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 pe...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.展开更多
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 desi...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).展开更多
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）...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.展开更多
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 ...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.展开更多
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 t...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.展开更多
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 ongoin...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.展开更多
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 es...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.展开更多
A lateral flow immunoassay(LFA)biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and other common respiratory viruses remains highly desired ...A lateral flow immunoassay(LFA)biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic.Here,we propose a multiplex LFA method for the on-site,rapid,and highly sensitive screening of multiple respiratory viruses,using a multilayered film-likefluorescent tag as the performance enhancement and signal amplification tool.This film-like three-dimensional(3D)tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS-COOH quantum dots(QDs)onto the surfaces of monolayer graphene oxide nanosheets,which can provide larger reaction interfaces and specific active surface areas,higher QD loads,and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications.The constructedfluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2,influenza A virus,and human adenovirus with low detection limits(8 pg/mL,488 copies/mL,and 471 copies/mL),short assay time(15 min),good reproducibility,and high accuracy.Moreover,our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples.展开更多
To reduce the false positive results caused by cross reactivity of the antibodies with other structural analogues,it is crucial to prepare a high specificity and sensitivity antibody against target for developing an a...To reduce the false positive results caused by cross reactivity of the antibodies with other structural analogues,it is crucial to prepare a high specificity and sensitivity antibody against target for developing an accurate immunoassay.In this study,tilmicosin(TM)was selected as a model molecule.Firstly,two-dimensional similarity,electrostatic potential energy,mulliken atomic charges and overlapping of different haptens with TM were calculated using Gaussian 09W and Discovery studio,and the newly designed TM-HS was selected as the optimal hapten.Furthermore,a monoclonal antibody(mAb 12C8)was produced with the half maximal inhibitory concentration(IC50)of 0.36 ng/mL,and negligible cross-reactivity(CR)with other antibiotics.Finally,a lateral flow immunoassay(LFA)for the detection of TM based on amorphous carbon nanoparticles(ACNPs)labeled mAb 12C8 was developed by the reflectance value under natural light.The recoveries of TM ranged from 83.18%to 103.25%with a coefficient of variation(CV)<12.47%.The results showed that the cut-off value of TM in milk samples was 1 ng/mL,and the limits of detection(LODs)for chicken muscle,bovine muscle,porcine muscle and porcine liver samples were 5.23,5.98,6.85 and 7.31μg/kg,respectively.In addition,40 real samples were tested by the LFA,and the detection results were consisted with that of high-performance liquid chromatography-UV detector(HPLC-UV).Those results indicated that the developed LFA is an accurate and useful tool for on-site screening of TM in milk and animal tissues.展开更多
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 ...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.展开更多
Lateral flow immunoassays(LFIAs) have been developed rapidly in recent years and used in a wide range of application at point-of-care-testing(POCT),where small biomolecules can be conveniently examined on a test strip...Lateral flow immunoassays(LFIAs) have been developed rapidly in recent years and used in a wide range of application at point-of-care-testing(POCT),where small biomolecules can be conveniently examined on a test strip.Compared with other biochemical detection methods such as ELISA(enzyme linked immunosorbent assay) or mass spectrometry method,LFIAs have the advantages of low cost,easy operation and short time-consuming.However,it suffers from low sensitivity since conventional LFIA can only realize qualitative detection based on colorimetric signals.With the increasing demand for more accurate and sensitive determination,novel nanomaterials have been used as labels in LFIAs due to their unique advantages in physical and chemical properties.Colloidal gold,fluorescent nano particles,SERSactive nanomaterials,magnetic nanoparticles and carbon nanomaterials are utilized in LFIAs to produce different kinds of signals for quantitative or semi-quantitative detection.This review paper first gives a description of the LFIA principles,and then focuses on the state-of-the-art nanomaterial labelling technology in LFIAs.At last,the conclusion and outlook are given to inspire exploration of more advanced nanomaterials for the development of future LFIAs.展开更多
A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized...A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.展开更多
Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the head...Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced,disease control by fungicide application solely based on the detection of visual symptoms is ineffective.To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control,we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments,MoT-6098 and MoT-6099,that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae(MoO)pathotype.Using polymerase chain reaction(PCR),we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh.To test the efficiency of the two markers,we first established a loop-mediated isothermal amplification(LAMP)method to detect MoT at isothermal conditions,without the use of a PCR machine.Following this,we used the Cas12a protein and guide RNAs(gRNAs)to target the MoT-6098 and MoT-6099 sequences.The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease(ssDNase)activity.We then combined targetdependent Cas12a ssDNase activation with recombinase polymerase amplification(RPA)and nucleic acid lateral flow immunoassay(NALFIA)to develop a method that accurately,sensitively,and cost-effectively detects MoT-specific DNA sequences in infected wheat plants.This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.展开更多
The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome ...The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.展开更多
As a typical triazole fungicide,penconazole(PEN)is widely used in agriculture but has been proven to be toxic.In this study,we designed a new hapten to prepare a highly sensitive and specific anti-PEN monoclonal antib...As a typical triazole fungicide,penconazole(PEN)is widely used in agriculture but has been proven to be toxic.In this study,we designed a new hapten to prepare a highly sensitive and specific anti-PEN monoclonal antibody(mAb)and established a gold nanoparticle-based lateral-flow immunoassay(LFIA)for the detection of PEN residues in watermelon and cucumber.The 50%inhibitory concentration(IC_(50))of the mAb was 0.42 ng/mL and the LFIA strip had a visual limit of detection(vLOD)of 2.5 ng/g and a cut-off value of 10 ng/g in watermelon and cucumbers.The calculated limit of detection(LOD)of the LFIA strip was 0.36 ng/g for watermelon and 0.29 ng/g for cucumber.The LFIA strip also gave a re-covery rate of 92.5%-109.0%for watermelon samples and 92.5%-106.7%for cucumber samples.These results using the LFIA strip are highly consistent with those seen using liquid chromatography-tandem mass spectrometry.Thus our developed LFIA strip represents a potentially reliable tool for the rapid on-site screening for PEN in watermelons and cucumbers.展开更多
The on-going global pandemic of coronavirus disease 2019(COVID-19)caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has been underway for about 11 months.Through November ...The on-going global pandemic of coronavirus disease 2019(COVID-19)caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has been underway for about 11 months.Through November 20,2020,51 detection kits for SARS-CoV-2 nucleic acids(24 kits),antibodies(25 kits),or antigens(2 kits)have been approved by the National Medical Products Administration of China(NMPA).Convenient and reliable SARS-CoV-2 detection assays are urgently needed worldwide for strategic control of the pandemic.In this review,the detection kits approved in China are summarised and the three types of tests,namely nucleic acid,serological and antigen detection,which are available for the detection of COVID-19 are discussed in detail.The development of novel detection kits will lay the foundation for the control and prevention of the COVID-19 pandemic globally.展开更多
A novel lateral flow immunoassay biosensor,combining the receptor-ligand reaction and antigen-antibody reaction was developed for the detection of β-lactam antibiotics in milk. The receptor protein, the carboxy-termi...A novel lateral flow immunoassay biosensor,combining the receptor-ligand reaction and antigen-antibody reaction was developed for the detection of β-lactam antibiotics in milk. The receptor protein, the carboxy-terminal domain of β-lactam sensor-transducer mutant from Bacillus.licheniformis, served as the recognition element. Colloidal gold(CG)-labelled receptor antibody-receptor ternary complex was applied as a signal transducer probe. The presence ofβ-lactams was identified through a color change in the test zone, where the reaction between the complex of CG/antireceptor antibody/receptor and β-lactam antigen on the test line could be inhibited by β-lactam residues in the sample,thus leading to a reduction in color signal. Based on the indirect labeling of the receptor with an antibody as a linker, the ability to detect 33 β-lactams was below or near the corresponding maximum residue limit. The proposed lateral flow immunoassay biosensor appears to be an excellent field-based screening tool for the qualitative screening of β-lactams in milk.展开更多
The outbreak of the novel coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),spread rapidly causing a severe global health burden.The standard COVID-19 diagnosis re...The outbreak of the novel coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),spread rapidly causing a severe global health burden.The standard COVID-19 diagnosis relies heavily on molecular tests to detect viral RNA in patient samples;however,this method is costly,requires highly-equipped laboratories,multiple reagents,skilled laboratory technicians,and takes 3-6 hours to complete.To overcome these limitations,we developed a plant-based production platform for the SARS-CoV-2 receptor-binding domain as an economical source of detection reagents for a lateral-flow immunoassay strip(LFIA)which is suitable for detection of IgM/IgG antibodies in human samples.Further,we validated the plant-produced SARS-CoV-2 receptor-binding domain-based LFIA as a useful diagnostic tool for COVID-19.A total of 51 confirmed COVID-19 serum samples were tested using the LFIA,and the obtained results were consistent with those from polymerase chain reaction assays,while providing sensitivity and specificity of 94.1%and 98%,respectively.The developed LFIA is rapid,scalable,user-friendly,and relatively inexpensive with a simple test procedure,making it useful for the routine monitoring of COVID-19 in clinical settings.This study was approved on March 19,2020 by the Ethics Committee of the Faculty of Medicine,Chulalongkorn University(COA No.354/2020 and IRB No.236/63).展开更多
基金supported by grants from Shanghai Agriculture Applied Technology Development Program,China(Grant No.:2020-02-08-00-08-F01456)the Key Research and Development Program of Zhejiang Province,China(Grant No.:2020C02024-2).
文摘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.
基金financially supported by the Ministry of Education and Science of the Russian Federation in the framework of increase Competitiveness Program of NUST ‘‘MISIS’’, implemented by a governmental decree dated 16th of March 2013, No. 211part of state assignment Organization of scientific researches (Project No. 16.6548.2017/BY)
文摘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).
基金supported by the national science and technology support program in the 12th Five Year Plan(2011BAK10B04 and 2011BAK10B01)the national natural science foundation of China(Grant No.31160323)the research program of the state key laboratory of food science and technology,Nanchang University(SKLF-ZZB-201306)
文摘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.
基金This work was supported by the National Key Research and Development Program of China(2019YFC1606300)the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2017BT01S174)the Guangdong Academy of Sciences Special Project of Implementing Innovation-Driven Development Capacity Building(2018GDASCX-0401).
文摘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.
基金This work was supported by the National Key Research and Development Program of China(2016YFD0500600)the Fund for Guangdong Enterprise Science and Technology Commissioner(GDKTP2020032200).
文摘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.
基金Funding for this study was provided through grants from the National Bio and Agro-Defense Facility(NBAF)Transition Fund from the State of Kansas,and the AMP Core of the Center of Emerging and Zoonotic Infectious Diseases(CEZID)from National Institute of General Medical Sciences(NIGMS)under award number P20GM130448.
文摘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.
基金the National Natural Science Foundation of China(No.22236002)National Key R&D Program(Nos.2023YFF1105003 and 2022YFA1207300).
文摘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.
基金supported by the National Natural Science Foundation of China(Nos.81830101 and 32200076)the National Science and Technology Major Project for Infectious Diseases Control(Nos.2018ZX10712001-010 and 2018ZX10101003-001)+1 种基金the Natural Science Foundation of Anhui Province(No.2208085MB29)The authors would like to thank Prof.Chengfeng Qin from Beijing Institute of Microbiology and Epidemiology for providing inactivated SARS-CoV-2 virions,and thank Ms.Le Zhao of National Center for Nanoscience and Technology for helping to conduct SEM analysis.
文摘A lateral flow immunoassay(LFA)biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic.Here,we propose a multiplex LFA method for the on-site,rapid,and highly sensitive screening of multiple respiratory viruses,using a multilayered film-likefluorescent tag as the performance enhancement and signal amplification tool.This film-like three-dimensional(3D)tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS-COOH quantum dots(QDs)onto the surfaces of monolayer graphene oxide nanosheets,which can provide larger reaction interfaces and specific active surface areas,higher QD loads,and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications.The constructedfluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2,influenza A virus,and human adenovirus with low detection limits(8 pg/mL,488 copies/mL,and 471 copies/mL),short assay time(15 min),good reproducibility,and high accuracy.Moreover,our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples.
基金supported by the Key Scientific and Technological Project of Henan Provincial Department of China(222102310162)the National Natural Science Foundation of China(32172298)+2 种基金the Young Talents Project of Henan Agricultural University(30501305)the Henan Postgraduate Joint Training Base Project(YJS2022JD16)the Program for Innovative Research Team(in Science and Technology),University of Henan Province(NO.23IRTSTHN023).
文摘To reduce the false positive results caused by cross reactivity of the antibodies with other structural analogues,it is crucial to prepare a high specificity and sensitivity antibody against target for developing an accurate immunoassay.In this study,tilmicosin(TM)was selected as a model molecule.Firstly,two-dimensional similarity,electrostatic potential energy,mulliken atomic charges and overlapping of different haptens with TM were calculated using Gaussian 09W and Discovery studio,and the newly designed TM-HS was selected as the optimal hapten.Furthermore,a monoclonal antibody(mAb 12C8)was produced with the half maximal inhibitory concentration(IC50)of 0.36 ng/mL,and negligible cross-reactivity(CR)with other antibiotics.Finally,a lateral flow immunoassay(LFA)for the detection of TM based on amorphous carbon nanoparticles(ACNPs)labeled mAb 12C8 was developed by the reflectance value under natural light.The recoveries of TM ranged from 83.18%to 103.25%with a coefficient of variation(CV)<12.47%.The results showed that the cut-off value of TM in milk samples was 1 ng/mL,and the limits of detection(LODs)for chicken muscle,bovine muscle,porcine muscle and porcine liver samples were 5.23,5.98,6.85 and 7.31μg/kg,respectively.In addition,40 real samples were tested by the LFA,and the detection results were consisted with that of high-performance liquid chromatography-UV detector(HPLC-UV).Those results indicated that the developed LFA is an accurate and useful tool for on-site screening of TM in milk and animal tissues.
基金Project supported by the National Key R&D Program of China(2017YFA0205100)。
文摘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.
基金financial support from the National Natural Science Foundation of China(51802060)Shenzhen Science and Technology Program(Grant No.:KQTD20170809110344233)+1 种基金Shenzhen Bay Laboratory(SZBL2019062801005)Natural Science Foundation of Guangdong Province(No.2019A1515010762)。
文摘Lateral flow immunoassays(LFIAs) have been developed rapidly in recent years and used in a wide range of application at point-of-care-testing(POCT),where small biomolecules can be conveniently examined on a test strip.Compared with other biochemical detection methods such as ELISA(enzyme linked immunosorbent assay) or mass spectrometry method,LFIAs have the advantages of low cost,easy operation and short time-consuming.However,it suffers from low sensitivity since conventional LFIA can only realize qualitative detection based on colorimetric signals.With the increasing demand for more accurate and sensitive determination,novel nanomaterials have been used as labels in LFIAs due to their unique advantages in physical and chemical properties.Colloidal gold,fluorescent nano particles,SERSactive nanomaterials,magnetic nanoparticles and carbon nanomaterials are utilized in LFIAs to produce different kinds of signals for quantitative or semi-quantitative detection.This review paper first gives a description of the LFIA principles,and then focuses on the state-of-the-art nanomaterial labelling technology in LFIAs.At last,the conclusion and outlook are given to inspire exploration of more advanced nanomaterials for the development of future LFIAs.
基金Financial supported by the Gansu ProvincialSci. & Tech. Department (1002NKDA037)
文摘A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.
基金The data that support the findings of this study have being submitted to GenBank and the accession numbers are JAAXMV000000000 and JAAXMU000000000.
文摘Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced,disease control by fungicide application solely based on the detection of visual symptoms is ineffective.To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control,we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments,MoT-6098 and MoT-6099,that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae(MoO)pathotype.Using polymerase chain reaction(PCR),we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh.To test the efficiency of the two markers,we first established a loop-mediated isothermal amplification(LAMP)method to detect MoT at isothermal conditions,without the use of a PCR machine.Following this,we used the Cas12a protein and guide RNAs(gRNAs)to target the MoT-6098 and MoT-6099 sequences.The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease(ssDNase)activity.We then combined targetdependent Cas12a ssDNase activation with recombinase polymerase amplification(RPA)and nucleic acid lateral flow immunoassay(NALFIA)to develop a method that accurately,sensitively,and cost-effectively detects MoT-specific DNA sequences in infected wheat plants.This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.
基金supported by grants from the Applied Basic Research Key Project of Wuhan Municipal Bureau of Science and Technology(2020020601012218)the Fundamental Research Funds for the Central Universities(HUST COVID-19 Rapid Response Call No.2020kfyXGYJ040).
文摘The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.
基金the National Key R&D Program(2019YFC1604604)the Suzhou Science and Technology Committee Program(SS202038)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(K YCX20_1841),China.
文摘As a typical triazole fungicide,penconazole(PEN)is widely used in agriculture but has been proven to be toxic.In this study,we designed a new hapten to prepare a highly sensitive and specific anti-PEN monoclonal antibody(mAb)and established a gold nanoparticle-based lateral-flow immunoassay(LFIA)for the detection of PEN residues in watermelon and cucumber.The 50%inhibitory concentration(IC_(50))of the mAb was 0.42 ng/mL and the LFIA strip had a visual limit of detection(vLOD)of 2.5 ng/g and a cut-off value of 10 ng/g in watermelon and cucumbers.The calculated limit of detection(LOD)of the LFIA strip was 0.36 ng/g for watermelon and 0.29 ng/g for cucumber.The LFIA strip also gave a re-covery rate of 92.5%-109.0%for watermelon samples and 92.5%-106.7%for cucumber samples.These results using the LFIA strip are highly consistent with those seen using liquid chromatography-tandem mass spectrometry.Thus our developed LFIA strip represents a potentially reliable tool for the rapid on-site screening for PEN in watermelons and cucumbers.
基金supported by the National Key Program for Infectious Disease of China(2018ZX10101002)the National Key Research and Development Program of China(2016YFD0500301,2020YFC0840900)。
文摘The on-going global pandemic of coronavirus disease 2019(COVID-19)caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has been underway for about 11 months.Through November 20,2020,51 detection kits for SARS-CoV-2 nucleic acids(24 kits),antibodies(25 kits),or antigens(2 kits)have been approved by the National Medical Products Administration of China(NMPA).Convenient and reliable SARS-CoV-2 detection assays are urgently needed worldwide for strategic control of the pandemic.In this review,the detection kits approved in China are summarised and the three types of tests,namely nucleic acid,serological and antigen detection,which are available for the detection of COVID-19 are discussed in detail.The development of novel detection kits will lay the foundation for the control and prevention of the COVID-19 pandemic globally.
基金financially supported by the National Key R&D Program of China (2019YFC1606603)。
文摘A novel lateral flow immunoassay biosensor,combining the receptor-ligand reaction and antigen-antibody reaction was developed for the detection of β-lactam antibiotics in milk. The receptor protein, the carboxy-terminal domain of β-lactam sensor-transducer mutant from Bacillus.licheniformis, served as the recognition element. Colloidal gold(CG)-labelled receptor antibody-receptor ternary complex was applied as a signal transducer probe. The presence ofβ-lactams was identified through a color change in the test zone, where the reaction between the complex of CG/antireceptor antibody/receptor and β-lactam antigen on the test line could be inhibited by β-lactam residues in the sample,thus leading to a reduction in color signal. Based on the indirect labeling of the receptor with an antibody as a linker, the ability to detect 33 β-lactams was below or near the corresponding maximum residue limit. The proposed lateral flow immunoassay biosensor appears to be an excellent field-based screening tool for the qualitative screening of β-lactams in milk.
基金This study was funded by Baiya Phytopharm Co.,Ltd.
文摘The outbreak of the novel coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),spread rapidly causing a severe global health burden.The standard COVID-19 diagnosis relies heavily on molecular tests to detect viral RNA in patient samples;however,this method is costly,requires highly-equipped laboratories,multiple reagents,skilled laboratory technicians,and takes 3-6 hours to complete.To overcome these limitations,we developed a plant-based production platform for the SARS-CoV-2 receptor-binding domain as an economical source of detection reagents for a lateral-flow immunoassay strip(LFIA)which is suitable for detection of IgM/IgG antibodies in human samples.Further,we validated the plant-produced SARS-CoV-2 receptor-binding domain-based LFIA as a useful diagnostic tool for COVID-19.A total of 51 confirmed COVID-19 serum samples were tested using the LFIA,and the obtained results were consistent with those from polymerase chain reaction assays,while providing sensitivity and specificity of 94.1%and 98%,respectively.The developed LFIA is rapid,scalable,user-friendly,and relatively inexpensive with a simple test procedure,making it useful for the routine monitoring of COVID-19 in clinical settings.This study was approved on March 19,2020 by the Ethics Committee of the Faculty of Medicine,Chulalongkorn University(COA No.354/2020 and IRB No.236/63).
