2‐D t‐ITP/CZE determination of clinical urinary proteins using a microfluidic‐chip capillary electrophoresis device

Abstract

To replace the time-consuming sample pretreatment procedure, a microfluidic chip-CE device incorporating on-chip sample desalting/preconcentration with transient isotachophoresis (ITP)/capillary zone electrophoresis (CZE) was fabricated to perform sequential on-chip sample pretreatment and CE determination of four urinary proteins in clinical samples. On-chip sample desalting, clean-up and analyte preconcentration enable removing interfering sample matrix prior to transferring analytes to separation capillary for transient ITP/CZE determination. Four important urinary proteins transferrin, β2-microglobulin, human serum albumin (HSA) and immunoglobulin G (IgG), investigated were shown to achieve quantitation limits sufficiently high to meet medical requirements, sensitivity enhancement up to 40-fold and detection limits down to 0.3, 0.05, 0.6, 0.5 mg/L, respectively. Compared to the stacking effect, the use of a large sample size was found to be the major factor for sensitivity enhancement. The method reliability is established by close to 100% recoveries and statistical agreement of results from the method developed with currently used clinical radio-immunoassay method for all four proteins investigated. Moreover, an assay time of less than 10 min is needed in the method developed as compared to 7 h for the radio-immunoassay method.

Transient Isotachophoresis Focusing of DNA and DNA-Protein Complexes Is Essentially Enhanced by Spontaneously Dissolved Aerial Carbon Dioxide in Electrolytes

“…Therefore, by assuming that dissolved aerial carbon dioxide is saturated in background electrolyte, we are able to calculate the concentrations of HCO 3 − and CO 3 2− using eqs 1−3 and listed equilibrium constants. The concentrations of HCO 3 − and CO 3 2− in 0.5× TG at pH 8.5 are estimated as 6.9 and 0.02 mM, respectively. Evidently, HCO 3 − is the major ionic species of carbonic acid, and may be involved in enhancing t-ITP focusing.…”

“…The concentrations of HCO 3 − and CO 3 2− in 0.5× TG at pH 8.5 are estimated as 6.9 and 0.02 mM, respectively. Evidently, HCO 3 − is the major ionic species of carbonic acid, and may be involved in enhancing t-ITP focusing. As a decrease in pH or an increase in acidity, the fraction of HCO 3 − decreased (Figure 2).…”

“…The solubility of carbon dioxide is known to be about 1.45 g/ L at 25°C (∼30 mM), 36,39 and the equilibrium constant for formation of carbonic acid and the pK a values of H 2 CO 3 and HCO 3 − are also known as those listed in eqs 1−3. Therefore, by assuming that dissolved aerial carbon dioxide is saturated in background electrolyte, we are able to calculate the concentrations of HCO 3 − and CO 3 2− using eqs 1−3 and listed equilibrium constants.…”

“…To test the above hypothesis, we further examined the effect of HCO 3 − on t-ITP focusing using NH 4 HCO 3 and Na 2 CO 3 . In these two experiments, either NH 4 HCO 3 (0−4.0 mM) or Na 2 CO 3 (0−2.0 mM) was directly added to the freshly prepared 0.5× TG buffer (pH 8.5).…”

“…Interestingly, the E value of the high-E zone increases up to 187 V/cm ( Figure 4D). In contrast, the E value of the high-E zone is only about 100−125 V/cm if no HCO 3 − ions are included in the background electrolyte (freshly prepared 0.5× TG buffer, pH 8.5) ( Figure 4B). Consequently, DNA molecules (in the enhanced high-E zone) migrate toward the positive end and the velocity of these molecules increases to 265 μm/s ( Figure 4C and Supporting Information video 2).…”