PNNL-13501 - Pacific Northwest National Laboratory

Integrated Microfabricated Devices for Rapid and Automated Identification
of Biological Agents
Study Control Number: PN98039/1285
Yuehe Lin, Keqi Tang, Richard D. Smith, Dean W. Matson
There is an enormous need for highly rapid and sensitive identification of microorganisms across a spectrum of research
areas, as well as in medicine and in clinical applications where there often is no available technological solutions to
provide the desired speed and reliability. The new technology developed from this project would find broad applications
in biological and medical research, as well as forensic and clinical applications, pathogen detection, and would also
advance DOE’s interest in biological research and bioremediation.
Project Description
This project involves the development and evaluation of
novel integrated microfabricated devices for the rapid and
automated identification of biological agents (pathogens)
and other medical and biological applications.
Microfabricated devices were developed for the rapid
fractionation and processing of small biological samples
(micro-biopsies). These integrated devices were
interfaced directly with mass spectrometers for analysis of
cellular components from complex matrices (cell lysates)
for identification of biological agents based upon the
detection of specific constituents (“biomarkers”). This
work will develop PNNL’s capability in the Bio-MEMS
area and provide a powerful tool for environmental health
research at the molecular level.
Introduction
During fiscal year 1998 and fiscal year 1999, we
successfully demonstrated on-line sample cleanup,
separation, and concentration of complex biological
samples (cell lysates) using microfabricated dual-dialysis
and isoelectric focusing devices. In fiscal year 2000, we
continued to modify and improve the design of these
components. The microfabricated dual-dialysis device,
combined with global ESI-MS/MS, was tested for use in
the characterization of microorganisms and biomarkers.
Specifically, the application of this method for detecting
the bacteriophage MS2 in the presence of a large excess
of E. coli was successfully demonstrated.
Results and Accomplishments
To expand the capability of microfabricated analytical
devices and allow for high-throughput parallel sample
processing, plastic microchips containing arrays of laser-
micromachined electrospray nozzles were developed.
These micronozzle arrays will be key components for
directly interfacing microfluidic sample separation and
cleanup devices with mass spectrometers.
The micronozzle arrays were fabricated from 1-mm thick
polycarbonate sheet material using PNNL’s Resonetics
Maestro UV excimer laser micromachining station. All
machining was performed using a Lumonics 848 excimer
laser operating at 248 nm. Through-holes were first
machined at a high demagnification factor (~35 times)
using a small circular mask in the laser beam. The laser
demagnification was then reduced to approximately five
times and a well was machined around the nozzle outlet.
Because of the inherent taper of the laser at low
demagnification factors, the nozzle tips produced by this
method had a conical cross section recessed into the end
of the chip. The openings of these micromachined
nozzles were flush with the end of the polycarbonate
chips, thereby reducing susceptibility to breakage or other
damage.
When the laser-machined micronozzle arrays were tested
for electrospray formation and stability, it was observed
that under a variety of conditions the solution exiting the
hole rapidly moistened the edge surface of the nozzle.
This wetting prevented the formation of a well-focused
electric field essential for the generation of a stable
electrospray. To inhibit the wetting process, the surfaces
of the polycarbonate microchips were treated with CF4 RF
plasma. After this plasma treatment, the surface property
of polycarbonate chip was changed from hydrophilic to
hydrophobic. The increased hydrophobicity of the treated
polycarbonate surface prevented the sample solution from
spreading over the edge surfaces of the nozzles and
helped to focus the electric field at the surface of the
liquid exiting the channel. Stable electrospray was
Analytical and Physical Chemistry 13