Dispensing Of Reagents Research Articles

High-Throughput Quality Control of DMSO Acoustic Dispensing Using Photometric Dye Methods

One high-throughput technology gaining widespread adoption in industry and academia is acoustic liquid dispensing, in which focused sound waves eject nanoliter-sized droplets from a solution into a recipient microplate. This technology allows for direct dispensing of small-molecule compounds or reagents dissolved in DMSO, while keeping a low final concentration of organic solvent in an assay. However, acoustic dispensing presents unique quality control (QC) challenges when measuring the accuracy and precision of small dispense volumes ranging from 2.5 to 100 nL. As part of an effort to develop a rapid and cost-effective QC method for acoustic dispensing of 100% DMSO, we implemented the first high-throughput photometric dual-dye-based QC protocol in the nanoliter volume range. This technical note validates the new photometric 100% DMSO QC method and highlights its cost-effectiveness when compared with conventional low-throughput fluorimetric QC methods. In addition, a potential software solution is described for the analysis, storage, and display of accumulated high-throughput QC data, called LabGauge. As the need for high-throughput QC grows, conventional low-throughput methods can no longer meet demand. Validated high-throughput techniques, such as the dual-dye photometric method, will need to be implemented.

Photocolorimetric Biosensor for Detection of Cholinergic Organophosphorus Compounds

To detect nerve agents in practice, the analytical methods such as gas, liquid and thin-layer chromatography, mass spectrometry or capillary electrophoresis are usually used. Apart from these analytical methods, we developed an analytical device (tape photocolorimetric biosensor) based on the modified Ellman's cholinesterase biochemical reaction for multidetection of cholinergic organophosphorus compounds. Enzyme butyrylcholinesterase was used as a biorecognizing component and its activity was evaluated by red, blue, green (RGB) sensor. This method eliminates errors in the evaluation and provides automatic data collection with their subsequent evaluation. The unique method of dosing allows appropriate dispensing of reagents in microlitres volumes and the whole system is simple to operate. Suitability of the constructed biosensors was evaluated using the six organophosphates (Tabun, sarin, Soman, cyclosin, VX and R33 compound). Biosensor showed the ability to measure substances at concentrations ranging between ~ 1×10-8 mg/l - 1×10-6 mg/l in the air, according to their inhibition effect. Defence Science Journal, 2012, 62(6), pp.399-403 , DOI:http://dx.doi.org/10.14429/dsj.62.2589

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Accurate dispensing of volatile reagents on demand for chemical reactions in EWOD chips

Digital microfluidic chips provide a new platform for manipulating chemicals for multi-step chemical synthesis or assays at the microscale. The organic solvents and reagents needed for these applications are often volatile, sensitive to contamination, and wetting, i.e. have contact angles of <90° even on the highly hydrophobic surfaces (e.g., Teflon® or Cytop®) typically used on digital microfluidic chips. Furthermore, often the applications dictate that the processes are performed in a gas environment, not allowing the use of a filler liquid (e.g., oil). These properties pose challenges for delivering controlled volumes of liquid to the chip. An automated, simple, accurate and reliable method of delivering reagents from sealed, off-chip reservoirs is presented here. This platform overcomes the issues of evaporative losses of volatile solvents, cross-contamination, and flooding of the chip by combining a syringe pump, a simple on-chip liquid detector and a robust interface design. The impedance-based liquid detection requires only minimal added hardware to provide a feedback signal to ensure accurate volumes of volatile solvents are introduced to the chip, independent of time delays between dispensing operations. On-demand dispensing of multiple droplets of acetonitrile, a frequently used but difficult to handle solvent due to its wetting properties and volatility, was demonstrated and used to synthesize the positron emission tomography (PET) probe [(18)F]FDG reliably.

Virtual microwells for digital microfluidic reagent dispensing and cell culture.

Digital microfluidic (DMF) liquid handling includes active (electrostatic) and passive (surface tension) mechanisms for reagent dispensing. Here we implement a simple and straightforward Teflon-AF liftoff protocol for patterning hydrophilic sites on a two-plate device for precise passive dispensing of reagents forming virtual microwells--an analogy to the wells found on a microtitre plate. We demonstrate here that devices formed using these methods are capable of reproducible dispensing of volumes ranging from ~80 to ~800 nL, with CVs of 0.7% to 13.8% CV. We demonstrate that passive dispensing is compatible with DMF operation in both air and oil, and provides for improved control of dispensed nano- and micro- litre volumes when compared to active electrostatic dispensing. Further, the technique is advantageous for cell culture and we report the first example of reagent dispensing on a single-plate DMF device. We anticipate this method will be useful for a wide range of applications--particularly those involving adherent cell culture and analysis.

Overview of Liquid Handling Instrumentation for High‐Throughput Screening Applications

Liquid handling in the laboratory has unique challenges specific to the types of research being performed. The devices employed for purposes of performing liquid handling can be broken down into three general categories: bulk reagent dispensers, transfer devices, and plate washers. An overview of these types of liquid handlers, as well as common features and relevance to high-throughput applications, are discussed in this article. Important topics such as sterility, ease of use, cost, and instrument design advantages and disadvantages are also covered. Curr. Protoc. Chem Biol. 1:43-54. © 2009 by John Wiley & Sons, Inc.

A channel-resolved approach coupled with magnet-captured technique for multianalyte chemiluminescent immunoassay

A concept of channel-resolved multianalyte immunoassay (MAIA) and a semi-automated flow-through chemiluminescent (CL) MAIA system coupled with magnet-captured technique were proposed for rapid quantitation of different analytes in a single run. Using α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carcinoma antigen 125 (CA 125) as model analytes. They were firstly incubated in the mixtures of capture antibodies-immobilized paramagnetic microspheres (PMs) and corresponding alkaline phosphatase-labeled antibodies under stir and pumped into three parallel detection channels, the PMs were simultaneously captured by magnet, and the CL signals from the three channels were then sequentially collected with the aid of optical shutters to perform quantitative detection. AFP, CEA and CA 125 could be rapidly assayed in the ranges of 1.0–40μg/l, 0.20–30μg/l and 1.0–50kU/l with the detection limits of 0.60μg/l, 0.080μg/l and 0.70kU/l at 3σ, respectively. After manual dispensing of specimen and reagents the whole assay process could be completed in 18min. The assay results of clinical serum samples with the proposed method were in acceptable agreement with the reference values. This system, based on the designed channel-resolved strategy and magnet-captured technique provides a semi-automated, reusable, simple, sensitive, rapid and low-cost approach for MAIA without using of expensive array detector.

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5 h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.

Hybrid Valve Structure for High-Throughput, Low-Volume Liquid-Handling Applications

A hybrid valve that integrates precision microfluidics for fluid routing, high-speed valving for fluid switching, and reagent-jetting devices for metering the fluid dispenses is described. The hybrid valve enables parallel switching between aspiration and dispense modes for multiple sample streams. This unique valve structure addresses many of the concerns with handling microscale volumes, including efficient use of samples, degradation of ink jet valves and speed of operation. A broad range of volumes can be manipulated with excellent reproducibility. The hybrid valve can be configured for a variety of applications. Pick-and-place aspiration and dispensing of unique reagents and rapid dispensing of a common reagent are possible. Together these features lead to higher-speed transfer of smaller volumes of reagent.

Highly parallel dispensing of chemical and biological reagents.

We present a technology for the highly parallel dispensing of a multitude of reagents. It allows one to dispense up to 96 different reagents simultaneously in a fixed array, in a volume range of 100 pL up to several nL. The pitch of the dispensed droplets can be as small as 500 microm. All channels are fired simultaneously, giving an unprecedented throughput. The system was originally developed for the high-throughput fabrication of microarrays, but can easily be adopted for other applications such as highly parallel filling of nanotiterplates. Based on our standard configuration we achieved droplets with 125- micro m in-flight diameter (1.2 nL) with a CV of <1%.

The μFill: A New 96-/384- Well Microplate Reagent Dispenser for HTS and Drug Discovery

Today's biomedical research requires instrumentation that is both functional and versatile. While high throughput screening (HTS) and drug discovery laboratories require instrumentation that can be automated, pilot assay laboratories may not necessarily need total automation. Towards that end, Bio-Tek has developed the μFill, a 96-/384-well microplate reagent dispenser capable of running stand-alone or computer-controlled as part of a robotics system. The μFill is compatible with both 96- and 384-well microplates, and using a specially designed adaptor can also dispense to deep-well microplates. It is capable of dispensing from 10–3000 μl for 96-well plates and 5–1500 μl for 384-well plates in 1-μl increments. The μFill can dispense 20 μl into a 96-well plate in four seconds and into a 384-well microplate in 12 seconds. The microprocessor-controlled syringe pump is based on a tested, low-maintenance design that requires no calibration, yet provides a high degree of accuracy and precision. The accuracy with an 80 μl dispense is within 1 μl with a percentage CV of less than 2%, and with a dispense volume of 20 μl, the percentage CV is still less than 5%. For those needing to dispense organic solvents or sterile aqueous solutions, a model that is autoclavable and has increased solvent resistance is also available. The programming allows for the control of flow rates from 225 μl/well/sec, for dispensing to cell cultures, and to 1000 μl/well/sec for rapid and vigorous reagent dispensing. The flexible software provides complete programming capabilities from the keypad. For more complete automation, robotics interfaces can be developed using ActiveX® software commands. The μFill's size, with a 14 times 14-inch footprint and a height of seven inches, allows it to be used almost anywhere.

The μFill: A New 96-/384- Well Microplate Reagent Dispenser for HTS and Drug Discovery

Today's biomedical research requires instrumentation that is both functional and versatile. While high throughput screening (HTS) and drug discovery laboratories require instrumentation that can be automated, pilot assay laboratories may not necessarily need total automation. Towards that end, Bio-Tek has developed the μFill, a 96-/384-well microplate reagent dispenser capable of running stand-alone or computer-controlled as part of a robotics system. The μFill is compatible with both 96- and 384-well microplates, and using a specially designed adaptor can also dispense to deep-well microplates. It is capable of dispensing from 10–3000 μl for 96-well plates and 5–1500 μl for 384-well plates in 1-μl increments. The μFill can dispense 20 μl into a 96-well plate in four seconds and into a 384-well microplate in 12 seconds. The microprocessor-controlled syringe pump is based on a tested, low-maintenance design that requires no calibration, yet provides a high degree of accuracy and precision. The accuracy wit...

Silicon based affinity biochips viewed with imagingellipsometry

In this paper we report on the fabrication of an affinity biochipwith a matrix of 900 targets for detection with imaging ellipsometry. Twomethods of fabrication of chips are shown: one based on wet etching of asilicon surface and the other on the preparation of so-called tension wellson the silicon surface. The dispensing of reagents and ligands wasperformed using a pipetting robot equipped with a micro-capillary, asyringe pump and micro-stepping motors. Measurements were performed on thechips in real time with carbohydrate model substances selected for sixcommon lectins. Affinity binding was shown for three of the tested modelsubstances.

Use of firefly luciferase in atp-related assays of biomass, enzymes, and metabolites

The kinetics of ATP reagents not affected by product inhibition or other forms of inactivation of luciferase during the measurement time has been clarified. Under these conditions the decay rate of the light emission expressed as percentage per minute is a measure of luciferase activity and can be given as the rate constant k (min-1), directly reflecting the degradation of ATP in the luciferase reaction. Three types of reagents with different analytical characteristics and different application possibilities have been identified. Stable light-emitting reagents are suitable for measurements of ATP down to 1000 amol. This is the only type of reagent suitable for monitoring ATP-converting reactions, i.e., assays of enzymes or metabolites, assays of oxidative phosphorylation, photophosphorylation, and so on. A higher luciferase activity resulting in a slow decay of the light emission by approximately 10% per minute (k = 0.1 min-1) gives a reagent suitable for measurements down to 10-100 amol. The slow decay of light emission allows use of manual luminometers without reagent dispensers. A further increase of the luciferase activity resulting in a decay rate of approximately 235% per min (k = 2.35 min-1) and only 10% of the light emission remaining after 1 min is suitable for measurements down to 1 amol corresponding to half a bacterial cell. With this type of flash reagent the total light emission can be calculated from two measurements of the light intensity on the decay part of the light emission curve. This new measure is not affected by moderate variations in luciferase activity, but only by changes in quantum yield and self-absorption of the light in the sample. Flash-type reagents require the use of reagent dispensers. The stringent requirements for ATP-free cuvettes, pipette tips, and contamination-free laboratory techniques make it unlikely that flash reagents would be useful in nonlaboratory surroundings. A potential application for this type of reagent is sterility testing. In general, it is concluded that one should select the ideal ATP reagent carefully for each application. Obviously the reagents used in a particular application do not have to match the decay rates given earlier exactly. However, various applications of the ATP technology and the properties of manual and automatic luminometers fall quite nicely into categories corresponding to the properties of the three reagents described. The rapidly growing interest in ATP technology has already resulted in the development of a greater variety of luminometers, from hand-held instruments to high-throughput systems. The continuation of efforts in both reagent and instrument development will undoubtedly result in many new applications.

Combinatorial Methods for the Synthesis of Aluminophosphate Molecular Sieves.

Automatic dispensing of reagents into autoclave blocks followed by synthesis, isolation, and automated structure analysis with X-ray diffractometry represents an efficient methodology for the combinatorial synthesis of microporous materials. The figure shows a typical diffractogram of as-synthesized AFI-type molecular sieves taken with a CCD camera.

Inexpensive Reagent Dispenser

An inexpensive reagent dispenser constructed from a disposable syringe and three-way valve.

An inexpensive, liquid reagent dispenser

It is expensive to dispose of liquid reagents, but it may also be expensive to invest in commercial dispensers. These authors offer a $5 solution to the problem.

Reagent Dispenser for Field Preservation of Water Samples

Reagent Dispenser for Field Preservation of Water Samples

Radioimmunoassay for digoxin with a fully automated continuous-flow system.

We describe a fully automated continuous-flow radioimmunoassay for digoxin with use of a Technicon AutoAnalyzer system. It makes use of antibodies linked to magnetizable particles; a magnet separates the bound and free fractions. The precise dispensing of reagents by the AutoAnalyzer system permits a small incubation volume (about 160 microliter) and the reproducible timing enables non-equilibrium conditions to be used. Thus a single sample is assayed in 15 min, with a sample throughput of 30/h. The standard curve ranges from 0.5 to 8.0 microgram/liter and is most precise between 1 and 3 microgram/liter, the between-assay coefficient of variation being less than 3%. There is no significant carryover between samples of high and low concentration, and results by the method correlate closely (r = 0.969) with those by an established manual assay in which charcoal separation is used.

Improved Reagent Dispenser for the Determination of Dissolved Oxygen in Sea Water

An improved dispenser for delivering the reagents used in the quantitative determination of dissolved oxygen in sea water is described.