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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.

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In the rapidly evolving field of life sciences and biomedicine, detecting low-abundance biomolecules, and ultraweak biosignals presents significant challenges. This has spurred a rapid development of analytical techniques aiming for increased sensitivity and specificity. These advancements, including signal amplification strategies and the integration of biorecognition events, mark a transformative era in bioanalytical precision and accuracy. A prominent method among these innovations is immuno-rolling circle amplification (immuno-RCA) technology, which effectively combines immunoassays with signal amplification via RCA. This process starts when a targeted biomolecule, such as a protein or cell, binds to an immobilized antibody or probe on a substrate. The introduction of a circular DNA template triggers RCA, leading to exponential amplification and significantly enhanced signal intensity, thus the target molecule is detectable and quantifiable even at the single-molecule level. This review provides an overview of the biosensing strategy and extensive practical applications of immuno-RCA in detecting biomarkers. Furthermore, it scrutinizes the limitations inherent to these sensors and sets forth expectations for their future trajectory. This review serves as a valuable reference for advancing immuno-RCA in various domains, such as diagnostics, biomarker discovery, and molecular imaging.

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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.

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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.

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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.

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The on-site detection of mancozeb in food samples holds immense value for food safety. A red-fluorescent europium complex (Eu-PYDC-Phen) has been prepared and employed as a fluorescence probe for mancozeb detection. The optimized probe suspension exhibits excellent detection performances, including a wide linear range (0-0.24 mM), low detection limit (65 nM), rapid response (2 mins) and high selectivity. Moreover, a portable detection platform was carefully designed, integrating the Eu-PYDC-Phen-based fluorescent test strips with smartphone color recognition software. This innovative platform enables visual and on-site detection of mancozeb in tomato, apple, and lettuce, achieving satisfactory recovery rates (90.34 to 106.50%). Furthermore, the integration of machine learning techniques based on hierarchical clustering algorithm has the potential to further improve the prediction and decision-making efficiency in mancozeb detection. This work provides an economical, convenient, and reliable strategy for on-site detection of pesticide in agricultural products, thereby making a meaningful contribution to food safety.

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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.

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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.

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Controlling glucose (Glu) intake is a "required course" for diabetics, thus quickly and precisely measuring the amount of Glu in food is crucial. For this purpose, a novel smartphone-assisted portable swab for the dual-mode visual detection of Glu was constructed combined the selectivity of natural enzymes with the controllable catalytic activity of nanozymes. Glu was specifically decomposed by glucose oxidase (natural enzyme) to produce H2O2, which was catalyzed by carbon dots (FeMn/N-CDs, nanozyme) to accelerate the reaction of o-phenylenediamine (OPD, colorless) to produce 2,3-diaminophenazine (DAP, yellow). As a result, the absorbance at 450 nm gradually increased with the increasing concentration of Glu, leading to a color change in the system from colorless to yellow. Meanwhile, the fluorescence of FeMn/N-CDs gradually decreased at 450 nm, while the fluorescence of DAP gradually increased at 550 nm, allowing for both ratiometric fluorescence and colorimetric dual-mode detection. Furthermore, natural enzyme and nanozyme together with OPD were co-loaded on the swabs to achieve cascade catalysis of Glu. The assembled portable swabs have detection ranges of 1-600 µM (LOD = 0.37 µM) and 4-1200 µM (LOD = 1.19 µM) for the colorimetric and fluorometric detection, respectively. The field test results on real samples demonstrated that the portable swabs have great promise for use in efficiently and accurately guiding the dietary intake of diabetics.

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In this study, the goal was to develop a method for detecting and classifying organophosphorus pesticides (OPPs) in bodies of water. Sixty-five samples with different concentrations were prepared for each of the organophosphorus pesticides, namely chlorpyrifos, acephate, parathion-methyl, trichlorphon, dichlorvos, profenofos, malathion, dimethoate, fenthion, and phoxim, respectively. Firstly, the spectral data of all the samples was obtained using a UV-visible spectrometer. Secondly, five preprocessing methods, six manifold learning methods, and five machine learning algorithms were utilized to build detection models for identifying OPPs in water bodies. The findings indicate that the accuracy of machine learning models trained on data preprocessed using convolutional smoothing + first-order derivatives (SG + FD) outperforms that of models trained on data preprocessed using other methods. The backpropagation neural network (BPNN) model exhibited the highest accuracy rate at 99.95%, followed by the support vector machine (SVM) and convolutional neural network (CNN) models, both at 99.92%. The extreme learning machine (ELM) and K-nearest neighbors (KNN) models demonstrated accuracy rates of 99.84% and 99.81%, respectively. Following the application of a manifold learning algorithm to the full-wavelength data set for the purpose of dimensionality reduction, the data was then visualized in the first three dimensions. The results demonstrate that the t-distributed domain embedding (t-SNE) algorithm is superior, exhibiting dense clustering of similar clusters and clear classification of dissimilar ones. SG + FD-t-SNE-SVM ranks highest among the feature extraction models in terms of performance. The feature extraction dimension was set to 4, and the average classification accuracy was 99.98%, which slightly improved the prediction performance over the full-wavelength model. As shown in this study, the ultraviolet-visible (UV-visible) spectroscopy system combined with the t-SNE and SVM algorithms can effectively identify and classify OPPs in waterbodies.

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Aims: Clostridium perfringens is one of the major anaerobic pathogen causing food poisoning and animal enteritis. With the rise of antibiotic resistance and the restrictions of the use of antibiotic growth promoting agents (AGPs) in farming, Clostridium enteritis and food contamination have become more common. It is time-consuming and labor-intensive to confirm the detection by standard culture methods, and it is necessary to develop on-site rapid detection tools. In this study, a combination of recombinase polymerase amplification (RPA) and lateral flow biosensor (LFB) was used to visually detect C. perfringens in chicken meat and milk. Methods and results: Two sets of primers were designed for the plc gene of C. perfringens, and the amplification efficiency and specificity of the primers. Selection of primers produces an amplified fragment on which the probe is designed. The probe was combined with the lateral flow biosensor (LFB). The reaction time and temperature of RPA-LFB assay were optimized, and the sensitivity of the assay was assessed. Several common foodborne pathogens were selected to test the specificity of the established method. Chicken and milk samples were artificially inoculated with different concentrations (1 × 102 CFU/mL to 1 × 106 CFU/mL) of C. perfringens, and the detection efficiency of RPA-LFB method and PCR method was compared. RPA-LFB can be completed in 20 min and the results can be read visually by the LFB test strips. The RPA-LFB has acceptable specificity and the lowest detection limit of 100 pg./µL for nucleic acid samples. It was able to stably detect C. perfringens contamination in chicken and milk at the lowest concentration of 1 × 104 CFU/mL and 1 × 103 CFU/mL, respectively. Conclusion: In conclusion, RPA-LFB is specific and sensitive. It is a rapid, simple and easy-to-visualize method for the detection of C. perfringens in food and is suitable for use in field testing work.

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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.

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Vibrio parahaemolyticus (V. parahaemolyticus) is a major foodborne pathogen, which can cause serious foodborne illnesses like diarrhoea. Rapid on-site detection of foodborne pathogens is an ideal way to respond to foodborne illnesses. Herein, we provide an electrochemical sensor for rapid on-site detection. This sensor utilized a pH-sensitive metal-oxide material for the concurrent isothermal amplification and label-free detection of nucleic acids. Based on a pH-sensitive hydrated iridium oxide oxyhydroxide film (HIROF), the electrode transforms the hydrogen ion compound generated during nucleic acid amplification into potential, so as to achieve a real-time detection. The results can be transmitted to a smartphone via Bluetooth. Moreover, HIROF was applied in nucleic acid device detection, with a super-Nernst sensitivity of 77.6 mV/pH in the pH range of 6.0-8.5, and the sensitivity showed the best results so far. Detection of V. parahaemolyticus by this novel method showed a detection limit of 1.0 × 103 CFU/mL, while the time consumption was only 30 min, outperforming real-time fluorescence loop-mediated isothermal amplification (LAMP). Therefore, the characteristics of compact, portable, and fast make the sensor more widely used in on-site detection.

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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.

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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.

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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.

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BACKGROUND: Rapid on-site detection of infectious diseases is considerably essential for preventing and controlling major epidemics and maintaining social and public safety. However, the complexity of the natural environment in which infectious disease pathogens exist severely disrupts the performance of on-site detection, and rapid detection can become meaningless because of the cumbersome sample pretreatment process. RESULT: Herein, a new detection platform based on a carbon sphere@Fe3O4 micromotor (CS@Fe3O4) in combination with a graphene field-effect transistor (GFET) was designed and used for the on-site detection of SARS-CoV-2 coronavirus pathogens. The CS@Fe3O4 micromotor, surface-modified with anti-SARS-CoV-2 coronavirus antibody, could move at a velocity of 79.4 µm/s in a solution containing hydrogen peroxide (H2O2) and exhibited capture rates of 67.9% and 36.2% for the SARS-CoV-2 pathogen in phosphate buffered saline (PBS) and soil solutions, respectively. After magnetic field separation, the captured micromotor was used for GFET detection, with detection limits of 4.6 and 15.6 ag/mL in PBS and soil solutions, respectively. SIGNIFICANCE AND NOVELTY: This detection platform can be employed to avoid complex sample pretreatment procedures and achieve rapid on-site detection of SARS-CoV-2 coronavirus pathogens in complex environments. This study introduces a novel approach for the on-site detection of infectious diseases.

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Phytophthora sojae is a devastating plant pathogen that causes soybean Phytophthora root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of Phytophthora root rot (P. sojae). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 P. sojae from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. µL-1, while the RPA-Cas12a assay achieved a detection limit of 10 pg. µL-1. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of P. sojae on the infected field soybean samples. This assay provides a simple, efficient, rapid (<1 h), and visual detection platform for diagnosing Phytophthora root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of Phytophthora root rot in the field or customs fields.

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An organic-inorganic hybrid nanoprobe, namely LML-D-SBA@Eu3+-Gd3+, was constructed, with SBA-15 acting as the carrier material, and luminol and Eu3+ acting as fluorescence channels to achieve ratiometric signals that eliminate external interference (accurate detection). Gd3+ was used as a sensitizer to amplify the red emission of Eu3+ (ultrasensitive detection). In TCs detection, the luminol emission at 428 nm was quenched due to the photoinduced electron transfer mechanism, and the Eu3+ emission at 617 nm was sensitized due to the synergistic energy transfer from TCs and Gd3+ to Eu3+. The fluorescence intensity at 617 and 428 nm showed ratiometric changes as indicated by notable color changes from blue to red. The detection limits for TC and OTC were 0.21 and 0.08 ng/mL, respectively. To realize a facile, rapid, and cost-effective detection, we constructed a portable intelligent sensing platform based on smartphones, and it demonstrated great potential for on-site detection of TCs.

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A portable and integrated electrochemical detection system has been constructed for on-site and real-time detection of chemical oxygen demand (COD). The system mainly consists of four parts: (i) sensing electrode with a copper-cobalt bimetallic oxide (CuCoOx)-modified screen-printed electrode; (ii) an integrated electrochemical detector for the conversion, amplification, and transmission of weak signals; (iii) a smartphone installed with a self-developed Android application (APP) for issuing commands, receiving, and displaying detection results; and (iv) a 3D-printed microfluidic cell for the continuous input of water samples. Benefiting from the superior catalytic capability of CuCoOx, the developed system shows a high detection sensitivity with 0.335 µA/(mg/L) and a low detection limit of 5.957 mg/L for COD determination and possessing high anti-interference ability to chloride ions. Moreover, this system presents good consistency with the traditional dichromate method in COD detection of actual water samples. Due to the advantages of cost effectiveness, portability, and point-of-care testing, the system shows great potential for water quality monitoring, especially in resource-limited remote areas.