Colin J. H. Brenan

Nanoliter high throughput quantitative PCR

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

Encapsulated polypyrrole actuators

Abstract Conducting polymer-based actuators undergo volumetric changes as they are oxidized or reduced, from which mechanical work can be obtained. Polypyrrole [Q. Pei, O. Inganas, Advanced Materials 4 (1992) 277; Q. Pei, O. Inganas, Journal of Physical Chemistry 96 (1992) 10508; E. Smela, O. Inganas, Q. Pei, I. Lundstrom, Advanced Materials 5 (1993) 630; E. Smela, O. Inganas, I. Lundstrom, Science 268 (1995) 1735; J.D. Madden, S.R. Lafontaine, I.W. Hunter, Proceedings – Micro Machine and Human Science 95, Nagoya, Japan, October 1995] and polyaniline-based actuators have attracted recent interest because of the high forces per cross-sectional area (stress) and relatively large strains generated at low activation voltages; however, with the notable exception of some bilayers, the operation of these actuators has largely been constrained to bulk liquid environments. Operation out of solution is clearly desirable for many potential applications. Linear actuators that contract in length like muscle fibers fully exploit the high forces produced by conducting polymers. Results presented here demonstrate the operation in air of polypyrrole linear actuators. These actuators are capable of generating stresses exceeding those of mammalian skeletal muscle.

Massively parallel microfluidics platform for storage and ultra-high-throughput screening

We have developed a novel microarray technology for performing very large numbers of biochemical, chemical and cell-based nanoliter volume synthesis, storage and screening operations in a massively parallel manner. The Living Chip is an array of precisely machined through-holes retaining nanoliters of fluid by capillary action. Sample loading, washing and recovery are operations that can be performed manually or with simple automation. Mixing between co- registered through-holes is achieved by stacking two or more precision aligned arrays and optical assay read-outs are in parallel with a CCD imaging system. An automated picker system transfers hits into lower density microtiter plates for further analysis. We will present result demonstrating massively parallel implementation of both homogeneous and inhomogeneous fluidic and cell-based assay systems and applications of the chip for drug compound library storage and management.

Nanoliter High-Throughput PCR for DNA and RNA Profiling

The increasing emphasis in life science research on utilization of genetic and genomic information underlies the need for high-throughput technologies capable of analyzing the expression of multiple genes or the presence of informative single nucleotide polymorphisms (SNPs) in large-scale, population-based applications. Human disease research, disease diagnosis, personalized therapeutics, environmental monitoring, blood testing, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency, are a few areas where such systems are in demand. This has stimulated the need for PCR technologies that preserves the intrinsic analytical benefits of PCR yet enables higher throughputs without increasing the time to answer, labor and reagent expenses and workflow complexity. An example of such a system based on a high-density array of nanoliter PCR assays is described here. Functionally equivalent to a microtiter plate, the nanoplate system makes possible up to 3,072 simultaneous end-point or real-time PCR measurements in a device, the size of a standard microscope slide. Methods for SNP genotyping with end-point TaqMan PCR assays and quantitative measurement of gene expression with SYBR Green I real-time PCR are outlined and illustrative data showing system performance is provided.

Raman spectral estimation via fast orthogonal search

A Fourier transform (FT) spectrometer measures the autocorrelation (interferogram) of radiation emitted from a source and estimates the optical power spectral density through application of the discrete Fourier transform (DFT) to the recorded interferogram. Although a widely used method, FT spectrometry suffers because its frequency resolution is limited to the sampling rate divided by the number of time-series data points. A large number of points are therefore required to resolve an optical spectrum properly. In this paper, it is shown that a noise-resistant technique known as fast orthogonal search (FOS) can be used to achieve accurate optical spectrum estimation. Further, it is shown that frequency accuracy comparable to the DFT applied to the full interferogram can be obtained with FOS even if the original interferogram is contaminated with noise and then reduced by a factor of up to 10 by irregularly spaced sampling. The FOS application presented here is for the estimation of Raman spectra from interferograms acquired with an FT Raman spectrometer.

Design and Characterization of a Visible-Light Fourier Transform Raman Spectrometer

We demonstrate the feasibility of Fourier transform (FT) Raman spectroscopy with visible wavelength excitation through design, construction, and characterization of a visible-light FT-Raman spectrometer. Our motivation to explore this approach stemmed from the need for a versatile Raman spectrometer for use in a confocal scanning laser Raman microscope we built. We discuss the spectrometer design features which contribute to efficient and reliable microscope operation and evaluate the spectrometer performance on the basis of a series of measurements chosen because of their impact on confocal microscope function. The measurements include the acquisition of representative Raman spectra from both solids and liquids, a demonstration of the independence of resolving power from input aperture diameter, the measurement of the absolute spectrometer optical efficiency curve, and an evaluation of the short- and long-term spectrometer noise processes.

Confocal Image Properties of a Confocal Scanning Laser Visible Light FT-Raman Microscope

The confocal Raman microscope is an instrument designed for acquisition of high-contrast volumetric Raman spectral images of three-dimensional chemical structures. Little effort, however, has gone into the investigation of the spatial imaging properties of this class of confocal microscope. In this paper we present experimental results, obtained with a confocal scanning laser visible light FT-Raman microscope we built, that demonstrates the high depth resolution and enhanced Raman image contrast intrinsic to the confocal Raman microscope design. We explore these microscope properties through a combination of experimental measurement and theory based on a paraxial wave diffraction model.

BioMEMS applied to the development of cell-based bioassay systems

Biological applications of MEMS technology (bioMEMS) is of increasing interest in the development of miniature and portable instrumentation for cell-based microassays and sensor applications. A major bioMEMS challenge is the physical incorporation of living cells into sensors and diagnostic devices and creation of the environmental conditions conducive for organization of differentiated cells into tissue-like structures. Our work towards these goals is illustrated by a tissue-based bioassay system we are developing based on a miniature cross-flow bioreactor constructed from of an array of cell-filled microchannels integrated into an environmentally-controlled polymer microfluidics manifold. We describe our microchannel array and manifold manufacturing methods and report on the in vitro culture of cell populations in the bioreactor.

Tissue modification with feedback: the smart scalpel

While feedback control is widespread throughout many engineering fields, there are almost no examples of surgical instruments that utilize a real-time detection and intervention strategy. This concept of closed loop feedback can be applied to the development of autonomous or semi- autonomous minimally invasive robotic surgical systems for efficient excision or modification of diseased tissue. Spatially localized regions of the tissue are first probed to distinguish pathological from healthy tissue based on differences in histochemical and morphological properties. Energy is directed to only the diseased tissue, minimizing collateral damage by leaving the adjacent healthy tissue intact. Continuous monitoring determines treatment effectiveness and, if needed, enables real-time treatment modifications to produce optimal therapeutic outcomes. The present embodiment of this general concept is a microsurgical instrument we call the Smart Scalpel, designed to treat skin angiodysplasias such as port wine stains. Other potential Smart Scalpel applications include psoriasis treatment and early skin cancer detection and intervention.

Volumetric Raman spectral imaging with a confocal Raman microscope: image modalities and applications

The Fourier transform Confocal Raman Microscope (FT-CRM) enables non-invasive 3D Raman spectroscopic analysis and visualization of chemically heterogeneous preparations. The instrument combines a confocal optical microscope with a visible light Fourier transform Raman spectrometer to acquire and analyze the Raman spectrum of light scattered from a voxel in the sample defined by the confocal optics. Scanning the sample relative to the confocal voxel and recording the Raman spectrum at each scan position generates a multi- dimensional data set encoding the spatially-varying compositional properties of the sample. We report here on the spatial and spectral FT-CRM image properties that includes recent work on correlation-based Raman spectroscopic imaging and application of parametric spectral estimators for robust Raman spectrum estimation.