Electrochemiluminescence Biosensor Based on a Duplex-Specific Nuclease and Dual-Output Toehold-Mediated Strand Displacement Cascade Amplification Strategy for Sensitive Detection of MicroRNA-499.

The timely and accurate diagnosis of acute myocardial infarction (AMI) is of great significance to reduce mortality and morbidity associated with the condition. Herein, we developed an electrochemiluminescence (ECL) biosensor for the detection of the potential AMI biomarker microRNA-499 (miRNA-499), which was based on duplex-specific nuclease-assisted target recycling and dual-output toehold-mediated strand displacement (TMSD). First, miRNA-499 was converted into a large amount of single-stranded DNA through the DSN-assisted target recycling, which was further incubated with the DNA triple-stranded complex (S) to implement TMSD cycles. Thus, the Ru(bpy)32+-labeled signal strands were released and captured by the capture probe on the electrode surface, resulting in an intense ECL signal. Owing to the prominent cascade signal amplification, the constructed biosensor exhibited a good linear response to miRNA-499 within the range of 100 aM-100 pM with a detection limit of 69.99 aM. Furthermore, it demonstrated superior selectivity, stability, and reproducibility. In addition, the biosensor was successfully applied to detect miRNA-499 in real human serum samples, demonstrating its potential for nucleic acid detection in the early diagnosis of diseases.

Room-Temperature Synthesized Iron/Cobalt Metal-Organic Framework Nanosheets with Highly Efficient Catalytic Activity toward Luminol Chemiluminescence Reaction.

Two-dimensional (2D) iron/cobalt metal-organic framework nanosheets (Fe/Co-MOF NSs) were synthesized via the cooperative self-assembly reaction of Fe3+/Co2+ and terephthalic acid at room temperature. The as-prepared 2D Fe/Co-MOF NSs display superior performance in catalysis of the chemiluminescence (CL) reaction between luminol and H2O2. The CL spectrum, UV-vis absorption spectroscopy, radical scavenger experiments, and electron spin resonance (ESR) spectroscopy are utilized to research the possible CL mechanism of the luminol-H2O2-Fe/Co-MOF NSs system. All results indicate that Fe/Co-MOF NSs present outstanding peroxidase-like activity and could catalyze H2O2 to produce 1O2, O2·-, and ·OH, which could react rapidly with the luminol anion radical and result in strong CL. With the highly efficient CL of the luminol-H2O2-Fe/Co-MOF NSs system, a sensitive sensor for the detection of dopamine (DA) is developed based on the inhibitory effect of DA on the CL intensity. Good linearity over the range of 50-800 nM is achieved with a limit of detection of 20.88 nM (S/N = 3). This research demonstrates that 2D Fe/Co-MOF NSs is a highly effective catalyst for luminol CL reaction and has great application potential in the CL field.

A Self-Driven Microfluidic Chip for Ricin and Abrin Detection.

Ricin and abrin are phytotoxins that can be easily used as biowarfare and bioterrorism agents. Therefore, developing a rapid detection method for both toxins is of great significance in the field of biosecurity. In this study, a novel nanoforest silicon microstructure was prepared by the micro-electro-mechanical systems (MEMS) technique; particularly, a novel microfluidic sensor chip with a capillary self-driven function and large surface area was designed. Through binding with the double antibodies sandwich immunoassay, the proposed sensor chip is confirmed to be a candidate for sensing the aforementioned toxins. Compared with conventional immunochromatographic test strips, the proposed sensor demonstrates significantly enhanced sensitivity (≤10 pg/mL for both toxins) and high specificity against the interference derived from juice or milk, while maintaining good linearity in the range of 10-6250 pg/mL. Owing to the silicon nanoforest microstructure and improved homogeneity of the color signal, short detection time (within 15 min) is evidenced for the sensor chip, which would be helpful for the rapid tracking of ricin and abrin for the field of biosecurity.

Auxiliary effect of CO2 on pyrolysis of oily sludge.

The efficient recovery and conversion of energy in oily sludge has great prospects. In this article, the main objective is to investigate the impact of the addition of CO2 during the pyrolysis of oily sludge on energy recovery and conversion by thermogravimetric analysis (TGA) and compare the effect with the traditional pyrolysis effect from the perspective of thermal conversion behavior, products composition, and kinetics analysis. The results of the experiment showed that in the CO2 atmosphere, the main weight loss temperature of oily sludge was mainly concentrated in the range of 300-500 °C, which is lower than the reaction temperature range of traditional pyrolysis. The yields of CO and H2 in the products have been greatly improved, and the highest proportion in the gas products can reach 19.29% and 22.38%, respectively. The Ea (activation energy) values of oily sludge were determined to be in the range of 40-120 kJ·mol-1 with the conversion between 0.2 and 0.8 via DAEM, KAS, Starink, and FWO methods, respectively, in which the FWO method has shown the strongest adaptability. The results of this study provide reference values for practical engineering applications.

Electrochemical Biosensing Platform Based on Toehold-Mediated Strand Displacement Reaction and DSN Enzyme-Assisted Amplification for Two-Target Detection.

Simultaneous detection of multiple targets is of great significance for accurate disease diagnosis. Herein, based on duplex-specific nuclease (DSN) assisted signal amplification and the toehold-mediated strand displacement reaction (TSDR), we constructed an electrochemical biosensor with high sensitivity and high specificity for dual-target detection. MiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc) and methylene blue (MB) with significant peak potential differentiation were used as the electrochemical signal probes. The elaborately designed hairpin probe H1, which was fixed on the electrode surface, could be hybridized with the target miRNA-141 to perform signal amplification by the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be detected by Fc signal changes at 0.41 V. The hairpin H1 can also combine with the MB-labeled signal probe (SP) output from miRNA-133a-induced TSDR, and the detection of miRNA-133a can be realized according to the response signal generated by MB at -0.26 V. The two sensing lines are independent of each other, and there is no mutual interference in the detection process. Therefore, two independent detection lines could be connected in series, and the simultaneous detection of two targets can be achieved on a single electrode. This novel detection strategy provides a new way to simultaneously detect different biomarkers.

Penicillium-Infected Apples Benefit Larval Development of Conogethes punctiferalis via Alterations of Their Gut Bacteria Community and Gene Expression.

Pathogenic microorganisms can impact the behavior and physiology of herbivores by direct or indirect means. This study demonstrated that yellow peach moth Conogethes punctiferalis larvae feeding on Penicillium-infected apples exhibited significantly longer body length and weight parameters compared to the control group. The sequencing of gut 16S rRNA showed a significant increase in the diversity and abundance of bacteria in the larvae feeding on Penicillium-infected apples. Additionally, transcriptomic sequencing of the larval gut indicated significant upregulation of genes related to digestion and cuticle formation after consuming Penicillium-infected apples. Furthermore, enzyme activity assays revealed notable changes in the trypsin and lipase activity. Consequently, these alterations in gut microbiota structure, diversity, and gene expression levels may underlie the observed growth and developmental variations in C. punctiferalis larvae mediated by pathogenic microorganisms. This study holds theoretical significance for a deeper understanding of the tripartite interaction among microorganisms, insects, and plants as well as for the development of novel pest control measures based on gut microbiota.

PLGA Nanoparticle-Based Dissolving Microneedle Vaccine of Clostridium perfringens ε Toxin.

Epsilon toxin (ETX) is an exotoxin produced by type B and D Clostridium perfringens that causes enterotoxemia or necrotic enteritis in animals such as goats, sheep, and cattle. Vaccination is a key method in preventing such diseases. In this study, we developed a new type of dissolving microneedle patch (dMN) with a nanoparticle adjuvant for enhanced immune response to deliver the rETXY196E-C protein vaccine. We chose FDA-approved poly(lactic-co-glycolic acid) (PLGA) to prepare nanospheres as the vaccine adjuvant and introduced dimethyldioctadecylammonium bromide (DDAB) to make the surface of PLGA nanoparticles (PLGA NPs) positively charged for antigen adsorption. PLGA NPs with a diameter of 100~200 nm, a surface ZETA potential of approximately +40 mV, and good safety were successfully prepared and could effectively adsorb rETXY196E-C protein. Using non-toxic and antibacterial fish gelatin as the microneedle (MN) matrix, we prepared a PLGA-DDAB dMN vaccine with good mechanical properties that successfully penetrated the skin. After immunization of subcutaneous (SC) and dMN, antibody titers of the PLGA and Al adjuvant groups were similar in both two immune ways. However, in vivo neutralization experiments showed that the dMN vaccines had a better protective effect. When challenged with 100 × LD50 GST-ETX, the survival rate of the MN group was 100%, while that of the SC Al group was 80%. However, a 100% protective effect was achieved in both immunization methods using PLGA NPs. In vitro neutralization experiments showed that the serum antibodies from the dMN and SC PLGA NPs groups both protect naive mice from 10 × LD50 GST-ETX attack after being diluted 20 times and could also protect MDCK cells from 20 × CT50 GST-ETX attack. In conclusion, the PLGA-DDAB dMN vaccine we prepared has good mechanical properties, immunogenicity, and protection, and can effectively prevent ETX poisoning. This provides a better way of delivering protein vaccines.

A lanthanide-based high-sensitivity fluorescence method for the on-site rapid detection of thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50-5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person.

A Thermostable Dissolving Microneedle Vaccine with Recombinant Protein of Botulinum Neurotoxin Serotype A.

BACKGROUND: As a Class A bioterrorism agent, botulinum neurotoxin serotype A (BoNT/A) carries the risk of being used by terrorists to cause mass poisoning. The microneedle (MN) patch has a great potential for application as a novel vaccine delivery method. The aim of this study is to develop a thermally stable, dissolving microneedle patch for the delivery of a recombinant protein vaccine using a recombinant C-terminal heavy chain of BoNT/A (Hc of BoNT/A, AHc) to prevent botulism. METHODS: Fish gelatin, a natural non-toxic and bacteriostatic material, was selected as the microneedle matrix for the preparation of the dissolving microneedle vaccine. Subsequently, the mechanical performance, bacteriostatic properties, vaccination effect, and stability of the microneedle patches were evaluated using instruments such as the displacement-force test station and optical coherence tomography (OCT) scanner. RESULTS: Fish gelatin matrix at high concentrations has good bacteriostatic properties, and excellent mechanical performance and vaccination effect, meeting the necessities of a vaccine. In both in vivo and in vitro neutralization experiments, MN vaccines containing different antigen doses achieved the same protective efficacy as subcutaneous vaccinations, protecting mice against 106 LD50 of BoNT/A injected intraperitoneally. Thermal stability analysis of the MN vaccines revealed that the fish gelatin matrix protected the AHc vaccine from protein denaturation even after 7 days of storage at 37 °C and enabled the vaccine patches to maintain good immunogenicity and protective efficacy even after 6 months of storage at room temperature. CONCLUSION: In this study, we successfully prepared a bacteriostatic MN patch using a fish gelatin matrix that not only has a good vaccination effect, but also obviates the need for a cold chain for the AHc vaccine, providing the possibility of rapid, painless, and large-scale vaccination.

[Microneedle-based percutaneous immunity: a review].

Microneedle percutaneous immunization is achieved by puncturing the stratum corneum of the skin with microneedles so that the vaccine is efficiently recognized by antigen-presenting cells to induce a specific immune response. Due to the advantages of efficient induction of immune response, low pain and easy storage, transdermal immunization by microneedles has been widely used for immunization of various vaccines in recent years. This review summarizes the materials of microneedles, application for transcutaneous immunization, as well as the challenges that need to be addressed.