Chip‐based immunoaffinity CE: Application to the measurement of brain‐derived neurotrophic factor in skin biopsies

Abstract

A chip-based immunocapillary electrophoresis system has been employed to measure the concentrations of brain-derived neurotrophic factor (BDNF) in human skin biopsies, taken during atopic inflammatory events. The device employs a replaceable immunoaffinity disk to which capture antibodies have been chemically immobilized. Homogenates obtained from microdissected human skin samples were injected into the system where the analyte of interest was captured in the immunoextraction port, thus allowing non-reactive materials to be removed prior to analysis. The captured analyte was labeled in situ with a red-emitting laser dye before being released from the capture antibody, separated by electrophoresis, and the resolved peaks detected by on-line laser-induced fluorescence. Comparison of this chip-based system to conventional immunoassay demonstrated good correlation when analyzing both standards and patient samples. The system was semi-automated resulting in a CE analysis within 1.5 min and a total of circa 5 min. Intra-and inter-assay CV's of 3.85 and 4.19 were achieved with circa 98.8% recovery of BDNF at a concentration of 100 pg/mL. The assay demonstrated clear differences between clinical stages of atopic dermatitis in human patients and could run 10-15 samples per hour. This system holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

“…The anti-chemokine antibodies were enzymatically digested as previously described [13,14]. Briefly, the antibodies were reduced to F(ab)′ 2 fragments using the Pierce IgG F(ab)′ 2 digestion kit according to the manufacturers instructions.…”

“…The digested antibody fragments were immobilized on an immunoaffinity insert as previously described [13,14]. Briefly, the insert was prepared by chemically modifying AP40 glass fiber filters (Millipore).…”

“…Saturation was determined in a similar manner by applying increasing concentrations of a chemokine mixture to the immunoaffinity disk until no further increase could be detected. In these studies a new disk was used for each analysis, although previous studies had shown that the disks were re-usable [14]. Additionally, concentrations of 10 pg, 100 pg and 500 pg of each chemokine were added to a saline extract of normal tissue and run throughout the system as described in Section 2.8.…”

Microfluidic, bead-based assay: Theory and experiments

“…Immunoassay-based techniques are of particular importance due to their high specificity for biomolecular targets, allowing the separation of analytes of interest from complex biological mixtures. The integration of immunoaffinity analytical systems in microfluidic devices provides enhanced reaction efficiency, reduced sample and reagent consumption, and lower costs [1, 2]. Furthermore, the development of these portable, disposable, and rapid devices that can be operated by minimally trained personnel may offer relatively sophisticated laboratory capabilities at the point-of-care, at home, and in resource poor regions.…”

Microdevices integrating affinity columns and capillary electrophoresis for multibiomarker analysis in human serum

“…SPE has been applied successfully in a microfluidic format;20, 21 however, nonspecific interactions like hydrophobic absorption alone do not provide high selectivity. To circumvent this shortcoming, enzymes or antibodies can be immobilized on the solid surface 21, 22. For instance, pisum sativum agglutinin has been immobilized on monolithic substrates to retain glycoproteins, which can be eluted in several fractions based on their affinities 23.…”