Versatile Platinum Nanoparticles-Decorated Phage Nanozyme Integrating Recognition, Bacteriolysis, and Catalysis Capabilities for On-Site Detection of Foodborne Pathogenic Strains Vitality Based on Bioluminescence/Pressure Dual-Mode Bioassay.

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 102-107 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

Red and far-red light improve the antagonistic ability of Trichoderma guizhouense against phytopathogenic fungi by promoting phytochrome-dependent aerial hyphal growth.

Light as a source of information regulates morphological and physiological processes of fungi, including development, primary and secondary metabolism, or the circadian rhythm. Light signaling in fungi depends on photoreceptors and downstream components that amplify the signal to govern the expression of an array of genes. Here, we investigated the effects of red and far-red light in the mycoparasite Trichoderma guizhouense on its mycoparasitic potential. We show that the invasion strategy of T. guizhouense depends on the attacked species and that red and far-red light increased aerial hyphal growth and led to faster overgrowth or invasion of the colonies. Molecular experiments and transcriptome analyses revealed that red and far-red light are sensed by phytochrome FPH1 and further transmitted by the downstream MAPK HOG pathway and the bZIP transcription factor ATF1. Overexpression of the red- and far-red light-induced fluffy gene fluG in the dark resulted in abundant aerial hyphae formation and thereby improvement of its antagonistic ability against phytopathogenic fungi. Hence, light-induced fluG expression is important for the mycoparasitic interaction. The increased aggressiveness of fluG-overexpressing strains was phenocopied by four random mutants obtained after UV mutagenesis. Therefore, aerial hyphae formation appears to be a trait for the antagonistic potential of T. guizhouense.

A microfluidic chip platform based on Pt nanozyme and magnetized phage composite probes for dual-mode detecting and imaging pathogenic bacteria viability.

The detection of pathogen viability is critically important to evaluate its infectivity. In the study, an integrated microfluidic chip based on dual-mode analytical strategy was developed to rapidly realize detection of bacteria activity (with Salmonella typhimurium, S.T, as a model analyte). Firstly, the composite probes, including deactivated phage modified magnetic beads and nano Pt-antimicrobial peptide (AMP) which can specifically recognize Gram-negative bacteria as nanozyme were prepared. When the composite probes are introduced into the chip together with target bacteria, after enrichment, oscillating and magnetic separation, they will conjugate with S.T and produce a magnetic sandwich complex. The complex can catalyze tetramethylbenzidine (TMB)-H2O2 to produce visible colorimetric signals which is correspondent to the total S.T content. Simultaneously, PtNPs in the complex can produce hydroxyl radical oxidation (∙OH) by decomposing H2O2. Under the synergistic action of ∙OH and AMP, the captured live S.T can be lysed to release ATP and emit bioluminescence signals which corresponds to the live S.T concentration. Therefore, the chip can simultaneously detect and image S.T at different viability in one test. The dual-mode assay demonstrated high sensitivity (≤33 CFU/mL), high specificity (identifying strain), signal amplification (5 folds) and short time (≤40min). The chip array can detect four samples in one test and exhibited advantages of high-integration, -sensitivity, -specificity and miniaturization, which are suitable to rapidly detect and image pathogen's viability in trace level. The replacement of phage probes can detect other bacteria. It has a wide prospect in pathogens screening.

A universal dual-mode hydrogel array based on phage-DNA probe for simultaneous rapid screening and precisely quantitative detection of Escherichia coli O157:H7 in foods by the fluorescent/microfluidic chip electrophoresis methods.

Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL-1 by the FL signal and 6 CFU mL-1 by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.

Femtomolar endogenous adenosine triphosphate-responded photoelectrochemical biosensor based on Au@Cu2O core-shell nanocubes for the ultrasensitive determination of Escherichia coli O157:H7 in foods.

Sensitive and precise determination of virulent foodborne pathogens is significant for food safety. Herein, an ultrasensitive photoelectrochemical (PEC) bioanalysis was developed using the endogenous adenosine triphosphate (ATP)-responded Au@Cu2O core-shell nanocubes (Au@Cu2O NCs) to measure Escherichia coli O157: H7 (E. coli O157:H7) in food. Briefly, the phage-functionalized gold wire was used to specifically recognize the target pathogen. With the bacteriolysis of lysozyme, the endogenous ATP molecules were emitted from the captured target bacteria and enriched by another ATP aptamer-modified gold wire. Following the exchange with complementary DNA (cDNA) chains, the bonded ATP would be released. It could simultaneously etch the Au@Cu2O NCs and compete with external circuit electrons to combine photogenerated holes on the Au@Cu2O NCs-modified screen-printed electrode. With the synergy of the two signal amplification mechanisms, a significant attenuation of photocurrent signal appeared even with femtomolar ATP. Therefore, the purpose of ultrasensitive determination of E. coli O157:H7 was realized, which depended on the endogenous ATP rather than exogenous signal probes. The proposed biosensor presented a good analysis performance within 10-106 CFU/mL with a detection limit of 5 CFU/mL. Besides, its specificity, repeatability, and stability were also investigated and acceptable. The detection results for food samples matched well with the results detected by the plate counting method. This work gives an innovative and sensitive signal amplification strategy for PEC bioassays in foodborne pathogens detection.

Ultrasensitive and Specific Phage@DNAzyme Probe-Triggered Fluorescent Click Chemistry for On-Site Detection of Foodborne Pathogens Using a Smartphone.

Rapid, specific, and on-site detection of virulent foodborne pathogenic strains plays a key role in controlling food safety. In this work, an ultrasensitive and specific Phage@DNAzyme signal probe was designed to detect foodborne pathogens. The proposed sensing probe was composed of the selected phage and functionalized DNAzyme, which realized the specific recognition of target foodborne pathogens at the strain level and the efficient catalysis of copper(II) based azide-alkyne cycloaddition (CuAAC) click reaction with fluorescent signal, respectively. As a proof of concept, the virulent Escherichia coli O157:H7 (E. coli O157:H7) as the representative analyte was first enriched and purified from the complex food samples by a 4-mercaptophenylboronic acid-modified gold slide. Following, the Phage@DNAzyme probes were specifically combined with the captured E. coli O157: H7 and catalyzed the click reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol with the assistance of Cu(II) to generate a visual fluorescent signal. Finally, the corresponding fluorescent signals were measured by a smartphone to quantify the target concentrations. Under optimized conditions, the bioassay exhibited a wide linear range from 102 to 108 CFU/mL and the detection limit was 50 CFU/mL (S/N = 3). It was further extended to the detection of another foodborne pathogen Salmonella typhimurium with satisfying sensing performances. This work gives a new path for developing rapid, specific, and on-site detection methods for trace levels of pathogenic strains in foods.

Tesla valve-assisted biosensor for dual-mode and dual-target simultaneous determination of foodborne pathogens based on phage/DNAzyme co-modified zeolitic imidazolate framework-encoded probes.

Sensitive and accurate detection of multiplex foodborne pathogens is crucial for food safety. In this work, a dual-mode and dual-target biosensor regulated by a Tesla valve was established for simultaneously determining Escherichia coli O157:H7 (E. coli) and Salmonella typhimurium (S. T). Two zeolitic imidazolate framework (ZIF-8) signal probes decorated with electroactive materials (ferrocene or methylene blue), DNAzyme, and different phages were synthesized to specifically recognize the targets and generate fluorescent/electrochemical dual-mode signals. In the presence of bacteria, they were captured and enriched on two individual working electrodes through the modified 4-mercaptophenylboric acid. The encoded signal probes added on different working electrodes could be conjugated with the corresponding target bacteria depending on the specificity of phages. Under the acidic condition, the DNAzyme could catalyze click chemistry for fluorescent signals. Simultaneously, the released ferrocene and methylene blue from ZIF-8 could generate electrochemical signals at different potentials. Benefiting from the flow regulation feature of the Tesla valve, the triggered fluorescent and electrochemical signals in the two individual electrodes would not influence each other, achieving simultaneous dual-mode and dual-target determination of foodborne pathogens. It depicted good linearity ranged 10-107 CFU mL-1. And the corresponding detection of limits were 5 CFU mL-1 and 8 CFU mL-1 for two bacteria, respectively. A low false positive was realized through the dual-mode strategy. The proposed biosensor can not only on-site, specifically, and sensitively determine E. coli and S. T, but also provide the wide prospect in rapid screening of other foodborne pathogens.

Simultaneous and rapid screening of live and dead E. coli O157:H7 with three signal outputs: An all-in-one biosensor using phage-apoferritin@CuO2 signal tags on MXenes-modified electrode platform.

Simultaneous detection of live and dead bacteria is a huge challenge for food safety. To solve this issue, an all-in-one biosensor for bacteria was developed using the phage-apoferritin@CuO2 (phage-Apo@CP) probe on an antimicrobial peptide (AMP)/MXenes-modified detection platform. With the specific recognition of AMP and phage-Apo@CP, the biosensor for the target Escherichia coli O157:H7 (E. coli O157:H7) presented multi-mode (bioluminescent, colorimetric, and electrochemical) signals to simultaneously measure live and dead bacteria. The bioluminescent signal caused by the adenosine triphosphate (ATP) from the bacteria was used to quantify live bacteria. The colorimetric and voltammetric signals triggered by ·OH and Cu2+ from the probe with the assistance of acid could rapidly screen and quantitative determination of total E. coli O157:H7 concentration. Thus, the dead one was obtained according to the total and live ones. All three signals could be mutually corrected to improve the accuracy. The biosensor was successfully used for on-site measurement of live and dead E. coli O157:H7 in food samples with the limit of detection of 30 CFU/mL for live ones and 6 CFU/mL for total bacteria within 50 min. This work presents a novel pathway for rapid and simultaneous quantification of both live and dead bacteria.

Dual-Mode Biosensor for Simultaneous and Rapid Detection of Live and Whole Salmonella typhimurium Based on Bioluminescence and Fluorescence Detection.

Both live and dead Salmonella typhimurium (S.T) are harmful to human health, but there are differences in pathological mechanism, dosage, and security. It is crucial to develop a rapid and simultaneous assay to distinguish and quantify live and dead S.T in foods. Herein, one dual-mode biosensor for simultaneous detection of live and dead S.T was fabricated based on two phage probes, using portable bioluminescence and fluorescent meter as detectors, respectively. Firstly, a magnetic phage capture probe (M-P1) and a phage signal tag (P2-S) labeled with SYTO 13 fluorescent dye were prepared, respectively. Both M-P1 and P2-S can specifically conjugate with S.T to form a magnetic sandwich complex. After magnetic separation, the isolated complex can emit a fluorescent signal under an excited 365 nm laser, which can reflect the total amount of S.T. Afterwards, the lysozyme was added to decompose the captured live S.T, which can release ATP and produce a bioluminescent signal corresponding to the live S.T amount. The dead S.T concentration can be deduced by the difference between total and live examples. The detection limit of 55 CFU/mL for total S.T and 9 CFU/mL for live ones was within 20 min. The assay was successfully employed in milk samples and prospectively for on-site screening of other dead and live bacteria, while changing the phages for the targets.

A novel aggregation-induced emission probe-linked phage sorbent assay for virulent bacteria strain imaging and on-site detection.

It was critically important to develop some sensitive, rapid, and specific imaging or detection methods for the virulent strain in food safety monitoring. In the study, a novel tetraphenyl mono-phenylboronic acid dye (TPE-PBA) with good aggregation-induced emission (AIE) features and high combining capacity towards bacteria was first synthesized. With TPE-PBA as a signal tag, a sandwich-type AIE probe-linked phage sorbent assay was developed for imaging and detecting virulent strains using Escherichia coli O157:H7 (E. coli O157:H7) as a representative. In the assay, phages for E. coli O157:H7 were firstly fixed on the bottom of a 96-well plate to specifically capture the strain, then the TPE-PBA signal tag was added and incubated with the captured strain to produce the phage/E. coli O157:H7/TPE-PBA complex. The complex could produce intensive AIE fluorescence being proportional to the amount of E. coli O157:H7 with a detection limit of 30 CFU mL-1 within 30 min. Simultaneously, the strain could be imaged in the plate with good anti-photobleaching and AIE effects. The results demonstrated the AIE-linked phage sorbent assay with a TPE-PBA signal tag could provide a suitable platform for rapid and specific detection and imaging of virulent strains. Therefore, it exhibited good application prospects in the on-site monitoring of food pathogens.

Portable ATP bioluminescence sensor with high specificity for live Escherichia coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation.

Live foodborne pathogens proliferate rapidly and do great harm to human health, which requires appropriate methods to supervise. In this work, a portable adenosine triphosphate (ATP) bioluminescence sensor with high specificity for live E. coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation was developed. In brief, the selected phages were directionally immobilized on the poly(diallyldimethylammonium chloride)-modified gold stir bar as the bioreceptor. Following the simple stir bar absorptive extraction and bio-proliferation in the Luria-Bertani medium, the number of captured E. coli O157:H7 exploded. Finally, it was quantified by a portable ATP bioluminescence sensor. Benefitting from the high specificity of phage and simple signal dual-amplification strategy, the proposed biosensor achieved the recognition of live bacteria at strain level with superior sensitivity. Also, the portable signal readout made it suitable for on-site detection. Under optimal conditions, this bioassay provided a detectable range of 102-107 CFU mL-1 with a low detection limit of 30 CFU mL-1 within 30 min. The detection results for real samples demonstrated that there were no differences between the assay and the plate counting method, while the detection time was largely shortened. Furthermore, the assay gives a novel path for the point-of-care test (POCT) of live E. coli strain, which is promising to be extended to other virulent strains measurement with corresponding phages.

The bZIP transcription factor ATF1 regulates blue light and oxidative stress responses in Trichoderma guizhouense.

In several filamentous fungi, incident light and environmental stress signaling share the mitogen-activated protein kinase (MAPK) HOG (SAK) pathway. It has been revealed that short-term illumination with blue light triggers the activation of the HOG pathway in Trichoderma spp. In this study, we demonstrate the crucial role of the basic leucine zipper transcription factor ATF1 in blue light responses and signaling downstream of the MAPK HOG1 in Trichoderma guizhouense. The lack of ATF1 severely impaired photoconidiation and delayed vegetative growth and conidial germination. Upon blue light or H2O2 stimuli, HOG1 interacted with ATF1 in the nucleus. Genome-wide transcriptome analyses revealed that 61.8% (509 out of 824) and 85.2% (702 out of 824) of blue light-regulated genes depended on ATF1 and HOG1, respectively, of which 58.4% (481 out of 824) were regulated by both of them. Our results also show that blue light promoted conidial germination and HOG1 and ATF1 played opposite roles in controlling conidial germination in the dark. Additionally, the lack of ATF1 led to reduced oxidative stress resistance, probably because of the downregulation of catalase-encoding genes. Overall, our results demonstrate that ATF1 is the downstream component of HOG1 and is responsible for blue light responses, conidial germination, vegetative growth, and oxidative stress resistance in T. guizhouense.

A Simple and Low-Cost Strategy to Improve Conidial Yield and Stress Resistance of Trichoderma guizhouense through Optimizing Illumination Conditions.

Light is perceived by photoreceptors in fungi and further integrated into the stress-activated MAPK HOG pathway, and thereby potentially activates the expression of genes for stress responses. This indicates that the precise control of light conditions can likely improve the conidial yield and stress resistance to guarantee the low cost and long shelf life of Trichoderma-based biocontrol agents and biofertilizers. In this study, effects of wavelengths and intensities of light on conidial yield and stress tolerance to osmotic, oxidative and pH stresses in Trichoderma guizhouense were investigated. We found that 2 µmol photons/(m2 × s) of blue light increased the conidial yield more than 1000 folds as compared to dark condition and simultaneously enhanced conidial stress resistance. The enhanced conidial stress resistance is probably due to the upregulated stress-related genes in blue light, which is under the control of the blue light receptor BLR1 and the MAP kinase HOG1.

Ultrasensitive microfluidic immunosensor with stir bar enrichment for point-of-care test of Staphylococcus aureus in foods triggered by DNAzyme-assisted click reaction.

This work demonstrated an ultrasensitive and simple microfluidic immunosensor for point-of-care test of Staphylococcus aureus (S. aureus) based on the stir bar enrichment and DNAzyme-assisted click reaction. Initially, S. aureus was enriched by the 4-mercaptophenylboronic acid-functionalized stir bar. The yolk antibody (immunoglobulin Y) and copper-labeled polydopamine nanoparticles were then specifically conjugated with the captured target. The Cu(II) was released under acidic conditions and effectively catalyzed the copper-catalyzed azide-alkyne cycloaddition (CuAAC) between the alkyne group-labeled DNAzyme and the streptavidin-biotin-azido with the assistance of DNAzyme. Finally, the DNAzyme-streptavidin complexes were detected by microfluidic chips to quantify S. aureus. Under optimum conditions, this immunosensor showed good detection performances toward S. aureus within 10 to 2.5 × 104 CFU/mL with a limit of detection of 3 CFU/mL. Moreover, the satisfying detection results of real samples of animal origin also implied that this immunosensor owned great potential in practical applications.

Optimized design for illusion device by genetic algorithm.

The illusion device developed from the scattering cancellation employs very simple homogeneous and isotropic materials, but this device is only valid for electrically small objects. In this paper, we prove that the illusion device optimized by genetic algorithm can be applied to large-scale occasions. For an electrically small target, an optimized core-shell illusion device can achieve better illusion effect than the analytical design based on the scattering cancellation. With the increase of the device size, the ability of the single-layered shell to manipulate the scattering is very limited. For a moderate-size target, two optimized multi-layered examples are presented: one is to make a dielectric cylinder appear as another dielectric target, and the other is to make a conducting cylinder behave like a double-negative-material target. The full-wave simulations are carried out to visualize the similar field distributions of the target and the optimized multi-layered design. This optimized design greatly widens the size application range of the illusion device and can also improve the illusion performance with simple material parameters.

Genome-wide analyses of light-regulated genes in Aspergillus nidulans reveal a complex interplay between different photoreceptors and novel photoreceptor functions.

Fungi sense light of different wavelengths using blue-, green-, and red-light photoreceptors. Blue light sensing requires the "white-collar" proteins with flavin as chromophore, and red light is sensed through phytochrome. Here we analyzed genome-wide gene expression changes caused by short-term, low-light intensity illumination with blue-, red- or far-red light in Aspergillus nidulans and found that more than 1100 genes were differentially regulated. The largest number of up- and downregulated genes depended on the phytochrome FphA and the attached HOG pathway. FphA and the white-collar orthologue LreA fulfill activating but also repressing functions under all light conditions and both appear to have roles in the dark. Additionally, we found about 100 genes, which are red-light induced in the absence of phytochrome, suggesting alternative red-light sensing systems. We also found blue-light induced genes in the absence of the blue-light receptor LreA. We present evidence that cryptochrome may be part of this regulatory cue, but that phytochrome is essential for the response. In addition to in vivo data showing that FphA is involved in blue-light sensing, we performed spectroscopy of purified phytochrome and show that it responds indeed to blue light.

Comprehensive analysis of the regulatory network of blue-light-regulated conidiation and hydrophobin production in Trichoderma guizhouense.

Conidia of Trichoderma guizhouense (Hypocreales, Ascomycota) are frequently applied to the production of biofertilizers and biocontrol agents. Conidiation of some Trichoderma species depends on blue light and the action of different blue light receptors. However, the interplay between different blue-light receptors in light signalling remained elusive. Here, we studied the functions of the blue light receptors BLR1 and ENV1, and the MAP kinase HOG1 in blue light signalling in T. guizhouense. We found that the BLR1 dominates light responses and ENV1 is responsible for photoadaptation. Genome-wide gene expression analyses revealed that 1615 genes, accounting for ~13.4% of the genes annotated in the genome, are blue-light regulated in T. guizhouense, and remarkably, these differentially expressed genes (DEGs) including 61 transcription factors. BLR1 and HOG1 are the core components of the light signalling network, which control 79.9% and 73.9% of the DEGs respectively. In addition, the strict regulation of hydrophobin production by the blue light signalling network is impressive. Our study unravels the regulatory network based on the blue light receptors and the MAPK HOG pathway for conidiation, hydrophobin production and other processes in T. guizhouense.

Fungal phytochrome chromophore biosynthesis at mitochondria.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

A new atypical short-chain dehydrogenase is required for interfungal combat and conidiation in Trichoderma guizhouense.

Hypocrealean Trichoderma are the most extensively studied facultative mycoparasites against phytopathogenic fungi. Aerial hyphae of Trichoderma guizhouense can rapidly proliferate over Fusarium oxysporum hyphae, cause sporadic cell death and arrest the growth of the host. The results of the present study demonstrated that a unique short-chain dehydrogenase/reductase (SDR), designated as TgSDR1, was expressed at a high level in T. guizhouense challenged by the hosts. Similar to other SDRs family members, the TgSDR1 protein contains a cofactor-binding motif and a catalytic site. The subcellular localization assay revealed that the TgSDR1::GFP fusion protein translocated to lipid droplets in mycelia and conidia. The data obtained using reverse genetic approach indicated that TgSDR1 is associated with antifungal ability, plays an important role in providing reducing equivalents in the form of NADPH and regulates the amino sugar and nucleotide sugar metabolism in T. guizhouense upon encountering a host. Moreover, the TgSDR1 deletion mutant was defective in conidiation. Thus, TgSDR1 functions as a key metabolic enzyme in T. guizhouense to regulate mycotrophic interactions, defence against other fungi, such as F. oxysporum, and conidiation.

Pressure-Based Biosensor Integrated with a Flexible Pressure Sensor and an Electrochromic Device for Visual Detection.

This work demonstrated a pressure-based biosensor integrated with a flexible pressure sensor and an electrochromic device for visual detection. Initially, a sandwich-type immunoreaction for target carcinoembryonic antigen (CEA, as a model analyte) was carried out using the capture antibody (cAb) and platinum nanoparticles-labeled detection antibody (PtNPs-dAb) in a reaction cell. The added hydrogen peroxide (H2O2) could be catalyzed by the PtNPs to generate oxygen (O2). In a sealed chamber, the pressure increased with the overflowing O2. Meanwhile, a skin-inspired flexible pressure sensor with excellent sensing performance was fabricated to monitor the pressure change in real time. Thus, the electrical signal of the pressure sensor could reveal the target concentration. Moreover, a voltage-regulated electrochromic device based on polyaniline (PANI) and tungsten oxide (WO3) was integrated into the platform to provide a visualized readout. According to the electrical signal of the pressure sensor, the electrochromic device would change its color from green to blue, which also revealed the target concentration and could be observed by the naked eye. Under optimal conditions, the biosensor presented a high sensitivity for CEA in a detectable range of 0.2-50 ng/mL. The limit of detection (LOD) was 94 pg/mL. The selectivity, reproducibility, and accuracy were also satisfying. Furthermore, this immunoassay gives a path for developing visualized biosensors in point-of-care settings.