On-site Detection Research Articles

Integrated Sensing Platform Validated for the Efficient and On-Site Screening of Amine-Containing Illicit Drugs.

Efficient and reliable technologies for the on-site detection of illicit drugs are important in drug-facilitated crime investigations. However, the development of such technologies is challenging. Based on the synthetic optimization, introducing a boron ester functional group to the two furanic indicators endows the stimulus-responsive properties synergistically. The ring-opening reaction of the indicators in the presence of amine-containing illicit drugs generated well-known donor-acceptor Stenhouse adducts, accompanied by strong color changes. A small-size and lightweight laminated sensor was integrated based on the outstanding ratiometric variations of the two active furanic indicators. A prototype platform was fabricated equipped with a circuit control, a mini pump, and a signal processing system. A user-friendly detection and efficient screening of amine-containing illicit drugs, including phenethylamines, amphetamines, cathinones, and tryptamines in the liquid states were conducted. The ratiometric response of the sensor was linear in the concentration range of 2.1-10.6 μg·mL-1 for methamphetamine·HCl and methcathinone ·HCl. The detection limits for the two illicit drugs at the sublevel (ng·mL-1) were found to be 8.4 and 9.0 ng·mL-1, respectively. Double-blind field tests and different illicit drugs were evaluated with good screening capability. Successful trials showed the potential applications of the developed prototype platform for efficient and on-site analytical determination.

SERS-integrated centrifugal microfluidic platform for the detection and quantification of Chemical Warfare Agents in single-component solution and mixtures

Chemical Warfare Agents (CWAs) are toxic chemicals. Among CWAs, nerve agents are lethal compounds at low concentrations, and, thus, there is the need to detect and quantify the danger. The combination of Surface-Enhanced Raman Spectroscopy (SERS) with centrifugal microfluidic (CM) systems is a possibility for a safer handling and a fast analysis of nerve agents.In this work, the SERS signal from single-component solutions and mixtures of Tabun (GA) and VX were analyzed with Au-capped silicon nanopillar (NP) SERS substrates integrated on a CM platform and recorded with a custom-built Raman System (RS). Univariate and multivariate analysis methods were applied for the quantification. Both GA and VX were detected and quantified individually with Partial Least Squares regression (PLSR) models, with LoD and LoQ values of 7.39 ppm and 22.17 ppm respectively for GA, and 7.13 ppm and 21.39 ppm for VX. Detection and quantification of GA and VX in mixtures was achieved with Supported Vector Machine (SVM), obtaining R2 of the prediction 0.87 and 0.95, respectively.The results obtained show the potential of the SERS-integrated CM platform combined with machine learning analysis as a reliable method for CWAs on-site detection and quantification and for national security applications in real-case scenarios.

A pyrene-based hydrogen-bonded framework with excimer induced ratiometric emission for discriminative luminescence detection of metal ions

Discriminatory fluorescence detection of various metal ions is of significant importance in environmental and health-related applications. A luminescent hydrogen-bonded organic framework (HOF) material, PFC-1 was synthesized using 1,3,6,8-tetra(4-carboxylbenzene) pyrene (H4TBAPy) as the organic building block. The fluorescence behavior of PFC-1 is influenced by the concentration of the suspension, demonstrating different emissions characteristic of monomer and excimer fluorescence, which are highly sensitive to the surrounding environment. This allows for the potential differentiation and sensing of different target analytes. PFC-1 showed discriminatory fluorescence sensing performance towards metal ions such as Al3+, Sc3+, Cr3+, and Cu2+, with changes in fluorescence intensity, emission peak shifts, and intensity ratio changes between monomer and excimer emissions. Furthermore, a smartphone-based detection strategy was proposed, leveraging color recognition capabilities of smartphones for onsite and real-time sensing. The work demonstrate that PFC-1 is a promising material for the development of portable, cost-effective fluorescent sensors for onsite and real-time detection of metal ions. The integration of PFC-1 with smartphone technology paves the way for practical applications in environmental monitoring, industrial processes, and healthcare diagnostics.

Instrument-free detection of natamycin in beer and wastewater based on Eu-MOF ratiometric fluorescence response

Excessive intake of natamycin (NAT) is harmful to human health, so it is of great significance and necessity to effectively detect the content of NAT in food. In this study, we synthesized a dual-emission fluorescence probe, Eu@UiO-66(OH)2/(COOH)2, which exhibited emission peaks at 439nm and 614nm. This probe was obtained through the europium post-modification of UiO-66 type metal-organic frameworks (Eu-MOFs), which consisted of two types of ligands, 2, 5-dihydroxyterephthalic acid and pyromellitic acid. As expected, this probe Eu@UiO-66(OH)2/(COOH)2 can specifically respond to NAT with distinguishable ratiometric fluorescence changed from purple-red to blue with low limit of detection (0.15μM, approximately 0.10mg/kg), wide linear range (0-100μM), fast response (less than 1min), good selectivity and interference resistance. Based on the ratiometric fluorescence responsiveness, Eu@UiO-66(OH)2/(COOH)2 had been successfully used for the quantitative detection of NAT in wastewater, beer, yoghurt, and juice, demonstrating good recovery through smartphone-based colorimetric analysis. Thus, this work provides a ratiometric fluorescent probe for rapid, instrument-free and on-site detection of NAT in real samples such as beverage and environmental water.

Dual-mode colorimetric-fluorometric immunoassay for foodborne parasitic infection

Immunoassays are the primary method used for detection of foodborne parasitic infection. The nanomaterials used in these assays often present challenges such as complex preparation requirements and low stability during long-term storage and transport. Herein, based on cost-effective K3[Fe(CN)6]-mediated in-situ generated CuFe Prussian blue analogues (CuFe-PBA) and quinoxaline derivatives with high fluorescence, a dual-mode immunoassay was proposed for foodborne parasitic infection. This platform is relied on the restoration of K3[Fe(CN)6] by ascorbic acid (AA). In addition to this process, K4[Fe(CN)6]-triggered the generation of CuFe-PBA, and the enhanced fluorogenic reaction of dehydrogenated AA and o-phenylenediamine (OPD) occur sequentially. Without complex probes preparation, the assay enabled the rapid on-site detection of ALP and foodborne parasitic infection through a colorimetric mode accessible via smartphone and offered enhanced sensitivity through a fluorescent mode. The method was used for the detection of ALP, achieving detection limits of 0.38 U/L and 0.03 U/L for colorimetric and fluorescence modes, respectively. Subsequently, the dual-mode immunoassay was applied to Trichinella spiralis infection. The sensitivity and specificity of both the colorimetric and fluorescence modes were 100 % in 950 pig serum samples. This immunosensor provides a universal analysis tool for the accurate, cost-effective and highly sensitive detection of foodborne parasitic infection

A nanopore-based label-free CRISPR/Cas12a system for portable and ultrasensitive detection of zearalenone

BackgroundFood safety has become a serious global concern. Therefore, there is a need for effective detection technologies in this field. Currently, the development of effective on-site detection techniques is extremely important for food safety. However, the traditional on-site detection methods currently lack effective signal amplification. Herein, the aim of this study was to construct a nanopore-based label-free CRISPR/Cas12a system for the detection of Zearalenone (ZEN). The method is expected to be highly sensitive for portable detection of ZEN in food. ResultsThe proposed strategy was mainly involved three steps, including the displacement of the target DNA, the triggering of the cleavage of hairpin DNA probes (probes 1) by the trans-cleavage of CRISPR/Cas12a, and the generation of a measurable nanopore current signal. The probes 1 and DNA after the cleavage of probes 1 (probes 2) produce different characteristic nanopore signals as they pass through the nanopore. The established method achieved a low limit of detection (LOD) of 6.52 fM for ZEN and a wide liner range under optimized conditions. Furthermore, the practical applicability of this method was verified in real maize samples and showed good recoveries (90.68–101.98 %) and low relative standard deviations (RSD) (9.21–9.72 %). Therefore, this method is a promising option for rapid and ultrasensitive detection of ZEN. Significance and noveltyThe study presented a portable nanopore-based CRISPR/Cas12a signal amplification detection system for the detection of ZEN in food, which had a low LOD and the advantages of rapid, portability, and on-site detection potential. In conclusion, the method presented a promising prospect and universal platform for the detection of ZEN and other mycotoxins, offering a novel insight into on-site food safety detection.

Towards portable kits for on-site colorimetric detection of aqueous hydrazine using piperidine-thiophene-barbituric acid push-pull probe.

Owing to the carcinogenicity and environmental risks as well as the wide industrial use of hydrazine, we report herein a colorimetric probe for its ratiometric detection in pure water. The developed probe possesses push-pull architecture with 2-(piperidyn-1-yl)thiophene as the donor, N,N'-dibutylbarbituric as the acceptor, and butadiene as the spacer. In contrast to weak solvatochromic behavior in organic solvents, the probe showed distinct optical photophysical properties in water resulting from the formation of nanoscopic aggregates. The probe underwent pronounced spectral changes upon the addition of hydrazine including an 11.5-fold decrease in absorbance and ~2.4-fold fluorescence quenching. The mechanistic investigation revealed the rapid formation of hydrazone upon the interaction of the probe with hydrazine via retro-Knoevenagel reaction as confirmed experimentally and corroborated with DFT calculations. The induced colorimetric and fluorometric changes were utilized in hydrazine sensing with excellent selectivity over other biologically relevant analytes with a detection limit of 0.76 µM in aqueous media. The practical utility of the probe was assessed in real-life natural water samples, while we have also developed a cost-effective portable kit for the on-site hydrazine detection both in the solution and vapor phases.

MNAzyme-Assisted Nucleic Acid Lateral Flow Assay for Cost-Effective, On-Site Mercury Detection.

Mercury ions (Hg2+) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection method for Hg2+ using a multicomponent nucleic acid enzyme (MNAzyme)-assisted nucleic acid lateral flow assay (NALFA). The MNAzyme, which was engineered to contain thymine-thymine mismatches, is responsive only to the presence of Hg2+ and exerts efficient cleavage activity on substrates that can be captured by the NALFA strip, and thus the proposed system enables the visual detection of Hg2+ in the NALFA strip. Our assay demonstrated sufficient detection sensitivity and specificity to meet the WHO standards, offering a good practical alternative for rapid environmental and public health monitoring.

Multifunctional performance of chitosan/soy protein isolation-based films impregnated carbon dots/anthocyanin derived from purple hull pistachio for tracking and extending the shelf life of fish

The present study aimed to develop a sustainable biopolymer film that exhibits active and intelligent properties. The chitosan/soy protein isolation (Cs/SPI) based film was prepared by blending purple hull pistachio anthocyanin (PHPA) with carbon dots (CDs), which were synthesized using a by-product from purple hull pistachio (PHP) extraction. PHPA and CDs were shown to be compatible with the Cs/SPI biopolymer films. The resulting matrix was a homogeneous film with improved physico-mechanical properties. The biopolymer films were characterized by their various desirable properties, including 100% UV protection, potent antibacterial activity against S.aureus and E. coli strains with inhibition zonoe about 22.1 ± 36±0.24 mm and 20.36 ± 0.21 mm, respectively, and strong antioxidant properties (DPPH; 82.3 ± 0.1 % and ABTS; 90.6 ± 0.1 %). The film was also found to exhibit distinguishable colorimetric responses to pH 2–12 buffers and volatile ammonia. The packaging test of CS/SPI/PHPA/PHP-CDs film in scenario one (intelligent assay) for evaluation through fish freshness monitoring showed that the film's color changed from light pink (fresh fish) to yellow/brownish (spoilage fish) during storage at 4 °C and 25 °C. Also, scenario two (active assay) at 4 °C showed the ability to increase the shelf life of fish up to 12 days. The study demonstrated that the biopolymer films have exhibited exceptional adaptability, making it a highly promising option for prompt and effective on-site detection of food quality. Furthermore, the film's capacity to prolong the shelf life of fish and reduce the rate of quality degradation during storage positions it as an outstanding contender for multifunctional film applications in the food industry.

Fluorescent aptasensor based on competitive recognition strategies and point-of-care testing system for brain natriuretic peptide detection

Brain natriuretic peptide (BNP) is the preferred biomarker for clinical analysis, widely used in early screening and prognostic monitoring of cardiovascular and neurological diseases. Due to the low concentration and short half-life of BNP in the blood, its sensitive detection remains a challenge that must be overcome. With the rapid development of molecular diagnosis and point-of-care testing (POCT), developing a BNP biosensor with high sensitivity, good stability, accuracy, speed, and low-cost is of great significance for clinical applications and emergency diagnosis. However, BNP in human blood exists in various forms, and traditional fluorescence sensors may lead to overestimation of the concentration. In this work, we constructed an “on” fluorescent aptasensor based on competitive recognition strategy for quantitative detection of BNP, and built a fluorescence sensing detection system based on smartphone to achieve rapid on-site detection. We designed a specific aptamer labeled with carboxyfluorescein (FAM) with a simple structure and high biological affinity for selective capture of BNP, and utilized the large specific surface area and excellent fluorescence quenching effect of carboxylated graphene oxide (GO-COOH) as a carrier and quencher for the aptasensor. By optimizing experimental parameters such as aptamer and GO-COOH concentrations, as well as quenching/recovery time, linear sensitive detection of BNP was achieved in the concentration range of 0.01 ng/mL–10 ng/mL, with a detection limit as low as 10 pg/mL, good specificity, and excellent application potential in artificial serum spiking experiments. Additionally, a stable and sensitive fluorescence signal collection system has been developed to meet the needs of portable detection, breaking through the usage environment of traditional fluorescence detection equipment and filling the gap of portable fluorescence biosensing. In summary, we have creatively proposed a fluorescent aptasensor that enables rapid and sensitive detection of BNP and the development of low-cost fluorescent biosensors. The combination with POCT has shown broad application prospects and also provides some ideas for the fluorescence detection of other protein biomarkers.

Evaluation of Fentanyl Exposure Effects on Butyrylcholinesterase Activity as a Tool for Future On-Site Detection Methods.

The prominence of fentanyl and fentanyl analogues or Fentanyl Related Substances (FRS) has driven a nationwide crisis of opioid overdoses, which significantly presents an issue for public health and safety. Originally developed for medical purposes, fentanyl and FRS have become critical contributors to opioid overdose deaths due to their distribution, availability, and potency. This study examined toxicodynamic properties between butyrylcholinesterase (BChE) and fentanyl analogues via Ellman's assay. The enzymatic function of BChE was significantly inhibited by each of the 5 fentanyl analogues tested, which indicates the potential for utilization of this interaction. This reaction can be immobilized for a portable, single-use kit to detect FRS directly from any surface on-site. This would immensely benefit society by reducing the frequency of exposure and overdoses by providing additional safety measures to law enforcement and first responders.

Dual-color immunochromatographic test strip for simultaneous sensitive detection of malachite green and leucomalachite green residues in fish and shrimp meat samples

A sensitive dual immunochromatographic test strip (dual-ICTS) was developed to detect malachite green (MG) and its metabolite, leucomalachite green (LMG), using two types of gold nanoparticles: round-shaped (red) and star-shaped (blue). The detection limits were determined to be 0.221 μg L−1 for MG and 0.214 μg L−1 for LMG, respectively. The dual-ICTS provided a cut-off value of 1.8 μg L−1 for MG and LMG detection. The dual-ICTS successfully detected MG and LMG in food samples, with recovery rates ranging from 86 % to 116 %. The dual-ICTS was evaluated by correlation analysis between the proposed assay and the well-established enzyme-linked immunosorbent assay in the MG and LMG detection. This is the first report on the development of the ICTS that can detect both MG and LMG at the same time within only 5 min, making it a sensitive and rapid tool for on-site detection.

Signal processing for miniature mass spectrometer based on LSTM-EEMD feature digging

Miniature mass spectrometers exhibit immense application potential in on-site detection due to their small size and low cost. However, their detection accuracy is severely affected by factors such as sample pre-processing and environmental conditions. In this study, we propose a data processing method based on long short-term memory-ensemble empirical mode decomposition (LSTM-EEMD) to improve the quality of on-site detection data from miniature mass spectrometers. The EEMD method can clearly decompose the different physical feature components in the small-scale spectrometer signals, while the LSTM method can adaptively learn the internal feature relationships of the signals. Thus, by combining the two, the parameters for the EEMD signal reconstruction can be optimized in an adaptive manner, obtaining the optimized coefficients. Compared to the previous EEMD feature enhancement approach, the LSTM-EEMD method not only significantly improves the coefficient of determination (R2) and relative standard deviation (RSD) of the data, enhancing the linear range, but also achieves fully adaptive processing throughout the workflow, greatly boosting the efficiency. By leveraging a miniature mass spectrometer, data for N-acetyl-l-aspartic acid (NAA), 2-Hydroxyglutarate (2-HG), and γ-Aminobutyric acid (GABA) in actual blood samples have been obtained. The experimental results demonstrate that the LSTM-EEMD method can markedly enhance the accuracy and usability of the biological sample data in practical testing, providing new perspectives and possibilities for research and applications in the relevant domain.

Microfluidic sensor integrated with selenite-enriched lanthanum hydroxide and in situ filtration for the on-site detection of the antibiotic trimethoprim in environmental samples

The improper disposal of antibiotics in water bodies and using contaminated wastewater in irrigation severely damage the environment. Despite efforts to monitor these contaminants, effective detection methods are limited. Here, we design and develop a novel microfluidic electrochemical (EC) sensor for on-site detection of trimethoprim (TMP) using a selenite-enriched lanthanum hydroxide (La(OH)3:SeOx) working electrode and a polyimide (PI)-filter integrated microfluidic channel (MFC), thus termed a “µTMP-chip”. For the first time, we introduced a new two-pronged strategy for enhancing TMP detection: i) incorporating selenite into the La(OH)3 lattice to improve charge transfer properties and ii) using a laser-processed PI filter in the MFC to trap and isolate complex biomasses. Material characterizations confirmed that incorporating selenite into the La(OH)3 lattice initiated La–O–Se bond formation and enhanced hybridization between the La 4f and O 2p orbitals. This process created holes in the O 2p valence band and improved the charge transfer properties, thus enhancing both sensitivity and selectivity. EC studies confirmed that when the PI filter is not used in the MFC, the µTMP-chip experiences a 15–45 % drop in efficiency. The scalable µTMP-chip offers cost-effective, highly reproducible TMP detection in soil and water.

Plasmonic Au-NPs photodecorated on NiCoLDH nanosheets as a flexible SERS sensor for the real-time detection of fipronil

Rapid extraction and detection of probe molecules from curved surfaces is critical for on-site and real-time detection. In this study, a flexible platform was developed using carbon cloth (CC) to create honeycomb-like nickel cobalt layered double hydroxides (NiCoLDH) nanosheets via a simple electrodeposition technique, which were then decorated with gold nanoparticles (Au-NPs) via photodeposition process. The Au-NPs/NiCoLDH/CC was designed as a surface-enhanced Raman scattering (SERS) substrate for detecting the broad-spectrum insecticide, Fipronil (FP). The fabricated sensor achieves a superior SERS activity due to the electrodeposited NiCoLDH, which provides the charge-transfer effect, and the photodeposited Au-NPs, which generate efficient SERS hotspots through the electromagnetic effect. The Density Functional Theory (DFT) calculation was used to estimate the optimal geometry and frontier molecular orbital diagrams of the FP molecules. The influence of electrodeposition time on NiCoLDH production and Au-NPs decorating quantity was investigated in detail. Furthermore, the flexible SERS sensor has excellent sensitivity, homogeneity, a low limit of detection (LOD), and high reproducibility for FP detection. Even after 40 cycles of bending and torsion, the SERS substrate maintained excellent mechanical endurance. Through a direct-sampling approach, FP molecules on the surfaces and mesocarp region of grapes and tomatoes were successfully detected with lower LOD. These findings highlight the outstanding potential of the produced flexible SERS sensor for real-time and on-site insecticide detection, making it valuable for food security analysis and monitoring.

Novel AIE and solvatochromism exhibiting TPE based organic fluorophore with excellent emission properties in solid and solution phase for latent fingerprint and metronidazole sensing in real samples

Organic molecules with extended aromaticity are important when designing probes with optical properties. Herein, we designed and synthesized a novel tetraphenylethylene (TPE) based organic molecule TC exhibiting excellent photophysical characteristics. A thorough and in-depth study of the optical properties of probe TC was performed including solvatochromism, aggregation and solid-state studies using ultraviolet–visible (UV Vis) and fluorescence spectroscopy. Also, its optical properties under a UV 365 nm laser were investigated. The compound exhibited aggregation induced emission (AIE) which was thoroughly investigated in a H2O/DMF system and later utilized in formulation of invisible ink and latent fingerprint sensing. Moreover, the excellent emission properties of probe TC were utilized for the highly selective detection of the antibiotic metronidazole (MET) in real samples including human serum and artificial urine. Furthermore, portable TLC and paper-based strips were designed for the on-site detection of MET. Additionally, the mechanism of selective sensing of MET was thoroughly investigated through spectroscopic studies including titration NMR, fluorescence, UV Vis studies and DFT studies. Photoinduced electron transfer (PET) was attributed as the plausible sensing mechanism for the detection of MET. Scanning electron microscopy (SEM) also supported the sensing of MET through change in morphology. DFT calculations involved the measurement of thermodynamic stability, reduced density gradient (RDG) analysis, molecular orbital, and charge transfer studies as well as analysis of quantum theory of atoms in molecules (QTAIM). All the studies supported the presence of non-covalent interactions between probe TC and MET.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus

Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/μl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.

Adjacent Fe-Pt atomic site synergistically boosting wearable biosensor performance in sweat analysis

Wearable electrochemical sensors have enormous potential in real-time and continuous monitoring of an individual’s physiological and biological state. In electrocatalysis, single-atom catalysts (SACs) with Fe-N4 active centers are attracting interest for their nearly complete metal utilization rate and unique unsaturated coordination configuration. Yet, their reliance on a single active site leads to a fixed adsorption mode for oxygen intermediates such as OH, greatly constraining design flexibility and catalytic performance. This study explores the synergistic effects between Fe-N4 and Pt-N4 active sites and their impact on the electrocatalytic activity towards uric acid (UA) and dopamine (DA). Proximal Pt-N4 atoms facilitate the activation of OH species on the Fe-N4 site and modulate the rehybridization of Fe d orbitals, thereby enhancing the adsorption of oxygen intermediates. This mechanism accelerates the overall electrocatalytic process for both UA and DA. Moreover, a non-swelling double network hydrogel is designed by combining chitosan with the P(AA-MEA)-Fe network. As a practical application, the non-swelling hydrogel channel, when combined with FeN4/PtN4 catalyst, successfully achieves real-time on-site detection of skin sweat biomarkers.

A dendrimer-based platform integrating surface-enhanced Raman scattering and class-incremental learning for rapidly detecting four pathogenic bacteria

Rapid monitoring of pathogens is crucial for preventing foodborne diseases, thus making it an urgent need to develop efficient, fast, and simple methods for on-site detection of multiple pathogens. With advances in SERS-based label-free biosensors and machine learning, progress is evident, yet challenges such as complex substrates and limited model interpretability persist. In this work, we reported a novel dendrimer-based platform that integrated surface-enhanced Raman scattering (SERS) with class-incremental learning (CIL) for quick and simultaneous detection of four pathogenic bacteria, namely Escherichia coli, Salmonella Paratyphi B, Pseudomonas aeruginosa, and Staphylococcus aureus. First, the poly(amidoamine) (PAMAM)-based gold nanoassemblies on silicon wafer (PGNAs/Si) was designed as a highly active SERS substrate with an easy fabrication process. Compared with naked gold nanoparticles, both sensitivity and repeatability were improved by introducing a controllable dendritic nanostructure with ultra-small internal nanogaps. The detection limits of four pathogens in water, food, and dietary supplement matrices were all on the order of 10 CFU/mL. Subsequently, to analyze the complex SERS spectra and distinguish samples with different pathogens, CIL models were established using the light gradient boosting machine (LightGBM) algorithm. Notably, the discriminative models demonstrated excellent classification performance with an accuracy of over 93.44 %. Further, the SHapley Additive exPlanations (SHAP) method was utilized to identify the key SERS features in accurately discriminating different pathogens. Together, these results demonstrate that the dendrimer-based platform, by integrating SERS with CIL, can rapidly detect four pathogenic bacteria, underscoring its potential for food safety testing or quality monitoring in medical supplies manufacturing.

Achieving precise dual detection: One-tube reverse transcription-recombinase aided amplification (RT-RAA) combined with lateral flow strip (LFS) assay for RNA and DNA target genes from pepper mild mottle virus and Colletotrichum species in crude plant samples

Ensuring the detection sensitivity of both RNA-derived and DNA-derived target genes in a single reaction has posed a significant challenge for on-site detection of plant pathogens. This challenge was addressed by developing a one-tube dual RT-RAA assay combined with LFS for the rapid on-site detection of pepper mild mottle virus (PMMoV) and four Colletotrichum species causing anthracnose in Solanaceous crops. By testing four different combinations of primer groups, two combinations were precisely adjusted within the dual RT-RAA system to balance amplification efficiency and maintain consistent levels of amplification in crude plant samples. Utilizing commercially accessible small-scale equipment and following a streamlined optimization strategy, the assay achieved a limit of detection of 0.32 copies/μL of target genes in the reaction. Importantly, it demonstrated no cross-reactivity with other plant pathogens, thereby affirming the high sensitivity and specificity of the developed dual RT-RAA-LFS detection assay. Moreover, the entire process took only 25 min from sample collection to the visible presentation of results. The assay was validated with 60 field samples and 10 seed samples, producing results consistent with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Notably, it successfully detected PMMoV in systemic leaves without visible symptoms three days post-inoculation, underscoring its effectiveness in early disease detection. This streamlined strategy offers a valuable approach for rapid, low-cost, and highly sensitive on-site simultaneous detection of RNA genome-contained PMMoV and DNA genome-contained Colletotrichum species.