One-pot microplate-based chemiluminescent assay coupled with catalytic hairpin assembly amplification for DNA detection.

Nowadays, considerable efforts are focused on advancing DNA detection methods, which are extremely important in clinical diagnostics, pathogen determination, gene therapy, and forensic analysis. A one-pot sensitive microplate-based chemiluminescent assay coupled with catalytic hairpin assembly (CHA) amplification for detection of a 35-mer DNA oligonucleotide was developed. To improve the assay sensitivity, a triple amplification strategy based on the application of CHA (1), streptavidin-polyperoxidase conjugate (Stp-polyHRP) (2), and an enhanced chemiluminescent reaction (3) was used. The one-pot format of the assay, where all steps of the DNA determination are performed in the same well without transfer of samples from one test tube to another, increased its precision. The proposed assay detected the target DNA in the fM range and distinguished the target DNA from related DNAs, demonstrating its high sensitivity and high selectivity. Moreover, the assay was applied successfully for the quantitative determination of the target in spiked samples of human plasma. A microplate format of the assay was convenient for the analysis of a large number of samples. This study provides a prospective tool for DNA detection. Graphical abstract.

The Efficiency of Color Space Channels to Quantify Color and Color Intensity Change in Liquids, pH Strips, and Lateral Flow Assays with Smartphones.

Bottom-up, end-user based feed, and food analysis through smartphone quantification of lateral flow assays (LFA) has the potential to cause a paradigm shift in testing capabilities. However, most developed devices do not test the presence of and implications of inter-phone variation. Much discussion remains regarding optimum color space for smartphone colorimetric analyses and, an in-depth comparison of color space performance is missing. Moreover, a light-shielding box is often used to avoid variations caused by background illumination while the use of such a bulky add-on may be avoidable through image background correction. Here, quantification performance of individual channels of RGB, HSV, and LAB color space and ΔRGB was determined for color and color intensity variation using pH strips, filter paper with dropped nanoparticles, and colored solutions. LAB and HSV color space channels never outperformed the best RGB channels in any test. Background correction avoided measurement variation if no direct sunlight was used and functioned more efficiently outside a light-shielding box (prediction errors < 5%/35% for color/color intensity change). The system was validated using various phones for quantification of major allergens (i.e., gluten in buffer, bovine milk in goat milk and goat cheese), and, pH in soil extracts with commercial pH strips and LFA. Inter-phone variation was significant for LFA quantification but low using pH strips (prediction errors < 10% for all six phones compared). Thus, assays based on color change hold the strongest promise for end-user adapted smartphone diagnostics.

Machine learning models for predicting risks of MACEs for myocardial infarction patients with different VEGFR2 genotypes.

Background: The development of prognostic models for the identification of high-risk myocardial infarction (MI) patients is a crucial step toward personalized medicine. Genetic factors are known to be associated with an increased risk of cardiovascular diseases; however, little is known about whether they can be used to predict major adverse cardiac events (MACEs) for MI patients. This study aimed to build a machine learning (ML) model to predict MACEs in MI patients based on clinical, imaging, laboratory, and genetic features and to assess the influence of genetics on the prognostic power of the model. Methods: We analyzed the data from 218 MI patients admitted to the emergency department at the Surgut District Center for Diagnostics and Cardiovascular Surgery, Russia. Upon admission, standard clinical measurements and imaging data were collected for each patient. Additionally, patients were genotyped for VEGFR-2 variation rs2305948 (C/C, C/T, T/T genotypes with T being the minor risk allele). The study included a 9-year follow-up period during which major ischemic events were recorded. We trained and evaluated various ML models, including Gradient Boosting, Random Forest, Logistic Regression, and AutoML. For feature importance analysis, we applied the sequential feature selection (SFS) and Shapley's scheme of additive explanation (SHAP) methods. Results: The CatBoost algorithm, with features selected using the SFS method, showed the best performance on the test cohort, achieving a ROC AUC of 0.813. Feature importance analysis identified the dose of statins as the most important factor, with the VEGFR-2 genotype among the top 5. The other important features are coronary artery lesions (coronary artery stenoses ≥70%), left ventricular (LV) parameters such as lateral LV wall and LV mass, diabetes, type of revascularization (CABG or PCI), and age. We also showed that contributions are additive and that high risk can be determined by cumulative negative effects from different prognostic factors. Conclusion: Our ML-based approach demonstrated that the VEGFR-2 genotype is associated with an increased risk of MACEs in MI patients. However, the risk can be significantly reduced by high-dose statins and positive factors such as the absence of coronary artery lesions, absence of diabetes, and younger age.

A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity.

CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.

Comparative Analysis of Chemical Profile and Biological Activity of Juniperus communis L. Berry Extracts.

Researchers are looking for the most effective ways to extract the bioactive substances of Juniperus communis L. berries, which are capable of displaying the greatest range of biological activity, namely antimicrobial potential "against phytopathogens", antioxidant activity and nematocidal activity. This study provides detailed information on the chemical activity, group composition and biological activity of the extracts of juniper berries of 1- and 2-year maturity (JB1 and JB2), which were obtained by using different solvents (pentane, chloroform, acetone, methanol and 70% ethanol) under various extraction conditions (maceration and ultrasound-assisted maceration (US)). Seventy percent ethanol and acetone extracts of juniper berries were analyzed via gas chromatography-mass spectrometry, and they contained monoterpenes, sesquiterpenes, polysaccharides, steroids, fatty acid esters and bicyclic monoterpenes. The antimicrobial activity was higher in the berries of 1-year maturity, while the acetone extract obtained via ultrasound-assisted maceration was the most bioactive in relation to the phytopathogens. Depending on the extraction method and the choice of solvent, the antioxidant activity with the use of US decreased by 1.5-1.9 times compared to the extracts obtained via maceration. An analysis of the nematocidal activity showed that the sensitivity to the action of extracts in Caenorhabditis elegans was significantly higher than in Caenorhabditis briggsae, particularly for the acetone extract obtained from the juniper berries of 1-year maturity.

Comparative study of magnetic beads and microplates as supports in heterogeneous amplified assay of miRNA-141 by using mismatched catalytic hairpin assembly reaction.

Magnetic beads (MBs) are often considered as an effective carrier in heterogeneous assays due to the simplicity of separation and washing, and the ability to increase and control the surface area. However, the effect of the MBs surface on the analytical parameters is poorly characterized and is often postulated from intuitive considerations. Herein, experimental evaluation through the comparison of MBs and microwell plate was carried out using the miRNA-141 (biomarker for cancer) as a target, the detection of which was performed by chemiluminescent assay with a homogeneous mismatched catalytic hairpin assembly (mCHA) reaction. The mCHA reaction produced double-stranded (ds) DNA labeled at one end with fluorescein (Flu) for capture with anti-Flu antibodies immobilized on a solid carrier, on the other end with biotin for recognition by streptavidin-polyperoxidase conjugate. The conditions of immobilization of anti-Flu antibody on MBs (a diameter of 440 nm) performed using a carbodiimide method were optimized by varying the antibody concentration in the reaction solution. It was shown that the dependence of chemiluminescent signal as a function of the concentration of anti-FluAb-MBs conjugates had a bell-shaped character. The maximum chemiluminescence was produced at the concentration of the conjugates of 2 × 109 particles/mL, with a surface area of 65 mm2. The identical surface area was used upon the assay performance with polystyrene microplates. Comparison of MBs- and microplate-assays for miRNA-141 determination showed that the obtained calibration curves and their detection limit values were the same and did not depend on the used carrier. The results showed that the choice of a carrier for heterogeneous assays should be guided by the convenience of the assay performance, not its surface area.

One-step label-free chemiluminescent assay for determination of exonuclease III activity towards hairpin oligonucleotides.

Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5'-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M-1, respectively. The coefficient of variation (CV) was lower than 6%.