Lab-on-a-chip (LOC) systems allow complex laboratory assays to be carried out on a single chip using less time,
reagents, and manpower than traditional methods. There are many chips addressing PCR and other DNA assays, but few
that address blood cell analysis. Blood analysis, particularly of the cellular component, is highly important in both
medical and scientific fields. Traditionally blood samples require a vial of blood, then several processing steps to
separate and stain the various components, followed by the preparations for each specific assay to be performed. A LOC
system for blood cell analysis and sorting would be ideal. The microfluidic-based system we have developed requires a
mere drop of blood to be introduced onto the chip. Once on chip, the blood is mixed with both fluorescent and magnetic
labels. The lab-on-a-chip device then uses a syringe drive to push the cells through the chip, while a permanent magnet
is positioned to pull the magnetically labeled white blood cells to a separate channel. The white blood cells, labeled with
different color fluorescent quantum dots (Qdots) conjugated to antibodies against WBC subpopulations, are analyzed
and counted, while a sampling of red blood cells is also counted in a separate channel. This device will be capable of
processing whole blood samples on location in a matter of minutes and displaying the cell count and should eventually
find use in neonatology, AIDS and remote site applications.
Point-of-care devices represent the future for medical technologies. Current diagnostic tools are cumbersome,
expensive, complicated, and often at risk for contamination. There is a need for cost effective, portable, closed-system,
high-speed cell screening and cell isolating device. A microfabricated, exponentially-staging, BioMEMS microfluidic
cytometer/cell sorting device offers these advantages over current technologies. A two-stage branched architecture
allows the study of inter-particle spacing, flow relations, pressure measurements, and cell behavior in an environment
where fluorescence detection is used to identify and analyze certain cellular characteristics. This device was
microfabricated using the polymer PDMS to transmit light effectively, to be inexpensive and disposable, and to be easy
to manipulate. For initial prototyping, an inverted fluorescent Nikon microscope provided the necessary excitation to
view the particles and cells. For the portable device, avalanche photo diodes (APDs) and light emitting diodes (LEDs)
are being incorporated into the device for the detection and excitation respectively. For low light level applications,
sigma-delta modulation methods are being applied to reduce noise susceptibility and to detect the APD signal more
efficiently. In addition, a data acquisition system (DAQ) has been designed that can effectively track signals from a cell
sorter using a digital signal processing (DSP) board and a laptop computer. Currently elastomeric valves for diverting
flow have been incorporated into the microfluidic chip. Measurements are being made of the effects of the microfluidics
valve structures, or the simple opening and closing of selected channels to divert flow and cells down specific channels
depending on their measured properties.
To produce a large increase in total throughput, a multi-stage microfluidics system (US Patent pending) is being
developed for flow cytometry and closed system cell sorting. The multi-stage system provides for sorting and re-sorting
of cohorts of cells beginning with multiple cells per sorting unit in the initial stages of the microfluidic device and
achieving single cell sorting at subsequent stages. This design theoretically promises increases of 2- or 3-orders of
magnitude in total cell throughput needed for cytomics applications involving gene chip or proteomics analyses of sorted
cell subpopulations.
Briefly, silicon wafers and CAD software were used with SU-8 soft photolithography techniques and used as a mold
to create Y-shaped, multi-stage microfluidic PDMS chips. PDMS microfluidic chips were fabricated and tested using
fluorescent microspheres driven through the chip by a microprocessor-controlled syringe drive and excited on an
inverted Nikon fluorescence microscope. Inter-particle spacings were measured and used as experimental data for
queuing theory models of multi-stage system performance.
A miniaturized electronics system is being developed for a small portable instrument. A variety of LED light sources,
waveguides, and APD detectors are being tested to find optimal combinations for creating an LED-APD configuration at
the entry points of the Y-junctions for the multi-stage optical PDMS microfluidic chips. The LEDs, APDs, and PDMS
chips are being combined into an inexpensive, small portable, closed system sorter suitable for operation inside a
standard biohazard hood for both sterility and closed system cell sorting as an alternative to large, expensive, and
conventional droplet-based cell sorters.
Trans-cellular calcium currents play a central role in the establishment of polarity in differentiating cells. Typically these
currents are measured and studied experimentally using ion selective glass microelectrodes. We have recently developed
an in silico cell electrophysiology lab-on-a-chip device with the specific science objectives of measuring these
transcellular calcium currents in an advanced throughput format. The device consists of 16 pyramidal pores on a silicon
substrate with four Ag/AgCl electrodes leading into each pore on the four poles. An SU-8 layer is used as the structural
and insulating layer and a calcium ion selective membrane is used to impart ion selectivity to the Ag/AgCl electrodes. In
this paper we demonstrate the utility of the cell electrophysiology biochip in measuring these transcellular calcium
currents from single cells using the model biological system Ceratopteris richardii. We monitored these fern spores
during germination and pharmacologically inhibited biophysical calcium transport. These results demonstrate the utility
and versatility of the in silico cell electrophysiology biochip. While this version of the biochip was engineered to fulfill
the specific science objectives of measuring trans-cellular calcium currents from Ceratopteris fern spores, the chip can
easily be modified for a variety of biomedical and pharmacological applications. Future
Conference Committee Involvement (1)
Smart Biomedical and Physiological Sensor Technology V
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