Improved solid‐phase microextraction device for use in on‐line immunoaffinity capillary electrophoresis

Abstract

A simple solid-phase microextraction device was fabricated for use in on-line immunoaffinity capillary electrophoresis (CE). The device, designed in the form of a four-part cross-shaped or cruciform configuration, includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes) permitting the confinement of beads or suitable microstructures. The surface of the beads in the analyte concentrator carried a molecular recognition adsorbing chemical or affinity ligand material. The improved cruciform configuration of the analyte concentrator-microreactor device, designed for use in on-line immunoaffinity CE, enables it to specifically trap, enrich, and elute an analyte from any biological fluid or tissue sample extract without any sample pretreatment except filtration, centrifugation, and/or dilution allowing the separation and characterization of target analyte(s) with improved speed, sensitivity, and lower cost than existing techniques. As a model system, Fab' fragments derived from a purified immunoglobulin G (IgG) antibody were covalently bound to controlled-porosity glass and used as constituents of the analyte-microreactor device. The high-specificity polyclonal antibodies employed in these experiments were individually raised against the acidic nonsteroidal anti-inflammatory drugs ibuprofen and naproxen, and the neuropeptides angiotensin II, and neurotensin. These compounds, which were present in simple and complex matrices were captured by and eluted from the analyte concentrator-microreactor using a 50 mM sodium tetraborate buffer solution, pH 9.0, followed by a 100 nL plug of 300 mM glycine buffer, pH 3.4. Two analyte concentrators were tested independently: one containing Fab' fragments derived from antibodies raised against ibuprofen and naproxen; the other containing Fab' fragments derived from antibodies raised against angiotensin II and neurotensin. Each resulting electropherogram demonstrated the presence of two eluted materials in less than 20 min. Immunoaffinity CE performed in a cruciform structure was simpler and faster than previously reported in the literature using on-line microextraction devices designed in a linear format. The new concentration-separation system operated consistently for many runs, maintaining reproducible migration times and peak areas for every analyte studied.

Capillary electrophoresis immunoassay using magnetic beads

“…So far, different approaches have been developed to prepare the immunosorbent phase. Open tubular [6][7][8][9][10][11], packed [12][13][14][15][16], and monolithic [17][18][19] capillaries are mostly used to immobilize the antibodies. For instance, Phillips' group described an immunoaffinity CE technique with Fab fragment covalently immobilized on the walls of a fused-silica capillary [9].…”

Microscale immunoaffinity SPE and MEKC in fast determination of testosterone in male urine

“…The hyphenated IA-SPE-PF-MEKC is related to immuno-ACE (IACE) in which the separation power of CE and specific interactions between an analyte and an antibody (fragment) are combined [29][30][31][32][33][34][35].…”

“…Previously, IACE analyses of gonadotropin-releasing hormone [32], naproxen, ibuprofen [33], some neuropeptides [29,30,33] recombinant cytokines [31], and cardiac troponin I [34] have been reported, making use of immobilized antibodies or antibody fragments. Those studies were focused on charged analytes and CZE, however.…”

A Miniaturized Transverse Flow Gating Interface for the On-line Coupling of Solid-phase Extraction with Capillary Electrophoresis

“…For such a purpose, a variety of interfaces have been designed, including such types as valve-loop, 7,8 T-split, 9,10 valve-loop-vial or T-piece, [11][12][13][14] two-leveled two cross, 15,16 flow-gating, 11,[17][18][19][20][21] and interfaces with integrated SPE. 22,23 With the first three types of interfaces, success of the coupling has been achieved to some extent, but some limitations have been recognized. Because of the large void volume resulting from the loop and the eluate flow path between the two capillaries, serious dilution and dispersion of the elution plug have occurred, thus leading to a loss of SPE efficiency, especially when a small elution volume is used.…”

“…15,16 For the interface with integrated SPE, as the SPE column is part of the CE system, some deleterious influences of the SPE sorbent may occur on the CE system during analysis. 22 Most of the aforementioned interfaces require relatively complicated switching procedures that may result in poor reproducibilities. 7,8,10 The flow-gating type is a cross-like interface, having the SPE column and CE capillary positioned on opposite sides of the cross with a proper gap.…”