Lateral Flow Immunoassay System Research Articles

One-Component Dual-Readout Aggregation-Induced Emission Nanobeads for Qualitative and Quantitative Detection of C-Reactive Protein at the Point of Care.

Fluorescent lateral flow immunoassay (LFA) systems are versatile tools for sensitive and quantitative detection of disease markers at the point of care. However, traditional fluorescent nanoparticle-based lateral flow immunoassays are not visible under room light, necessitate an additional fluorescent reader, and lack flexibility for different application scenarios. Herein, we report a dual-readout LFA system for the rapid and sensitive detection of C-reactive protein (CRP) in clinical samples. The system relied on the aggregation-induced emission nanobeads (AIENBs) encapsulated with red AIE luminogen, which possesses both highly fluorescent and colorimetric properties. The AIENB-based LFA in the naked-eye mode was able to qualitatively detect CRP levels as low as 8.0 mg/L, while in the fluorescent mode, it was able to quantitatively measure high-sensitivity CRP (hs-CRP) with a limit of detection of 0.16 mg/L. The AIENB-based LFA system also showed a good correlation with the clinically used immunoturbidimetric method for CRP and hs-CRP detection in human plasma. This dual-modal AIENB-based LFA system offers the convenience of colorimetric testing and highly sensitive and quantitative detection of disease biomarkers and medical diagnostics in various scenarios.

Dual-color MoS2@QD nanosheets mediated dual-mode lateral flow immunoassay for flexible and ultrasensitive detection of multiple drug residues

The sensitive on-site monitoring of multiple agricultural drugs in complex real media is critical to human health but remains a challenge. In this paper, we reported the design of film-like dual-mode nanolabel (MTQD), using molybdenum disulfide (MoS2) nanosheet-loaded numerous quantum dots (QDs) to generated co-enhanced colorimetric-fluorescence signals, and demonstrated its powerful capabilities in competitive lateral flow immunoassay (LFA) for flexible, ultrasensitive, and multiplex detection of four kinds of common agricultural drugs. Three layers of red and green QDs were continuously coated onto the surface of MoS2 to suppress the inner filter effect of two-dimensional nanomaterials effectively and greatly improve the specificity of the LFA system for multitarget analysis. The dual-mode LFA combined with a low-cost dual-fluorescence reading instrument can flexibly and simultaneously monitor two pesticides (clothianidin, carbendazim) and two veterinary drugs (kanamycin, chloramphenicol) in complex food and experimental samples with low detection limits of 0.1 ng/mL level in the observation of colorimetric signals and showed at least a 46-fold improvement in sensitivity in the sensing of fluorescence signals. This colorimetric dual-fluorescence platform greatly broadens the application range of existing LFA techniques without increasing testing time, thus showing great application prospects for the point-of-care testing of small-molecule pollutants in different situations.

Plasmonic Approach to Fluorescence Enhancement of Mesoporous Silica-Coated Gold Nanorods for Highly Sensitive Influenza A Virus Detection Using Lateral Flow Immunosensor.

Rapid diagnostic tests based on the lateral flow immunoassay (LFI) enable early identification of viral infection, owing to simple interpretation, short turnaround time, and timely isolation of patients to minimize viral transmission among communities. However, the LFI system requires improvement in the detection sensitivity to match the accuracy of nucleic acid amplification tests. Fluorescence-based LFIs are more sensitive and specific than absorption-based LFIs, but their performance is significantly affected by fundamental issues related to the quantum yield and photobleaching of fluorophores. Metal-enhanced fluorescence (MEF), which is a plasmonic effect in the vicinity of metallic nanoparticles, can be an effective strategy to improve the detection sensitivity of fluorescence-based LFIs. The key factors for obtaining a strong plasmonic effect include the distance and spectral overlap of the metal and fluorophore in the MEF system. In this study, MEF probes were designed based on core-shell nanostructures employing a gold nanorod core, mesoporous silica shell, and cyanine 5 fluorophore. To optimize the efficiency of MEF probes incorporated on the LFI platform (MEF-LFI), we experimentally and theoretically investigated the distance dependence of plasmonic coupling between cyanine 5 and gold nanorods by adjusting the shell thickness, resulting in significant fluorescence enhancement. The proposed MEF-LFI enabled highly sensitive detection of influenza A virus (IAV) nucleocapsid protein with a detection limit of 0.52 pg mL-1 within 20 min and showed high specificity and accuracy for determining IAV clinical samples. Overall, our findings demonstrate the potential of this method as an effective tool for molecular diagnosis under emergency conditions.

Rapid and sensitive detection of E. coli O157:H7 by lateral flow immunoassay and silver enhancement

The aim of this study was to develop a sensitive lateral flow immunoassay (LFIA) for the rapid detection of Escherichia coli (E. coli) O157:H7, a pathogen contributor to diseases and fatalities worldwide. Au nanoparticles with high stability, uniform size, and shape were synthesized and coated with heterobifunctional PEG polymer with carboxyl groups, and they were bioconjugated to be used as label in sandwich-LFIA. Then, a silver enhancement strategy was developed as an accessible, rapid, and cost-effective approach for signal amplification to reduce the limit of detection (LOD). The optimal results were achieved when a solution of silver nitrate and hydroquinone/citrate buffer was added to the strips for 4 min. This led to a decrease in the visual LOD from 2 × 106 (CFU mL−1) to 2 × 103 (CFU mL−1), resulting in a threefold improvement in sensitivity compared to the conventional LFIA system. The specificity of the system was evaluated by using non-target bacteria (E. coli BL21 and E. coli T515) and its reliability was determined by testing commercial food samples (milk, tap water, and orange juice), demonstrating its effectiveness for quickly detecting pathogenic bacteria in food products.Graphical

How Exactly Do AIEgens Target Bacteria? Leveraging the Targeting Mechanism to Design Sensitive Fluorescent Immunosensors.

Aggregation-induced emission luminogens (AIEgens) are promising candidates for bacterial imaging and detection because they can "Light-Up" pathogenic bacteria without complicated labeling or washing steps. However, there have been few in-depth analyses of the intrinsic mechanism underlying their utility as fluorescence probes for targeting bacteria. Therefore, using large-scale molecular dynamics simulations, we investigated the mechanism of their bacterial "Light-Up" behavior with N,N-diphenyl-4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl) aniline functionalized with 1-bromoethane (TBP-1). We propose that the triphenylamine motif of TBP-1, rather than the positively charged pyridine group, first contacts the cell membrane. After TBP-1 completely inserts into the cell membrane, the hydrophobic triphenylamine motif localizes in the hydrophobic core of the cell membrane, restricting the molecular variation of TBP-1, which induces the fluorescent "turn-on" and bacterial "Light-Up." On this basis, we established a heterogeneous lateral flow immunoassay (LFIA) for the detection of foodborne pathogens. The LFIA system showed improved sensitivity with a limit of detection as low as 103 CFU mL-1 and strong specificity. Our protocol opened an effective shortcut to the design of more efficient AIEgens and novel AIEgens-based immunoassays.

Development of a High Sensitive Multiplex Lateral Flow Immunoassay (LFIA) System for Rapid Detection of Methicillin-Resistant Staphylococcus Aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worldwide concern as an epidemic bacterium and a cause of nosocomial and community-acquired infections. One of the major problems in the prevention and treatment of infections caused by MRSA strains is their multi-drug resistant trait, which causes the spread of infections and increases the mortality rate. Therefore, a rapid and accurate method is needed to identify MRSA strains, initiate appropriate antibiotic therapy, and control its infection. The aim of this study was to develop a twin lateral flow immunoassay system to detect methicillin-resistant Staphylococcus aureus (MRSA). First, BSA blocked AuNPs-anti-peptidoglycan antibody and AuNPs-anti-BSA antibody were used to detect Staphylococcus aureus (S. aureus). Then, AuNPs-anti-PBP2a antibody was used to specifically detect MRSA. Sensitivity, specificity and limit of detection of this twin immunoassay system were assessed using MRSA, methicillin susceptible S. aureus and clinical samples. Results were compared to those of cefoxitin disc diffusion (FOX30) and Polymerase Chain Reaction (PCR) as gold standards. The Limit of Detection (LOD) of this twin system were 103 and 104 CFU/ml for the first and second strips, respectively. Sensitivity and specificity of this innovative assay in detecting MRSA were 92.30 and 97.36%, compared to FOX30 and PCR, respectively. High rates of sensitivity and specificity of this initiative system show its high potentials for rapid and accurate detection of MRSA.

Introduction of multilayered magnetic core–dual shell SERS tags into lateral flow immunoassay: A highly stable and sensitive method for the simultaneous detection of multiple veterinary drugs in complex samples

Direct, convenient, and sensitive monitoring of the residues of multiple drugs in complex environments is important but remains a challenge. Here, we report a surface-enhanced Raman scattering (SERS)-based multiplexed lateral flow immunoassay (LFA) that supports the simultaneous and sensitive detection of commonly used drugs kanamycin, ractopamine, clenbuterol, and chloramphenicol in unprocessed complex samples through the dual signal amplification strategy of numerous efficient hotspots and magnetic enrichment. Multilayered magnetic-core dual-shell nanoparticles (MDAu@Ag) with controllable subtle nanogaps were fabricated via the polyethyleneimine-mediated layer-by-layer (LBL) assembly of two layers of Au@Ag satellites onto superparamagnetic Fe3O4 cores and conjugated with specific antibodies as multifunctional tags in the LFA system for rapid capture, separation, and quantitative analysis. Two Raman reporters were embedded in internal nanogaps and modified on the surface of MDAu@Ag for the simultaneous and ultrasensitive detection of four targets on two test lines, which greatly simplified the fabrication and signal reading of SERS-LFA. The proposed assay can rapidly detect multiple drug residues in 35 min with detection limits down to pg/mL level. Moreover, the MDAu@Ag–based SERS-LFA demonstrated better stability, higher throughput, and superior sensitivity (at least 400 times) than traditional colloidal gold immunochromatography, showing its great potential in the field of point-of-care testing.

Facile, Rapid, and Low-Cost Detection for Influenza Viruses and Respiratory Syncytial Virus Based on a Catalytic DNA Assembly Circuit

Influenza viruses and respiratory syncytial virus (RSV) have contributed to severe respiratory infections, causing huge economic and healthcare burdens. To achieve rapid and precise detection of influenza viruses and RSV, we proposed a catalytic hairpin assembly (CHA) combined with the lateral flow immunoassay (CHA–LFIA) detection method. The presence of the target RNA triggers the initiation of CHA circuits. H1/H2 complexes, the amplified signal products, which were labeled with digoxin and biotin, were detected with a highly sensitive lateral flow immunoassay system. The sensitivity of the CHA–LFIA system to influenza A and B viruses and RSV reached up to 1, 1, and 5 pM, respectively. In addition, this method exhibited excellent capability for differentiating between target RNA and base-mismatched RNA. The results demonstrated that an enzyme-free, rapid, highly sensitive, and specific method had been developed to detect influenza A and B viruses and RSV.

Lateral Flow Immunoassay with Quantum-Dot-Embedded Silica Nanoparticles for Prostate-Specific Antigen Detection.

Prostate cancer can be detected early by testing the presence of prostate-specific antigen (PSA) in the blood. Lateral flow immunoassay (LFIA) has been used because it is cost effective and easy to use and also has a rapid sample-to-answer process. Quantum dots (QDs) with very bright fluorescence have been previously used to improve the detection sensitivity of LFIAs. In the current study, a highly sensitive LFIA kit was devised using QD-embedded silica nanoparticles. In the present study, only a smartphone and a computer software program, ImageJ, were used, because the developed system had high sensitivity by using very bright nanoprobes. The limit of PSA detection of the developed LFIA system was 0.138 ng/mL. The area under the curve of this system was calculated as 0.852. The system did not show any false-negative result when 47 human serum samples were analyzed; it only detected PSA and did not detect alpha-fetoprotein and newborn calf serum in the samples. Additionally, fluorescence was maintained on the strip for 10 d after the test. With its high sensitivity and convenience, the devised LFIA kit can be used for the diagnosis of prostate cancer.

Ultrasensitive and Simultaneous Detection of Two Specific SARS-CoV-2 Antigens in Human Specimens Using Direct/Enrichment Dual-Mode Fluorescence Lateral Flow Immunoassay.

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the “hook effect.” The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.

Anti-SARS-CoV-2 IgG and IgM detection with a GMR based LFIA system

Since December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused millions of deaths and seriously threatened the safety of human life; indeed, this situation is worsening and many people are infected with the new coronavirus every day. Therefore, it is very important to understand patients’ degree of infection and infection history through antibody testing. Such information is useful also for the government and hospitals to formulate reasonable prevention policies and treatment plans. In this paper, we develop a lateral flow immunoassay (LFIA) method based on superparamagnetic nanoparticles (SMNPs) and a giant magnetoresistance (GMR) sensing system for the simultaneously quantitative detection of anti-SARS-CoV-2 immunoglobulin M (IgM) and G (IgG). A simple and time-effective co-precipitation method was utilized to prepare the SMNPs, which have good dispersibility and magnetic property, with an average diameter of 68 nm. The Internet of Medical Things-supported GMR could transmit medical data to a smartphone through the Bluetooth protocol, making patient information available for medical staff. The proposed GMR system, based on SMNP-supported LFIA, has an outstanding advantage in cost-effectiveness and time-efficiency, and is easy to operate. We believe that the suggested GMR based LFIA system will be very useful for medical staff to analyze and to preserve as a record of infection in COVID-19 patients.

Self-Assembling Allochroic Nanocatalyst for Improving Nanozyme-Based Immunochromatographic Assays

Paper-based rapid diagnostic tests, such as immunochromatographic assays, namely lateral flow immunoassay (LFA), are valuable alternatives for biomarker detection compared to traditional laboratory-based tests, but these assays need further refinement to consolidate their biosensing capabilities. Nanozyme integration into LFA systems may provide a reliable means of improving the analytic sensitivity of LFA tests. Due to the involvement of multiple liquid-handling steps, the quantitative accuracy is compromised, hence hindering the use of untrained personnel point-of-care use. Self-assembling allochroic nanocatalyst (SAN) assemblies satisfy these LFA quality measures by optimizing analyte-antibody reporting performance and by intrinsically catalyzing chromogen activation, thereby reducing the number of liquid handling steps involved during sample analysis. In SANs, the hydrophobic chromogens serve as peroxidase substrates that self-assemble into nanoparticles at high loading fractions. These features demonstrate the potential for SAN-LFAs to be a valuable patient point-of-care (POC) test. Herein, we describe the SAN fabrication process and employ SAN-LFAs to detect cardiac troponin I-troponin C (cTnI-TnC) and myoglobin (Myo) levels present in plasma samples. Using SAN-LFAs, the limits of detection for cTnI-TnC and Myo were 0.012 ng/mL and 0.2 ng/mL respectively. We also demonstrate SAN compatibility with blood samples and stability under long-term storage conditions. The successful utlization of SANs in LFA-based biomarker detection may inspire these nanocatalysts to be integrated into similar immunochromatographic testing methods.

Rapid, Quantitative, High-Sensitive Detection of Escherichia coli O157:H7 by Gold-Shell Silica-Core Nanospheres-Based Surface-Enhanced Raman Scattering Lateral Flow Immunoassay.

Escherichia coli O157:H7 is regarded as one of the most harmful pathogenic microorganisms related to foodborne diseases. This paper proposes a rapid-detection biosensor for the sensitive and quantitative analysis of E. coli O157:H7 in biological samples by surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFIA). A novel gold-shell silica-core (SiO2/Au) nanosphere (NP) with monodispersity, good stability, and excellent SERS activity was utilized to prepare high-performance tags for the SERS-based LFIA system. The SiO2/Au SERS tags, which were modified with two layers of Raman reporter molecules and monoclonal antibodies, effectively bind with E. coli O157:H7 and form sandwich immune complexes on the test lines. E. coli O157:H7 was quantitatively detected easily by detecting the Raman intensity of the test lines. Under optimal conditions, the limit of detection (LOD) of the SiO2/Au-based SERS-LIFA strips for the target bacteria was 50 cells/mL in PBS solution, indicating these strips are 2,000 times more sensitive than colloidal Au-based LFIA strips. Moreover, the proposed assay demonstrated high applicability in E. coli O157:H7 detection in biological samples, including tap water, milk, human urine, lettuce extract and beef, with a low LOD of 100 cells/mL. Results indicate that the proposed SERS-based LFIA strip is applicable for the sensitive and quantitative determination of E. coli O157:H7.

Quantitative and simultaneous detection of two inflammation biomarkers via a fluorescent lateral flow immunoassay using dual-color SiO2@QD nanotags.

Anon-site detection strategy is reportedbased on dual-color SiO2@quantum dot (QD)-integrated lateral flow immunoassay (LFA) strip to realize the quantitative and simultaneous detection of C-reactive protein (CRP) and procalcitonin (PCT) in serum. The dual-color SiO2@QD nanotags with monodispersity and excellent luminescence were synthesized using polyethyleneimine-mediated electrostatic adsorption of dense red CdSe/ZnS-COOH (excitation/emission 365/625nm) or green CdSe/ZnS-COOH (excitation/emission 365/525nm) QDs on the surface of 180nm SiO2 spheres and were conjugated with anti-PCT and anti-CRP monoclonal antibodies, as stable and fluorescent-enhanced QD nanotags in the LFA system. The use of SiO2@QDs with two different fluorescent signals causedthe sensitivity and specificity of themultiplex LFA system. As a result, the proposed assay provided a wide logarithmic determinationrange with a CRP quantitative range of 0.5-103ng/mL and PCT quantitative range of 0.05-103ng/mL. The limits of detection (LODs) of CRP and PCT reached 0.5 and 0.05ng/mL, respectively. The SiO2@QD-based LFA showed great potential as rapid detection tool for the simultaneous monitoring of CRP and PCT in serum sample.

A Lateral Flow Immunoassay for Prostate‐Specific Antigen Detection Using Silica‐Coated CdSe@ZnS Quantum Dots

Protein detection technologies are important for early disease diagnosis because of their advantages over biopsy testing. Considering the diagnostic potential of prostate‐specific antigen (PSA) in prostate cancer, in this study, we present a lateral flow immunoassay (LFIA) for successful detection of PSA by using silica‐coated CdSe@ZnS quantum dots (QD@SiO2). The QD@SiO2 were synthesized using a reverse microemulsion method. The average size of the synthesized nanoparticles (NPs) was 51.76 nm, and their quantum yield (QY) was relatively high (36.5%). PSA detection was performed using LFIA on a strip. The QD@SiO2 surface was modified by conjugating the anti‐PSA antibody to detect the PSA. At concentrations above 1.0754 ng/mL, PSA was detected using the designed LFIA system without any nonspecific binding. The proposed QD@SiO2 has other bioapplications as well.

Instrumentation-Free Semiquantitative Immunoanalysis Using a Specially Patterned Lateral Flow Assay Device.

In traditional colorimetric lateral flow immunoassay (LFI) using gold nanoparticles (AuNPs) as a probe, additional optical transducers are required to quantify the signal intensity of the test line because it presents as a single red-colored line. In order to eliminate external equipment, the LFI signal should be quantifiable by the naked eye without the involvement of optical instruments. Given this objective, the single line test zone of conventional LFI was converted to several spots that formed herringbone patterns. When the sandwich immunoassay was performed on a newly developed semi-quantitative (SQ)-LFI system using AuNPs as an optical probe, the spots were colorized and the number of colored spots increased proportionally with the analyte concentration. By counting the number of colored spots, the analyte concentration can be easily estimated with the naked eye. To demonstrate the applicability of the SQ-LFI system in practical immunoanalysis, microalbumin, which is a diagnostic marker for renal failure, was analyzed using microalbumin-spiked artificial urine samples. Using the SQ-LFI system, the calibration results for artificial urine-based microalbumin were studied, ranging from 0 to 500 μg/mL, covering the required clinical detection range, and the limit of detection (LOD) value was calculated to be 15.5 μg/mL. Thus, the SQ-LFI system provides an avenue for the realization of an efficient quantification diagnostic device in resource-limited conditions.

Time-resolved fluorescence resonance energy transfer-based lateral flow immunoassay using a raspberry-type europium particle and a single membrane for the detection of cardiac troponin I

Herein, we report a novel lateral flow immunoassay (LFIA) system for detecting cardiac troponin I (cTnI) in serum using the time-resolved fluorescence resonance energy transfer (TR-FRET) technique and the fusion 5 membrane. The fusion 5 membrane is used as a strip for LFIA, and it is constructed without additional matrices (such as a sample or conjugation pad). Although this strategy for constructing the LFIA strip is quite simple and cost-effective, LFIA is still not suitable for the analysis of biomarkers that require high sensitivity, such as cTnI. Therefore, the highly sensitive TR-FRET technique is integrated with a fusion 5 membrane-based LFIA strip. To accomplish this, a microparticle covered with europium chelate-contained silica nanoparticles is synthesized as a raspberry-type particle and used as a fluorescence donor. A gold nanorod (GNR) is used as a fluorescence acceptor particle. In the TR-FRET-based LFIA system, the competitive immunoassay should be performed to satisfy the condition required for the FRET phenomenon to occur. Therefore, the fluorescence signal is proportional to the cTnI concentration, ensuring a quantitative analysis of cTnI can be accomplished by measuring the fluorescence signal between the raspberry-type europium particles and GNR. Using the developed TR-FRET-based LFIA system, sensitive detection of cTnI is successfully achieved with a limit of detection of 97 pg/mL in human serum. Moreover, because the result can be obtained using one matrix (the fusion 5 membrane), the developed LFIA system can be employed in cTnI diagnosis with a simple manufacturing process.

A lateral flow-based portable platform for determination of reproductive status of cattle

Our objective was to develop and validate a tool integrating a disposable fluorescence-based lateral flow immunoassay (LFIA) coupled with a portable imaging device for estimating circulating plasma concentrations of progesterone (P4). First, we developed and optimized a competitive LFIA test strip to measure P4 in bovine plasma. The LFIA design included a sample pad, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test and control lines, and a cellulose-fiber absorbent pad. To perform a test, 20 µL of plasma and 50 µL of running buffer were added on the sample pad. After 3 min, 45 µL of running buffer was added to initiate sample flow. After allowing 15 min to stabilize the colorimetric signal, strips were introduced in an LFIA portable reader wirelessly linked to a laptop to determine P4 concentration based on test-to-control-line signal (T/C ratio). In a series of experiments (n = 6), the ability of the LFIA to differentiate plasma samples with ≥1 or <1 ng/mL of P4 was evaluated. For each experiment, a calibration curve was constructed using plasma with known concentrations of P4 (0.1 to 3.7 ng/mL; n = 5). The resulting linear equation was then used to determine a T/C ratio cutoff to differentiate samples with ≥1 or <1 ng/mL of P4. In addition, to evaluate the ability of the platform to assign samples to P4 concentration groups without a calibration curve for individual batches, we performed a receiver operating characteristic analysis to identify a single cutoff value for T/C ratio that could potentially be used for all batches. Overall, calibration curves showed a linear relationship between T/C ratio and P4 levels (mean coefficient of determination = 0.74; range 0.42 to 0.99). Next, plasma samples from lactating dairy cows (n = 58) were tested in triplicate to determine the ability of the LFIA system to differentiate plasma samples with ≥1 or <1 ng/mL of P4 using a RIA for P4 as reference test. Overall, the LFIA assay correctly classified 90% of the samples, with 97% sensitivity, 83% specificity, 85% positive predictive value, and 96% negative predictive value. Agreement between the tests was substantial (kappa = 0.79; 95% confidence interval 0.64 to 0.95). When using a single cutoff value for T/C ratio selected by receiver operating characteristic analysis, sensitivity and specificity to determine CL presence were 97 (95% confidence interval 82 to 99) and 79% (95% confidence interval 60 to 92), respectively. These data suggest that the developed portable LFIA system can accurately differentiate plasma samples with ≥1 or <1 ng/mL of P4.

Ovalbumin antibody-based fluorometric immunochromatographic lateral flow assay using CdSe/ZnS quantum dot beads as label for determination ofT-2 toxin.

This work describes an anti-ovalbumin antibody-based lateral flow immunoassay (LFI) for T-2 toxin. The antibody uses acoating antigen as a bifunctional element for universality and introduces preincubation to improve the detection limits of the method. T-2 toxin and ovalbumin-modified T-2 toxin competitively binds on the anti-T-2 toxin monoclonal antibody modified on CdSe/ZnS quantum dot beads during preincubation. The modified T-2 toxin acts as a bifunctional element that forms immuno complexes during preincubation and combines with anti-ovalbumin antibody coated in the test line through the ovalbumin terminal. Fluorescence is detected at 610nm on the test zone following photoexcitation at 365nm. It has a reverse dose-effect relationship with the amount of T-2 toxin. The calibration plot is linear inthe 20-110fgmL-1T-2 toxin concentration range, and the limit of detection (LOD) is 10fgmL-1, which is lower by 8-fold than that of the traditional LFI system (LOD 80fgmL-1) and one order of magnitude than those of LFIs with labels of colloidal gold nanoparticles (LOD 150fgmL-1) or fluorophores (LOD 190ngmL-1). Universality was verified through aflatoxin B1 detection using the established ovalbumin antibody-based LFI system (LOD 10fgmL-1). The performance of the method was compared with that of established systems and a commercial ELISA kit (LOD 360fgmL-1). Graphical abstractSchematic representation of ovalbumin antibody-based immunochromatographic lateral flow assay for T-2 toxin. Preincubation is introduced for high sensitivity. T-2- anti-ovalbumin acts as a bi-functional element for universality. CdSe/ZnS quantum dot beads act as label. Fluorometric signal is detected at 610nm.

Enhanced Immunoadsorption on Imprinted Polymeric Microstructures with Nanoengineered Surface Topography for Lateral Flow Immunoassay Systems.

Lateral flow immunoassay devices have revolutionized the style of on-site disease detection and point-of-care testing in the past few decades. The surface nanotopography of a solid substrate is a dominant parameter in the efficiency of antibody immobilization, but precise control over surface roughness has not been fully investigated. Here we presented lateral flow immunoassay platforms with nanometer-scale surface roughness, reproducibly engineered using thermal nanoimprinting lithography, and investigated the effects of surface nanotopography on immunoadsorption and immunoassay performance. We fabricated three types of imprinted polycarbonate sheets with microcone array structures having different degrees of surface roughness using three types of molds fabricated by micromachining or laser ablation. The structures fabricated by laser-ablated nickel mold exhibited numerous bumps measuring several tens of nanometers, which enhanced antibody adsorption. We performed sandwich immunoassays of C-reactive protein in serum samples and achieved highly sensitive detection with a detection limit of ∼0.01 μg mL-1 and a broad dynamic range. The present results provide useful information on the remarkable effect of nanoengineered surfaces on biomolecule adsorption, and the platforms presented here will widen the applicability and versatility of lateral flow immunoassay devices.