Homogeneous Assay Research Articles

Generic strategy for the synthesis of highly specific Au/MIP nanozymes and their application in homogeneous assays

A novel strategy for creating peroxidase-mimicking nanozymes (Au/MIP) using gold-core nanoparticles is reported, enabling sensitive, specific colorimetric amphetamine detection in complex samples as a robust alternative to traditional assays.

A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in the early stage of infection. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro). The test system consists of a peptide that fluoresces when cleaved by SARS PLpro that is active in crude, unprocessed lysates from human tongue scrapes and saliva. Test results are obtained in 30 minutes or less using widely available fluorescence plate readers, or a battery-operated portable instrument for on-site testing. Proof-of-concept was obtained in a study on clinical specimens collected from patients with COVID-19 like symptoms who tested positive (n = 10) or negative (n = 10) with LIAT RT-PCR using nasal mid turbinate swabs. When saliva from these patients was tested with in-house endpoint RT-PCR, 17 were positive and only 5 specimens were negative, of which 2 became positive when tested 5 days later. PLpro activity correlated in 17 of these cases (3 out of 3 negatives and 14 out of 16 positives, with one invalid specimen). Despite the small number of samples, the agreement was significant (p value = 0.01). Two false negatives were detected, one from a sample with a late Ct value of 35 in diagnostic RT-PCR, indicating that an active infection was no longer present. The PLpro assay is easily scalable and expected to detect all viable SARS-CoV-2 variants, making it attractive as a screening and surveillance tool. Additionally, we show feasibility of the platform as a new homogeneous phenotypic assay for rapid screening of SARS-CoV-2 antiviral drugs and neutralizing antibodies.

P110 Zasocitinib (TAK-279) exhibits high levels of TYK2 inhibition and no inhibition of JAK 1/3 when compared with licensed TYK2 and JAK inhibitors

Abstract Zasocitinib (TAK-279) is an oral, allosteric, highly selective, tyrosine kinase 2 (TYK2) inhibitor in late-stage clinical development for the treatment of immune-mediated inflammatory diseases, including psoriasis. Zasocitinib was developed using AI-assisted compound design strategy to create a TYK2 inhibitor with next-generation selectivity and >1 million-fold greater affinity for TYK2 than for Janus kinase (JAK) 1/2/3. This study compared the potency and selectivity of zasocitinib for TYK2 vs. JAK1/3 with licensed TYK2/JAK inhibitors. The inhibitory constants (Kis) of zasocitinib and deucravacitinib for JAK1 and TYK2 Janus homology 2 (JH2) domains were determined using a homogeneous time-resolved fluorescence assay. Fresh whole-blood cultures from 12 to 13 healthy volunteers characterized concentration–response (percentage inhibition) curves for TYK2- (IL-12/18 IFN-γ) and JAK1/3- (IL-2 pSTAT5) dependent pathways for zasocitinib and licensed TYK2/JAK inhibitors. Statistical modelling determined the relationship between concentration and percentage inhibition, and half-maximal inhibitory concentration (IC50) values were estimated. Drug plasma concentrations at clinically relevant/approved doses were simulated using published population pharmacokinetic models. Time over plasma concentrations >IC50 and percentage daily inhibition were compared between drugs for each pathway. The Kis of zasocitinib and deucravacitinib for the JAK1 JH2 domain were >15 000 and 1 nM, respectively. The Kis of zasocitinib and deucravacitinib for the TYK2 JH2 domain were 0.0087 and 0.0115 nM, respectively. Simulated plasma concentrations were above TYK2 IC50 for 24 h for zasocitinib 30 mg once daily (QD) vs. 3 h for deucravacitinib 6 mg QD, and 0 h for baricitinib 4 mg QD, tofacitinib 10 mg twice daily and upadacitinib 30 mg QD. TYK2 percentage daily inhibition was 91% for zasocitinib 30 mg and 23% for deucravacitinib 6 mg. Baricitinib 4 mg, upadacitinib 30 mg and tofacitinib 10 mg showed minimal TYK2 inhibition (0–8%). Simulated plasma concentrations of zasocitinib 30 mg and deucravacitinib 6 mg did not reach JAK1/3 IC50; those of baricitinib 4 mg, upadacitinib 30 mg and tofacitinib 10 mg were above JAK1/3 IC50 for 24 h. JAK1/3 percentage daily inhibition was 0% for zasocitinib 30 mg and 3% for deucravacitinib 6 mg vs. 91–97% for baricitinib 4 mg, upadacitinib 30 mg and tofacitinib 10 mg. Selective TYK2 inhibition offers a distinct cytokine receptor inhibition profile. Zasocitinib showed greater and longer selective inhibition of TYK2-mediated signalling vs. deucravacitinib at its clinical dosage, without affecting JAK1/3-mediated signalling.

Identification of RdRp-NiRAN/JAK1 Dual-Target Drugs for COVID-19 Treatment.

Inhibition of virus replication and inflammatory response is important for the treatment of severe COVID-19 patients. RNA-dependent RNA polymerase (RdRp) is indispensable for SARS-CoV-2 replication, and Janus kinase (JAK) 1 inhibitors exert immunosuppressive effects. RdRp/JAK1 dual-target drugs are expected to ameliorate the severity of the COVID-19 disease. The N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain of RdRp is a pseudokinase, and it has structural similarities with JAK1. Herein, we evaluated the inhibitory effects of triphosphate forms of 31 nucleoside drugs in the DrugBank database on the NiRAN domain and JAK1 through a combination of theoretical and experimental methods. By analyzing the three properties of 31 nucleoside drugs (total hydrophobic surface area, number of hydrophobic atoms, and molecular weight), these drugs met the application rule of our developed molecular docking with conformer-dependent charges (MDCC). Based on the MDCC method combined with molecular dynamics simulations, Azvudine and Citicoline among these 31 drugs showed stronger predicted binding affinities with the NiRAN domain as well as JAK1 compared to the reference drug Remdesivir. Further experimental verification, including a thermal shift assay and homogeneous time-resolved fluorescence assay, demonstrated that Azvudine was an RdRp-NiRAN/JAK1 dual-target drug. This work provided a previously unexplored mechanism of Azvudine for COVID-19 treatment and proposed a design concept for RdRp-NiRAN/JAK1 dual-target nucleoside drugs.

Validation and Optimization of a Stable Isotope-Labeled Substrate Assay for Measuring AGAT Activity.

L-arginine: glycine amidinotransferase (AGAT) gained academic interest as the rate-limiting enzyme in creatine biosynthesis and its role in the regulation of creatine homeostasis. Of clinical relevance is the diagnosis of patients with AGAT deficiency but also the potential role of AGAT as therapeutic target for the treatment of another creatine deficiency syndrome, guanidinoacetate N-methyltransferase (GAMT) deficiency. Applying a stable isotope-labeled substrate method, we utilized ARG 15N2 (ARG-δ2) and GLY 13C215N (GLY-δ3) to determine the rate of 1,2-13C2,15N3 guanidinoacetate (GAA-δ5) formation to assess AGAT activity in various mouse tissue samples and human-derived cells. Following modification and optimization of the assay, we analyzed AGAT activity in several mouse organs. The Km and Vmax of AGAT in mouse kidney for GLY-δ3 were 2.06 mM and 6.48 ± 0.26 pmol/min/mg kidney, and those for ARG-δ2, they were 2.67 mM and 2.17 ± 0.49 pmol/min/mg kidney, respectively. Our results showed that mouse kidneys had the highest levels of enzymatic activity, followed by brain and liver, with 4.6, 1.8, and 0.4 pmol/min/mg tissue, respectively. Both the heart and muscle had no detectable levels of AGAT activity. We noted that due to interference with arginase in the liver, performing the enzyme assay in liver homogenates required the addition of Nor-NOHA, an arginase inhibitor. In immortalized human cell lines, we found the highest levels of AGAT activity in RH30 cells, followed by HepaRG, HAP1, and HeLa cells. AGAT activity was readily detectable in lymphoblasts and leukocytes from healthy controls. In our assay, AGAT activity was not detectable in HEK293 cells, in human fibroblasts, and in the lymphoblasts of a patient with AGAT deficiency. Our results demonstrate that this enzyme assay is capable of accurately quantifying AGAT activity from both tissues and cells for diagnostic purposes and research.

Transdermal Delivery of Tofacitinib Citraste via Mannose-Decorated Transferosomes Loaded with Tofacitinib Citrate in Arthritic Joints.

Transdermal drug delivery systems are a promising option for the treatment of rheumatoid arthritis (RA) because they can lower systemic adverse effects of immunosuppressants. Janus kinase (JAK) inhibitors were found to be effective for the treatment of RA by inhibiting the JAK-STAT pathway and preventing autoimmune joint destruction. The aim of this study is to deliver tofacitinib (a JAK 1 and 3 inhibitor) through mannose-decorated transferosomes (MDTs) directly to inflamed joints. Transferosomes are composed of phospholipids, Cremophor A25, PEG400, Labrafac lipophile, and oleic acid to enhance the permeation of tofacitinib and control nanovesicle size (∼70-200 nm). Permeation through rat skin was evaluated, where the skin permeation of MDTs (Q24: 38.8 ± 9.82 μg/cm2) and flux (0.5311 ± 0.072 μg/cm2/h) were significantly greater than those of the uncoated transferosomes (Q24 of T1: 1.522 ± 0.329 μg/cm2, Q24 of T2: 3.5002 ± 0.998 μg/cm2, and Q24 of T3: 18.226 ± 5.25 μg/cm2). In addition, MDTs seem to permeate the skin intact, as shown by the transmission electron microscopy (TEM) images of the recipient buffer removed from the Franz diffusion cell. A histopathology assay was performed during the in vivo evaluation of MDTs in an arthritic rat model, in which, significantly less inflammation was observed when MDTs were applied directly to the joint compared to when applied to the dorsal skin and untreated arthritic joints. Furthermore, significantly lower tumor necrosis factor-α (TNFα), IL-6, and IL-1β levels (P < 0.05) were detected by enzyme-linked immunosorbent assay (ELISA) in homogenates of the joints treated with MDTs than in untreated arthritic joints. In conclusion, this study proposed effective MDTs that could deliver tofacitinib directly to inflamed joints possibly by targeting the macrophages circulating in the proximity of the site of inflammation.

Effect of Zinc on improving silver diamine fluoride-derived tooth discoloration in vitro.

Silver Diamine Fluoride (SDF) is effective for arresting dental caries, presenting a valuable non-invasive treatment option in dentistry. Despite its therapeutic advantages, a significant drawback is the tooth discoloration that follows its application, which can affect patient acceptance. Addressing this aesthetic concern without diminishing the treatment's efficacy remains challenging in dental practice. This study explores strategies to improve the aesthetic outcomes of SDF treatments. This in vitro study assessed the efficacy of Zinc in reducing SDF-induced discoloration on dentin blocks and examined its impact on the physical properties of dentin, including hardness and roughness. Dentin blocks were pre-treated with various concentrations of Zinc, followed by SDF application. Color changes were analyzed using Image J software, and cytotoxicity was evaluated using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. Dentin surface characteristics, including micro-hardness and roughness, were assessed using scanning electron microscopy. The study results revealed a dose-dependent efficacy of Zinc in reducing discoloration caused by SDF on dentin, with higher Zinc concentrations showing better improvement in color outcomes. The application of a 20M Zinc solution prior to SDF treatment significantly reduced discoloration compared to SDF alone, measured on day 14. Additionally, no significant changes in the hardness or roughness of etched dentin were observed in Zinc + SDF group compared to the SDF alone group. Zinc treatments demonstrated a desirable outcome on mucosal cytotoxicity, comparable to that of the negative control. Zinc significantly reduced SDF-induced tooth discoloration in a dose-dependent manner without affecting the etched dentin's micro-hardness and roughness, potentially improving patient acceptance, especially in visible areas of the mouth. Further research is warranted to validate the effectiveness of this zinc-enhanced treatment protocol in vivo. Zinc-enhanced SDF treatments could enhance patient acceptance, especially in visible areas of the mouth, offering an improved option for caries management.

Gold Nanoprobes for Robust Colorimetric Detection of Nucleic Acid Sequences Related to Disease Diagnostics.

Gold nanoparticles (AuNPs) are highly attractive for applications in the field of biosensing, particularly for colorimetric nucleic acid detection. Their unique optical properties, which are highly sensitive to changes in their environment, make them ideal candidates for developing simple, rapid, and cost-effective assays. When functionalized with oligonucleotides (Au-nanoprobes), they can undergo aggregation or dispersion in the presence of complementary sequences, leading to distinct color changes that serve as a visual signal for detection. Aggregation-based assays offer significant advantages over other homogeneous assays, such as fluorescence-based methods, namely, label-free protocols, rapid interactions in homogeneous solutions, and detection by the naked eye or using low-cost instruments. Despite promising results, the application of Au-nanoprobe-based colorimetric assays in complex biological matrices faces several challenges. The most significant are related to the colloidal stability and oligonucleotide functionalization of the Au-nanoprobes but also to the mode of detection. The type of functionalization method, type of spacer, the oligo-AuNPs ratio, changes in pH, temperature, or ionic strength influence the Au-nanoprobe colloidal stability and thus the performance of the assay. This review elucidates characteristics of the Au-nanoprobes that are determined for colorimetric gold nanoparticles (AuNPs)-based nucleic acid detection, and how they influence the sensitivity and specificity of the colorimetric assay. These characteristics of the assay are fundamental to developing low-cost, robust biomedical sensors that perform effectively in biological fluids.

Effect of Zinc on Improving Silver Diamine Fluoride-derived Tooth Discoloration in Vitro.

Silver Diamine Fluoride (SDF) is effective for arresting dental caries, presenting a valuable non-invasive treatment option in dentistry. Despite its therapeutic advantages, a significant drawback is the tooth discoloration that follows its application, which can affect patient acceptance. Addressing this aesthetic concern without diminishing the treatment's efficacy remains challenging in dental practice. This study explores strategies to improve the aesthetic outcomes of SDF treatments. This in vitro study assessed the efficacy of Zinc in reducing SDF-induced discoloration on dentin blocks and examined its impact on the physical properties of dentin, including hardness and roughness. Dentin blocks were pre-treated with various concentrations of Zinc, followed by SDF application. Color changes were analyzed using Image J software, and cytotoxicity was evaluated using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. Dentin surface characteristics, including micro-hardness and roughness, were assessed using scanning electron microscopy. The study results revealed a dose-dependent efficacy of Zinc in reducing discoloration caused by SDF on dentin, with higher Zinc concentrations showing better improvement in color outcomes. The application of a 20M Zinc solution prior to SDF treatment significantly reduced discoloration compared to SDF alone, measured on day 14. Additionally, no significant changes in the hardness or roughness of etched dentin were observed in Zinc + SDF group compared to the SDF alone group. Zinc treatments demonstrated a desirable outcome on mucosal cytotoxicity, comparable to that of the negative control. Zinc significantly reduced SDF-induced tooth discoloration in a dose-dependent manner without affecting the etched dentin's micro-hardness and roughness, potentially improving patient acceptance, especially in visible areas of the mouth. Further research is warranted to validate the effectiveness of this zinc-enhanced treatment protocol in vivo.

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

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&amp;lt;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 &amp;lt;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 &amp;gt;= 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 &amp;lt;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 (&amp;gt;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 &amp;lt; 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 &amp;gt;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.

PROTAC Beyond Cancer- Exploring the New Therapeutic Potential of Proteolysis Targeting Chimeras.

In the realm of oncology, the transformative impact of PROTAC (PROteolysis TAgeting Chimeras) technology has been particularly pronounced since its introduction in the 21st century. Initially conceived for cancer treatment, PROTACs have evolved beyond their primary scope, attracting increasing interest in addressing a diverse array of medical conditions. This expanded focus includes not only oncological disorders but also viral infections, bacterial ailments, immune dysregulation, neurodegenerative conditions, and metabolic disorders. This comprehensive review explores the broadening landscape of PROTAC application, highlighting ongoing developments and innovations aimed at deploying these molecules across a spectrum of diseases. Careful consideration of the design challenges associated with PROTACs reveals that, when appropriately addressed, these compounds present significant advantages over traditional therapeutic approaches, positioning them as promising alternatives. To evaluate the efficacy of PROTAC molecules, a diverse array of assays is employed, ranging from High-Throughput Imaging (HTI) assays to Cell Painting assays, CRBN engagement assays, Fluorescence Polarization assays, amplified luminescent proximity homogeneous assays, Timeresolved fluorescence energy transfer assays, and Isothermal Titration Calorimetry assays. These assessments collectively contribute to a nuanced understanding of PROTAC performance. Looking ahead, the trajectory of PROTAC technology suggests its potential recognition as a versatile therapeutic strategy for an expansive range of medical conditions. Ongoing progress in this field sets the stage for PROTACs to emerge as valuable tools in the multifaceted landscape of medical treatments.

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.