Microassays for Enzyme Activity

DESCRIPTION (provided by applicant): Longitudinal metabolic and physiological studies in small animal models of disease will greatly benefit from new methods of easy, rapid enzyme assay in very small sample volumes (sub-microliter). Phase 1 demonstrated the feasibility of adapting silicon microsampling and assay devices, originally developed for blood glucose monitoring, to enzyme activity measurement. A novel technique, "Diffusive Enzyme Activity Measurement" was developed. An electrochemical redox-mediated serum cholinesterase assay was the example, requiring no on-chip biologicals (providing a potentially long shelf-life). Silicon microchips with novel diffusional microfluidics were designed and fabricated. These disposable microchips have integrated silicon microneedles capable of penetrating the skin and taking a blood sample painlessly and reliably. In Phase II, this technique will be generalized to other enzymes. A computer simulation model will be used, experimental work will develop assays for the families of cholinesterases and lactate oxidizing enzymes, and a prototype readout instrument will be built. The enzymes chosen for Phase ll kinetic constants are covering many orders of magnitude, to probe the range of applicability of this new analytical method. Potential commercial applications include enzyme activity measurements on sub-microliter liquid samples, such as blood drawn from experimental animals (or humans), tissue extract specimens, purified enzymes, and drug discovery research and development.