| 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 |