Development of a CMOS Array for Toxicity Monitoring |
Collaborators |
Dr. Dave Mathine: Optical Sciences | |
Dr. Ray Runyan: Cell Biology and Anatomy | |
Dr. Bob Arnold: Chemical and Environmental Engineering | |
Dr. Dan Liebler: Pharmacy and Toxicology | |
Matt Scholz: Cell Biology and Anatomy | |
Amruta Kulkarni: Electrical and Computer Engineering | |
Cherry Yu: Electrical and Computer Engineering |
Overview of Talk |
Part I: Introduction and Background | |
Part II: Current Project | |
Part III: Progress to Date | |
Part I: Introduction and Background |
Goal: Online Toxicity Testing |
Role of DNA |
Nucleic Acid Review |
Project Logic |
If : | |
some genes regulate the abundance of proteins they express in response to toxic exposure | |
then: | |
we should be able to assay for the presence of toxins in a sample by studying the expression of these genes. One way of doing this is to monitor mRNA levels in the cell. | |
Does TCE affect gene expression in the heart? |
Pregnant rats were exposed to 110 ppm TCE in drinking water | |
Rat embryos were collected at day 11 when heart valves were forming | |
mRNA was extracted from treated and control embryos and converted to cDNA |
Does TCE affect gene expression in the heart? |
cDNAs were compared to look for up- and down-regulated gene expression | |
80 differentially-expressed clones were identified and sequenced | |
Several genes identified at 110 ppm TCE were affected at 100 ppb | |
Improvements in Methodology |
Toxicants producing developmental and cellular defects alter gene expression. | ||
Before the development of cDNA microarrays, genetic expression could only be monitored one gene at a time | ||
cDNA microarrays on glass substrates permit monitoring of thousands of genes in one experiment |
Slide 12 |
Microarray Measurement of Differential Gene Expression |
Power of Approach |
Obtain information about differential gene expression across diverse set of arrayed molecules in a single experiment | |
Drawbacks of Conventional Technology |
Target DNA binds non-specifically and yields background noise | |
Labeling procedure is inherently inefficient and 90% of target is lost in process | |
Uniform hybridization temperature used to decouple non-homologous targets from probes fails to account for variations in target-to-probe binding energies | |
Insensitive to subtle changes in gene expression (< 1.5-fold) | |
Requires large, expensive equipment and is arduous | |
Part II: Current Project |
Slide 17 |
CMOS Microarray |
A set of DNA molecules (“probes”) arrayed on the electrodes of a Complementary Metal-Oxide Semiconductor (CMOS) chip | |
Electrical gradients created on the chip surface do the work of printing and hybridizing | |
Inexpensive technology that will replace traditional DNA microarrays |
State of the Art |
Features of the Prototype Chip (a-version) |
AutoCAD Illustration of New Prototype Chip (b-version) |
Procedural Overview |
Modify DNA probes with linker for attachment to gold electrodes | |
“Print” probes onto electrodes | |
Hybridize mRNA target molecules to probe sites | |
Detect binding between targets and their homologous probes |
Microarray on a Chip |
DNA Microarray |
DNA Microarray |
DNA Microarray |
Advantages |
First proposed microarray capable of direct detection of mRNA (no reverse transcription necessary) | |
Quantitative and promises extreme sensitivity | |
CMOS platform facilitates automation and upgrading | |
Relatively inexpensive |
Potential Applications |
Toxicological studies | |
Researching disease pathways | |
Drug development | |
Proteomics | |
Health assessment | |
Agricultural research |
Slide 29 |
Experimental Set-up |
Progress to Date |
Research Ahead |
Target transport: electrically address complex mixtures of mRNA targets to each probe site for selective binding | |
Hybridization: Improve binding stringency electrically | |
Detection: Determine limits of sensitivity | |
Optimization: refine above steps in an ongoing fashion |
Thank You |