Mixing Of Reagents Research Articles

A success story in a low-resource setting: Advancing cancer diagnostics in underserved region

Abstract Introduction/Objective Shefaa Al Orman Hospital (SOH) is the first oncology hospital in Luxor. Since its inception in 2016, the hospital has served approximately 45,000 patients from Luxor and other governorates. The hospital’s lab provides excellent routine laboratory services. However, there is a pressing demand for molecular genetics and cytogenetics, that is necessary for diagnosis of hematological malignancies. This strategic expansion faced several challenges: lack of expertise and the need for special expensive equipment and reagents. Methods/Case Report The project started with collaboration with oncologists to convene on a simplified panel with the aim of prioritizing clinically relevant tests. At first, these investigations were referred to a specialized oncology hospital in Cairo. This entailed 2 problems: transport of samples led to deterioration of the quality of some samples and delay in results reporting. In the second phase, experienced consultants from Cairo were enlisted to provide training and mentorship to local staff. Infrastructure setup included the allocation of dedicated laboratory space and the procurement of specialized equipment. Test method selection was used to achieve successful transition. In cytogenetics, we started with FISH technique which, while more expensive, gave rapid and reliable results. After gaining experience, the technique of conventional karyotyping was established. On the other hand, in molecular genetics, we resorted to custom made reagent mix preparation, instead of commercial kits. This approach, while more laborious, is less expensive. Results (if a Case Study enter NA) With the gradual successful in-house setup of these tests, there has been a notable reduction of out-sourced samples to 5%. This has led to shorter result turnaround times. Cytogenetics and FISH improved from 4 to 1 week, while molecular genetics decreased from 4 to 2 weeks. Moreover, the local processing of samples has minimized the risk of sample damage during transportation, ensuring the integrity of test results. Conclusion The establishment of the Molecular Genetics and Cytogenetics Laboratory at SOH represents a significant milestone. The hospital has successfully overcome logistical challenges and enhanced diagnostic capabilities for hematological malignancies, contributing to better patient outcomes and satisfaction. Moving forward, the continued investment in infrastructure, personnel training, and quality assurance measures will further strengthen the hospital’s capacity to provide high-quality cancer care to the community.

Developmental validation of a multiplex qPCR assay for simultaneous quantification of nuclear and mitochondrial DNA

Quantification of human DNA is key in forensic genetics. A more accurate estimate of the amount of DNA is essential for planning and optimising genotyping assays, as is evaluating the presence of PCR inhibitory substances and DNA degradation status. Multiplex qPCR assays are helpful in forensics because they can quantify different targets simultaneously, thus saving valuable samples, time, and labour. The aim of this study was to highlight the challenges in the developmental validation of a multiplex real-time PCR assay and the drawbacks encountered in translating a previously described and validated assay (SD quants) to a different technology by modifying the dye probes and reagent mix to be used in a different instrument. We developed a TaqMan probe-based multiplex qPCR using reagents and fluorescent probes adapted for the Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany). The initial assay combined two mitochondrial DNA (mtDNA) and two nuclear DNA (nDNA) targets, with amplification products of different sizes (mtDNA = 69 and 143 bp; nDNA = 71 and 181 bp), to estimate the DNA degradation status and an internal positive control (IPC) to detect potential inhibitors. During the initial testing of the assay, we observed an interaction between the 69 bp mtDNA target and the 71 bp nDNA target probe, and experiments were conducted to resolve this issue without success. We removed the small nDNA target (71 bp) and changed from a 5-plex to a 4-plex qPCR assay (qMIND). The final tetraplex assay was tested on 105 forensic samples and/or small amounts of degraded DNA, such as bones, teeth, fingernails, formalin-fixed paraffin-embedded tissues (FFPE), and hair shaft samples. The quantification results were compared with data acquired from the same samples using another commercially available quantification system commonly used in forensic laboratories. In addition, the short tandem repeat (STR) profiles were investigated to determine their correlation with the quantitative values obtained. Overall, the qPCR assay was robust and reliable for DNA quantification in samples commonly used in forensic practice.

Numerical investigation of centrifugal passive mixing in Omega shaped microchannel and comparison with fixed platform

The extraction of DNA from isolated cells is crucial for diagnosing and treating various diseases, including cancer, which has become a leading cause of death in recent years. Circulating tumor cells (CTCs) in the blood can be separated from other blood cells, allowing for DNA extraction and analysis for further research. Microfluidic methods are more cost-effective than conventional DNA extraction techniques and have significant potential for high-throughput mixing in lab-on-a-chip (LOC) and lab-on-a-disk (LOD) devices without damaging DNA. Chemical methods for cell lysis, where the mixing of reagents is essential, are particularly efficient on these platforms due to the generation of secondary flows. This is especially true in microchannels with a serpentine design, which enhances mixing quality—a critical parameter for cell lysis efficiency. This study presents and compares the mixing capabilities of the two microchannel platforms (LOC and LOD). Additionally, we introduce a new Omega-shaped serpentine microchannel that combines features of both straight and curved serpentine channels. The mixing quality across all geometries and both platforms has been investigated. Our findings indicate that the centrifugal platform is the most effective for mixing at low inlet velocities. Furthermore, we examine the number of stages in the Omega channel and conclude that 7 Ω stages are sufficient for achieving high mixing quality (∼100 %).

B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process

Abstract Background Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput. Methods The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis. Results To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days. Conclusions The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Non-enzymatic Determination of Glucose in Artificial Urine Using 3D-µPADs through Silver Nanoparticles Formation

Patients with diabetes often experience blood glucose fluctuations, making monitoring crucial. Traditional blood sampling methods pose risks of infection and pain. An alternative non-invasive approach using urine tests has been explored. Recent studies highlight microfluidic paper-based analytical devices (µPADs) as convenient, simple, and easily fabricated tools for non-invasive glucose measurement. This study aims to develop a concept of measuring glucose in artificial urine using 3D-µPADs in a non-enzymatic manner by utilizing glucose as a reducing agent for silver nanoparticle (AgNPs) formation. Embedding three-dimensional connectors in µPADs links the sample and detection zones to limit reagent mixing and improve glucose detection resolution. The optimal conditions were NaOH 10 M, starch 1%, and AgNO3 30 mM, with sample and detection zone volumes of 10 and 9 µL, respectively. The fifth reaction sequence involved AgNO3 in the detection zone and a solution of glucose, NaOH, and starch in the sample zone at 1:1:1 volume ratio. The reagent drying time was 15 min, with immobilization once and reaction time of 9 min. The method showed excellent linearity (R2 = 0.9905), precision (%RSD = 4.27%), accuracy (77.32–92.58%), and limit of detection (11.11 mg/dL).

Comparative experimental and numerical study of mixing efficiency in 3D-printed microfluidic droplet generators: T junction, cross junction, and asymmetric junctions with varying angles

In droplet-based microfluidics, rapid mixing during droplet formation enhances reaction uniformity, eliminating the need for micromixers. In this research, we conducted a comprehensive study by first employing a series of two-dimensional (2D) numerical simulations, followed by experimental investigations using 3D-printed microfluidic chips. We compared the mixing efficiency, droplet diameter, and droplet eccentricity of three different types of droplet generators: T junction, cross junction, and asymmetric droplet generators with various angles. Regarding mixing efficiency, we observed that the asymmetric droplet generators outperformed the cross junction by 30 % but fell slightly short of the mixing efficiency achieved by the T junction (1 %). Additionally, while the mixing index in the asymmetric generators closely matched that of the T junction, these asymmetric generators produced smaller droplets by 72 %. Increasing the angle in asymmetric droplet generators resulted in enhanced mixing efficiencies and an increase in droplet diameters. The asymmetric junction with a 30° angle could achieve a mixing efficiency of up to 80 %. Additionally, an analysis of the dispersed phase flow rate revealed that higher flow rates lead to larger droplet sizes and reduced mixing efficiencies. The asymmetric droplet generators improve mixing efficiency facilitating rapid reagent mixing, all while maintaining a small droplet diameter.

AN AGAROSE GEL-BASED APPROACH FOR THE REMOVAL OF METALLIC-BASE INK STAIN FROM THE PAPER SURFACES OF A 17TH CENTURY BOOK

Removing ink stains from the pages of ancient and valuable books is a very challenging and sometimes impossible task. Only in some circumstances, restorers are able to improve the appearance of an ink-stained page by lightening the spots using an ink eradicator. This paper reports on an approach for the removal of ink stains from a 17th century book using a combination of two commercial houseware products, the rust remover for textile and bleach. The products were chosen on the basis of their active ingredients such as hydrofluoric acid (HF), which facilitates the removal of metal ions contained in the inks (such as iron and chromium) and sodium hypochlorite (NaClO), known for its whitening abilities. The products were first tested by direct application to one of the several stained pages as a control experiment. The ink stain was successfully removed from the book, but by SEM analysis the page results in a clear detachment of the cellulose fibers from the paper pulp, which reflect a significant change in the paper fine structure. Moreover, the mixing of chemicals occurring during this procedure generated toxic vapors and the entire protocol could only be performed under a chemical fume hood. A new procedure was developed to mitigate these negative effects. Each of the two commercial products was dispersed in a 2% agarose solution shortly before its gelification and the two gels thus obtained were applied consecutively on the surface of the paper to be cleaned. The results showed an effective removal of the ink stain from treated page, avoiding irreparable damage to the paper structure and improving the working conditions of the restorers. Hence, the proposed gel cleaning procedure offers three main advantages: i) a simple and practical tool for effective removal of metallic ink stains from the paper surface; ii) reduction of damage caused by aggressive reagents (HF and NaClO) contained in cleaning products for a better conservation of paper fibers; iii) a great decrease of the risk of accidentally exposing the operators to harmful toxic vapor (produced by accidental mixing of aforementioned chemical reagents).

Possibilities of using nested PCR-RFLP for taxonomic identification of L3 larvae of the family Trichostrongylidae, Leiper, 1912

The purpose of the research is to apply molecular genetic research methods to identify the taxonomic affiliation of gastrointestinal parasitic sheep nematodes of the family Trichostrongylidae using nested PCR followed by the restriction fragment length polymorphism (RFLP) analysis.Materials and methods. Parasitic nematodes, L3 Strongylata larvae obtained from incubated fecal samples of sheep. The genomic DNA was isolated using a commercial kit for DNA extraction from micro-quantities of tissues (Synthol, Moscow) as per the manufacturer’s guidelines. For DNA amplification, a T-100 Bio-Rad thermal cycler and a commercial Eurogen Master Mix reagent kit were used. The PCR regime was performed according to the WAAVP guidelines, 2006. The restriction endonuclease Rsa I of amplified Trichostrongylidae fragments was performed according to guidelines of the enzyme manufacturer (Sibenzyme, Novosibirsk).Results and discussion. To determine the taxonomic affiliation of Strongylata larvae isolated after incubation of feces from sheep, molecular genetic studies were performed using nested PCR followed by the restriction fragment length polymorphism (RFLP) analysis. This method makes it possible to identify, with the least effort, the genotypes of three species of Strongylata Haemonchus contortus, Trichostrongylus colubriformis, and Teladorsagia circumcincta at the larval stage.

An automated syringe-based PoC RT-LAMP LFB platform for infectious disease detection from saliva

Decentralized Point-of-Care (PoC) diagnostics hold momentous potential for rapid and accessible viral infection disease detection. Presented is a unique design application of an easy-to-use (plug-and-play) platform for viral detection. The platform leverages a simplified multiplex Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Lateral Flow Biosensor (LFB) assay with a lyophilized master mix, eliminating the need for RNA isolation or special reporting equipment. A user-friendly Saliva Measuring Tube (SMT) ensures accurate saliva volume self-collection, and a Syringe-based PoC (SPoC) platform automates sample treatment, reagent mixing, and temperature control using readily available components and consumables. The platform’s performance was evaluated by multiplexed detection of the SARS-CoV-2 N2 target gene and human ACTB gene from saliva samples. The SPoC platform achieved a detection limit of spiked 500 copies/mL for SARS-CoV-2 and consistent internal control readout. The presented PoC system offers a promising initial step for further development toward a decentralized solution for viral infection testing.

Saliva-STAT: Sample-to-answer saliva test for COVID-19

Highly accessible and highly accurate diagnostics are necessary to combat rapidly-spreading infectious diseases, such as the recent COVID-19 pandemic. While lateral flow antigen tests have become pervasive, they are insufficiently sensitive to detect early or asymptomatic disease. Nucleic acid amplification tests provide the needed sensitivity, but accessibility of these tests continues to be a challenge due to the need for precise sample processing steps. Here we report a sample-to-answer test for saliva samples (saliva-STAT) that utilizes a battery-powered handheld instrument and a low-cost easily-manufacturable sample cassette to perform a nucleic acid amplification test for viral pathogens. To enable a completely automated assay, we leverage thermally responsive alkane partitions (TRAPs) and paramagnetic beads for virus purification and concentration, as well as reagent addition and mixing. Notably, the saliva STAT easily accommodates directly-dispensed saliva samples (in contrast to microfluidic devices), which is necessary for self-testing. Using the saliva-STAT platform, we demonstrate detection of down to 0.2 copies/μL of SARS-CoV-2 virus in saliva samples. We envision that the saliva-STAT could be used in walk-in clinics, mobile clinics, public testing locations, and in the home. With minor adjustments to the assay, the saliva-STAT platform can easily be adapted for other respiratory viruses, such as Influenza.

Development of a Portable Residual Chlorine Detection Device with a Combination of Microfluidic Chips and LS-BP Algorithm to Achieve Accurate Detection of Residual Chlorine in Water.

Chlorine is widely used for sterilization and disinfection of water, but the presence of excess residual chlorine in water poses a substantial threat to human health. At present, there is no portable device which can achieve accurate, rapid, low-cost, and convenient detection of residual chlorine in water. Therefore, it is necessary to develop a device that can perform accurate, rapid, low-cost, and convenient detection of residual chlorine in water. In this study, a portable residual chlorine detection device was developed. A microfluidic chip was studied to achieve efficient mixing of two-phase flow. This microfluidic chip was used for rapid mixing of reagents in the portable residual chlorine detection device, reducing the consumption of reagents, detection time, and device volume. A deep learning algorithm was proposed for predicting residual chlorine concentration in water, achieving precise detection. Firstly, the microfluidic chip structure for detecting mixed reagents was optimized, and the microfluidic chip was fabricated by a 3D-printing method. Secondly, a deep learning (LS-BP) algorithm was constructed and proposed for predicting residual chlorine concentration in water, which can realize dual-channel signal reading. Thirdly, the corresponding portable residual chlorine detection device was developed, and the detection device was compared with residual chlorine detection devices and methods in other studies. The comparison results indicate that the portable residual chlorine detection device has high detection accuracy, fast detection speed, low cost, and good convenience. The excellent performance of the portable residual chlorine detection device makes it suitable for detecting residual chlorine in drinking water, swimming pool water, aquaculture and other fields.

Droplet Microfluidics for High-Throughput Screening and Directed Evolution of Biomolecules.

Directed evolution is a powerful technique for creating biomolecules such as proteins and nucleic acids with tailor-made properties for therapeutic and industrial applications by mimicking the natural evolution processes in the laboratory. Droplet microfluidics improved classical directed evolution by enabling time-consuming and laborious steps in this iterative process to be performed within monodispersed droplets in a highly controlled and automated manner. Droplet microfluidic chips can generate, manipulate, and sort individual droplets at kilohertz rates in a user-defined microchannel geometry, allowing new strategies for high-throughput screening and evolution of biomolecules. In this review, we discuss directed evolution studies in which droplet-based microfluidic systems were used to screen and improve the functional properties of biomolecules. We provide a systematic overview of basic on-chip fluidic operations, including reagent mixing by merging continuous fluid streams and droplet pairs, reagent addition by picoinjection, droplet generation, droplet incubation in delay lines, chambers and hydrodynamic traps, and droplet sorting techniques. Various microfluidic strategies for directed evolution using single and multiple emulsions and biomimetic materials (giant lipid vesicles, microgels, and microcapsules) are highlighted. Completely cell-free microfluidic-assisted in vitro compartmentalization methods that eliminate the need to clone DNA into cells after each round of mutagenesis are also presented.

Rapid on-site amplification and visual detection of misfolded proteins via microfluidic quaking-induced conversion (Micro-QuIC)

Protein misfolding diseases, such as prion diseases, Alzheimer’s, and Parkinson’s, share a common molecular mechanism involving the misfolding and aggregation of specific proteins. There is an urgent need for point-of-care (POC) diagnostic technologies that can accurately detect these misfolded proteins, facilitating early diagnosis and intervention. Here, we introduce the microfluidic quaking-induced conversion (Micro-QuIC), a novel acoustofluidic platform for the rapid and sensitive detection of protein misfolding diseases. We demonstrate the utility of our technology using chronic wasting disease (CWD) as a model system, since samples from wild white-tailed deer are readily accessible, and CWD shares similarities with human protein misfolding diseases. Acoustofluidic mixing enables homogeneous mixing of reagents in a high-Reynolds-number regime, significantly accelerating the turnaround time for CWD diagnosis. Our Micro-QuIC assay amplifies prions significantly faster than the current gold standard, real-time quaking-induced conversion (RT-QuIC). Furthermore, we integrated Micro-QuIC with a gold nanoparticle-based, naked-eye detection method, which enables visual discrimination between CWD-positive and CWD-negative samples without the need for a bulky fluorescence detection module. This integration creates a rapid, POC testing platform capable of detecting misfolded proteins associated with a variety of protein misfolding diseases.

Rapid detection of FadA in Fusobacterium nucleatum using the quantitative LAMP colorimetric phenol red method in stool samples from colorectal cancer patients

The study aimed to develop a quantitative colorimetric loop-mediated isothermal amplification technique using the phenol red indicator (QLAMP-PhR) for detecting Fusobacterium nucleatum (Fn) levels in colorectal cancer (CRC) patients and healthy individuals. QLAMP-PhR assays were conducted on 251 stool samples specific for the Fn FadA gene. Six primers were synthesized and utilized with master mix reagents, and a phenol red indicator was employed to enhance the QLAMP-PhR technique. A standard quantitative analysis curve was generated using a logarithmic function (absorbance vs. concentration) by serially diluting the copy number of genomic DNA templates (Fn ATCC25586). The CRC group exhibited a significantly higher abundance of Fn compared to the healthy control group (P < 0.001). These findings suggest that the QLAMP-PhR technique effectively identifies Fn specifically by its gene for the key virulence factor FadA. Additionally, ideas for developing a real-time QLAMP-PhR test were presented. Compared to the traditional polymerase chain reaction (PCR) technique, QLAMP-PhR offers several advantages including rapidity, simplicity, specificity, sensitivity, and cost-effectiveness method that can quantitatively screen for Fn presence in normal populations. The QLAMP-PhR method represents a sensitive and specific amplification assay for the rapid detection of the Fn pathogen. To the best of our knowledge, this study is the first to report the application of QLAMP-PhR for detecting FadA in Fn.

Combinatorial sample preparation platform for droplet-based applications in microbiology

Droplet microfluidics has demonstrated immense potential in microbiological studies due to its unique features, such as miniaturization, compartmentalization, and parallelization. Multiplexing droplet content allows the investigation of various experimental conditions in a highly parallelized manner. Yet, droplet library generation and tracking remain challenging in high-throughput screening. The introduction of distinct reagents into droplets necessitates precise control over droplet flow in a microfluidic chip, limiting the throughput to a few reagents. Additionally, tracking individual droplets is complex due to their fast dynamics. To address these challenges, we have developed a multiplexing platform for automated sample preparation, enabling on-demand merging and mixing of reagents for fine-tuning the sample compositions for droplet generation. A coding space with 169 optical barcodes can be realized by the pairwise combination of four fluorescence dyes at six concentration levels to encode droplet populations as required by the experimental design. A machine-learning algorithm has been employed to identify distinct droplet populations. As proof of concept, we conducted an antibiotic susceptibility assay on an E. coli strain to screen for susceptibility of four antibiotics and determine minimum inhibitory concentrations in one experiment. Utilizing the on-demand sample preparation, optical barcodes, and machine-learning analysis, our setup provides a rapid, straightforward, and reliable multiplexing capability for numerous microbial and biochemical applications.

Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation

Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption–desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations.

Systematic evaluation of the influence of Taq enzyme choice and amplification conditions on targeted environmental DNA assay performance and detection in field samples

AbstractTargeted environmental DNA (eDNA) studies mainly rely on quantitative real‐time polymerase chain reaction (qPCR) to amplify extremely low concentrations of DNA present in environmental samples. Understanding factors that influence targeted eDNA assay performance and detection in field samples, such as Taq DNA polymerase enzyme type and thermocycle protocol, is critical for the interpretation of qPCR results. We completed a systematic performance evaluation of five distinct targeted eDNA assays (eANFI6, eFISH1, eANBO5, eGLIN1, and eLICA3 targeting sablefish, general fish, Boreal toad, wood turtle, and the American bullfrog) by subjecting the same samples to analysis by five different Taq enzyme reagent mixes (Immolase, Environmental Master Mix (EMM), Amplitaq, QIAcuity, and QuantiNova) and the commonly used 2‐step (95°C, 60°C) and 3‐step (95°C, 64°C, 72°C) thermocycle protocols. We evaluated assay performance using a standardized dilution series of synthetic dsDNA target sequences and calculated limits of detection (LOD) and quantification (LOQ) and 95% confidence intervals for each combination of Taq enzyme and thermocycle protocol. All assays performed within acceptable performance criteria as defined by the Canadian national standard for targeted eDNA assays. Based on data generated by synthetic dsDNA fragments, the eDNA assays performed comparably regardless of the enzyme reagent mix and protocol used, except for eANFI6 and eGLIN1 using EMM and the 3‐step protocol, where no amplification was observed. On freshwater field samples, EMM and Immolase performed best. On marine field samples, Immolase, EMM, Qiacuity, and QuantiNova performed equally well, although EMM failed to amplify some samples. The work reveals that an enzyme reaction mix or thermocycle protocol can affect the result of an eDNA assay, but the appropriate choice also depends on the nature of the field sample. It is therefore imperative that these are considered when selecting appropriate reaction conditions and that they are clearly reported.

Fabrication of Size‐Coded Amphiphilic Particles with a Configurable 3D‐Printed Microfluidic Device for the Formation of Particle‐Templated Droplets

AbstractCompartmentalizing an aqueous media into numerous nanoliter‐scale droplets has substantially improved the performance of amplification assays. Particle‐templated droplets or dropicles offer a user‐friendly workflow for creating uniform volume compartments upon simple mixing of reagents and particles using common laboratory apparatus. Amphiphilic shape‐coded particles are demonstrated to spontaneously hold aqueous droplets within hydrophilic cavities for multiplexed diagnostic assays. Here, a configurable 3D‐printed microfluidic device is proposed for the tunable fabrication of amphiphilic size‐coded particles. The device is configured with multiple outlet tubings of different diameters and photomasks of variable slit lengths to fabricate a wide range of size‐coded particles. a range of unique particle codes are fabricated using a single reconfigurable device. The cross‐sectional profile of the particles is further engineered by tuning the flow rate ratios of precursor streams to vary the inner and outer diameters of the particles and the thicknesses of the inner hydrophilic and outer hydrophobic layers. A range of cavity diameters and particle lengths enabled dropicle volumes of ≈1 nL to ≈30 nL. The fabricated particles are characterized by their ability to hold uniform droplet volumes and to orient themselves facing upward or sideways in a well plate based on their aspect ratios.

Determination of Trace Levels of Fenbendazole in Milk, Yogurt and Curd Using Surface Enhanced Raman Spectroscopy (SERS)

A simple, rapid and sensitive surface enhanced Raman spectroscopy (SERS) method is described for the determination of fenbendazole (FBZ) residues in milk and fermented dairy products using yogurt and curd as examples. An extraction method was used to purify the samples from interfering components of the matrix. 5-Nitrothiabendazole, which has similar physicochemical properties, was used as an internal standard. The detection limits for milk were 1.8 nM, 8.8 nM for yogurt, and 13.5 nM for curd. Using partial least squares (PLS), a calibration model was built for the determination of fenbendazole from 100 to 1000 nM. The root-mean square error of calibration (RMSEC) and the root-mean square error of prediction (RMSEP) were 39.4 and 46.5 nM, respectively. The recovery rate was from 97% to 120% and the relative standard deviation was less than 5.0%. The difference between this technique from the widely used liquid chromatography–mass spectrometry (LC–MS) method is in the reduction in time for preliminary sample preparation and significantly lower costs for reagents and consumables. The article examines the influence of order when mixing reagents in a SERS experiment, which is an important tool for optimizing the sensitivity and reproducibility of the method. It was shown for the first time that when various alkaline earth metal chlorides were used for agglomeration of silver nanoparticles (AgNPs), a change in the ratio of peak intensities of the two analytes in the solution was observed. This phenomenon may be used to increase the selectivity of SERS analysis of multicomponent samples.