Colorimetric detection of alkaline phosphatase based on the off-on effect of light-responsive oxidase mimicking activity of covalent organic framework (Cu-TpBpy-COF) under near-neutral condition.

A colorimetric strategy has been developed for the detection of alkaline phosphatase (ALP) activity based on the off-on effect of the catalytic activity of light-responsive oxidase mimics covalent organic framework (Cu-TpBpy-COF) in near-neutral condition. Cu-TpBpy-COF can effectively catalyze the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to form a blue oxidized product (oxTMB) with an absorption peak at 652 nm. Cu2+ is the active center of Cu-TpBpy-COF and pyrophosphate (PPi) can form a complex with Cu2+ to weaken the catalytic activity of Cu-TpBpy-COF. In the presence of ALP, PPi is hydrolyzed into orthophosphates (Pi) with low affinity to Cu2+, thus resulting in absorbance restoration. The absorbance at 652 nm is related to ALP activity in the linear range 10-150 U·L-1 with a detection limit of 7.17 U·L-1. The recoveries of ALP in serum samples are in the range 94.7~107.0% with relative standard deviations (RSD) lower than 5%. The decisive role of Cu2+ on the enhancing catalytic activities of Cu-TpBpy-COF in neutral condition was verified by TpBpy-COF and TpBD-COF as controls, in which the main difference between them is that TpBpy-COF contains pyridine nitrogen. Upon Cu2+ modification, Cu-TpBpy-COF has better catalytic activity than TpBpy-COF in a broader pH range because of the in situ generation of Cu+ under irradiation.

Preparation of D-histidine modified zeolitic imidazolate framework-90 coated capillary column and its application in open-tube capillary electrochromatography enantioseparation.

Metal-organic framework materials are a class of novel crystalline porous materials with regular pore structures formed by covalent bonding between metal centers and organic functional groups. Metal-organic framework materials have attracted great interest in analytical chemistry due to their unique properties such as good stability and permanent porosity. In this work, D-histidine was used to carry out chiral modification of zeolitic imidazolate framework-90 under mild conditions, and the D-histidine modified zeolitic imidazolate framework-90 coated capillary column was prepared. This chiral capillary column was used to separate epinephrine, norepinephrine, terbutaline, and tryptophan enantiomers. Under optimum conditions, baseline separations were achieved. The intra-day, inter-day, and inter-column relative standard deviations (n = 3) of the four pairs of enantiomeric migration times were 0.15%-0.56%, 0.74%-2.40%, and 1.93%-3.18%, respectively. Moreover, the D-histidine modified zeolitic imidazolate framework-90 coated capillary could be reused for at least 150 runs without significant changes in the separation efficiency and migration time.

Generation of 3'-OH terminal-triggered encoding of multicolor fluorescence for simultaneous detection of different DNA glycosylases.

Uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG) are the important DNA glycosylases for initiating the repair of DNA damage, and the aberrant expression of DNA glycosylases is closely associated with various diseases, such as Parkinson's disease, several cancers, and human immunodeficiency. The simultaneous detection of UDG and hAAG is helpful for the study of early clinical diagnosis. However, the reported methods for multiple DNA glycosylase assay suffer from the application of an expensive single-molecule instrument, labor-tedious magnetic separation, and complicated design. Herein, we develop a simple fluorescence method with only three necessary DNA strands for the selective and sensitive detection of multiple DNA glycosylase activity based on the generation of 3'-OH terminal-triggered encoding of multicolor fluorescence. The method can achieve the detection limits of 5.5 × 10-5 U/mL for UDG and 3.3 × 10-3 U/mL for hAAG, which are lower than those of the reported fluorescence methods. Moreover, it can be further used to detect multiple DNA glycosylases in the human cervical carcinoma cell line (HeLa cells), normal human renal epithelial cells (293 T cells), and biological fluid and measure the enzyme kinetic parameters of UDG and hAAG.

Characterization of the Ligand Exchange Reactions on CdSe/ZnS QDs by Capillary Electrophoresis.

The continuous development of semiconductor quantum dots (QDs) in biochemical research has attracted special attention, and surface functionalizing becomes more important to optimize their performance. Ligand exchange reactions are commonly used to modify the surface of QDs for their biomedical applications. However, the kinetics of ligand exchange for semiconductor QDs remain fully unexplored. Here, we describe a simple and rapid method to characterize the ligand exchange reactions on CdSe/ZnS QDs by capillary electrophoresis (CE). The results of ultraviolet-visible absorption spectra, fluorescence spectra, and Fourier transform infrared spectroscopy indicated the successful implementation of the ligand exchange process. The dynamics of ligand exchange of OA-coated CdSe/ZnS QDs with 4-mercaptobenzoic acid was monitored by CE, and the observed ligand exchange trends were fitted with logistic functions. When the ligand exchange reactions reached equilibrium, the ligand density of QDs can be quantified by CE. It is anticipated that CE will be a new powerful technique for quantitative analysis of the ligand exchange reactions on the surface of QDs.

Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase.

DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies. Thus, the detection of DG activity is essential for biomedical research and clinical diagnosis. Herein, we develop a facile fluorometric method based on the base excision repair (BER) mediated cascading triple-signal amplification for the sensitive detection of DG. The presence of human alkyladenine DNA glycosylase (hAAG) can initiate the cleavage of the substrate at the mismatched deoxyinosine site by endonuclease IV (Endo IV), resulting in the breaking of the DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate strand displacement amplification (SDA) to release primers. The released primers can further bind to a circular template to induce an exponential primer generation rolling circle amplification (PG-RCA) reaction, producing a large number of primers. The primers that resulted from the SDA and PG-RCA reaction can induce the subsequent recycling cleavage of signal probes, leading to the generation of a fluorescence signal. Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification, this method exhibits a low detection limit of 0.026 U mL-1 and a large dynamic range of 4 orders of magnitude for hAAG. Moreover, this method has distinct advantages of simplicity and low cost, and it can further quantify the hAAG activity from HeLa cell extracts, holding great potential in clinical diagnosis and biomedical research.

A peptide-WS2 nanosheet based biosensing platform for determination of ß-secretase and screening of its inhibitors.

ß-Secretase (BACE1) is an important drug target in the treatment of Alzheimer's disease (AD). Therefore, sensitive detection of BACE1 is essential for AD treatment and drug discovery. In this work, a facile and sensitive fluorescence biosensing platform was developed for BACE1 detection. This sensing platform was constituted based on the interaction between a WS2 nanosheet and a peptide sequence. In the absence of BACE1, a FAM-labeled peptide substrate could be adsorbed on the surface of the WS2 nanosheet, thereby quenching its fluorescence. However, in the presence of BACE1, the hydrolysis of the peptide substrate by BACE1 triggers could occur with the subsequent release of short FAM-linked peptide fragments which could not be adsorbed on the surface of the WS2 nanosheet. This resulted in weak fluorescence quenching, thus restoring the fluorescence signal. By measuring the change in the fluorescence of the FAM-labeled peptide substrate, the fluorescence sensing platform based on the WS2 nanosheet could monitor BACE1. The proposed WS2 nanosheet-based platform exhibited excellent specificity and high sensitivity with a detection limit of 66 pM for BACE1. Importantly, we also demonstrated that this platform was suitable for the screening of BACE1 inhibitors. The proposed sensing platform not only provides a novel strategy for the BACE1 assay but also offers a potential tool for screening drugs.

A label-free colorimetric biosensor for sensitive detection of vascular endothelial growth factor-165.

Sensitive detection of a low abundant protein is essential for biomedical research and clinical diagnostics. Herein, we develop a label-free colorimetric biosensor for the sensitive detection of recombinant human vascular endothelial growth factor-165 (VEGF165). This biosensor consists of an aptamer-based hairpin probe, an assistant DNA-trigger duplex and a linear template. In the presence of VEGF165, the specific binding of VEGF165 with the aptamer-based hairpin probe results in the opening of a hairpin probe and the opened hairpin probe subsequently hybridizes with the single-stranded region of the assistant DNA-trigger duplex to initiate the strand displacement amplification (SDA) to yield abundant triggers. The released triggers can further function as the primers to anneal with the hairpin probe and lead to the opening of the hairpin structure, which subsequently hybridizes with the assistant DNA-trigger duplex to initiate the next round of SDA reaction and generates more triggers. Large amounts of triggers could be generated by the synergistic operation of dual SDA reaction, and the obtained triggers can initiate a new round of SDA reaction to yield numerous G-quadruplex DNAzymes, which subsequently catalyze the conversion of ABTS2- to ABTS˙- by H2O2 to yield a color change with the assistance of a cofactor hemin. In contrast, in the absence of target VEGF165, the hairpin probe, the assistant DNA-trigger duplex and the linear template can stably coexist in solution, and thus no color change is observed because no trigger can initiate SDA to generate the G-quadruplex DNAzyme. This biosensor has a low detection limit of 1.70 pM and a dynamic range over 3 orders of magnitude from 24.00 pM to 11.25 nM. Moreover, the biosensor shows excellent specificity toward the target VEGF165 and the entire reaction can be carried out in an isothermal manner without the involvement of a high precision thermal cycler, making the current assay extremely cost effective.

Detection of biogenic amines in C57BL/6 mice brain by capillary electrophoresis electrokinetic supercharging.

Ischemic stroke is caused when blood flow to the brain is stopped and is a major cause of death and long term disability across the globe. Excessive release of neurotransmitters is triggered in the brain by ischemia that mediates neuronal damage and causes ischemic injury. In this study, a simple, sensitive, and on-line preconcentration capillary electrophoresis method based on electrokinetic supercharging (EKS) was developed for the determination of the biogenic amines including dopamine (DA), epinephrine (E), and norepinephrine (NE) in C57BL/6 mice brain. Under the optimized conditions, the analytes were concentrated and detected within 10 min. The detection limits for the analytes ranged from 0.42 to 0.57 ng mL(-1) for a mice brain matrix. With the proposed method, the analyses of three neurochemical amines in C57BL/6 mice brain tissue during cerebral ischemic/reperfusion had been performed successfully.

Determination of enantiomers by FESI-sweeping with an acid-labile sweeper in nonaqueous capillary electrophoresis.

In this work, a facile and highly efficient on-line concentration strategy based on a coupling of field enhanced sample injection (FESI) and sweeping was developed for the determination of trace enantiomers (propranolol, PL) by nonaqueous capillary electrophoresis (NACE). In this FESI-sweeping method, the use of a sample of high acidity and low conductivity (pH* = 2.5, 4.0 µS cm(-1)) allowed for a large amount of analyte injection. Then, the concentration of the analytes was carried out by sweeping based on the interaction of an acid-labile anionic selector, di-n-butyl L-tartrate-boric acid complex acid, and cationic analytes. Simultaneously, the concentrated analytes were released and focused at the boundary of the acid sample solution and separation buffer due to the decomposition of the selector in the acid sample solution. Under the optimum conditions, a 21,000-fold sensitivity enhancement upon normal capillary zone electrophoresis (CZE) was achieved for PL enantiomers. The detection limits of R-propranolol and S-propranolol were 0.26 ng mL(-1) and 0.31 ng mL(-1), respectively. Eventually, the FESI-sweeping method was applied to detect PL enantiomers in plasma, saliva, and urine.

Recent developments in chiral analysis of ß-blocker drugs by capillary electromigration techniques.

The latest developments in chiral analysis of ß-blocker drugs by capillary electromigration techniques are reviewed in this article. Following the previous review by Aturki et al. [Electrophoresis 2011, 32, 2602-2628], this review includes the papers published during the period from January 2011 to December 2013. During this time, some novel chiral selectors were reported and applied to improve the enantioseparation of ß-blocker drugs and structurally related compounds. These chiral selectors include CDs and their derivatives, macrocyclic antibiotics, tartrate complexs, the monolithic molecularly imprinted polymer, and the polymeric surfactants. In addition, this article summarizes the methodological improvements for enhancing sensitivity in chiral analysis of ß-blockers and structurally related compounds by CE. The involved authors described the use of online sample preconcentration techniques to increase the detection sensitivity in the enantiomeric analysis of a broad range of samples.

Sensitive enantioanalysis of ß-blockers via field-amplified sample injection combined with water removal in microemulsion electrokinetic chromatography.

In this study, an on-line sample preconcentration technique, field-amplified sample injection combined with water removal by electroosmotic flow (EOF) pump, was applied to realize a highly sensitive chiral analysis of ß-blocker enantiomers by MEEKC. The introduction of a water plug in capillary before the electrokinetic injection provided the effective preconcentration of chiral compounds. And then the water was moving out of the column from the injection end under the effect of the EOF, which avoided dilution of the stacked ß-blocker enantiomers concentration suffering from the presence of water in separation buffer. Moreover, the addition of H3 PO4 and methanol in the sample solution greatly improved the enhancement efficiency further. Under optimized conditions, more than 2700-fold enhancement in sensitivity was obtained for each enantiomer of bupranolol (BU), alprenolol (AL), and propranolol (PRO) via electrokinetic injection. LODs were 0.10, 0.10, 0.12, 0.11, 0.02, and 0.02 ng/mL for S-BU, R-BU, S-AL, R-AL, S-PRO, and R-PRO, respectively. Eventually, the proposed method was successfully applied to the determination of BU, AL, and PRO in serum samples with good recoveries ranging from 93.4 to 98.2%.

Construction of dual exponential amplification accompanied by multi-terminal signal output method for convenient detection of tumor biomarker FEN1 activity.

As an important 5'-nuclease in DNA replication and damage repair, Flap endonuclease 1 (FEN1) has been considered as a potential tumor biomarker due to its overexpression in different human cancer cells. Here, we developed a convenient fluorescent method based on dual enzymatic repairing exponential amplification accompanied by multi-terminal signal output to realize the rapid and sensitive detection of FEN1. In the presence of FEN1, the double-branched substrate could be cleaved to produce 5' flap single strand DNA (ssDNA) which subsequently was used as a primer to initiate the dual exponential amplification (EXPAR) to generate abundant ssDNAs (X' and Y'), then the ssDNAs can respectively hybridize with the 3' and 5' ends of the signal probe to form partially complementary double strands (dsDNAs). Subsequently, the signal probe on the dsDNAs could be digested under the assistance of Bst. polymerase and T7 exonuclease, as well as releasing the fluorescence signals. The method displayed high sensitivity with the detection limit of 9.7 × 10-3 U mL-1 (1.94 × 10-4 U) and also exhibited good selectivity towards FEN1 under the challenge from complicated samples including extracts of normal and cancer cells. Furthermore, it was successfully applied to screen FEN1 inhibitors, holding great promise in the screening of potential drugs targeting FEN1. This sensitive, selective and convenient method could be used for FEN1 assay without the complicated nanomaterial synthesis/modification, showing great potential in FEN1- related prediction and diagnosis.

Enantioseparation in capillary eletrochromatography by covalent organic framework coating prepared in situ.

Chiral covalent organic frameworks (CCOFs) have recently exhibited particularly promising potential as effective chiral stationary phases (CSPs) for open tubular capillary electrochromatography (OT-CEC) enantioseparation. However, it remains difficult to synthesis of CCOFs and preparation of CCOFs coated capillary under mild reaction conditions. In this work, we designed and fabricated a CCOF (CB-DA-COF) with high chemical stability and high specific surface area at room temperature. Then, through one-step in situ growth method, the chiral CB-DA-COF coated capillary was fabricated at room temperature for the first time. This method requires neither pre-modification to the capillary by organic molecular building units nor harsh reaction conditions, and the preparation time of the CCOF coating was significantly shortened (within 2 h). This chiral CB-DA-COF coated capillary showed excellent enantioseparation ability and stability. Under optimal conditions, rapid enantioseparation (within 5 min) could be achieved for six enantiomers including terbutaline, propranolol, phenylephrine, verapamil, norepinephrine and isoprenaline. And, no significant change was observed in enantioseparation efficiency after over 200 runs. The relative standard deviations (RSDs) of the analyte's migration time for intra-day, inter-day and column-to-column were within the range of 0.8-3.5% (n = 5), 1.5-4.7% (n = 3) and 4.3-8.3% (n = 3), respectively. In addition, the enantioseparation mechanism was studied, which indicated that binding energy between of enantiomers and chiral site were the main factors for enantioseparation.

A Reverse Transcription Recombinase-Aided Amplification Method for Rapid and Point-of-Care Detection of SARS-CoV-2, including Variants.

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its emergence of variants needs rapid and point-of-care testing methods for a broad diagnosis. The regular RT-qPCR is time-consuming and limited in central laboratories, so a broad and large-scale screening requirement calls for rapid and in situ methods. In this regard, a reverse transcription recombinase-aided amplification (RT-RAA) is proposed here for the rapid and point-of-care detection of SARS-CoV-2. A set of highly conserved primers and probes targeting more than 98% of SARS-CoV-2 strains, including currently circulating variants (four variants of concerns (VOCs) and three variants of interest (VOIs)), was used in this study. With the preferred primers, the RT-RAA assay showed a 100% specificity to SARS-CoV-2 from eight other respiratory RNA viruses. Moreover, the assay here is of a high sensitivity and 0.48 copies/µL can be detected within 25 min at a constant temperature (42 °C), which can be realized on portable equipment. Furthermore, the RT-RAA assay demonstrated its high agreement for the detection of SARS-CoV-2 in clinical specimens compared with RT-qPCR. The rapid, simple and point-of-care RT-RAA method is expected to be an appealing detection tool to detect SARS-CoV-2, including variants, in clinical diagnostic applications.

Sensitive homogeneous fluorescent detection of DNA glycosylase by target-triggering ligation-dependent tricyclic cascade amplification.

Abnormal DNA glycosylases are concerned with the aging process as well as numerous pathologies in humans. Herein, a sensitive fluorescence method utilizing target-induced ligation-dependent tricyclic cascade amplification reaction was developed for the detecting DNA glycosylase activity. The presence of DNA glycosylase triggered the cleavage of damaged base in hairpin substrate, successively activating ligation-dependent strand displacement amplification (SDA) and exponential amplification reaction (EXPAR) for the generation of large amount of reporter probes. The resultant reporter probes bound with the signal probes to form stable dsDNA duplexes. And then the signal probes could be digested circularly in the dsDNA duplexes by T7 exonuclease, leading to the generation of an enhanced fluorescence signal. Due to the high efficiency of tricyclic cascade amplification and the low background signal deriving from the inhibition of nonspecific amplification, this method exhibited a detection limit of 0.14 U/mL and a dynamic range from 0.16 to 8.0 U/mL. Moreover, it could be applied for detecting DNA glycosylase activity in human serum with good selectivity and high sensitivity, and even quantifying other types of enzyme with 5'-PO4 residue cleavage product by rationally designing the corresponding substrate. Importantly, this method could be performed in homogenous solution without any complicated separation steps, providing a new strategy for DNA glycosylase-related biomedical research.

Stable and Reusable Light-Responsive Reduced Covalent Organic Framework (COF-300-AR) as a Oxidase-Mimicking Catalyst for GSH Detection in Cell Lysate.

Covalent organic frameworks (COFs), as one of the most significant members of the porous organic frameworks, have been well used in the photocatalysis owing to their outspread π-conjugated framework, high crystallinity and regular pore structure. Herein, after reducing the labile imine-linked COF-300 to the more stable amine-linked COF-300-AR, we for the first time demonstrated that COF-300-AR was the light-responsive oxidase mimic. COF-300-AR exhibited excellent oxidase-mimicking activity under purple light stimulation (λ = 400 nm), which can catalyze the oxidation of classical substrates such as 3,3',5,5'-tetramethylbenzydine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by the formation of •OH and O2•- free radicals in the presence of dissolved oxygen. The COF-300-AR oxidase mimic has outstanding advantages of easy light control, high stability, good reusability, and highly catalytic oxidation capacity and has been applied to detect glutathione (GSH) levels in HL60 cells with good selectivity and high sensitivity. This study will broaden the sensing applications of COFs and offer a promising build block for the construction of artificial enzymes.

Extremely large volume electrokinetic stacking of cationic molecules in MEKC by EOF modulation with strong acids in sample solutions.

In this report, a novel facile way of online preconcentration of trace levels of analytes in capillary electrophoresis is presented. The proposed strategy is based on the combination of strong acidic phosphate as sample buffers with borate separation buffer containing sodium dodecyl sulfate. When injection voltage is applied, the continuous introduction of low pH sample causes the apparent bulk flow inside the capillary gradually slows down. Finally at a certain point, it reaches the same magnitude as that of the oppositely migrating anionic micelles, thus the frontier of the micelle zone becomes stagnant. This steady state can be maintained for a very long time so that essentially extremely large volume of sample solutions can be injected into the capillary, and the cationic analytes may be efficiently stacked at the neutralized micelle zone. A theoretical model was proposed and preconcentration conditions of two model analytes, matrine and oxymatrine, were optimized with the aid of the model. Under optimized conditions, more than 1000-fold increase in sensitivity was obtained as compared with the normal hydrodynamic injection without sample stacking. The limits of detection for oxymatrine and matrine were 0.81 and 0.18 ng/mL, respectively, using photodiode array UV detection at wavelength 211 nm.

Development of a cascade isothermal amplification approach for the sensitive detection of DNA methyltransferase.

DNA methyltransferase (MTase) is an important epigenetic modification enzyme responsible for DNA methylation, and the dysregulation of DNA MTase activity is associated with various diseases in humans. Herein, we take advantage of the DNA lesion repair mechanism in vivo to develop a new fluorescence approach for the specific and sensitive detection of DNA methyltransferase (DNA MTase) on the basis of the DNA lesion repair-directed cascade isothermal amplification. Due to the high amplification efficiency of the uracil repair-mediated exponential isothermal amplification reaction (EXPAR), the efficient cleavage of endonuclease IV (Endo IV)-induced cyclic catalysis, and the low background signal caused by single uracil repair-mediated inhibition of nonspecific amplification, this approach exhibits high sensitivity with a detection limit of 0.014 U mL-1 for pure Dam MTase and 0.61 × 10-6 mg mL-1 for Dam MTase in E. coli cells and it can be further applied for the screening of DNA MTase inhibitors. More importantly, this approach can be applied to detect other DNA MTases by designing appropriate substrates, showing great potential in biomedical research and clinical diagnosis.

Target-mediated hyperbranched amplification for sensitive detection of human alkyladenine DNA glycosylase from HeLa cells.

Human alkyladenine DNA glycosylase (hAAG) is an important protein enzyme which can specifically recognize and initiate the repair of a variety of alkylated purines and hypoxanthine, and the dysregulation of hAAG activity is associated with various human diseases. Although there are several methods focusing on hAAG detection, they share common defects such as time-consuming protocols, laborious operation or requirement of expensive analytical instruments. Herein, taking advantage of the high amplification efficiency of hyperbranched signal amplification and the low background signals by modifying NH2 at 3' terminus of hairpin substrate and signal probe to prevent the terminal deoxynucleotidyl transferase (TdT)-activated nonspecific amplification, a fluoresence method for sensitive detection of hAAG was established using TdT-activated Endonuclease IV (Endo IV)-assisted hyperbranched signal amplification. This method exhibits high sensitivity with a limit of detection of 0.090 U/mL for pure hAAG and shows a large dynamic range of 3 orders of magnitude from 0.1 to 50 U/mL, and it can be applied for accurate detection of hAAG in complicated HeLa nuclear extract. Moreover, the method can be used for discrimination of hAAG from other DNA glycosylases, holding great potential in hAAG-related biomedical research and clinical diagnosis.

Rapid and mild fabrication of protein membrane coated capillary based on supramolecular assemble for chiral separation in capillary electrochromatography.

Exploration of the simple and stable coating methods is of great significance in capillary electrochromatography (CEC). In this work, a lysozyme assemble supramolecular membrane coated capillary column was developed for CEC chiral separation. Taking advantage of phase transformation of lysozyme, the coating process was achieved within 1.0 h thus to a large extent reduced the capillary preparation time and simplified the coating procedure. The successful fabrication of the supramolecular membrane coated capillary was verified by scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), fluorescence imaging, and electroosmotic flow (EOF). The separation capacity of the coated capillary was evaluated by analysis of different chiral analytes, including chiral amine drug and neurotransmitters, and good enantioseparation efficiency was achieved for the three pairs of enantiomers. For three consecutive runs, the relative standard deviations (RSD) for the migration time of the analytes for intra-day (n = 3), inter-day (n = 3) and column-to-column (n = 3) were in the range of 0.7-1.5%, 2.7-3.6%, and 4.5-5.8%, respectively. Additionally, the supramolecular membrane coated capillary column could run consecutively 100 times without observable change in the separation efficiency, proving the feasibility of the coating method based on the adhesion of the protein-based supramolecular membrane.