Application of Capillary Electrophoresis with Laser-Induced Fluorescence to Immunoassays and Enzyme Assays

Abstract

Capillary electrophoresis using laser-induced fluorescence detection (CE-LIF) is one of the most sensitive separation tools among electrical separation methods. The use of CE-LIF in immunoassays and enzyme assays has gained a reputation in recent years for its high detection sensitivity, short analysis time, and accurate quantification. Immunoassays are bioassay platforms that rely on binding reactions between an antigen (analyte) and a specific antibody. Enzyme assays measure enzymatic activity through quantitative analysis of substrates and products by the reaction of enzymes in purified enzyme or cell systems. These two category analyses play an important role in the context of biopharmaceutical analysis, clinical therapy, drug discovery, and diagnosis analysis. This review discusses the expanding portfolio of immune and enzyme assays using CE-LIF and focuses on the advantages and disadvantages of these methods over the ten years of existing technology since 2008.

Keywords: CE-LIF; chip-based CE-LIF assay; enzyme assay; immunoassay.

Figure 1

Chemical structures and labeling reactions of naphthalene-2,3-dicarboxylaldehyde (NDA), 5-furoylquinoline-3-carboxyaldehyde (FQ), and (4-carboxylbenzoyl) quinoline-2-carboxaldehyde (CBQA).

Figure 2

Capillary electrophoresis using laser-induced fluorescence detection (CE-LIF) detection of thrombin using tetramethylrhodamine TMR labeled Toggle-25. (A) Electropherograms of Toggle-25-TMR in the presence of varying concentrations of thrombin. (B) The relationship between the peak area of complex and the concentrations of thrombin. Reproduced under permission of [66].

Figure 3

Electropherograms of immunocomplexes of fluorescent cyclic citrullinated peptides (F-CCP) with (a) anti-cyclic citrullinated peptides (CCP) antibodies in PBS, (b) anti-CCP antibodies in Fetal Bovine Serum, and (c) human IgG. (d) Electropherogram of patient samples without F-CCP treatment. Arrows indicate the peak from the immunocomplex of F-CCP and anti-CCP antibodies. Reproduced with the permission of [60].

Figure 4

Time course study of enzymatic reaction. Extracted media from gastric cancer MKN-1 cells treated with 2 µg/mL F-ERK, 0.01 µg/mL PKCδ, 50 mM Adenosine triphosphate ATP, and incubated at different time intervals. (a) 3 h incubation; (b) 6 h incubation; (c) 12 h incubation; (d) 24 h incubation. Reproduced with the permission of [59].

Figure 5

Microfluidic devices used for parallel electrophoretic enzyme assays. (a) Design of microfluidic network containing 16 parallel separation channels, (b) design of a 36-channel network, (c) photograph of a finished 36-channel chip, (d) bright-field image of the detection area on the 36-channel chip, (e) repetitive units of the microfluidic network. The Electrokinetic injection procedure is described in experimental section. Reproduced with the permission of [105].

Figure 6

Electropherograms obtained by CE-LIF analysis for (A) S₃ or 5-FAM-Ala-Ala-Ala-Phe-Tyr-Asp-OH and (B) S₄ or 5-FAM-Arg-Glu-Ala-Val-Val-Tyr-OH hydrolysis by HNE. Reproduced with the permission of [108].