Automated and Rapid ADAMTS13 Testing Using Chemiluminescence: Utility for Identification or Exclusion of TTP and Beyond.

Thrombotic thrombocytopenic purpura (TTP) is a prothrombotic condition caused by a significant deficiency of the enzyme, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In the absence of adequate levels of ADAMTS13 (i.e., in TTP), plasma VWF accumulates, in particular as "ultra-large" VWF multimers, and this leads to pathological platelet aggregation and thrombosis. In addition to TTP, ADAMTS13 may be mildly to moderately reduced in a range of other conditions, including secondary thrombotic microangiopathies (TMA) such as those caused by infections (e.g., hemolytic uremic syndrome (HUS)), liver disease, disseminated intravascular coagulation (DIC), and sepsis, during acute/chronic inflammatory conditions, and sometimes also in COVID-19 (coronavirus disease 2019)). ADAMTS13 can be detected by a variety of techniques, including ELISA (enzyme-linked immunosorbent assay), FRET (fluorescence resonance energy transfer) and by chemiluminescence immunoassay (CLIA). The current report describes a protocol for assessment of ADAMTS13 by CLIA. This protocol reflects a rapid test able to be performed within 35 min on the AcuStar instrument (Werfen/Instrumentation Laboratory), although certain regional approvals may also permit this testing to be performed on a BioFlash instrument from the same manufacturer.

Effect of storage temperatures simulating transport conditions of nasopharyngeal swabs on the results of a chemiluminescence immunoassay (CLIA) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.

PURPOSE: Chemiluminescence Immunoassay (CLIA) is high throughput, rapid diagnostic test which has recently come up for the detection of SARS-CoV-2 antigen. The present study evaluated performance of CLIA antigen test in nasopharyngeal swab samples stored at different temperatures for 7 days to simulate the transport conditions and transit time across the country from remote peripheral laboratories to central facilities. MATERIALS AND METHODS: Limit of detection (LOD), sensitivity and specificity of VITROS® SARS-CoV-2 antigen assay was determined using Real-time reverse transcriptase PCR (rRT-PCR) confirmed SARS-CoV-2 positive and negative samples. To detect the effect of storage temperatures on VITROS ®SARS-CoV-2 antigen results, samples were stored at 4 â€‹°C, 25 â€‹°C & 37 â€‹°C for 7 days followed by detection of SARS-CoV-2 nucleocapsid antigen and compared with N-gene rRT-PCR. RESULTS: The VITROS® SARS-CoV-2 antigen test was found to have a sensitivity and specificity of 78.9% and 100% respectively with high sensitivity of 88.1% for samples with Ct â€‹< â€‹30. The LOD of VITROS assay was equivalent to 3800 copies of RNA per reactions as compared to 72 copies per reaction for rRT-PCR. We observed that more than 80% of samples with <30 Ct values could be detected by VITROS SARS-CoV-2 antigen assay at day 7 even when stored at 37 â€‹°C. For samples with Ct values between 26 and 30, on day 7 the positivity rate of N-antigen at 4 â€‹°C was 90.9% and 37 â€‹°C was 63.6%. CONCLUSIONS: CLIA testing can be carried out for the detection of SARS-CoV-2 N-protein in NP-swab samples transported in cold chain even with 7 days transit time, particularly for Ct â€‹< â€‹30 samples which represents cases with higher transmissibility. As drop in positivity for VITROS assay was lower as compared to rRT-PCR on day 7 in cold chain-maintained samples, the assay can be useful to screen samples received from remote peripheral areas before performing rRT-PCR.

Development of an Interactive Touchless Technology Based on Static-Electricity-Induced Luminescence.

Touchless technology has garnered significant interest in recent years because of its effectiveness in combating infectious diseases such as the novel coronavirus (COVID-19). The goal of this study was to develop an inexpensive and high-precision touchless technology. A base substrate was coated with a luminescent material that emitted static-electricity-induced luminescence (SEL), and it was applied at high voltage. An inexpensive web camera was used to verify the relationship between the non-contact distance to a needle and the applied-voltage-triggered luminescence. The SEL was emitted at 20-200 mm from the luminescent device upon voltage application, and the web camera detected the SEL position with an accuracy of less than 1 mm. We used this developed touchless technology to demonstrate a highly accurate real-time detection of the position of a human finger based on SEL.

Rapid point-of-care detection of SARS-CoV-2 RNA with smartphone-based upconversion luminescence diagnostics.

Accurate COVID-19 screening via molecular technologies is still hampered by bulky instrumentation, complicated procedure, high cost, lengthy testing time, and the need for specialized personnel. Herein, we develop point-of-care upconversion luminescence diagnostics (PULD), and a streamlined smartphone-based portable platform facilitated by a ready-to-use assay for rapid SARS-CoV-2 nucleocapsid (N) gene testing. With the complementary oligo-modified upconversion nanoprobes and gold nanoprobes specifically hybridized with the target N gene, the luminescence resonance energy transfer effect leads to a quenching of fluorescence intensity that can be detected by the easy-to-use diagnostic system. A remarkable detection limit of 11.46 fM is achieved in this diagnostic platform without the need of target amplification, demonstrating high sensitivity and signal-to-noise ratio of the assay. The capability of the developed PULD is further assessed by probing 9 RT-qPCR-validated SARS-CoV-2 variant clinical samples (B.1.1.529/Omicron) within 20 min, producing reliable diagnostic results consistent with those obtained from a standard fluorescence spectrometer. Importantly, PULD is capable of identifying the positive COVID-19 samples with superior sensitivity and specificity, making it a promising front-line tool for rapid, high-throughput screening and infection control of COVID-19 or other infectious diseases.

The Luminescence Hypothesis of Olfaction.

A new hypothesis for the mechanism of olfaction is presented. It begins with an odorant molecule binding to an olfactory receptor. This is followed by the quantum biology event of inelastic electron tunneling as has been suggested with both the vibration and swipe card theories. It is novel in that it is not concerned with the possible effects of the tunneled electrons as has been discussed with the previous theories. Instead, the high energy state of the odorant molecule in the receptor following inelastic electron tunneling is considered. The hypothesis is that, as the high energy state decays, there is fluorescence luminescence with radiative emission of multiple photons. These photons pass through the supporting sustentacular cells and activate a set of olfactory neurons in near-simultaneous timing, which provides the temporal basis for the brain to interpret the required complex combinatorial coding as an odor. The Luminescence Hypothesis of Olfaction is the first to present the necessity of or mechanism for a 1:3 correspondence of odorant molecule to olfactory nerve activations. The mechanism provides for a consistent and reproducible time-based activation of sets of olfactory nerves correlated to an odor. The hypothesis has a biological precedent: an energy feasibility assessment is included, explaining the anosmia seen with COVID-19, and can be confirmed with existing laboratory techniques.

A performance comparison of two (electro) chemiluminescence immunoassays for detection and quantitation of serum anti-spike antibodies according to SARS-CoV-2 vaccination and infections status.

The information provided by SARS-CoV-2 spike (S)-targeting immunoassays can be instrumental in clinical-decision making. We compared the performance of the Elecsys® Anti-SARS-CoV-2 S assay (Roche Diagnostics) and the LIAISON® SARS-CoV-2 TrimericS IgG assay (DiaSorin) using a total of 1176 sera from 797 individuals, of which 286 were from vaccinated-SARS-CoV-2/experienced (Vac-Ex), 581 from vaccinated/naïve (Vac-N), 147 from unvaccinated/experienced (Unvac-Ex), and 162 from unvaccinated/naïve (Unvac-N) individuals. The Roche assay returned a higher number of positive results (907 vs. 790; p = 0.45; overall sensitivity: 89.3% vs. 77.6%). The concordance between results provided by the two immunoassays was higher for sera from Vac-N (Ï°: 0.58; interquartile ranges [IQR]: 0.50-0.65) than for sera from Vac-Ex (Ï°: 0.19; IQR: -0.14 to 0.52) or Unvac-Ex (Ï°: 0.18; IQR: 0.06-0.30). Discordant results occurred more frequently among sera from Unvac-Ex (34.7%) followed by Vac-N (14.6%) and Vac-Ex (2.7%). Antibody levels quantified by both immunoassays were not significantly different when <250 (p = 0.87) or <1000 BAU/ml (p = 0.13); in contrast, for sera ≥1000 BAU/ml, the Roche assay returned significantly higher values than the DiaSorin assay (p < 0.008). Neutralizing antibody titers (NtAb) were measured in 127 sera from Vac-Ex or Vac-N using a S-pseudotyped virus neutralization assay of Wuhan-Hu-1, Omicron BA.1, and Omicron BA.2. The correlation between antibody levels and NtAb titers was higher for sera from Vac-N than those from Vac-Ex, irrespective of the (sub)variant considered. In conclusion, neither qualitative nor quantitative results returned by both immunoassays are interchangeable. The performance of both assays was found to be greatly influenced by the vaccination and SARS-CoV-2 infection status of individuals.

A CLEIA Antigen Assay in Diagnosis and Follow-Up of SARS-CoV-2-Positive Subjects.

This study includes 259 consecutive nasopharyngeal swabs which tested positive for a molecular SARS-CoV-2 test and 77 subjects who were followed longitudinally, with nasopharyngeal swabs performed weekly until clinical recovery and a negative result for the molecular test were reached. All swabs were also tested with a Lumipulse SARS-CoV-2 chemiluminescence enzyme immunoassay (CLEIA) antigen assay. The antigen test was positive in 169 (65.3%) out of the 259 subjects, while no antigen was detected in 90 subjects (34.7%). In the antigen-positive subjects, clinical status moved slightly toward a more frequent presence of symptoms. Longitudinal follow-up shows how the time of negativization has a faster kinetic in the antigenic test than in the molecular test. Antigenic test result values, considered as a time-dependent covariate and log-transformed, were highly associated with the time to negative swab, with good prediction ability. Receiver operating characteristic (ROC) curve analysis showed a very good discrimination ability of antigenic tests in classifying negative swabs. The optimal cutoff which jointly maximized sensitivity and specificity was 1.55, resulting in an overall accuracy of 0.75, a sensitivity of 0.73, and a specificity of 0.83. After dichotomizing the antigenic test according to the previously determined cutoff value of 1.55, the time-dependent covariate Cox model again suggests a highly significant association of antigenic test values with the time to negative swab molecular: a subject with an antigenic test value lower than 1.55 had almost a 13-fold higher probability to also result negative in the molecular test compared to a subject with an antigenic test value higher than 1.55. IMPORTANCE Our work explores the possibility of using a sensible and reliable antigenic test in a wider range of SARS-CoV-2 diagnostic and clinical applications. Furthermore, this tool seems particularly promising in follow-up with infected subjects, because while the molecular test frequently yields the persistence of low positivities, raising yet unanswered questions, this antigenic test shows more uniform and faster negativization during the evolution of the infection, somehow paralleling the dynamics of infectivity. Although more data will be required to definitely prove it, we believe these findings might be of great interest.

Time-Resolved Persistent Luminescence Encoding for Multiplexed Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

Capable of precise simultaneous multitarget identifications within a minimized sample, optical multiplexing is vital for accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) while remaining spectral crowding and background interfering. In merits of an autofluorescence-free background and high-capability throughput, a persistent luminescence (PersL) lifetime/color binary encoding strategy was herein proposed for SARS-CoV-2 diagnosis. Based on luminescence resonance energy transfer processes, the intense lifetimes and representative emissions of PersL nanoplatforms were rationally manipulated to create a temporal coding dimension within a wide seconds-to-minutes range through three individual channels. Particularly, at least four populations of barcoding in a certain channel were successfully decoded by a purpose-built time-resolved PersL technology. As a proof-of-concept, functionalized PersL nanoplatforms were further well developed for the simultaneous quantification of five-plex SARS-CoV-2 biomarkers with limits of detection in the subnanomolar range. Remarkably, PersL nanoplatforms enabled a highly differentiable discrimination of multitargets at various concentrations of ultralow background and high-fidelity resolutions, thereby advancing a powerful tool for optical multiplexing in biomedical applications.

Discovery of a Phage Peptide Specifically Binding to the SARS-CoV-2 Spike S1 Protein for the Sensitive Phage-Based Enzyme-Linked Chemiluminescence Immunoassay of the SARS-CoV-2 Antigen.

The COVID-19 pandemic has led to a global crisis with devastating effects on public healthcare and the economy. Sensitive detection of SARS-CoV-2 is the key to diagnose and control its spread. The spike (S) protein is an abundant viral transmembrane protein and a suitable target protein for the selective recognition of SARS-CoV-2. Here, we report that with bovine serum albumin prescreening, a specific phage peptide targeting SARS-CoV-2 S1 protein was biopanned with the pIII phage display library. The identified phage #2 expressing the peptide (amino acid sequence: NFWISPKLAFAL) shows high affinity to the target with a dissociation constant of 3.45 ± 0.58 nM. Furthermore, the identified peptide shows good specificity with a binding site at the N-terminal domain of the S1 subunit through a hydrogen bond network and hydrophobic interaction, supported by molecular docking. Then, a sandwiched phage-based enzyme-linked chemiluminescence immunoassay (ELCLIA) was established by using phage #2 as a bifunctional probe capable of SARS-CoV-2 S1 antigen recognition and signal amplification. After optimizing the conditions, the proposed phage ELCLIA exhibited good sensitivity, and as low as 78 pg/mL SARS-CoV-2 S1 could be detected. This method can be applied to detect as low as 60 transducing units (TU)/mL SARS-CoV-2 pseudovirus in 50% saliva. Therefore, specific phage peptides have good prospects as powerful biological recognition probes for immunoassay detection and biomedical applications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulins using chemiluminescence immunoassay and its correlation with neutralizing antibodies.

BACKGROUND: Neutralizing antibodies (NAbs) against SARS-CoV-2 infection have a pivotal role in protective immune response; however, their measurement requires specialized facilities. We evaluated the degree of correlation between NAbs and anti-SARS-CoV-2 IgG/total Ig antibodies detected by chemiluminescent immunoassay in asymptomatic and previously symptomatic SARS-CoV-2 patients. METHODS: A total of 1241 participants (previously symptomatic patients and asymptomatic individuals), who were screened for SARS-CoV-2 infection by RT-PCR or serology, were enrolled in our study. Sera were analyzed for the presence of anti-spike-1(S1)-SARS-CoV-2 IgG/total Ig antibodies, using Ortho Clinical Diagnostics, USA. A signal/cut-off value (S/CO) ≥ 1 was considered reactive. NAbs were measured in 103 random samples from groups using microneutralization assay, with titer ≥ 1:10 being considered positive. RESULTS: Asymptomatic (n = 229) and 261 previously symptomatic individuals with positive serology and negative RT-PCR were finally included. Significant higher anti-S1-IgG titers were seen in asymptomatic individuals (P < 0.0001). Conversely, anti-S1-total Ig titers were significantly higher in previously symptomatic (P < 0.0001). NAbs were detected in both groups, however, higher titers were seen in previously symptomatic patients. There is a correlation between NAbs and both IgG/total anti-S1-SARS-CoV-2 antibodies (r = 0.47, P < 0.0001 and r = 0.49, P < 0.0001, respectively). IgG and total Ig could predict a neutralization titer of ≥ 1:160 at S/CO >4.44 and >65 with AUC 0.69 and 0.67, respectively. CONCLUSION: Asymptomatic SARS-CoV-2 infection can produce comparable antibodies response to previously symptomatic individuals, however higher neutralization activity was seen in the previously symptomatic. Anti-S1-SARS-CoV-2 IgG/total Ig antibodies showed a correlation with neutralization activity and can be used to estimate the presence of protective immunity.

Conditionally designed luminescent DNA crystals doped by Ln3+(Eu3+/Tb3+) complexes or fluorescent proteins with smart drug sensing property.

In this work, a designed porous DNA crystal with high intrinsic biocompatibility was used as the scaffold material to load fluorescent guest molecules to detect anti-cancer drugs. It is shown here that the synthesized crystals have the characteristics consistent with the designed large solvent channels, and can therefore accommodate guest molecules such as fluorescent proteins that cannot be accommodated by less porous crystals. Eu(TTA)3phen and Tb(acac)3phen lanthanide complexes were individually noncovalently loaded into the porous crystals, resulting in hybrid luminescent DNA crystals. Emodin, an anti-cancer, anti-tumor, anti-inflammatory drug, was found to quench lanthanide complexes in solution or in crystals. Notably, emodin is the active ingredient of Lianhua Qingwen Capsule, an anti-COVID-19 drug candidate. Therefore, the porous DNA crystals reported here have potential applications as a biocompatible and theranostic delivery biomaterial for functional macromolecules.

Performance evaluation of an automatic chemiluminescence immune platform for SARS-CoV-2 neutralizing antibody after vaccination in real world.

OBJECTIVE: Reliable high-throughput serological assays for SARS-CoV-2 antibodies present an important role in the strength and duration of immunity after vaccination. The study investigated the analytical and clinical performances of neutralizing antibodies (NTAb) assay by chemiluminescent (CLIA), and SARS-CoV-2 neutralizing antibody after vaccination in real world. METHODS: The analytical performances of CLIA for SARS-CoV-2 NTAb were evaluated, followed by the sensitivity and specificity identified with a PRNT test from 50 volunteers. Then, a cohort of vaccine recipients (n = 37) were tracked with SARS-CoV-2 NTAb assay at prior to vaccination, one, three and six months post two doses. In real world, a total of 737 cases were recruited from physical examination center in Shenzhen Luohu People's Hospital (from Jun to August 2021) to analyze vaccination status. RESULTS: Serological assays on the CLIA were found with excellent characteristics including imprecision, repeatability and linearity. Besides, it was robust to icterus, lipemia and hemolysis. The good sensitivity and specificity were obtained at 98% and 100%, respectively. NTAb results showed a high correlation with PRNT50 titers (r 0.61). Until July 2021, the BBIBP-CorV (76.3%) and Sinovac CoronaVac (20.5%) were the predominant vaccines injection in Shenzhen, China. Adolescent less than 18 years was the main unvaccinated group (52.1%). The seropositive rate of inactive SRAR-CoV-2 vaccines exceeded 97% after inoculation. The NTAb generated by Sinovac CoronaVac with the schedule of 0-56 days was found significantly lower than that by BBIBP-CorV (P < 0.001). The follow-up of NTAb changes in a cohort and the dynamic variation of NTAb in real world disclosed steep downward by almost three times for NTAb level occurred at three months post twice vaccinations. The seropositive ratio was at least 50% over 6 months. CONCLUSIONS: SARS-CoV-2 neutralizing antibodies assay show excellent analytical and clinical performances, and a high correlation with neutralizing activity. Anti-epidemic measures and the urgent trial of SARS-CoV-2 vaccine was calling for adolescents.

Fully Automated Chemiluminescence Microarray Analysis Platform for Rapid and Multiplexed SARS-CoV-2 Serodiagnostics.

Lateral-flow immunoassays and laboratory diagnostic tests like enzyme-linked immunosorbent assays (ELISAs) are powerful diagnostic tools to help fight the COVID-19 pandemic using them as antigen or antibody tests. However, the need emerges for alternative bioanalytical systems that combine their favorable features─simple, rapid, and cost-efficient point-of-care (POC) analysis of lateral-flow immunoassays and higher reliability of laboratory tests─while eliminating their disadvantages (limited sensitivity and specificity of lateral-flow assays and prolonged time and work expenditure of laboratory analysis). An additional need met by only a few tests is multiplexing, allowing for the analysis of several immunorecognition patterns at the same time. We herein present a strategy to combine all desirable attributes of the different test types by means of a flow-based chemiluminescence microarray immunoassay. Laminated polycarbonate microarray chips were developed for easy production and subsequent application in the fully automated microarray analysis platform MCR-R, where a novel flow cell design minimizes the sample volume to 40 µL. This system was capable of detecting IgG antibodies to SARS-CoV-2 with 100% sensitivity and specificity using recombinant antigens for the SARS-CoV-2 spike S1 protein, nucleocapsid protein, and receptor binding domain. The analysis was accomplished within under 4 min from serum, plasma, and whole blood, making it also useful in POC settings. Additionally, we showed the possibility of serosurveillance after infection or vaccination to monitor formerly unnoticed breakthrough infections in the population as well as to detect the need for booster vaccination after the natural decline of the antibody titer below detectable levels. This will help in answering pressing questions on the importance of the antibody response to SARS-CoV-2 that so far remain open. Additionally, even the sequential detection of IgM and IgG antibodies was possible, allowing for statements on the time response of an infection. While our serodiagnostic application focuses on SARS-CoV-2, the same approach is easily adjusted to other diseases, making it a powerful tool for future serological testing.

Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.

REGEN-COV® antibody cocktail bioanalytical strategy: comparison of LC-MRM-MS and immunoassay methods for drug quantification.

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV® (RONAPREVE® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.

Label-Free Immunoassay for Sensitive and Rapid Detection of the SARS-CoV-2 Antigen Based on Functionalized Magnetic Nanobeads with Chemiluminescence and Immunoactivity.

Direct detection of SARS-CoV-2 in biological specimens is often challenging due to the low abundance of viral components and lack of enough sensitivity. Herein, we developed a new type of chemiluminescent functionalized magnetic nanomaterial for sensitive detection of the SARS-CoV-2 antigen. First, HAuCl4 was reduced by N-(aminobutyl)-N-(ethylisoluminol) (ABEI) in the presence of amino magnetic beads (MB-NH2) to generate ABEI-AuNPs, which were directly assembled on the surface of MB-NH2. Then, Co2+ was modified onto the surface to form MB@ABEI-Au/Co2+ (MAA/Co2+). MAA/Co2+ exhibited good chemiluminescence (CL) and magnetic properties. It was also found that it was easy for the antibody to be connected with MAA/Co2+. Accordingly, MAA/Co2+ was used as a sensing interface to construct a label-free immunoassay for rapid detection of the N protein in SARS-CoV-2. The immunoassay showed a linear range from 0.1 pg/mL to 10 ng/mL and a low detection limit of 69 fg/mL, which was superior to previously reported methods for N protein detection. It also demonstrated good selectivity by virtue of magnetic separation, which effectively removed a sample matrix after immunoreactions. It was successfully applied for the detection of the N protein in spiked human serum and saliva samples. Furthermore, the immunoassay was integrated with an automatic CL analyzer with magnetic separation to detect the N protein in patient serums and rehabilitation patient serums with satisfactory results. Thus, the CL immunoassay without a complicated labeling procedure is sensitive, selective, fast, simple, and cost-effective, which may be used to combat the COVID-19 pandemic. Finally, the CL quenching mechanism of the N protein in the immunoassay was also explored.

Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay.

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

Performance evaluation of automated chemiluminescence immunoassay based antigen detection - Moving towards more reliable ways to predict SARS-CoV-2 infection.

Real-time reverse transcription- polymerase chain reaction (RT-PCR) has been the most reliable armoury for the diagnosis of COVID-19, considered to be the reference standard but fails to reproduce the correct predictability about the infectivity of the disease every time. Antigen detection however puts foothold in this aspect even though lacks in sensitivity, especially conventional Rapid Antigen Tests (RATs). Recently developed Chemiluminescence Immunoassay (CLIA) based antigen detection tests are promising and displayed better sensitivity. In the current study we have evaluated VITROS® SARS-CoV-2 Ag Test CLIA Kit, which was tested on 148 patient's samples attended to a tertiary care centre for testing of SARS-CoV-2. The performance of the kit was evaluated in comparison to RT-PCR and RAT and found to be a good test for antigen detection, best within the first few days of infection. The test has shown sensitivity of 94.3 % and specificity of 100 % in samples with corresponding Ct values of ≤25 by RT-PCR, which corresponds to high viral load and can predict ability of spreading the disease by the patients. With the results being semiquantitative along with improved sensitivity it can replace RATs for antigen detection for screening, provided good laboratory set up is included under consideration.

The Influence of UV Light on Photodegradation of Acetylsalicylic Acid.

Photodegradation of the aqueous solutions of acetylsalicylic acid, in the absence (ASA) and the presence of excipients (ASE), is demonstrated by the photoluminescence (PL). A shift of the PL bands from 342 and 338 nm to 358 and 361-397 nm for ASA and ASE in solid state and as aqueous solutions was reported. By exposure of the solution of ASA 0.3 M to UV light, a decrease in the PL band intensity was highlighted. This behavior was revealed for ASA in the presence of phosphate buffer (PB) having the pH equal to 6.4, 7, and 8 or by the interaction with NaOH 0.3 M. A different behavior was reported in the case of ASE. In the presence of PB, an increase in the intensity of the PL band of ASE simultaneously with a change of the ratio between the intensities of the bands at 361-364 and 394-397 nm was highlighted. The differences between PL spectra of ASA and ASE have their origin in the presence of salicylic acid (SAL). The interaction of ASE with NaOH induces a shift of the PL band at 405-407 nm. Arguments for the reaction of ASA with NaOH are shown by Raman scattering and FTIR spectroscopy.