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Chinese Journal of Chromatography
2020, Vol. 38, No. 10
Online: 08 October 2020

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Volume 38,Number 10 Content 2020, 38 (10):  0-0.  Abstract ( 16 )   PDF (4987KB) ( 30 )

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Capillary electrophoresis-mass spectrometry and its application to proteomic analysis LIANG Yu, ZHANG Lihua, ZHANG Yukui 2020, 38 (10):  1117-1124.  DOI: 10.3724/SP.J.1123.2020.03005 Abstract ( 347 )   HTML ( 29 )   PDF (2925KB) ( 174 )   Proteomic analysis plays an important role in basic biological studies and precision medicine. However, real samples contain numerous proteins with a wide dynamic distribution range. Such high complexity of the samples has a drastic effect on the identification coverage of proteins. Consequently, with advancements in mass spectrometry (MS) technology, concomitant improvements in separation technologies for simplifying the sample should be critical. With the advantages of small sample loading volume, high separation efficiency, and high speed, capillary electrophoresis (CE) coupled to MS has been gained much attention in the field of proteomics research. A nanoflow sheath liquid interface and a sheathless interface have been developed and commercialized, boosting the development of the CE-MS technology. Capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and capillary electrochromatography (CEC) have been successfully combined with MS, and CZE-MS has widespread application. In proteomic research, the "bottom-up" strategy, which is based on the separation and identification of enzymatic peptides, is widely applied. With the limit of detection as low as 1 zmol for peptides, CE-MS has been successfully applied to single-cell proteomic analysis. Besides, CE is complementary to reversed-phase liquid chromatography (RPLC), providing a new approach for the separation and identification of peptides with similar hydrophobic properties (especially, post-translational peptides). The "top-down" strategy, which is based on the separation and identification of intact proteins, can directly provide more accurate and complete information about proteins. For protein separation, CE is advantageous in terms of the high separation resolution and high protein recovery, thereby improving the sensitivity and coverage of protein identification. Native MS enables successful identification and characterization of protein complexes under nondenaturing conditions. Because of the good compatibility of CE with MS, attempts have been made to use CE coupled with native MS for the separation and identification of protein complexes. In this review, the development of the CE-MS technology is first reported, including a robust and sensitive CE-MS interface, and a separation mode coupled to MS. Then, the application of the CE-MS technology to "bottom-up", "top-down" and native MS analysis is discussed. The superiority of CE-MS in proteomic analysis is also emphasized. Finally, the promising future prospects of CE-MS are discussed.

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Recent progress in capillary electrophoresis-based high-sensitivity proteomics YANG Yun, TIAN Ruijun 2020, 38 (10):  1125-1132.  DOI: 10.3724/SP.J.1123.2020.03003 Abstract ( 132 )   HTML ( 13 )   PDF (883KB) ( 66 )   In recent years, proteomic techniques have undergone rapid progress in terms of sample pretreatment, separation, and mass spectrometry (MS) detection. The current MS-based proteomic techniques can be used to identify up to 10000 proteins both qualitatively and quantitatively within a few hours. However, the current mainstream proteomic approaches do not fulfill the need to analyze minute amounts of biological samples, especially rare cells and single mammalian cells. Capillary electrophoresis (CE)-based separation offers many advantages, such as narrow peaks, high separation efficiency, and low sample requirement, which make it an ideal separation approach for combination with high-resolution MS. We have reviewed the state-of-the-art development of integrated and online sample preparation methods and nanoscale liquid chromatography-mass spectrometry (nanoLC-MS) for high-sensitivity proteomics, and described the associated challenges. Integrated and online sample preparation methods can minimize sample loss and improve lysis and digestion efficiencies. The simple and integrated spintip-based proteomics technology (SISPROT) developed by our group has shown robust performance for the comprehensive profiling of various types of samples and the sensitive analysis of small numbers of cells, down to a few hundred. A few groups have applied integrated/online sample preparation methods, such as nanodroplet processing in one pot for trace samples (nanoPOTS) and integrated proteome analysis system for one cell (iPAD-1), to achieve the identification of hundreds of proteins from a single HeLa cell. We propose that one of the key technical challenges in this field is that the performance of current nanoLC separation techniques cannot match modern high-resolution MS techniques, with ultrahigh scan rates of over 40 Hz; therefore, the insufficient chromatographic performance results in reduced utilization of MS/MS scan capacity. Wide chromatographic peaks result in insufficient precursors to trigger MS/MS scans and redundant sampling, irrespective of whether dynamic exclusion has been enabled. In view of the above mentioned technical challenges, we have focused on discussing the unique technical advantages and potential opportunities of CE-MS, which mainly include the following. (1) High-performance capillary electrophoresis (HPCE) separation for minute amounts of tryptic peptide samples. Capillary electrochromatography can further improve the column capacity limit of HPCE. (2) CE-MS interfaces for high-sensitivity proteomics. Although sheath liquid interfaces have proven versatile and robust and are currently more commonly used, sheathless interfaces can significantly enhance the signal/noise ratio owing to decreased analyte dilution and background noise. Thus, sheathless interfaces are potentially more suitable for ultrasensitive proteomics. (3) Synergetic utilization of HPCE separation and MS detection at high scan rates. The most promising way to fully utilize the ultrahigh scan rates of modern high-resolution MS is to enhance the quality of peptide separation. Narrower peptide peaks in HPCE separation may greatly reduce redundant sampling and boost sensitivity. Overall, we anticipate that, after further improvement, CE-MS-based proteomics will be more widely applied to proteomic analysis of minute amounts of biological samples, such as single mammalian cells. Furthermore, more sensitive data acquisition modes, such as data-independent acquisition, may be used for global proteomic profiling, and parallel reaction monitoring may be used to target a limited number of important proteins. Matching between runs and machine learning algorithms may improve the accuracy of proteomic analysis of minute amounts of samples.

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Application of affinity capillary electrophoresis in the study of protein-DNA interactions YU Fangzhi, ZHANG Dapeng, YUAN Zheng, ZHAO Qiang, WANG Hailin 2020, 38 (10):  1133-1142.  DOI: 10.3724/SP.J.1123.2020.03007 Abstract ( 145 )   HTML ( 22 )   PDF (1942KB) ( 74 )   Protein-DNA interactions play essential roles in various biological events that determine the cell fate. Research on the molecular mechanism of protein-DNA interactions has helped elucidate diverse fundamental life processes, thereby providing theoretical guidance for establishing clinical treatment and screening potential drug of target diseases. Furthermore, well-known protein-DNA interactions have been utilized to develop advanced bioengineering and bioanalytical techniques, therefore providing robust technical support for related research. Hence, it is important to establish sensitive and rapid analytical methods to study protein-DNA interactions. High-performance capillary electrophoresis (CE) has been widely used in many research fields such as chemistry, life sciences, and environmental sciences, mainly due to its advantages including ultra-high separation efficiency, extremely low sample consumption, and short analysis time. For instance, affinity capillary electrophoresis (ACE) has become an important analytical tool for investigating molecular interactions. In this paper, we review the applications of ACE in studying protein-DNA interactions since it was first proposed in 1992, addressing previous significant work in this field. Three major aspects have been summarized in this review: (1) brief introduction to the development of ACE technique; (2) applications of ACE in the fundamental research on the molecular mechanism of protein-DNA interactions; and (3) applications of well-known protein-DNA interactions in CE-based detection of target molecules and reactions. In the first aspect, along with the concept and separation modes of ACE, general strategies to enhance the analytical ability of ACE are briefly introduced. In the second aspect, the applications of ACE in studying several important protein-DNA interactions involving transcription factors (e.g., GCN4), DNA repair proteins (e.g., UvrA, UvrB, and RecA), and methylated DNA-binding proteins (MBDs) are reviewed. In the third aspect, the applications of well-known molecular interactions (e.g., antigen-antibody, aptamer-target, etc.) to facilitate CE-based detection of target molecules (e.g., DNA adducts, DNA methylation, microRNA, single nucleotide polymorphism, etc.) and target reactions (e.g., DNA strand exchange) are addressed. Finally, we prospect and discuss the advancements of ACE that can be established in future studies. The following two aspects should be improved in future ACE analysis: (1) the advantages of extremely low volume consumption and short analysis time should be fully utilized to develop sensitive and high-throughput CE platforms for the assessment of rare biological samples and massive uncertain samples, respectively; (2) ACE should be combined with other advanced techniques, such as DNA sequencing and mass spectrometry, to rapidly screen and identify the precise interacting sites of unknown protein-DNA interactions.

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Advances in on-line enzyme assays by sequence analysis-based capillary electrophoresis TIAN Miaomiao, YANG Li 2020, 38 (10):  1143-1153.  DOI: 10.3724/SP.J.1123.2020.05008 Abstract ( 72 )   HTML ( 13 )   PDF (4070KB) ( 43 )   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.

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Advances in application of capillary electrophoresis in pharmaceutical analysis XU Xu, CHEN Gang, LIU Hao 2020, 38 (10):  1154-1169.  DOI: 10.3724/SP.J.1123.2020.03012 Abstract ( 232 )   HTML ( 32 )   PDF (2522KB) ( 139 )   Capillary electrophoresis (CE) is mainly applied in pharmaceutical analysis. All CE separation modes and detection methods show their characteristics and application abilities in the separation and analysis of different drug samples. The present review provides a brief cross section of new advances in CE application in pharmaceutical analysis, including for small molecular drugs and related substances (including chiral drug separation), traditional Chinese medicine and natural products, in vivo pharmaceutical analysis, and biological product analysis. However, the research on physical and chemical constant determination, affinity capillary electrophoresis and binding constant research (drug and receptor interaction, etc.), clinical biomarker analysis, metabolomics, and microchip CE analysis are not included. According to traditional pharmaceutical analysis developments, the recent advances in CE in compliance with pharmaceutical analysis regulatory requirements include CE capacitively coupled contactless conductivity detection (C4 D), improved detection sensitivity and precision, CE-sodium dodecyl sulfate (CE-SDS), imaged capillary isoelectric focusing (icIEF), antibody analysis, and so on. Combined with the references, this review also discusses the current requirements in the field of traditional pharmaceutical analysis, as well as the status, challenges and opportunities of CE in it. Some suggestions on CE application as a complementary analysis method for chemical drugs and traditional Chinese medicine analysis are put forward, and the characteristics and ability of CE in biological product analysis is expected to further development. The new development of CE-MS and improvement of CE repeatability may greatly expand the field of application of CE in the future. This review covers the improvements published between January 2017 and February 2020, as well as some important CE papers published in 2016.

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Methods and techniques of capillary electrophoresis for drug screening QIAN Xin, TIAN Yan, LUO Xinxin, PAN Jingmiao, DENG Suya, HUANG Yike, FU Qifeng, XIA Zhining 2020, 38 (10):  1170-1178.  DOI: 10.3724/SP.J.1123.2020.05040 Abstract ( 85 )   HTML ( 17 )   PDF (1218KB) ( 55 )   Capillary electrophoresis (CE) shows enormous potential for application in new drug research and development. Because of the aqueous medium employed as the running buffer in CE, drug screening can be carried out in an environment similar to that in physiological testing media. Drug screening methods based on CE are different from other instrumental measurements in vitro. CE can not only sustain the biological activity of the screened molecules and ligands, but also help evaluate the interactions between the receptors and the ligands. Based on these interactions, some important pharmacological parameters related to drug screening, such as the association constant Kb , bonding rate constant Kon , and dissociation rate constant Koff , can be determined by CE. Thus, CE is an effective tool for simulating and predicting the entire interaction process between receptors and drugs in vivo. In this review, the history of CE for drug screening is revisited. The theories, common methods for drug screening by CE, and some application examples and related technologies are reviewed. The methods of drug screening by means of affinity CE and kinetic CE are introduced. Some selected studies on different ligands at the molecular and cellular level are reported, along with examples several types of drugs. Techniques based on a combination of CE with mass spectrometry and chemiluminescence are reviewed, with focus on the screening of candidate drugs and active compounds from traditional Chinese medicine. The application prospect of drug screening by CE combined with a DNA-encoded compound library is introduced. This paper discusses the core of the fraction collection step in CE and emphasizes the significance of combining CE with systematic evolution of ligands by exponential enrichment. In conclusion, various optional methods for CE drug screening would pave the way for new concepts related to drug screening and evaluation in the future.

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Research progress on analysis of human papillomavirus by microchip capillary electrophoresis LIN Xuexia, WANG Chenjing, LIN Jin-Ming 2020, 38 (10):  1179-1188.  DOI: 10.3724/SP.J.1123.2020.05016 Abstract ( 104 )   HTML ( 16 )   PDF (2510KB) ( 60 )   Human papillomavirus (HPV), is a common spherical DNA virus that can lead to six types of cancers later in life, which has recently garnered human's attention. Microchip capillary electrophoresis (MCE) has provided simple, fast, portable, and sensitive HPV typing assay assisted by a variety of signal amplification technologies. This review presents the latest research progress of MCE in routine HPV typing assays, including both of the MCE techniques and MCE combined with the nucleic acid amplification techniques for HPV assay. The introduction on the former part concerns the MCE system, the MCE chips design and electrophoretic separation methods. The typical MCE system includes high voltage power supply, microfluidic chip of separation, sample injection, electrolyte cell, detection unit and so on. Four different chips are reviewed, containing straight separation channel, T-channel, serpentine channel and dual channel, because these microchips are the most used in the last decade. Furthermore, the high integration and high throughput on a single chip are often integrated the sample preparation unit on a chip. The advantages and disadvantages of different designed microchips are introduced at the same time. The separation methods of chip electrophoresis are briefly introduced. With the development and application of MCE for HPV detection, the separation time is greatly shortened from a few hours to several minutes. The review on the second part gives the comments on various kinds of nucleic acid amplification technologies coupled with MCE for HPV assay. Firstly, the comparative analysis is given on the polymerase chain reaction (PCR) combined with MCE, loop-mediated isothermal amplification (LAMP), PCR combined with restriction fragment length polymorphism (RFLP) for HPV DNA detection, and nucleic acid sequence based amplification (NASBA) for the detection of HPV mRNA, nested PCR and so on. Secondly, the reviews on the other methods beside MCE are also summarized, including the PCR coupled with Fourier transform-infrared spectroscopy (FT-IR spectroscopy), nanotechnology, DNA probes combined with electrochemical methods, reductive Cu(Ⅰ) particles catalyzed Zn-doped MoS2 quantum dots and T7 exonuclease with electrochemiluminescence, LAMP with CRISPR/Cas12a based lateral. In these non-MCE methods, the electrochemical sensing, e. g., impedimetric detection, pulse voltammetry method and flow biosensor, is an ideal method due to its low background signal and excellent time control ability. Finally, although MCE technologies have been developed and the developed instruments are applied in recent years, there are still some challenges in MCE techniques, methods and applications. The first challenge faced in the application of MCE technique in HPV typing assay is that the MCE device can not be well utilized for the detection of HPV with high resolution and high sensitivity, because MCE can not do signal amplification of HPV nucleic acid. The second challenge is that even though some researchers have successfully integrated PCR and MCE on one chip, the technique still faces difficulty for wide application and there is still no really integrated PCR-MCE chip for HPV detection. The third one is the MCE technique is lack for the manufacture of miniaturized and automatic instrument. At the end of review, the authors' insights are given on the development of automatic, fast, high stable and reliable detection in the HPV typing via the portable MCE device.

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Mass transport properties and applications of nanochannels LI Zhongqiu, WU Zengqiang, XIA Xinghua 2020, 38 (10):  1189-1196.  DOI: 10.3724/SP.J.1123.2020.04029 Abstract ( 77 )   HTML ( 10 )   PDF (2773KB) ( 72 )   With the development of materials science, micro/nano processing technologies, and mass transport theories at the micro/nano scale, nanochannel-based technology has been receiving increasing attention. Nanochannels can be classified as biological and artificial. The size of a nanochannel is typically 1-100 nm, which greatly enhances the interaction between the channel surface and substances inside the channel, inducing several special mass transport properties such as ion selectivity, ionic current rectification, and resistive current pulse. Ion selectivity is caused by the electrostatic interaction between ions and the surface charge of the nanochannel, ionic current rectification arises from the asymmetric distribution of the electrochemical potential inside the nanochannel, and a resistive current pulse is generated by the blocking of the nanochannel during the transport of ions/molecules. By taking advantage of these mass transport properties, nanochannels can be applied to various fields. For example, gated ion transport can be realized by modifying the surface of the nanochannel with functional groups; single-molecule sensing can be realized using sub-nanoscale channels; the separation of ions, molecules, or nanoparticles can be realized by regulating the interaction between the nanochannel and transport substances; and various forms of energy, such as light, heat, and salt gradient, can drive charge separation within the nanochannel and be converted into electricity by harnessing the ion selectivity of the nanochannel. However, despite the achievements in nanochannel-based technology, in-depth exploration of the mass transport properties at the nanoscale and further expansion of its application remain to be realized. For the development of this technology, four major issues need to be addressed: fabrication of a single-atom-thick nanochannel/nanopore membrane, precise control of the nanochannel structure, effective regulation of the surface properties of the nanochannel, and enrichment and development of mass transport theories. Nanochannel-based technology requires interdisciplinary efforts at the intersection of chemistry, materials science, and nanotechnology, and shows good promise for solving basic problems in biology, environment, and energy. Herein, we briefly describe the mass transport properties of nanochannels and some emerging developments and applications, and finally provide a brief outlook on this field.

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Research progress of electrically-driven force based online rapid separation and enrichment techniques LIU Yulan, CHEN Yali, XIAO Xiaohua, XIA Ling, LI Gongke 2020, 38 (10):  1197-1205.  DOI: 10.3724/SP.J.1123.2020.07026 Abstract ( 56 )   HTML ( 9 )   PDF (1567KB) ( 57 )   Sample preparation is a critical step in complex sample analysis, which enables analyte isolation and preconcentration from a complex matrix. Therefore, sample preparation is an effective approach to enhance the sensitivity, selectivity, and accuracy of the analytical method. However, the transfer of target analytes from a random state in the original sample matrix to a highly ordered pre-analysis state involves an entropy reduction process that cannot occur spontaneously. Therefore, sample preparation is always a time-consuming, labor-intensive, and error-prone process. Introducing additional energy or reducing the entropy of the system can enhance the separation and enrichment effects as well as accelerate sample preparation. The introduction of an electric field into an online sample preparation system can not only introduce additional energy into the system, but also drive the directional migration of the sample among the separation, enrichment, and detection processes, ensuring that the entropy reduction progresses smoothly. These advantages of electrically-driven force based online separation and enrichment techniques make them effective for accelerating sample preparation. Typically, there are four acceleration strategies in electrically-driven force based online separation and enrichment techniques: (1) the additional energy of the electric field is added into the system to accelerate mass transfer and energy exchange; (2) electrically-driven flows, including electroosmotic flow and electrophoretic flow, are applied to drive the directional migration of the sample among the separation, enrichment, and detection processes, ensuring that sample preparation and analysis are executed smoothly; (3) the online integration technique is applied to enhance the automaticity of the entire sample preparation and analysis processes, and reduce errors from manual operation; (4) device miniaturization or size reduction methods such as microextraction are applied to enhance the sample preparation efficiency and reduce the time consumed. This review summarizes the progress in electrically-driven force based online rapid separation and enrichment techniques in the last ten years. In this specific research area, more than one hundred research papers are published each year, and can be classified into three types based on the electrically-driven force based online rapid separation and enrichment techniques considered: capillary, microchip, and membrane extraction. Among these, over 50% of the studies focused on electrically-driven capillary force based online rapid separation and enrichment techniques. By applying a high-voltage electric field at the two ends of a capillary, charged species in the capillary can migrate along the direction of the medium. This makes the electrically-driven capillary technique not only fast, highly efficient, and low-cost, but also effective for rapid sample preparation. Typically, two modes of electrically-driven capillary force based online rapid separation and enrichment techniques are employed: online capillary electrophoretic separation and enrichment, and online microextraction-capillary electrophoresis. Device miniaturization from the capillary to microchip through microelectronic mechanical systems and microfluidics enables small-amount sample preparation and analysis, and is also advantageous due to being rapid and efficient, as well as low energy- and sample-consuming. The specific easy-integration trait of microchip devices enables the online integration of multi-step sample preparation and analysis. On the other hand, the controllable electrically-driven force can be used for both, directional flow transfer between different functional units in the microchip as well as for dynamic control of the electrically fluid pump and valve. Online microchip electrophoretic separation and enrichment, and online microextraction-microchip electrophoresis, are two common modes of microchip electrically-driven force based online rapid separation and enrichment techniques. In electrically-driven membrane extraction online separation and enrichment techniques, a supporting liquid membrane is used to eliminate the matrix interference, enabling large-scale real sample application. These techniques have received increasing attention in the research area of electrically-driven force based online separation and enrichment. Overall, rapid separation and enrichment techniques are highly desired in complex sample analysis, and electrically-driven force based online approaches offer significant application potential, especially in food, the environment, and medicine.

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Advances in capillary-based immobilized enzyme microreactor based on DNA-directed immobilization SONG Jiayi, LI Mengqi, SHEN Hao, ZHOU Zixin, HE Wenting, SU Ping, YANG Yi 2020, 38 (10):  1206-1210.  DOI: 10.3724/SP.J.1123.2020.05035 Abstract ( 84 )   HTML ( 12 )   PDF (1340KB) ( 46 )   Life processes such as metabolism and energy conversion are catalyzed by biological enzymes. The changes of enzymatic activity in organisms can lead various diseases. Thus, it is imperative to develop novel methods of analyzing enzymatic activities for gaining deeper insights into metabolic processes, disease diagnosis, and drug development. Capillary electrophoresis (CE) has the advantages of high separation efficiency, fast analysis speed, and simple operation; moreover, it requires less sample and can be combined with a variety of detection methods. Therefore, CE has attracted increasing attention for enzyme analysis. Enzyme analysis based on CE mainly includes off-line mode and on-line mode. In the off-line mode, the enzyme and substrate are incubated outside the capillary, and then the product is introduced into the CE for analysis. In the on-line mode, the capillary is not only used as a separation channel, but also as an enzyme reaction site. Therefore, the on-line mode facilitates all steps of enzymatic hydrolysis, separation, and detection within a capillary. In the on-line mode, homogeneous analysis method, electrophoretically mediated microanalysis (EMMA), and heterogeneous analysis method, immobilized enzyme microreactor (IMER), were developed. The on-line enzyme analysis method of IMER combined with capillary electrophoresis (CE-IMER) was developed into a mainstream enzyme analysis method. CE-IMER combines the advantages of immobilized enzyme and CE. By immobilizing the free enzyme in capillary, it can not only significantly improve the stability and reusability of enzyme, but also enables the automatic enzyme analysis at nanoscale. This can significantly reduce the cost of enzyme analysis. Although, there are numerous methods to prepare new IMER for enzyme analysis by CE, preparing CE-IMER with good performance, reusability, large enzyme loading, and high degree of automation is the focus of research in this field. DNA-directed immobilization (DDI) makes use of the complementary base pairs (A-T, C-G) of DNA molecules to specifically immobilize biomacromolecules under mild physiological conditions. The enzyme can be immobilized on the carrier surface by DDI and the short double helix DNA molecules possess strong mechanical strength and physicochemical stability. This can form an enzyme microarray, reduce the resistance of mass transfer, improve the contact between enzyme and substrate, and promote the enzymatic analysis process. Compared with the traditional immobilization methods of adsorption, crosslinking, encapsulation, and covalent bonding, DDI can be operated under mild physiological conditions. Further, this can significantly reduce the influence of the immobilization process on the activity, conformation, and stability of the enzyme. Meanwhile, the reversible immobilization process of DDI can regenerate the surface of the carrier, thereby significantly reducing the economic and time cost of IMER preparation. Therefore, DDI is an ideal method to prepare IMER. In this article, the preliminary research and progress of our research group in the field of IMER preparation by DDI technology are presented. At present, the research on the preparation of novel IMER based on DNA nanotechnology, such as DDI, is in the initial stage and there is much scope for development and research. Based on the previous studies, we can focus on the following aspects: (1) building a more efficient catalytic IMER cascade reaction system by immobilizing target enzymes in specific regions of the capillary based on DDI; (2) aiming at the problems existing in the preparation of IMER, such as stability, enzymatic activity, and enzyme immobilization capacity, while taking advantages of DNA structure and nanomaterials to prepare novel IMERs to promote the wide application of CE-IMER in enzyme analysis.

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Unified theory of electroseparations CHEN Yi 2020, 38 (10):  1211-1216.  DOI: 10.3724/SP.J.1123.2020.07037 Abstract ( 49 )   HTML ( 11 )   PDF (1060KB) ( 49 )   Linear electroseparations in vacuum, gas phase and liquid phase have created mass spectrometry, ion mobility spectrometry, electrophoresis and capillary electrophoresis. However, they have not systematized yet, developing independently, with their own theory, not related to each other. In this paper, unified motion equations were derived from the second law of Newtonian mechanics combined with electrostatic theory. From them, the general sub-equations of motion and measurement modes of all the above-mentioned methods were deduced and discussed briefly. These equations are not only helpful to systematize these existing methods but also are usable to deduce and discover novel electro-separation modes.

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Super stable capillary electrophoresis: theory and examples CHEN Yi 2020, 38 (10):  1217-1223.  DOI: 10.3724/SP.J.1123.2020.07038 Abstract ( 52 )   HTML ( 15 )   PDF (3232KB) ( 77 )   Capillary electrophoresis is often plagued by unstable and unrepeatable results. In this paper, some novel modes are derived theoretically and illustrated by separation examples, as expected these modes can resist the change of some conditions under certain prerequisites and obtain super stable electropherograms. They are weighted mobility spectrometry, migrated charge spectrometry, charge density spectrometry, molar charge density spectrometry, diffusion coefficient spectrometry, liquid-phase mass spectrometry, and their ratio spectrometry. The first four are real-time measurement modes while the others are post-experimental modes. These modes need to develop new devices and algorithm, but they are all highly promising and worth of further exploitation.

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Selenium speciation in watermelon by g-C3 N4 enrichment combined with capillary electrophoresis-inductively coupled plasma-mass spectrometry FENG Jinsu, CAO Yupin, MO Guichun, TANG Lifu, DENG Biyang 2020, 38 (10):  1224-1231.  DOI: 10.3724/SP.J.1123.2020.06015 Abstract ( 51 )   HTML ( 9 )   PDF (953KB) ( 44 )   Selenium is one of the essential trace elements in the human body, and it plays a critical role in human health. In this work, 2.0 g melamine was placed in an alumina crucible, which was heated in a box-type resistance furnace for 2 h at 600 ℃, at the heating rate of 3 ℃/min, and then cooled to room temperature. After cooling, yellow graphite phase carbon nitride (g-C3 N4 ) nanosheets were obtained. Subsequently, 500 mg of the nanosheets was dispersed in 50 mL water with ultrasonication for 10 h in order to remove the residual un-exfoliated g-C3 N4 nanoparticles and large-sized nanosheets. The obtained suspension was centrifuged at about 10000 r/min, followed by drying at 60 ℃ to produce g-C3 N4 . The prepared g-C3 N4 was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), and field emission-environmental scanning electron microscopy (SEM) analyses. Given that the selenium content in actual samples is very low, high sensitivity, and accuracy are imperative for selenium detection. The combination of capillary electrophoresis (CE) with inductively coupled plasma-mass spectrometry (ICP-MS) can greatly improve the sensitivity, accuracy, and speed of the analysis. A novel method based on CE-ICP-MS was established for the determination of selenourea (SeUr), L-selenocystine (SeCys2 ), DL-selenomethionine (SeMet), selenite (Se(Ⅳ)), selenate (Se(Ⅵ)), and selenoethionine (SeEt) in watermelon. The selenium species in watermelon were extracted by ultrasonication with pepsin as an extractant and g-C3 N4 enrichment. The enrichment factor of g-C3 N4 ranged from 12 to 29. Six selenium species were completely separated within 11 min in a 100-cm-long capillary with 100 μm internal diameter, at an applied voltage of 22 kV, using a buffer solution of 8 mmol/L NaH2 PO4 -12 mmol/L H3 BO3 -0.2 mmol/L cetyl trimethyl ammonium bromide (CTAB; pH 9.2). The interference in the selenium detection was eliminated using a dynamic reaction cell with CH4 . The linear correlation coefficients of all the selenium species were greater than 0.9995. Under the optimal conditions, the limits of detection (3 σ, σ for standard deviation, as Se) for SeUr, SeCys2 , SeMet, Se(Ⅳ), Se(Ⅵ), and SeEt were 6.2, 30, 11, 8.2, 48, and 5.5 ng/L, respectively. The linear range (as Se) for SeUr, SeCys2 , SeMet, Se(Ⅳ), Se(Ⅵ), and SeEt were 0.017-20 μg/L, 0.091-50 μg/L, 0.032-40 μg/L, 0.023-60 μg/L, 0.015-75 μg/L, and 0.015-30 μg/L, respectively. The recoveries ranged from 96.0% to 106%, and the relative standard deviations (RSDs; n =5) were less than 3%. The developed method is simple, rapid, and sensitive, and it is also suitable for the detection of selenium species in other food and environmental samples.

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Determination of phenylethyl alcohol in rose dew by micellar capillary electrochromatography WANG Weifeng, ZHANG Ying, YANG Junli 2020, 38 (10):  1232-1237.  DOI: 10.3724/SP.J.1123.2020.03029 Abstract ( 70 )   HTML ( 10 )   PDF (880KB) ( 49 )   Rose dew has emerged as one of the superior products in the field of skin care after rose essential oil. However, at present, there is no quality control standard for rose dew. To this end, a micellar capillary electrochromatography (MEKC) method was developed to determine the amount of phenylethyl alcohol, one of the characteristic components of rose dew. The factors affecting the MEKC performance, including the concentrations of borax and sodium dodecyl sulfate (SDS), separation voltage, injection conditions, and detection conditions, were optimized. The capillary length was selected as 48.5 cm, and the effective capillary length was 40 cm. The new capillary was treated successively with methanol, sodium hydroxide (NaOH) solution, and deionized water for 10 min, 60 min, and 30 min when it was used for the first time. Under the running process, the capillary was flushed with 0.5 mol/L NaOH, deionized water, and running buffer solution (10 mmol/L Na2 B2 O7 +15 mmol/L SDS) for 2 min, 2 min, and 3 min each. Between two runs, the capillary was balanced with the running buffer solution for 5 min. Sample injection was performed under a pressure of 5 kPa for 5 s. The separation voltage was set at a positive value of 20 kV. The capillary was maintained at a constant temperature of 20 ℃ using an air refrigeration system. A photo-diode array (PDA) detector with a detection wavelength range of 190-600 nm was coupled to the capillary for monitoring the target molecule, and the optimum wavelength was fixed at 208 nm. Under the optimized conditions, the rose dew samples could be separated and detected within 7 min. The linearity for phenylethyl alcohol detection was found to be 0.50 to 1000 mg/L, with a correlation coefficient (r 2 ) of 0.9990. The limit of detection (LOD, S/N =3) and limit of quantification (LOQ, S/N =10) of the method were calculated to be 0.091 mg/L and 0.35 mg/L, respectively. The accuracy was tested by spiking phenylethyl alcohol into the rose dew samples at mass concentrations of 10, 100, and 500 g/L. The recoveries ranged from 98.1% to 102.7%, and the relative standard deviations (RSD; n =3) were less than 2.8%. This MEKC method is fast, sensitive, inexpensive, and highly effective for the determination of phenylethyl alcohol in rose dew. With the advantages of good stability, anti-matrix interference ability, and high sensitivity, this method represents a simple, sensitive, accurate, and robust strategy for the quality control of rose dew and related products.

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Determination of sulfation degree of heparin and low molecular weight heparins by capillary electrophoresis SHEN Yuting, KANG Jingwu 2020, 38 (10):  1238-1242.  DOI: 10.3724/SP.J.1123.2020.05032 Abstract ( 93 )   HTML ( 15 )   PDF (853KB) ( 63 )   Heparin is composed of a highly sulfated linear saccharide and is widely used as an anticoagulant. Low molecular weight heparins (LMWHs) are derived from the unfractionated heparin (UFH) by enzymatic or chemical degradation. LMWHs have largely replaced heparin as an anticoagulant for treatment and prevention of thrombosis because of the advantages of less bleeding, greater bioavailability, and more predictable anticoagulant effects in comparison to heparin. Enoxaparin, produced by the alkaline degradation of UFH through β-eliminative cleavage, represents the most commonly used LMWH. The structural characteristics of LMWHs differ from their parent heparin not only in terms of molecular weight but also in the sulfation degree as a result of losing the sulfate ester groups during the manufacturing process. The resulting compositional variation directly leads to a fluctuation in anticoagulant activity. In vitro functional assays showed that there is a wide variation in anticoagulant activity among the various LMWHs from different manufacturers owing to slight differences in the manufacturing process. This will directly affect heparin drug safety. In order to ensure the stability of product quality, it is necessary to develop a method for detecting the degree of heparin sulfation to monitor the stability of UFH and processing conditions. During the last two decades, various analytical methods based on chromatography or NMR have been developed for structural characterization of UFH and LMWHs. However, the reported methods require expensive equipment and professional data processing. These limitations make it difficult to apply the current methods to quality control via sulfation degree determination. Herein, we report a simple and robust method for the detection of the sulfation degree of UFH and LMWHs. The determination is based on the separation of building blocks of heparin obtained by exhaustive digestion of UFH and LMWHs in a mixture of heparinases. A mixed solution of heparinase Ⅰ, Ⅱ, and Ⅲ was prepared to give a final content of 0.13 IU/mL for each enzyme. The digestion of enoxaparin and heparin samples was performed at 25 ℃ for 48 h. By using a capillary electrophoresis (CE) method, a total of 18 oligosaccharides building blocks of heparin, including ten disaccharides, one trisaccharide, three tetrasaccharides, and four 1, 6-anhydro derivatives, can be baseline separated. Then, the compositions of enoxaparin and UFH can be precisely determined. Based on the assumption that the molar extinction coefficient of each oligosaccharide at UV 232 nm is the same, the concentration of each oligosaccharide can be conveniently replaced by their peak area, and the accurate number of sulfate ester groups in each disaccharide unit can be determined, hence the average sulfation degree (SD). The developed method allows us to compare the sulfation degree data between the enoxaparin batches from the different manufacturers to evaluate the composition similarity. Herein, eight batches of commercially available enoxaparin from two manufacturers and four batches of UFH source materials were measured. Each sample was measured in triplicate, and the average values as well as the relative standard deviations (RSD) were calculated. The total sulfation degree (T-SD), the individual degree of N-sulfation (N-SD) and O-sulfation (O-SD) data were also determined and compared. A significant difference was observed in the SD of the products from the different manufacturers, which indicated that our method can be used as one of the quantitative compositional analysis methods for quality control of LMWHs and UFH. The variation in terms of the sulfation degree of enoxaparin products from different manufacturers can be precisely identified using this method. This allows us to determine the detailed compositional differences between products from the different manufacturers. The obtained satisfactory data show that high fluctuation in the sulfation degree of UFH could transmit to the final enoxaparin products. The consistency of the products can also be evaluated by using these methods. The CE method has several advantages for quantitative compositional analysis of LMWHs, such as high separation efficiency, high sensitivity, automation, short analysis time and low consumption of both sample and reagents. It has a good application potential in the quality control heparin production.

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Determination of the anticoagulant activity of low molecular weight heparins by micellar electrokinetic chromatography combined with on-column enzymatic reaction ZHANG Mingyu, KANG Jingwu 2020, 38 (10):  1243-1248.  DOI: 10.3724/SP.J.1123.2020.07010 Abstract ( 40 )   HTML ( 5 )   PDF (943KB) ( 33 )   Supporting Information Low molecular weight heparins (LMWHs) have largely replaced heparin for the treatment and prevention of thrombosis because of their various advantages over unfractionated heparins (UFHs) such as less bleeding, greater bioavailability, and more predictable anticoagulant effects. For special groups of patients, such as pregnant women, children, and patients with renal failure, it is necessary to monitor the anticoagulant activity of LMWHs in the blood. The traditional method used to determine the anticoagulant activity of heparin is the coagulation test. However, the results are various from different laboratories and different reagents. In contrast, the chromogenic substrate method is more accurate, sensitive and is easy to automate. Here, a method for the determination of the anticoagulant activity of LMWHs was developed by using a capillary-electrophoresis-based substrate chromogenic method. In this method, micellar electrokinetic chromatography (MEKC) was used in combination with electrophoretically mediated microanalysis to determine the anti-factor Xa (FXa) activity of LMWHs. The inhibition was measured by employing a chromogenic peptide substrate (CPS) with a p -nitroaniline (p -NA) moiety as the chromophore. The injection end of the capillary was used as a microreactor in which solutions of LMWHs, antithrombin Ⅲ (ATⅢ), FXa and CPS were successively injected and mixed by using diffusion, the transverse diffusion of laminar flow profiles and applied voltage. The reaction product p -NA was separated from unreacted CPS and sample matrix by using the MEKC mode with discontinuous background electrolyte system. The produced p -NA was baseline separated from the other components and detected at 380 nm to obtain maximum sensitivity. The amount of p -NA was inversely proportional to the activity of LMWHs in the sample. To improve the accuracy of quantification and the method repeatability of methods, nitrofurantoin (NF) was selected as the internal standard, which was added to the solution of CPS. The method was validated and used to measure a set of samples. The method is characterized by automation, good repeatability, high sensitivity, and cost-effectiveness. Additionally, the method does not interfere by the sample matrix, and thus can be used to monitor the anticoagulant activity of LMWHs in plasma.

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Identification of the relative age of iron gall ink in handwriting by capillary electrophoresis using the Fe(Ⅱ)/Fe(Ⅲ) ratio CAI Yu, CAO Chengxi, ZHUO Xianyi, LI Honggen, FAN Liuyin 2020, 38 (10):  1249-1255.  DOI: 10.3724/SP.J.1123.2020.05033 Abstract ( 51 )   HTML ( 9 )   PDF (897KB) ( 45 )   Identifying the relative age of iron gall ink in the handwriting on a questioned file is highly significant for court science, because it serves as important evidence for solving criminal cases and in confirming the authenticity of historical documents. This is because many criminal cases involve analysis of forged documents to conclude whether an entire document is as old as purported, or whether the entire text in the document was written at the same time. In this paper, a novel approach based on capillary electrophoresis (CE) to estimate the relative age of iron gall ink-written texts is discussed. Two kinds of chelating agents, 1, 10-phen and CDTA, were used for the simultaneous determination of Fe(Ⅱ) and Fe(Ⅲ) by CE. The stability constants of [Fe(Ⅱ)-(phen)3 ]2+ and [Fe(Ⅱ)-CDTA]2- complexes are log β3 =21.3 and log K =18.2, respectively, while the corresponding values of [Fe(Ⅲ)-(phen)3 ]3+ and [Fe(Ⅲ)-CDTA]- complexes are log β3 =14.1 and log K =29.3. First, specific binding between the two kinds of chelating agents and the ferrous/ferric ions was investigated. The results confirmed specific binding between Fe(Ⅱ) and 1, 10-phen as well as that between Fe(Ⅲ) and CDTA. Preliminary studies also showed that Fe(Ⅱ) in the iron gall ink was relatively stable in the ink tank due to the low pH of commercial inks; hence, the oxidation of Fe(Ⅱ) in the tank was considered to be negligible. However, when the gall ink was exposed on a paper, sulfuric acid in the ink was gradually consumed by the cellulose of paper, thus causing gradual oxidation of Fe(Ⅱ) in the written text. Changes in the peak area ratio of Fe(Ⅱ) and Fe(Ⅲ) with aging were monitored: the older the ink in the writing, the smaller is the Fe(Ⅱ)/Fe(Ⅲ) ratio. Hence, the Fe(Ⅱ)/Fe(Ⅲ) ratio could be used for estimating the relative age of iron gall ink in writing. The Fe(Ⅱ)/Fe(Ⅲ) ratio was determined by CE, and the ratio extracted from the questioned handwriting ink was compared with that extracted from the entire document to confirm whether the entire text written at the same time. The keys to the success of this technique are establishing a suitable procedure for extracting the Fe(Ⅱ) and Fe(Ⅲ) species in the handwriting ink and a CE separation procedure. The optimized sample pretreatment procedure is as follows: (1) an ink-drawn line of 1 cm length was cut and placed in a 2 mL Eppendorf tube; (2) then, 0.5 mL of 5.0 mmol/L 1, 10-phen was added to the EP tube for chelation with Fe(Ⅱ), and the mixture was subjected to vibration on a vortex mixer; (3) within 60 s, 0.5 mL of 20 mmol/L CDTA was added to the sample tube for chelation with Fe(Ⅲ); (4) the tube was strongly vibrated for 10 min on a vortex mixer; (5) after centrifugation at 10000 r/min for 15 min, the supernatant was decanted into another tube for CE analysis. The optimized conditions for the CE analysis are as follows: 100 mmol/L of pH 9.2 H3 BO3 -Na2 B4 O7 buffer, 20 kV applied voltage, sample injection (1.379 kPa, 5s), fused-silica capillary dimensions 40.2 cm×75 μm i.d. (30 cm to the detector), and 254 nm detection wavelength. Meanwhile, small amounts of 1, 10-phen and CDTA were added to the buffer solution to ensure stability of the formed complexes during the CE run in the capillary and to maintain the metal ions in their original oxidation state. Finally, two kinds of iron gall ink samples were tested to evaluate the applicability of the developed method. The Fe(Ⅱ)/Fe(Ⅲ) ratios of ink sample 1 and ink sample 2 changed from 1.79 to 0.45 and from 2.67 to 0.3, respectively, from the 1st day to the 75th day after writing. The results demonstrate that the developed method can be used to highlight fraudulent insertion of information and provide important guidance for the forensic analysis of the relative age of gall ink in handwriting.