表面改性微芯片电泳分离分析保健品中的功效成分

doi:10.3724/SP.J.1123.2023.08019

摘要/Abstract

摘要：

微芯片电泳(microchip electrophoresis, MCE)分离效率高、试剂和样品消耗量少、易实现多操作单元集成,在食品、环境和药物等领域应用广泛。环烯烃共聚物(cycloolefin copolymer, COC)等聚合物微芯片材料成本低、制作简便,但电泳过程中通道表面易发生非特异性吸附,且电渗流不稳定,限制了其应用。这些不足可通过COC表面改性解决。本文采用静态涂层和动/静态涂层联合策略,研制通道表面改性COC微芯片,结合激光诱导荧光(laser-induced fluorescence, LIF)检测,发展了简单高效的MCE-LIF分离分析方法,用于保健品中功效成分检测。通过负电荷涂层微通道表面改性方法,可以提高MCE对阳离子氨基酸的分离效果,而通过动/静态涂层结合的微通道表面改性方法,可以提高MCE对阴离子氨基酸的分离效果。负电荷涂层由COC微通道表面的疏水氨基酸吸附、戊二醛固定化和亲水氨基酸功能化构建,正电荷涂层由COC微通道表面的缬氨酸吸附、羧基活化和乙二胺功能化构建,动态涂层通过向微通道引入含有羟丙基甲基纤维素与十二烷基硫酸钠的缓冲液自动形成。实验研究了表面改性微通道的理化性质和电泳分离的影响因素,并将所建立的方法用于儿童保健品中赖氨酸和γ-氨基丁酸以及运动饮料中天冬氨酸与牛磺酸MCE-LIF的检测,儿童保健品中赖氨酸和γ-氨基丁酸的加标回收率为84.8%~118%,相对标准偏差(RSD)≤7.2%,运动饮料中天冬氨酸与牛磺酸的加标回收率为97.5%~118%, RSD≤6.4%。分析结果与HPLC方法的测定结果吻合,该法在保健品中氨基酸分离分析中有应用前景。

关键词: 表面改性, 微芯片, 电泳, 环烯烃共聚物, 保健品

Abstract:

Microchip electrophoresis (MCE) is widely applied in food, environment, medicine, and other fields, owing to its high separation efficiency, low consumption of reagents and samples, and ease of integrating multiple operating units. Polymer microchip materials like cycloolefin copolymer (COC) are low-cost and easy to fabricate. However, their practical applications are limited by the non-specific adsorption on channel surface during electrophoresis and the instability of electroosmotic flow. These shortcomings can be solved by COC surface modification. In this study, a static coating and dynamic/static coating combined strategy was used to develop a channel-surface-modified COC microchip. Combined with laser-induced fluorescence (LIF) detection, a MCE-LIF separation and analysis method was developed for detecting functional components in health care products. The separation performance of MCE was improved by the static coating microchannel surface modification method. The static coating was constructed by hydrophobic amino acid adsorption, glutaraldehyde immobilization, and hydrophilic amino acid functionalization on the COC microchannel surface. The separation performance of MCE was improved by microchannel surface modification combined with dynamic/static coating. The static coating was constructed by valine adsorption, carboxyl activation, and ethylenediamine functionalization on the COC microchannel surface. The dynamic coating is automatically formed by introducing a buffer solution containing hydroxypropyl methylcellulose and sodium dodecyl sulfate into the microchannel. The physical and chemical properties of surface-modified microchannels and the factors governing electrophoretic separation were studied. Combined with LIF detection, the MCE-LIF separation and analysis of lysine and γ-aminobutyric acid present in children’s health care products, as well as aspartic acid and taurine in sport drinks, were developed. The recoveries of lysine and γ-aminobutyric acid in children’s health care products were 84.8%-118%, and the relative standard deviations (RSDs) were less than 7.2% (n=3). The recoveries of aspartic acid and taurine in sport drinks were 97.5%-118%, and the RSDs were less than 6.4% (n=3). The analysis results are consistent with the HPLC results, and the method has potential for application in the separation and analysis of anionic amino acids in health care products.

Key words: surface modification, microchips, electrophoresis, cycloolefin copolymer (COC), health care products

中图分类号:

O658

刘惠珍, 陈雅莉, 夏凌, 肖小华, 李攻科. 表面改性微芯片电泳分离分析保健品中的功效成分[J]. 色谱, 2023, 41(10): 937-948.

LAU Waichun, CHEN Yali, XIA Ling, XIAO Xiaohua, LI Gongke. Surface-modified microchip electrophoretic separation and analysis of functional components in health care products[J]. Chinese Journal of Chromatography, 2023, 41(10): 937-948.

图/表 12

图 1 (a)芯片电泳、(b)负电荷涂层及(c)正电荷涂层COC表面改性过程的示意图

Fig. 1 Schematics of (a) the microchip electrophoresis, surface modification processes of (b) negative charge coating and (c) positive charge coating on the surface of COC COC: cycloolefin copolymer; GA: glutaraldehyde; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxy succinimide; EDA: ethylenediamine.

图 2 (a)静态涂层改性COC表面XPS光谱图, (b)C 1s、(c)N 1s、(d) O 1s峰拟合图,(e)负电荷涂层COC板上接触角,(f)COC微通道中空气-水界面轮廓图以及静态涂层(g)涂覆前和(h)涂覆后COC通道表面对氨基酸的吸附

Fig. 2 (a) Survey XPS spectrum and the deconvolution analysis of (b) C 1s, (c) N 1s, and (d) O 1s XPS spectra of static coating COC surface, (e) contact angle on COC plate and (f) profiles of air-water interface in COC microchannel with negative charge coating unmodified and modified, adsorption to amino acids on COC channel surfaces with static coating (g) unmodified and (h) modified XPS: X-ray photoelectron spectroscopy.

图 3 疏水氨基酸种类对(a)Arg峰形及(b)H的影响,亲水氨基酸种类对(c)Arg峰形及(d)H的影响

Fig. 3 Effects of hydrophobic amino acid types on (a) the peak shape and (b) theoretical plate height (H) of arginine,effects of hydrophilic amino acid on (c) the peak shape and (d) H of arginine For b and d: n=3.

图 4 分离电压φ对(a)u以及(b)H的影响,(c)不同分离距离和(d)不同分离电压下Lys和GABA的分离度(n=3)

Fig. 4 Effects of operating voltage (φ) on (a) the fluidic transport rate (u) and (b) the H of Lys and GABA, measured Rs on (c) different separation distances (L) and (d) different φ of Lys and GABA (n=3)

表 1 儿童保健品中Lys和GABA的分析(n=3)

Table 1 Analysis of Lys and GABA in children’s health products (n=3)

Analyte	This method					Original with HPLC method/(mg/kg)	Relative error/%
Analyte	Original/(mg/kg)	Spiked/(mg/kg)	Found/(mg/kg)	Recovery/%	RSD/%	Original with HPLC method/(mg/kg)	Relative error/%
Lys	3.08±0.03	3.00	6.51	107	3.6	3.05±0.02	0.98
		6.00	10.8	118	2.1
		12.0	15.7	104	1.0
GABA	2.53±0.15	3.00	4.69	84.8	7.2	2.32±0.04	9.1
		6.00	8.25	96.7	7.2
		12.0	16.6	114	2.6

图 5 儿童保健品及3.00 mg/kg加标样品的电泳图

Fig. 5 Electropherograms of original and 3.00 mg/kg spiked children’s health products BGE: 0.1 mmol/L sodium tetraborate; φ: 800 V; separation distance: 2.0 cm.Peaks: 1. Lys; 2. GABA.

图 6 静态涂层改性COC表面(a)XPS光谱图和(b)C 1s、(c)N 1s、(d)O 1s峰拟合图,(e)正电荷涂层COC板上接触角和(f)COC微通道中空气-水界面轮廓,静电涂层(g)涂覆前和(h)涂覆后COC通道表面对Asp和Tau的吸附

Fig. 6 (a) Survey XPS spectrum and the deconvolution analysis of (b) C 1s, (c) N 1s and (d) O 1s XPS spectra of static coating COC surface, (e) contact angle on COC plate and (f) profiles of air-water interface in COC microchannel with positive charge coating unmodified and modified, adsorption to aspartate and taurine on COC channel surfaces with static coating (g) unmodified and (h) modified

图 7 HPMC与SDS的质量分数对(a)Asp与(b)Tau u的影响,HPMC与SDS的质量分数对(c)Asp与(d)Tau σ2的影响(n=3)

Fig. 7 Effect of mass fractions of HPMC and SDS on the u of (a) aspartate acid and (b) taurine, and effect of mass fractions of HPMC and SDS on the total peak width σ2 of (c) aspartate acid and (d) taurine (n=3)

图 8 φ对(a)Asp和(b)Tau u的影响及φ对(c)Asp和(d)Tau H的影响(n=3)

Fig. 8 Effect of φ on the u of (a) aspartic acid and (b) taurine, and effect of φ on H of (c) aspartic acid and (d) taurine (n=3) For a and b, 1. dynamic only; 2. dynamic & static; 3. static only. For c and d, 1. dynamic & static; 2. static only; 3. dynamic only.

图 9 (a)涂层类型对Asp和Tau分离的影响和(b)不同分离距离、(c)不同分离电压下测得的Asp和Tau的分离度

Fig. 9 (a) Effect of the coating type on the separation resolution of aspartic acid and taurine, measured Rs of aspartic acid and taurine under (b) different separation distances and (c) different φ For b and c: n=3. Peaks: 1. aspartic acid; 2. taurine.

图 10 运动饮料及8.00 mg/kg加标样品的电泳图

Fig. 10 Electropherograms of original and 8.00 mg/kg spiked sport drink samples BGE: 0.1 mmol/L sodium tetraborate containing 0.005%(mass fraction) HPMC and 0.015% (mass fraction) SDS; φ: 800 V; L: 1.5 cm.Peaks: 1. aspartic acid; 2. taurine.

表 2 运动饮料中Asp和Tau的分析(n=3)

Table 2 Analysis of aspartic acid and taurine in sport drink (n=3)

Analyte	This method					Original with HPLC method/(mg/kg)	Relative error/%
Analyte	Original/(mg/kg)	Spiked/(mg/kg)	Found/(mg/kg)	Recovery/%	RSD/%	Original with HPLC method/(mg/kg)	Relative error/%
Asp	9.89±0.55	8.00	18.2	102	1.0	9.75±0.15	1.4
		16.0	25.3	97.5	6.4
		32.0	43.9	105	3.6
Tau	15.9±0.8	8.00	24.2	101	4.1	14.6±0. 1	9.4
		16.0	37.4	117	2.6
		32.0	56.6	118	3.0

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