Conventional Lateral Flow Immunoassay Research Articles

One-step copper deposition-induced signal amplification for multiplex bacterial infection diagnosis on a lateral flow immunoassay device

The lateral flow immunoassay (LFIA) is predominant in rapid diagnostic tests owing to its cost-effectiveness and operational simplicity. However, the conventional LFIA exhibits limited sensitivity and is susceptible to human variance for the result readout, impacting result interpretation. In this study, we introduced a novel one-step copper deposition-induced signal amplification lateral flow immunoassay (osa-LFIA) that markedly enhances the detection sensitivity for Staphylococcus aureus (protein A) and Pseudomonas aeruginosa (exotoxin A). Utilizing gold nanoparticles (AuNPs) as a catalyst, this approach employs ascorbic acid to reduce Cu2+ to Cu0, depositing on AuNPs at the test line and amplifying the signal. A user-friendly design features a three-dimensional paper structure incorporating pre-dried reagents, enabling a streamlined, efficient testing process. The osa-LFIA significantly lowers detection limits to 3 ng mL−1 for protein A and 10 ng mL−1 for exotoxin A, offering a tenfold improvement over conventional LFIA. Additionally, we developed a portable grayscale detection device, achieving less than 10% error in quantitative analysis compared to the data acquired and analyzed in the lab. This entire process, from detection to signal amplification, is completed in just 20 min. For the clinical trial, we utilized the osa-LFIA to test synovial fluid samples infected with Staphylococcus aureus. We also successfully detected different concentrations of the exotoxin A in parallel, with a recovery value of 96%–110%. Our findings demonstrate the osa-LFIA's potential as a rapid, highly sensitive, and simple-to-use diagnostic tool for detecting various pathogens, significantly advancing the field of rapid diagnostic testing.

Smartphone-based lateral flow immunoassay for sensitive determination of lactate dehydrogenase at the point of care

Lactate dehydrogenase (LDH), a prevalent enzyme involved in anaerobic glycolysis, is released into body fluids following cell damage and has long been a general marker of tissue injury. However, due to its lack of selectivity and the advent of more accurate biomarkers, the clinical utility of LDH has been largely limited to confirming hemolysis. LDH has been recognized as a valuable prognostic biomarker for various cancers, making its monitoring crucial during cancer management. Traditional LDH methods include spectrophotometric analysis of NADH at 340 nm, native electrophoresis, or enzyme-linked immunosorbent assay. This study presents the first lateral flow immunoassay (LFIA) for the smartphone-based quantification of serum LDH levels at the point of care. Highly-affinity and specific antibodies have been produced, with 5 nM equilibrium dissociation constant and no cross-reactivity with human serum albumin and human immunoglobulin G. Utilizing carbon nanoparticles as signal transducers significantly enhanced the quantification limit 55-fold, compared to the conventional gold nanoparticles-based LFIA, achieving a quantification limit of 1.5 ng mL−1. The developed assay demonstrated a mean recovery rate of 115 ± 21 % when evaluating LDH-spiked serum samples. This method can be an interesting home-testing tool for monitoring cancer progression or therapy effectiveness.

A highly sensitive dual-mode lateral flow immunoassay based on plasmonic hollow Ag/Au nanostars enhancing light absorption

The conventional lateral flow immunoassay (LFIA) based on gold nanoparticles (Au NPs) is limited by low sensitivity due to the insufficient brightness of Au NPs. To address this problem, noble metal nanomaterials with localized surface plasmon resonance (LSPR) and synthetic tunability are potential signal outputs for LFIA, which can achieve better optical properties by adjusting the preparation conditions. Herein, this study prepared the hollow silver/gold nano-stars (HAg/Au NSts) as LFIA signal output via the galvanic replacement method. HAg/Au NSts with anisotropic hollow alloy nanostructures exhibit a wide visible light absorption band and great NIR thermal conversion efficiency (η = 37.32 %), which endows them with enhanced colorimetric and photothermal signals. Further, we constructed a colorimetric-photothermal (CM-PT) dual-signal HAg/Au NSts-LFIA and chose staphylococcal enterotoxin B as the target analyte. The linear range of HAg/Au NSts-LFIA is 0.19–100 ng mL−1, and the limit of detection (LOD) is up to 0.29 ng mL−1 and 0.09 ng mL−1 in the colorimetric and photothermal modes respectively. Compared with the conventional Au NPs-LFIA, HAg/Au NSts-CM/PT-LFIA effectively improved the detection performance of LFIA. In addition, HAg/Au NSts-LFIA also showed satisfactory sensitivity (vLOD = 0.78 ng mL−1) and recovery (89.06–114.74 %) in milk and pork samples. Therefore, this work provides a new shape design idea for noble metal nanomaterials in biosensor applications.

"Three-in-One": Ultrasensitive Lateral Flow Immunoassay Driven by Magnetic Enrichment and Photothermal Signal Amplification.

Conventional lateral flow immunoassay (LFIA) usually suffers from poor antimatrix interference, unsatisfactory sensitivity, and lack of quantitative ability for target analyte detection in food matrices. In response to these limits, here, multifunctional nanomaterial ZnFe2O4 nanoparticles (ZFOs) were developed and integrated into LFIA for powerful magnetic separation/enrichment and colorimetric/photothermal target sensing. Under optimum conditions, the detection for clenbuterol (CL) with magnetic enrichment achieves 9-fold higher sensitivity compared to that without enrichment and 162-fold higher sensitivity compared to that based on traditional colloidal golds. Attributing the improved performances of ZFOs, CL can be detected at ultralow levels in pork and milk with 10 min of immunoreaction time. The vLODs were 0.01 μg kg-1 for two modes, and the cutoff values of CL were about 5 and 3 μg kg-1, respectively. More importantly, the enrichment ZFO-mediated LFIA (ZE-LFIA) exhibits a similar limit of detection (LOD) in both buffer solution and food matrix, demonstrating a universal resistance to the food matrix. The multitudinous performance merits of this ZE-LFIA with high sensitivity, matrix tolerance, accuracy, and specificity have ensured a broad application potential for target detection of clenbuterol and can serve as an experience for other veterinary drug residues' detection.

An ultrasensitive nanozyme-based immunochromatographic platform for quantitative detection of NT-proBNP using V2(Sn0.8Pt0.2)C MAX phase

terminal pro-brain natriuretic peptide (NT-proBNP) has been demonstrated to be as a sensitive and specific biomarker for the heart failure (HF) management, while the development of timely and accurate point-of-care testing methods for frequent quantitative monitoring of NT-proBNP remains challenging. Nanozymes are gaining increasing attention as an alternative to natural enzymes for colorimetric lateral flow immunoassay (LFIA). However, the development of nanozymes with high catalytic activity and colorimetric signal amplification still poses challenges. Herein, we develop a novel nanozyme system with peroxidase (POD)-like activity by incorporating Pt into the A layer of V2SnC MAX phase. V2(Sn0.8Pt0.2)C MAX displays enhanced brightness in colorimetric signal and remarkably high peroxidase mimicking activity (2542.9 U mg−1), which contributes to the improvement of LFIA sensitivity through catalytic amplification. Density functional theory (DFT) calculations reveal that the presence of Pt effectively lowers the energy barrier for H2O2 adsorption at the A site in V2SnC. Specifically, V2(Sn0.8Pt0.2)C-immunolabeled LFIA strips were developed for the detection of NT-proBNP, exhibiting an unprecedented limit of detection at 0.0016 ng mL−1, which is remarkably enhanced by 1437-fold compared to conventional Au nanoparticles-based LFIA methods. Furthermore, these LFIA strips also demonstrated excellent performance in detecting human plasma-containing samples with a sensitivity of 0.02 ng mL−1 within a linear detection range of 0.02–71.64 ng mL−1. These findings suggest that V2(Sn0.8Pt0.2)C holds great potential as a signal reporter for the development of highly sensitive LFIA assays aimed at accurately monitoring HF progression and prognosis.

A novel integrated lateral flow immunoassay platform for the detection of cardiac troponin I using hierarchical dendritic copper-nickel nanostructures

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI). Herein, we report a novel integrated lateral flow immunoassay (LFIA) platform for highly sensitive point-of-care testing (POCT) of cTnI using hierarchical dendritic copper-nickel (HD-nanoCu-Ni) nanostructures. The electrodeposited HD-nanoCu-Ni film (∼22 μm thick) on an ITO-coated glass substrate exhibits superior capillary action and structural integrity. These properties enable efficient sample transport and antibody immobilization, making it a compelling alternative to conventional multi-component paper-based LFIA test strips, which are often plagued by structural fragility and susceptibility to moisture damage. The biofunctionalized HD-nanoCu-Ni substrates were laser-etched with lateral flow channels, including a sample loading/conjugate release zone, a test zone, and a control zone. Numerical simulations were used to further optimize the design of these channels to achieve optimal fluid flow and target capture. The HD-nanoCu-Ni LFIA device utilizes a fluorescence quenching based sandwich immunoassay format using antibody-labeled gold nanoparticles (AuNPs) as quenchers. Two different fluorescent materials, fluorescein isothiocyanate (FITC) and CdSe@ZnS quantum dots (QDs), were used as background fluorophores in the device. Upon the formation of a sandwich immunocomplex with cTnI on the HD-nanoCu-Ni device, introduced AuNPs led to the fluorescence quenching of the background fluorophores. The total assay time was approximately 15 min, demonstrating the rapid and efficient nature of the HD-nanoCu-Ni LFIA platform. For FITC, both inner filter effect (IFE) and fluorescence resonance energy transfer (FRET) contributed to the AuNP-mediated quenching. In the case of CdSe@ZnS QDs, IFE dominated the AuNP-induced quenching. Calibration curves were established based on the relationship between the fluorescence quenching intensity and cTnI concentration in human serum samples, ranging from 0.5 to 128 ng/mL. The limits of detection (LODs) were determined to be 0.27 ng/mL and 0.40 ng/mL for FITC and CdSe@ZnS QDs, respectively. A method comparison study using Passing-Bablok regression analysis on varying cTnI concentrations in human serum samples confirmed the equivalence of the HD-nanoCu-Ni LFIA platform to a commercial fluorescence cTnI LFIA assay kit, with no significant systematic or proportional bias observed.

Popcorn-like bimetallic palladium/platinum exhibiting enhanced peroxidase-like activity for signal enhancement in lateral flow immunoassay

BackgroundThe lateral flow immunoassay (LFIA) is widely employed as a point-of-care testing (POCT) technique. However, its limited sensitivity hinders its application in detecting biomarkers with low abundance. Recently, the utilization of nanozymes has been implemented to enhance the sensitivity of LFIA by catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). The catalytic performance of nanozymes plays a crucial role in influencing the sensitivity of LFIA. ResultsThe Cornus officinalis Sieb. et Zucc-Pd@Pt (CO–Pd@Pt) nanozyme with good peroxidase-like activity was synthesized herein through a facile one-pot method employing Cornus officinalis Sieb. et Zucc extract as a reducing agent. The morphology and composition of the CO–Pd@Pt nanozyme were characterized using TEM, SEM, XRD, and XPS. As a proof of concept, the as-synthesized CO–Pd@Pt nanozyme was utilized in LFIA (CO–Pd@Pt-LFIA) for the detection of human chorionic gonadotropin (hCG). Compared to conventional gold nanoparticles-based LFIA (AuNPs-LFIA), CO–Pd@Pt-LFIA demonstrated a significant enhancement in the limit of detection (LOD, 0.08 mIU/mL), which is approximately 160 times lower than that of AuNPs-LFIA. Furthermore, experiments evaluating accuracy, precision, selectivity, interference, and stability have confirmed the practical applicability of CO–Pd@Pt-LFIA for hCG content determination. SignificanceThe present study presents a novel approach for the synthesis of bimetallic nanozymes through environmentally friendly methods, utilizing plant extracts as both protective and reducing agents. Additionally, an easily implementable technique is proposed to enhance signal detection in lateral flow immunoassays.

Enhancing antibody detection sensitivity in lateral flow immunoassays using endospores of Bacillus subtilis as signal amplifiers

Antibody detection is the critical first step for tracking the spread of many diseases including COVID-19. Lateral flow immunoassay (LFIA) is the most commonly used method for rapid antibody detection because it is easy-to-use and inexpensive. However, LFIA has limited sensitivity when gold nanoparticles (AuNPs) are used as the signals. In this study, the endospores of Bacillus subtilis were used in combination with AuNP in a LFIA to detect antibodies. The endospores serve as a signal amplifier. The detection limit was about 10−8 M for anti-beta galactosidase antibody detection whereas the detection limit of conventional LFIA is about 10−6 M. Furthermore, the proposed methods have no additional user steps compared with the traditional LFIA. This method, therefore, improved the sensitivity 100-fold without compromising any advantages of LFIA. We believe that the proposed method will be useful for detection of antibodies against HIV, Zika virus, SARS-CoV-2, and so on.

Membrane-Free Lateral Flow Assay with the Active Control of Fluid Transport for Ultrasensitive Cardiac Biomarker Detection.

Membrane-based lateral flow immunoassays (LFAs) have been employed as early point-of-care (POC) testing tools in clinical settings. However, the varying membrane properties, uncontrollable sample transport in LFAs, visual readout, and required large sample volumes have been major limiting factors in realizing needed sensitivity and desirable precise quantification. Addressing these challenges, we designed a membrane-free system in which the desirable three-dimensional (3D) structure of the detection zone is imitated and used a small pump for fluid flow and fluorescence as readout, all the while maintaining a one-step assay protocol. A hydrogel-like protein-polyelectrolyte complex (PPC) within a polyelectrolyte multilayer (PEM) was developed as the test line by complexing polystreptavidin (pSA) with poly(diallyldimethylammonium chloride) (PDDA), which in turn was layered with poly(acrylic acid) (PAA) resulting in a superior 3D streptavidin-rich test line. Since the remainder of the microchannel remains material-free, good flow control is achieved, and with the total volume of 20 μL, 7.5-fold smaller sample volumes can be used in comparison to conventional LFAs. High sensitivity with desirable reproducibility and a 20 min total assay time were achieved for the detection of NT-proBNP in plasma with a dynamic range of 60-9000 pg·mL-1 and a limit of detection of 56 pg·mL-1 using probe antibody-modified fluorescence nanoparticles. While instrument-free visual detection is no longer possible, the developed lateral flow channel platform has the potential to dramatically expand the LFA applicability, as it overcomes the limitations of membrane-based immunoassays, ultimately improving the accuracy and reducing the sample volume so that finger-prick analyses can easily be done in a one-step assay for analytes present at very low concentrations.

PEI-Mediated Assembly of Fe3O4 onto SiO2-Encapsulated CsPbBr3 for Highly Sensitive Fluorescent Lateral Flow Immunoassay.

The conventional lateral flow immunoassay (LFIA) method using colloidal gold nanoparticles (Au NPs) as labeling agents faces two inherent limitations, including restricted sensitivity and poor quantitative capability, which impede early viral infection detection. Herein, we designed and synthesized CsPbBr3 perovskite quantum dot-based composite nanoparticles, CsPbBr3@SiO2@Fe3O4 (CSF), which integrated fluorescence detection and magnetic enrichment properties into LFIA technology and achieved rapid, sensitive, and convenient quantitative detection of the SARS-CoV-2 virus N protein. In this study, CsPbBr3 served as a high-quantum-yield fluorescent signaling probe, while SiO2 significantly enhanced the stability and biomodifiability of CsPbBr3. Importantly, the SiO2 shell shows relatively low absorption or scattering toward fluorescence, maintaining a quantum yield of up to 74.4% in CsPbBr3@SiO2. Assembly of Fe3O4 nanoparticles mediated by PEI further enhanced the method's sensitivity and reduced matrix interference through magnetic enrichment. Consequently, the method achieved a fluorescent detection range of 1 × 102 to 5 × 106 pg·mL-1 after magnetic enrichment, with a limit of detection (LOD) of 58.8 pg·mL-1, representing a 13.3-fold improvement compared to nonenriched samples (7.58 × 102 pg·mL-1) and a 2-orders-of-magnitude improvement over commercial colloidal gold kits. Furthermore, the method exhibited 80% positive and 100% negative detection rates in clinical samples. This approach holds promise for on-site diagnosis, home-based quantitative tests, and disease procession evaluation.

A single-step, digital immunoassay based on serial imaging and image processing

Single-step immunoassays have attracted much attention compared with conventional multi-step sandwich immunoassays, owing to their user-friendliness, minimization of potential errors (e.g., false signals) arising from multistep procedures, and reduced diagnosis time. However, developing a single-step immunoassay with high sensitivity and accuracy has remained challenging. In this study, we developed a single-step, digital immunoassay by applying an imaging technique to a typical sandwich immunoassay, leading to high sensitivity and accuracy. The imaging technology—composed of serial imaging of a sensing surface, stacking of the obtained images, and particle counting using an algorithm—enabled the differentiation of bound and unbound conjugates (detection antibody-conjugated polystyrene beads) without washing. Stationary bound conjugates became more distinct after stacking over 30 images, while randomly drifting unbound conjugates disappeared, enabling the counting of only individual bound labels. The platform efficiently detected SARS-CoV-2 nucleocapsid protein in the concentration range of 10 pg mL–1 to 1 ng mL–1 (limit of detection, 0.87 pg mL–1), which was 103 times more sensitive than that of a conventional lateral flow immunoassay. This immunoassay could be a promising method for highly sensitive and accurate point-of-care biosensors.

Integration of a hamper pad on test strips for improved sensitivity of carbendazim detection

Lateral flow immunoassays (LFIAs) are widely used to determine carbendazim (CBZ) residues in food products due to their advantages of low cost, ease and rapid use, on-site detection capability. However, conventional LFIAs have low detection sensitivity. Although improvements have been made to increase the sensitivity, it is not sufficient. Here, a hamper pad, polyvinyl alcohol coated on a nitrocellulose membrane, was integrated to enhance the sensitivity of LFIA for CBZ detection. The hamper pad was inserted between the conjugated and nitrocellulose pads to delay the flow rate, thereby increasing the possibility of the antibody and target analyte binding. This platform exhibited a fourfold sensitivity increase in CBZ detection compared with the conventional LFIA, and its limit of detection was 1.6 ng/mL. In addition, a single-step operation was successfully applied to detect CBZ in rice (white rice, brown rice, sticky rice, and paddy) and soybean samples, with acceptable recoveries of 93.6%–120.0%. This novel device was compared to the standard high-performance liquid chromatography method, which shows high accuracy with a Kappa coefficient of 0.91. Therefore, improved sensitivity with a rapid, simple, and inexpensive device could facilitate the detection of CBZ residues in agricultural products for on-field screening and improved user-friendliness.

Dual-Mode Lateral Flow Immunoassay Based on "Pompon Mum"-Like Fe3O4@MoS2@Pt Nanotags for Sensitive Detection of Viral Pathogens.

Lateral flow immunoassay (LFIA) has been widely used for the early diagnosis of diseases. However, conventional colorimetric LFIA possesses limited sensitivity, and the single-mode readout signal is easily affected by the external environment, leading to insufficient accuracy. Herein, multifunctional Fe3O4@MoS2@Pt nanotags with a unique "pompon mum"-like structure were triumphantly prepared, exhibiting excellent peroxidase (POD)-like activity, photothermal properties, and magnetic separation capability. Furthermore, the Fe3O4@MoS2@Pt nanotags were used to establish dual-mode LFIA (dLFIA) for the first time, enabling the catalytic colorimetric and photothermal dual-mode detection of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein (SARS-CoV-2 NP) and influenza A (H1N1). The calculated limits of detection (cLODs) of SARS-CoV-2 NP and H1N1 were 80 and 20 ng/mL in catalytic colorimetric mode and 10 and 8 ng/mL in photothermal mode, respectively, demonstrating about 100 times more sensitive than the commercial colloidal Au-LFIA strips (1 ng/mL for SARS-CoV-2 NP; 1 μg/mL for H1N1). The recovery rates of dLFIA in simulated nose swab samples were 95.2-103.8% with a coefficient of variance of 2.3-10.1%. These results indicated that the proposed dLFIA platform showed great potential for the rapid diagnosis of respiratory viruses.

Trifunctional Nanocomposites with Colorimetric Magnetic Catalytic Activities Labels in Sandwich Immunochromatographic Detection of Escherichia coli O157:H7.

The emergence of nanoenzymes has catalyzed the robust advancement of the lateral flow immunoassay (LFIA) in recent years. Among them, multifunctional nanocomposite enzymes with core-shell architectures are considered preferable for promoting the sensing ability due to their good biocompatibility, precise control over size, and surface properties etc. Herein, we developed a dual-channel ensured lateral flow immunoassay (DFLIA) platform utilizing a magnetic, colorimetric, and catalytic multifunctional nanocomposite enzyme (Fe3O4@TCPP@Pd) [TCPP, Tetrakis (4-carboxyphenyl) porphyrin] for the ultrasensitive and highly accurate rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). Fe3O4@TCPP@Pd-mAb exhibits superior performance compared to traditional AuNPs, including enhanced sensitivity and an extended linear detection range, benefiting from its high brightness signal, strong magnetic separation ability, and high peroxidase activity (Vmax = 2.32 μM S1-). Moreover, the Fe3O4@TCPP@Pd-labeled mAb probe exhibited exceptional stability and high affinity toward E. coli O157:H7 (with an affinity constant of approximately 1.723 × 109 M-1), indicating its potential for the efficient capture of the pathogen. Impressively, the developed Fe3O4@TCPP@Pd-DFLIA achieved ultrasensitive detection for E. coli O157:H7 with pre- and postcatalytic naked-eye detection sensitivities of 255 cfu/mL and 77 cfu/mL, respectively, representing an approximately 41-fold improvement over the conventional AuNP-based LFIA and also possessed good specificity and reproducibility [relative standard deviation (RSD) < 10%]. Additionally, the established DFLIA exhibited satisfactory recoveries in detecting pork and milk samples, further validating the reliability of this platform for immunoassays and demonstrating its potential for utilization in bioassays and clinical diagnostics.

A one-step immunoassay based on switching peptides for diagnosis of porcine epidemic diarrhea virus (PEDV) using screened Fv-antibodies.

In this study, a one-step immunoassay for porcine epidemic diarrhea virus (PEDV) based on Fv-antibodies and switching peptides was developed, and the assay results of PEDV were obtained by just mixing samples without any further reaction or washing steps. The Fv-antibodies with binding affinity to the spike protein of PEDV were screened from the Fv-antibody library using the receptor-binding domain (RBD) of the spike protein as a screening probe. Screened Fv-antibodies with binding affinities to the RBD antigen were expressed, and the binding constants (KD) were calculated to be 83-142 nM. The one-step immunoassay for the detection of PEDV was configured as a displacement immunoassay using a fluorescence-labeled switching peptide. The one-step immunoassay based on switching peptides was performed using PEDV, and the limit of detection (LOD) values for PEDV detection were estimated to be Ct = 39.7-36.4. Compared with the LOD value for a conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay showed a remarkably improved LOD for the detection of PEDV. Finally, the interaction between the screened Fv-antibodies and the PEDV RBD was investigated using docking simulations and compared with the amino acid sequences of the receptors on host cells, such as aminopeptidase N (APN) and angiotensin-converting enzyme-2 (ACE-2).

Redox Cycling Amplified Electrochemical Lateral-Flow Immunoassay: Toward Decentralized Sensitive Insulin Detection.

While paper-based lateral-flow immunoassays (LFA) offer considerable promise for centralized diagnostic applications, the analytical capability of conventional LFA remains constrained due to the low sensitivity of its common optical detection strategy. To address these issues, we report a simple electrochemical LFA (eLFA) with nanocatalytic redox cycling for decentralized insulin detection. Simultaneous binding of insulin with detection antibodies and capture antibodies through the capillary flow at the LFA platform and signal amplification through the rapid nanocatalytic reduction of [Fe(CN)6]3- (Fe3+) with Au nanoparticles (AuNP) and ammonia-borane (AB), coupled to electrochemical redox cycling reactions involving Fe3+, AuNP, and AB on the carbon working electrode, offer higher sensitivity than conventional colorimetric LFA and enzymatic redox cycling. The resulting integrated eLFA strip allows the detection of low insulin concentrations (LOD = 12 pM) and offers considerable promise for highly sensitive decentralized assays of different biological fluids (saliva and serum) without additional pretreatment or washing steps.

Modified polysulfone membrane facilitates rapid separation of plasma from whole blood for an effective anti-SARS-CoV-2-IgM diagnosis

During the outbreak of coronavirus, RT-PCR was the premier gold standard method for severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) diagnosis. However, the sophisticated procedure of RT-PCR persuades researchers to develop sustainable point-of-need immunoassay methods for tracing unwitting carriers of SARSCoV-2. Herein, by fabricating a modified polysulfone (MPSF) membrane, we developed an integrated radial flow immunoassay (IRFIA) platform as a point-of-care system, capable of multiplying the immunoassays at a short run time. The target molecule is the SARSCoV-2 IgM in separated plasma. Although the lateral flow immunoassay kits for the rapid identification of Covid-19 have already been commercially developed but, the proposed method is superior to the conventional lateral flow immunoassay. In the newly designed membrane system, we have combined the five membranes of prevalent lateral flow immunoassay (LFIA) strips in one polymeric membrane. The MPSF membrane is capable of separating plasma from whole blood sample, which will reduce the interference of red colour of hemoglobin with generated signal and enhance the immunoassay precision. The efficiency of plasma separation, reached the mean value of 97.34 v/v% in 5 s. Furthermore, the gel electrophoresis results of the separated plasma contrasted with centrifuged plasma sample, demonstrated more efficient separation by the membrane. Using the MPSF membrane, signal generation time reduced from about 20 min in conventional rapid test strip for Covid-19 to about 7 min in IRFIA platform. The sensitivity and specificity of the membrane platform were determined to be 89% and 90%, respectively and a Kappa coefficient of 0.79 showed reliable agreement between the RT-PCR and the membrane system.

Integrated lateral flow immunoassays using trimethylsilyl cellulose barriers for the enhanced sensitivity of COVID-19 diagnosis

Lateral flow immunoassays (LFIAs) serve as rapid and convenient tools for disease screening. However, there is room for enhancing diagnostic sensitivity. Therefore, this paper introduces a novel material combined with lateral flow immunoassays to improve the sensitivity of COVID-19 detection and diagnosis. Trimethylsilyl cellulose (TMSC) was printed on the device between the test and control line to delay the flow of the solution, which resulted in a more excellent binding of the immunoassay. These delay-LFIAs (d-LFIAs) were developed for the diagnosis of COVID-19, targeting the detection of immunoglobulins G and M, as well as the COVID-19 antigen. Under optimized conditions, the d-LFIA increased the sensitivity by up to 5 times compared to that of conventional LFIAs. Moreover, it exhibited the ability to detect IgG, IgM, and SARS-CoV-2 antigens as low as 1 ng/mL (limit of detection) by the naked eye. In addition, the application of this device to real samples yielded highly consistent results with those obtained from other LFIAs and standard SARS-CoV-2 diagnostic methods (such as enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction) diagnosis. Therefore, we expect this device to be a new platform for highly sensitive, easy, and affordable immunoassay strip testing to certify future emerging diseases.

Recent Advances in Quantum Dot-Based Lateral Flow Immunoassays for the Rapid, Point-of-Care Diagnosis of COVID-19.

The COVID-19 pandemic has spurred demand for efficient and rapid diagnostic tools that can be deployed at point of care to quickly identify infected individuals. Existing detection methods are time consuming and they lack sensitivity. Point-of-care testing (POCT) has emerged as a promising alternative due to its user-friendliness, rapidity, and high specificity and sensitivity. Such tests can be conveniently conducted at the patient's bedside. Immunodiagnostic methods that offer the rapid identification of positive cases are urgently required. Quantum dots (QDs), known for their multimodal properties, have shown potential in terms of combating or inhibiting the COVID-19 virus. When coupled with specific antibodies, QDs enable the highly sensitive detection of viral antigens in patient samples. Conventional lateral flow immunoassays (LFAs) have been widely used for diagnostic testing due to their simplicity, low cost, and portability. However, they often lack the sensitivity required to accurately detect low viral loads. Quantum dot (QD)-based lateral flow immunoassays have emerged as a promising alternative, offering significant advancements in sensitivity and specificity. Moreover, the lateral flow immunoassay (LFIA) method, which fulfils POCT standards, has gained popularity in diagnosing COVID-19. This review focuses on recent advancements in QD-based LFIA for rapid POCT COVID-19 diagnosis. Strategies to enhance sensitivity using QDs are explored, and the underlying principles of LFIA are elucidated. The benefits of using the QD-based LFIA as a POCT method are highlighted, and its published performance in COVID-19 diagnostics is examined. Overall, the integration of quantum dots with LFIA holds immense promise in terms of revolutionizing COVID-19 detection, treatment, and prevention, offering a convenient and effective approach to combat the pandemic.

A universal boronate affinity capture-antibody-independent lateral flow immunoassay for point-of-care glycoprotein detection

Protein glycosylation and other post-translational modifications are involved in many biological processes including growth, development and immune responses, and glycoproteins are also known as biomarkers for cancer, diabetes and cardiovascular diseases. In traditional lateral flow immunoassay (LFIA) for glycoprotein detection, capture antibody (CA) is often required to label targets. However, the production of CA is complicated and expensive, restricting the wide application of LFIA. In this study, we developed a universal boronate affinity CA-independent LFIA method for glycoprotein detection. 4-Mercaptophenylboronic acid (4-MPBA)-modified Au nanoparticles (namely 4-MPBA-AuNPs) were used as LFIA labels, which could generate colorimetric signal and showed outstanding capability to bind glycoprotein. Compared with CA, 4-MPBA molecular as a glycoprotein recognition element had more prominent advantages, e.g., low cost, easy availability and good quality controllability. Take carcinoembryonic antigen (CEA) as model glycoprotein, the limit of detection of this CA-independent LFIA was 1.25 ng/mL by naked eyes, which was 8-fold lower than conventional CA-dependent sandwich LFIA. Significantly, the developed 4-MPBA-AuNPs-based CA-independent LFIA successfully detected 23 CEA-positive samples from 64 suspected human serum samples within 50 min in a nonlaboratory environment, with a 100% accuracy compared to clinical detection method. Therefore, this diagnostic platform could provide an effective tool for point-of-care glycoprotein detection with excellent reproducibility and high specificity.