Bead-based immunoassays with microelectrode detection

Farrell, Svetlana; Ronkainen-Matsuno, Niina J.; Halsall, H. Brian; Heineman, William R.
June 2004
Analytical & Bioanalytical Chemistry;Jun2004, Vol. 379 Issue 3, p358
Academic Journal
The suitability of a microelectrode as the detector for a small-volume, bead-based enzyme-labeled immunoassay for later use in a microfluidic device was investigated. The microelectrode helps to overcome consumption of the electroactive species by the electrode (depletion) that is encountered with macroelectrodes such as the rotating disk electrode (RDE) and allows the volume of the detection cell to be reduced. Microelectrodes also allow the chemical reactions to be monitored in real time due to the electrodes’ close proximity to the assay site. A bead-based sandwich immunoassay for mouse IgG was developed with alkaline phosphatase (AP) as the enzyme label, p-aminophenyl phosphate (PAPP) as the enzyme substrate, and microelectrode detection. The diffusion coefficient of the product of enzymatic hydrolysis, p-aminophenol (PAP), was determined to be 7.2±0.9×10-6 cm2 s-1. The detection limits were determined for free (0.52 ng mL-1) and bead-bound AP (10 ng mL-1). The number of binding sites for AP per bead was calculated to be 9.6×104 molecules/bead, and under saturation conditions the minimum detectable number of beads was 2500. Lower detection limits could be achieved with the microelectrode than the RDE while maintaining similar reproducibility. The microelectrode also made it possible to work with lower sample volumes (down to 10 μL) than with the RDE (minimum volume of 40 μL). Depletion of PAP was not observed with the microelectrode. The results obtained here with a microelectrode showed great promise for later use of microelectrodes in microfluidic devices with limited sample volumes. RDE detection cannot be used in a microfluidic system due to its complex set-up that includes a motor for rotation.


Related Articles

  • Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling. Shang, Jing; Zrazhevskiy, Pavel; Postupna, Nadia; Keene, C. Dirk; Montine, Thomas J.; Gao, Xiaohu // Scientific Reports;9/4/2015, p13651 

    In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes...

  • Apoenzymes of Alkaline Phosphatases of Different Origin for Determining of Trace Zinc(II). Zhavoronkova, A. M.; Muginova, S. V.; Shekhovtsova, T. N. // Journal of Analytical Chemistry;Jun2003, Vol. 58 Issue 6, p594 

    The applicability of the apoenzymes of three alkaline phosphatases of different origin (bacterial from E. coli and animal from chicken intestine or the small intestine of a Greenland seal) to the determination of zinc(II) was studied. The alkaline apophosphatase from seal intestine was found to...

  • Heat Inactivation in the Study of Human Alkaline Phosphatases. Posen, Solomon; Neale, Francis C.; Clubb, John S. // Annals of Internal Medicine;Jun65, Vol. 62 Issue 6, p1234 

    Investigates the heat stability of serum alkaline phosphate in normal subjects, in subjects with increased osteoblastic activity and in subjects with various hepatobiliary disorders. Medical history of the patients in the study; Attempts to identify the sources of human serum alkaline...

  • Effects of land use on water column bacterial activity and enzyme stoichiometry in stream ecosystems. Williams, Clayton; Scott, Andrew; Wilson, Henry; Xenopoulos, Marguerite // Aquatic Sciences;Jun2012, Vol. 74 Issue 3, p483 

    Fifty streams, located in southern Ontario, Canada, were visited in September 2008 to investigate the effect of varying land use, land cover, and associated resource inputs on water column bacterial abundance (BACT), production (BP), and extracellular enzyme activity and stoichiometry. Principle...

  • Interfering effect of bilirubin on the determination of alkaline phosphatase. Zhong Wang; Hongyan Guo; Ying Wang; Fanjun Kong; Rui Wang // International Journal of Clinical & Experimental Medicine;2014, Vol. 7 Issue 11, p4244 

    Objective: This study was designed to evaluate whether the high concentration of bilirubin is able to interfere the determination of alkaline phosphatase (ALP). Methods: Clinical tests evaluating the interfering substance of bilirubin of various concentrations on the determination of ALP were...

  • Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane. Azam, Md.; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki // Analytical & Bioanalytical Chemistry;Dec2011, Vol. 401 Issue 4, p1211 

    In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran...

  • Acidifying Composts from Vegetable Crop Wastes To Prepare Growing Media for Containerized Crops. Carri�n, C.; De la Fuente, R. Garc�a; Fornes, F.; Puchades, R.; Abad, M. // Compost Science & Utilization;Winter2008, Vol. 16 Issue 1, p20 

    The effects of adding five rates of powdered elemental sulfur (S0) or ferrous sulfate (FeSO4.7H2O) on the acidification process of three alkaline composts prepared with residual vegetable crop biomass (melon, C1; pepper, C2; and zucchini, C3) were investigated. Composts reacted differently to...

  • Mineralization process during acellular cementogenesis in rat molars: a histochemical and immunohistochemical study using fresh-frozen sections. Yamamoto, Tsuneyuki; Domon, Takanori; Takahashi, Shigeru; Anjuman, Khan; Fukushima, Chifumi; Wakita, Minoru // Histochemistry & Cell Biology;Mar2007, Vol. 127 Issue 3, p303 

    This study was designed to detect tissue non-specific alkaline phosphatase (TNSALP) by Azo-dye staining, calcium by glyoxal bis (2-hydroxyanil) (GBHA) staining, bone sialoprotein (BSP) and osteopontin (OPN) by immunoperoxidase staining in developing rat molars, and also to discuss the...

  • Modal behavior of the Kv1.1 channel conferred by the Kvβ1.1 subunit and its regulation by dephosphorylation of Kv1.1. Singer-Lahat, D.; Dascal, N.; Lotan, I. // Pflugers Archiv European Journal of Physiology;Jan2000, Vol. 439 Issue 1/2, p18 

    Modulation of fast-inactivating voltage-gated K+ channels can produce plastic changes in neuronal signaling. Previously, we showed that the voltage-dependent K+ channel composed of brain Kv1.1 and Kvβ1.1 subunits (αβ channel) gives rise to a current that has a fast-inactivating and a...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics