Spaceflight Therapeutic Drug Monitoring

		Astronauts have been taking medications during spaceflight since the first Mercury mission. Common ailments requiring treatment include space motion sickness, sleeplessness, congestion, and pain.  The rigors of spaceflight and microgravity induce a number of physiologic changes that affect drug metabolism and, therefore, the efficacy of medications in some cases. In collaboration with the Pharmacotherapeutics Laboratory at Johnson Space Center we are developing a portable device based on a microfluidic platform for therapeutic drug monitoring that can be taken on missions to the space station and beyond.
This device will analyze biofluids (blood, urine and saliva) taken from astronauts during spaceflight allowing for proper dosing and to give insight into the effects of microgravity on drug metabolism. Solid phase extraction using commercially available sorbents is being developed on-chip for sample cleanup and preconcentration and will be integrated with an electrophoretic separation and appropriate detection method. The figure shows an envisioned integrated device using fluorescence detection.

MEKC drugs

Micellar electrokinetic chromatography (MEKC) is a well established separation technique used to separate neutral molecules in an electric field using a pseudostationary phase such as SDS. We are applying MEKC to the separation of drugs and metabolites on a microchip. This work is being done in conjunction with the Spaceflight Therapeutic Drug Monitoring project with the goal of integrating the MEKC separation with SPE of small molecules from biofluids.

The figure shows electropherograms of an NBD labeled pseudoephedrine (PE) elution fraction after microchip extraction (5 sec pressure injection. 40 mM borate pH 9.5, 60 mM SDS 30 kV separation voltage). a) NBD-PE reaction followed by extraction b) PE spiked into urine followed by NBD labeling and extraction.

Microchip Isoelectric Focusing (mIEF)

Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS–glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids. Guillo, C.; Karlinsey, J.; and Landers, J. P.  "On-chip pump for pressure mobilization of the focused zones following microchip isoelectric focusing" Lab Chip, 2007, 7, 112 - 118.

Protein Separation

The overall goal of this project is to develop microfluidic devices for rapid protein detection, purity determination, and charge heterogeneity.  Microdevices have proven useful in analytical and clinical chemistry providing much faster analyses, with sensitivity similar to conventional methods.  Application of these devices to electrophoretic separations has been well studied, but has mostly focused on DNA separations.  Protein separations on microchips have been more limited.  First, UV absorbance detection is normally used with proteins, but is not easily coupled with microdevices.  The development of microchip MS interfaces provides a new detection method for proteins, which has renewed interest in protein separations on microchips, although significant challenges remain.  
  
One of the biggest challenges to the use of capillary electrophoresis for protein separation is the propensity of the charged proteins to adsorb onto the negatively charged capillary walls.  This sticking may be eliminated via SDS-protein separations, however, when coated with negatively-charged SDS, all proteins have the same charge to mass ratio, giving them all the same electrophoretic mobility.  Sieving gels are therefore required to perform separations in the presence of SDS. Unfortunately, with this method information related to the charge or mass heterogeneity of the proteins is lost.  A major subgoal of this project is to evaluate the use of both semi-permanent and dynamic type coatings to determine which provide the adsorption prevention necessary to enable a free zone (CZE) separation, thus retaining information on the protein charge and mass heterogeneity.  The coatings will also be evaluated for their compatibility with MS detection, for eventual incorporation into microdevice CE-MS systems.

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