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Real-time digital polymerase chain reaction (qdPCR) provides enhanced precision in the field of molecular diagnostics by integrating absolute quantification with process information. However, the optimal reaction conditions are traditionally determined through multiple iterative of experiments. Therefore, we proposed a novel approach to precisely determine the optimal reaction conditions for qdPCR using a standard process, employing real-time fluorescence monitoring within microwells. The temperature-sensitive fluorophore intensity presented the real temperature of each microwell. This enabled us to determine the optimal denaturation and annealing time for qdPCR based on the corresponding critical temperatures derived from the melting curves and amplification efficiency, respectively. To confirm this method, we developed an ultrathin laminated chip (UTL chip) and chose a target that need to be absolutely quantitative. The UTL chip was designed using a fluid‒solid‒thermal coupling simulation model and exhibited a faster thermal response than a commercial dPCR chip. By leveraging our precise determination of reaction conditions and utilizing the UTL chip, 40 cycles of amplification were achieved within 18 min. This was accomplished by precisely controlling the denaturation temperature at 2 s and the annealing temperature at 10 s. Furthermore, the absolutely quantitative of DNA showed good correlation (R2 > 0.999) with the concentration gradient detection using the optimal reaction conditions with the UTL chip for qdPCR. Our proposed method can significantly improve the accuracy and efficiency of determining qdPCR conditions, which holds great promise for application in molecular diagnostics. [Display omitted] [ABSTRACT FROM AUTHOR]

For digital polymerase chain reaction (PCR), data classification is always a crucial task. The dynamic real-time amplification process information of each partition is always ignored in typical digital PCR analysis, which can easily lead to inaccurate outcomes. In this work, an integrated device that offers real-time chip-based digital PCR analysis was established. In addition, an enhanced process-based classification model (PAM) was built and trained. And then the device and the analytical model were employed in classification tasks for different concentrations of Epstein-Barr Virus (EBV) plasmid quantification assays. The results indicated that the real-time analysis device achieved a linearity of 0.97, the classification method was able to distinguish the false-positive curves, and the recognition error of positive wells was decreased by 64.4% compared with typical static analysis techniques when low concentrations of samples were tested. With these advantages, it is supposed that the real-time digital PCR analysis apparatus and the improved classification method can be employed to enhance the performance of digital PCR technology. • An integrated device supporting real-time digital polymerase chain reaction (RT-dPCR) was established. • An improved data classification method for RT-dPCR was proposed based on the amplification process of each subsample. • The improved classification method would lead to a more accurate result than traditional static analysis methods. [ABSTRACT FROM AUTHOR]

Anticoagulation therapy with heparin is an effective treatment against thrombosis. Heparin tends to cause spontaneous bleeding and requires regular monitoring during therapy. Most high-sensitivity heparin sensors have focused on the concentration detection in clarified buffer solution. However, the pharmacodynamics of heparin vary depending on individual patient or disease, while potency detection with high sensitivity and dynamic range outperforms concentration detection in clinical diagnosis. In this study, a novel heparinase-linked differential time (HLDT) method was established with a two-zone of Graphene modified Carbon (GR-C) sensor, which was utilized to evaluate heparin potency in whole blood. It was based on electrochemical measurement of clotting time shifting associated with presence or absence of heparinase. Heparinase inhibits the anticoagulant ability of heparin by forming a heparin–antithrombin–thrombin complex during coagulation. And the intensity and peak time of electrochemical current were associated with thrombin activity and clotting on the electrode. The results demonstrated that the sensor had high selectivity for heparin potency in 10 μL of whole blood with a detection limit of 0.1 U/mL, and the linear detection range was 0.1–5 U/mL. The coefficient of variation (CV) of the peak time was less than 5%, and linear correlation between the GR-C sensor and the TEG-5000 instrument was 0.987. Thus, the HLDT method has better clinical application due to its good repeatability, high sensitivity and wide range in heparin potency evaluation. • A novel assay, the heparinase-linked differential time (HLDT) method, was developed. • The HLDT method is based on heparinase preventing thrombin inhibition by heparin • Heparin potency in 10 μL whole blood was measured using the HLDT method. • The detection range was 0.1–5 U/mL and detection accuracy was 0.1 U/mL. • R2 = 0.987 between the GR-C heparin sensor and TEG-5000 for 15 patient groups. [ABSTRACT FROM AUTHOR]