Development of a Fluorescent Immunochromatographic Assay Based on Quantum Dot-Functionalized Two-Dimensional Monolayer Ti3C2 MXene Nanoprobes for the Simultaneous Detection of Influenza A Virus and SARS-CoV-2.

Accurate and rapid detection of the influenza A virus (FluA) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can effectively control their spread. We developed a colorimetric and fluorescent dual-functional two-channel immunochromatographic assay (ICA) biosensor to simultaneously detect the above-mentioned viruses. A unique two-dimensional Ti3C2-QD immunoprobe was established by adsorbing dense quantum dots (QDs) onto the light green monostromatic Ti3C2 MXene surface, resulting in light green colorimetric and superior fluorescence signals and guaranteeing high sensitivity, stability, and excellent liquidity for ICA detection. Rapid visual screening for FluA and SARS-CoV-2 infections was applicable via a green colorimetric signal. Sensitive and quantitative detection of viruses in their early stages of infection was performed by using the fluorescence signal. Our proposed Ti3C2-QD-ICA biosensor can simultaneously detect 1 ng/mL or 2.4 pg/mL FluA and 1 ng/mL or 6.2 pg/mL SARS-CoV-2 via its colorimetric or fluorescence signals, respectively, with a short testing time (20 min), good reproducibility, specificity, and accuracy. In addition, this method demonstrated sensitivity higher than that of the conventional AuNP-based ICA method in throat swab samples. Hence, our proposed Ti3C2-QD-ICA method can be potentially applied for the rapid, ultrasensitive, and multiplex detection of respiratory viruses.

Evaluación de un inmunoensayo para la medida de anticuerpos anti-tiroglobulina y anti-tiroperoxidasa / Evaluation of an immunoassay for the antithyroglobulin and antithyroperoxidase measuring

Introducción: La medición de los anticuerpos frente a tiroglobulina (ATG) y peroxidasa tiroidea (ATPO) es de interés para identificar pacientes con tiroiditis autoinmune.Este estudio pretende evaluar un inmunoensayo comercial de electroquimioluminiscencia para ATG y ATPO, estudiando la imprecisión, la linealidad, sensibilidad analítica, evaluación del arrastre, e influencia de interferentes endógenos.Material y métodos: La imprecisión se evaluó usando tres soluciones con diferentes concentraciones de analitos, analizándose 20 veces en la misma serie analítica y durante 20 días consecutivos, calculando el coeficiente de variación. Para el estudio de linealidad se combinaron una muestra con elevada concentración de analitos y un diluyente, obteniéndose concentraciones intermedias que se analizaron por triplicado. El límite de detección se calculó haciendo 20 determinaciones de una muestra de diluyente. El arrastre se evaluó analizando una muestra con alta concentración de anticuerpos seguida por otra con concentraciones muy bajas. El estudio de interferencias se realizó añadiendo a mezclas de suero hemolizado, Intralipid 30% y bilirrubina.Resultados: Las imprecisiones totales obtenidas (%) fueron 26,63, 9,53, y 14,9 para ATG y 21,19, 14,82 y 5,77 para ATPO. La linealidad queda definida por las ecuaciones de regresión: Y=6.61+1.01X(ATG) y Y=16.37+0.97X(ATPO). El límite de detección fue 17,17 para ATG y 5 para ATPO. El arrastre no fue significativo. La hemólisis produjo interferencia significativa en ambos ensayos.Conclusiones: Las imprecisiones obtenidas son comparables a las declaradas por el fabricante. La sensibilidad analítica cumple las especificaciones del fabricante. El comportamiento de ambos ensayos es lineal y no se halla arrastre significativo. La hemólisis interfiere ambos ensayos. (AU)

Introduction: The measuring of antibodies against thyroglobulin (ATG) and thyroperoxydase (ATPO) is useful for identifying patients with autoimmunethyroiditis. This study tries to assess an electrochemiluminescent immunoassay for ATG and ATPO, evaluating imprecision, linearity, analytic sensitivity, carry-over and the influence of endogenous interferents.Material and methods: Imprecision was assessed using three pools with different analytes concentrations, performing within run and between run 20 times. Fort the linearity study a sample containing high analyte concentration and a solvent devoid of analyte were combined, obtaining intermediates concentrations, which were analyzed by triplicate. The limit of detection was calculated analyzing 20 times a sample devoid of analyte. Carry-over was evaluated analyzing a sample with a high antibody concentration followed by other one containing low antibody concentration. The interference study was carried-out adding hemolyzed, Intralipid 30% and bilirubin into sera pool.Results: Total imprecision obtained (%) were 26.63, 9.53, and 14.9 for ATG and 21.19, 14.82, and 5.77 for ATPO. Linearity was defined for the following regression equations: Y=6.61+1.01X (ATG) and, Y=16.37+0.97X (ATPO). The limit of detection was 17.17 for ATG and 5 for ATPO. Carry-over was not significant. Hemolysis caused significant interference in both assays.Conclusions: Imprecision obtained were similar to the manufacturer declared ones. Analytic sensibility complies the manufacturer’s specifications. The behavior of both assays was linear and significant carry-over was not found. Hemolysis interferes in both assays. (AU)

Gold-silver alloy hollow nanoshells-based lateral flow immunoassay for colorimetric, photothermal, and SERS tri-mode detection of SARS-CoV-2 neutralizing antibody.

Although many approaches have been developed for the quick assessment of SARS-CoV-2 infection, few of them are devoted to the detection of the neutralizing antibody, which is essential for assessing the effectiveness of vaccines. Herein, we developed a tri-mode lateral flow immunoassay (LFIA) platform based on gold-silver alloy hollow nanoshells (Au-Ag HNSs) for the sensitive and accurate quantification of neutralizing antibodies. By tuning the shell-to-core ratio, the surface plasmon resonance (SPR) absorption band of the Au-Ag HNSs is located within the near infrared (NIR) region, endowing them with an excellent photothermal effect under the irradiation of optical maser at 808 nm. Further, the Raman reporter molecule 4-mercaptobenzoic acid (MBA) was immobilized on the gold-silver alloy nanoshell to obtain an enhanced SERS signal. Thus, these Au-Ag HNSs could provide colorimetric, photothermal and SERS signals, with which, tri-mode strips for SARS-CoV-2 neutralizing antibody detection were constructed by competitive immunoassay. Since these three kinds of signals could complement one another, a more accurate detection was achieved. The tri-mode LFIA achieved a quantitative detection with detection limit of 20 ng/mL. Moreover, it also successfully detected the serum samples from 98 vaccinated volunteers with 79 positive results, exhibiting great application value in neutralizing antibody detection.

An ultrasensitive NIR-IIa' fluorescence-based multiplex immunochromatographic strip test platform for antibiotic residues detection in milk samples.

INTRODUCTION: Widely used in livestock breeding, residues of antibiotic drugs in milk have become a threat to food safety and human health. Current rapid detection technologies using colorimetric immunochromatographic strip tests (IST) lack the necessary sensitivity for on-site trace monitoring. Fluorescence-based detection in the near-infrared IIa' (NIR-IIa') region (1000 âˆ¼ 1300 nm) has enormous potential due to greatly minimized auto-fluorescence and light scattering. OBJECTIVES: The aim of this work is to develop an ultrasensitive IST platform using NIR-IIa' fluorescent nanoparticles as labels for multiplex antibiotic residues detection in milk. METHODS: NIR-IIa' fluorescent nanoparticles were assembled by encapsulating synthesized NIR-IIa' fluorophores into carboxyl - modified polystyrene nanoparticles. The NIR-IIa' nanoparticles were subsequently used as labels in an IST platform to detect sulfonamides, quinolones, and lincomycin simultaneously in milk. A portable fluorescent reader was fabricated to provide on-site detection. To further validate the developed IST platform, the detection was compared with LC-MS/MS in 22 real milk samples. RESULTS: Fluorescent nanoparticles were synthesized with low energy emission (1030 nm) and large Stokes shift (>250 nm) showing a much higher signal-to-noise ratio compared with fluorophores emitting in the NIR-I region. The developed IST platform yielded a highly sensitive, simultaneous quantification of sulfonamides, quinolones, and lincomycin in milk with detection limits of 46.7, 27.6 and 51.4 pg/mL, respectively, achieving a wide detection range (up to 50 ng/mL). The IST platform showed good accuracy, reproducibility, and specificity with the portable fluorescent reader which could rapidly quantify in 10 s. These results were better than reported immunochromatographic assays using fluorescent labels, and remarkably, showed a higher recognition ability than LC-MS/MS for real samples. CONCLUSION: The utility of NIR-IIa' fluorescence-based IST platform for the fast, sensitive, and accurate detection of antibiotics in milk was demonstrated, successfully verifying the potential of this platform in detecting trace materials in complex matrices.

Thermometric lateral flow immunoassay with colored latex beads as reporters for COVID-19 testing.

Temperature sensing is a promising method of enhancing the detection sensitivity of lateral flow immunoassay (LFIA) for point-of-care testing. A temperature increase of more than 100 °C can be readily achieved by photoexcitation of reporters like gold nanoparticles (GNPs) or colored latex beads (CLBs) on LFIA strips with a laser power below 100 mW. Despite its promise, processes involved in the photothermal detection have not yet been well-characterized. Here, we provide a fundamental understanding of this thermometric assay using non-fluorescent CLBs as the reporters deposited on nitrocellulose membrane. From a measurement for the dependence of temperature rises on the number density of membrane-bound CLBs, we found a 1.3-fold (and 3.2-fold) enhancement of the light absorption by red (and black) latex beads at 520 nm. The enhancement was attributed to the multiple scattering of light in this highly porous medium, a mechanism that could make a significant impact on the sensitivity improvement of LFIA. The limit of detection was measured to be 1 × 105 particles/mm2. In line with previous studies using GNPs as the reporters, the CLB-based thermometric assay provides a 10× higher sensitivity than color visualization. We demonstrated a practical use of this thermometric immunoassay with rapid antigen tests for COVID-19.

Europium (III) chelate nanoparticle-based lateral flow immunoassay strips for rapid and quantitative detection of cystatin C in serum.

The concentration of Cys C in the patient's serum can reflect the level of glomerular filtration rate and indicate the occurrence of renal failure. The establishment of a simple and rapid analytical method to quantitatively monitor the concentration of Cys C in serum could help timely detection of renal failure. In this study, we have developed an Eu (III) chelate nanoparticles based lateral flow immunoassay to fulfill real-time monitoring of Cys C concentration in serum within 15 min. This method was performed as a sandwich immunoassay with a wide detection range (0.05-10 µg/mL) and a low limit of detection (24.54 ng/mL). The intra and inter-assay coefficients of variation were 8.31-8.61% and 8.92-9.95%, respectively. Furthermore, the application of this method was evaluated by comparing the determined results with those obtained by chemiluminescence immunoassay, exhibiting a satisfactory correlation (R2 = 0.9830). The developed LFIA method with satisfactory analytical performance has great potential for real-time monitoring of renal failure and self-detection for the high-risk population.

Highly Stable Buffer-Based Zinc Oxide/Reduced Graphene Oxide Nanosurface Chemistry for Rapid Immunosensing of SARS-CoV-2 Antigens.

The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1-10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.

An electronic biosensor based on semiconducting tetrazine polymer immobilizing matrix coated on rGO for carcinoembryonic antigen.

Point-of-care devices are expected to play very critical roles in early diagnosis and better treatment of cancer. Here, we report the end-to-end development of novel and portable biosensors for detecting carcinoembryonic antigen (CEA), a cancer biomarker, almost instantly at room temperature. The device uses reduced graphene oxide (rGO) as the base conducting layer and a novel poly[(1,4-phenylene)-alt-(3,6-(1,2,4,5-tetrazine)/3,6-(1,2,4,5-dihydrotetrazine))] (PhPTz) as an immobilizing matrix for the CEA antibodies. Judiciously introduced nitrogen-rich semiconducting PhPTz brings multiple advantages to the device-(1) efficiently immobilizes anti-CEA via synergistic H-bonding with peptide and N-glycal units and (2) transports the charge density variations, originated upon antibody-antigen interactions, to the rGO layer. The CEA was dropped onto the anti-CEA/PhPTz/rGO devices at ambient conditions, to facilitate binding and the change in current flowing through the sensors was measured. A response of 2.75-33.7 µA was observed when the devices were tested for a broad range of concentrations (0.25 pg/mL to 800 ng/mL) of CEA. A portable read-out circuit was assembled using Arduino UNO and a voltage divider circuit, and a simple algorithm was developed for the classification of the CEA concentrations. The prediction accuracy of the interfacing electronics along with the algorithm was found to be 100%.

Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a serious problem in the horse industry, and controlling EP is critical for international horse trading. EP is caused by two apicomplexan protozoan parasites, Theileria equi and Babesia caballi. Rapid and accurate methods that are suitable for detecting these parasites in the field are crucial to control the infection and spread of EP. In this study, we developed a card to detect antibodies against T. equi and B. caballi based on two colloidal gold immunochromatographic strips according to the principle of the double-antigen sandwich. The proteins equi merozoite antigen 1 (EMA1) and rhoptry protein BC48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. On the strip, the purified EMA1 or BC48 protein labeled with colloidal gold was used as the detector, and nitrocellulose membranes were coated with EMA1 or BC48 and the corresponding MAb as the test and control lines, respectively. The protocol takes 10 to 15 min and requires no specialized equipment or chemical reagents, and one test can detect two EP pathogens in one card. Specificity tests confirmed there was no cross-reactivity with sera positive for common equine pathogens. Using a commercial competitive enzyme-linked immunosorbent assay (cELISA) kit for comparison, 476 clinical samples were tested with the card. The coincidence rates were 96.43% and 97.90% for T. equi and B. caballi, respectively. The field trial feedback was uniformly positive, suggesting that this diagnostic tool may be useful for controlling the spread of T. equi and B. caballi. IMPORTANCE Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. EP is considered a reportable disease by the World Organization for Animal Health (OIE). The accurate diagnosis and differentiation of T. equi and B. caballi are very important for the prevention, control, and treatment of EP. Therefore, we developed a double-antigen sandwich colloidal gold immunochromatography assay (GICG) to detect T. equi and B. caballi. Two GICG strips were assembled side by side on one card for the detection of T. equi and B. caballi, and the two EP pathogens could be detected in one test. This method was simple, rapid, and specific for the detection of EP; therefore, compared to the previous methods, this method is more suitable for pathogen diagnosis in the field.

All Fiber-Optic Immunosensors Based on Elliptical Core Helical Intermediate-Period Fiber Grating with Low-Sensitivity to Environmental Disturbances.

An all fiber-optic immunosensor based on elliptical core helical intermediate-period fiber grating (E-HIPFG) is proposed for the specific detection of human immunoglobulin G (human IgG). E-HIPFGs are all-fiber transducers that do not include any additional coating materials or fiber architectures, simplifying the fabrication process and promising the stability of the E-HIPFG biosensor. For human IgG recognition, the surface of an E-HIPFG is functionalized by goat anti-human IgG. The functionalized E-HIPFG is tested by human IgG solutions with a concentration range of 10-100 µg/mL and shows a high sensitivity of 0.018 nm/(µg/mL) and a limit of detection (LOD) of 4.7 µg/mL. Notably, the functionalized E-HIPFG biosensor is found to be insensitive to environmental disturbances, with a temperature sensitivity of 2.6 pm/°C, a strain sensitivity of 1.2 pm/µÎµ, and a torsion sensitivity of -23.566 nm/(rad/mm). The results demonstrate the considerable properties of the immunosensor, with high resistance to environmental perturbations, indicating significant potential for applications in mobile biosensors and compact devices.

Design and Optimisation of Elliptical-Shaped Planar Hall Sensor for Biomedical Applications.

The magnetic beads detection-based immunoassay, also called magneto-immunoassay, has potential applications in point-of-care testing (POCT) due to its unique advantage of minimal background interference from the biological sample and associated reagents. While magnetic field detection technologies are well established for numerous applications in the military, as well as in geology, archaeology, mining, spacecraft, and mobile phones, adaptation into magneto-immunoassay is yet to be explored. The magnetic field biosensors under development tend to be multilayered and require an expensive fabrication process. A low-cost and affordable biosensing platform is required for an effective point-of-care diagnosis in a resource-limited environment. Therefore, we evaluated a single-layered magnetic biosensor in this study to overcome this limitation. The shape-induced magnetic anisotropy-based planar hall effect sensor was recently developed to detect a low-level magnetic field, but was not explored for medical application. In this study, the elliptical-shaped planar hall effect (EPHE) sensor was designed, fabricated, characterized, and optimized for the magneto-immunoassay, specifically. Nine sensor variants were designed and fabricated. A customized measurement setup incorporating a lock-in amplifier was used to quantify 4.5 µm magnetic beads in a droplet. The result indicated that the single-domain behaviour of the magnetic film and larger sensing area with a thinner magnetic film had the highest sensitivity. The developed sensor was tested with a range of magnetic bead concentrations, demonstrating a limit of detection of 200 beads/µL. The sensor performance encourages employing magneto-immunoassay towards developing a low-cost POCT device in the future.

Spectral image contrast-based flow digital nanoplasmon-metry for ultrasensitive antibody detection.

BACKGROUND: Gold nanoparticles (AuNPs) have been widely used in local surface plasmon resonance (LSPR) immunoassays for biomolecule sensing, which is primarily based on two conventional methods: absorption spectra analysis and colorimetry. The low figure of merit (FoM) of the LSPR and high-concentration AuNP requirement restrict their limit of detection (LOD), which is approximately ng to µg mL-1 in antibody detection if there is no other signal or analyte amplification. Improvements in sensitivity have been slow in recent for a long time, and pushing the boundary of the current LOD is a great challenge of current LSPR immunoassays in biosensing. RESULTS: In this work, we developed spectral image contrast-based flow digital nanoplasmon-metry (Flow DiNM) to push the LOD boundary. Comparing the scattering image brightness of AuNPs in two neighboring wavelength bands near the LSPR peak, the peak shift signal is strongly amplified and quickly detected. Introducing digital analysis, the Flow DiNM provides an ultrahigh signal-to-noise ratio and has a lower sample volume requirement. Compared to the conventional analog LSPR immunoassay, Flow DiNM for anti-BSA detection in pure samples has an LOD as low as 1 pg mL-1 within only a 15-min detection time and 500 µL sample volume. Antibody assays against spike proteins of SARS-CoV-2 in artificial saliva that contained various proteins were also conducted to validate the detection of Flow DiNM in complicated samples. Flow DiNM shows significant discrimination in detection with an LOD of 10 pg mL-1 and a broad dynamic detection range of five orders of magnitude. CONCLUSION: Together with the quick readout time and simple operation, this work clearly demonstrated the high sensitivity and selectivity of the developed Flow DiNM in rapid antibody detection. Spectral image contrast and digital analysis further provide a new generation of LSPR immunoassay with AuNPs.

Tailoring noble metal nanoparticle designs to enable sensitive lateral flow immunoassay.

Lateral flow immunoassay (LFIA) with gold nanoparticles (AuNPs) as signal reporters is a popular point-of-care diagnostic technique. However, given the weak absorbance of traditional 20-40 nm spherical AuNPs, their sensitivity is low, which greatly limits the wide application of AuNP-based LFIA. With the rapid advances in materials science and nanotechnology, the synthesis of noble metal nanoparticles (NMNPs) has enhanced physicochemical properties such as optical, plasmonic, catalytic, and multifunctional activity by simply engineering their physical parameters, including the size, shape, composition, and external structure. Using these engineered NMNPs as an alternative to traditional AuNPs, the sensitivity of LFIA has been significantly improved, thereby greatly expanding the working range and application scenarios of LFIA, particularly in trace analysis. Therefore, in this review, we will focus on the design of engineered NMNPs and their demonstration in improving LFIA. We highlight the strategies available for tailoring NMNP designs, the effect of NMNP engineering on their performance, and the working principle of each engineering design for enhancing LFIA. Finally, current challenges and future improvements in this field are briefly discussed.

Automated liquid handling robot for rapid lateral flow assay development.

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.

An ultrasensitive colloidal gold immunosensor to simultaneously detect 12 beta (2)-adrenergic agonists.

In this study, we first prepared a selective monoclonal antibody against 12 beta (2)-adrenergic agonists (Salbutamol, Clenbuterol, Brombuterol, Clenpenterol, Mabuterol, Carbuterol, Cimbuterol, Mapenterol, Pirbuterol, Terbutaline, Cimaterol, and Clenproperol). Then three haptens were designed and derived, among which, haptenS3 used the amino group of the salbutamol analog to derive a carboxyl group containing a spacer, which is unique to this study. The half-maximal inhibitory concentration (IC50) values were 0.35 ng/mL (Salbutamol), 0.42 ng/mL (Clenbuterol), 0.78 ng/mL (Brombuterol), 0.88 ng/mL (Clenpenterol), 1.34 ng/mL (Mabuterol), 1.38 ng/mL (Carbuterol), 1.71 ng/mL (Cimbuterol), 2.24 ng/mL (Mapenterol), 2.25 ng/mL (Pirbuterol), 2.27 ng/mL (Terbutaline), 3.49 ng/mL (Cimaterol), and 4.89 ng/mL (Clenproperol). We further developed a monoclonal antibody-based colloidal gold immunochromatographic test strip for screening and detecting 12 beta (2)-adrenergic agonists in swine urine and lamb samples. The immunochromatographic method developed in this study is a suitable tool for the on-site rapid detection and screening of beta (2)-adrenergic agonists in swine urine and lamb samples.

Amperometric detection of tumor suppressor protein p53 via pencil graphite electrode for fast cancer diagnosis.

Cancer occupies the second place in terms of worldwide mortality. Early and fast diagnosis of cancer helps clinicians to expand therapeutic approaches ultimately leading towards early diagnosis of cancer patients. In the present work, we delineated an amperometric immunosensor to diagnose cancer to detect p53, a biomarker for cancer. The immunosensor was fabricated by immobilizing anti-p53 antibodies onto the pencil graphite electrode (PGE). The immobilization of probe was studied by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The immunosensor was optimized for pH, incubation temperature, antibody concentration, incubation time and antigen concentration. The developed immunosensor, showed a linear range between 10 pgmL-1 to 10 ngmL-1 with a detection limit (LOD) of 10 pgmL-1. p53 antigen was analyzed by measuring current under optimal conditions. The occurrence of p53 was determined in sera of prostate, breast, colon and lung cancer patients by the present immunosensor. The lower incubation time i.e., fast response and lower LOD demonstrated an improved p53 immunosensor for early diagnosis of cancer.

Three-dimensional protein microarrays fabricated on reactive microsphere modified COC substrates.

Fabrication of three-dimensional (3D) surface structures for the high density immobilization of biomolecules is an effective way to prepare highly sensitive biochips. In this work, a strategy to attach polymeric microspheres on a cyclic olefin copolymer (COC) substrate for the preparation of a 3D protein chip was developed. The COC surface was firstly functionalized by the photograft technique with epoxy groups, which were subsequently converted to amine groups. Then monodisperse poly(styrene-alt-maleic anhydride) (PSM) copolymer microspheres were prepared by self-stabilized precipitation polymerization and deposited as a single layer on a modified COC surface to form a 3D surface texture. The surface roughness of the COC support undergoes a significant increase from 1.4 nm to 37.1 nm after deposition of PSM microspheres with a size of 460 nm, and the modified COC still maintains a transmittance of more than 63% at the fluorescence excitation wavelengths (555 nm and 647 nm). The immobilization efficiency of immunoglobulin G (IgG) on the 3D surface reached 75.6% and the immobilization density was calculated to be 0.255 µg cm-2, at a probe protein concentration of 200 µg mL-1. The 3D protein microarray can be rapidly blocked by gaseous ethylenediamine within 10 minutes due to the high reactivity of anhydride groups in PSM microspheres. Immunoassay results show that the 3D protein microarray achieved specific identification of the target protein with a linear detection range from 6.25 ng mL-1 to 250 ng mL-1 (R2 > 0.99) and a limit of detection of 8.87 ng mL-1. This strategy offers a novel way to develop high performance polymer-based 3D protein chips.

Laser-patterned paper-based flow-through filters and lateral flow immunoassays to enable the detection of C-reactive protein.

We report the use of a laser-based fabrication process in the creation of paper-based flow-through filters that when combined with a traditional lateral flow immunoassay provide an alternative pathway for the detection of a pre-determined analyte over a wide concentration range. The laser-patterned approach was used to create polymeric structures that alter the porosity of the paper to produce porous flow-through filters, with controllable levels of porosity. When located on the top of the front end of a lateral flow immunoassay the flow-through filters were shown to block particles (of known sizes of 200 nm, 500 nm, 1000 nm and 3000 nm) that exceed the effective pore size of the filter while allowing smaller particles to flow through onto a lateral flow immunoassay. The analyte detection is based on the use of a size-exclusive filter that retains a complex (∼3 µm in size) formed by the binding of the target analyte with two antibodies each of which is tagged with different-sized labels (40 nm Au-nanoparticles and 3 µm latex beads), and which is larger than the effective pore size of the filter. This method was tested for the detection of C-reactive protein in a broad concentration range from 10 ng/ml to 100,000 ng/ml with a limit-of-detection found at 13 ng/ml and unlike other reported methods used for analyte detection, with this technique we are able to counter the Hook effect which is a limiting factor in many lateral flow immunoassays.

Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.