NIR-responsive photoelectrochemical sensing platform for the simultaneous determination of tetrodotoxin and okadaic acid in Nassariidae.

Simultaneous detection of tetrodotoxin (TTX) and okadaic acid (OA) is important for seafood safety. In this work, a novel paper electrode-based near-infrared (NIR) light-responsive photoelectrochemical (PEC) immunosensor was constructed using Ag2S quantum dots (QDs) and NaYF4: Yb, Er upconversion nanoparticles (UCNPs) matched with BiOI for the simultaneous detection of TTX and OA in aquatic products. A low-cost, easily prepared gold nanoparticle-functionalized paper-based screen-printed electrode with six channels was designed to immobilize OA and Ab1 of TTX. Correspondingly, PEC signal immunoprobes (BiOI@UCNPs-Ab and Ab2-Ag2S QDs) with NIR-light response were introduced to construct competitive-type and sandwich-type PEC immunosensors for OA and TTX, respectively. Under optimal conditions, the linear ranges for TTX and OA were 0.001-100 and 0.001-80 ng mL-1, respectively, and the detection limits were 5 and 7 pg mL-1, respectively. The proposed sensor was successfully used for the simultaneous analysis of TTX and OA in Nassariidae samples.

"Fluorescence-wavelength" label-free POCT tandem with "fluorescence-photothermal" nanobody-immunosensor for detecting BSA and ß-lactoglobulin.

Two carbon dots (CDs) (λEm = 525 nm, G-CDs and λEm = 640 nm, R-CDs) were synthesized from citric acid and urea. The bovine serum albumin (BSA) responsiveness of the R-CDs was used to develop a "fluorescence-wavelength" label-free point of care testing (POCT) for the detection of the milk quality marker BSA with the detection limit (LOD) of 4.89 µg/mL for fluorescence mode and 3.38 µg/mL for wavelength mode. In addition, R-CDs were found to have hydroxyl radical (·OH)-dependent fluorescence quenching properties, and a "fluorescence-photothermal" immunosensor based on nanobodies was constructed by introducing the fluorescence signal of R-CDs@BSA and the photothermal signal of oxTMB for the detection of ß-lactoglobulin (ß-LG) with the LOD of 0.034 ng/mL for fluorescence mode and 0.075 ng/mL for photothermal mode. The tandem detection of POCT and immunosensor enables the simultaneous and highly sensitive detection of BSA and ß-LG after only simple dilution of less than 5 µL of sample.

Development of an immunochromatographic assay for the rapid screening of torasemide in health food.

Torasemide is a new loop diuretic agent added illegally to health foods for weight loss, which can result in serious health risks for consumers. A rapid and sensitive immunochromatographic assay for detection of torasemide (ICA) based on a new monoclonal antibody (mAb) was developed. The mAb IC50 for torasemide was 0.93 ng/mL, and the mAb did not cross-react with other analogues. In PBS, the cut-off value and limit of detection were 1 ng/mL and 0.11 ng/mL, respectively, with a linear range between 0.61 and 6.13 ng/mL. In slimming tablet and capsule samples, the cut-off value was 5 ng/g. Recoveries were 101.1% ± 1.7%-106.1% ± 1.3% in tablet samples and 101.2% ± 2.2%-109.1% ± 3.9% in capsule samples, with coefficients of variation 2.1%-3.1% and 1.8%-3.6%, respectively, consistent with existing LC-MS/MS methods. Therefore, the ICA is suitable for use in slimming tablet and capsule samples.

Magnetic-encoded fluorescent nanospheres-based competitive immunoassay for near-simultaneous detection of four mycotoxins in wheat.

Simultaneous detection of mycotoxins is important for food safety. In this study, a magnetic-encoded fluorescent nanosphere-based competitive immunosensor (cFMEIS) with 2×2 array was first developed for simultaneous detection of aflatoxin B1 (AFB1), ochratoxin (OTA), deoxynivalenol (DON), and zearalenone (ZEN) in wheat. Specifically, magnetic nanoparticles with strong and weak responses were conjugated with mycotoxin antigens as capture probes. Fluorescent nanoparticles doped with europium ion (Eu3+) and terbium ion (Tb3+) with red and green emission were coupled with mycotoxin antibodies as signal probes. Using a magnetic field, immune complexes were sequentially separated in a complex system and fluorescently detected. The detection limits of AFB1, DON, OTA, and ZEN were 0.032, 0.141, 0.097, and 0.376 µg/kg, respectively. The recoveries in the certified reference material of wheat flour ranged from 81.6 to 120.0 %. Owing to its high accuracy, selectivity, and sensitivity, the cFMEIS shows great promise as an efficient and sensitive multitarget sensor for mycotoxins.

Ultrasensitive immunosensor for multiplex detection of cancer biomarkers carcinoembryonic antigen (CEA) and yamaguchi sarcoma viral oncogene homolog 1 (YES1) based on eco-friendly synthesized gold nanoparticles.

Cancer is one of the most extensive diseases with the highest mortality rate, accounting for almost 10 million deaths in 2020. The most common cancers are breast, lung, colon and rectum and prostate cancers. Of these, lung cancer, accounted for about 1.8 million of all cancer deaths (25%) in 2020. Detection of cancer relies on presence of biomarkers such as DNA molecules, proteins and metabolites released by cancerous cells into the circulation. Carcinoembryonic antigen (CEA) is one of the biomarkers that has been used for the detection of lung cancer. However, CEA is not specific to lung cancer since it is also manifested in gastric cancer, pancreatic cancer, colorectal cancer, and breast cancer. Recently, v-YES1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1) was described as a specific biomarker for lung cancer. The detection of both CEA and YES1 would give more precise and authentic information for detecting lung cancer. This is because detection of a single tumor marker usually limits the precision in tumor diagnosis, due to the fact that several cancers have more than one marker linked with their prevalence. Whereas traditional methods have been used for the detection of CEA, electrochemical immunosensors have attracted considerable attention owing to their profound advantages, including fast response, miniaturization, high selectivity, low sample requirements and magnificent sensitivity. The fabrication of a multiplex and simultaneous immunosensor is met with challenge of preparation of distinguishable immunoprobes with different redox activities. This can be addressed by incorporation of electroactive Nano metals into the sensing platform. In this study, gold nanoparticles were used for the fabrication of an ultrasensitive sandwich electrochemical multiplex immunosensor for simultaneous detection of CEA and YES1. Under optimized conditions, the electrochemical immunosensor detection limit for YES1 and CEA was found to be 0.0022 and 0.0034 ng/mL respectively within a linear range of 0.1-50 ng/mL. The proposed immunosensor proved to be stable for up to 2 weeks and had negligible cross reactivity towards various interfering compounds in human plasma. This study reports that gold nanoparticles can be bio synthesized using shade dried Mangifera indica leaves extract. The bio-synthesized gold nanoparticles coupled with thiolated protein G can be used for fabrication of a multiplex immunosensor for detection of CEA and YES1. The proposed immunosensor can provide a new approach for early diagnosis of circulating cancer biomarkers and holds great promise for application in clinical diagnosis.

Highly bright and stable CsPbBr3 perovskite nanocrystals with amphiphilic polymer binding based dual-readout lateral flow immunoassay for detection of carcinoembryonic antigen.

Rapid and highly sensitive detection of tumor marker (TM) is critical for the early diagnosis and treatment of cancers. Herein, utilizing highly bright and water-stable CsPbBr3 perovskite nanocrystals (NCs) capped with amphiphilic polymer ligand of octylamine-modified polyacrylic acid (OPA) and gold nanoparticles (AuNPs) as reporters, a lateral flow immunoassay (LFIA) strip is developed for fluorescence and colorimetric dual-mode detection of carcinoembryonic antigen (CEA). The prepared CsPbBr3 NCs capped by an amphiphilic polymeric of OPA ligand showed high stability and bright fluorescence. Moreover, the AuNPs immunoprobes were captured with CEA antigen and quench the green fluorescence of CsPbBr3/OPA NCs on the T line due to the inner filter effect (IFE). Therefore, CEA could be quantitative analyzed by the dual-readout of fluorescence and colorimetric signal. The detection limits of CEA can reach as low as 0.023 ng/mL and 0.027 ng/mL for the fluorescence and colorimetric mode, respectively. Good specificity and reproducibility were also demonstrated for this method. Finally, the CsPbBr3/OPA NCs-based LFIA showed good accuracy in detection of CEA level from clinical serum samples. This work firstly enables the application of CsPbBr3 perovskite NCs in a LFIA, displaying great potential in point-of-care clinical diagnosis.

Magnetic copper silicate and boronic acid-conjugated AuNCs@keratin-based electrochemical/fluorescent dual-sensing for carcinoembryonic antigen.

Boronic Acid Sensitivity, selectivity, and reliability are of great importance for tumor diagnosis. Herein, we proposed a novel electrochemical and fluorescent dual-sensing strategy to detect carcinoembryonic antigens (CEA). To this end, monodisperse spindle-like magnetic copper silicate (FeOx@C@CS) was prepared with multiple active sites to immobilize the CEA antibody. Moreover, magnetic properties improved the anti-interference ability and sensitivity to endow the assay for complex samples. In addition, boronic acid-conjugated gold nanocluster (AuNCs@keratin-BA) was prepared as an electrochemical and fluorescent dual-signal indicator. Thus, the sandwich structure of FeOx@C@CS/CEA/AuNCs@keratin-BA was formed for electrochemical/fluorescent dual-modality assay. Under optimal conditions, the quantitation range of 12.5 fg mL-1-37.5 pg mL-1 and detection limit of 4.3 fg mL-1 were obtained for the electrochemical strategy. The fluorescence detection owned the linear range of 0.05 pg mL-1-7.5 pg mL-1 with a detection limit of 0.025 pg mL-1. Dual-modality assay improved the accuracy and efficiency of CEA detection to meet the requirement of tumor diagnosis, while chemical identification and signal transduction lay an important foundation for engineering advanced nanomaterials for clinical applications.

Electrochemical immunosensor based on hollow Pt@Cu2O as a signal label for dual-mode detection of procalcitonin.

As a reliable biomarker to evaluate the severity of sepsis, sensitive and accurate detection of procalcitonin (PCT) is essential. In this study, a dual-mode electrochemical immunosensor based on Au/ZIF-8 as substrate and Pt@Cu2O as signal label was constructed for the detection of PCT. By loading Au nanoparticles onto rhombic dodecahedral ZIF-8, the substrate (Au/ZIF-8) has large specific surface area and can immobilize antibody (Ab1) by Au-N bonds. Meanwhile, hollow Pt@Cu2O nanocomposite with excellent peroxidase-like activity and electrocatalytic activity were synthesized as signal label. In the process of electrochemical testing, Pt@Cu2O catalyzed the reduction of hydrogen peroxide (H2O2) and further promotes the oxidation of hydroquinone (HQ) to achieve the synergistic amplification of electrochemical signals. The proposed immunosensor detected PCT by amperometric i-t and differential pulse voltammetry (DPV) tests with a good linear response and low limit of detection (i-t: 0.70 fg/mL and DPV: 0.40 fg/mL) in the range of 10 fg/mL∼100 ng/mL. The immunosensor exhibited excellent sensitivity and accuracy, indicating the potential application of this method for PCT detection.

Electrochemical magneto-immunoassay for detection of ADAM10 Alzheimer's biomarker using gold nanoparticles as label.

Alzheimer's disease (AD), a neurodegenerative and progressive illness with no known cure, is the most frequent cause of dementia in older adults. Dementia in AD is usually preceded by a stage of cognitive decline known as mild cognitive impairment (MCI). MCI has gained attention as an ideal target for prevention and early interventions, considering its reversible characteristic. Here, we propose a magneto-immunoassay based on a low-cost screen-printed electrode for detecting soluble ADAM10 in plasma samples, a potential biomarker for early AD diagnosis. We present a sandwich immunoassay using magnetic beads modified with antibodies to capture ADAM10 from plasma samples and using gold nanoparticles (AuNPs) as an electrochemical label. The assay was designed to accurately detect ADAM10 in diluted plasma with a limit of detection (LoD) of 32.5 pg/mL and a dynamic linear range of 10.0-1000.0 pg/mL. Twenty-three plasma samples from the elderly, including patients with AD, MCI, and healthy subjects (negative control), were analyzed by the magneto-immunoassay and enzyme-linked immunosorbent assay (ELISA), and the ADAM10 levels correlated. This work shows the potential of this protein as a biomarker in the early diagnosis and progression of AD and provides an interesting disposable device with capabilities for applications as point-of-care (PoC) to measure ADAM10 levels.

Bioscreening specific peptide-expressing phage and its application in sensitive dual-mode immunoassay of SARS-CoV-2 spike antigen.

Biorecognition components with high affinity and selectivity are vital in bioassay to diagnose and treat epidemic disease. Herein a phage display strategy of combining single-amplification-panning with non-amplification-panning was developed, by which a phage displaying cyclic heptapeptide ACLDWLFNSC (peptide J4) with good affinity and specificity to SARS-CoV-2 spike protein (SP) was identified. Molecular docking suggests that peptide J4 binds to S2 subunit by hydrogen bonding and hydrophobic interaction. Then the J4-phage was used as the capture antibody to establish phage-based chemiluminescence immunoassay (CLIA) and electrochemical impedance spectroscopy (EIS) analytical systems. The as-proposed dual-modal immunoassay platform exhibited good sensitivity and reliability in SARS-CoV-2 SP and pseudovirus assay. The limit of detection for SARS-CoV-2 SP by EIS immunoassay is 0.152 pg/mL, which is dramatically lower than that of 42 pg/mL for J4-phage based CLIA. Further, low to 40 transducing units (TU)/mL, 10 TU/mL SARS-CoV-2 pseudoviruses can be detected by the proposed J4-phage based CLIA and electrochemical immunosensor, respectively. Therefore, the as-developed dual mode immunoassays are potential methods to detect SARS-CoV-2. It is also expected to explore various phages with specific peptides to different targets for bioanalysis.

Comparison of Direct and Extraction Immunoassay Methods With Liquid Chromatography-Tandem Mass Spectrometry Measurement of Urinary Free Cortisol for the Diagnosis of Cushing's Syndrome.

Background: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing's syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Methods: Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected. A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing-Bablok regression and Bland-Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Results: Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r=0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r=0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LC-MS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8-1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Conclusions: Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Performance of plasma Aß42/40, measured using a fully automated immunoassay, across a broad patient population in identifying amyloid status.

BACKGROUND: Plasma biomarkers have emerged as promising screening tools for Alzheimer's disease (AD) because of their potential to detect amyloid ß (Aß) accumulation in the brain. One such candidate is the plasma Aß42/40 ratio (Aß42/40). Unlike previous research that used traditional immunoassay, recent studies that measured plasma Aß42/40 using fully automated platforms reported promising results. However, its utility should be confirmed using a broader patient population, focusing on the potential for early detection. METHODS: We recruited 174 participants, including healthy controls (HC) and patients with clinical diagnoses of AD, frontotemporal lobar degeneration, dementia with Lewy bodies/Parkinson's disease, mild cognitive impairment (MCI), and others, from a university memory clinic. We examined the performance of plasma Aß42/40, measured using the fully automated high-sensitivity chemiluminescence enzyme (HISCL) immunoassay, in detecting amyloid-positron emission tomography (PET)-derived Aß pathology. We also compared its performance with that of Simoa-based plasma phosphorylated tau at residue 181 (p-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL). RESULTS: Using the best cut-off derived from the Youden Index, plasma Aß42/40 yielded an area under the receiver operating characteristic curve (AUC) of 0.949 in distinguishing visually assessed 18F-Florbetaben amyloid PET positivity. The plasma Aß42/40 had a significantly superior AUC than p-tau181, GFAP, and NfL in the 167 participants with measurements for all four biomarkers. Next, we analyzed 99 participants, including only the HC and those with MCI, and discovered that plasma Aß42/40 outperformed the other plasma biomarkers, suggesting its ability to detect early amyloid accumulation. Using the Centiloid scale (CL), Spearman's rank correlation coefficient between plasma Aß42/40 and CL was -0.767. Among the 15 participants falling within the CL values indicative of potential future amyloid accumulation (CL between 13.5 and 35.7), plasma Aß42/40 categorized 61.5% (8/13) as Aß-positive, whereas visual assessment of amyloid PET identified 20% (3/15) as positive. CONCLUSION: Plasma Aß42/40 measured using the fully automated HISCL platform showed excellent performance in identifying Aß accumulation in the brain in a well-characterized cohort. This equipment may be useful for screening amyloid pathology because it has the potential to detect early amyloid pathology and is readily applied in clinical settings.

FeNC nanozyme-based electrochemical immunoassay for sensitive detection of human epidermal growth factor receptor 2.

The proof-of-concept of sensitive electrochemical immunoassay for the quantitative monitoring of human epidermal growth factor receptor 2 (HER2) is reported. The assay is carried out on iron nitrogen-doped carbon (FeNC) nanozyme-modified screen-printed carbon electrode using chronoamperometry. Introduction of target HER2 can induce the sandwiched immunoreaction between anti-HER2 monoclonal antibody-coated microplate and biotinylated anti-HER2 polyclonal antibody. Thereafter, streptavidin-glucose oxidase (GOx) conjugate is bonded to the detection antibody. Upon addition of glucose, 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized through the produced H2O2 with the assistance of GOx and FeNC nanozyme. The oxidized TMB is determined via chronoamperometry. Experimental results revealed that electrochemical immunosensing system exhibited good amperometric response, and allowed the detection of target HER2 as low as 4.5 pg/mL. High specificity and long-term stability are acquired with FeNC nanozyme-based sensing strategy. Importantly, our system provides a new opportunity for protein diagnostics.

Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity.

The profound understanding and detailed evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (SCoV2-S) protein and specific antibody interaction mechanism is of high importance in the development of immunosensors for COVID-19. In the present work, we studied a model system of immobilized SCoV2-S protein and specific monoclonal antibodies by molecular dynamics of immune complex formation in real time. We simultaneously applied spectroscopic ellipsometry and quartz crystal microbalance with dissipation to reveal the features and steps of the immune complex formation. We showed direct experimental evidence based on acoustic and optical measurements that the immune complex between covalently immobilized SCoV2-S and specific monoclonal antibodies is formed in two stages. Based on these findings it was demonstrated that applying a two-step binding mathematical model for kinetics analysis leads to a more precise determination of interaction rate constants than that determined by the 1:1 Langmuir binding model. Our investigation showed that the equilibrium dissociation constants (KD) determined by a two-step binding model and the 1:1 Langmuir model could differ significantly. The reported findings can facilitate a deeper understanding of antigen-antibody immune complex formation steps and can open a new way for the evaluation of antibody affinity towards corresponding antigens.

Comparative assessment of the SjSAP4-incorporated gold immunochromatographic assay for the diagnosis of human schistosomiasis japonica.

Background: Schistosomiasis, a disease caused by parasites of the genus Schistosoma, remains a global public health threat. This study aimed to validate the diagnostic performance of a recently developed gold immunochromatographic assay (GICA) for the detection of S. japonicum infection in a rural endemic area of the Philippines. Methods: Human clinical samples were collected from 412 subjects living in Laoang and Palapag municipalities, Northern Samar, the Philippines. The presence of Schistosoma-specific antibodies in serum samples was tested with the SjSAP4-incorporated GICA strips and the results were converted to fully quantitative data by introducing an R value. The performance of the established GICA was further compared with other diagnostic tools, including the Kato-Katz (KK) technique, point-of-care circulating cathodic antigen (POC-CCA), droplet digital (dd) PCR, and enzyme-linked immunosorbent assays (ELISAs). Results: The developed GICA strip was able to detect KK positive individuals with a sensitivity of 83.3% and absolute specificity. When calibrated with the highly sensitive faecal ddPCR assay, the immunochromatographic assay displayed an accuracy of 60.7%. Globally, the GICA assay showed a high concordance with the SjSAP4-ELISA assay. The schistosomiasis positivity rate determined by the GICA test was similar to those obtained with the SjSAP4-ELISA assay and the ddPCR assay performed on serum samples (SR_ddPCR), and was 2.3 times higher than obtained with the KK method. Conclusion: The study further confirms that the developed GICA is a valuable diagnostic tool for detecting light S. japonicum infections and implies that this point-of-care assay is a viable solution for surveying endemic areas of low-intensity schistosomiasis and identifying high-priority endemic areas for targeted interventions.

The instantly blocking-based fluorescent immunochromatographic assay for the detection of SARS-CoV-2 neutralizing antibody.

Introduction: At present, there is an urgent need for the rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs) to evaluate the ability of the human body to resist coronavirus disease 2019 (COVID-19) after infection or vaccination. The current gold standard for neutralizing antibody detection is the conventional virus neutralization test (cVNT), which requires live pathogens and biosafety level-3 (BSL-3) laboratories, making it difficult for this method to meet the requirements of large-scale routine detection. Therefore, this study established a time-resolved fluorescence-blocking lateral flow immunochromatographic assay (TRF-BLFIA) that enables accurate, rapid quantification of NAbs in subjects. Methods: This assay utilizes the characteristic that SARS-CoV-2 neutralizing antibody can specifically block the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2) to rapidly detect the content of neutralizing antibody in COVID-19-infected patients and vaccine recipients. Results: When 356 samples of vaccine recipients were measured, the coincidence rate between this method and cVNT was 88.76%, which was higher than the coincidence rate of 76.97% between cVNT and a conventional chemiluminescence immunoassay detecting overall binding anti-Spike-IgG. More importantly, this assay does not need to be carried out in BSL-2 or 3 laboratories. Discussion: Therefore, this product can detect NAbs in COVID-19 patients and provide a reference for the prognosis and outcome of patients. Simultaneously, it can also be applied to large-scale detection to better meet the needs of neutralizing antibody detection after vaccination, making it an effective tool to evaluate the immunoprotective effect of COVID-19 vaccines.

Rapid and Specific Detection of B. melitensis Targeting BMEI1661 Gene Using Loop-mediated Isothermal Amplification (LAMP) Combined With Lateral Flow immunoassay (LFIA).

B. melitensis is the most pathogenic zoonotic species of Brucella transmitted to animals through fetal secretions, placenta, and vaginal discharges of infected animals and humans by ingesting unpasteurized milk, dairy products, and raw meat. Early detection of B. melitensis is essential for timely intervention and control of the disease. The gold standard diagnostic methods, such as culture, are time-consuming and may take several weeks aiding to the disease spread. Loop-mediated isothermal amplification assay (LAMP) is widely used to detect infectious pathogens. LAMP can be utilized as a rapid point-of-care test, but has lower specificity which can be enhanced by combining this test with lateral flow immunoassay. No point-of-care test is available for detecting Brucella melitensis in clinical samples. Herein, we developed a LAMP coupled with lateral flow immunoassay (LFIA) for the specific detection of B. melitensis. The sensitivity of LAMP-LFIA was found to be 12.1 fg of genomic DNA isolated from the organism, which is 100-fold more sensitive to conventional PCR and equally sensitive to Real-time (RT-PCR). Moreover, the assay demonstrated high specificity when tested against other Brucella and non-Brucella species. The infective dose of B. melitensis is relatively low for humans, which may remain undetected by conventional PCR, but will be detected using the new technique.

Computer vision-assisted smartphone microscope imaging digital immunosensor based on click chemistry-mediated microsphere counting technology for the detection of aflatoxin B1 in peanuts.

Aflatoxin B1 is a carcinogenic contaminant in food or feed, and it poses a serious health risk to humans. Herein, a computer vision-assisted smartphone microscope imaging digital (SMID) immunosensor based on the click chemistry-mediated microsphere counting technology was designed for the detection of aflatoxin B1 in peanuts. In this SMID immunosensor, the modified polystyrene (PS) microspheres were used as the signal probes and were recorded by a smartphone microscopic imaging system after immunoreaction and click chemistry reaction. The number of PS probes is adjusted by aflatoxin B1. The customized computer vision procedure was used to efficiently identify and count the obtained PS probes. This SMID immunosensor enables sensitive detection of aflatoxin B1 with a linear range from 0.001 ng/mL to 500 ng/mL, providing a simple, sensitive, and portable tool for food safety supervision.

One-step selective layer assemble: A versatile approach for the development of a SARS-CoV-2 electrochemical immunosensor.

The appearance of new viruses and diseases has made the development of rapid and reliable diagnostic tests crucial. In light of it, we proposed a new method for assembling an electrochemical immunosensor, based on a one-step approach for selective layer formation. For this purpose, a mixture containing the immobilizing agent (polyxydroxybutyrate, PHB) and the recognition element (antibodies against SARS-CoV-2 nucleocapsid protein) was prepared and used to modify a screen-printed carbon electrode with electrodeposited graphene oxide, for the detection of SARS-CoV-2 nucleocapsid protein (N-protein). Under optimum conditions, N-protein was successfully detected in three different matrixes - saliva, serum, and nasal swab, with the lowest detectable values of 50 pg mL-1, 1.0 ng mL-1, and 50 pg mL-1, respectively. Selectivity was assessed against SARS-CoV-2 receptor-binding domain protein (RBD) and antibodies against yellow fever (YF), and no significant response was observed in presence of interferents, reinforcing the suitability of the proposed one-step approach for selective layer formation. The proposed biosensor was stable for up to 14 days, and the mixture was suitable for immunosensor preparation even after 60 days of preparation. The proposed assembly strategy reduces the cost, analysis time, and waste generation. This reduction is achieved through miniaturization, which results in the decreased use of reagents and sample volumes. Additionally, this approach enables healthcare diagnostics to be conducted in developing regions with limited resources. Therefore, the proposed one-step approach for selective layer formation is a suitable, simpler, and a reliable alternative for electrochemical immunosensing.

Triple strategy-enhanced immunochromatographic assay based on APCB and AIEFM for the ultrasensitive detection of AFM1.

Aflatoxin M1 (AFM1) is highly toxic, widely distributed, and difficult to monitor, posing a serious threat to human health. Therefore, a highly sensitive, rapid, convenient, and low-cost detection method must be urgently established. In this study, a triple strategy-enhanced immunochromatographic assay (ICA) was developed to satisfy these detection requirements. First, a turn-on signal output mode of the fluorescence quenching ICA substituted the turn-off mode of the traditional ICA for sensitive response to trace AFM1, with the limit of detection (LOD) reduced by approximately 4.9-fold. Then, a novel Au and polydopamine (PDA) cogrowth chrysanthemum-like blackbody was prepared as the quenching probe to reduce the background signal. This probe combined the excellent properties of Au nanoparticles with PDA. Thus, its fluorescence quenching constant was higher than that of single Au and PDA nanoparticles by 25.8- and 4.9-fold, respectively. Furthermore, an aggregation-induced emission fluorescence microsphere with a 5.7-fold higher relative quantum yield than a commercial fluorescence microsphere was selected as the signal output carrier to improve the signal-to-noise ratio. The integration of the above triple strategies established a 53.4-fold sensitivity-enhanced fluorescence quenching ICA (LOD = 0.9 pg/mL) for detecting AFM1 in milk, providing a strong technical guarantee for the safety monitoring of milk products.