Dispensing Of Reagents Research Articles

3D Printer-Controlled syringe pumps for Dual, Active, Regulable and Simultaneous dispensing of reagents. Manufacturing of immunochromatographic test strips

Lateral flow immunoassays (LFIA) are widely used worldwide for the detection of different analytes because they combine multiple advantages such as low production cost, simplicity, and portability. They allow biomarkers detection without requiring infrastructure or highly trained personnel. In this work we propose solutions for the manufacturing process of LFIA at laboratory-scale, particularly to the controlled and active dispensing of the reagents in the form the Test Lines (TL) and the Control Lines (CL). To accomplish this task, we adapted a 3D printer to also control Syringe Pumps (SP), since the proposed adaptation of a 3D printer is easy, free and many laboratories already have it within their infrastructure. In turn, the standard function of the 3D printer can be easily restored by disconnecting the SPs and reconnecting the extruder. Additionally, the unified control of the 3D printer enables dual, active, regulable and simultaneous dispensing, four features that are typically found only in certain high-cost commercial equipment. With the proposed setup, the challenge of dispensing simultaneously at least 2 lines (CL and TL) with SPs controlled by a 3D printer was addressed, including regulation in the width of dispensed lines within experimental limits. The construction of a LFIA for the detection of leptospirosis is shown as a practical example of the automatized reagent dispensing.

Drip Detection System based on STM32 Microcontroller

This design of the drip detection system is based on the STM32 microcontroller, making it highly practical and innovative. The system combines multiple sensors and control modules to achieve high-precision detection and monitoring of intravenous drip infusions. By integrating various sensors such as a drip rate detection module, a temperature sensor, and a water level detection module, the system can comprehensively monitor the status of the intravenous drip, including drip rate, liquid level, and temperature. Through data processing and control by the STM32 microcontroller, the system can real-time monitor the drip rate, liquid level, and temperature of the intravenous drip, and display these data on an OLED screen, providing intuitive information feedback. After conducting tests and analysis on the system, its stability and reliability have been verified. With its high-precision drip detection capability, the system can meet the needs of life science experiments for droplet detection. This system is widely applicable in various fields such as medical, chemistry, and biology. In the medical field, it can be used for precise drug delivery, thereby improving treatment effectiveness. In chemical and biological experiments, it can be used for accurate dispensing of liquid reagents, ensuring the accuracy of experimental results. The system features an OLED display, providing a user-friendly interface, and also comes with button operations, enabling users to conveniently set and operate system parameters. Overall, this drip detection system based on the STM32 microcontroller not only possesses a high level of technological sophistication but also has broad prospects for practical applications. It provides an efficient and accurate solution for droplet detection and will play a significant role in related fields.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

Open-source perfusion system for medium-scale fabrication of demineralized bone matrix chip grafts

Demineralized bone matrix (DBM) is considered one of the most reliable bone tissue grafts for regular surgical use, as it provides a scaffold that is structurally like native bone, and that enhances bone regeneration. However, commercially available DBM products are not suited for surgical restitutions of large bones. Therefore, each Tissue Bank is urged to implement their own demineralization protocol, which usually does not meet the high demand for bone grafting. In this project, we developed an open source system for medium-scale manufacturing of DBM grafts from human cadaveric donors to automate the demineralization protocol and improve its reproducibility. The device consists in (1) unidirectional flow reaction chamber, where the demineralization protocol takes place; (2) automated syringe pump, which controls the reagent́s inlet and vacuum; and (3) reagent dispenser, for the management of the reagents need for the demineralization protocol. Validation of the device included histological analysis, DNA quantification temperature regulation, electrochemiluminescence and colorimetric protocols, followed by the optimization of physicochemical parameters based on Response Surface Methodology. The results showed values of residual lipids and calcium within standardized ranges, and the maintenance of the structural integrity of the DBM, demonstrating the capacity of the system to support the proposed demineralization protocol.

Single-step measurement of cell-free DNA for sepsis prognosis using a thread-based microfluidic device.

Cell-free DNA (cfDNA) content in plasma has been studied as a biomarker for sepsis. Recent publications show that the cfDNA content in sepsis patients entering intensive care unit who were likely to survive had a total cfDNA concentration of 1.16 ± 0.13μg/mL compared to 4.65 ± 0.48μg/mL of non-survivors. Current methods for measuring cfDNA content in plasma were designed to amplify and measure low concentrations of specific DNA, making them unsuitable for low-cost measurement of total cfDNA content in plasma. Here, we have developed a point of care (POC) device that uses a thread silicone device as a medium to store a fluorescent dye which eliminates the need for preparatory steps, external aliquoting and dispensing of reagents, preconcentration, and external mixing while reducing the detection cost. The device was paired with a portable imaging system with an excitation filter at 472 ± 10nm and an emission filter of 520 ± 10nm that can be operated with just 100mA current supply. The device was demonstrated for use in the quantification of buffered cfDNA samples in a range 1-6μg/mL with a sensitivity of 5.72 AU/μg/mL and with cfDNA spiked in plasma with a range of 1-3μg/mL and a sensitivity of 5.43 AU/μg/mL. The results showed that the device could be used as a low-cost, rapid, and portable POC device for differentiating between survivors and non-survivors of sepsis within 20min.

Multi-reagents dispensing centrifugal microfluidics for point-of-care testing

Point-of-care testing (POCT) has shown great advantages for public health monitoring in resource-limited settings. However, developing of POCT tools with automated and accurate quantitative dispensing of multiple reagents and samples is challenging. Here, we demonstrate a novel multi-reagents dispensing centrifugal microfluidics (MDCM) that allows rapid and automated dispensing of multiple reagents and samples with high throughput and accuracy. The MDCM was designed with multiple aliquoting units with the hydrophobic valve at different radial positions. All reagents and samples were loaded simultaneously, dispensed in parallel by centrifugation at low speed, and then introduced into the reaction chamber sequentially by centrifugation at high speed. Two MDCM chips are demonstrated, including a uniform concentration generator and a gradient concentration generator. The concentration coefficient of variation (CV) among the independent reaction chambers was lower than 0.56%, and the theoretical quantitative concentration gradient was strongly correlated with the actual concentration gradient (R2 = 0.9938). We have successfully applied the MDCM to loop-mediated isothermal amplification (LAMP)-based nucleic acid detection for multiple infectious pathogens and antimicrobial susceptibility testing (AST) for kanamycin sulfate against E. coli. To further extend the applications, the MDCM has also been applied to bicinchoninic acid (BCA) protein assays with online calibration, reducing the detection time from 2 h to 10 min with a twenty-fold reduction in reagent consumption. These results indicated that the MDCM is a high potential platform for POCT.

A Novel Method to Gently Mix and Uniformly Suspend Particulates for Automated Assays.

The SpinVessel system provides a methodology using pulsed radial flow to gently mix and uniformly suspend particulates (cells, magnetic beads, silica beads, and microcarrier beads) for automated assays. SpinVessels are well suited for aliquoting on robotic liquid handlers and with robotic reagent dispensers, as well as manually. The SpinVessel system combines two critical features: (1) special internal side fins and projections in the bottom of the vessels and (2) an instrument that quickly spins the vessels and repeatedly reverses the spin direction. This rapid reversing motion sends multiple pulses of fluid up the side walls of the SpinVessel, creating a circular radial flow pattern. We tested five different particulates and six different SpinVessels with volume capacities varying from 50 mL to 1200 mL. SpinVessels are compatible with either single-, 8-, 12-, 96-, or 384-channel pipettors or with siphon tubing on robotic reagent dispensers. Experiments have demonstrated high viability of cells and undamaged morphology of microcarrier beads even after hours of constant agitation. The uniformity of aliquots collected at various vertical depths and horizontally across the SpinVessels demonstrated that cells, magnetic beads, and silica beads were uniformly suspended throughout the height and breadth of the SpinVessels, and uniformity of samples was consistent from the beginning to the end of the aliquoting procedure. Only 5 min of mixing is required to resuspend settled particulates. This novel mixing methodology has many applications in laboratory automation where particulate aliquot uniformity and/or particulate integrity are important to automating assays.

Low-cost, open-source 3D printed antibody dispenser for development and small-scale production of lateral flow assay strips

We present a low cost, 3D printed open-source antibody dispenser that can be easily built and used for the development of lateral flow assay (LFA) strips. The fabrication of LFA strips need dispensing of antibodies or antigens in a linear fashion and commercially available dispensers typically cost from few thousands to few tens of thousands of US dollars. In this paper, an antibody dispenser was built by using 3D printed and commercially available parts, which cost no more than 30 USD. This paper presents a detailed instruction on how to assemble the printer and how to achieve a specific line width for the dispensed antibody. By using syringe needles of different gauges, line width ranging from 0.23 to 1.8 mm can be dispensed, and by varying the speed controller, the dispensing needle’s speed can be varied between 2.8 and 3.8 cm/s. We demonstrate uniform dispensing of anti-C-reactive protein (CRP) antibody and anti-rabbit antibody to draw a test line and a control line, which are used for the detection of CRP. The dispenser can also be equipped with two dispensing needles to allow simultaneous dispensing of multiple reagents, which can be useful for LFA strip development.

Diagnostic performance of McMaster, Wisconsin, and automated egg counting techniques for enumeration of equine strongyle eggs in fecal samples

Fecal egg counts are the cornerstone of equine parasite control programs. Previous work led to the development of an automated, image-analysis-based parasite egg counting system. The system has been further developed to include an automated reagent dispenser unit and a custom camera (CC) unit that generates higher resolution images, as well as a particle shape analysis (PSA) algorithm and machine learning (ML) algorithm. The first aim of this study was to conduct a comprehensive comparison of method precision between the original smartphone (SP) unit with the PSA algorithm, CC/PSA, CC/ML, and the traditional McMaster (MM) and Wisconsin (MW) manual techniques. Additionally, a Bayesian analysis was performed to estimate and compare sensitivity and specificity of all five methods. Feces were collected from horses, screened with triplicate Mini-FLOTAC counts, and placed into five categories: negative (no eggs seen), > 0 - ≤ 200 eggs per gram (EPG), > 200 - ≤ 500 EPG, > 500 - ≤ 1000 EPG, and > 1000 EPG. Ten replicates per horse were analyzed for each technique. Technical variability for samples > 200 EPG was significantly higher for MM than CC/PSA and CC/ML (p < 0.0001). Biological variability for samples> 0 was numerically highest for CC/PSA, but with samples > 200 EPG, MM had a significantly lower CV than MW (p = 0.001), MW had a significantly lower CV than CC/PSA (p < 0.0001), CC/ML had a significantly lower CV than both MW and SP/PSA (p < 0.0001, p = 0.0003), and CC/PSA had a significantly lower CV than CC/SP (p = 0.0115). Sensitivity was> 98 % for all five methods with no significant differences. Specificity, however, was significantly the highest for CC/PSA, followed numerically by SP/PSA, MM, CC/ML, and finally MW. Overall, the automated counting system is a promising new development in equine parasitology. Continued refinement to the counting algorithms will help improve precision and specificity, while additional research in areas such as egg loss, analyst variability at the counting step, and accuracy will help create a complete picture of its impact as a new fecal egg count method.

Miniaturization and optimization of 384-well compatible RNA sequencing library preparation.

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

TRTH-28. HIGH THROUGHPUT SCREENING OF NOVEL HISTONE DEACETYLASE INHIBITORS FOR EPIGENETIC THERAPY OF PRIMARY BRAIN TUMORS

Abstract Histone deacetylases (HDACs) are crucial regulators of epigenetic and posttranslational modifications and therefore represent a promising therapeutic target in cancer cells that harbor distinct epigenomes from normal cells. To exploit the therapeutic potential of HDAC inhibitors (HDACi) we synthesized a unique in-house library of more than 200 inhibitors. For the evaluation of the antitumor effects of the compound library in brain tumor cell lines, we successfully established an optimal screening workflow. The screening procedure was streamlined by automated dispensing of cell lines, reagents and inhibitors using state of the art equipment. The drugs were evaluated for their effect on tumor cell viability in a panel of cell lines derived from different brain tumor entities (8 glioblastoma, 10 medulloblastoma and 6 atypical teratoid/rhabdoid tumor cell lines) and compared to 5 normal control tissues. Corresponding dose-response profiles were generated using an optimized bioinformatics workflow. In addition to our in-house library commercially available and clinically used HDACi (e.g. Vorinostat) were included. The semi-automated setup enabled the miniaturization of the assay format to 384-well plates. In combination with additional modifications, a remarkable increase of the overall throughput was realized, while generating accurate and reproducible results. Based on this workflow, we created a unique and comprehensive data set and could thereby identify various HDACi acting universally across brain tumors or being specifically active in distinct tumor entities. Promising candidates are currently being further characterized, e.g. Panobinostat showed activity in the majority of brain tumor models at low nanomolar concentrations. The growing importance of HDAC inhibitors is reflected by an increasing number of HDACi in clinical trials, with four HDACi already approved by the FDA for treating lymphomas and multiple myelomas. Our study supports the finding that HDACi are valid therapeutic agents and that selected inhibitors are promising candidates for future epigenetic therapy of primary brain tumors.

Validation of Simultaneous Analytical Method of Veterinary Drugs in Foods Using New Automatic Pretreatment Equipment.

New automatic pretreatment equipment (FASVED; Food Automatic Analytical Systems for Veterinary Drugs) was developed. FASVED consists of ten main units: reagent dispenser, homogenizer, transfer hand, lid opening/closing device, centrifugal separator, pipette, shaker, column purification device, centrifugal evaporator and cooling device, and it is capable of freely combining operations by these units. A validation study was performed on two methods for determination of 178 veterinary drugs in livestock products, swine muscle, egg and shrimp, according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. The numbers of analytes that satisfied the criteria of the guideline were 148 in swine muscle, 160 in egg and 151 in shrimp.

Evaporation-Driven Bioassays in Suspended Droplets.

The microtiter plate has been an essential tool for diagnostics, high-throughput screening, and biological assays. We present an alternative platform to perform bioassays in a microplate format that exploits evaporation to drive assay reactions. Our method consists of droplets suspended on plastic pillars; reactions occur in these droplets instead of the wells. The pillars are fabricated by milling, and the rough surface created by this fabrication method pins the droplet to a constant contact line during the assay and also acts as a hydrophobic surface. Upon evaporation, natural convection arising from Marangoni currents mixes solutions in the droplet, which speeds up assay reactions, decreases assay times, and increases limits of detection. As a proof of concept we implemented two colorimetric assays to detect glucose and proteins in only 1.5 μL, without any external devices for mixing and with a digital microscope as a readout mechanism. Our platform is an ideal alternative to the microtiter plate, works with different volumes, is compatible with commercially available reagent dispensers and plate-readers, and could have broad applications in diagnostics and high-throughput screening.

Simultaneous metering and dispensing of multiple reagents on a passively controlled microdevice solely by finger pressing.

In this work, we report a novel design of a passively controlled, finger-driven microfluidic circuit for the metering and delivery (MaD) of a liquid reagent. The proposed design modularized the fluidic circuit for a single reagent's MaD so that it can be multiplexed conveniently for the MaD of an arbitrary number of reagents solely by finger pressing. The microdevice has comparable accuracy with pipettes and we have demonstrated its applicability in the preparation of biochemical assays. The proposed design of the modularized, structurally "stackable" fluidic circuit provides a reference in designing future single-pressure-source-driven, passively controlled multi-liquid handling microfluidic platforms.

Designing innovative therapies for neuropathic pain: pros and cons of target-based drug discovery.

Designing innovative therapies for neuropathic pain: pros and cons of target-based drug discovery.

Application Areas.

Application Areas.

Next generation 1536-well oligonucleotide synthesizer with on-the-fly dispense

Here we report the development of our Next Generation Automated Multiplexed Oligonucleotide Synthesizer (NG-1536-AMOS), capable of producing 1536 samples in a single run using a multi-well filtered titer plate. With the potential to synthesize up to 3456 samples per plate, we converted the BioRAPTR Flying Reagent Dispenser into an open-well system where spent reagents are drained to waste under vacuum. During synthesis, reagents are delivered on-the-fly to each micro-titer well at volumes ≤5μl with plate speeds up to 150mm/s. Using gas-phase cleavage and deprotection, a full plate of 1536 60mers may be processed with same-day turnaround with an average yield per well at 3.5nmol. Final product at only $0.00277/base is eluted into a low-volume collection plate for immediate use in downstream application (e.g. Biomek FX for versatile sample handling). Also, crude oligonucleotide quality is comparable to that of commercial synthesis instrumentation, with an error rate on the NG-1536-AMOS platform of 1.53/717 bases. Furthermore, mass spectral analysis on strands synthesized up to 80 bases showed high purity with an average coupling efficiency of 99.5%.

Cell-Free Protein Synthesis in Miniaturized Array Devices and Effects of Device Orientation

Cell-free protein synthesis (CFPS) has been used as an alternative to cell-based recombinant technology for protein production in academic and industrial labs. The continuous-exchange format generally has higher expression yield by constantly supplying a nutrient solution and removing inhibitory by-products through a porous membrane. Because of the concern of possible membrane clogging by large molecules in the CFPS solution, we investigated the effects of membrane orientation on protein synthesis. We fabricated a miniaturized array device called Vertical-I with its membrane oriented vertically in reference to the table surface and found that the protein synthesis yield in the Vertical-I device was 144% higher than the Horizontal Device reported previously. The reaction time was also faster; β-glucuronidase reached the synthesis yield plateau after 2 h in the Vertical-I device versus 4 h in the Horizontal Device. Possible clogging of membrane pores was confirmed by fluorescein diffusion measurement. Using these results, we designed a device called Vertical-II that would fit into a 96 well plate holder for compatibility with commercial reagent dispensers and microplate readers. The experimentally optimized device increased protein expression 406% over the Horizontal Device and consumes 5 times fewer reagents than a commercial device, showing the potential for high-throughput protein synthesis.