Homogeneous Assay Research Articles

B-296 Assessment of Clozapine Assay on The Automated, High-performance Beckman AU Analyzer

Abstract Background Treatment-resistant schizophrenia (TRS) affects approximately one in three patients with schizophrenia. The timely initiation of clozapine therapy is crucial for achieving remission in TRS patients. However, long turnaround times (TATs) associated with sending clozapine tests to reference laboratories cause significant challenges, leading to delays in treatment initiation and compromising patient outcomes. To address this issue, we implemented in-house clozapine testing on Beckman AU analyzers, aiming to facilitate early clozapine initiation and enhance confidence in prescribing and monitoring clozapine therapy for TRS patients. Methods The Beckman AU clozapine assay is a homogenous competitive nanoparticle agglutination assay. Precision and linearity were assessed for the Beckman AU DxC700 clozapine assay using manufacturer-provided standards. We conducted a correlation study involving 31 samples analyzed on both Beckman AU DxC 700 and LC-MS/MS methods. Data analysis was performed using Deming and Linear regression methods. Additionally, we analyzed 356 clozapine tests performed in-house on Beckman AU DxC700 between March 15th and Sept 10th, 2023, and compared them with 1399 clozapine tests sent to a reference laboratory using the LC-MS/MS method from March 13th, 2020, to March 13th, 2023. Turnaround time was evaluated. Results The Beckman AU DxC700 clozapine assay exhibited excellent performance across the measuring range, with intra-assay and inter-assay coefficients of variation ranging from 1.7-3.6% and 2.2-5.3%, respectively. The assay demonstrated linearity over the five recommended ranges (68-1500 ng/mL) without significant bias observed. The Beckman AU DxC700 and LC-MS/MS clozapine assays displayed excellent correlation (R2=0.948, slope=0.941, intercept=6.3). Implementation of in-house clozapine testing led to a significant reduction in total turnaround time, from 3922 minutes to 116 minutes. The time from sample receipt to results also decreased substantially, from 3894 minutes to 32 minutes. Conclusions The clozapine assay was validated and routinely measured in-house using the Beckman AU DxC700 automated assay. Moreover, we validated the correlation by confirming that the results from Beckman AU DxC had around 6% lower bias comparing with LC-MS/MS. Turnaround time serves as a critical quality indicator for evaluating testing process effectiveness and efficiency, as well as clinician and patient satisfaction. The implementation of in-house clozapine testing significantly improved pre-analytical, analytical, and post-analytical Turnaround times.

A-214 Evaluate LDL-C using NIH equation 2, Friedewald equation, and direct homogeneous measurement

Abstract Background It is essential to accurately assess Low-density lipoprotein cholesterol (LDL-C) levels, a key marker for cardiovascular disease often used for treatment recommendations. The Friedewald equation has been used for many years to calculate plasma LDL-C levels, but it has limitations. Therefore, before implementing the NIH equation, we compared LDL-C results from NIH Equation 2 to the Friedewald formula and then to Atellica direct homogeneous assay LDL-C results. Methods A retrospective study of 1200 randomly selected patients out of 53,258 data points pulled from the laboratory information (LIS) system between November 2021 and October 2023. The study evaluated triglycerides, LDL-C, total cholesterol, and HDL-C measurements. The LDL-C calculated using NIH equation 2 has been set in the LIS as a non-reportable test component since November 1, 2021. The results obtained from NIH equation 2 and Friedewald equation were compared. LDL-C levels were measured using an Atellica direct homogeneous assay for an additional 75 specimens. Deming regression analysis compared measured and calculated LDL-C using both formulae. Results The LDL-C calculated using the Friedewald equation has a mean of 106.1 mg/dL and SD 40.1 mg/dL, while the NIH equation has a mean of 108.5 mg/dL and SD 39.9 mg/dL. The average difference between both equations is 2.5 mg/dL, and there is a significant difference between both calculations using paired t-tests (p-value<0.0001). We first compared the calculated LDL-C derived from the Friedewald formula to the NIH equation 2. The regression analysis equation for the Friedewald formula was y = -2.7 + 1.001 X (r = 0.99). When the Triglyceride values <150 mg/dL (N=879), the average difference is 1.3, and the regression equation is -0.45+1.01X(r=0.99). However, when the Triglyceride value >= 150 mg/dL (N=321), the average bias is 5.4 mg/dL, and the regression equation is -11.2+1.05X(r=0.99). The Friedewald formula was compared to the measured LDL-C in 75 specimens; the regression analysis for the Friedewald formula was y = -19.6 + 1.06 X (r = 0.91, p-value <0.0001). The 75 measured LDL-C results were also compared with the NIH formula. The regression analysis of NIH LDL-C with measured LDL-C was y = -5.6 + 0.981 x (r = 0.90, p-value = 0.0002). Both formulae showed a linear relationship against measured LDL-C. The NIH formula outperformed the Friedewald formula; bias difference -7.8(-6.8%) compared to the Friedwald equation bias -12.9(-12.0%). If the NIH equation had been used during this period, an additional 1,116 patients would have had LDL-C results. This is because the Friedewald equation should not be used if the triglyceride value is above 400 mg/dL. Out of the additional patients, 513 (46%) had a high LDL (>100 mg/dL). Conclusions The NIH formula performed better than the Friedewald formula, with a less negative bias when both calculations were compared to direct homogenous measurement. The average difference between the two formulas showed the lowest value when triglyceride < 150 mg/dL. Moving to NIH equation 2 will benefit our population as we had 2% of our patients in the past two years have triglyceride >400 mg/dL.

A-215 Feasibility of a unique homogenous assay for lipoprotein(a) particle number without bias from apo(a) size isoforms

Abstract Background Lipoprotein(a) [Lp(a)] is an LDL-like particle with apolipoprotein(a) [apo(a)] covalently attached to apolipoprotein B. Elevated Lp(a) is associated with increased risk of atherosclerotic heart disease. The structural homology of Lp(a) and plasminogen and the variable number of apo(a) kringle 4 type 2 subunits (k4t2) between individuals, up to 51 repeats and a variable MW of 300-800 kDa, presents an analytical challenge. `Selection of calibrators of specific isoform size does not fix the problem, despite manufacturer’s claims to the contrary. Methods We have developed goat antisera to human apo(a)-specific peptides and a prototype immunoturbidimetric assay for Lp(a). We selected and synthesized 10 peptide sequences (15-30 aa each) distinct from the sequences found in k4t2 regions and plasminogen. Peptides were prepared attached to KLH (GenScript) and combined to immunize goats following a standard protocol including monthly boosters (Nittobo). Next, we assessed various sample volumes, reagent formulations and antisera dilutions to establish parameters on the Randox Imola analyzer. Cross-reactivity with recombinant k4t2 (40 mg/dL) and plasminogen (200 mg/dL, Athens Research) was assessed by comparison to the LOD (using delipidated plasma blank and low concentration samples). Results Titers to human Lp(a)) were positive to 1:6400 dilution of antisera, showing exceptional reactivity. Working formulation/parameters were 10 µL sample, 100 µL PBS, pH 7.4/PEG6000/detergent and 95 µL R2 (antisera). Sample/R1 was incubated for 5-minutes before addition of R2; reaction was monitored at 340 nm/37°C. LOD and linearity were also assessed. No cross-reactivity with k4t2 or plasminogen up to the concentrations tested was observed. Linearity to 330 nmol/L and LOD of 18 nmol/L were found. Conclusions The use of this antisera/assay eliminates the bias associated with apo(a) size polymorphisms and allows for the accurate measurement of Lp(a) particle number. More work is needed to optimize assay parameters and formulation and comprehensively validate assay performance.

Analyte-induced hindrance in the RCA-assisted CRISPR/Cas12a system for homogeneous protein assays

Heterogeneous assays, such as enzyme-linked immunosorbent assays, have become indispensable for in vitro diagnostics. However, the simple, sensitive, and accurate detection is limited by their multiple washing and incubation steps, and limited amplification methods. In this study, we design a novel approach utilizing analyte-induced hindrance within the rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for simple and highly sensitive homogenous protein detection. Streptavidin (SA) and digoxin antibody (anti-Dig) are employed as representative detection models. The specific recognition of target proteins using primers modified with small molecules hinders the RCA process, preventing the activation of Cas12a′s trans-cleavage activity, thereby leading to a reduction in fluorescence intensity. Our developed platform exhibites exceptional detection performance characterized by high sensitivity, robust specificity, and significant potential for application in complex samples. By expanding the recognition elements, this platform can evolve into a versatile clinical diagnostic tool with universal applicability. In addition, this platform provides a novel direction for quantifying ultralow-concentration disease biomarkers in clinical practice.

Development of immunochromatographic and homogeneous assay based on quantum dot-functionalized polystyrene nanoprobes for the qualitative and quantitative screening of respiratory viruses

Accurately differentiating respiratory diseases caused by viruses is challenging because of the similarity in their early or clinical symptoms. Moreover, different infection sources require different treatments. However, the current diagnostic methods have limited differentiating efficiency and sensitivity. We developed a dual-system immunosensor with a bilayer fluorescent label as a signal amplifier for the on-site, sensitive, and accurate identification of multiple respiratory viruses (RVs). The nanomaterial, comprising a polystyrene (PS) nanosphere core encapsulated by two layers of CdSe@ZnS-COOH quantum dots (QDs), outperforms the conventional color and fluorescent labels in RV detection. The dual-system detection platform, comprising a PS@DQD-based lateral flow immunoassay (LFIA) and a PS@DQD-based homogeneous sensor, enables qualitative and quantitative screening of multiple respiratory viruses within 10 and 30 min, respectively, depending on the specific detection requirements for different application scenarios. This remarkable method provides 51.2 to 1000 times sensitivity improvement over commercial antigen detection kits and greater than 12.5 to 100 times improvement over QD-based immunosensors. Furthermore, we comprehensively evaluated the specificity, reproducibility, and stability of the integrated dual-system detection platform, demonstrating its reliability. Remarkably, the respiratory viral testing was validated using biological samples, thus illustrating its promise and convenience in the detection of respiratory viruses.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

Evaluation of a Novel Detection Method for Allergen-Specific IgE Antibodies with IgE Receptor Crosslinking Using Rat Food Allergy Model.

The specific detection of serum IgE antibodies specific to allergens (sIgE Abs) that can crosslink the plural high-affinity IgE receptor (FcεRIα) molecules on the surface of mast cells or basophils with a multivalent allergen can reduce the false-positive diagnoses observed in chemiluminescent and fluorescence enzyme immunoassays for type-I allergic patients. In this study, we detected sIgE Abs to the egg-allergen ovalbumin (OVA) and the wheat-allergen gluten in the sera of rats sensitized with each allergen using an amplified luminescence proximity homogeneous assay by crosslinking (AlphaCL). OVA and gluten were reacted with each sIgE Ab in the sera. Then, acceptor and donor beads labeled with the human FcεRIα were added to the reacted solution. The luminescence intensity for anti-OVA IgE Abs in the sera with the removal of IgG Abs was observed in five of seven (71.4%) of the sensitized rats, whereas no signals were observed in any of the unsensitized rats. The AlphaCL could also detect anti-gluten sIgE Abs in the sera of sensitized rats, but not of unsensitized rats. In conclusion, we successfully detected sIgE Abs in the sera of rats sensitized to two allergens using the AlphaCL. This detection method has the potential to be used as a new diagnostic tool for type-I allergic patients.

Integrated CRISPR/Cas12a with terminal protection strategy for homogeneous protein assay

Developing appropriate protein assay methods is necessary, as protein assays play an important role in molecular diagnosis, drug screening, and biomedical research. This article presents a universal homogeneous method based on the CRISPR/Cas12a system and the terminal protection strategy for protein assays. Initially, a single-stranded DNA (ssDNA) labeled with a small molecule at its 3’-end was designed to respond to the target protein and serve as an activator of Cas12a. In the absence of the target protein, small-molecule-linked ssDNA was effectively cleaved using exonuclease I (Exo I). The lack of the activator resulted in the trans-cleavage inactivity of Cas12a, leading to low fluorescence in the detection solution. However, in the presence of the target protein, the specific binding of the target protein to the small molecules on the activator protected the activator against digestion by Exo I. The remaining intact activator triggered the trans-cleavage activation of Cas12a, producing strong fluorescence. Based on this principle, our method achieved fluorescence enhanced detection of the target protein. After experimental condition optimization, our method achieved highly sensitive and specific protein detection in two models: folate receptor and streptavidin. The excellent performance of the fluorescence method highlights the applicability and effectiveness of the CRISPR/Cas12a system in protein assays, and broadening its application scope.

Light-activated oxidative capacity of isoquinoline alkaloids for universal, homogeneous, reliable, colorimetric assays with DNA aptamers

Aptamers are good affinity receptors for bio-assays, while colorimetric method is suitable for point-of-care sensing via direct visualization. But previously aptamers often need complex re-engineering for colorimetric measurement at the cost of affinity and performance. Here isoquinoline alkaloids are found to own unique light-activated oxidative capacity, which can be specifically triggered by unmodified aptamers. This feature is universal for two alkaloids to efficiently oxidize four chromogenic substrates with obvious color changes. Based on a dye-displacement process, we have developed a novel light-activated aptamer system for the colorimetric assay of estradiol. It shows a good sensitivity with a detection limit of 326 nM, and this homogeneous assay is reliable to avoid artifacts in previous heterogeneous scheme. Besides, it is proven to be a universal design to assay other two targets. Significantly, they do not employ any aptamers re-engineering but only simply use their parental aptamers. Therefore, this light-activated oxidative capacity of isoquinoline alkaloid can serve as an ideal tool for colorimetric assay of various targets based on aptamer's specific recognition.

Development of a Quick and Highly Sensitive Amplified Luminescent Proximity Homogeneous Assay for Detection of Saxitoxin in Shellfish.

Saxitoxin (STX), an exceptionally potent marine toxin for which no antidote is currently available, is produced by methanogens and cyanobacteria. This poses a significant threat to both shellfish aquaculture and human health. Consequently, the development of a rapid, highly sensitive STX detection method is of great significance. The objective of this research is to create a novel approach for identifying STX. Therefore, amplified luminescent proximity homogeneous assay (AlphaLISA) was established using a direct competition method based on the principles of fluorescence resonance energy transfer and antigen-antibody specific binding. This method is sensitive, rapid, performed without washing, easy to operate, and can detect 8-128 ng/mL of STX in only 10 min. The limit of detection achieved by this method is as low as 4.29 ng/mL with coefficients of variation for the intra-batch and inter-batch analyses ranging from 2.61% to 3.63% and from 7.67% to 8.30%, respectively. In conclusion, our study successfully establishes a simple yet sensitive, rapid, and accurate AlphaLISA method for the detection of STX which holds great potential in advancing research on marine biotoxins.

Enzymatic biofuel cells-based self-powered electrochemical biosensor for detection of 17β-estradiol based on dual-enzyme cascade amplification and bioconjugate

Self-powered electrochemical biosensors (SPEB) based on enzymatic biofuel cells (EBFC) have garnered significant attention due to their distinctive characteristic of not necessitating the source of external power supply. This study developed a novel EBFC-based SPEB by incorporating dual-enzyme cascade amplification and bioconjugate for extremely precise detection of 17β-estradiol (E2). The carbon nanocages/gold nanoparticles composite was employed as substrate to immobilize bilirubin oxidase (BOD) and complementary ssDNA (csDNA) to form bioconjugate. The carbon nanotubes/gold nanoparticles composite was used in the EBFC device as the electrode material, through which glucose oxidase (GOD) and E2 aptamer were wired to form the bioanode and biocathode, respectively. The biocathode further linked with bioconjugate via a hybridization reaction between E2 aptamer and csDNA. The bioconjugate integrates the recognition and signal probes for homogeneous electrochemical assay of targets, which released from biocathode once the target E2 was recognized and hindered the transfer of electrons produced by GOD on bioanode. Thus, the open-circuit voltage (EOCV) of the EBFC-based SPEB dramatically decreased, exhibiting exceptional sensitivity for the detection of E2 over a concentration range from 10 pg mL−1 to 20 ng mL−1, with a detection limit of 3.3 pg mL−1 (S/N = 3). Furthermore, the prepared EBFC-based SPEB showed significant potential for implementation as a viable prototype for a mobile and an on-site bioassay system in food and environmental safety applications.

Monoterpene-chalcone conjugates and diarylheptanoids isolated from the seeds of Alpinia katsumadai Hayata with cytotoxic activity

Five undescribed monoterpene-chalcone conjugates (1-5), one undescribed hypothetical precursor of diarylheptanoid (6), two undescribed diarylheptanoids (7–8), and fourteen known compounds (9–22) were isolated from the seeds of Alpinia katsumadai. Their structures were elucidated through the interpretation of HRESIMS, NMR, ECD, and X-ray diffraction data. MTT assays on human cancer cell lines (HepG2, A549, SGC7901, and SW480) revealed that compounds 3–8, 11, and 13 exhibited broad-spectrum antiproliferative activities with IC50 values ranging from 3.59 to 21.78 μM. B cell lymphoma 2 was predicted as the target of sumadain C (11) by network pharmacology and verified by homogeneous time-resolved fluorescence assay and molecular docking.

Portable Device with Nicking Enzyme Enhanced Special RCA on μPADs toward Sensitive Detection of High-Risk HPV Infection.

Development of an accurate, rapid, and cost-effective portable device is in high demand for point-of-care molecular diagnosis toward disease screening. Here we report a one-pot homogeneous isothermal assay that leverages nicking endonuclease and minimum secondary structured rolling circle amplification (N-MSSRCA) for fast and sensitive quantification of nucleic acids on distance microfluidic paper-based analytical devices (dμPAD) by a portable custom-made fluorescence detector. Human papillomavirus (HPV) oncogenic E7 mRNA as the biomarker for cervical cancer was used as the model analyte. N-MSSRCA integrates ligase for target recognition, the nicking enzyme for primer generation, and the dual function of the Phi29 DNA polymerase for both on- and off-loop amplification. The proposed method was capable of detecting 1 and 10 fM of the analyte using the microplate reader and portable detector with dμPAD, respectively, with ∼1 h assay time. A cohort study of 40 cervical swab samples shows N-MSSRCA reached positive and negative predictive values of 87.5% and 93.5% using the portable detector with dμPAD, compared to 91.67% and 100% using the microplate reader. N-MSSRCA demonstrates potential in early screening of high-risk HPV infection as a generic strategy to detect various nucleic acids in point-of-care scenarios.

Glycyrrhetinic acid blocks SARS-CoV-2 infection by activating the cGAS-STING signalling pathway.

Traditional Chinese medicine (TCM) played an important role in controlling the COVID-19 pandemic, but the scientific basis and its active ingredients are still weakly studied. This study aims to decipher the underlying anti-SARS-CoV-2 mechanisms of glycyrrhetinic acid (GA). GA's anti-SARS-CoV-2 effect was verified both in vitro and in vivo. Homogeneous time-resolved fluorescence assays, biolayer interferometry technology, and molecular docking were employed to examine interactions of GA with human stimulator of interferon genes (hSTING). Immunofluorescence staining, western blot, and RT-qPCR were used to investigate nuclear translocation of interferon regulatory factor 3 (IRF3) and levels of STING target genes. Pharmacokinetics of GA was studied in mice. GA could directly bind to Ser162 and Tyr240 residues of hSTING, thus up-regulating downstream targets and activation of the STING signalling pathway. Such activation is crucial for limiting the replication of SARS-CoV-2 Omicron in Calu-3 cells and protecting against lung injury induced by SARS-CoV-2 Omicron infection in K18-ACE2 transgenic mice. Immunofluorescence staining and western blot indicated that GA increased levels of phosphorylated STING, phosphorylated TANK-binding kinase-1, and cyclic GMP-AMP synthase (cGAS). Importantly, GA increased nuclear translocation of IRF3. Pharmacokinetic analysis of GA in mice indicated it can be absorbed into circulation and detected in the lung at a stable level. Activation of the cGAS-STING pathway through the GA-STING-IRF3 axis is essential for the antiviral activity of GA in mice, providing new insights into the potential translation of GA for treating SARS-CoV-2 in patients.

Nanobody-enhanced split-luciferase technology for innovative detection of the liver cancer biomarker alpha-fetoprotein

Liver cancer is one of the most common cancers and the third leading cause of cancer deaths worldwide. Diagnosis and screening for liver cancer rely on the alpha-fetoprotein (AFP) biomarker. This study aimed to pioneer a novel assay for AFP detection utilizing a tri-part split-luciferase system in conjunction with nanobodies targeting AFP. The strategy involved fusing anti-AFP nanobodies P5 and P15 to the split-nanoluciferase components β9 and β10, respectively. Upon binding to AFP and in the presence of the third nanoluciferase component Δ11S, the proximity-induced reassembly of split-nanoluciferase components triggers luciferase activation and luminescence emission. Following expression in a bacterial system, purification, and assay implementation, the developed assay exhibited high sensitivity in detecting AFP within a linear range of 1–20 ng/ml, with a Limit of Detection (LOD) of 0.5 ng/ml. The assay results were in line with those obtained from ELISA, indicating its efficiency. This study highlights the specificity of the homogenous assay developed with nanobodies for AFP, offering a reliable and user-friendly test for AFP detection.

Serum anti‑KIAA0513 antibody as a common biomarker for mortal atherosclerotic and cancerous diseases.

Numerous antibody biomarkers have been reported for cancer and atherosclerosis-related diseases. The major complications of atherosclerosis and diabetes mellitus (DM) are acute ischemic stroke (AIS), cardiovascular disease (CVD) and chronic kidney disease (CKD). Cancer development is accompanied by arterial disorders, such as angiogenesis and atherosclerosis, and DM is a risk factor for the development of certain types of cancer. Atherosclerosis-related diseases and cancers are therefore interrelated and could be detected using a common biomarker. In the present study, the initial screening using the protein array method identified KIAA0513 as an antigen recognized by serum IgG antibodies in patients with atherosclerosis. The amplified luminescent proximity homogeneous assay-linked immunosorbent assay revealed significantly higher serum antibody levels against recombinant KIAA0513 protein in patients with AIS, transient ischemic attack (TIA), DM, CVD, obstructive sleep apnea syndrome (OSAS), CKD and solid cancers, such as esophageal, gastric, colon, lung and breast cancers, compared with healthy donors. A receiver operating characteristic (ROC) analysis revealed that the highest areas under the ROC curves of anti-KIAA0513 antibodies were obtained for esophageal cancer, nephrosclerosis-type CKD and DM. Spearman's correlation analysis revealed that serum anti-KIAA0513 antibody levels were associated with maximum intima-media thickness and plaque score, which are indices of atherosclerosis and stenosis. Serum anti-KIAA0513 antibody markers appear to be useful for diagnosing AIS, TIA, DM, CVD, OSAS, CKD and solid cancers, and may reflect common arterial alterations leading to atherosclerotic and cancerous diseases.

Apollon: a deoxyribozyme that generates a yellow product.

Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR. A bimolecular version of Apollon catalyzed multiple turnover phosphorylation and amplified the colorimetric signal. Engineered versions of Apollon could detect oligonucleotides with specific sequences as well as several different types of nucleases in homogenous assays that can be performed in a single tube without the need for washes or purifications. We anticipate that Apollon will be particularly useful to reduce costs in high-throughput screens and for applications in which specialized equipment is not available.

Aurora: a fluorescent deoxyribozyme for high-throughput screening.

Fluorescence facilitates the detection, visualization, and tracking of molecules with high sensitivity and specificity. A functional DNA molecule that generates a robust fluorescent signal would offer significant advantages for many applications compared to intrinsically fluorescent proteins, which are expensive and labor intensive to synthesize, and fluorescent RNA aptamers, which are unstable under most conditions. Here, we describe a novel deoxyriboyzme that rapidly and efficiently generates a stable fluorescent product using a readily available coumarin substrate. An engineered version can detect picomolar concentrations of ribonucleases in a simple homogenous assay, and was used to rapidly identify novel inhibitors of the SARS-CoV-2 ribonuclease Nsp15 in a high-throughput screen. Our work adds an important new component to the toolkit of functional DNA parts, and also demonstrates how catalytic DNA motifs can be used to solve real-world problems.

Evaluation of 13 Formulae for Calculated LDL-C Using Direct Homogenous Assay in a South Indian Population.

LDL cholesterol (LDL-C) is regarded as a significant therapeutic target and a known risk factor for atherosclerosis. It can be calculated using the results of the other lipid tests or tested directly. Despite its shortcomings, the Friedewald formula is most frequently utilized since it is simple and practical. Until now, several formulae have been proposed for calculating LDL-C; however, their accuracy has not been evaluated across different populations. We sought to evaluate the validity of calculated LDL-C by comparing the findings with values acquired by the direct homogeneous technique, utilizing 13 distinct formulae from the literature. This study was a retrospective observational study conducted for a year at SRIHER, Chennai, Tamil Nadu, India. From the total 25 043 patients who had their serum lipid profile tested, 16 314 participants had their fasting blood sugar and fasting lipid profile measured simultaneously, and they were chosen for the research. The de Cordova, Chen, Martin/Hopkins (initial), and Teerakanchana equations correlated well with the direct LDL-C assay. When the dataset was stratified according to triglycerides, the Chen and Martin/Hopkins initial equations had the better measurement of agreement compared to other equations. The Martin/Hopkins initial equation outperformed all the other equations when the whole dataset irrespective of the triglyceride population was considered. Our study suggests that the Martin/Hopkins initial equation outperformed all the other equations and can be used as an alternative to direct LDL-C measurement in a South Indian population.

AlphaLISA-Based Immunoassay for Detection of Troponin T in Serum of Patients with Acute Myocardial Infarction.

Survival and prognosis of patients with acute myocardial infarction (AMI) are highly dependent on rapid and accurate diagnosis of myocardial damage. Troponin T is the primary diagnostic biomarker and is widely used in clinical practice. Amplified luminescent proximity homogeneous assay (AlphaLISA) may provide a solution to rapidly detect a small amount of analyte through molecular interactions between special luminescent donor beads and acceptor bead. Here, a double-antibody sandwich assay was introduced into AlphaLISA for rapid detection for early diagnosis of AMI and disease staging evaluation. The performance of the assay was evaluated. The study found that the cTnT assay has a linear range of 48.66 to 20,000 ng/L with a limit of detection of 48.66 ng/L. In addition, the assay showed no cross-reactivity with other classic biomarkers of myocardial infarction and was highly reproducible with intra- and inter-batch coefficients of variation of less than 10%, notably, only 3min was taken, which is particularly suitable for clinical diagnosis. These results suggest that our method can be conveniently applied in the clinic to determine the severity of the patient's condition.