively reduces the transfer resistance of photogenerated carriers and improves the separation efficiency of photogenerated carriers. The degradation rates of NiWO4/WO3-4 Z-scheme heterojunction microspheres to MB dye are 1.8 and 3.2 times higher than that of pure WO3·2H2O and WO3 microspheres, respectively. Combined with the Mott-Schottky curve and the active species capture experiments, a possible Z-scheme photogenerated carrier transfer mechanism is proposed. This study provides a method for the development and design of Z-scheme heterojunction photocatalysts in the field of wastewater purification.

Botulinum toxin as an ultrasensitive reporter for bacterial and SARS-CoV-2 nucleic acid diagnostics.

The rapid identification of pathogenic microorganisms plays a crucial role in the timely diagnosis and treatment strategies during a global pandemic, especially in resource-limited area. Herein, we present a sensitive biosensor strategy depended on botulinum neurotoxin type A light chain (BoNT/A LC) activated complex assay (BACA). BoNT/A LC, the surrogate of BoNT/A which embodying the most potent biological poisons, could serve as an ultrasensitive signal reporter with high signal-to-noise ratio to avoid common strong background response, poor stability and low intensity of current biosensor methods. A nanoparticle hybridization system, involving specific binding probes that recognize pathogenic 16S rRNAs or SARS-CoV-2 gene site, was developed to measure double-stranded biotinylated target DNA containing a single-stranded overhang using Fluorescence Resonance Energy Transfer (FRET)-based assay and colorimetric method. The method is validated widely by six different bacteria strains and severe acute respiratory related coronavirus 2 (SARS-CoV-2) nucleic acid, demonstrating a single cell or 1 aM nucleic acid detecting sensitivity. This detection strategy offers a solution for general applications and has a great prospect to be a simple instrument-free colorimetric tool, especially when facing public health emergency.

Cas14a1-mediated nucleic acid detectifon platform for pathogens.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) based biosensing system provides a novel genomic diagnostic tool for pathogenic detection. However, most of the discovered Cas effectors have poor single strand DNA (ssDNA) target recognition capability with the constraint of protospacer adjacent motif (PAM) sites, which are not suitable for universal pathogenic diagnosis. Herein, we developed a highly sensitive and specific fluorescence tool for bacterial detection by utilizing the unique collateral cleavage activity of a Cas14a1-mediated nucleic acid detection platform (CMP). We combine CMP with molecular amplification to build a CRISPR-Cas based bioanalysis technique, offering fast nucleic acid detection with high sensitivity and specificity. This technique can identify different species of pathogens in milk samples with excellent accuracy. The CMP technique is a promising platform for pathogenic genomic diagnostic in biomedicine and food safety field.

Combining tag-specific primer extension and magneto-DNA system for Cas14a-based universal bacterial diagnostic platform.

Nucleic acid-based diagnosis using CRISPR-Cas associated enzymes is essential for rapid infectious disease diagnosis and treatment strategies during a global pandemic. The obstacle has been blossomed CRIPSR-Cas based tools that can monitor wide range of pathogens in clinical samples with ultralow concentrations. Here, a universal nucleic acid magneto-DNA nanoparticle system was exploited for the detection of pathogenic bacteria, based on the collateral cleavage activity of CRISPR-Cas14a and tag-specific primer extension. In the system, the target nucleic acids were amplificated and be separated from mixtures by streptavidin-coated magnetic bead. The collateral cleavage activity of CRISPR-Cas14a can be activated via the tag sequence on the target product. Consequently, the fluorophore quencher reporter can be activated by CRISPR-Cas14a, leading to the increasing response. The exploited universal bacterial diagnostic can distinguish six different bacteria strains with 1 cfu/mL or 1 aM sensitivity, which may provide new strategies to construct fast, accurate, cost-effective and sensitive diagnostic tools in environments with limited resources.

Cas12a-Activated Universal Field-Deployable Detectors for Bacterial Diagnostics.

Field-deployable detectors of disease biomarkers provide a simple and fast analysis of clinical specimens. However, most of the existing field-deployable diagnostics have poor sensitivity and are not suitable for the detection of biomarkers with low abundance. Herein, we report a highly sensitive and rapid colorimetric readout paper-based assay for pathogen detection by integrating the unique collateral activity of a Cas12a-activated universal field-deployable detector (CUFD). The collateral effect of Cas12a results in a nonspecific destruction of a fluorophore biotin-labeled ssDNA reporter for the CUFD. This technique can quantify seven different kinds of pathogens in blood samples without any purification procedure, with sensitivity as low as 10 aM for the Shigella dysenteriae DNA. This CUFD technique has significant potential for the detection of pathogenic DNA as well as other types of DNA or RNA targets at the point-of-care application.

Enzymatic characterization and functional analysis of EcChi3C from ridgetail white prawn Exopalaemon carinicauda.

Chitinase belongs to the glycosyl hydrolases family 18 and plays key role in the development and pathogen resistance of crustaceans. In this study, the enzymatic characterization of chitinase 3C (EcChi3C) of Exopalaemon carinicauda was analyzed. In addition, we analyzed the expression profiles of EcChi3C at different tissues and different molting stages. In the all tested tissues, it was predominantly expressed in hepatopancreas, and then stomach, but poor in other tissues. In all tested molting periods, it was mainly expressed in intermolt and molting stages, but poor in other stages. The results of molting, mortality and the uropod ultrastructure of prawns after being injected with EcChi3C dsRNA were in accordance with those of the control group. In addition, there is no difference for endopodite morphology between the survival and dead individuals in experimental group. After being challenged with bacteria, the expression of EcChi3C was up-regulated significantly at 12 h and followed with a comeback at 96 h. These results suggest that EcChi3C is an important immune related gene but not a necessary gene in the molting process of E. carinicauda.

CRISPR/Cas9-Mediated Genome Editing and Mutagenesis of EcChi4 in Exopalaemon carinicauda.

The development of the type II clustered regularly interspaced short palindromic repeats (CRISPR) system has resulted in the revolution of genetic engineering, and this technology has been applied in the genome editing of various species. However, there are no reports about target-specific genome editing in shrimp. In this research, we developed a microinjection method for the ridgetail white prawn Exopalaemon carinicauda and successfully applied CRISPR/Cas9 technology to the genome editing of E. carinicauda Through coinjection of mRNA of Cas9 nuclease and gRNA specialized for E. carinicauda chitinase 4 (EcChi4), shrimps with indel mutations were obtained. Further analysis showed that the mutations could be transmitted to the next generation. This is the first time that site-specific genome editing has been successfully demonstrated in a decapod, and will further contribute to the study of functional genomics in decapods.