Biological, Bioanalytical and Clinical Chemistry
Almost every aspect of the biochemical, biomedical and clinical sciences involves separation of species in complex matrices. Electrophoresis has been a benchmark technique for separation and characterization of biologically-active species. Instead of using conventional slab gel electrophoretic approaches, electrophoresis in micron-scale capillaries using applied fields as high as 30,000 volts, results in unprecedented resolution with unique selectivities and short analysis times. As a result of the microscalar nature of the capillary, only microliters of reagent are consumed by analysis with only a few nanoliters of samples injected for analysis. These characteristics, as well as the ability for on-line detection with laser-induced fluorescence sensitivities in the attomole (10-18 moles) range, made capillary electrophoresis (CE) appealing as a replacement for electrophoretic gels in the biomedical and clinical arenas. We have demonstrated the potential impact of CE on clinical diagnostics through the development of new CE-based assays for measuring kidney function, detecting multiple sclerosis and viral infections, screening for lymphoma, as well as for diagnosing drug abuse and alcoholism.
Figure 1. A – Schematic of capillary electrophoresis instrumentation. B – CE separation of human serum for the diagnosis of alcoholism.
While the diagnostic impact of standard CE technology is clear, an alternative platform for electrophoresis in microscalar structures has evolved in the form of microchip electrophoresis. The use of microfabricated glass devices containing etched capillary-like channels provides an electrophoretic platform akin to CE but with more flexibility. “Microchip electrophoresis” allows for analysis times to be decreased by an order of magnitude over times achievable by CE (as fast as 10-200 seconds) and two orders of magnitude faster than gel electrophoresis. This provides obvious value to clinical diagnostic laboratories in terms of more rapid turn around time and capability for high throughput screening. We have demonstrated this with the detection T-cell and B-cell lymphoma in a separation remarkably faster than with conventional means.
Figure 2. Demonstration of microchip electrophoresis as a technique for rapid diagnosis of T-Cell lymphoma. Sample T1 shows a negative sample, which is represented by the smear after 100 seconds of separation. T4 is a positive sample with a sharp peak.
With a program focused on the application of miniaturized electrophoretic technology to the clinical and forensic sciences, our current efforts involve broadening the scope of applications for microchip technology. This involves addressing issues associated with integrating functions other than ‘separation’ onto microchips. For example, we are focused on defining approaches for integrating DNA sample preparation into microchips. PCR amplification of DNA carried out using infrared-mediated thermocycling for rapid on-chip amplification and rapid DNA extraction using microchamber-bound solid phases are two examples of our integration efforts.
Figure 3. A – Demonstration of IR-mediated PCR in a polyimide microchip. Total time necessary for thermocycling was 220 seconds. B – Elution profile of DNA in mSPE chip.
The successful integration of DNA extraction and amplification will lead to the development of an “Integrated Diagnostic” or ID-chip, which we ultimately hope will improve laboratory medicine. Efforts are also underway to 1) define better detection systems using acouto-optic technology, 2) develop multichannel devices for high-throughput analysis using this optical technology, 3) explore proteomic aspects of disease using multi-dimensional microchips for protein separations, and 4) apply the relevant methods to forensic applications.
Solid Phase Extraction of DNA from Biological Samples in a Post-based High Surface Area Poly (methyl methacrylate) (PMMA) Microdevice.Reedy CR, Price CW, Begley M, Sniegowski J, Ferrance JP, Landers JP. Lab Chip. 11(9):1603-11 (Journal Cover) (2011).
Dual-Domain Microchip-Based Process for Volume Reduction Solid Phase Extraction of Nucleic Acids from Dilute, Large Volume Biological Samples. Reedy CR, Hagan KA, Strachan BC, Higginson JJ, Bienvenue JM, Greenspoon SA, Ferrance JP, Landers JP. Anal Chem. 82:5669-78 (2010).
A simple method for the evaluation of microfluidic architecture using flow quantitation via a multiplexed fluidic resistance measurement. Leslie DC, Melnikoff BA, Marchiarullo DJ, Cash DR, Ferrance JP, Landers JP. Lab Chip. 10:1960-6 (2010).
Acoustic differential extraction for forensic analysis of sexual assault evidence.Norris JV, Evander M, Horsman-Hall KM, Ferrance JP, Nilsson J, Laurell T, Landers JP. Anal Chem. 81(15):6089-95 (2009).
Frequency specific Flow Control in Microfluidic Circuits with Passive Elastomer Features. Leslie DC, Easley CJ, Seker E, Karlinsey J, Utz M, Begley MR, and Landers JP. Nature Physics. 5(1); 231-235 (2009). [Editor’s Choice Science, 2009; News and Views in Nature Physics. 5:178, 2009; Research Highlight in Lab-on-a-chip. 9, 861–862, 2009].
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