色谱 ›› 2021, Vol. 39 ›› Issue (8): 921-926.DOI: 10.3724/SP.J.1123.2021.02021

• 技术与应用 • 上一篇    

多材料3D打印技术制作用于毛细管电泳的非接触电导/激光诱导荧光二合一检测池

张丕旺1, 杨立业1, 刘强1, 陆善贵2, 梁英1, 张敏1,*()   

  1. 1.桂林电子科技大学生命与环境科学学院, 广西 桂林 541004
    2.品创检测(广西)有限公司, 广西 桂林 541004
  • 收稿日期:2021-02-22 出版日期:2021-08-08 发布日期:2021-06-29
  • 通讯作者: 张敏
  • 作者简介:*Tel:(0773)2305125,E-mail: zhangmin@guet.edu.cn.
  • 基金资助:
    国家自然科学基金(21966012);国家自然科学基金(21705028);广西自然科学基金重点项目(2017GXNSFDA198043);广西八桂青年学者项目

Multimaterial 3D-printed contactless conductivity/laser-induced fluorescence dual-detection cell for capillary electrophoresis

ZHANG Piwang1, YANG Liye1, LIU Qiang1, LU Shangui2, LIANG Ying1, ZHANG Min1,*()   

  1. 1. School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin 541004, China
    2. Pinchuang Testing (Guangxi) Corporation, Guilin 541004, China
  • Received:2021-02-22 Online:2021-08-08 Published:2021-06-29
  • Contact: ZHANG Min
  • Supported by:
    National Natural Science Foundation of China(21966012);National Natural Science Foundation of China(21705028);Guangxi Natural Science Foundation(2017GXNSFDA198043);Guangxi Bagui Young Scholar Program

摘要:

利用多材料3D打印技术研制了用于毛细管电泳(CE)的二合一检测池,实现了电容耦合非接触电导(C4D)与共聚焦激光诱导荧光(LIF)两种检测方法在毛细管柱上同一位置同时检测。3D打印的检测池采用了导电的复合聚乳酸(PLA)材料制作C4D的屏蔽层,采用普通的绝缘PLA材料支撑C4D金属管电极并隔离屏蔽层。两根金属管电极通过“打印-暂停-打印”的方式嵌入到检测池中,两电极被2 mm厚的导电屏蔽层隔开,在屏蔽层中有一直径为1 mm的圆形通孔用于LIF检测。该检测池与带流通式进样接口的自组装CE系统联用,用于同时检测无机离子和异硫氰酸荧光素(FITC)标记的氨基酸。研究优化了C4D激励信号频率与电泳缓冲液浓度,选用的电泳缓冲溶液为10 mmol/L 3-吗啉丙烷-1-磺酸(MOPS)与10 mmol/L二(2-羟乙基)亚氨基三(羟甲基)甲烷(Bis-Tris)的混合溶液,选用C4D激励频率为77 kHz。二合一检测池应用于内径为25 μm的毛细管时,C 4D对Na+、K+和Li+的检出限分别为2.2、2.0和2.6 μmol/L; LIF对荧光素和FITC的检出限分别为7.6和1.7 nmol/L。两种检测方法的相对标准偏差在0.3%至4.5%之间(n=3),工作曲线的相关系数r 2≥0.9904。采用3D打印技术可以在实验室内实现复杂结构的制作,降低了制作的成本,且便于方法的推广和改进。

关键词: 毛细管柱上检测, 组合检测, 电容耦合非接触电导检测, 激光诱导荧光, 毛细管电泳

Abstract:

Dual detection, which simultaneously employs two complementary detection methods, is a useful approach to enhance the selectivity and sensitivity of capillary electrophoresis (CE). Through dual detection, multiple classes of analytes with different structural and chemical characteristics can be sensitively detected using a single CE method. In addition, the comigrating peaks can be distinguished by comparing the signal outputs of two detectors with different selectivities. Typically, dual detection is achieved by coupling two detectors in series along a capillary. However, in this approach, it is inconvenient to evaluate the signal outputs of the two detectors. The two detectors present differences in their corresponding effective capillary lengths and dead volumes of the detection cell. Therefore, detectors that combine two or three detection methods in a single detection point are proposed to address this issue.
In this work, to fabricate a combined detector in a simple and low-cost manner, multimaterial 3D printing technology is employed. A two-in-one detection cell that combines capacitively coupled contactless conductivity detection (C4D) and confocal laser-induced fluorescence (LIF) detection was fabricated by 3D printing functional materials. In 3D printing, conductive composite polylactic acid (PLA, Proto-pasta) filaments and normal nonconductive PLA filaments were employed. The conductive material was used to build a C4D shielding layer that was electrically grounded. The nonconductive PLA was used as an electrical insulator placed between the shielding layer and C4D electrodes, which were two stainless-steel tubes (0.4 mm i.d. and 5 mm length). To embed the electrodes into the nonconductive material, a “print-pause-print” approach was applied. After building two chambers for housing electrodes using nonconductive PLA, the 3D printing was paused, following which the two electrodes were manually installed. Printing was then resumed, and the remaining part was built. The two electrodes were 2 mm apart, and the gap between them was filled with a conductive material for shielding to eliminate stray capacitance. A through-hole (1 mm i.d.) was placed between the middle conductive shielding layer for LIF detection. The size of the detection cell was 60 mm×29 mm×7.2 mm. The cell was screwed onto an XYZ stage to precisely align the light path of LIF detection, which was realized using a TriSep TM-2100LIF detector equipped with a 473 nm laser. C4D detection was achieved using a TraceDec detector equipped with a ChipCE adaptor. The two-in-one detector was coupled with a lab-made CE system that had a flow-through injection interface.
Use of the detection cell allows the simultaneous detection of inorganic cations and fluorescein isothiocyanate (FITC)-labeled amino acids. The C4D excitation frequency and buffer concentration were then optimized. A mixture of 10 mmol/L 3-(N-morpholino)propanesulfonic acid (MOPS) and 10 mmol/L bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris) was selected as the background electrolyte as a compromise of C4D signal-to-noise ratio (S/N) and separation efficiencies of amino acids. The C4D excitation frequency was set to 77 kHz with S/N=233±8 for 200 μmol/L Na +. The baseline separation of Na+, K+, Li+, FITC, fluorescein, histidine (His), lysine (Lys), tryptophan (Trp), phenylalanine (Phe), alanine (Ala), and glycine (Gly) was achieved with a 25 μm i.d.×365 μm o.d.×45 cm (35 cm effective length) capillary and -10 kV separation voltage. The limits of detection (LODs) of C 4D for Na+, K+, and Li+were 2.2, 2.0, and 2.6 μmol/L, respectively. The LODs of LIF for fluorescein and FITC were 7.6 and 1.7 nmol/L, respectively. The relative standard deviations (RSDs) of the two detection methods were within the range of 0.3%-4.5% (n=3). The r 2 of the calibration curves was ≥0.9904. Thus, 3D printing technology is a simple and low-cost approach to implement complex designs, including those that are difficult to fabricate by traditional “workshop” technologies.

Key words: on-capillary detection, dual detection, capacitively coupled contactless conductivity detection (C4D), laser induced fluorescence (LIF), capillary electrophoresis (CE)

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