Chemiluminescence detection in integrated post‐separation reactors for microchip‐based capillary electrophoresis and affinity electrophoresis

Abstract

Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 microm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 microm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 microg/mL) with increasing mouse IgG (0-60 microg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were +/- 5.8 and +/- 1.3 %, respectively.

Integration of a flow-type chemiluminescence detector on a glass electrophoresis chip

“…Since these characters of CL match the demands of miniaturization and integration, CL will be a promising detection method for -TAS. Mangru and Harrison [21] first realized CL detection on a microchip, Y-shaped reaction zone junction was adopted. Horseradish peroxidase (HRP) and fluorescein-conjugated HRP were separated and detected with a luminol CL system.…”

Biomedical applications of protein chips

“…One tool is enzyme-linked immunosorbent assay (ELISA) in a high throughput array format that can be used for detection of infectious agents or as a diagnostic for cancers [38]. Mangru and Harrison [35] performed an IgG immunoassay using chemiluminescence detection in a post-separation reactor capillary electrophoresis chip. Schneider et al [20] developed an optical chip immunoassay to analyze human chronic gonadotropin (hCG) in undiluted, whole blood where better control of nonspecific binding effects is expected with improved surface blocking.…”

“…Although the different protein chip formats [1][2][3], and fabrication technologies [4][5][6][7][8][9][10][11][12] are not described, it is noteworthy to mention that a protein chip system faces challenges that include the attachment of proteins in their proper conformations [7,9,11,[13][14][15][16][17], the production of stable capture agents that selectively and uniformly bind to target proteins under a defined set of conditions on the chip surface [7,16,[18][19][20][21][22][23][24][25], the alternative use of molecular tags that resolves steric problems or inaccessibility of binding sites [26][27][28][29][30][31], expression and purification of proteins in their native conformations in high-throughput [32,33], and the detection of protein interactions [3,11,12,[18][19][20][34]…”

Effect of Mixing Modes on Chemiluminescent Detection of Epinephrine with Lucigenin by an FIA System Fabricated on a Microchip

“…Recently, the CL method has been applied to detection in microchip capillary electrophoresis. 4,5 Wu et al applied the luminol and peroxyoxalate CL methods to the detection of H2O2 in the integrated FIA system with gravity as a driving force. 6 In the FIA system, the reaction for detection is preferred to be complete within the residence time of the reactants in the flow cell.…”