Microchip Capillary Electrophoresis Research Articles

Design of experiments for micellar electrokinetic chromatography method development for the monitoring of water-soluble vitamins in cell culture medium.

Biopharmaceutical production takes place in complex processes which should be thoroughly understood. Therefore, the iConsensus project focuses on developing a monitoring platform integrating several process analytical technology tools for integrated, automated monitoring of the biopharmaceutical process. Water-soluble vitamin monitoring using (microchip) capillary electrophoresis (CE) is part of this platform. This work comprises the development of conventional CE methods as the first part towards integrated vitamin monitoring. The vitamins were divided based on their physical-chemical properties to develop two robust methods. Previously, a method for the analysis of cationic vitamins (pyridoxine, pyridoxal, pyridoxamine, thiamine and nicotinamide) in cell culture medium was developed. This work focused on the development of a micellar electrokinetic chromatography method for anionic and neutral vitamins (riboflavin, d-calcium pantothenate, biotin, folic acid, cyanocobalamin and ascorbic acid). By employing multivariate design of experiments, the background electrolyte (BGE) could be optimised within one experiment testing only 11 BGEs. The optimised BGE conditions were 200mM borate with 77mM sodium dodecyl sulphate at a pH of 8.6. Using this BGE, all above-mentioned cationic, anionic and neutral vitamins could be separated in clean samples. In cell culture medium, most anionic and neutral vitamins could be separated. Combining the two methods allows for analysis of cationic, anionic and neutral vitamins in cell culture medium samples. The next step towards integrated vitamin monitoring includes transfer to microchip CE. Due to the lack of fast and reliable methods for vitamin monitoring, the developed capillary methods could be valuable as stand-alone at-line process analytical technology solutions as well.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications-Updated and completely revised edition.

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

Determination of rhein and physcion in rhubarb by microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection.

Microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection was developed for the separation and detection of physcion and rhein in rhubarb. In contrast to the conventional capillary electrophoresis method, ammonium acetate-dimethyl sulfoxide was used as the basic buffer system in this method. The effects of background buffer, buffer apparent pH*, buffer concentration, water ratio, sample preparation method, and separation voltage on separation and detection were investigated. Optimized separation and detection conditions were obtained: the buffer consisted of 20mmol/L of ammonium acetate in hydro-organic solvent composed dimethyl sulfoxide, formamide, and water mixed at 60/20/20 (v/v/v) ratio. The separation voltage was 1.9kV. Under these conditions, the physcion, rhein, and other components of rhubarb can be completely separated within 150 s. Under the methodological verification, good linearity (R ≥ 0.9995) for physcion and rhein, and low limits of detection (0.085μg·mL-1 and 0.077μg·mL-1 , respectively), satisfactory peak area precisions, migration time precisions (1.74%-3.09%), and accuracy (recovery rate 97.8% and 101.4%) were achieved. It is shown that the proposed method is simple, efficient, fast, sensitive, simple instrument, consumes few samples, has low operating cost, and is linear.

Micro-extraction, pre-concentration, and microfluidic-based separation of organophosphate insecticides followed by the miniaturized electrochemical detection system

Introduction: A new analytical method based on the coupling of microextraction and microfluidics was developed and investigated for the pre-concentration, separation, and electrochemical detection of fenitrothion (FT) and parathion (PA) at the sub-ppm concentrations. Method: In the first step, the microchip capillary electrophoresis technique was used to serve as a separation and detection system. Analytes were injected into the 40 mm long microchannel with 10 mm sidearms. Then, they were separated by applying a direct electrical field (+1800V) between buffer and detection reservoirs. 2-(n-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 5) was used as the running buffer. The electrochemical detection was performed using three Pt microelectrodes with the width of working, counter, and reference electrodes (50, 250, and 250 µm, respectively) in the out channel approach. Results: The system was devised to have the optimum detection potential equal to -1.2 V vs. Pseudo Ref. electrode. The dimensions of the SU-8 channel have 20 µm depth and 50 µm width. In the second step, an air-assisted liquid-liquid microextraction technique was used for the extraction and preconcentration of analytes from human blood plasma. Then, 1, 2 di-bromoethan was used as extractant solvent, the analytes were preconcentrated, and the sedimented solvent (50 µL) was evaporated in a 60 ˚C water bath followed by substitution of running buffer containing 10% ethanol. The optimum extraction cycles were found 8 with adding 1% NaCl to the aqueous phase. Analyzing time of the mentioned analytes was less than 100s, and the precision range was 3.3 – 8.2 with the linear range of 0.8 – 100 ppm and 1.2 - 100 ppm for FT and PA, respectively. The extraction recoveries were about 91% and 87% for FT and PA, respectively. The detection limits for FT and PA were 240 and 360 ppb, respectively. Finally, the reliability of the method was investigated by GC-FID. Conclusion: The proposed method and device were validated and can be used as in situ and portable detection system for detecting fenitrothion and parathion insecticides.

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid-2018-2022).

This review gives a wide overview of recent advances and applications of capillary electrophoresis and microchip capillary electrophoresis methods in the fields of proteomics and peptidomics in the period from mid-2018 up to the end of 2022. The methodological topics covering sample preparation and concentration techniques, hyphenation of capillary electrophoresis methods with mass spectrometry, and multidimensional separations by on-line or off-line coupled different capillary electrophoresis and liquid chromatography techniques are described and new developments in both bottom-up and top-down approaches in proteomics are presented. In addition, various applications of capillary electrophoresis methods in proteomic and peptidomic studies are demonstrated. They include monitoring of protein posttranslational modifications and applications in biological and biochemical research, clinical peptidomics and proteomics, and food analysis.

Systematic analytical workflow for characterisation and identification of partially reduced species in monoclonal antibody manufacturing

Fragmentation is a major degradation pathway ubiquitous to all therapeutic monoclonal antibody (mAb) and therefore, monitored throughout the manufacturing process. Here, we describe a three-step approach to 1) detect, 2) confirm and 3) characterize partially reduced fragment species in an immunoglobulin G1 (IgG1) mAb with prolonged hold time of harvested cell culture fluid (HCCF). Microchip capillary electrophoresis (MCE) and high-performance size exclusion chromatography (HPSEC) were used as fast and efficient screening methods to detect fragmentation. HPSEC was found to be underestimating fragmentation levels. To confirm and characterize the fragments, capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) was employed. Interestingly, the absence of fragments in the reduced CE-SDS analysis suggested partial reduction of disulphide bonds contributing to fragmentation in this mAb lot. This was further confirmed using reverse phase high performance liquid chromatography (RP-HPLC) coupled with mass spectrometry, which established the presence of heavy-heavy-light (HHL), heavy-heavy (HH), light-light dimer (LL), light chain (LC) and half antibody (HL) fragments with good mass accuracy. In this study, we demonstrated a readily applicable systematic strategy to support process development and investigate anomalous events in manufacturing. An additional highlight of this work is the data-driven comprehensive comparison of modern and conventional analytical techniques for fragment analysis.

Lock-in Amplifier Dairy Sensor for Detection of Ciprofloxacin

This work introduces a sensor for dairy antibiotic detection of ciprofloxacin in milk. We provide an important tool for antibiotic detection in milk and the main contributions of this work are as follows. We introduce a technique based on florescence spectroscopy and lock-in amplification for sensing ciprofloxacin in milk that is capable of detection below the regulatory limit. We compare the system against one without the integrated lock-in amplification, as in traditional fluorescence sensors. We provide microchip capillary electrophoresis results and place our work in context for future integration with microchip capillary electrophoresis.

On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

Point-of-care test for rapid assessment of blood lithium levels in women with bipolar disorder during perinatal period

IntroductionDetermination of lithium levels in serum has become a standard of care due to its narrow therapeutic rang, thus an immediate test for determination of blood lithium may contribute to minimize toxicit, to avoid relapse and to ensure treatment adherence. This is particularly relevant during pregnancy and early postpartum because pharmaokinetic changes in renal physiology.ObjectivesThe aim of this study is verify Medimate point-of-care method performance and systematically compare it with the routine laboratory measurement of lithium.MethodsThis cross-sectional method comparison study was conductec in the Unit of Perinatal Mental Health in the Hospital Clinic of Barcelona. Pearson and Bland-Altman analyses were performed to assess the accuracy, precision and correlation between the capillary electrophoresis technology (Medimate MiniLab) and the ion selective electrode (ISE) potentiometry method (AVL 9180).ResultsTwenty-five women with bipolar disorder in treatment with lithium during perinatal period were enrolled, corresponding to 75 blood specimens for analyses. Correlation (r), mean difference (bias), and 95% limit of agreement (LOA) of the point-of-care method [r=0.917; bias 0.0021 (95% LOA; 0.440, 0.619) mEq/L], showed that difference between ISE method and capillary electrophoresis technology was not statistically significant.ConclusionsConsidering the practicality, the microchip capillary electrophoresis technology provides a simple and highly affordable way of measuring lithium levels in a single drop of blood outside the clinical laboratory. The Medimate point-of-care system (POC) appears well adapted for the rapid and specific detection of lithium as an alternative to the current ISE procedure.DisclosureNo significant relationships.

Inside Front Cover: Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines

DOI: 10.1002/elps.202100272 The inside cover picture shows a method to measure the purity of messenger RNA (mRNA) in lipid nanoparticle (LNP) vaccines by microchip capillary electrophoresis. An LNP containing mRNA is depicted in the top left which undergoes treatment to solubilize the LNP and release mRNA, as shown in the bottom left. The mRNA is introduced into a separation channel containing a gel-sieving matrix that separates the mRNA by size, as the smaller molecules travel faster than larger molecules, followed by fluorescence detection, as shown in the bottom right. An electropherogram of the separation of just over one minute duration is shown in the top right. The mRNA purity is calculated as the percent peak area of the main peak (∼49 s).

Using multi-material fused deposition modeling (FDM) for one-step 3D printing of microfluidic capillary electrophoresis with integrated electrodes for capacitively coupled contactless conductivity detection

For the first time, the fused deposition modeling (FDM) 3D printing method was used to fabricate a microchip capillary electrophoresis (MCE) with channel dimensions (58 × 65 µm) below 100 µm. Moreover, electrodes to conduct capacitively coupled contactless conductivity detection (C4D) were integrated into the MCE using three conductive thermoplastic filaments containing graphene, carbon black, and copper. After optimizing the parameters for multi-material printing, the MCE-C4D device was fabricated in a single (one step) 3D printing process using an FDM 3D printer equipped with two extrusion nozzles. An optimization design method allowed finding that the distance (gap) between the electrodes of around 1 mm provided the maximum response of the C4D. The optimized frequency of the excitation signal applied in the C4D electrodes depended on the composition of the conductive filament used to print the electrodes. The optimized frequencies for the electrodes printed with thermoplastic polymers containing copper and carbon black were 268 and 384 kHz, respectively. The MCE-C4D successfully separated and detected a mixture of K+, Na+, and Li+ with good resolution, efficiency, precision, linearity, and limits of detections (LOD) of 10.2, 11.6, and 14.1 µmol L-1, respectively.

A simple and reliable microfabrication process for a programmable microvalve array

We describe our reliable methodology for fabricating a complex programmable microvalve array (PMA) and its integration with a glass microcapillary electrophoresis chip. This methodology is applicable to any device that requires multilayered PDMS, multiple alignment processes, selective PDMS bonding, and multilayered integration with downstream sensing systems. Along with the detailed step-by-step process, we discuss essential quality assurance checks that can be performed throughout fabrication to assist in troubleshooting and maximizing chip yield.•Comprehensive instructions for designing and fabricating a programmable microvalve array.•Selective bonding of PDMS and glass by microcontact printing.•Numerous quality control procedures to boost chip yield.

Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines.

Messenger RNA (mRNA)‐based vaccines are advantageous because they can be relatively quicker and more cost efficient to manufacture compared to other traditional vaccine products. Lipid nanoparticles have three common purposes: delivery, self‐adjuvanting properties, and mRNA protection. Faster vaccine development requires an efficient and fast assay to monitor mRNA purity and integrity. Microchip CE is known to be a robust technology that is capable of rapid separation. Here, we describe the development and optimization of a purity and integrity assay for mRNA‐based vaccines encapsulated in lipid nanoparticles using commercial microchip‐based separation. The analytical parameters of the optimized assay were assessed and the method is a stability indicating assay.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified.Graphical abstract.

Robust and easy-to-use microchip electrophoresis within sub-millimeter channels for fast and highly efficient separation

Microchip capillary electrophoresis (MCE) is a powerful technique for rapid separation; however, its acceptance in routine laboratories is still limited. Compromises caused by the efforts for solving different problems, such as reducing its cost of fabrication and ensuring high separation efficiency, undermine the competitiveness of this technology compared to other separation techniques. Contrary to the conventional pursuit of narrow microchannels, this study investigated the suitability of microchips with channels at the sub-millimeter level, targeting the simplification of the overall operation, cost reduction, and robustness improvement. To this effect, we considered the influence of pressurized flow and Joule heating on the separation. The suppression of pressurized flow with viscous solutions was confirmed through a combination of simulations and experimental results, indicating that the buffer viscosity was enough for successful separation. We fabricated channels of 200 μm × 230 μm using computer numerical controlled (CNC) machining and obtained theoretical plate numbers of 4.8 × 105 m−1 and 5.3 × 105 m−1 for fluorescein isothiocyanate (FITC) labeled small molecules and DNA fragments, respectively, with a buffer viscosity of 168 mPa s (0.5 % hydroxypropyl methylcellulose, HPMC). These values are comparable with that of narrow-bore microchips. Furthermore, we did not observe any deleterious effects with low-conductivity buffers. We investigated the rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA (cfDNA), which gave a limit of detection (LOD) as low as 2.3 ng mL−1. Owing to the significant reduction in cost, ease of operation, and fast separation capabilities demonstrated in this work, MCE can be a viable alternative to the usual slab gel electrophoresis running in most biological laboratories.

PE-9: Validation of Sodium Concentration in Urine By POCT: Feasibility of the Medimate in Patients with an Ileostomy

Introduction: Excessive loss of water and sodium leading to dehydration is a frequent complication in patients with an ileostomy or those with a (near) short bowel syndrome (1). To maintain electrolyte and fluid balances in these patients, measurement of the sodium concentration in a single portion of urine can be a simple and helpful tool. In general a sodium of over > 20 mmol/L is considered to rule out significant dehydration and/or sodium depletion. A direct measurement of sodium concentration in urine using the new Medimate Minilab enables patients to monitor their own sodium status at home. In this study, the performance characteristics of the Medimate Minilab were evaluated. Method: The Medimate Minilab (Medimate BV) is a self test to measure sodium concentration in urine. The technology is based on microchip capillary electrophoresis combined with conductivity detection, consisting of disposable lab-chips, the Medimate Lab-Chip and a measurement device, the Medimate Multireader ®) (2). For validation, 50 ml of urine was collected and mixed with 5 ml of lithium-containing buffer freshly collected urine was used to determine the limit of quantification (LOQ), imprecision and agreement with the Cobas 8000 ISE Roche system. Results: Imprecision of urine samples was low: a maximum CV of 6,1% was determined in repeated measurements during five days (Table 1). The smallest concentration of sodium that can be reliably measured with an acceptable imprecision was 17 mmol/L (Figure 1). The agreement with Sodium on Cobas 8000 ISE system showed an overall positive bias of 23% (Figure 2, 95 CI of +13% -+33%), with substantial deviation in individual points (R2=0,96).The device was easy to handle, and found to be suitable for patients in a home setting. It is of essential importance that instructions are adequately followed, such as filling the cup to 55 ml of urine, to avoid false low results. Conclusion: Reproducibility of results of individual samples was high. - The stability of the buffer must be optimized - The influence of the expired shelf life of the buffer on the positive bias of sodium in urine should be further investigated - Interferences in individual points must be re-evaluated Figure 1.: LOQ of Sodium in Urine Nine urine samples ranging from 17,7 mmol/l-260 mmol/l were measured in triplo and the average and CV (%) were determined.Figure 2.: Agreement of Sodium in urine Urine samples were measured on 5 consecutive days on both Cobas 8000 system and the Medimate device. Demming regression analysis: y=1,23x-3.5 Note: The buffer solution exceeded the shelf-life. The influence of the buffer on the positive bias will be part of further investigation.

Design-Based Learning Framework for Introducing Factorial Experimental Design and Lab-on-a-Chip Separations in an Instrumental Chemistry Laboratory

Lab-on-a-chip devices are a critical and emerging chemical technology. New pedagogical tactics that effectively capture both analytical chemistry concepts along with the design elements unique to these devices are necessary for effective integration in the undergraduate chemistry curriculum. This article develops a design-based learning laboratory exercise to concurrently introduce analytical miniaturization, chemical separations, and formal factorial design of experiment methods in an Introduction to Instrumental Analysis course. A new student-accessible lab-on-a-chip fabrication platform using a benchtop laser cutter allows for rapid, iterative, and safe production of student-designed microchip capillary electrophoresis devices in minutes. Student teams used a full factorial experimental design approach to efficiently screen three experimental variables of their choice when evaluating the separation speed of their own lab-on-a-chip devices. This laboratory exercise has been successfully implemented on three occasions over the past several years at Colorado College. Both reflective and performance-based assessment methods suggest this exercise is an effective pedagogical approach for realizing the intended learning outcomes.

Specific Analysis of α-2,3-Sialylated N-Glycan Linkage Isomers by Microchip Capillary Electrophoresis-Mass Spectrometry.

Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6- and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.

Rapid Analysis for Staphylococcus aureus via Microchip Capillary Electrophoresis.

Staphylococcus aureus (S. aureus) is one of the most common pathogens for nosocomial and community infections, which is closely related to the occurrence of pyogenic and toxic diseases in human beings. In the current study, a lab-built microchip capillary electrophoresis (microchip CE) system was employed for the rapid determination of S. aureus, while a simple-to-use space domain internal standard (SDIS) method was carried out for the reliable quantitative analysis. The precision, accuracy, and reliability of SDIS were investigated in detail. Noted that these properties could be elevated in SDIS compared with traditional IS method. Remarkably, the PCR products of S. aureus nuc gene could be identified and quantitated within 80 s. The theoretical detection limit could achieve a value of 0.066 ng/μL, determined by the using SDIS method. The current work may provide a promising detection strategy for the high-speed and highly efficient analysis of pathogens in the fields of food safety and clinical diagnosis.

Microchip Electrophoresis Assay for Calmodulin Binding Proteins.

The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in-vitro crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.