Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor.
This paper describes a microquantitative method for glucose determination in situ of living cells in real-time. In this novel technique adherent cells are cultured onto microcarrier beads and packed into a renewable microcolumn within a microsequential injection lab-on-valve system (microSI-LOV). Glucose sensing is performed through the use of a two-step, NAD-linked enzymatic process. The course of the assay is monitored in real-time, by absorbance of NADH at 340 nm. The microsequential assay based on plug/nozzle design has a linear dynamic range for glucose of 0.1 to 5.6 mM. The design of the (microSI-LOV) system allows the assay to be carried out using only 40 microL of the enzyme reagent and 3 microL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopore beads. Rapid cellular glucose consumption, in this technique, is facilitated by a high cell density, which allows a large number of cells (10(4)-10(5)) to be retained in a very small volume (3 microL). In turn, this cell density results in the rapid depletion of glucose from the cell medium over short time periods (< 2 min). In conjunction with the assay development, the plug/nozzle design and its ramifications on mixing in general are presented and discussed.
Schulz
Craig M
CM
University of Washington, Department of Chemistry, Seattle, WA 98195, USA. cschulz@uwashington.edu
Ruzicka
Jaromir
J
D000818:Animals; D019149:Bioreactors; D002477:Cells / Q000378:metabolism*; D004798:Enzymes; D017022:Flow Injection Analysis; D005947:Glucose / Q000032:analysis*; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D009243:NAD / Q000378:metabolism*
D004798:Enzymes; D009243:NAD; D005947:Glucose