Advances in on-line enzyme assays by sequence analysis-based capillary electrophoresis

doi:10.3724/SP.J.1123.2020.05008

Abstract

Abstract:

Due to unique advantages such as short analysis time, high separation efficiency and sensitivity, easy automation, extremely low sample and reagent volume requirements, and the ability to utilize several detection methods, capillary electrophoresis (CE) is used as a high-efficiency separation technique, and has been developed as a powerful tool for on-line enzyme assays. On-line enzyme assays based on CE have been applied to almost all aspects of enzyme assays over the past two decades, including the evaluation of enzyme activities and kinetics, identification and characterization of enzyme inhibitors and activators, detection of enzyme substrates, investigation of enzyme-mediated metabolic pathways, and proteome analysis. One potential use of enzyme assays is in tracing enzymatic reactions from beginning to the end at high temporal resolution. Measurements of enzyme reactions at high temporal resolution can result in more accurate estimates of reaction mechanisms and reaction rate constants, which is vitally important for improving understanding of the functions of enzymes in metabolism and for identifying the potential use of enzymes in clinical diagnostics. Furthermore, high-throughput online enzyme analysis is of great importance for the analysis of enzyme reactions and enzyme inhibition reactions. The development of accurate, rapid and high-throughput enzyme inhibition screening methods is especially important for accelerating the development of new drugs. Electrophoretically mediated microanalysis (EMMA) and CE-integrated immobilized enzyme microreactor (IMER) are the two most used techniques for online CE enzyme assays. The EMMA technique utilizes different electrophoretic mobilities of enzymes and substrates to initiate reactions within the capillary and to separate the components of the reaction mixture for the final in-capillary quantification. In a CE-integrated IMER, the enzyme is bound to the capillary surface or to a suitable carrier attached to the capillary through physical adsorption, cross-linking, covalent bonding or other methods. The enzyme reactor is usually located at one end of the capillary; the enzyme-catalyzed reaction occurs when the substrates pass through the enzyme reactor and the substrates/products of the enzymatic reaction are separated and online detected by CE at the downstream end of the capillary. In both either techniques, the samples are usually introduced into the capillary by electrokinetic injection or by hydrodynamic injection. Because both injection methods require that the capillary inlet be physically moved from the sample container to the running buffer for CE analysis after each sample injection, it is unlikely that EMMA or microreactor techniques can be successfully used to perform sequential online analysis. Therefore, a CE sequence analysis technique based on rapid sequential injection has been developed as another powerful method for online enzyme analysis. Compared with the widely used electrokinetic and hydrodynamic injection methods used in traditional CE online enzyme analysis methods, rapid sequence injection methods can achieve sequential injection without any physical disturbance of the capillary inlet, allowing for the successful performance of online enzyme assays with high temporal resolution and at high throughput. A rapid, sequential, and automatic sample introduction system is an important part of online enzyme analysis based on CE sequence analysis. Several sequential injection methods such as optical-gating injection, flow-gated injection, two-dimension diffusion injection, flow injection and droplet microfluidics combined with CE have been developed to successfully perform online enzyme assays with high temporal resolution and high throughput. In this paper, we will review recently developed CE online enzyme assays and inhibition studies based on rapid sequential injection. We review the progress and applications of various sequential sample injection approaches that have been developed for sequential on-line CE analysis of enzyme reactions at high temporal resolution and high-throughput screening of enzyme inhibitors, including optical-gating injection, flow gated injection, two-dimension diffusion injection, flow injection and droplet microfluidics.

Key words: capillary electrophoresis (CE), sequential injection, temporal resolution, high throughput screening, on-line enzyme assay, review

TIAN Miaomiao, YANG Li. Advances in on-line enzyme assays by sequence analysis-based capillary electrophoresis[J]. Chinese Journal of Chromatography, 2020, 38(10): 1143-1153.

Figures/Tables 8

Table 1 Summary of on-line enzyme assays by CE based on sequential injection

Sequential injection method	Enzyme	Time resolution/sample throughput	Detection	Application	Ref.
LIF: laser induced fluorescence detection; UV-Vis: ultraviolet-visible detection; -: not mentioned.
Optical gated injection	β-galactosidase	30 s	LIF	enzyme kinetics and inhibition	[9, 10]
	trypsin	5 s	LIF	enzyme kinetics and inhibition	[11]
	L-asparaginase	12 s	UV-Vis	enzyme kinetics	[12]
	carboxypeptidase Y	50 s	LIF	peptide Sequencing	[13]
	D-amino acid oxidase	-	LIF	enzyme kinetics and inhibition	[14]
Flow gated injection	glutathione reductase	10 s	LIF	enzyme kinetics	[15]
Two-dimension diffusion	glutamate pyruvate transaminase	30 s	UV-Vis	enzyme kinetics	[16]
injection	glucose-6-phosphate	40 s	UV-Vis	enzyme kinetics	[17]
	dehydrogenase
	alanine amino transferase	30 s	UV-Vis	inhibitor screening	[18]
	alamine racemase	40 s	UV-Vis	enzyme kinetics	[19]
Flow injection	β-galactosidase	5 sample/min	LIF	inhibitor high throughput screening	[20]
Droplet microfluidics	GTPase	12 sample/min	LIF	enzyme assay with high throughput	[21]
	protein kinase	8 sample/min	LIF	inhibitor high throughput screening	[22]
	sirtuin 5	30 sample/min	LIF	inhibitor high throughput screening	[23]
	sirtuin 5	6 sample/min	LIF	inhibitor high throughput screening	[24]

Fig. 1 Schematic diagram of (a) the OGPB-LIF instrumentfor sequential CE analysis, (b) the loading end of the capillary and (c) evolution of the substrate and product peaks as a function of time in study of immobilized trypsin cleavage reaction[11] The inset of Fig. 1a shows an enlarged view of the optically gated vacancy capillary electrophoresis (OGVCE) region, the distance between the photobleachingposition (F1 ) and the detection position (F2 ) on the capillary is the effective separation distance. OGPB: optical-gated photobleaching injection.

Fig. 2 (a) Schematic diagram of the OGPB-UV-Vis setup for sequential CE analysis, (b) electropherogram for the 20 sequential analysis of Asn and Asp standard mixture and (c) evolution of the substrate and product peaks as a function of reaction time in the study of L- asparaginase-catalyzed reaction[12] The inset of Fig. 2c shows the measured Michaelis-Menten diagram obtained from the sequential CE enzyme assay.

Fig. 3 (a) Schematic diagram of the flow-gated injection coupled to microchip electrophoresis for sequential CE analysis and (b) evolution of GSH* peaks as a function of time in the study of a glutathione reductase-catalyzed enzymatic reaction[15] GSH* : ThioGlo-1-GSH adduct; GND: ground; DP: detection point.

Fig. 4 (a) Scheme diagram for on-line sequential analysisby capillary electrophoresis based on two-dimension diffusion injection and (b) electropherogram for the 20 sequential analysis of Ala and Glu standard mixture[16]

Fig. 5 Evolution of Ala and Glu peaks as a function of time in the study of a glutamate pyruvate transaminase-catalyzed enzymatic reaction[16] Electrophoretogram of substrate and product corresponding to different enzymatic reaction time: a. 0-2.5 min; b. 2.5-5.0 min; c. 5.0-7.5 min; d. 7.5 min-10.0 min; e. 10.0-12.5 min; f. 12.5-15.0 min; g. 20.0-22.5 min.

Fig. 6 Schematic diagram of CE combined with flow injection system[20]

Fig. 7 (a) Schematic of polydimethylsiloxane(PDMS)-glasshybrid microfluidic device for analysis of segmented flow samples and (b) inhibitory effect of 140 small molecules against protein kinase A[22]

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