Digital Loop-mediated Isothermal Amplification Research Articles

Integrated Droplet-Based Digital Loop-Mediated Isothermal Amplification Microfluidic Chip with Droplet Generation, Incubation, and Continuous Fluorescence Detection

This study integrated sample partition, incubation, and continuous fluorescence detection on a single microfluidic chip for droplet-based digital Loop-Mediated Isothermal Amplification (LAMP) of nucleic acids. This integration eliminated the need to transfer reactions between different platforms, avoiding sample contamination and loss. Prior to the reaction, filling the channels with an oil phase and adding a glass cover slip on top of the chip overcame the problem of bubble generation in the channels during the LAMP reaction due to heating. Additionally, using two fluorescence intensity thresholds enabled simultaneous detection and counting of positive and negative droplets within a single fluorescence detection channel. The chip can partition approximately 6000 droplets from a 5 µL sample within 10 min, with a droplet diameter of around 110 µm and a coefficient of variation (CV) value of 0.82%. Staphylococcus aureus was quantified via the proposed platform. The results demonstrated a highly accurate correlation coefficient (R = 0.9998), and the detection limit reached a concentration of 1.7 × 102 copies/µL. The entire process of the droplet digital LAMP reaction, from droplet generation to incubation to quantitative results, took a maximum of 70 min.

Nucleic Acid Plate Culture: Label-Free and Naked-Eye-Based Digital Loop-Mediated Isothermal Amplification in Hydrogel with Machine Learning.

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (i.e., short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white "colony" spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

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.

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.

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.

Hydrogel Digital LAMP with Suppressed Nonspecific Amplification for Rapid Diagnostics of Fungal Disease in Fresh Fruits.

Fungal disease, mainly caused by Alternaria alternata infection, can generate severe economic losses and health hazards. However, rapid nucleic acid test without nonspecific reaction still remains challenging. Here, we reported the hydrogel digital loop-mediated isothermal amplification (HdLAMP) with suppressed nonspecific amplification for rapid diagnosis of fungi in fresh fruits. The introduction of hydrogel offered a simple platform to achieve absolute quantification. By breaking the 3'end G-C anchor, the nonspecific amplification of primers could be suppressed, while the specific positive reaction in HdLAMP was not affected. This method could be applied for A. alternata detection in 9 min with excellent performances in speed, specificity, reproducibility, sensitivity, and detection limit down to a single copy. Finally, the real diseased jujubes during postharvest storage were successfully diagnosed as an A. alternata infection. HdLAMP promotes the molecular diagnosis of fungal diseases and broadens the application of hydrogels in the agricultural and food industry.

An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/μL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

Single-molecule RNA capture-assisted droplet digital loop-mediated isothermal amplification for ultrasensitive and rapid detection of infectious pathogens

To minimize and control the transmission of infectious diseases, a sensitive, accurate, rapid, and robust assay strategy for application on-site screening is critical. Here, we report single-molecule RNA capture-assisted digital RT-LAMP (SCADL) for point-of-care testing of infectious diseases. Target RNA was captured and enriched by specific capture probes and oligonucleotide probes conjugated to magnetic beads, replacing laborious RNA extraction. Droplet generation, amplification, and the recording of results are all integrated on a microfluidic chip. In assaying commercial standard samples, quantitative results precisely corresponded to the actual concentration of samples. This method provides a limit of detection of 10 copies mL−1 for the N gene within 1 h, greatly reducing the need for skilled personnel and precision instruments. The ultrasensitivity, specificity, portability, rapidity and user-friendliness make SCADL a competitive candidate for the on-site screening of infectious diseases.

Design of a Digital LAMP Detection Platform Based on Droplet Microfluidic Technology.

Loop-mediated isothermal amplification (LAMP) is a rapid and high-yield amplification technology for specific DNA or RNA molecules. In this study, we designed a digital loop-mediated isothermal amplification (digital-LAMP)-functioning microfluidic chip to achieve higher sensitivity for detection of nucleic acids. The chip could generate droplets and collect them, based on which we could perform Digital-LAMP. The reaction only took 40 min at a constant temperature of 63 °C. The chip enabled highly accurate quantitative detection, with the limit of detection (LOD) down to 102 copies μL-1. For better performance while reducing the investment of money and time in chip structure iterations, we used COMSOL Multiphysics to simulate different droplet generation ways by including flow-focusing structure and T-junction structure. Moreover, the linear structure, serpentine structure, and spiral structure in the microfluidic chip were compared to study the fluid velocity and pressure distribution. The simulations provided a basis for chip structure design while facilitating chip structure optimization. The digital-LAMP-functioning chip proposed in the work provides a universal platform for analysis of viruses.

A 3D Scalable Chamber-Array Chip for Digital LAMP.

As an absolute quantification method at the single-molecule level, digital PCR (dPCR) offers the highest accuracy. In this work, we developed a 3D scalable chamber-array chip that multiplied the number of partitions by stacking chamber-array layers and realized digital loop-mediated isothermal amplification to quantify DNA molecules. It greatly increases the number of partitions to improve the performance of dPCR without increasing the chip size, the operation workflow complicity, and operation time. For the three-chamber-array-layer chip which contains 200,000 reactors of a 0.125 nL volume, it has been proved that the reagent filling and partition were finished within 3 min, and the whole detection could be finished within 1 h. The method demonstrated that it could be scalable to a six-chamber-array layer, which contains 400,000 reactors without increasing the size of the chip and the complication of filling/partition workflow but only takes an additional hour for scanning. Due to its potential for high throughput, low cost, and simple operation, our device may significantly expand the clinical application range of dPCR.

Auto Flow-Focusing Droplet Reinjection Chip-Based Integrated Portable Droplet System (iPODs).

Droplet microfluidics provides powerful tools for biochemical applications. However, precise fluid control is usually required in the process of droplet generation and detection, which hinders droplet-based applications in point-of-care testing (POCT). Here, we present a droplet reinjection method capable of droplet distribution without precise fluid control and external pumps by which the droplets can be passively aligned and detected one by one at intervals. By further integrating the surface-wetting-based droplet generation chip, an integrated POrtable Droplet system (iPODs) is developed. The iPODs integrates multiple functions such as droplet generation, online reaction, and serial reading. Using the iPODs, monodisperse droplets can be generated at a flow rate of 800 Hz with a narrow size distribution (CV <2.2%). Droplets are kept stable, and the fluorescence signal can be significantly identified after the reaction. The spaced droplet efficiency in the reinjection chip is nearly 100%. In addition, we validate digital loop-mediated isothermal amplification (dLAMP) within 80 min with a simple operation workflow. The results show that iPODs has good linearity (R2 = 0.999) at concentrations ranging from 101 to 104 copies/μL. Thus, the developed iPODs highlights its potential to be a portable, low-cost, and easy-to-deploy toolbox for droplet-based applications.

CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification.

Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report color-encoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification.

Bent-Capillary-Centrifugal-Driven Monodisperse Droplet Generator with Its Application for Digital LAMP Assay.

We developed a bent-capillary-centrifugal-driven (BCCD) monodisperse droplet generator, which could achieve a perfect combination of driving and segmentation for the dispersed phase only using a rotating bent capillary immersed in the continuous phase (mineral oil). The sample could flow continuously to the bent-capillary outlet to form the droplet precursors, which were segmented into homogeneous droplets in the continuous phase. Through the investigation of influence factors on droplet size and stability, we found that the droplet size could be conveniently controlled by the rotational speed of the bent capillary. The droplet volumes could be adjusted with the range from 34 pL to 1 μL, and the coefficient variations (CVs) were less than 3%. Meanwhile, the BCCD droplet generator could realize the controllable droplet output with a high-efficiency sample utilization of 99.75 ± 1.15%, which offered a significant advantage in reducing the waste of precious samples in the droplet generation process. We validated this system with a digital loop-mediated isothermal amplification (dLAMP) assay for the absolute quantification of Mycobacterium tuberculosis complex nucleic acids. The results demonstrated that the BCCD droplet generator was easy to build, was of low cost, and was convenient to operate, as well as avoided sample loss and cross-contamination by coupling with a 96-well plate. Overall, the present platform, as a simple chip-free droplet generator, will provide an especially valuable droplet generation solution for biochemical applications based on droplets.

Droplet Encoding-Pairing Enabled Multiplexed Digital Loop-Mediated Isothermal Amplification for Simultaneous Quantitative Detection of Multiple Pathogens.

Despite the advantages of digital nucleic acid analysis (DNAA) in terms of sensitivity, precision, and resolution, current DNAA methods commonly suffer a limitation in multiplexing capacity. To address this issue, a droplet encoding-pairing enabled DNAA multiplexing strategy is developed, wherein unique tricolor combinations are deployed to index individual primer droplets. The template droplets and primer droplets are sequentially introduced into a microfluidic chip with a calabash-shaped microwell array and are pairwise trapped and merged in the microwells. Pre-merging and post-amplification image analysis with a machine learning algorithm is used to identify, enumerate, and address the droplets. By incorporating the amplification signals with droplet encoding information, simultaneous quantitative detection of multiple targets is achieved. This strategy allows for the establishment of flexible multiplexed DNAA by simply adjusting the primer droplet library. Its flexibility is demonstrated by establishing two multiplexed (8-plex) droplet digital loop-mediated isothermal amplification (mddLAMP) assays for individually detecting lower respiratory tract infection and urinary tract infection causative pathogens. Clinical sample analysis shows that the microbial detection outcomes of the mddLAMP assays are consistent with those of the conventional assay. This DNAA multiplexing strategy can achieve flexible high-order multiplexing on demand, making it a desirable tool for high-content pathogen detection.

Fabrication of planar monolayer microreactor array for visual statistical analysis and droplet-based digital quantitative analysis in situ.

Planar monolayer microreactor arrays (PMMRAs) make droplet-based numerical measurements and statistical analysis cheap and easy. However, PMMRAs are typically produced in complex microfluidic devices and, moreover, still requires stringent control to reduce droplet loss during heating. In this paper, a simple, reliable, and flexible method for fabricating PMMRAs in a 96-well plate is described in detail by using simple materials and low-cost equipment. The partitioned droplets spontaneously assemble into PMMRAs in the plates, and this distribution is maintained even after incubation. This is advantageous for in situ analysis based on an individual droplet in droplet digital loop-mediated isothermal amplification (ddLAMP) and does not require the transfer of positive droplets. Precise and reproducible quantification of classical swine fever virus (CSFV) extracts was executed in these PMMRAs to verify its availability. Our results demonstrate that the proposed approach not only provides a flexible and controllable execution scheme for droplet-based nucleic acid quantification in resource-limited laboratories but also opens new perspectives for numerous analytical and biochemical applications using droplets as versatile plastic microreactors.

Picoliter Droplet Generation and Dense Bead-in-Droplet Encapsulation via Microfluidic Devices Fabricated via 3D Printed Molds.

Picoliter-scale droplets have many applications in chemistry and biology, such as biomolecule synthesis, drug discovery, nucleic acid quantification, and single cell analysis. However, due to the complicated processes used to fabricate microfluidic channels, most picoliter (pL) droplet generation methods are limited to research in laboratories with cleanroom facilities and complex instrumentation. The purpose of this work is to investigate a method that uses 3D printing to fabricate microfluidic devices that can generate droplets with sizes <100 pL and encapsulate single dense beads mechanistically. Our device generated monodisperse droplets as small as ~48 pL and we demonstrated the usefulness of this droplet generation technique in biomolecule analysis by detecting Lactobacillus acidophillus 16s rRNA via digital loop-mediated isothermal amplification (dLAMP). We also designed a mixer that can be integrated into a syringe to overcome dense bead sedimentation and found that the bead-in-droplet (BiD) emulsions created from our device had <2% of the droplets populated with more than 1 bead. This study will enable researchers to create devices that generate pL-scale droplets and encapsulate dense beads with inexpensive and simple instrumentation (3D printer and syringe pump). The rapid prototyping and integration ability of this module with other components or processes can accelerate the development of point-of-care microfluidic devices that use droplet-bead emulsions to analyze biological or chemical samples with high throughput and precision.

Loop-Mediated Isothermal Amplification-Based Microfluidic Platforms for the Detection of Viral Infections.

Easy-to-use, fast, and accurate virus detection method is essential for patient management and epidemic surveillance, especially during severe pandemics. Loop-mediated isothermal amplification (LAMP) on a microfluidic platform is suitable for detecting infectious viruses, regardless of the availability of medical resources. The purpose of this review is to introduce LAMP-based microfluidic devices for virus detection, including their detection principles, methods, and application. Facing the uncontrolled spread of viruses, the large-scale deployment of LAMP-based microfluidic platforms at the grassroots level can help expand the coverage of nucleic acid testing and shorten the time to obtain test reports. Microfluidic chip technology is highly integrated and miniaturized, enabling precise fluid control for effective virus detection. Performing LAMP on miniaturized systems can reduce analysis time, reagent consumption and risk of sample contamination, and improve analytical performance. Compared to traditional benchtop protocols, LAMP-based microfluidic devices reduce the testing time, reagent consumption, and the risk of sample contamination. In addition to simultaneous detection of multiple target genes by special channel design, microfluidic chips can also integrate digital LAMP to achieve absolute quantification of target genes.

TriD-LAMP: A pump-free microfluidic chip for duplex droplet digital loop-mediated isothermal amplification analysis

Digital nucleic acid amplification techniques are powerful and attractive approaches for providing sensitive and absolute quantification in biology. Among these, digital loop-mediated isothermal amplification (dLAMP) shows the potential for field detection, since its robustness and independence from thermal cycling. The key of dLAMP is to generate a large number of individual droplets or microwells. However, an auxiliary precision pump is always required for sample digitalization. In addition, current systems for droplet dLAMP usually need to transfer the droplets after digitalization or amplification. Herein, we developed and evaluated a pump-free microfluidic chip for duplex droplet dLAMP (TriD-LAMP) detection. This chip was designed based on step emulsification and contains a droplet generation zone and a droplet storage zone. Droplets are formed through the step due to the difference in Laplace pressure. There are 64 parallel nozzles that could generate tens of thousands of uniform droplets manually (variation <5%). The storage zone for droplets collection was previously filled with oil containing fluorosurfactant that keeps the droplets from fusing and evaporation during the amplification. Therefore, this custom chip is able to perform all stages of the dLAMP process without transferring droplets. Combined with the optimized fluorescent probe method, the chip achieves accurate quantification of the E. coli DNA down to 19.8 copies/μL. As a proof of concept, the simultaneous quantification of two targets was successfully realized on this custom chip. Conclusively, this integrated, pump-free TriD-LAMP chip can provide a promising tool for multiple targets detection in clinical diagnostics and point-of-care applications.

Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection.

Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.

Self-Assembled Inkjet Printer for Droplet Digital Loop-Mediated Isothermal Amplification

Developing rapid and inexpensive diagnostic tools for molecular detection has been pushed forward by the advancements of technical aspects. However, attention has rarely been paid to the molecular detection methodology using inkjet printing technique. Herein, we developed an approach that employed a self-assembled inkjet printer as the enabling technology to realize droplet digital loop-mediated isothermal amplification in a low-cost and practical format. An inkjet printer is a self-assembled tool for the generation of discrete droplets in controllable volumes from a picoliter to a nanoliter. A microfluidic chip serves as a droplets reservoir to perform droplet digital LAMP assays. The inkjet printer approach successfully quantified the HPV16 from CaSki cells. This self-assembled and practical inkjet printer device may therefore become a promising tool for rapid molecular detection and can be extended to on-site analysis.