Development of a push-pull microdialysis sampling technique for the quantitative determination of proteins

Abstract

An on-line push-pull sampling technique has been developed for continuous analysis of proteins of molecularweight from 5.7 to 67 kDa. The characteristics of the system include gradient elution with a total cycle time of 21 min, membrane stabihb,, unattended automatic operation, and adjustment of the sampling mode and extraction fraction (the ratio of the concentration of analyte in the clialysate to that in the sample) by varying the effective dialysis length. The push and pull flow rates were adjusted in a mannerwhich enabled three different modes of operation. When push-pull microclialysis was compared with conventional microclialysis sampling, significantly higher extraction fractions were obtained for all five model proteins studied. The technique has been applied to the quantification of proteins in cell samples. On-line fractionation enabled complementary MS identification of the proteins present.

Microdialysis of proteins: Performance of the CMA/20 probe

“…They used much slower buffer flow rates because of the longer diffusion times for macromolecules versus small molecules. The same group later used "push-pull microdialysis" (Kjellstrom et al, 2000) in which buffer is simultaneously pumped onto the probe but also pulled by vacuum out of the outlet of the probe. This causes water to traverse the probe walls, thus adding ultrafiltration, i.e.…”

Effect of pumping methods on transmembrane pressure, fluid balance and relative recovery in microdialysis

“…Employing ultrafiltration in the microdialysis procedure, e.g. to create a transmembrane pressure (TMP) and hence a net fluid flow through the membrane, either using a pull pumping system [13,19] or creating a hydrostatic pressure difference [20] can increase the protein recovery. However, this will cause a nonzero permeate flux and the dialysate will gain fluid, which is not desirable for most in vivo applications.…”

Utilizing transmembrane convection to enhance solute sampling and delivery by microdialysis: Theory and in vitro validation

“…Kjellström et al [21] proposed exploiting this propensity by controlling the perfusate and dialysate flow rates with separate “push” and “pull” pumps, respectively. With the same 100-kDa membrane as in our measurements, these authors obtained higher extraction fractions sampling five model proteins with inward ultrafiltration than by conventional diffusional microdialysis.…”

“…Quantitative protocols should probably include determination of mass and fluid transport characteristics for each probe when encountering substantial transmembrane convection. For probes with high molecular weight cutoff membranes and attendant high hydraulic conductivities, ultrafiltration is difficult to avoid without the use of separate pumps for the afferent and effluent streams [7, 21]. The current analysis provides a basis for assessing the effect of ultrafiltration on extraction fractions, whether unintended or deliberately imposed for the purpose of enhancing sampling or delivery capabilities.…”