David L. Cunningham

Automated fluid mixing in glass capillaries

A fast method and compact device for mixing sub-microliter fluid samples contained in glass capillaries is presented. The fluid is rapidly moved back and forth by air volume displacement driven by a piezo-ceramic actuator. Rapid mixing of different fluids is achieved via diffusion between the main fluid volume in the capillary and the thin fluid film it deposits on the capillary wall through its motion. Bubbles in the fluid are processed out of the capillary by use of an asymmetric velocity profile. A simple analysis model is used to optimize the design of the device and to elucidate the mechanisms involved in mixing. The mixing time is found to be inversely proportional to the fraction of the fluid volume that is left in the film layer for each cycle, which is determined by the wetting properties and the viscosity. The mixing time is therefore controlled by the dead-air volume of the system, the fluid volume, the capillary size, and the displacement limits of the piezo-ceramic actuator, in addition to the intrinsic properties of the fluid being mixed. The device described can mix two 1 μl water solutions in under 3 s. The possible shear breakage of DNA in solution is investigated, and λ-DNA is found to remain intact at aggressive mixing parameters. No evidence of aerosol contamination in polymerase chain reaction reactions was found to date.

Sample preparation in glass capillaries for high-throughput biochemical analyses

A capillary-based fluid handling system, the ACAPELLA-5K (ASK), has been developed to prepare precise mixtures of sample and reagents in glass capillaries at a rate of 5000 preparations in 8 hours. Following a user-defined protocol the system architecture enables sample aspiration, reagent dispensing, mixing, and thermal cycling, with intermediate imaging steps for in-process monitoring of critical fluid delivery steps. A serial pipeline process is used to provide flexibility, reproducibility, and reliability for the reactions prepared. A typical reaction comprises a 0.5 /spl mu/L sample aspirated from a microplate well followed by 1 to 8 reagents dispensed in 40-100 pL droplet volumes using piezoelectric reagent dispensers. Reactions are then mixed to prepare a 2.0 /spl mu/L final reaction volume. Reaction volumes from 0.5-2 /spl mu/L have been demonstrated, representing performance comparable to the state-of-the-art in a majority of core sequencing facilities. A5K has been extensively tested in the preparation of deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) and DNA sequencing reactions. Other applications of the A5K platform of technologies include quantitation of minimal residual disease, protein crystallography, and potential for application in drug discovery, forensics, and DNA analysis of environmental samples.

A real-time PCR analyzer compatible with high-throughput automated processing of 2-/spl mu/L reactions in glass capillaries

This paper describes the development of a real-time polymerase chain reaction testbed for the analysis of up to 48 1- to 2-/spl mu/L reactions in glass capillaries. It is suitable for high-throughput experimentation when used as a downstream module for ACAPELLA-5K, an automated, high-precision, fluid-handling system developed in our laboratory over the past five years. The testbed features a laser-induced fluorescence scanner with high-sensitivity photomultiplier tubes for the detection of three spectral wavelengths. The scanner has a detection threshold of 1-nM fluorescein in a 1-/spl mu/L detection volume. Peltier-effect thermoelectric elements are used to control thermal cycles with a slew rate of 3/spl deg/C/s, and a set point tracking accuracy of /spl plusmn/0.5/spl deg/C. Analyzer design and performance are presented. Experimental results show a successful detection of 3 copies//spl mu/L of the albumin gene in serial dilutions of human genomic deoxyribonucleic-acid. Detection levels down to 4 copies//spl mu/L of the t(14;18) translocation associated with the cancer follicular lymphoma, was demonstrated in RL-7 cells. Note to Practitioners-Real-time polymerase chain reaction is a thermally driven in-vitro replication of specific segments of DNA, with simultaneous monitoring or reaction product by means of fluorescence detection. This process is important in biochemical analysis for the detection and/or estimation of gene concentration in DNA samples. By increasing the concentration of an initially undetectable gene and monitoring the kinetic history of the reaction, an estimate of the initial concentration can be computed. Although there exists a plethora of commercial real-time PCR instruments, none of these instruments are suitable for low-volume (single-digit microliters) reagents and high-throughput processing while preserving a high degree of fidelity to obtain reliable, repeatable results, and versatility (support for different fluorogenic probes, different operating modes, etc.). The A5K-automated fluid handling and sample preparation system addresses the high-throughput reaction preparation in small-volume glass capillaries. The testbed presented here is a downstream processing module for A5K. To address the challenges posed by the use of low-volume, elongated reaction vessels, we employ a scanning optical system with a novel inline illumination method to address up to 48 capillaries. We empirically demonstrate that the systems performance is comparable to that of existing commercial real-time PCR platforms, and verify its repeatability and versatility. Future work will address the development of a more compact instrument that can be directly integrated with A5K.

ACAPELLA-5K, a high-throughput automated genome and chemical analysis system

A capillary-based fluid handling system bas been developed to process 5000 samples in 8 hours. The system takes deoxyribonucleic acid (DNA) or other chemical samples and automatically processes them as specified in a user-defined protocol with aspiration, dispensing, mixing, thermal cycling, and imaging steps. A serial pipeline process is used to provide flexibility, reproducibility, and reliability for the samples prepared. This laboratory automation system dispenses 40-100 pL droplet volumes using piezoelectric reagent dispensers and prepares 1 or 2-/spl mu/L final reaction volumes, a 3 to 5-fold reduction in reagent usage over current (2003) state-of-the-art manual and automated instrumentation. Extensive testing of the system has been performed with the University of Washington Genome Center. Applications for ACAPELLA-5K include DNA sequencing, diagnostics, minimal residual disease quantification, drug discovery, environmental testing, forensics, protein crystallography, Polymerase Chain Reaction (PCR) and so on.

ACAPELLA-1K: a biomechatronic fluid handling system for genome analysis that processes 1000 samples in 8 hours

An automated biomechatronic submicroliter fluid handling system for processing deoxyribonucleic acid (DNA) has been developed in the Genomation Laboratory, Department of Electrical Engineering, University of Washington, Seattle. This first generation system, ACAPELLA-1K, can process 1000 samples in 8 h in preparation for DNA sequencing using sample volumes ten times smaller than current state-of-the art manual and automated instrumentation. The system is based upon a proof-of-concept system that was developed by the Genomation Laboratory. The ACAPELLA-1K is the first integration of modules for fluid aspiration, dispensing, mixing, transport, and thermal processing that have been designed and developed with corporate partners Orca Photonic Systems, Inc., Redmond, WA, and Engineering Arts, Mercer Island, WA. These modules, comprising piezoceramic actuators, pneumatic pumps, linear mechanisms, thermal controllers, optical sensors, electronics, computer control, and software, are described in detail. Processing statistics are presented and successful experimental results are presented.

Mobile SO2 and NO2 DIAL Lidar system for enforcement use

A self-contained mobile differential absorption lidar (DIAL) system intended for measuring SO2 and NO2 concentrations from stationary combustion sources has been completed for enforcement use. The system uses tunable Ti:sapphire laser technology, with nonlinear conversion to the blue and UV absorption wavelengths. Separate tunable laser oscillators at slightly offset wavelengths are pumped on alternate pulses of a 20 Hz doubled Nd:YAG pump laser; the outputs are amplified in a common amplifier, doubled or tripled, and transmitted toward a target region via a two-mirror beam director. Scattered atmospheric returns are collected in a 0.27-m-diameter telescope, detected with a filtered photomultiplier, and digitized and stored for analysis. Extensive software-based control and display windows are provided for operator interaction with the system. The DIAL system is built into a small motor coach. Gasoline- powered electrical generation, laser cooling, and air conditioning services are present. Separate computers are provided for simultaneous data collection and data analysis activities, with shared data base access. A laser printer supplies hardcopy output. The system includes the capability for automatic data collection at a series of scanner angles, and computer processing to present results in a variety of formats. Plumes from coal-fired and mixed-fuel-fired combusters have been examined for NO2 and SO2 content. Noise levels of a few parts per million are reached with averaging times of less than one minute.

Design of a compact high-sensitivity aerosol profiling lidar

The design of an unusually compact and rugged backscatter lidar system is described in detail. This lidar system, called AROL- 2, was designed for routine monitoring of the boundary layer, tropospheric aerosols, and clouds. In order to support the maximum possible range of observations with minimum investment of labor, the system was specifically designed to be mobile, rugged, and easy to set up and deploy. The lidar system is housed in a weatherproof enclosure, in order to allow operation at sites without sophisticated laboratory facilities. In order to provide maximum flexibiliity and sensitivity in a relatively small package, the lidar system is equipped with both analog and photon counting channels, with two polarization channels to allow for quantitative measurementsof depolarization behavior. Substantial effort has been expended to produce a lidar that is flexible, easy to use, and robust in the field environment.

Real-Time Fluorescence Detection of DNA in 5 µl Capillary Channels For Minimal Residual Disease Quantification Using the Acapella-5K High-Throughput Automated Analysis System

This paper describes a system for quantification of template DNA in a sample using fluorescence detection of DNA amplification during a real-time PCR reaction in a 5 µl glass capillary channel. The capillary system allows aliquots to be drawn from small samples efficiently and with no cross contamination when many samples are processed in an automated high-throughput sample preparation system, the Acapella-5K.

Design and application of an advanced laser spectrometer for industrial process monitoring

Summary form only given. Vacuum deposition of metallic and metal oxide films is economically important in manufacturing products such as semiconductors, superconducting materials, optical component coatings, dielectric insulating coatings, and high-performance composite materials. Because of the high value of improved process measurements, along with the availability of new lasers and optical components, we have undertaken the development of an advanced laser spectrometer for metal vapor monitoring in vacuum deposition.

Design and performance of a compact backscatter lidar for boundary-layer profiling

We have designed a very compact, low cost lidar system designed for profiling of aerosols in the planetary boundary layer. Our design emphasizes portability, reliability, ease-of-use, and the lowest possible cost. Our goal is an instrument which can provide easy and reliable characterization of the boundary layer for users in operational meteorology or air quality management. The lidar transmitter is a diode laser array. As compared with more conventional laser transmitters, the diode array offers overwhelming advantages in compactness, reliability, and cost. The emission wavelength of the AlGaAs diode array is well matched to the peak sensitivity of silicon avalanche photodiodes. The transmitter and receiver are polarization multiplexed through a common aperture. A relatively large (37 cm) optical aperture compensates for the low transmitter peak power. Since the main mission of this particular lidar is measurement in and around major urban areas where aerosol loadings are generally high, the modest sensitivity is not a severe limitation. The transmitter beam is eye- safe at all ranges. Control and data acquisition are managed by a portable computer. To demonstrate the capabilities of the design, we show simulated results under a wide variety of atmospheric conditions.