Flexible, Parameter-Controlled Nanoliter Pipetting on a Tecan xyz Platform

Cost pressure and rising throughput requirements are important drivers for assay miniaturization. Typical examples for the trend “smaller is better” are found in BioChip applications and in High Throughput Screening (HTS), which is evolving from the 96-well standard to high-density microplates with 384, 864, 1536 or more wells. These applications require the automated pipetting of liquids in the submicroliter volume range, a difficult task for traditional automated liquid handling systems based on syringe pumps. Tecan developed a new device for the accurate pipetting of volumes in the nanoliter range. Based on ink-jet printer technology, this device allows the exact control of the volume of the ejected droplets via a set of parameters. The integration of this new technology into Tecan's flexible xyz-platforms allows an easy use of this powerful technology for several applications. Results such as volume range, accuracy and precision are discussed.     

Simultaneous monitoring of oxygen consumption and acidification rates of a single zebrafish embryo during embryonic development within a microfluidic device

A microfluidic device with a light modulation system was developed to simultaneously measure the oxygen consumption rate (OCR) and acid extrusion rate (AER) of a single zebrafish embryo during embryonic development. The device combines two components: an array of acrylic microwells containing two sensing layers as the dual luminescent sensor for oxygen (O₂) and acid (pH) detection, and a microfluidic module with pneumatically actuated glass lids to controllably seal the microwells. The continuous blue LED and modulated UV LED lights were simultaneously used to excite the dual luminescent sensor, with the emission detected by a single photodetector. The detection signals were then split into DC and AC components to measure the time variations in fluorescence intensity and phosphorescence lifetime for pH and O₂ detection, respectively. We have successfully measured the OCR and AER of a single developing zebrafish embryo inside a sealed microwell from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage of 48 hpf. We also demonstrated the measurement of the OCR and AER of a single 48 hpf zebrafish that experienced acute hypoxia by using our device to monitor the transition between aerobic and anaerobic metabolism. We observed that the AER began to significantly increase, while the OCR rapidly decreased after 20 min of hypoxia, indicating the time point of transition where the non-mitochondrial metabolism subsequently dominated the energy production. Our proposed methodology provides the potential for studying the bioenergetic metabolism in a developing organism that relates mitochondrial physiology and disease.     

Automated High Throughput Purification of BigDye™ Terminator Fluorescent DNA Sequencing Reactions Using Wizard® MagneSil™ Paramagnetic Particles

We describe a reagent system and robotic methods for the purification of BigDye™ Terminator sequencing reactions prior to automated fluorescent sequence analysis. The methods use MagneSil™ paramagnetic particles to isolate sequencing extension products from unincorporated dye-labeled terminators and exchanges sequencer loading solution for reaction buffer. Processed samples give usable data that is greater than 98% accurate from primer plus 5–15 bases to over 700 bases. Typical Phred greater than 20 quality scores range from 600 to over 700 bases. This process has been adapted to a number of liquid handling robotic platforms in both 96- and 384-well formats. One method using a single POD Beckman Biomek® FX can process up to four plates in approximately 40 minutes.     

High-Throughput Automated Injection of Individual Biological Cells

The ability of efficiently delivering soluable/insoluable drug compounds or biomolecules into individual biological cells and quantifying their cellular responses is important for genetics, proteomics, and drug discovery. This paper presents a fully automated system for zebrafish embryo injection, which overcomes the problems inherent in manual injection, such as human fatigue and large variations in success rates due to poor reproducibility. Based on ldquolooking-then-movingrdquo control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute. Besides a high injection speed that compares favorably with that of a highly proficient injection technician, a vacuum-based embryo holding device enables fast immobilization of a large number of zebrafish embryos, shortening the embryo patterning process from minutes to seconds. The recognition of embryo structures from image processing identifies a desired destination inside the embryo for material deposition, together with precise motion control resulting in a success rate of 100%. Carefully tuning suction pressure levels as well as injection and retraction speeds produced a high survival rate of 98%. The quantitative performance evaluation of the automated system was based on the continuous injection of 250 zebrafish embryos. The technologies can be extended to other biological injection applications such as the injection of mouse embryos, Drosophila embryos, and C. elegans to enable high-throughput biological and pharmaceutical research.     

Miniaturization of an Enzyme Assay (β-Galactosidase) in the 384- and 1536-Well Plate Format

Miniaturization is one way to realize today's demands in the drug discovery process by moving from the standard 96-well plate to higher density microplate formats. In this article we describe the adaptation of a fluorescence-based enzyme assay to the challenges of the 384- and 1536-well plate format. The liquid-handling was realized by the automated micropipettor CyBi-Well™ 96/384/1536* (CyBio AG - formerly JENOPTIK Bioinstruments Gmbh - Jena, Germany). On the basis of optimized liquid-handling parameters pipetting routines were established to perform an enzyme assay (β-galactosidase) in the microplate formats of higher density. Finally, the experimental results were compared to those obtained in the well-established 96-well format.

In the enzyme assay, the bioconversion of the substrate Fluorescein-di-(β-D-galactopyranoside) (FDG), occurred as a linear function of the β-galactosidase concentration comparably in all three assay formats. We conclude that miniaturization using the higher density 384- and 1536-well plate formats is advantageous as the next evolutionary step in HTS, especially using enzyme assays. A careful individual adaptation procedure for each microplate format and assay at the basis of the optimized liquid-handling parameters is essential. CyBi-Well™ 96/384/1536 proves to be a powerful tool for a careful adaptation of the liquid-handling procedures of biological assays especially also in the 384- and 1536-well formats.     

Automation of cell-based drug absorption assays in 96-well format using permeable support systems

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.     

Automated High-Throughput Purification of PCR Products Using WizardMagneSil™ Paramagnetic Particles

Here we describe a reagent system and robotic methods for the purification of PCR^(a) fragments from other contaminating amplification reaction components. The methods use the MagneSil™ paramagnetic particle chemistry^(b) to isolate double stranded DNA fragments from 150bp to 23kbp. Purified fragments are eluted in water ready for downstream applications such as cloning, fluorescent DNA sequencing and microarray printing. This method has been adapted to a number of liquid handling robotic platforms, including the Biomek^® FX and Biomek^® 2000 Laboratory Automation Workstations, in both 96 and 384-well formats.     

Increasing Productivity Through a Combination of Automation and Robotics: A Case Study of Assay Services

The Assay Services Department at Genentech, Inc., is a service laboratory that performs drug level measurement and antibody testing in support of pre-clinical animal studies and human clinical studies. As the number of Genentech products has increased, so have the number of studies, resulting in an annual increase in the number of samples generated and an increased demand for assay support. Assay Services has addressed this by increasing the efficiency and productivity of sample handling and assaying through the automation of various procedures. All sample dilutions are now done by automated dilutors, reducing the number of reassays and virtually eliminating the repetitive stress of manual sample dilutions. In addition, two complete ELISA robot stations have been in use over the last two years that have increased throughput by increasing the number of plates per run (a robot can assay ten to fifteen 96-well microtiter plates per run), and by allowing assays to run overnight without requiring the presence of lab personnel. The net gain from the automation efforts has been to double the number of samples run without increasing the number of lab personnel.     

The 104-well microplate

The 96-well microplate is a ubiquitous tool in the laboratory; its use is so extensive that in a limited number of situations it can be restrictive. Consider the situation where 96 samples need analysis or a downstream process in which the 96-well format leaves no space for additional standards or controls in the upstream 96-well processing. Consequently, plates are split or sample number reduced thereby incurring additional cost for plates, reagents, standards, controls, sample tracking, data files, and time to analyze the entire plate. A simple solution is proposed with the development of a companion 8 × 13-array microplate. The 104-well microplate was developed within the American National Standards Institute/Society for Biomolecular Science standards as to plate geometry and dimension, including well spacing (9 mm) with the exception that the columns have been shifted 4.5mm to the left to accommodate the 13th column. The extra column allows for additional standards/controls without modifying chemistry, incorporating additional plates or changing to a 384-well plate. We show negligible difference (-0.0003 optical density) when comparing mean absorbance readings in 96- and 104-well format. We demonstrate use of the 104-well plate in a 96-well environment by incorporating it in an enzyme-linked immunosorbent assay on a standard liquid handler. Results from the assay show no difference between formats (y=1.039x-0.004, r=0.997). Although the 104 plate was not created to supplant the 96-well standard, we conclude that the 104 plate can be incorporated into the 96-well environment without significant change in existing systems.     

The Planning and Establishment of a Sample Preparation Laboratory for Drug Discovery

Nature has always been a productive source of new drugs. With the advent of HTS, it has now become possible to rapidly screen large sample collections. In addition to seeking greater diversity from natural products sources (microorganisms, plants, etc.), fractionation of the crude extracts prior to screening is becoming a more important part of our efforts.As sample preparation protocols become more involved, automation can help achieve and maintain a desired sample throughput. To address the needs of our screening program, two robotic systems were designed.The first system processes crude extracts all the way to 96-well plates, containing solutions suitable for screening in biological and biochemical assays. The system can dissolve crude extracts, fractionate them on SPE cartridges, dry and weigh each fraction, redissolve them to a known concentration, and prepare mother plates. The second system replicates mother plates into a number of daughter plates.     

Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations

Acoustic droplet ejection (ADE) gently and precisely aliquots nanoliter and picoliter liquid volumes without any physical contact with the solution being transferred. The technology is very automation-friendly, as it is compatible with conventional microplates. Focused energy from an acoustic transducer induces droplet ejection into an inverted standard microplate. The commercial system transfers low-nanoliter volumes of dimethyl sulfoxide–dissolved compound libraries and thereby enables cell-based assays to be performed in 1536-well plates.     

Microsystems for controlled genetic perturbation of live Drosophila embryos: RNA interference, development robustness and drug screening

A combination of genetic regulation and microenvironmental perturbation might prove especially useful for identifying the fundamental mechanisms responsible for development biology and critical bio-chemical networks. This could transform our understanding of genomics, proteomics, and drug discovery and lead to personalized drugs and molecular diagnostics for improved healthcare. With the complete sequencing of the Drosophila melanogaster, there has been a growing interest in correlating its genetics to human disease. The Drosophila embryo is an excellent whole-animal model, and is ideal for high throughput analysis. This review highlights research on the recent development of miniature tools to study critical cellular processes in Drosophila embryogenesis. First, we discuss the use of micro-injectors to conduct chip-level RNA interference experiments on self-assembled Drosophila embryos in order to determine how genes contribute to the growth and function of a developing organism. Second, we review recent progress on perturbing the microenvironment as a complementary approach to perturbing the genetic components of the development network. We designed a bio-compatible microfluidic device that automatically positions live embryos within a fluid-filled imaging channel and exposes embryos to constant or biphasic temperature or different drug concentrations. We have demonstrated that a thermal gradient applied across live Drosophila embryos results in asynchronous cellularization. Additionally, preliminary drug screening experiments were carried out on live Drosophila embryos in both 96-well plates and microfluidic channels. Time-lapse differential interference contrast microscopic images were taken to show the effects of colchicine (399.40 Da) and cytochalasin D (507.62 Da) on cellularization.     

Development and validation of a higher-throughput equilibrium dialysis assay for plasma protein binding

We describe the practical aspects of developing a semiautomated, higher-throughput plasma protein binding (PPB) assay. The assay has a capacity of 32 PPB measurements per screen using triplicate incubations per measurement, and it is flexible with respect to the number of compounds and the number of plasma types used. The described method is based on the 48-well format rapid equilibrium dialysis (RED) device in combination with a robotic liquid handling platform and quantitative bioanalysis. The RED device method was optimized with respect to equilibration time. Method validation was performed by comparison of results from the semiautomated RED PPB assay with both of those obtained using an alternative, manual equilibrium dialysis method and with literature values. Propranolol and warfarin were used as control compounds. We have modeled the effect of dialysis membrane leakage on the measured unbound fraction and implemented a test for measuring protein content in the buffer compartment to confirm the integrity of each insert of the RED device. With the described method, it is possible to screen a relatively large number of compounds for PPB in a drug discovery environment.     

基于改进型AdaBoost网络模型的斑马鱼胚胎血管识别

基于图像特征与改进型AdaBoost网络模型,对斑马鱼节间血管的识别进行了研究.对3组斑马鱼胚胎荧光图像训练集的节间血管进行正负样本选取,使用Haar-like特征图提取图像特征,通过Ada-Boost网络模型对所提取的特征训练形成级联分类器,根据识别效果,调整改进网络的系数得到改进型级联分类器,最终实现了节间血管的精确识别和统计.实验结果表明:对于节间血管提取的准确率和识全率分别达到了93.8%和91.1%,说明该算法检测准确率高,对不同组别图像均有稳定的检测效果.     

Motion analytics of zebrafish using fine motor kinematics and multi-view trajectory

Zebrafish is a useful animal model for studying human diseases such as muscle disorders. However, manual monitoring of fish motion is time-consuming and prone to subjective variations. In this paper, an automatic fish motion analytics framework is proposed. The proposed framework could be exploited to help validate zebrafish models of transgenic zebrafish that express human genes carrying mutations which lead to muscle disorders, thus affecting their ability to swim normally. To differentiate between wild-type (normal) and transgenic zebrafish, the proposed framework consists of two approaches to exploit discriminative spatial–temporal kinematic features which are extracted to represent zebrafish movements. First, the proposed approach studies precise quantitative measurements of motor movement abnormalities using a camera with the capability to record videos with high frames rates (up to 1,000 frames per second). This differs from previous works, which only tracked each fish as a single point over time. Second, the proposed approach studies multi-view spatial–temporal swimming trajectories. This differs from previous works which typically only considered single-view analysis of fish swimming trajectories. The proposed motion features are then incorporated into a supervised classification approach to identify abnormal fish movements. Experimental results have shown that the proposed approach is capable of differentiating between wild-type and transgenic zebrafish, thus helping to validate the zebrafish models.     

Drying Saucer: A 96-well Format Microplate Evaporator

A multi-plate evaporator, capable of simultaneously drying twelve 96-well format deep (1.2 mL) microplates using heated Nitrogen is discussed.A method for drying 96-well format samples with good temperature regulation and uniformity is developed for this purpose. Maximum temperature for Methanol-Water and Methyl-Ethyl-Ketone (MEK) samples is limited to 40 (C. Reduction of current drying times is desired to increase overall process throughput. Current technology requires more than 12 hours to evaporate 96-well deep microplate plates with Methanol-Water (50/50 concentration) and 4 hours for the MEK samples.This paper describes a device which reduces drying time to 4 hours for Methanol-Water samples and 0.75 hours for MEK samples.     

Automated RNA Isolation Using the Ambion RNAqueous™-96 Kit with the Packard MutiPROBEII HT Liquid Handling System

Automated isolation of total RNA from cultured cells was accomplished by adapting Ambion's solid-phase total RNA isolation kit, RNAqueous-96, for use with the MultiPROBE II liquid handling system from Packard Instruments.Cultured cells (HeLa S3 adherent cells and K562 cells in suspension) were lysed, DNase treated, washed, and then eluted from the 96-well filter plate.Total RNA recovered was analyzed for yield, intactness, purity, and ability to serve as a substrate for real-time RT-PCR.     

Assay Development in High Density MicroWell® Plates: Use of Well Geometries, Format, Surface Modification and Optical Properties to Achieve Optimal Assay Performance.

While high density MicroWell plates, including 384 well and 1536 well plates, offer advantages of low volume reaction or growth vessels in a standardized footprint allowing robotic manipulation, the greatest benefits lie in the increased well surface area to reaction volume ratios. This surface area to volume benefit can be utilized to optimize assay development by utilizing the molecular surface as a reactant. The well geometry and optical properties are also enhanced in high-density plates, allowing microscopic imaging and increased signal detection for fluorescence and luminescence.The molecular characteristics of high density MicroWell plates were examined in regard to base materials and induced surface modification. Various modification methods including injectionmolding parameters, exposure to high and low energy sources and exposure to specific physical conditions were studied. The surfaces were molecularly characterized and then utilized as reactants in high throughput and low volume in vitro assays. Polystyrene plates exhibited a natural relatively hydrophobic surface due to the hydrocarbon backbone of the polystyrene along with the repeating benzene ring. Exposure to direct electrical discharge, and several forms of irradiation in the presence of air and oxygenated atmosphere resulted in surfaces exhibiting various levels of oxidation. These levels were measured and the molecular species were calculated.These specific surfaces were utilized as reactants in solid phase, cell based and cell culture assays. Specific surfaces demonstrated specific efficacy in regard to particular applications. For example, a highly charged surface exhibiting hydroxyl, carbonyl, and some carboxyl moieties was best for adherent cell culture. Utilizing passive adsorption procedures, a variant surface exhibiting more hydroxyl and carbonyl groups did not bind IgG well but did bind phospholipids, providing a surface “high-binding” for lipids while maintaining low background due to non-specific adsorption. A surface, commonly referred to as “high binding,” having slightly lower surface energy than the other two studied and characterized by aromatic hydrocarbons, hydroxyl and some few carbonyl groups did not bind hydrophobic antigens, but did bind glycoproteins such as IgG very well under slightly basic, passive conditions. The geometries of wells were also examined. Well geometries contributed significantly by eliminating reagent wicking or creeping, enhancing detection of signals, and increasing the active surface area to reagent volume, allowing the utilization of decreased substrate concentrations along with low reagent volumes without loss of sensitivity.     

Dispensing Precision for the SciClone™ Automated Liquid Handling Workstation for 96-Channel Pipetting

One of the first steps in drug discovery involves identification of novel compounds that interfere with therapeutically relevant biological processes.

Identification of ‘lead’ compounds in all therapeutic areas included in a drug discovery program requires labor-intensive evaluation of numerous samples in a battery of therapy targeted biological assays. To accelerate the identification of ‘lead’ compounds, Janssen Research Foundation (JRF) has developed in the past an automated high throughput screening (HTS) based on the unattended operation of a custom Zymark tracked robot system. Automation of enzymatic and cellular assays was realized with this system adapted to the handling of microtiter plates. The microtiter plate technology is the basis of our screening. All compounds within our chemical library are stored and distributed in micronic tube racks or microtiter plates for screening. An efficient in-house developed mainframe based laboratory information management system supported all screening activities. Our experience at JRF has shown that the preparation of test compounds and serial dilutions has been a rate-limiting step in the overall screening process. In order to increase compound throughput, it was necessary both to optimize the robotized assays and to automate the compound supply processes. In HTS applications, one of the primary requirements is highly accurate and precise pipetting of microliter volumes of samples into microplates. The SciClone™ is an automated liquid handling workstation capable of both 96- and 384-channel high precision pipetting. For high throughput applications, the SciClone™ instrumentation is able to pipette a variety of liquid solutions with a high degree of accuracy and precision between microplates (inter-plate variability) and tip-to-tip (intra-plate variability) within a single plate. The focus of this presentation is to review the liquid handling performance of the SciCloneTM system as a multipurpose instrument for pipetting aqueous or organic solutions, and virus suspensions into 96- and 384-well microplates. The capabilities of the system and the resulting benefits for our screening activities will be described.     

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 x 14-inch footprint and a height of seven inches, allows it to be used almost anywhere.