One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples.

Salmonella enterica is a common foodborne pathogen that can cause food poisoning in humans. The organism also infects and causes disease in animals. Rapid and sensitive detection of S. enterica is essential to prevent the spread of this pathogen. Traditional technologies for the extraction and detection of this pathogen from complex food matrices are cumbersome and time-consuming. In this study, we introduced a novel strategy of biphasic assay integrated with an accelerated strand exchange amplification (ASEA) method for efficient detection of S. enterica without culture or other extraction procedures. Food samples are rapidly dried, resulting in a physical fluidic network inside the dried food matrix, which allows polymerases and primers to access the target DNA and initiate ASEA. The dried food matrix is defined as the solid phase, while amplification products are enriched in the supernatant (liquid phase) and generate fluorescence signals. The analytical performances demonstrated that this strategy was able to specifically identify S. enterica and did not show any cross-reaction with other common foodborne pathogens. For artificially spiked food samples, the strategy can detect 5.0 × 101 CFU mL-1S. enterica in milk, 1.0 × 102 CFU g-1 in duck, scallop or lettuce, and 1.0 × 103 CFU g-1 in either oyster or cucumber samples without pre-enrichment of the target pathogen. We further validated the strategy using 82 real food samples, and this strategy showed 92% sensitivity. The entire detection process can be finished, sample-to-answer, within 50 min, dramatically decreasing the detection time. Therefore, we believe that the proposed method enables rapid and sensitive detection of S. enterica and holds great promise for the food safety industry.

Boron nitride nanoplate-based strand exchange amplification with enhanced sensitivity and rapidity for quantitative detection of Staphylococcus aureus in food samples.

Staphylococcus aureus is one of the most common foodborne pathogens that can cause serious food poisoning and infectious diseases in humans. Standard identification approaches include nucleic acid amplification, but current amplification tools suffer from low amplification efficiency, resulting in the risk of low sensitivity and long detection time. Herein, boron nitride nanoplates (BNNPs) were chosen as an additive for enhancing the sensitivity and rapidity of strand exchange amplification (SEA), thereby successfully expanding the application of nucleic acid detection for detecting Staphylococcus aureus in food samples. As a result, SEA based on boron nitride nanoplates (BNNP-SEA) was employed for sensitive and rapid detection of foodborne pathogen Staphylococcus aureus. Compared with classical SEA, the BNNP-based SEA assay was more than 10-fold sensitive, and the detection time was reduced by 15 minutes. The optimized BNNP-based SEA shows a wide linear range from 40 pg to 50 ng in a diluted solution of the target DNA with a low detection limit of 40 pg. Moreover, the BNNP-based SEA achieves the quantitative detection of Staphylococcus aureus in different food samples (pork, beef, mutton, duck, milk and shrimp). In contrast to the classical SEA, the BNNP-based SEA method enabled sensitive and rapid detection of Staphylococcus aureus in the above food samples at concentrations as low as 5 × 103 CFU mL-1. The BNNP-based SEA assay is specific, sensitive and reliable, offering a valuable diagnostic technology for routine analysis in food safety research.

Electrical potential-assisted DNA-RNA hybridization for rapid microRNA extraction.

Analysis of microRNAs (miRNAs) is important in cancer diagnostics and therapy. Conventional methods used to extract miRNA for analysis are generally time-consuming. A novel approach for rapid and sensitive extraction of miRNAs is urgently need for clinical applications. Herein, a novel strategy based on electrical potential-assisted DNA-RNA hybridization was designed for miRNA extraction. The entire extraction process was accomplished in approximately 3 min, which is much shorter than the commercial adsorption column method, at more than 60 min, or the TRIzol method, at more than 90 min. Additionally, the method offered the advantages of simplicity and specificity during the extraction process by electrical potential-assisted hybridization of single-stranded DNA and RNA. Taking let-7a as an example, satisfactory results were achieved for miRNA extraction in serum, demonstrating the applicability in miRNA nucleic acid amplification.

Recent advances and perspectives of enzyme-based optical biosensing for organophosphorus pesticides detection.

The overuse or abuse of organophosphorus pesticides (OPs) can bring about severe contamination problems in foodstuff and the environment, which will seriously threaten human health and the ecosystem's cycle. Hence, it is in high demand to establish sensitive, portable, specific, and cost-effective methods for monitoring OPs to control food safety, protect the ecosystem, and prevent disease. The optical biosensor with enzyme as bio-recognition elements has been an effective alternative for OPs detection. Herein, we firstly introduce various enzymes, sensing mechanisms, advantages and disadvantages used as bio-recognition elements in optical sensing for OPs detection. Then, we review various optical biosensing strategies based on enzymes as recognition elements that were ingeniously designed and successfully utilized for OPs detection, with a particular emphasis on photoluminescence (PL), chemiluminescence (CL), electrochemiluminescence (ECL), and colorimetric (CM) biosensing strategies. We not only highlight the state-of-art developments and the construction strategies of the enzyme-based optical biosensing method but also summarize the existing deficiencies, current challenges, and the future perspectives of OPs detection.

Catalytic hairpin assembly indirectly covalent on Fe3O4@C nanoparticles with signal amplification for intracellular detection of miRNA.

Fluorescence resonance energy transfer, a promising method for in situ imaging of miRNA in living cells, has intrinsic limitation on sensitivity and selectivity. Herein, a fluorescent amplification strategy based on catalyzed hairpin assembly indirectly covalent on Fe3O4@C nanoparticles via short single-stranded DNA was investigated for cellular miRNA detection in living cells, integrating non-enzyme target-active releasing for amplifying the signal output, highly quenching efficiency of Fe3O4@C nanoparticles with low background, ssDNA assisted fluorescent group-fueled chain releasing from Fe3O4@C nanoparticles with enhanced fluorescence response. The designed platform exhibits highly sensitive in a wide linear concentration range of 0.450 pM-190 pM and is highly specific for miRNA-20a detection with the ability of discriminating one mistake base. Additionally, the CHA-Fe3O4@C was successfully applied in imaging visualization of miRNA-20a in the living cell. The strategy provides a promising bioassay approach for clinical research.

Environmental and intercellular Pb2+ ions determination based on encapsulated DNAzyme in nanoscale metal-organic frameworks.

With the merits of low cost, simple synthesis procedure, and high affinity for metal ions, deoxyribozyme (DNAzyme) have played important roles in metal ions detection. However, the intracellular applications of DNAzyme are limited because of enzymatic degradation and inefficient cellular uptake. To address these problems, GR-5 as model DNAzyme was encapsulated into zeolitic imidazolate frameworks-8 (ZIF-8) nanoparticles by biomimetic mineralization. The positively charged ZIF-8 with high DNAzyme loading capacity retained their ability to enter cells. Compared with free DNAzyme, the biomimetic mineralization synthesis method has greatly improved the stability of pristine DNAzyme. The as-synthesized DNAzyme@ZIF-8 composite exhibited good stability resisting DNase I, and was used as a sensitive fluorescent nanoprobe for Pb2+ determination and successfully achieved selective and sensitive determination for Pb2+ at λex/λem = 494/522 nm in real samples. The linear range for the determination of Pb2+ is 50 to 500 nM. Moreover, the highly active DNAzyme delivered by ZIF-8 allows noninvasive imaging of Pb2+ measurement in living cells. This strategy will extend the suitability of functional nucleic acids for in vitro and in vivo bioanalysis and bioimaging. Graphical abstract.

Energy-transfer-mediated oxygen activation in carbonyl functionalized carbon nitride nanosheets for high-efficient photocatalytic water disinfection and organic pollutants degradation.

Polymeric photocatalysts are promising candidates for water purification, however their catalytic performance are still unsatisfactory due to the fast charge recombination that leads to low reactive oxygen radicals production. In this study, a conceptual energy-transfer-mediated photocatalytic oxygen activation system over polymeric carbon nitride without the need of electron-hole separation is proposed, exhibiting remarkable singlet oxygen triggered bacteria inactivation performance as well as organic pollutants degradation. By structure and excitonic effect modulation, the oxygen activation process changes from the traditional electron-transfer mechanism to the final energy-transfer pathway, leading to the selective generation of singlet oxygen with high efficiency. The generated singlet oxygen is found to fervently attack the bacteria membrane, creating irreparable pores or holes on the cell membrane for cytoplasmic contents leaking out to accelerate bacteria destruction. The work demonstrated here offers a new photocatalytic oxygen activation pathway for achieving high-efficient reactive oxygen species generation performance without the need of charge separation.

Selection and characterization of DNA aptamers for constructing colorimetric biosensor for detection of PBP2a.

Rapid and accurate diagnosis of methicillin-resistant staphylococcus aureus (MRSA) is vital for patient treatment, control of infection and monitoring epidemiology. Penicillin binding proteins (PBP2a), as an important marker protein of MRSA, has been proposed as the screening test target for tolerant bacteria of MRSA. However, current technologies based on PBP2a activity or PBP2a immunoassays were suboptimal specificity and sensitivity. In this report, the selection and characterization of DNA aptamers that binds to PBP2a was described. The DNA aptamer is with high affinity and selectivity to binding with PBP2a. Furthermore, utilizing the switched mimicking peroxidase for gold nanoparticles loaded graphene oxide (GO/Au) nanomaterials based on the effect between GO/Au and DNA, a powerful strategy was set out for designing aptamer-based colorimetric biosensor for detection of PBP2a. In this strategy, the employment of biosensor based on GO/Au and PBP2a aptamer greatly improved the detection sensitivity and selectivity with limit of detection as low as 20 nM. Accordingly, the reversible nanozyme inhibition/activation approach may be universally applicable for the biomedical diagnosis.

Enhanced Perfluorooctanoic Acid Degradation by Electrochemical Activation of Sulfate Solution on B/N Codoped Diamond.

Electrochemical oxidation based on SO4•- and •OH generated from sulfate electrolyte is a cost-effective method for degradation of persistent organic pollutants (POPs). However, sulfate activation remains a great challenge due to lack of active and robust electrodes. Herein, a B/N codoped diamond (BND) electrode is designed for electrochemical degradation of POPs via sulfate activation. It is efficient and stable for perfluorooctanoic acid (PFOA) oxidation with first-order kinetic constants of 2.4 h-1 and total organic carbon removal efficiency of 77.4% (3 h) at relatively low current density of 4 mA cm-2. The good activity of BND mainly originates from a B and N codoping effect. The PFOA oxidation rate at sulfate electrolyte is significantly enhanced (2.3-3.4 times) compared with those at nitrate and perchlorate electrolytes. At sulfate, PFOA oxidation rate decreases slightly in the presence of •OH quencher while it declines significantly with SO4•- and •OH quenchers, indicate both SO4•- and â€¢OH contribute to PFOA oxidation but SO4•- contribution is more significant. On the basis of intermediates analysis, a proposed mechanism for PFOA degradation is that PFOA is oxidized to shorter chain perfluorocarboxylic acids gradually by SO4•- and •OH until it is mineralized.