On-site rapid detection of multiple pesticide residues in tea leaves by lateral flow immunoassay

The application of pesticides (mostly insecticides and fungicides) during the tea-planting process will undoubtedly increase the dietary risk associated with drinking tea. Thus, it is necessary to ascertain whether pesticide residues in tea products exceed the maximum residue limits. However, the complex matrices present in tea samples comprise a major challenge in the analytical detection of pesticide residues. In this study, nine types of lateral flow immunochromatographic strips (LFICSs) were developed to detect the pesticides of interest (fenpropathrin, chlorpyrifos, imidacloprid, thiamethoxam, acetamiprid, carbendazim, chlorothalonil, pyraclostrobin, and iprodione). To reduce the interference of tea substrates on the assay sensitivity, the pretreatment conditions for tea samples, including the extraction solvent, extraction time, and purification agent, were optimized for the simultaneous detection of these pesticides. The entire testing procedure (including pretreatment and detection) could be completed within 30 min. The detected results of authentic tea samples were confirmed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which suggest that the LFICS coupled with sample rapid pretreatment can be used for on-site rapid screening of the target pesticide in tea products prior to their market release.

Development of a multiplex recombinase polymerase amplification coupled with lateral flow dipsticks for the simultaneous rapid detection of Salmonella spp.,Salmonella Typhimurium and Salmonella Enteritidis

Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cannot provide fast mobile on-site systems to detect Salmonella in food.Materials and Methods:A visual method was established based on multiple recombinase polymerase amplification(RPA)coupled with lateral flow dipsticks(LFD)for the simultaneous detection of Salmonella spp.,Salmonella Enteritidis and Salmonella Typhimurium in vitro and food.Results:The optimal volume and temperature for the multiplex RPA-LFD method were determined to be 25μL and 38°C,respectively.The reaction process was completed within 25 min and the results were observed visually.The limits of detection(LODs)were 2.8×10^(2),5.9×10^(2),and 7.6×10^(2) CFU/mL for Salmonella spp.,S.Enteritidis and S.Typhimurium,respectively.Meanwhile,the results of the established method showed no cross-reactivity between the Salmonella cells and other common foodborne bacteria,which was highly specific for Salmonella.More importantly,the developed method exhibited good performance in artificially contaminated chicken samples with the LODs of 2.8×10^(3),5.9×10^(3),and 7.6×10^(3) CFU/mL for Salmonella spp.,S.Enteritidis,and S.Typhimurium,respectively.Finally,the application of the multiple RPA-LFD methods in retailed food samples displayed that this method was effective and practical for the detection of Salmonella spp.in food.Conclusion:The developed multiplex RPA-LFD method provides a new sensitive and rapid alternative for the specific detection of Salmonella spp.and its important serovars in food.

Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay

Lateral flow immunoassay(LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation.However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications.In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis.LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5–10 ng mL^(-1) with the limit of detection of 0.1 ng mL^(-1), which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method,which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents(antibodies).

Lateral Flow Immunoassay for Quantitative Detection of Ractopamine in Swine Urine

A strip reader based lateral flow immunoassay （LFIA） was established for the rapid and quantitative detection of ractopamine （RAC） in swine urine. The ratio of the optical densities （ODs） of the test line （AT） to that of the control line （Ac） was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration （IC50） of 0.59＋0.06 ng/mL. The limit of detection （LOD） of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.

Quantum Dot Nanobeads-Labelled Lateral Flow Immunoassay Strip for Rapid and Sensitive Detection of Salmonella Typhimurium Based on Strand Displacement Loop-Mediated Isothermal Amplification

Rapid,sensitive,point-of-care detection of pathogenic bacteria is important for food safety.In this study,we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip(QBs-labelled LFIAS)combined with strand displacement loop-mediated isothermal amplification(SD-LAMP)for quantitative Salmonella Typhimurium(ST)detection.Quantum dot nanobeads(QBs)served as fluorescence reporters,providing good detection efficiency.The customizable strand displacement(SD)probe was used in LAMP to improve the specificity of the method and prevent by-product capture.Detection was based on a sandwich immunoassay.A fluorescence strip reader measured the fluorescence intensity(FI)of the test(T)line and control(C)line.The linear detection range of the strip was 10^(2)–10^(8) colony forming units(CFU)·mL^(-1).The visual limit of detection was 10^(3) CFU·mL^(-1),indicating that the system was ten-fold more sensitive than AuNPs-labelled test strips.ST specificity was analyzed in accordance with agarose gel outputs of polymerase chain reaction(PCR)and SD-LAMP.We detected ST in foods with an acceptable recovery of 85%–110%.The method is rapid,simple,almost equipment-free,and suitable for bacterial detection in foods and for clinical diagnosis.

Rapid and easy determination of morphine in chafing dish condiments with colloidal gold labeling based lateral flow strips

In order to enhance the flavor of chafing dish and increase the attraction of consumers,the poppy shell is reported to be illegally added to the condiments of chafing dish.In this research,a rapid,simple,and convenient method based on the classic immunochromatographic lateral flow strip(LFS)with gold nanoparticles(GNPs)labeling was developed for easy monitoring of morphine(MOP),an effective component of poppy shell.Under optimized conditions,this developed LFS can well realize the detection of target MOP in the condiments of chafing dish in less than 10 min without any complicated pre-treatments.The limit of detection(LOD)can be achieved as low as 0.1 ppb for standard MOP or the MOP spiked condiments of chafing dish.All these results of the research strongly demonstrate that this established LFS method can be successfully applied in practical rapid and accurate on-site screening of poppy shell in condiments of chafing dish.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus

Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this study.Recombinase-aided amplification(RAA)is used to amplify template DNA,and lateral flow dipstick(LFD)is used to interpret the results after the amplification is completed.The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid.In addition,30 clinical samples were tested to evaluate the performance of the RAA assay.Results The RAA-LFD assay was completed within 15 min at 37°C,including 10 min for nucleic acid amplification and 5 minutes for LFD reading results.The detection limit of this assay was found to be 200 copies per reaction.And there was no cross-reactivity with other swine viruses.Conclusion A highly sensitive,specific,and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Performance of simultaneous nitrification and denitrification in lateral flow biological aerated filter

A new wastewater treatment facility—lateral flow biological aerated filter (LBAF) was developed aiming at solving energy consumption and operational problems in wastewater treatment facilities in small towns. It has the function of nitrification and removing organic substrate. In this study, we focused on the denitrification performance of LBAF and its possible mechanism under thorough aeration. We identified the existence of simultaneous nitrification and denitrification (SND) by analyzing nitrogenous compounds along the flow path of LBAF and supportive microbial microscopy, and studied the effects of air/water ratio and hydraulic loading on the performance of nitrogen removal and on SND in LBAF to find out the optimal operation condition. It is found that for saving operation cost, aeration can be reduced to some degree that allows desirable removal efficiency of pollutants, and the optimal air/water ratio is 10:1. Hydraulic loading less than 0.43 m h?1 hardly affects the nitrification and denitrification performance; whereas higher hydraulic loading is unfavorable to both nitrification and denitrification, far more unfavorable to denitrification than to nitrification.

Development of a chromatographic lateral flow immunoassay for detection of African swine fever virus antigen in blood

African swine fever(ASF)is a highly lethal disease of domestic and wild swine caused by African swine fever virus(ASFV).The disease currently circulates in Africa,Europe,Asia and on the island of Hispaniola.The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses.No effective vaccine is available for ASF,making rapid and accurate detection of ASFV essential for disease mitigation strategies.Currently available diagnostics for ASFV possess significant limitations related to assay performance,deployability,and/or turn-around time;therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity.A chromatographic lateral flow immunoassay(LFIA)was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein.The assay requires only water to perform and provides results in 25 min,making it well-suited for field use.The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection,though it was unable to detect a genotype X ASFV strain.Diagnostic sensitivity correlated with clinical disease severity,body temperature,and viral DNA levels,and was over 90%in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus.The LFIA also showed a robust diagnostic specificity of over 98%,which is essential to field testing for a high consequence to foregin animal disease.The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates,making it a promising candidate for use as a field-deployable antigen detection assay.Additional evaluation using field samples and different virus strains is required to further assess the utility of this rapid diagnostic test.

Development of an accurate lateral flow immunoassay for PEDV detection in swine fecal samples with a filter pad design

Porcine epidemic diarrhea virus(PEDV),as the main causative pathogen of viral diarrhea in pigs,has been reported to result in high morbidity and mortality in neonatal piglets and cause significant economic losses to the swine industry.Rapid diagnosis methods are essential for preventing outbreaks and transmission of this disease.In this study,a paper-based lateral flow immunoassay for the rapid diagnosis of PEDV in swine fecal samples was developed using stable color-rich latex beads as the label.Under optimal conditions,the newly developed latex bead-based lateral flow immunoassay(LBs-LFIA)attained a limit of detection(LOD)as low as 10^(3.60) TCID_(50)/mL and no cross-reactivity with other related swine viruses.To solve swine feces impurity interference,by adding a filtration unit design of LFIA without an additional pretreatment procedure,the LBs-LFIA gave good agreement(92.59%)with RT-PCR results in the analysis of clinical swine fecal samples{n=108),which was more accurate than previously reported colloidal gold LFIA(74.07%)and fluorescent LFIA(86.67%).Moreover,LBs-LFIA showed sufficient accuracy(coefficient of variance[CV]<15%)and stable(room temperature storage life>56 days)performance for PEDV detection,which is promising for on-site analysis and user-driven testing in pig production system.

Recent progress on lateral flow immunoassays in foodborne pathogen detection

Foodborne pathogens cause diseases in humans.The traditional methods of detecting foodborne pathogens are time-consuming.The lateral flow immunoassay(LFIA)has become a widely used detection platform for onsite testing of various foodborne pathogens due to its time-efficiency,cost-effectiveness,portability,and ease of use.With the development of novel nanomaterials,the sensitivity of the LFIA has improved tremendously compared with traditional colorimetric LFIA sensors.This review first summarizes the principles and corresponding formats of the LFIA.Then,a detailed classification of nanomaterial label(e.g.,metallic,carbon and selenium,fluorescent,and magnetic nanoparticles)synthesis,signal amplification strategy,and detection principles are discussed as related to food safety.Subsequently,the LFIA used in the detection of pathogenic bacteria,including Escherichia coli,Vibrio parahaemolyticus,Staphylococcus aureus,Listeria monocytogenes,and Salmonella,are classified and summarized.Multiple signal modes have been explored that improve the sensitivity of foodborne pathogen detection.Further improvement should focus on the design and preparation of high signal-to-noise ratio nanomaterials to achieve highly sensitive detection,and multitarget and multimode sensing.

Hydrodynamic behaviour of the lateral flow biological aerated filter

Pulsed signal experiment was carried out to determine the hydrodynamic behaviours of lateral flow biological aerated filter(LBAF). With the analysis of experimental results, LBAF is viewed as an approximate plug flow reactor, and hydraulic retention time distribution function was derived based on LBAF. The results show that flow rate and aeration strength are two critical factors which influence flow patterns in LBAF reactor. The hydrodynamic behaviour analysis of LBAF is the theoretical basis of future research on improving capacity factor and developing kinetic model for the reactor.

Performances of lateral flow biological aerated filter in treating domestic wastewater

A new biological aerated filter?lateral flow biological aerated filter(LBAF) is developed. The effects of air/water ratio, hydraulic loading and the length of LBAF on pollutants removal efficiency are tested. The results show that under optimal technological conditions when hydraulic loading is 0.43 m3 m?2 h?1 and air/water ratio is 10:1, the average removal efficiencies of COD, SS, NH3-N, and TN reach 88.01%, 95.18%, 78.97% and 52.58%, respectively. An LBAF has a large pollutants handling capacity; is less liable to be blocked, and has a longer operation cycle in comparison with a traditional BAF.

Performance of alpha-defensin lateral flow test after synovial fluid centrifugation for diagnosis of periprosthetic knee infection

BACKGROUND The quantitative alpha-defensin enzyme-linked immunosorbent assay(ELISA)demands a prior synovial fluid centrifugation,whereas this processing is not routinely required prior to the alpha-defensin lateral flow test.AIM To evaluate whether a prior synovial fluid centrifugation could lead the lateral flow performance to achieve comparable results to ELISA during periprosthetic joint infection(PJI)diagnosis.METHODS Fifty-three cases were included in this study:22 classified as PJI and 31 classified as aseptic cases,according to Musculoskeletal Infection Society 2013 criteria.Synovial fluid samples were submitted to centrifugation,and the supernatant was evaluated by ELISA and lateral flow tests.The sensitivity(SE),specificity(SP)and accuracy of each method were calculated as well as the agreement between those two methods.RESULTS In all of the 31 samples from aseptic patients,alpha-defensin ELISA and lateral flow tests showed negative results for infection.Regarding the 22 infected patients,the lateral flow test was positive in 19 cases(86.4%)and the ELISA was positive in 21(95.5%).Sensibility,SP and accuracy were,respectively,86.4%(95%CI:65.1%-97.1%),100%(95%CI:88.8%-100%)and 93.2%(95%CI:82.8%-98.3%)for the lateral flow test and 95.5%(95%CI:77.2%-99.9%),100%(95%CI:88.8%-100%)and 98.1%(95%CI:89.9%-100%)for ELISA.An agreement of 96.2%between those methods were observed.No statistical difference was found between them(P=0.48).CONCLUSION Alpha-defensin lateral flow test showed high SE,SP and accuracy after a prior synovial fluid centrifugation,achieving comparable results to ELISA.Considering the lower complexity of the lateral flow and its equivalent performance obtained in this condition,a prior centrifugation might be added as a valuable step to enhance the PJI diagnosis.

NIR-Ⅱfluorescence lateral flow immunosensor based on efficient energy transfer probe for point-of-care testing of tumor biomarkers

Fluorescence lateral flow immunoassay(LFA)has emerged as a powerful tool for rapid screening of various biomarkers owing to its simplicity,sensitivity and flexibility.It is noteworthy that fluorescent probe mainly determines the analytical performance of LFA.Due to the emission and excitation wavelengths are located in the visible region,most fluorophores are inevitably subject to light scattering and background autofluorescence.Herein,we reported a novel LFA sensor based on the second near-infrared(NIR-Ⅱ)fluorescent probe with excellent anti-interference capability.The designed NIR-Ⅱprobe was the Nd^(3+)and Yb^(3+)doped rare earth nanoparticles(RENPs)by employing Nd^(3+)as energy donor and Yb^(3+)as energy acceptor,which of the donor-acceptor energy transfer(ET)efficiency reached up to 80.7%.Meanwhile,relying on the convenient and effective encapsulation strategy of poly(lactic-co-glycolic acid)(PLGA)microspheres to RENPs,the surface functionalized NIR-Ⅱprobe(RE@PLGA)was obtained for subsequent bioconjugation.Benefiting from the optical advantages of NIR-Ⅱprobe,this proposed NIR-ⅡLFA displayed a good linear relationship ranging from 7 ng/mL to 200 ng/mL for the detection ofα-fetoprotein(AFP),an important biomarker of hepatocellular carcinoma(HCC).The limit of detection(LOD)was determined as low as 3.0 ng/m L,which was of 8.3 times lower than clinical cutoff value.It is promising that LFA sensor based on this efficient RENPs probe provides new opportunities for high sensitive detection of various biomarkers in biological samples.

Quantitative assessment of the breast cancer marker HER2 using a gold nanoparticle-based lateral flow immunoassay

Human epidermal growth factor receptor 2(HER2)is an important biomarker for detection and treatment of breast cancer.In this study,we developed monoclonal antibodies against the extracellular domain(ECD)of HER2 and established a rapid and accurate lateral flow immunoassay(LFIA)for use in community medical institutions.The gene sequence of human HER2-ECD was obtained from the National Center for Biotechnology Information(NCBI)to construct the expression plasmid.HER2-ECD protein expressed in HEK293F cells was used to immunize BALB/c mice.The monoclonal antibodies were produced in mouse ascites and isolated by hybridoma cell screening.Antibodies were analyzed for purity by SDS-PAGE(sodium dodecyl sulphate-polyacrylamide gel-electrophoresis)and affinity was assessed by enzyme-linked immunosorbent assay(ELISA)while subtypes were detected using the commercial kits.The HER2-ECD test strip was prepared based on the sandwich method and evaluated using a portable detection instrument.The affinity of the paired antibodies,4D8 and 8D9,both reached 1×108 L/mol.Both antibodies specifically recognized the HER2-ECD protein in serum.The limit of detection(LOD)of the gold nanoparticle(AuNP)-based LFIA was 1.7 ng/mL with a detection range of 1.7-400 ng/mL,and the performance of the HER2-ECD strip correlated well with that of a Siemens chemiluminescent immunoassay(CLIA)kit.In conclusion,the paired antibodies were successfully prepared with high affinity and specificity.The AuNP-based LFIA of HER2-ECD provides a fast and accurate method to detect the concentration of HER2-ECD in serum samples for clinical use in community medical institutions,and could contribute to determining the progress of the disease or the effectiveness of treatment.

Distance-based lateral flow biosensor for the quantitative detection of bacterial endotoxin

Bacterial endotoxin(a type of lipopolysaccharide,LPS)that acts as the strongest immune stimulant exhibits high toxicity to human health.The golden standard detection methods rely heavily on the use of a large amount of tachypleus amebocyte lysate(TAL)reagents,extracted from the unique blue blood of legally protected horseshoe crabs.Herein,a cost-effective distance-based lateral flow(D-LAF)sensor is demonstrated for the first time based on the coagulation cascade process of TAL induced by endotoxin,which causes the generation of gel-state TAL.The gelation process can increase the amount of trapped water molecules and shorten the lateral flow distance of the remaining free water on the pH paper.The water flow distance is directly correlated to the concentration of endotoxin.Noteworthy,the D-LAF sensor allows the detection of endotoxin with the reduced dosage of TAL reagents than the golden standard detection methods.The detection limit of endotoxin is calculated to be 0.0742 EU/mL.This method can be applied to the detection of endotoxin in real samples such as household water and clinical injection solution with excellent performance comparable to the commercial ELISA kit.

A recombinase polymerase amplification with lateral flow assay for rapid on-the-spot detection of Aeromonas salmonicida

Aeromonas salmonicida is a common pathogen of salmonid fishes that poses a significant threat to the fresh water and marine culture industry,potentially resulting in huge economic losses.To prevent and control fish diseases caused by A.salmonicida,rapid and effective diagnostic approaches must be developed,and which are important for routine monitoring and clinical care.By combining recombinase polymerase amplification(RPA)technology with a visible lateral flow strip(RPA-LF),we have enhanced both the precision of RPA detection and the convenience of real-time monitoring.In this study,we introduce a robust method for detecting A.salmonicida using RPA-LF.This assay specifically targets the ASA_1441 gene of A.salmonicida,ensuring high specificity,without cross-reactivity with other prevalent fresh water or marine pathogens.The optimal amplification temperature of the RPA assay was 39℃.Its sensitivity extends to as low as 100 fg of purified DNA,representing more than 1000-fold higher sensitivity than conventional PCR methods.Furthermore,to enhance the usability of the RPA-LF assay,we developed a rapid sample preparation method using cellulose dipsticks for nucleic acid extraction.This method achieves a limit of detection(LOD)as low as 1.67 CFU/μL and completes the entire process within 20 min.In conclusion,our findings present a rapid and precise tool for monitoring A.salmonicida infection in aquaculture and marine culture.This advancement offers valuable insights for effective disease prevention and control strategies.

Development of a lateral flow immunochromatography assay for the detection of fluoroacetamide in blood samples

Fluoroacetamide(FAM)has been employed as a rodenticide for an extended duration,leading to a multitude of inci-dents involving human ingestion poisoning.Currently,FAMs have been prohibited by nations globally;however,there are still instances of their illegal usage.Conventional instrument methods are characterized by their time-consuming nature and complex operational procedures,rendering them inadequate for meeting urgent diagnostic needs in patients with acute FAM poisoning.Therefore,there is an immediate need to develop a prompt,user-friendly,and precise immunoassay method for the diagnosis of acute poisoning induced by FAM.A lateral flow immunochro-matography assay(LFIA)was developed in this study for the visual detection of FAMs in blood samples,representing the first report of such an approach.The method exhibited a cut-off value of 0.5 mg/mL under the optimized condi-tions,enabling the entire FAM detection process in blood samples to be completed within a mere 8 min without any pretreatment requirements.Notably,the results were easily discernible by visual inspection alone.These results indi-cate that the developed LFIA holds great promise as a convenient and rapid diagnostic tool for FAM poisoning diag-nosis,thereby offering valuable support for subsequent treatment strategies.

One-step polymerized lanthanide-based polystyrene microsphere for sensitive lateral flow immunoassay

Lateral flow immunoassays(LFIAs)provide a powerful tool for rapid real-time assay of cancer biomarkers,which is vital for cancer detection and treatment follow-up.Lanthanide-based LFIAs is one of the most widely used methods,especially Eu(Ⅲ)chelates,which possess distinctive and attractive characteristics,such as time-resolved fluorescence and large Stokes shift.Herein,we adopted a new onestep mini-emulsion polymerization method to synthesize carboxyl-modified fluorescent microsphere(OS-EuCM),which shows good stability,resistance to non-specific adhesion and uniform particle size distribution compared with traditional microspheres synthesized through the swelling method.Benefiting from the above advantages,OS-EuCM was successfully used in LFIAs to detect tumor marker Alpha-fetoprotein with high sensitivity and selectivity in concentration as high as 320 ng/mL,as well as a detection limit of 0.683 ng/mL This lanthanide-based microsphere holds great potential for rapid pointof-care screening and clinical application.