Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

CdS/Bi2S3/NiS ternary heterostructure-based photoelectrochemical immunosensor for the sensitive detection of carbohydrate antigen 125.

The sensitive, accurate and rapid detection of carbohydrate antigen 125 (CA125) is essential for the early diagnosis and clinical management of ovarian cancer, but there is still challenge. Herein, a photoelectrochemical (PEC) immunosensor based on CdS/Bi2S3/NiS ternary sulfide heterostructured photocatalyst was presented for the detection of CA125. The CdS/Bi2S3/NiS was synthesized by a one-step hydrothermal approach. The heterojunction comprising of CdS and Bi2S3 could separate photogenerated carriers, the introduced narrow bandgap NiS could act as electron-conducting bridge to facilitate the transfer of interfacial photogenerated electrons, thereby improving the photoelectric conversion efficiency. Due to their synergistic effect, the photocurrent response produced by the composite was up to 14.6 times of pure CdS. On the basis, a PEC immunosensor was constructed by introducing the CA125 antibody through thioglycolic acid linkage. It was found that the resulting immunosensor showed good performance. Under the optimized conditions, its linear detection range was as wide as 1 pg mL-1-50 ng mL-1, and the detection limit was low to 0.85 pg mL-1. Furthermore, we experimentally tested its anti-interference, stability and reproducibility, and satisfactory results were achieved. The practicable feasibility of the sensor was confirmed by testing serum sample. Thus this work provided a simple, fast and enough sensitive approach for CA125 monitoring.

Atomically Co-dispersed nitrogen-doped carbon for sensitive electrochemical immunoassay of breast cancer biomarker CA15-3.

The development of an ultrasensitive and precise measurement of a breast cancer biomarker (cancer antigen 15-3; CA15-3) in complex human serum is essential for the early diagnosis of cancer in groups of healthy populations and the treatment of patients. However, currently available testing technologies suffer from insufficient sensitivity toward CA15-3, which severely limits early large-scale screening of breast cancer patients. We report a versatile electrochemical immunoassay method based on atomically cobalt-dispersed nitrogen-doped carbon (Co-NC)-modified disposable screen-printed carbon electrode (SPCE) with alkaline phosphatase (ALP) and its metabolite, ascorbic acid 2-phosphate (AAP), as the electrochemical labeling and redox signaling unit for sensitive detection of low-abundance CA15-3. During electrochemical detection by differential pulse voltammetry (DPV), it was found that the Co-NC-SPCE electrode did not have a current signal response to the AAP substrate; however, it had an extremely favorable response current to ascorbic acid (AA). Based on the above principle, the target CA15-3-triggered immunoassay enriched ALP-catalyzed AAP produces a large amount of AA, resulting in a significant change in the system current signal, thereby realizing the highly sensitive detection of CA15-3. Under the optimal AAP substrate concentration and ALP catalysis time, the Co-NC-SPCE-based electrochemical immunoassay demonstrated a good DPV current for CA15-3 in the assay interval of 1.0 mU/mL to 10,000 mU/mL, with a calculated limit of detection of 0.38 mU/mL. Since Co-NC-SPCE has an excellent DPV current response to AA and employs split-type scheme, the constructed electrochemical immunoassay has the merits of high preciseness and anti-interference, and its clinical diagnostic results are comparable to those of commercial kits.

Photoelectrochemical immunoassay of cardiac troponin I based on ZnTCPP/CdIn2S4 type-II heterojunction and co-catalyzed precipitation biolabeling.

CdIn2S4 and zinc tetrakis(4-carboxyphenyl)porphyrin (ZnTCPP) were synthesized by hydrothermal method, and an organic dye-sensitized inorganic semiconductor ZnTCPP/CdIn2S4 type II heterojunction was constructed on a fluorine-doped tin oxide (FTO) substrate electrode. A sandwich immunostructure for signal-attenuation photoelectrochemical (PEC) detection of cardiac troponin I (cTnI) was constructed using the ZnTCPP/CdIn2S4/FTO photoanode and a horseradish peroxidase (HRP)-ZnFe2O4-Ab2-bovine serum albumin (BSA) immunolabeling complex. The bioenzyme HRP and the HRP-like nanozyme ZnFe2O4 can co-catalyze the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 to produce an insoluble precipitate on the photoanode, thus notably reducing the anodic photocurrent for quantitative determination of cTnI. Under the optimal conditions, the photocurrent at 0 V vs. SCE in 0.1 M phosphate buffer solution (pH 7.40) containing 0.1 M ascorbic acid was linear with the logarithm of cTnI concentration from 500 fg mL-1 to 50.0 ng mL-1, and the limit of detection (LOD, S/N = 3) is 0.15 pg mL-1. Spiked recoveries were 95.1% ~ 104% for assay of cTnI in human serum samples.

An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma.

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Multiplex Bead Assay for the Serological Surveillance of Measles and Rubella.

There is increasing interest in evaluating antibody responses to multiple antigen targets in a single assay. Immunity to measles and rubella are often evaluated together because immunity is provided through combined vaccines and because routine immunization efforts and surveillance for measles and rubella pathogens are combined in many countries. The multiplex bead assay (MBA) also known as the multiplex immunoassay (MIA) described here combines the measurement of measles- and rubella-specific IgG antibodies in serum quantitatively according to international serum standards and has been successfully utilized in integrated serological surveillance.

Hollow versatile Ag@Pt alloy nanoparticles with nanozyme activity for detection and photothermal sterilization of Helicobacter pylori.

In view of a large number of people infected with Helicobacter pylori (H. pylori) with great harm followed, there is an urgent need to develop a non-invasive, easy-to-operate, and rapid detection method, and to identify effective sterilization strategies. In this study, highly specific nanoprobes with nanozyme activity, Ag@Pt nanoparticles (NPs) with the antibody, were utilized as a novel lateral flow immunoassay (LFIA). The optical label (Ag@Pt NPs) was enhanced by the introduction of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) and compared with a gold nanoparticles (Au NPs) optical label. Under the optimal condition, Ag@Pt-LFIA and TMB-enhanced Ag@Pt-LFIA for H. pylori were successfully established, two of which were over twofold and 100-fold more sensitive than conventional visual Au NP-based LFIA, respectively. Furthermore, Ag@Pt NPs with the antibody irradiated with NIR laser (808 nm) at a power intensity of 550 mW/cm2 for 5 min exhibited a remarkable antibacterial effect. The nanoprobes could close to bacteria through effective interactions between antibodies and bacteria, thereby benefiting photothermal sterilization. Overall, Ag@Pt NPs provide promising applications in pathogen detection and therapeutic applications.

Microfluidic Cartridge for Bead-Based Affinity Assays.

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.

Enhancing the Efficiency of Conventional Surface Immunoassays Within Standard Labware Using Microscale Flows.

In this chapter, we present the design and fabrication of a device and implementation of a protocol to realize increased efficiency of immunoassays within microtiter plates. The device, WellProbe, is a 3D-structured probe that can be used to deliver precise flows at the bottom of standard well plates to establish concentric areas of shear stress intensities using hydrodynamically confined flows. The protocols involve both operation and data analysis.

Fe-single-atom catalysts boosting electrochemiluminescence via bipolar electrode integrated with its peroxidase-like activity for bioanalysis.

Multifunctional single-atom catalysts (SACs) have been extensively investigated as outstanding signal amplifiers in bioanalysis field. Herein, a type of Fe single-atom catalysts with Fe-nitrogen coordination sites in nitrogen-doped carbon (Fe-N/C SACs) was synthesized and demonstrated to possess both catalase and peroxidase-like activity. Utilizing Fe-N/C SACs as dual signal amplifier, an efficient bipolar electrode (BPE)-based electrochemiluminescence (ECL) immunoassay was presented for determination of prostate-specific antigen (PSA). The cathode pole of the BPE-ECL platform modified with Fe-N/C SACs is served as the sensing side and luminol at the anode as signal output side. Fe-N/C SACs could catalyze decomposition of H2O2 via their high catalase-like activity and then increase the Faraday current, which can boost the ECL of luminol due to the electroneutrality in a closed BPE system. Meanwhile, in the presence of the target, glucose oxidase (GOx)-Au NPs-Ab2 was introduced through specific immunoreaction, which catalyzes the formation of H2O2. Subsequently, Fe-N/C SACs with peroxidase-like activity catalyze the reaction of H2O2 and 4-chloro-1-naphthol (4-CN) to generate insoluble precipitates, which hinders electron transfer and then inhibits the ECL at the anode. Thus, dual signal amplification of Fe-N/C SACs was achieved by increasing the initial ECL and inhibiting the ECL in the presence of target. The assay exhibits sensitive detection of PSA linearly from 1.0 pg/mL to 100 ng/mL with a detection limit of 0.62 pg/mL. The work demonstrated a new ECL enhancement strategy of SACs via BPE system and expands the application of SACs in bioanalysis field.

Rapid, on-site quantitative determination of mycotoxins in grains using a multiple time-resolved fluorescent microsphere immunochromatographic test strip.

The label probe plays a crucial role in enhancing the sensitivity of lateral flow immunoassays. However, conventional fluorescent microspheres (FMs) have limitations due to their short fluorescence lifetime, susceptibility to background fluorescence interference, and inability to facilitate multi-component detection. In this study, carboxylate-modified Eu(III)-chelate-doped polystyrene nanobeads were employed as label probes to construct a multiple time-resolved fluorescent microsphere-based immunochromatographic test strip (TRFM-ICTS). This novel TRFM-ICTS facilitated rapid on-site quantitative detection of three mycotoxins in grains: Aflatoxin B1 (AFB1), Zearalenone (ZEN), and Deoxynivalenol (DON). The limit of detection (LOD) for AFB1, ZEN, and DON were found to be 0.03 ng/g, 0.11 ng/g, and 0.81 ng/g, respectively. Furthermore, the TRFM-ICTS demonstrated a wide detection range for AFB1 (0.05-8.1 ng/g), ZEN (0.125-25 ng/g), and DON (1.0-234 ng/g), while maintaining excellent selectivity. Notably, the test strip exhibited remarkable stability, retaining its detection capability even after storage at 4 °C for over one year. Importantly, the detection of these mycotoxins relied solely on simple manual operations, and with a portable reader, on-site detection could be accomplished within 20 min. This TRFM-ICTS presents a promising solution for sensitive on-site mycotoxin detection, suitable for practical application in various settings due to its sensitivity, accuracy, simplicity, and portability.

Tracking epithelial-mesenchymal transition in breast cancer cells based on a multiplex electrochemical immunosensor.

Epithelial-mesenchymal transition (EMT) promotes tumor cell infiltration and metastasis. Tracking the progression of EMT could potentially indicate early cancer metastasis. A key characteristic of EMT is the dynamic alteration in the molecular levels of E-cadherin and N-cadherin. Traditional assays have limited sensitivity and multiplexing capabilities, relying heavily on cell lysis. Here, we developed a multiplex electrochemical biosensor to concurrently track the upregulation of N-cadherin expression and reduction of E-cadherin in breast cancer cells undergoing EMT. Small-sized gold nanoparticles (Au NPs) tagged with redox probes (thionin or amino ferrocene) and bound to two types of antibodies were used as distinguishable signal tags. These tags specifically recognized E-cadherin and N-cadherin proteins on the tumor cell surface without cross-reactivity. The diphenylalanine dipeptide (FF)/chitosan (CS)/Au NPs (FF-CS@Au) composites with high surface area and good biocompatibility were used as the sensing platforms for efficiently fixing cells and recording the dynamic changes in electrochemical signals of surface proteins. The electrochemical immunosensor allowed for simultaneous monitoring of E- and N-cadherins on breast cancer cell surfaces in a single run, enabling tracking of the EMT dynamic process for up to 60 h. Furthermore, the electrochemical detection results are consistent with Western blot analysis, confirming the reliability of the methodology. This present work provides an effective, rapid, and low-cost approach for tracking the EMT process, as well as valuable insights into early tumor metastasis.

Proximity extension assay in cerebrospinal fluid identifies neurofilament light chain as biomarker of neurodegeneration in sporadic cerebral amyloid angiopathy.

BACKGROUND: Sporadic cerebral amyloid angiopathy (sCAA) is a disease characterised by the progressive deposition of the amyloid beta (Aß) in the cerebral vasculature, capable of causing a variety of symptoms, from (mild) cognitive impairment, to micro- and major haemorrhagic lesions. Modern diagnosis of sCAA relies on radiological detection of late-stage hallmarks of disease, complicating early diagnosis and potential interventions in disease progression. Our goal in this study was to identify and validate novel biomarkers for sCAA. METHODS: We performed a proximity extension assay (PEA) on cerebrospinal fluid (CSF) samples of sCAA/control participants (n = 34/51). Additionally, we attempted to validate the top candidate biomarker in CSF and serum samples (n = 38/26) in a largely overlapping validation cohort, through analysis with a targeted immunoassay. RESULTS: Thirteen proteins were differentially expressed through PEA, with top candidate NFL significantly increased in CSF of sCAA patients (p < 0.0001). Validation analyses using immunoassays revealed increased CSF and serum NFL levels in sCAA patients (both p < 0.0001) with good discrimination between sCAA and controls (AUC: 0.85; AUC: 0.79 respectively). Additionally, the CSF: serum NFL ratio was significantly elevated in sCAA (p = 0.002). DISCUSSION: Large-scale targeted proteomics screening of CSF of sCAA patients and controls identified thirteen biomarker candidates for sCAA. Orthogonal validation of NFL identified NFL in CSF and serum as biomarker, capable of differentiating between sCAA patients and controls.

Retrospective analysis of canine fecal flotation and coproantigen immunoassay hookworm positive results in Greyhounds and other dog breeds.

Recent studies demonstrated that Greyhounds are commonly infected with Ancylostoma caninum and these infections have been shown to be resistant to anthelmintics. This study evaluated samples submitted to a commercial reference laboratory (IDEXX Laboratories) for canine fecal flotation zinc sulfate centrifugation and coproantigen immunoassay between January 1, 2019, and July 30, 2023 for evidence that Greyhounds were more often positive for Ancylostoma spp. (hookworms) compared to other breeds. The purpose of the study was to determine if Greyhounds were more likely to be hookworm-positive compared to other breeds, if Greyhounds on preventives with efficacy against hookworm infections are more likely to test positive than other breeds, if their infections take longer to resolve, to estimate how long this takes and to assess whether the proportion of hookworm positive tests for all breeds is increasing over time. Records of 25,440,055 fecal results were obtained representing 17,671,724 unique dogs. Of these, 49,795 (∼0.3%) were Greyhounds. The overall odds ratio (OR) of 15.3 (p < 0.001) suggests that Greyhounds are at significantly higher risk than other breeds for hookworm positive float findings, and the OR of 14.3 (p < 0.001) suggests significantly higher risk for hookworm antigen positive results. The median time to negative testing event from the Turnbull distribution estimate was in the interval of 1-2 days for other breeds and 71-72 days for Greyhounds. These results provide evidence that anthelmintic resistant A. caninum strains may be having population-level impacts on the frequency and duration of infections in Greyhounds. The findings have broader health implications beyond Greyhounds as MADR A. caninum strains could spread to other breeds and even pet owners.

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

BACKGROUND: The 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. RESULTS: The 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. SIGNIFICANCE: The 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.

Using Temporally and Spatially Resolved Measurements to Improve the Sensitivity of Fluorescence-Based Immunoassays.

Detecting low concentrations of biomarkers is essential in clinical laboratories. To improve analytical sensitivity, especially in identifying fluorescently labeled molecules, typical optical detection systems, consisting of a photodetector or camera, utilize time-resolved measurements. Taking a different approach, magnetic modulation biosensing (MMB) is a novel technology that combines fluorescently labeled probes and magnetic particles to create a sandwich assay with the target molecules. By concentrating the target molecules and then using time-resolved measurements, MMB provides the rapid and highly sensitive detection of various biomarkers. Here, we propose a novel signal-processing algorithm that enhances the detection and estimation of target molecules at low concentrations. By incorporating both temporally and spatially resolved measurements using human interleukin-8 as a target molecule, we show that the new algorithm provides a 2-4-fold improvement in the limit of detection and an ~25% gain in quantitative resolution.

Disposable graphene-oxide screen-printed electrode integrated with portable device for detection of SARS-CoV-2 in clinical samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis is the need of the hour, as cases are persistently increasing, and new variants are constantly emerging. The ever-changing nature of the virus leading to multiple variants, has brought an imminent need for early, accurate and rapid detection methods. Herein, we have reported the design and fabrication of Screen-Printed Electrodes (SPEs) with graphene oxide (GO) as working electrode and modified with specific antibodies for SARS-CoV-2 Receptor Binding Domain (RBD). Flexibility of design, and portable nature has made SPEs the superior choice for electrochemical analysis. The developed immunosensor can detect RBD as low as 0.83 fM with long-term storage capacity. The fabricated SPEs immunosensor was tested using a miniaturized portable device and potentiostat on 100 patient nasopharyngeal samples and corroborated with RT-PCR data, displayed 94 % sensitivity. Additionally, the in-house developed polyclonal antibodies detected RBD antigen of the mutated Omicron variant of SARS-CoV-2 successfully. We have not observed any cross-reactivity/binding of the fabricated immunosensor with MERS (cross-reactive antigen) and Influenza A H1N1 (antigen sharing common symptoms). Hence, the developed SPEs sensor may be applied for bedside point-of-care diagnosis of SARS-CoV-2 using miniaturized portable device, in clinical samples.

Nanobodies-based colloidal gold immunochromatographic assay for specific detection of parathion.

Parathion is one of organophosphorus pesticide, which has been prohibited in agricultural products due to its high toxicity to human beings. However, there are still abuse cases for profit in agricultural production. Hence, we established nanobodies-based colloidal gold immunochromatographic assay (GICA) in which nanobodies (Nbs) as an excellent recognition element, greatly improving the stability and sensitivity of ICA. Under the optimal conditions, the developed Nbs-based GICA showed a cut-off value of 50 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 2.39 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.15 ng/mL which was significantly 50-fold higher sensitivity than the commercial mAb-ICA. Additionally, this method exhibited good recoveries for the detection of cabbage, cucumber, and orange samples and excellent correlation with the UPLC-MS/MS method. The results showed that this method developed in this work based on nanobody can be used in practical detection of parathion in foods and nanobody is novel prospective antibody resource for immunoassays of chemical contaminants.