On-site Detection Of Pathogens Research Articles

A pH ultra-sensitive hydrated iridium oxyhydroxide films electrochemical sensor for label-free detection of Vibrio parahaemolyticus

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.

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses.

Isothermal nucleic acid amplification-based lateral flow testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative of addressing the existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

A simple graphene oxide-based DNA purification strategy for plant pathogen detection.

The on-site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor-intensive and time-consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL-1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO-based DNA purification method with the loop-mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. We present a feasible, rapid, simple and low-cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant-microbial microenvironments. © 2024 Society of Chemical Industry.

Nanotopology-Enabled On-Site Pathogen Detection for Managing Atopic Dermatitis.

Atopic dermatitis (AD), a prevalent skin condition often complicated by microbial infection, poses a significant challenge in identifying the responsible pathogen for its effective management. However, a reliable, safe tool for pinpointing the source of these infections remains elusive. In this study, a novel on-site pathogen detection that combines chemically functionalized nanotopology with genetic analysis is proposed to capture and analyze pathogens closely associated with severe atopic dermatitis. The chemically functionalized nanotopology features a 3D hierarchical nanopillar array (HNA) with a functional polymer coating, tailored to isolate target pathogens from infected skin. This innovative nanotopology demonstrates superior pathogenic capture efficiency, favorable entrapment patterns, and non-cytotoxicity. An HNA-assembled stick is utilized to directly retrieve bacteria from infected skin samples, followed by extraction-free quantitative loop-mediated isothermal amplification (direct qLAMP) for validation. To mimic human skin conditions, porcine skin is employed to successfully capture Staphylococcus aureus, a common bacterium exacerbating AD cases. The on-site detection method exhibits an impressive detection limit of 103 cells mL-1. The HNA-assembled stick represents a promising tool for on-site detection of bacteria associated with atopic dermatitis. This innovative approach enables to deepen the understanding of AD pathogenesis and open avenues for more effective management strategies for chronic skin conditions.

Electrochemical biosensors for pathogenic microorganisms detection based on recognition elements.

The worldwide spread of pathogenic microorganisms poses a significant risk to human health. Electrochemical biosensors have emerged as dependable analytical tools for the point-of-care detection of pathogens and can effectively compensate for the limitations of conventional techniques. Real-time analysis, high throughput, portability, and rapidity make them pioneering tools for on-site detection of pathogens. Herein, this work comprehensively reviews the recent advances in electrochemical biosensors for pathogen detection, focusing on those based on the classification of recognition elements, and summarizes their principles, current challenges, and prospects. This review was conducted by a systematic search of PubMed and Web of Science databases to obtain relevant literature and construct a basic framework. A total of 171 publications were included after online screening and data extraction to obtain information of the research advances in electrochemical biosensors for pathogen detection. According to the findings, the research of electrochemical biosensors in pathogen detection has been increasing yearly in the past 3years, which has a broad development prospect, but most of the biosensors have performance or economic limitations and are still in the primary stage. Therefore, significant research and funding are required to fuel the rapid development of electrochemical biosensors. The overview comprehensively evaluates the recent advances in different types of electrochemical biosensors utilized in pathogen detection, with a view to providing insights into future research directions in biosensors.

NFC-enabled potentiostat and nitrocellulose-based metal electrodes for electrochemical lateral flow assay

Rapid detection of pathogens at the point-of-need is crucial for preventing the spread of human, animal and plant diseases which can have devastating consequences both on the lives and livelihood of billions of people. Colorimetric, lateral flow assays consisting of a nitrocellulose membrane, are the preferred format today for low-cost on-site detection of pathogens. This assay format has, however, historically suffered from poor analytical performance and is not compatible with digital technologies. In this work, we report the development of a new class of digital diagnostics platform for precision point-of-need testing. This new versatile platform consists of two important innovations: i) A wireless and batteryless, microcontroller-based, low-cost Near Field Communication (NFC)-enabled potentiostat that brings high performance electroanalytical techniques (cyclic voltammetry, chronoamperometry, square wave voltammetry) to the field. The NFC-potentiostat can be operated with a mobile app by minimally trained users; ii) A new approach for producing nitrocellulose membranes with integrated electrodes that facilitate high performance electrochemical detection at the point-of-need. We produced an integrated system housed in a 3D-printed phone case and demonstrated its use for the detection of Maize Mosaic Virus (MMV), a plant pathogen, as a proof-of-concept application.

Development of Loop-Mediated Isothermal Amplification Assays for the Rapid and Accurate Diagnosis of Exserohilum turcicum for Field Applications.

Northern corn leaf blight (NCLB), caused by Exserohilum turcicum, is one of the most devastating foliar diseases of maize. Rapid and accurate diagnosis for this disease is urgently needed but still limited. Here, we establish a field-deployable diagnostic method to detect E. turcicum based on loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was used to search for the specific sequences belonging to E. turcicum by comparing the whole genome sequence between E. turcicum and other known maize pathogens. Five LAMP primer sets were designed based on specific and single-copy fragments of E. turcicum. Post-LAMP analyses indicated that only the primer set, Et9468_set1, was the most suitable, producing a ladder-like amplification pattern in the agarose gel electrophoresis and a strong fluorescence signal in the presence of SYBR Green I. The LAMP assay using Et9468_set1 primers demonstrated a high level of specificity in distinguishing E. turcicum from six other common fungal pathogens of maize, as well as 12 more fungal and oomycete strains including the epiphytic fungi from maize leaves and other crop pathogens. Moreover, it exhibited remarkable sensitivity by detecting five copies per reaction, which was approximately 104 times more sensitive compared with conventional PCR. The LAMP assay successfully detected E. turcicum in field maize leaves without DNA extraction, demonstrating its suitability for rapid on-spot detection of NCLB. Our study provides a direct LAMP diagnostic method to detect E. turcicum, which enables on-site pathogen detection in the field and the development of preventive strategies for NCLB management.

Instrument-free and rapid Vibrio parahaemolyticus biosensing based on coupled dual-signal of paper-needle and LnMOF

At present, most reported studies on paper-based sensing devices for food-borne pathogens usually rely on a single signal as the basis for judging the results, accompanied with inaccurate detection results. In this study, we used the lanthanide MOFs being covalently modified with aptamers (Apt-LnMOFs) as the sensing and recognition element of Vibrio parahaemolyticus (VP), and the paper-based needle shape device (paper-needle) as the visual signal output carrier. Finally, an accurate detection strategy for VP was successfully constructed. In terms of signal output, the two signal styles of Apt-LnMOFs diffusion distance on the paper-needle and R value (measured by the RGB analysis software of smartphone) are coupled to jointly improve the accuracy of detection results. The method is simple and fast, and the detection accuracy in real samples is 91%. This strategy is very suitable for on-site rapid detection of pathogens, and does not require large professional instruments.

A LAMP-based hydrogen ion selective electrochemical sensor for highly sensitive detection of Mycoplasma pneumoniae.

A concise and rapid detection method for Mycoplasma pneumoniae is urgently required due to its severe impact on human health. To meet such a need, this study proposed and constructed an innovative point-of-care testing (POCT) platform that consists of a hydrogen ion-selective loop-mediated isothermal amplification (H+-LAMP) sensor and an electrochemical detection device. The H+-LAMP sensor successfully integrated the working and reference electrodes and converted the H+ generated during the LAMP process into an electrochemical signal. High sensitivity and stability for pathogen detection were also achieved by treating the working electrode with an electrodeposited polyaniline solid contact layer and by using an ion-selective membrane. As a result, the sensor shows a sensitivity of 68.26 mV per pH, a response time of less than 2 s, and a potential drift of less than 5 mV within one hour, which well meets the urgent need. The results also demonstrated that the detection limit for Mycoplasma pneumoniae was lowered to 1 copy per μL, the nucleic acid extraction and detection process could be completed in 30 minutes, and the impact of interfering ions on the sensor was negligible. Validation with 20 clinical samples yielded satisfactory results. More importantly, the storage lifespan of such an electrochemical sensor is over seven days, which is a great advantage for on-site pathogen detection. Therefore, the hydrogen ion-selective sensor constructed in this investigation is particularly suitable as a core component for instant pathogen detection platforms.

An intelligent readable and capture-antibody-independent lateral flow immunoassay based on Cu2-xSe nanocrystals for point-of-care detection of Escherichia coli O157:H7.

Escherichia coli (E. coli) O157:H7 is a common foodborne pathogen which can cause serious harm. It is particularly important to establish a simple and portable method to achieve on-site pathogen detection. In this study, a capture-antibody-independent lateral flow immunoassay (LFIA) was constructed based on Cu2-xSe nanocrystals (Cu2-xSe NCs) for rapid detection of E. coli O157:H7. Cu2-xSe NCs can not only be regarded as the "nano-antibody" for the recognition of E. coli O157:H7 through electrostatic adsorption, but also as nanozymes that show good peroxidase-like catalytic activity. The formed compound of E. coli O157:H7 and Cu2-xSe NCs would be captured by a detection antibody on the T line due to the specific recognition of the antibody and E. coli O157:H7. Then, Cu2-xSe NCs could catalyze the oxidation of TMB by H2O2 to generate oxTMB, thereby generating blue bands. Meanwhile, we developed a mobile app for rapid data analysis. Under the optimal reaction conditions, E. coli O157:H7 could be detected within 70 min. The detection limit of this method was 2.65 × 105 CFU mL-1 with good specificity and stability. Additionally, it could achieve on-site rapid detection of E. coli O157:H7 in environmental water samples, providing a promising biosensor for portable pathogen detection.

Trends and challenges in electroanalytical biosensing methodologies for infectious viral diseases

Viral pandemic diseases have disruptive global consequences leading to millions of deaths and a severe impact on the global economy. Inadequate preventative protocols have led to an overwhelming demand for intensive care leading to uncontrollable burdens and even breakdown of healthcare sectors across many countries. The rapid detection of viral disease helps in the understanding of the relevant intricacies, helping to tackle infection with improved guidelines. Portable biosensor devices offer promising solutions by facilitating on-site detection of viral pathogens. This review summarizes the latest innovative strategies reported using electroanalytical methods for the screening of viral antigens. The structural components of viruses and their categories are presented followed by the various recognition elements and transduction techniques involved in biosensors. Core sections focus on biosensors reported for viral genomic detection(DNA and RNA) and antigenic capsid protein. Strategies for addressing the challenges of electroanalytical biosensing of viral components are also presented. The advantages, and disadvantages of biorecognition elements and nanozymes for the detection of viral disease are highlighted. Such technical insights will help researchers working in chemistry, and biochemistry as well as clinicians working in medical diagnostics.

A real-time fluorogenic recombinase polymerase amplification microfluidic chip (on-chip RPA) for multiple detection of pathogenic microorganisms of penaeid shrimp

Pathogenic microorganisms pose a significant threat to global shrimp production, often resulting in widespread reductions in production across continents. In this study, we developed a centrifugal microfluidic chip integrating real-time fluorogenic recombinase polymerase amplification (on-chip RPA) for the parallel detection of five important pathogenic microorganisms: acute hepatopancreatic necrosis disease-causing Vibrio spp. (VAHPND), white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), and Enterocytozoon hepatopenaei (EHP). This method enabled the parallel analysis of six genetic markers from a single sample through a single pipetting operation for sample injection. It also allowed for the highly automated detection of up to four samples simultaneously using a chip format. The on-chip RPA reaction was performed in a reduced system with a volume as low as 5 μL at a temperature of 39 °C for 20 min. This assay achieved a limit of detection (LOD) of 10 copies/μL of the recombinant plasmid of the target genes. The coefficient of variation (CV) for the time to positive (Tp) value was less than 0.10, even using concentrations at the LOD for multiple pathogens. This indicates a robust reproducibility among replicates. The clinical sensitivity and specificity for detecting field shrimp samples were 96.4% and 100%, respectively, compared with polymerase chain reaction (PCR) combined with DNA sequencing. On-chip RPA assays allow for low reagent consumption, ease of use, accelerated analysis, the ability to analyze multiple samples and genetic targets, and applicability for on-site detection and routine monitoring of multiple pathogens in shrimp aquaculture.

An Integrated Microfluidic Biosensing System Based on a Versatile Valve and Recombinase Polymerase Amplification for Rapid and Sensitive Detection of Salmonella typhimurium.

Detecting foodborne pathogens on-site is crucial for ensuring food safety, necessitating the development of rapid, cost-effective, highly sensitive, and portable devices. This paper presents an integrated microfluidic biosensing system designed for the rapid and sensitive detection of Salmonella typhimurium (S. typhimurium). The biosensing system comprises a microfluidic chip with a versatile valve, a recombinase polymerase amplification (RPA) for nucleic acid detection, and a customized real-time fluorescence detection system. The versatile valve combines the functions of an active valve and a magnetic actuation mixer, enabling on-demand mixing and controlling fluid flow. Quantitative fluorescence is processed and detected through a custom-built smartphone application. The proposed integrated microfluidic biosensing system could detect Salmonella at concentrations as low as 1.0 × 102 copies/µL within 30 min, which was consistent with the results obtained from the real-time quantitative polymerase chain reaction (qPCR) tests. With its versatile valve, this integrated microfluidic biosensing system holds significant potential for on-site detection of foodborne pathogens.

Ultrasensitive and Specific Phage@DNAzyme Probe-Triggered Fluorescent Click Chemistry for On-Site Detection of Foodborne Pathogens Using a Smartphone.

Rapid, specific, and on-site detection of virulent foodborne pathogenic strains plays a key role in controlling food safety. In this work, an ultrasensitive and specific Phage@DNAzyme signal probe was designed to detect foodborne pathogens. The proposed sensing probe was composed of the selected phage and functionalized DNAzyme, which realized the specific recognition of target foodborne pathogens at the strain level and the efficient catalysis of copper(II) based azide-alkyne cycloaddition (CuAAC) click reaction with fluorescent signal, respectively. As a proof of concept, the virulent Escherichia coli O157:H7 (E. coli O157:H7) as the representative analyte was first enriched and purified from the complex food samples by a 4-mercaptophenylboronic acid-modified gold slide. Following, the Phage@DNAzyme probes were specifically combined with the captured E. coli O157: H7 and catalyzed the click reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol with the assistance of Cu(II) to generate a visual fluorescent signal. Finally, the corresponding fluorescent signals were measured by a smartphone to quantify the target concentrations. Under optimized conditions, the bioassay exhibited a wide linear range from 102 to 108 CFU/mL and the detection limit was 50 CFU/mL (S/N = 3). It was further extended to the detection of another foodborne pathogen Salmonella typhimurium with satisfying sensing performances. This work gives a new path for developing rapid, specific, and on-site detection methods for trace levels of pathogenic strains in foods.

Copper: DNA extraction and solid phase detection agent for all-in-one molecular diagnostic device coupled with isothermal amplification.

In this study, an all-in-one poly(methyl methacrylate) (PMMA) device integrating two novel techniques - DNA extraction employing a CuSO4/H2O2 system and DNA detection utilizing solid phase copper tape - coupled with loop-mediated isothermal amplification (LAMP) is developed for on-site pathogen detection. The CuSO4/H2O2 system, also known as Fenton-like reaction, is used to produce hydroxyl radicals, which can disrupt bacterial membranes via lipid peroxidation and release DNA at room temperature. The released DNA is subsequently amplified by LAMP reaction. The acidic environment resulting from the production of hydrogen ions in the presence of target DNA in the LAMP reaction can stimulate the color change on copper tape due to the corrosion, while the innate alkaline environment in a negative sample not containing target DNA cannot stimulate the corrosion. The fabricated PMMA device integrates all the functionalities necessary for molecular diagnostics such as DNA extraction, amplification, and detection, and a carbon paste-based heater is fabricated for LAMP reaction. Using the PMMA device, Enterococcus faecium was detected as low as 4.67×102CFU/mL within 90min. E. faecium spiked in milk was successfully detected using the all-in-one PMMA device. The equipment-free techniques for decentralized diagnostics and naked-eye readout of results coupled with the portable heater serves as a promising solution for point-of-care testing particularly in a resource-limited environment.

Polyaniline-based 3D network structure promotes entrapment and detection of drug-resistant bacteria

Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin.Graphical

Research progress of visual detection in rapid on-site detection of pathogen nucleic acid.

Nucleic acid detection is widely used in pathogen screening and detection due to its high sensitivity and specificity. With the increase of detection requirements and the development of amplification technology, nucleic acid detection methods are gradually developing towards simple, fast and low-cost. Quantitative polymerase chain reaction (qPCR), as the "gold standard" for nucleic acid detection, relies on expensive equipment and professional operators, which is not suitable for rapid on-site detection of pathogens. The visual detection method without relying on excitation light source or complex equipment can present the detection results in a more intuitive and portable way after combining with rapid and efficient amplification technology, which has the potential of point-of-care testing (POCT). This paper focuses on the reported application of amplification technology and CRISPR/Cas technology in visual detection and compares their advantages and disadvantages, so as to provide reference for POCT strategy based on pathogen nucleic acid.

Capture, sterilization and determination platform of foodborne pathogen based on Halbach ring-mediated magnetic filter

Herein, an ingenious Halbach ring-mediated magnetic filter (HRMF) for capture, sterilization, and detection of foodborne pathogenic bacteria is developed. A composite material with magnetic nanoparticles (MNP) as the core and coated with polydopamine (PDA) and polyethylenimine (PEI) is synthesized, namely MNP@PDA@PEI. Under a Halbach ring, MNP@PDA@PEI can form chains to generate HRMF. Due to the positively charged of PEI, the HRMF could capture the negatively charged bacteria from solution via electrostatic adsorption. Within the HRMF, sterilization performance could be achieved due to the photothermal effect of MNP@PDA@PEI after 5 min of near-infrared light irradiation. More importantly, the number of bacteria could be calculated by bioluminescence method for measuring ATP, which are released from bacteria after photothermal sterilization. Listeria monocytogenes is chosen as a typical bacterium to evaluate the function of HRMF. The time of whole process has been greatly reduced to 38 min. The capture efficiency is 98.63%, and sterilization rate can achieve 99.96% in real samples. The limit of detection is 10.04 CFU/mL, representing a 10-fold decrease compared with bioluminescence method alone. The proposed platform might open a new way for on-site real-time detection of foodborne pathogens, and hold potential to nondestructive sterilization in the food processing industry.

Dual-mode colorimetric-electrochemical biosensor for Vibrio parahaemolyticus detection based on CuO2 nanodot-encapsulated metal-organic framework nanozymes

Rapid and accurate detection of Vibrio parahaemolyticus (VP) in a variety of testing environments is critical for preventing the spread of foodborne illnesses. Therefore, a dual-mode colorimetric-electrochemical biosensor was prepared for the rapid and sensitive detection of VP. Phenylboronic acid-decorated CuO2 nanodot-mediated organic metal-organic framework (CP@MOF) nanozymes were constructed to produce colorimetric and electrochemical signals. Furthermore, an electrode surface was fabricated using tetrahedral DNA nanostructure-functionalized poly(amidoamine) dendrimer-Au nanoparticles to specifically bind VP. Under acidic environments, the CP@MOF nanozymes disintegrated, enabling the simultaneous release of Cu2+ and H2O2. This in turn causes a Fenton-type reaction, generating a hydroxyl radical, which then oxidizes 3,3′,5,5′-tetramethylbenzidine, thereby enabling visual detection of VP. Moreover, the produced copper ions can be used for the electrochemical detection of VP. This dual-mode biosensor achieved an accuracy of 88.7 %, which was higher than that of single-mode assays (81.1 %). Hence, this dual-mode assay may facilitate the effective on-site detection of pathogens in shrimp samples.

Electrostatic Adsorption of Dense AuNPs onto Silica Core as High-Performance SERS Tag for Sensitive Immunochromatographic Detection of Streptococcus pneumoniae

Streptococcus pneumoniae (S. pneumoniae) is a prominent pathogen of bacterial pneumonia and its rapid and sensitive detection in complex biological samples remains a challenge. Here, we developed a simple but effective immunochromatographic assay (ICA) based on silica-Au core-satellite (SiO2@20Au) SERS tags to sensitively and quantitatively detect S. pneumoniae. The high-performance SiO2@20Au tags with superior stability and SERS activity were prepared by one-step electrostatic adsorption of dense 20 nm AuNPs onto 180 nm SiO2 core and introduced into the ICA method to ensure the high sensitivity and accuracy of the assay. The detection limit of the proposed SERS-ICA reached 46 cells/mL for S. pneumoniae and was 100-fold more sensitive than the traditional AuNPs-based colorimetric ICA method. Further, considering its good stability, specificity, reproducibility, and easy operation, the SiO2@20Au-SERS-ICA developed here has great potential to meet the demands of on-site and accurate detection of respiratory pathogens.