Urinary dengue NS1 detection on Au-decorated ZnO nanowire platform.

Biodetection for non-invasive diagnostics of fluids, especially urine, remains a challenge to scientists due to low target concentrations. And biological complexes of the detection target may contain contaminants that also interfere with any assay. Dengue non-structural 1 protein (Dengue NS1) is an important biomarker for dengue hemorrhagic fever and dengue shock syndrome. Here, we developed an Au-decorated nanowire platform and applied it with a sandwich fluorophore-linked immunosorbent well plate assay (FLISA) to detect Dengue NS1 in urine. For the platform, we fabricated zinc oxide (ZnO) nanowires to provide a high surface area and then coated them with gold nanoparticles (ZnO/Au nanowires) to simply modify the Dengue NS1 antibody and enhance the fluorescence intensity. Our platform employs a sandwich FLISA that exhibits high sensitivity, specifically detecting Dengue NS1 with a limit of detection (LOD) of 1.35 pg/mL. This LOD was 4500-fold lower than the LOD of a commercially available kit for Dengue NS1 enzyme-linked immunosorbent assay. We believe that our ZnO/Au nanowire platform has the potential to revolutionize the field of non-invasive diagnostics for dengue.

The serodiagnositic value of Chlamydia trachomatis antigens in antibody detection using luciferase immunosorbent assay.

Introduction: Among the different antigens used in the detection of anti-Chlamydia trachomatis antibodies, significant differences in sensitivity and specificity have been observed. Further evaluation of C. trachomatis antigens in antibody detection is urgently needed for the development and application of C. trachomatis serologic assays. Methods: Chlamydia trachomatis antigens Pgp3, TmeA, InaC, and HSP60 were selected and used in luciferase immunosorbent assay (LISA). The detection results obtained from well-defined C. trachomatis positive and negative samples were compared with the commercial C. trachomatis ELISA (Mikrogen) for performance evaluation. Results: Pgp3, TmeA, InaC, and HSP60-based LISA showed sensitivity of 92.8, 88.8, 90.4, and 94.4%, and specificity of 99.2, 99.2, 99.2, and 92%, respectively. ROC analysis indicated that Pgp3-based LISA showed similar performance to Mikrogen ELISA (AUC 0.986 vs. 0.993, p = 0.207). Furthermore, four C. trachomatis antigens achieved strong diagnostic efficiency, i.e., positive likelihood ratios [+LR] ≥ 10 in C. trachomatis-infected women and negative likelihood ratios [-LR] ≤ 0.1 in C. trachomatis negative low exposure risk children, but only Pgp3 and TmeA showed strong diagnostic value in general adults. In addition, Pgp3, TmeA, and InaC, but not HSP60, achieved high performance, i.e., both positive predictive value (PPV) and negative predictive value (NPV) ≥ 90.9%, and showed no significant cross-reactivity with anti-Chlamydiapneumoniae. Conclusion: Three C. trachomatis species-specific antigens Pgp3, TmeA, and InaC show superior performance in the detection of anti-C. trachomatis antibody, indicating the potential to be used in developing C. trachomatis serologic tests.

Chimeric AQP4-based immunosorbent for highly-specific removal of AQP4-IgG from blood.

Anti-aquaporin-4 autoantibodies (AQP4-IgG) are implicated in the pathogenesis of neuromyelitis optica spectrum disorders (NMOSD), and their removal from the blood circulation is considered to be an effective method for acute treatment. An ideal extracorporeal AQP4-IgG removal system should have high specificity, which means that it can selectively remove AQP4-IgG without affecting normal immunoglobulins. However, the conventional tryptophan immobilized column lacks sufficient specificity and cannot achieve this goal. In this study, we successfully prepared a fusion protein chimeric AQP4, which consists of the complete antigenic epitopes of human AQP4 and the constant region of scaffold protein DARPin. Chimeric AQP4 was expressed and purified from Escherichia coli, and then immobilized on agarose gel as a ligand for selective capture of AQP4-IgG immunosorbent. The prepared immunosorbent had a theoretical maximum adsorption capacity of 20.48 mg/g gel estimated by Langmuir isotherm. In vitro plasma perfusion tests demonstrated that the chimeric AQP4 coupled adsorbent had remarkable adsorption performance, and could eliminate more than 85 % of AQP4-IgG under the gel-to-plasma ratio of 1:50. Moreover, it exhibited high specificity because other human plasma proteins were not adsorbed in the dynamic adsorption experiment. These results suggest that the chimeric AQP4 coupled immunosorbent can provide a new approach for specific immunoadsorption (IA) treatment of NMOSD.

Biparatopic Nanobody-Based Immunosorbent for the Highly Selective Elimination of Tumor Necrosis Factor-α.

Removing the overexpressed TNF-α by hemoperfusion positively affects clinical treatments for diseases such as autoimmune disease and sepsis. However, clearance ratios of adsorbents targeting TNF-α were limited by the extremely low concentration of TNF-α (mostly <1000 ng/L in sepsis) and hydrophobic interactions. In this work, biparatopic nanobodies (NbC21) with a high affinity of 19.9 pM, which bind to two distinct sites of TNF-α, were constructed as high-affinity ligands for the immunosorbent. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm was up to 18.22 mg/g gel. The prepared immunosorbent (NbC21-sorbent) had an outstanding TNF-α clearance ratio of approximately 96% during the dynamic adsorption test, with a sorbent-to-serum ratio of 1:1000. Additionally, it demonstrated favorable hemocompatibility and a prolonged storage capability. The results indicated that the biparatopic nanobody immunosorbent exhibited significant potential for clinical applications as it met the stringent criteria for both efficacy and safety.

Ultrasensitive and Rapid Detection of Procalcitonin via Waveguide-Enhanced Nanogold-Linked Immunosorbent Assay for Early Sepsis Diagnosis.

Sepsis, a life-threatening inflammatory response, demands economical, accurate, and rapid detection of biomarkers during the critical "golden hour" to reduce the patient mortality rate. Here, we demonstrate a cost-effective waveguide-enhanced nanogold-linked immunosorbent assay (WENLISA) based on nanoplasmonic waveguide biosensors for the rapid and sensitive detection of procalcitonin (PCT), a sepsis-related inflammatory biomarker. To enhance the limit of detection (LOD), we employed sandwich assays using immobilized capture antibodies and detection antibodies conjugated to gold nanoparticles to bind the target analyte, leading to a significant evanescent wave redistribution and strong nanoplasmonic absorption near the waveguide surface. Experimentally, we detected PCT for a wide linear response range of 0.1 pg/mL to 1 ng/mL with a record-low LOD of 48.7 fg/mL (3.74 fM) in 8 min. Furthermore, WENLISA has successfully identified PCT levels in the blood plasma of patients with sepsis and healthy individuals, offering a promising technology for early sepsis diagnosis.

NLISA versus enzyme-linked immunosorbent assay: Nanozyme-linked immunosorbent array based on platinum sub-nanocluster nanozyme for α-fetoprotein detection.

Rapid and accurate identification of tumor metabolic markers is important for early tumor diagnosis and individualized treatment. Here, a stable monodisperse sub-nanometer platinum (Pt) material was developed as a highly efficient nanozyme with a specific activity of peroxidase as high as 20.86 U mg-1 through the growth of in situ domain-limited Pt quantum dots via the polymer polyvinylpyrrolidone. Further, the synthesis of large quantities of Pt-loaded SiO2 (Pt-SiO2 ) was determined by silylation reaction and used for naked eye colorimetric testing of human alpha-fetoprotein (AFP). In particular, the immunization incubation process occurred in preprepared microplates. A nanozyme-based immunomodel was constructed in the presence of the target AFP, and a chromogenic reaction occurred with exogenous hydrogen peroxide and the chromogenic substrate tetramethylbenzidine. On optimization of experimental conditions, the dynamic working response range for AFP was found to be 0.05-20 ng mL-1 , with a limit of detection of 38.7 pg mL-1 . This work provides a new strategy to design efficient nanozyme-based enzyme-linked immunochromatographic platforms to meet the practical use of replacing natural enzymes.

Development of fluorescence-linked immunosorbent assay for rapid detection of Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. KEY POINTS: • A fluorescence-linked immunosorbent assay-based S. aureus detection method • Simultaneous quantification of a maximum of 96 samples within 5 h • Application of the novel system to diagnosis clinical isolates.

Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling.

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules.

CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high trans-cleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive small-molecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of small-molecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.

Quercetin Alleviates Asthma-Induced Airway Inflammation and Remodeling through Downregulating Periostin via Blocking TGF-ß1/Smad Pathway.

INTRODUCTION: The aim of the study was to discuss whether the anti-asthmatic effect of quercetin is related to periostin and the downstream molecular pathway of quercetin's anti-asthmatic effect. METHODS: We constructed asthmatic mice, sensitized by ovalbumin, and administrated different treatments into mice according to the experimental design. In this study, we mainly observed the inflammatory response, airway fibrosis, and airway hyperresponsiveness in asthmatic mice. Pathological stains (H&E, PAS, and Masson) were performed. We also detected the inflammation factors and fibrosis-related cytokines by enzyme-linked immunosorbent serologic assay. In addition, we also explored the level of periostin by enzyme-linked immunosorbent serologic assay and Western blot. At the same time, TGF-ß1/Smad pathway was also determined by Western blot. RESULTS: A high expression of periostin was found in asthmatic mice, and quercetin decreases periostin content in bronchoalveolar lavage fluid. Quercetin and OC-20 inhibit airway inflammation response, airway fibrosis, and airway hyperreactivity. Quercetin downregulated TGF-ß1/Smad pathway in the lung tissues of asthmatic mice. Anti-asthma role of quercetin is related to periostin. Then deeper mechanical study revealed that inhibiting TGF-ß1 could improve asthmatic symptoms, and quercetin exerted the protective effect on asthmatic mice through inhibition of TGF-ß1/Smad pathway. CONCLUSION: Quercetin provided a protective role against asthma via periostin, manifested by mild inflammatory infiltration, reduced goblet cell proliferation, and reduced airway fibrosis. TGF-ß1/Smad pathway is an important transduction system, participating in the protective effect of quercetin on asthma.

A rapid and highly sensitive immunosorbent assay to monitor helicases unwinding diverse nucleic acid structures.

Helicases are crucial enzymes in DNA and RNA metabolism and function by unwinding particular nucleic acid structures. However, most convenient and high-throughput helicase assays are limited to the typical duplex DNA. Herein, we developed an immunosorbent assay to monitor the Werner syndrome (WRN) helicase unwinding a wide range of DNA structures, such as a replication fork, a bubble, Holliday junction, G-quadruplex and hairpin. This assay could sensitively detect the unwinding of DNA structures with detection limits around 0.1 nM, and accurately monitor the substrate-specificity of WRN with a comparatively less time-consuming and high throughput process. Remarkably, we have established that this new assay was compatible in evaluating helicase inhibitors and revealed that the inhibitory effect was substrate-dependent, suggesting that diverse substrate structures other than duplex structures should be considered in discovering new inhibitors. Our study provided a foundational example for using this new assay as a powerful tool to study helicase functions and discover potent inhibitors.

Pd@Pt nanoparticle-linked immunosorbent assay for quantification of Collagen type II.

The evaluation of specific protein content in engineered tissues provides a gateway for developing regenerative medicine treatments. Since collagen type II, the major component of articular cartilage, is critical for the blossoming field of articular cartilage tissue engineering, the interest in this protein is growing rapidly. Accordingly, the need for quantification of collagen type II is increasing as well. In this study, we provide recent results for a new quantifying nanoparticle sandwich immunoassay technique for collagen type II. Since mesoporous palladium@platinum (Pd@Pt) nanoparticles have peroxidase-like catalytic activities, these nanoparticles were utilized in an enzyme-linked immunosorbent assay (ELISA)-like format to circumvent the need for traditional enzymes. These nanoparticles were easily conjugated with anti-collagen type II antibodies by the natural affinity interaction and used to develop a direct sandwich ELISA-like format for nanoparticle-linked immunosorbent assays. Using this method, we obtained a limit of detection of 1 ng mL-1, a limit of quantification of 9 ng mL-1. and a broad linear range between 1 ng mL-1 and 50 µg mL-1 for collagen type II with an average relative standard deviation of 5.5%, useable over a pH range of 7 - 9 at least. The assay was successfully applied to quantify collagen type II in cartilage tissues and compared with the results of commercial ELISAs and gene expression by reverse transcription-quantitative polymerase chain reaction. This method provides a thermally stable and cost-efficient alternative to traditional ELISAs. It also extends the application of nanoparticle-linked immunosorbent assays, thereby providing the potential to quantify other proteins and apply the technology in the medical, environmental, and biotechnology industry fields.

Ps-Pt nanozyme-based synergistic signal amplification biosensor for highly sensitive colorimetric detection of protein.

Immunosorbent assay is one of the most popular immunological screening techniques which has been widely used for the clinical diagnosis of alpha-fetoprotein (AFP). While traditional immunosorbent assay (ELISA) suffers from low detection sensitivity due to its low intensity of colorimetric signal. To improve the sensitivity of AFP detection, we developed a new and sensitive immunocolorimetric biosensor by combining Ps-Pt nanozyme with terminal deoxynucleotidyl transferase (TdT)-mediated polymerization reaction. The determination of AFP was achieved by measuring the visual color intensity produced by the catalytic oxidation reaction of the 3,3',5,5'-tetramethylbenzidine (TMB) solution with Ps-Pt and horseradish peroxidase (HRP). Owing to the synergistic catalysis of Ps-Pt and horseradish peroxidase HRP enriched in polymerized amplification products, this biosensor exhibited a significant color change within 25 s in the presence of 10-500 pg/mL AFP. This proposed method allowed for the specific detection of AFP with a detection limit of 4.30 pg/mL and even 10 pg/mL target protein could be distinguished clearly by visual observation. Furthermore, this biosensor could be applied to analysis of AFP in the complex sample and could be easily extended to the detection of other proteins.

Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay.

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

Recent advances in quantum dot-based fluorescence-linked immunosorbent assays.

Fluorescence immunoassays have been given considerable attention among the quantitative detection methods in the clinical medicine and food safety testing fields. In particular, semiconductor quantum dots (QDs) have become ideal fluorescent probes for highly sensitive and multiplexed detection due to their unique photophysical properties, and the QD fluorescence-linked immunosorbent assay (FLISA) with high sensitivity, high accuracy, and high throughput has been greatly developed recently. In this manuscript, the advantages of applying QDs to FLISA platforms and some strategies for their application to in vitro diagnostics and food safety are discussed. Given the rapid development of this field, we classify these strategies based on the combination of QD types and detection targets, including traditional QDs or QD micro/nano-spheres-FLISA, and multiple FLISA platforms. In addition, some new sensors based on the QD-FLISA are introduced; this is one of the hot spots in this field. The current focus and future direction of QD-FLISA are also discussed, which provides important guidance for the further development of FLISA.

Double-Enzyme Active Vanadium Nanospheres-Mediated Ratiometric Multicolor Immunosensors for Sensitive Detection of the T-2 Toxin.

Owing to its high throughput, simplicity, and rapidity, enzyme-linked immunosorbent assay (ELISA) has attracted much attention in the field of immunoassays. However, the traditional ELISA usually affords a single signal readout and the labeling ability of the enzyme used is poor, resulting in low accuracy and a limited detection range. Herein, a vanadium nanospheres (VNSs)-mediated competitive ratio nanozymes-linked immunosorbent assay (VNSs-RNLISA) was created for the sensitive detection of the T-2 toxin (T-2). As the key to the biosensor, the VNSs with superoxide dismutase-like and peroxidase-like dual-enzyme mimetic activities were synthesized by a one-step hydrothermal method, which oxidized 1,1-diphenyl-2-picryl-hydrazyl fading and catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) color development. Therefore, T-2 could not only be qualitatively measured with the naked eye but also be quantitatively evaluated by monitoring the ratio of absorbance at 450 and 517 nm wavelengths. Moreover, the characterization of a VNSs-labeled antibody probe showed strong dual-enzymatic activity, excellent stability, and high affinity with T-2 [the affinity constant (ka) was approximately 1.36 × 108 M-1], which can significantly improve the detection sensitivity. The limit of detection of VNSs-RNLISA was 0.021 ng/mL, which was approximately 27-fold more sensitive than the single signal nanozymes-linked immunosorbent assay (0.561 ng/mL). Besides, the change in the ratio of absorbance (Δ450/Δ517) decreased linearly in a range of 0.22-13.17 ng/mL, outperforming the detection range of a single-mode nano-enzyme-linked immunosorbent assay using TMB by a factor of 1.6 times. Furthermore, the VNSs-RNLISA was successfully used to identify T-2 in maize and oat samples, with recoveries ranging from 84.216 to 125.371%. Overall, this tactic offered a promising platform for the quick detection of T-2 in food and might broaden the application range of the enzyme-linked immunosorbent assay.

Well-Based Multiplex Food Allergen Colorimetric ELISA.

Food allergy is a well-recognized and significant health hazard around the world. At least 160 food groups have been identified that present allergenic reactions or other sensitivities and intolerance in humans. Enzyme-linked immunosorbent assay (ELISA) is an accepted platform for identifying the nature of the food allergy and its severity. It is now possible to simultaneously screen patients for allergic sensitivity and intolerance to multiple allergens using multiplex immunoassays. This chapter describes the preparation and utility of a multiplex allergen ELISA for the assessment of food allergy and sensitivity in patients.

Sensitivity and performance of three novel quantitative assays of SARS-CoV-2 nucleoprotein in blood.

To assess if SARS-CoV-2 (COVID-19) systemic disease can be determined by available nucleoprotein assays, we compared the performance of three commercial SARS-CoV-2 nucleoprotein (N) assays in plasma. A total of 272 plasma samples collected in the period November-December 2021 were analyzed by the methods Simoa SARS CoV-2 N Protein Advantage Kit [Quanterix Simoa], Solsten SARS-CoV-2 Antigen enzyme immunosorbent assay (ELISA) [Solsten ELISA], and Elecsys SARS-CoV-2 Antigen electrochemiluminescence immunoassay [Elecsys ECLIA]. Additionally, a dilution series of inactivated virus culture was analyzed by the three assays. The SARS CoV-2 PCR-status was not known for the patients. Linear correlation in the pairwise correlation between assays as well as linearity of dilution series of inactivated virus culture was estimated by Spearman score. Sensitivity and specificity were estimated by pairwise comparison. The three assays showed poor agreement on patient samples with regards to concentration. Performance on virus culture was excellent but with different level of detection (LOD). Positive vs negative results show comparable sensitivity and specificity of Quanterix Simoa and Solsten ELISA, with a higher LOD in Elecsys ECLIA and thus lower sensitivity and high specificity. N by all tested assays can be used as a marker for systemic COVID-19 disease.

A visual CRISPR/dCas9-mediated enzyme-linked immunosorbent assay for nucleic acid detection with single-base specificity.

Specific and economical nucleic acid detection is crucial for molecular diagnoses in resource-limited settings. Various facile readout approaches have been developed for nucleic acid detection, but they have limited specificity. Herein, nuclease-dead Cas9 (dCas9)/sgRNA was used as an excellent DNA recognition probe system to develop a visual clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated enzyme-linked immunosorbent assay (ELISA) for specific and sensitive detection of cauliflwer mosaic virus 35s (CaMV35S) promoter in genetically modified (GM) crops. In this work, the CaMV35S promoter was amplified with biotinylated primers, and then precisely bound with dCas9 in the presence of sgRNA. The formed complex was captured by antibody-coated microplate and bound to a streptavidin-labeled horseradish peroxidase probe for the visual detection. Under the optimal conditions, dCas9-ELISA could detect CaMV35s promoter as low as 12.5 copies µL-1. Moreover, the proposed method was capable to distinguish the target sequence with single-base specificity. Coupled with one-step extraction and recombinase polymerase amplification, dCas9-ELISA can identify actual GM rice seeds within 1.5 h from sampling to results without expensive equipment and technical expertise. Therefore, the proposed method offers a specific, sensitive, rapid and cost-effective detection platform for molecular diagnoses.