Molecular tests for infectious diseases and genetic anomalies, which account for significant global morbidity and mortality, are central to nucleic acid analysis. In this study, we present a digital ...droplet LAMP (ddLAMP) platform that offers a cost-effective and portable solution for such assays. Our approach integrates disposable 3D-printed droplet generator chips with a consumer smartphone equipped with a custom image analysis application for conducting ddLAMP assays, thereby eliminating the necessity for expensive and complicated photolithographic techniques, optical microscopes, or flow cytometers. Our 3D printing technique for microfluidic chips facilitates rapid chip fabrication in under 2 h, without the complications of photolithography or chip bonding. The platform’s heating mechanism incorporates low-powered miniature heating blocks with dual resistive cartridges, ensuring rapid and accurate temperature modulation in a compact form. Instrumentation is further simplified by integrating miniaturized magnification and fluorescence optics with a smartphone camera. The fluorescence quantification benefits from our previously established RGB to CIE-xyY transformation, enhancing signal dynamic range. Performance assessment of our ddLAMP system revealed a limit of detection at 10 copies/μL, spanning a dynamic range up to 104 copies/μL. Notably, experimentally determined values of the fraction of positive droplets for varying DNA concentrations aligned with the anticipated exponential trend per Poisson statistics. Our holistic ddLAMP platform, inclusive of chip production, heating, and smartphone-based droplet evaluation, provides a refined method compatible with standard laboratory environments, alleviating the challenges of traditional photolithographic methods and intricate droplet microfluidics expertise.
The ability to simultaneously heat and image samples using transmitted light is crucial for several biological applications. However, existing techniques such as heated stage microscopes, thermal ...cyclers equipped with imaging capabilities, or non-contact heating systems are often bulky, expensive, and complex. This work presents the development and characterization of a Miniaturized Optically-clear Thermal Enclosure (MOTE) system—an open-source, inexpensive, and low-powered modular system—capable of convectively heating samples while simultaneously imaging them with transmitted light. We develop and validate a computational fluid dynamics (CFD) model to design and optimize the heating chamber. The model simulates velocity and temperature profiles within the heating chamber for various chamber materials and sizes. The computational model yielded an optimal chamber dimension capable of achieving a stable temperature ranging from ambient to 95 °C with a spatial discrepancy of less than 1.5 °C, utilizing less than 8.5 W of power. The dual-functionality of the MOTE system, enabling synchronous heating and transmitted light imaging, was demonstrated through the successful execution of paper-based LAMP reactions to detect λ DNA samples in real-time down to 10 copies/µL of the target concentration. The MOTE system offers a promising and flexible platform for various applications, from molecular diagnostics to biochemical analyses, cell biology, genomics, and education.
Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in ...resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in ...point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers.
Accurate quantification of immunoglobulin G (IgG) levels is vital for understanding immune status and diagnosing various medical conditions. Lateral flow assays (LFAs) offer rapid and convenient ...diagnostic tools, but their sensitivity has been a limitation. Our research introduces a refined method incorporating europium nanoparticles, enhancing both sensitivity and accuracy of LFAs in human IgG measurement. Utilizing a unique sandwich format, carboxylate-modified polystyrene Eu (III) chelate microparticles (CM-EUs) acted as the primary reporters. The concentrations of both detection and capture antibodies on the strip were optimized to bolster the LFA’s quantitative performance. The subsequent calibration curve between the IgG concentration and the measured intensity ratio (VR) established the linearity and analytical sensitivity of our method with a high correlation coefficient (r = 0.99) and an impressively low limit of detection (LoD = 0.04 ng/mL). Our precision assessment, segmented into intra-assay and inter-assay evaluations, showcases the method’s consistency and reproducibility. The LFA assay’s stability was established by demonstrating its resistance to degradation and affirming its potential for extended storage without a dip in performance. The study’s findings underscore the potential of this method to contribute to diagnostic medicine and improve patient care.
We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to ...isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone’s motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging.
Carbon dots are zero-dimensional nanomaterials that have garnered significant research interest due to their distinct optical properties, biocompatibility, low fabrication cost, and eco-friendliness. ...Recently, their light-to-heat conversion ability has led to several novel photothermal applications. In this minireview, we categorize and describe the photothermal application of carbon dots along with methods incorporated to enhance their photothermal efficiency. We also discuss the possible mechanisms by which the photothermal effect is realized in these carbon-based nanoparticles. Taken together, we hope to provide a comprehensive landscape highlighting several promising research directions for using carbon dots for photothermal applications.
Stereolithography based 3D printing of microfluidics for prototyping has gained a lot of attention due to several advantages such as fast production, cost-effectiveness, and versatility over ...traditional photolithography-based microfabrication techniques. However, existing consumer focused SLA 3D printers struggle to fabricate functional microfluidic devices due to several challenges associated with micron-scale 3D printing. Here, we explore the origins and mechanism of the associated failure modes followed by presenting guidelines to overcome these challenges. The prescribed method works completely with existing consumer class inexpensive SLA printers without any modifications to reliably print PDMS cast microfluidic channels with channel sizes as low as ~75 μm and embedded channels with channel sizes as low ~200 μm. We developed a custom multi-resin formulation by incorporating Polyethylene glycol diacrylate (PEGDA) and Ethylene glycol polyether acrylate (EGPEA) as the monomer units to achieve micron sized printed features with tunable mechanical and optical properties. By incorporating multiple resins with different mechanical properties, we were able to achieve spatial control over the stiffness of the cured resin enabling us to incorporate both flexible and rigid components within a single 3D printed microfluidic chip. We demonstrate the utility of this technique by 3D printing an integrated pressure-actuated pneumatic valve (with flexible cured resin) in an otherwise rigid and clear microfluidic device that can be fabricated in a one-step process from a single CAD file. We also demonstrate the utility of this technique by integrating a fully functional finger-actuated microfluidic pump. The versatility and accessibility of the demonstrated fabrication method have the potential to reduce our reliance on expensive and time-consuming photolithographic techniques for microfluidic chip fabrication and thus drastically lowering our barrier to entry in microfluidics research.
Paper-based diagnostics offer a promising alternative to traditional diagnostic methods for point-of-care use due to their low cost, ease of use, portability, rapid results, versatility, and low ...environmental impact. While paper-based serology tests in the form of lateral flow assays can provide rapid test results for past pathogen exposure, they currently lack the accuracy and sensitivity offered by molecular diagnostic tests such as the polymerase chain reaction (PCR). Loop-mediated isothermal amplification (LAMP)—an isothermal nucleic acid amplification test (NAAT)—provides PCR-like performance while simultaneously reducing the instrumentation and assay complexity associated with PCR. In this review, we discuss a newly emerging class of paper-based LAMP platforms that integrates the versatility of paper microfluidics with the accuracy of NAATs. Since its first adoption in 2015, we have discussed all paper-based LAMP platforms in terms of the paper substrates, reagent incorporation techniques, paper platform design, heating hardware, detection methods, and sensitivity and specificity of paper-based LAMP assays. We conclude by identifying the current challenges and future prospects of paper-based NAATs.
Porous mineral formations near subsea alkaline hydrothermal vents embed microenvironments that make them potential hot spots for prebiotic biochemistry. But, synthesis of long-chain macromolecules ...needed to support higher-order functions in living systems (e.g., polypeptides, proteins, and nucleic acids) cannot occur without enrichment of chemical precursors before initiating polymerization, and identifying a suitable mechanism has become a key unanswered question in the origin of life. Here, we apply simulations and in situ experiments to show how 3D chaotic thermal convection—flows that naturally permeate hydrothermal pore networks—supplies a robust mechanism for focused accumulation at discrete targeted surface sites. This interfacial enrichment is synchronized with bulk homogenization of chemical species, yielding two distinct processes that are seemingly opposed yet synergistically combine to accelerate surface reaction kinetics by several orders of magnitude. Our results suggest that chaotic thermal convection may play a previously unappreciated role in mediating surface-catalyzed synthesis in the prebiotic milieu.
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