UV Laser Micromachining of Polymers for Microfluidic Applications

We have recently begun to explore the use of UV laser ablation micromachining to construct microfluidic devices in polymers. This technique can create microchannels rapidly and modify the resulting polymer surface in a single step. By ablating under different atmospheres, it is possible to alter both the surface chemistries and physical surface morphologies of the microchannels. We have employed electroosmotic flow measurements, chemical mapping, and optical microscopy to characterize the microfluidic devices. In addition, we have studied the parameters affecting the ablation, such as the laser wavelength, laser fluence, laser firing repetition rate, and the material being ablated.     

Selective Ion Extraction for High Throughput Screening with a Microfluidic Enzyme Assay

This proof-of-concept paper describes the application of selective ion extraction to an assay of protein kinase A on a microfluidic chip platform. Selective ion extraction is a flux balance technique, where a combination of independent pressure control and voltage are used to selectively extract one ion from a mixture. The assay product is completely separated and diverted into a separate channel from the waste stream containing the unconverted substrate and enzyme. By detecting only product, background noise generated by the substrate is removed which increases the signal to noise ratio and assay sensitivity. This technique is intended for adapting kinase or protease assays with low conversion rates to an on-chip reaction format for HTS screening.     

Microfluidic Enabling Platform for Cell-based Assays

We have developed proprietary technologies for executing continuous flow assays in biochips which mimic human capillaries. Such technologies are integral to the rapidly growing laboratory instrumentation sector for applications in drug discovery, biotechnology, medical diagnostics and environmental studies. A common link between all sectors is the movement toward miniaturization to increase throughput, accuracy and efficiency in the development of new drugs. The miniaturization process itself leads to a demand for new instruments and tools capable of handling microlitre quantities of biological fluids and reagents, thus, we present an instrument which is capable of doing so in the form of a microfluidic enabling platform.     

Transferring Industrial Automation Technology to the Laboratory

Over the past few years, there has been much discussion about transferring industrial technology to laboratories. While it is easy to look at the superficial similarities, it is more important to examine the different requirements of different industries. In this way, it is possible to identify the technologies and techniques that can be successfully transferred to the laboratory to improve performance.This paper takes three very different industries - the bakery, High Throughput Screening (HTS) and mobile phone assembly and examines their different requirements. These industries have been selected from among the many sectors where the RTS Group operates - thus allowing real data from a number of situations to be used.One of the most important areas in automation design is the relationship between flexibility and throughput. This paper focuses on this relationship and its influence over machine configuration when comparing the requirements of the different industries.     

Automated Lab-on-a-Chip Analysis of DNA Fragments

The design of a fully automated system for the analysis of DNA fragments in 96-well plates is described. Microfluidic technology is used to integrate sample loading, electrophoretic analysis, and fragment detection onto a miniature lab-on-a-chip device. The microfluidic chip operates in an instrument platform that automates sample access, data collection, and data reporting. Each microfluidic chip provides sizing and concentration values for more than 1000 DNA samples.     

Cellular Telephone-Aided Real-Time Reporting System for Emergency Laboratory Test Results

The authors established a system for real-time reporting of clinical laboratory test results utilizing the mailing function of a cellular telephone service. The reporting system is composed of systems for sample transportation and automatic analysis, verification of test results, and reporting of test results. The introduction of the system has completely eliminated time delays at all stages of the laboratory testing process from start of testing to reporting of the test result. This has enabled doctors to receive test results in a timely manner wherever they are and to respond promptly to the patient condition.     

Development of a μ-concentrator Using Dielectrophoretic Forces

We present the simulation, development and experimental validation of a μ-concentrator based on dielectrophoresis, DEP.In a first step dielectrophoretic force fields of various electrodes are computed and compared. The simulation results for various electrode dimensions may serve as a general design rule for DEP devices. Favorable electrode designs were realized in gold on glass substrates. The performance of the DEP chips is validated by concentration of E.-Coli bacteria, a separation efficiency of 99.93% was achieved. Furthermore, we outline how the combination of forced convection and DEP allows for bacteria separation at increased flow rates.     

Converting a Protease Assay to a Caliper® Format LabChip System

The push for higher throughput screening coupled with the desire to use smaller volumes of material has sparked the development of new technologies. Caliper Technologies, Corp. (Mountain View, CA) has designed a microfluidics chip with unique properties yet to be fully exploited. The translation from a traditional plate-based assay to a microfluidic chip format has provided insights into assay development, screening data requirements, and the technology itself. Running a screen with this new technology presented challenges in throughput, signal acquisition from slow-conversion enzymes, the provision for a negative control, the translation of a time series into a single data point per compound, reagent adhesion in the channels, and fluid property mismatches. Overcoming these obstacles has resulted in a simple, robust system with significant savings in reagent use. Measures to improve throughput and generalize the system will be discussed.     

Automated Purification of Transfection-Grade Plasmid DNA Using Wizard MagneSil Tfx System

We describe a reagent system and robotic protocol for the isolation of highly purified plasmid DNA from cultured cells. The method is based on the Wizard® MagneSil™ Plasmid Purification System, which purifies sequencing-grade plasmid DNA. Two modifications to the sequencing-grade system were made to create the Wizard MagneSil Tfx™ System. This system provides lower endotoxin and other contaminant levels, giving higher quality plasmid for transfection applications. The Wizard MagneSil Tfx™ System uses MagneSil™ Paramagnetic Particles (PMPs) to clear lysate and bind plasmid, eliminating the need for filtration devices. The endotoxin removal step uses MagneSil™ (PMPs) and a guanidine/isopropanol wash to remove RNA and protein. One 96-well plate may be processed in 45 minutes on the Beckman Biomek® FX robotic workstation. We provide data showing DNA yield, contaminant levels, and transfection efficiency for 5 commonly used cell lines. Comparisons with other systems are also shown.     

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.     

The First Automated High Content Screening System

With biological discovery and technology advancing in parallel, AstraZeneca (AZ) announced that it would soon take delivery of what is thought to be the first fully automated high-throughput high-content screening system. This custom designed assay platform from RTS Life Science International (RTS) automates the IN Cell Analyzer 3000 sub cellular analysis system from Amersham Biosciences. RTS has integrated its advanced scheduling system with the imaging tool to enable AZ to evaluate the effect of drug compounds on cellular processes.     

Automated Purification of Plasmid DNA Using Paramagnetic Particles

We describe a reagent system and robotic method for purifying plasmid DNA for restriction digestion, PCR, and fluorescent sequencing applications. The method uses two types of Wizard® MagneSilTM paramagnetic particles. Following lysis and neutralization procedures, the first particle type binds and removes cell debris; the second type is then used to bind plasmid DNA from the cleared lysate. The particles are then washed to eliminate unwanted contaminants. Purified plasmid DNA is then eluted from the particles with nuclease free water. When using a cell mass of approximately 4 O.D.₆₀₀, the yield is 10–12μg of DNA when using high copy number plasmid. When used in BigDye® terminator sequencing, these DNA templates typically yield read lengths greater than 700 bases and Phred 20 scores of 600 to 750 bases. This purification method has been adapted for use on several robotic platforms in a 96-well format.     

A Study of a High-Throughput Plasmid DNA Purification System

An automated process that incorporates Millipore's Plasmid Miniprep₉₆ Montáge™ Kit with the Apogent Discoveries PlateMate Plus® and Tango™ automated high-throughput dispensing systems has been developed for purifying plasmid DNA. To test the efficacy of this process, parameters such as the reproducibility and consistency of the purified DNA quantity and quality as well as the purification speed were analyzed. The purification time for two plates of the Plasmid Miniprep₉₆ Kit (192 samples) was approximately 60 minutes using a PlateMate Plus equipped with 96 disposable tips and the Tango system equipped with 96 RB (resin bead) syringes. High uniformity and consistency in DNA yields (determined by spectrophotometric analysis) and quality (determined by gel electrophoresis analysis) among the different wells were observed. The purified plasmid DNA samples sequenced at an exceptional level with an average PHRED Q > 20 of 819 ± 25.*Millipore and Montage are the trademarks of Millipore Corporation     

Applicatons of Ink-Jet Printing Technology to BioMEMS and Microfluidic Systems

Applications of microfluidics and MEMS (micro-electromechanical systems) technology are emerging in many areas of biological and life sciences. Non-contact microdispensing systems for accurate, high-throughput deposition of bioactive fluids can be an enabling technology for these applications. In addition to bioactive fluid dispensing, ink-jet based microdispensing allows integration of features (electronic, photonic, sensing, structural, etc.) that are not possible, or very difficult, with traditional photolithographic-based MEMS fabrication methods. Our single fluid and multifluid (MatrixJet™) piezoelectric microdispensers have been used for spot synthesis of peptides, production of microspheres to deliver drugs/biological materials, microprinting of biodegradable polymers for cell proliferation in tissue engineering applications, and spot deposition for DNA, diagnostic immunoassay, antibody and protein arrays. We have created optical elements, sensors, and electrical interconnects by microdeposition of polymers and metal alloys. We have also demonstrated the integration of a reversed phase microcolumn within a piezoelectric dispenser for use in the fractionation of peptides for mass spectrometer analysis.     

Global observability of a class of nonlinear discrete time systems

It is shown that discrete time systems whose dynamics are governed by piecewise monotone maps of the interval are globally observable under a general class of observations. The results rest on the fact that the dynamics are ergodic.     

A Fully Automated High-throughput Nucleic Acid Purification System Using Silica Coated Magnetic Bead Technology

The design of a fully automated high-throughput system for the purification of sequencing templates is described. Hardware, software, and chemistries have been optimized to suit the needs of high-throughput laboratories involved in genome sequencing projects. Using this system, up to 5760 samples (60 x 96-well plates) can be purified in less than 16 hours during a single unattended run. The system can also be configured to perform sequencing reaction setup for all 60 plates following template purification, extending total run times to < 24 hours. Final sequencing reactions are prepared in 384- well microplates.