Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn2+. NH2-modified S. aureus-specific aptamer was loaded on the surface of Fe3O4 MNPs (apt-Fe3O4 MNPs) and MnO2 NPs (apt-MnO2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe3O4 MNPs/S. aureus/apt-MnO2 NPs complex and then MnO2 NPs were etched to release Mn2+ to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn2+ through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy.

Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5\' end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/μL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases.

Lead (Pb) is a heavy metal known for its adverse effects on both human health and the environment. In recent years, the industrial utilization of Pb2+ has surged, underscoring the imperative need for efficient measurement methods. In this study, a rapid and simple photochemical method was used to synthesize thioglycolic acid (TGA)-stabilized CdTe/ZnSe core-shell quantum dots (QDs). These CdTe/ZnSe QDs emit vibrant green fluorescence and exhibit remarkable quenching in the presence of Pb2+ ions. This property enables the development of an on-site on/off sensor without the necessity of additional modifications. The proposed sensor possesses an outstanding sensitivity to Pb2+, with a detection limit and linear range of 31.8 nM and 50 nM-10 µM, respectively. Importantly, the selectivity of this fluorescence-based sensor was validated by analyzing various positively and negatively charged ions. Furthermore, the developed sensor showed reliable performance against real river, agricultural, and tap water, as confirmed by Inductively Coupled Plasma (ICP) analysis. Additionally, CdTe/ZnSe QDs immobilized on glass slides were successfully employed for on-site water sample analysis, providing a versatile solution for environmental monitoring.

Buprofezin (BUP) is an insect growth regulator widely used in agriculture to control hemipteran pests, particularly the melon aphid, Aphis gossypii, due to its efficiency and low toxicity. Although approved by the Chinese government, its maximum residue limit (MRL) in food is strictly regulated, and conventional techniques for detecting BUP have several limitations. Our study reports successful BUP detection using a supramolecular fluorescent probe DP@ALB, constructed with chalcone-based fluorescent dye DP and albumin as the host. The probe offers advantages such as low cost, visual signal output with high fluorescence color variation, rapid response, and high sensitivity. Additionally, portable test strips enable convenient on-site BUP detection and simplifying field monitoring of spiked real samples. The study achieves precise qualitative and quantitative BUP analysis in grape fruit, groundwater, and soil with satisfactory recoveries. Further, the biological applicability of sensor for the in vitro detection of BUP in L929 living cells was demonstrated. This research breakthrough overcomes the limitations of traditional analytical methods, offering an efficient and reliable approach for food and environmental monitoring and pesticide residue detection.

Reliable and sensitive virus detection is essential to prevent airborne virus transmission. The polymerase chain reaction (PCR) is one of the most compelling and effective diagnostic techniques for detecting airborne pathogens. However, most PCR diagnostics rely on thermocycling, which involves a time-consuming Peltier block heating methodology. Plasmonic PCR is based on light-driven photothermal heating of plasmonic nanostructures to address the key drawbacks of traditional PCR. This study introduces a methodology for plasmonic PCR detection of air-sampled influenza virus (H1N1). An electrostatic air sampler was used to collect the aerosolized virus in a carrier liquid for 10 min. Simultaneously, the viruses collected in the liquid were transferred to a tube containing gold (Au) nanorods (aspect ratio = 3.6). H1N1 viruses were detected in 12 min, which is the total time required for reverse transcription, fast thermocycling via plasmonic heating through gold nanorods, and in situ fluorescence detection. This methodology showed a limit of detection of three RNA copies/μL liquid for H1N1 influenza virus, which is comparable to that of commercially available PCR devices. This methodology can be used for the rapid and precise identification of pathogens on-site, while significantly reducing the time required for monitoring airborne viruses.

As an important ROS species participating in various physiological and pathological processes, high level of hypochlorite (ClO-) poses significant health and safety concerns, necessitating efficient detection methods. Herein, this study introduces a water-soluble fluorescent nanoprobe Nano-SJD, effectively detect ClO- in both food samples and living cells. The small molecular probe SJD with N, N-dimethylthiocarbamyl (DMTC) as recognition moiety was constructed based on a naphthalene derivative. To further improve the water solubility, SJD was assembled with an amphiphilic copolymer (mPEG-DSPE) to prepare a water soluble fluorescent nanoprobe Nano-SJD. Fortunately, the nanoprobe preserves the excellent properties of small molecules and performs very well optical response to ClO- in aqueous solution, possessing the advantages including ultra-rapid response (within 1 s), minimal interference, low detection limits (0.39 μM) and good pH stability. What\'s more important, we have also developed smartphone-compatible test paper strips for convenient on-site detection of ClO- in real-water samples. Additionally, the robust fluorescent imaging behavior of Nano-SJD for visualization of ClO- in living cells highlights its broad potential in biosystem applicability.

Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.

Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method\'s simplicity, sensitivity, and specificity. This efficacy confirms the platform\'s suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.

Simple and rapid molecular detection technologies for authenticating animal species are urgently needed for food safety and authenticity. This study established a new direct-fast quantitative polymerase chain reaction (qPCR) detection technology for beef to achieve rapid and on-site nucleic acid detection in food. This technology can complete nucleic acid extraction in 4 min using a new type of food nucleic acid-releasing agent, followed by direct amplification of the DNA sample by fast qPCR in 25 min. The results indicated that direct-fast qPCR can specifically identify beef and can also identify 0.00001% of beef components in artificially simulated meat mixtures, with a detection precision variation coefficient of <4%. This method can be used to effectively identify beef in different food samples. As a simple, fast, and accurate molecular detection technology for beef, this method may provide a new tool for the on-site detection of beef components in food.

Organophosphorus compounds are widely distributed and highly toxic to the environment and living organisms. The current detection of organophosphorus compounds is based on a single-mode method, which makes it challenging to achieve good portability, accuracy, and sensitivity simultaneously. This study designed a multifunctional microfluidic chip to develop a dual-mode biosensor employing a DNA hydrogel as a carrier and aptamers as recognition probes for the colorimetric/electrochemical detection of malathion, an organophosphorus compound. The biosensor balanced portability and stability by combining a microfluidic chip and target-triggered DNA hydrogel-sensing technologies. Moreover, the biosensor based on target-triggered DNA hydrogel modified microfluidic developed in this study exhibited a dual-mode response to malathion, providing both colorimetric and electrochemical signals. The colorimetric mode enables rapid visualization and qualitative detection and, when combined with a smartphone, allows on-site quantitative analysis with a detection limit of 56 nM. The electrochemical mode offers a broad linear range (0.01-3000 μM) and high sensitivity (a limit of detection of 5 nM). The two modes could validate each other and improve the accuracy of detection. The colorimetric/electrochemical dual-mode biosensor based on target-triggered DNA hydrogel modified microfluidic chip offers a portable, simple, accurate, and sensitive strategy for detecting harmful environmental and food substances.