Digital Lamp Research Articles

Improving assay feasibility and biocompatibility of 3D cyclic olefin copolymer microwells by superhydrophilic modification via ultrasonic spray deposition of polyvinyl alcohol

Sample partitioning is a crucial step towards digitization of biological assays on polymer microfluidic platforms. However, effective liquid filling into microwells and long-term hydrophilicity remain a challenge in polymeric microfluidic devices, impeding the applicability in diagnostic and cell culture studies. To overcome this, a method to produce permanent superhydrophilic 3-dimensional microwells using cyclic olefin copolymer (COC) microfluidic chips is presented. The COC substrate is oxidized using UV treatment followed by ultrasonic spray coating of polyvinyl alcohol solution, offering uniform and long-term coating of high-aspect ratio microfeatures. The coated COC surfaces are UV-cured before bonding with a hydrophobic pressure-sensitive adhesive to drive selective filling into the wells. The surface hydrophilicity achieved using this method remains unchanged (water contact angle of 9°) for up to 6 months and the modified surface is characterized for physical (contact angle & surface energy, morphology, integrity of microfeatures and roughness), chemical composition (FTIR, Raman spectroscopy) and coating stability (pH, temperature, time). To establish the feasibility of the modified surface in biological applications, PVA-coated COC microfluidic chips are tested for DNA sensing (digital LAMP detection of CMV), and biocompatibility through protein adsorption and cell culture studies (cell adhesion, viability, and metabolic activity). Kidney and breast cells remained viable for the duration of testing (7 days) on this modified surface, and the coating did not affect the protein content, morphology or quality of the cultured cells. The ultrasonic spray coated system, coating with 0.25 % PVA for 15 cycles with 0.12 A current after UV oxidation, increased the surface energy of the COC (naturally hydrophobic) from 22.04 to 112.89 mJ/m2 and improved the filling efficiency from 40 % (native untreated COC) to 94 % in the microwells without interfering with the biocompatibility of the surface, proving to be an efficient, high-throughput and scalable method of microfluidic surface treatment for diagnostic and cell growth applications.

Development of a self-powered digital LAMP microfluidic chip (SP-dChip) for the detection of emerging viruses.

Point-of-care (POC) diagnostics have emerged as a crucial technology for emerging pathogen detections to enable rapid and on-site detection of infectious diseases. However, current POC devices often suffer from limited sensitivity with poor reliability to provide quantitative readouts. In this paper, we present a self-powered digital loop-mediated isothermal amplification (dLAMP) microfluidic chip (SP-dChip) for the rapid and quantitative detection of nucleic acids. The SP-dChip utilizes a vacuum lung design to passively digitize samples into individual nanoliter wells for high-throughput analysis. The superior digitization scheme is further combined with reverse transcription loop-mediated isothermal amplification (RT-LAMP) to demonstrate dLAMP detection of Zika virus (ZIKV). Firstly, the LAMP assay is loaded into the chip and passively digitized into individual wells. Mineral oil is then pipetted through the chip to differentiate each well as an individual reactor. The chip did not require any external pumping or power input for rapid and reliable results to detect ZIKA RNA as low as 100 copies per μL within one hour. As such, this SP-dChip offers a new class of solutions for truly affordable, portable, and quantitative POC detections for emerging viruses.

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.

Ultrasensitive quantification of pathogens in milliliters of beverage by filtration-based digital LAMP

The quantitative detection of pathogens in milliliters of beverage sample requires complex preprocessing. To achieve rapid and ultrasensitive quantification of pathogens in large volume food sample, we developed a filtration-based interfacial digital LAMP (idLAMP) system, which consists of a nanoporous membrane for filtration and nanoporous hydrogel for digital amplification. Digital counting of single bacteria at the membrane surface under nanoconfinement could be achieved. The idLAMP successfully accomplished the quantitative detection of Escherichia coli in 100 mL water samples within 30 min, with wide dynamic range from 0.09 to 900 cells/mL. This technique could also be well applied to the quantification of Escherichia coli and Salmonella typhi in real beverage samples (juice, tea drinks, carbonated drinks and alcoholic drinks) without tedious sample pretreatments. With facile operation, higher specificity and sensitivity and better end-point analysis capabilities, the system has great potential in quantitative counting of single bacteria in large-volume food samples.

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

Seeking optimized infectious pathogen detection tools is of primary importance to lessen the spread of infections, allowing prompt medical attention for the infected. Among nucleic-acid-based sensing techniques, loop-mediated isothermal amplification is a promising method, as it provides rapid, sensitive, and specific detection of microbial and viral pathogens and has enormous potential to transform current point-of-care molecular diagnostics. In this review, the advances in LAMP-based point-of-care diagnostics assays developed during the past few years for rapid and sensitive detection of infectious pathogens are outlined. The numerous detection methods of LAMP-based biosensors are discussed in an end-point and real-time manner with ideal examples. We also summarize the trends in LAMP-on-a-chip modalities, such as classical microfluidic, paper-based, and digital LAMP, with their merits and limitations. Finally, we provide our opinion on the future improvement of on-chip LAMP methods. This review serves as an overview of recent breakthroughs in the LAMP approach and their potential for use in the diagnosis of existing and emerging diseases.

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.

A reinforced PDMS mold for hot embossing of cyclic olefin polymer in the fabrication of microfluidic chips.

Hot embossing is a cost-effective and flexible fabrication technology with high replication accuracy for feature sizes as small as 50 nm. Here we develop a reinforced polydimethylsiloxane (PDMS) mold for hot embossing of cyclic olefin polymer (COP) sheets in the fabrication of microfluidic chips and demonstrate the method by fabricating chips for automated sample digitization in digital nucleic acid assays. The PDMS is hardened by adding an investment powder as a dopant and is constrained with an aluminum frame to prevent lateral expansion during hot pressing. The reinforced PDMS mold demonstrated excellent performance in hot embossing (180 °C, 103 kPa, 5 min) for micropatterning COP sheets, with highly reproducible features as small as 10 μm (width of draining channel). In contrast, the microscale features were inconsistent and distorted when omitting either the investment powder or frame from the PDMS mold. COP chips were assembled by thermally bonding patterned and unpatterned COP sheets. We tested the performance of the COP chip for automated sample digitization in a digital LAMP assay used to quantify human papillomavirus-18 (HPV-18) DNA. A mixture of nucleic acid amplification reagents was loaded into the main channel of the chip using a syringe pump, then the solution was spontaneously partitioned into chambers (∼0.6 nL), which were then isolated by flowing oil through the chip. The digital LAMP assay produced accurately absolute quantitation of DNA at concentrations ranging from 10 to 1000 copies per μL. The strategy presented here provides a simple, low-cost method to prepare molds for hot embossing, which facilitates rapid validation of microfluidic designs.

Digital loop-mediated isothermal amplification detection technology and its application progress

Digital loop-mediated isothermal amplification (digital LAMP, dLAMP) is a novel nucleic acid amplification technique developed in recent years. It divides the target nucleic acid and LAMP reagent into a large number of independent detection regions, and uses a highly active chain replacement DNA polymerase and four specially designed primers for rapid amplification under isothermal conditions, which provides a good platform for quantitative detection of target nucleic acids. The advantages of high accuracy, high sensitivity, absolute quantification, high tolerance to inhibitors and simple instrumentation make the dLAMP technique very promising in molecular diagnosis, especially in rapid detection of pathogenic microorganisms, it shows a good application prospect in the fields of clinical diagnosis, food safety and environmental monitoring. Certainly, the development of dLAMP still faces some challenges, such as how to avoid non-specific amplification in multiple primer designs, multi-target nucleic acids and simultaneous detection of a large number of samples. With the development of dLAMP technology, this technology will greatly enrich the future development of molecular diagnostics. Applying rapid and effective molecular diagnostic techniques to the diagnosis of pathogenic microorganisms has important social significance for disease prevention and control.

Pipette-Tip-Enabled Digital Nucleic Acid Analyzer for COVID-19 Testing with Isothermal Amplification.

Herein, a pipette-tip-enabled digital nucleic acid analyzer for high-performance COVID-19 testing is demonstrated. This is achieved by digital loop-mediated isothermal amplification (digital LAMP or dLAMP) using common laboratory equipment and materials. It is shown that simply fixing a glass capillary inside conventional pipette tips enables the generation of monodisperse, water-in-oil microdroplets with benchtop centrifugation. It is shown that using LAMP, the ORF1a/b gene, a standard test region for COVID-19 screening, can be amplified without a thermal cycler. The amplification allows counting of fluorescent microdroplets so that Poisson analysis can be performed to allow quantification with a limit of detection that is 1 order of magnitude better than those of nondigital techniques and comparable to those of commercial dLAMP platforms. It is envisioned that this work will inspire studies on ultrasensitive digital nucleic acid analyzers demanding both sensitivity and accessibility, which is pivotal to their large-scale applications.

Accurate Bulk Quantitation of Droplet Digital Polymerase Chain Reaction.

Droplet digital PCR provides superior accuracy for nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource settings or clinical laboratories. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout of the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR.

Detection of 14 High-Risk Human Papillomaviruses Using Digital LAMP Assays on a Self-Digitization Chip.

Cervical cancer is the fourth-leading cause of cancer deaths among women worldwide and most cases occur in developing countries. Detection of high-risk (HR) HPV, the etiologic agent of cervical cancer, is a primary screening method for cervical cancer. However, the current gold standard for HPV detection, real-time PCR, is expensive, time-consuming, and instrumentation-intensive. A rapid, low-cost HPV detection method is needed for cervical cancer screening in low-resource settings. We previously developed a digital loop-mediated isothermal amplification (dLAMP) assay for rapid, quantitative detection of nucleic acids without the need for thermocycling. This assay employs a microfluidic self-digitization chip to automatically digitize a sample into an array of nanoliter wells in a simple assay format. Here we evaluate the dLAMP assay and self-digitization chip for detection of the commonly tested 14 high-risk HPVs in clinical samples. The dLAMP platform provided reliable genotyping and quantitative detection of the 14 high-risk HPVs with high sensitivity, demonstrating its potential for simple, rapid, and low-cost diagnosis of HPV infection.

A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach.

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.

Slip formation of a high-density droplet array for nucleic acid quantification by digital LAMP with a random-access system.

Digital nucleic acid analysis (digital NAA) is an important tool for the precise quantification of nucleic acids. Various microfluidic-based approaches for digital NAA have been developed, but most methods require complex auxiliary control instruments, cumbersome device fabrication, or inconvenient preparation processes. A SlipChip is a microfluidic device that can generate and manipulate liquid partitions through simple movements of two microfluidic plates in close contact. However, the traditional SlipChip requires accurate alignment of microfeatures on different plates; therefore, the dimensions of the microwells and density of partitions can be constrained. Here, we developed a droplet array SlipChip (da-SlipChip) that can form droplets of various sizes at high density in a single slipping step. This process does not require precise overlapping microfeatures on different plates; therefore, the design flexibility and partition density can be significantly increased. We quantified SARS-CoV-2 nucleic acids extracted from the COVID-19 pseudovirus by digital loop-mediated isothermal amplification (LAMP) on a da-SlipChip with 21 696 of 0.25 nL droplets, and the results were in good agreement with those of the commercial digital PCR method of Stilla. Furthermore, we demonstrated a random-access system with a single-throughput fluorescence imager and a stackable thermal control instrument with nine independent heating modules. This random-access system with the da-SlipChip can greatly improve the total throughput and flexibility for digital isothermal nucleic acid quantification and significantly reduce the total waiting time.

Петлевая LAMP амплификация нуклеиновых кислот. I. Два десятилетия развития и совершенствования

Over the two decades that have passed since the development of loop amplification (Loop AMPlification, LAMP), that carry out to detect specific nucleic acid under isothermal conditions, it has undergone quite a lot of improvements. This review presents data represented methodological bases of about a hundred variations of the LAMP, classified according to the methods of detecting both target DNA products (lamplicons) and by-products (pyrophosphate and protons), taking into account the specificity of the processes, and according to the purpose of certain LAMP options and implementation, including microfluidics. Particular attention is paid to quantitative LAMP amplification and promising digital LAMP. The prospects for the development of the method are presented.

Rotational scan digital LAMP for accurate quantitation of nucleic acids.

Digital quantitation of nucleic acids is precise and sensitive because of its molecular-level resolution. However, only several quantitation formats are common, especially pertaining to how one obtains digital signals from multiple droplets. Here we present rotational scan digital loop-mediated amplification, termed RS-dLAMP. Droplets generated by centrifugation undergo isothermal loop-mediated amplification (LAMP), and self-tile by gravitation into a tubular space between two coaxial cylinders, which are then rotated and scanned to acquire droplet fluorescence signals. RS-dLAMP is quantitatively comparable to commercial digital PCR, yet has higher throughput. Moreover, by sealing the sample throughout analysis, RS-dLAMP eliminates contamination, facilitating point-of-care diagnosis and other applications.

High-performance detection of Mycobacterium bovis in milk using digital LAMP

This paper described a high-performance molecular test for the detection of Mycobacterium bovis (M. bovis) based on digital loop-mediated isothermal amplification (dLAMP). M. bovis is a persistent pathogen that causes zoonotic tuberculosis and can infect both animals and human beings. The detection of M. bovis in milk samples is critical for effective control and prevention of zoonotic diseases but there lacks effective and sensitive methods. Here, we developed a convenient and low-cost system for M. bovis detection in milk, which incorporated automated DNA extraction and dLAMP by interfacial emulsification technique. Versus real-time PCR, dLAMP provides higher accuracy and sensitivity for direct M. bovis detection in milk, offering a limit of detection of 14 CFU/mL within 2 h. The dLAMP system can become a powerful platform for the detection of pathogens in complex samples and provide more reliable guidance for food safety testing, epidemiological research and clinical diagnosis.

A centrifugal microfluidic emulsifier integrated with oil storage structures for robust digital LAMP.

Centrifugal droplet-based microfluidic devices have been applied to biomedical analysis and diagnostics recently. However, in centrifugal droplet-based microfluidic devices, droplets are tightly packed (i.e., the oil film between neighbouring droplets is thin). Therefore, droplet coalescence usually occurs especially during thermal incubation process. To preserve individual droplets in the devices, we report a new design for monodisperse droplet generation and storage that exploits a centrifugal configuration for droplet emulsification and oil-storage structures (OSSs) for regulation of the thickness of oil film between neighbouring droplets. The centrifugal emulsifier was well designed to ensure uniform droplet generation. Meanwhile, the OSSs could store oil during centrifugal emulsification while release oil before thermal incubation, which "loosen" tightly packed droplets to prevent droplets from coalescing. In this paper, the working process of OSS was analysed, and its shape and size were optimized. Then, the optimized OSSs were integrated into a centrifugal emulsifier for droplet digital loop mediated isothermal amplification (ddLAMP) by which detection of JAK2 V617F mutation within myeloproliferative neoplasms with a dynamic range of 101 to 104 copies per μL was achieved. We anticipate that the simplicity and robustness of our system make it attractive as an inexpensive and easy-to-operate device for DNA amplification, particularly applicable in point-of-care settings.

An LED-Driven AuNPs-PDMS Microfluidic Chip and Integrated Device for the Detection of Digital Loop-Mediated Isothermal DNA Amplification.

The sensitive quantification of low-abundance nucleic acids holds importance for a range of clinical applications and biological studies. In this study, we describe a facile microfluidic chip for absolute DNA quantifications based on the digital loop-mediated isothermal amplification (digital LAMP) method. This microfluidic chip integrates a cross-flow channel for droplet generation with a micro-cavity for droplet tiling. DNA templates in the LAMP reagent were divided into ~20,000 water-in-oil droplets at the cross-flow channel. The droplets were then tiled in the micro-cavity for isothermal amplification and fluorescent detection. Different from the existing polydimethylsiloxane (PDMS) microfluidic chips, this study incorporates gold nanoparticles (AuNPs) into PDMS substrate through silica coating and dodecanol modification. The digital LAMP chip prepared by AuNPs-PDMS combines the benefits of the microstructure manufacturing performance of PDMS with the light-to-heat conversion advantages of AuNPs. Upon illumination with a near infrared (NIR) LED, the droplets were stably and efficiently heated by the AuNPs in PDMS. We further introduce an integrated device with a NIR heating unit and a fluorescent detection unit. The system could detect HBV (hepatitis B virus)-DNA at a concentration of 1 × 101 to 1 × 104 copies/μL. The LED-driven digital LAMP chip and the integrated device; therefore, demonstrate high accuracy and excellent performance for the absolute quantification of low-abundance nucleic acids, showing the advantages of integration, miniaturization, cost, and power consumption.

Smartphone-Based Droplet Digital LAMP Device with Rapid Nucleic Acid Isolation for Highly Sensitive Point-of-Care Detection.

While advances in microfluidics have enabled rapid and highly integrated detection of nucleic acid targets, the detection sensitivity is still unsatisfactory in the current POC (point-of-care) detection systems, especially for low abundance samples. In this study, a chip that integrates rapid nucleic acid extraction based on IFAST (immiscible phase filtration assisted by surface tension) and digital isothermal detection was developed to achieve highly sensitive POC detection within 60 min. Based on the interface theory, the factors influencing the interface stability of the IFAST process were studied, and the IFAST nucleic acid extraction conditions were optimized to increase the nucleic acid extraction recovery rate to 75%. Spiral mixing channel and flow-focusing droplet generation structure were designed to achieve the mixing and sample partitioning by applying negative pressure. A portable microdroplet fluorescence detection device was developed based on smartphone imaging. Validation tests were carried out for quantification of low-abundance cfDNA and detection of mutations.

Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP.

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP. In this work, we improve the SD chip design to increase sample loading efficiency, speed, and completeness, and test a range of well volumes and numbers. We demonstrate the diagnostic capability of this platform by applying it to quantifying human papillomavirus 18 gene.