Quantification Of Nucleic Acids Research Articles

Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration

Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/μL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets.

Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.

Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.

Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Abstract 3667: Nucleic acid quantification in uveal melanoma aqueous humor

Abstract Introduction: Uveal melanoma (UM) is the most common primary adult intraocular cancer. Though radiation brachytherapy is often employed for UM treatment, half of UM patients develop systemic metastases. In large tumors with macular involvement, the disease and its treatment can lead to blindness. Clinically, UM prognosis is estimated by tumor size, location, American Joint Cancer Commission (AJCC) staging, and histologic findings. Prognostic UM biomarkers have been established but lesions <2 mm in height, particularly near the fovea, are difficult to biopsy, and thus physicians often do not perform biopsies on such tumors. Therefore, the use of aqueous humor (AH) as a liquid biopsy is a promising methodology. The purpose of the study was to quantify analytes in AH samples collected at diagnosis and post-brachytherapy from UM eyes with varying tumor sizes and AJCC Stages to analyze correlations between analyte (dsDNA, microDNA, and protein) concentrations and tumor features. Methods: This case series study included 119 UM AH samples from 66 UM eyes and 16 control AH samples collected from age-matched non-UM glaucoma (GLC) eyes analyzed for unprocessed analytes using Qubit fluorescence assays. Results: Most UM AH samples contained quantifiable analyte concentrations (dsDNA: 94.1%, miRNA: 88.0%, protein: 95.2%) and at higher concentrations than GLC controls (dsDNA, P=0.008; miRNA, P<0.0001; protein, P=0.007). In samples taken at diagnosis, concentrations were higher at more advanced AJCC stages; comparing most advanced Stage III (n=8) to least advanced Stage I (n=24), median dsDNA was 4 times greater (P<0.0001), protein was 3 times greater (P<0.0001), and miRNA was 2 times greater (P=0.001). Analytes were quantifiable in >70% of pre-brachytherapy samples from eyes with tumors <2 mm tall (n=17). Height was positively associated with analyte concentrations at diagnosis (dsDNA: R=0.43, P=0.0007; miRNA: R=0.35, P=0.01; protein: R=0.39, P=0.005). In paired samples (n=53), all analyte concentrations were significantly higher post-therapy compared to diagnosis pre-therapy (Ps<0.01). Conclusions: Although AH analytes were also quantifiable in smaller, less advanced tumors, AH samples from eyes with larger, more advanced tumors had higher analyte concentrations. The detection of prognostic biomarkers in AH may allow for early UM recognition and guide personalized treatment. Citation Format: Christina Chang, Sarah Pike, Mark Reid, Chen-Ching Peng, Benjamin Xu, Jesse Berry, Liya Xu. Nucleic acid quantification in uveal melanoma aqueous humor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3667.

Assumption-free analysis for amplification-based quantitative nucleic acid detection.

The accurate detection and quantification of biological species that are rarely present but potentially devastating is of paramount importance for the life sciences, biosecurity, food safety, and environmental monitoring. Consequently, there has been significant interest in the sensitive and accurate detection of nucleic acids, leveraging both chemical and biological methods. Among these, quantitative polymerase chain reaction (qPCR) is regarded as the gold standard due to its sensitivity and precision in identifying specific nucleic acid targets. Despite the widespread adoption of qPCR for nucleic acid detection, the analysis of qPCR data typically depends on the use of calibrated standard curves and a threshold method to interpret signal measurements. In this study, we use a stochastic simulation to show the limitations of the threshold method due to its assumptions on amplification kinetics. We propose a new approach for the absolute quantification of nucleic acids that overcomes these limitations by reconstructing the efficiency profile across amplification cycles and using cumulative amplification folds to build a standard curve, thus avoiding the constant efficiency assumption. Our method, validated through experiments with nucleic acid amplification in the presence of potent inhibitors, demonstrates improved accuracy in quantifying nucleic acids, avoiding the systematic errors of the threshold method. This innovation enhances the reliability of nucleic acid quantification, especially where traditional methods struggle with kinetic variability.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Amplification-free quantitative detection of genomic DNA using lateral flow strips for milk authentication

With the globalization and complexity of the food supply chain, the market is becoming increasingly competitive and food fraudulent activities are intensifying. The current state of food detection faced two primary challenges. Firstly, existing testing methods were predominantly laboratory-based, requiring complex procedures and precision instruments. Secondly, there was a lack of accurate and efficient quantitative detection methods. Taking cow's milk as an example, this study introduced a novel method for nucleic acid quantification in dairy products, based on lateral flow strips (LFS). The core idea of this method is to design single-stranded DNA (ssDNA) probes to hybridize with mitochondrial genes, which are abundant, stable, and species-specific in dairy products, as detection targets. Drawing inspiration from the principles of nucleic acid amplification, this research innovatively established a new DNA hybridization method, named LAMP-Like Hybridization (HybLAMP-Like). Leveraging the denaturation and DNA polymerization functions of the bst enzyme, efficient binding of the probe and template strand was achieved. This method eliminated the need for nucleic acid amplification, simplifying the procedure and mitigating aerosol contamination, thereby ensuring the accuracy of the detection results. The method exhibited exceptional sensitivity, capable of detecting extremely low to 12.5 ng in visual inspection and 3.125 ng when using a reader. In terms of practicality, it could achieve visual detection of cow's milk content as low as 1% in adulterated dairy products. When combined with a portable LFS reader, it also enabled precise quantitative analysis of milk adulteration.

Use of Droplet Digital Polymerase Chain Reaction to Identify Biomarkers for Differentiation of Benign and Malignant Renal Masses.

Several microRNAs (miRNAs) have been identified as cell-free biomarkers for detecting renal cell carcinoma (RCC). Droplet digital polymerase chain reaction (ddPCR) is a unique technology for nucleic acid quantification. It has the potential for superior precision, reproducibility, and diagnostic performance in identifying circulating miRNA biomarkers compared to conventional quantitative real-time PCR (qRT-PCR). This study aims to evaluate the performance of ddPCR compared to qRT- PCR in identifying miRNA biomarkers that differentiate malignant from benign renal masses. Potential biomarkers of RCC were identified from a literature review. RNA was extracted from the plasma of 56 patients. All the samples underwent analysis via ddPCR as well as qRT-PCR, and expression levels were recorded for the following miRNAs: miR-93, -144, -210, -221, and -222. Tumors were grouped into low-grade ccRCC, high-grade ccRCC, papillary RCC, and benign masses (primarily angiomyolipoma). The miRNA miR-210 (p = 0.034) and the combination of miRs-210 and miR-222 (p = 0.003) were expressed at significantly higher rates among those with RCC than those with benign masses, as measured by ddPCR. Using the combination of miR-210 and miR-222, ddPCR identified significant differences between the subgroups: papillary RCC versus benign (p = 0.03), low-grade ccRCC versus benign (p = 0.026), and high-grade ccRCC versus benign (p = 0.002). The only significant difference between these subgroups using qRT-PCR was between high-grade ccRCC and benign (p = 0.045). All the AUCs were significant when comparing each RCC subgroup with benign for both PCR technologies. Using a combination of miR-210 and miR-222, ddPCR identified significant differences between benign and malignant renal masses that were not identified as significant by conventional qRT-PCR.

Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

Analytical validation of the DropXpert S6 system for diagnosis of chronic myelocytic leukemia.

Digital PCR is a powerful method for absolute nucleic acid quantification and is widely used in the absolute quantification of viral copy numbers, tumor marker detection, and prenatal diagnosis. However, for most of the existing droplet-based dPCR systems, the droplet generation, PCR reaction, and droplet detection are performed separately using different instruments. Making digital PCR both easy to use and practical by integrating the qPCR workflow into a superior all-in-one walkaway solution is one of the core ideas. A new innovative and integrated digital droplet PCR platform was developed that utilizes cutting-edge microfluidics to integrate dPCR workflows onto a single consumable chip. This makes previously complex workflows fast and simple; the whole process of droplet generation, PCR amplification, and droplet detection is completed on one chip, which meets the clinical requirement of "sample in, result out". It provides high multiplexing capabilities and strong sensitivity while all measurements were within the 95% confidence interval. This study is the first validation of the DropXpert S6 system and focuses primarily on verifying its reliability, repeatability, and consistency. In addition, the accuracy, detection limit, linearity, and precision of the system were evaluated after sample collection. Among them, the accuracy assessment by calculating the absolute bias of each target gene yielded a range from -0.1 to 0.08, all within ±0.5 logarithmic orders of magnitude; the LOB for the assay was set at 0, and the LoD value calculated using probit curves is MR4.7 (0.002%); the linearity evaluation showed that the R2 value of the BCR-ABL was 0.9996, and the R2 value of the ABL metrics calculated using the ERM standard was 0.9999; and the precision evaluation showed that all samples had a CV of less than 4% for intra-day, inter-day, and inter-instrument variation. The CV of inter-batch variation was less than 7%. The total CV was less than 5%. The results of the study demonstrate that dd-PCR can be applied to molecular detection and the clinical evaluation of CML patients and provide more precise personal treatment guidance, and its reproducibility predicts the future development of a wide range of clinical applications.

A ddPCR platform based on a microfluidic chip with a dual-function flow-focusing structure for sample-to-result DNA quantification analysis.

Droplet digital PCR (ddPCR) is a powerful method for absolute nucleic acid quantification with high precision and accuracy. However, complicated operational steps have hampered the use and diffusion of ddPCR. Therefore, an automated, easy-to-use, low-sample-consumption, and portable ddPCR platform is urgently needed. This paper proposes a microfluidic ddPCR platform based on a microfluidic chip that can realize the sample-to-result function by switching the rotary valve, achieving the dual function of the flow-focusing structure for droplet generation and readout. Sample, generation oil, and analysis oil were pre-added to the reservoirs. Droplets were generated due to focusing flow, and after passing through the integrated temporary storage bin in the rotary valve, the droplets and oil subsequently entered the collecting tube, improving the droplet-to-oil volume ratio for enhanced thermal cycle performance. Droplets with an average diameter of 107.44 μm and a CV of 2.38% were generated using our chip under the optimal pressures. High-performance thermal cycling was achieved through improvements of the droplet-to-oil volume ratio of the sample, the integrated heating lid, the pure copper heating base, and the temperature-controlling algorithm. Gradient quantification experiments were conducted for the HER2 and CEP17 genes extracted from breast cancer cells, yielding strong linear correlations with R2 values of 0.9996 for FAM and 0.9989 for CY5. Moreover, pronounced linearity was obtained between the detected concentrations of HER2 and CEP17, indicated by a slope of 1.0091 and an R2 of 0.9997, signifying consistent HER2 : CEP17 ratios across various sample dilutions. The outcomes of the quantitative analysis, encompassing the dynamic range and the consistency of the HER2 : CEP17 ratio using our ddPCR platform, meet the standards required for breast cancer assessment and therapy. Our ddPCR platform is automated, portable, and capable of stable droplet generation, high-efficiency amplification, realization of the sample-to-result function based on dual-function flow-focusing structure, and accuracy absolute quantification, underscoring its significant potential for ddPCR analysis in clinical diagnostics.

Gen Mutasyonunun Belirlenmesinde Rekombinaz Polimeraz Çoğaltım Tekniği Optimizasyonu Çalışmaları ve Sonuçları

Single nucleotide polymorphisms (SNPs) in human genes are very significant genetic changes and PCR (polymerase chain reaction) or NGS (next-generation sequencing) are extensively employed in SNP analysis. Thanks to the studies on the progress of new technologies, interest in the isothermal nucleic acid amplification approach has increased. As one of these methods, recombinase polymerase amplification (RPA) represents an attractive option for point-of-care nucleic acid quantification. The target SNPs selected within the scope of the study are mutations identified in the PIK3CA gene region (E542K, E545K), and DNA samples which were evaluated about PIK3CA mutations were isolated from the cancer cells MCF7, BT474, and also SKBr3. The optimization studies for the RPA reaction conditions were carried out for parameters such as assay time, temperature, primer, and also magnesium acetate concentration. According to the results of the reaction optimization studies, in which the RPA products can be obtained in the most efficient way, the assay time was determined as 20 min; the temperature as 40°C; the primer concentration as 10 µM and the MgOAc concentration as 140 mM.

An Integrated ddPCR Lab-on-a-Disc Device for Rapid Screening of Infectious Diseases.

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient-dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection.

Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study.

Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip's inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 101-105 copies/μL (R2 = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.

Absolute quantification of nucleic acid on digital microfluidics platform based on superhydrophobic–superhydrophilic micropatterning

Digital microfluidics (DMF) shows great potential in the manipulation of individual droplets. However, the limited number of electrode control wirings for droplet control intrinsically restrains the massive generation of pL/nL individual droplets, fundamentally hindering the realization of high throughput applications on DMF such as digital nucleic acid amplification and analysis. This work demonstrates on-chip high throughput digital loop-mediated isothermal amplification (dLAMP) on a DMF platform based on a functional substrate with super-wettability contrast. Exploiting the passive dispensing technique as empowered by electrowetting on dielectric (EWOD) effect, a total of 1818 individual droplets with high uniformity are generated on the super-hydrophilic microarray within 1 min. Separation within the oil phase without tubes and valves can avoid evaporation, ensuring a precise total reaction volume. Absolute quantification of λDNA with a dynamic range of 1–1000 copies/μL is achieved by counting the amount of positive microdroplets after on-chip incubation, demonstrating the strong potential for rapid, low-cost, reliable, and quantitative nucleic acid analysis with high accuracy.