Aptamer-mediated double strand displacement amplification with microchip electrophoresis for ultrasensitive detection of Salmonella typhimurium.

Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium, releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis.

3D printed microfluidic devices for integrated solid-phase extraction and microchip electrophoresis of preterm birth biomarkers.

BACKGROUND: Preterm birth (PTB) is a leading cause of neonatal mortality, such that the need for a rapid and accurate assessment for PTB risk is critical. Here, we developed a 3D printed microfluidic system that integrated solid-phase extraction (SPE) and microchip electrophoresis (µCE) of PTB biomarkers, enabling the combination of biomarker enrichment and labeling with µCE separation and fluorescence detection. RESULTS: Reversed-phase SPE monoliths were photopolymerized in 3D printed devices. Microvalves in the device directed sample between the SPE monolith and the injection cross-channel in the serpentine µCE channel. Successful on-chip preconcentration, labeling and µCE separation of four PTB-related polypeptides were demonstrated in these integrated microfluidic devices. We further show the ability of these devices to handle complex sample matrices through the successful analysis of labeled PTB biomarkers spiked into maternal blood serum. The detection limit was 7 nM for the PTB biomarker, corticotropin releasing factor, in 3D printed SPE-µCE integrated devices. SIGNIFICANCE: This work represents the first successful demonstration of integration of SPE and µCE separation of disease-linked biomarkers in 3D printed microfluidic devices. These studies open up promising possibilities for rapid bioanalysis of medically relevant analytes.

Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.

Electrophoretic Determination of L-Carnosine in Health Supplements Using an Integrated Lab-on-a-Chip Platform with Contactless Conductivity Detection.

The health supplement industry is one of the fastest growing industries in the world, but there is a lack of suitable analytical methods for the determination of active compounds in health supplements such as peptides. The present work describes an implementation of contactless conductivity detection on microchip technology as a new strategy for the electrophoretic determination of L-carnosine in complex health supplement formulations without pre-concentration and derivatization steps. The best results were obtained in the case of +1.00 kV applied for 20 s for injection and +2.75 kV applied for 260 s for the separation step. Under the selected conditions, a linear detector response of 5 × 10-6 to 5 × 10-5 M was achieved. L-carnosine retention time was 61 s. The excellent reproducibility of both migration time and detector response confirmed the high precision of the method. The applicability of the method was demonstrated by the determination of L-carnosine in three different samples of health supplements. The recoveries ranged from 91 to 105%. Subsequent analysis of the samples by CE-UV-VIS and HPLC-DAD confirmed the accuracy of the obtained results.

[Surface-modified microchip electrophoretic separation and analysis of functional components in health care products].

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

[Advances in microchip electrophoresis for the separation and analysis of biological samples].

Microchip electrophoresis is a separation technology that involves fluid manipulation in a microchip; the advantages of this technique include high separation efficiency, low sample consumption, and fast and easy multistep integration. Microchip electrophoresis has been widely used to rapidly separate and analyze complex samples in biology and medicine. In this paper, we review the research progress on microchip electrophoresis, explore the fabrication and separation modes of microchip materials, and discuss their applications in the detection and analysis of biological samples. Research on microchip materials can be mainly categorized into chip materials, channel modifications, electrode materials, and electrode integration methods. Microchip materials research involves the development of silicon, glass, polydimethylsiloxane and polymethyl methacrylate-based, and paper electrophoretic materials. Microchannel modification research primarily focuses on the dynamic and static modification methods of microchannels. Although chip materials and fabrication technologies have improved over the years, problems such as high manufacturing costs, long processing time, and short service lives continue to persist. These problems hinder the industrialization of microchip electrophoresis. At present, few static methods for the surface modification of polymer channels are available, and most of them involve a combination of physical adsorption and polymers. Therefore, developing efficient surface modification methods for polymer channels remains a necessary undertaking. In addition, both dynamic and static modifications require the introduction of other chemicals, which may not be conducive to the expansion of subsequent experiments. The materials commonly used in the development of electrodes and processing methods for electrode-microchip integration include gold, platinum, and silver. Microchip electrophoresis can be divided into two modes according to the uniformity of the electric field: uniform and non-uniform. The uniform electric field electrophoresis mode mainly involves micro free-flow electrophoresis and micro zone electrophoresis, including micro isoelectric focusing electrophoresis, micro isovelocity electrophoresis, and micro density gradient electrophoresis. The non-uniform electric field electrophoresis mode involves micro dielectric electrophoresis. Microchip electrophoresis is typically used in conjunction with conventional laboratory methods, such as optical, electrochemical, and mass spectrometry, to achieve the rapid and efficient separation and analysis of complex samples. However, the labeling required for most widely used laser-induced fluorescence technologies often involves a cumbersome organic synthesis process, and not all samples can be labeled, which limits the application scenarios of laser-induced fluorescence. The applications of unlabeled microchip electrophoresis-chemiluminescence/dielectrophoresis are also limited, and simplification of the experimental process to achieve simple and rapid microchip electrophoresis remains challenging. Several new models and strategies for high throughput in situ detection based on these detection methods have been developed for microchip electrophoretic systems. However, high throughput analysis by microchip electrophoresis is often dependent on complex chip structures and relatively complicated detection methods; thus, simple high throughput analytical technologies must be further explored. This paper also reviews the progress on microchip electrophoresis for the separation and analysis of complex biological samples, such as biomacromolecules, biological small molecules, and bioparticles, and forecasts the development trend of microchip electrophoresis in the separation and analysis of biomolecules. Over 250 research papers on this field are published annually, and it is gradually becoming a research focus. Most previous research has focused on biomacromolecules, including proteins and nucleic acids; biological small molecules, including amino acids, metabolites, and ions; and bioparticles, including cells and pathogens. However, several problems remain unsolved in the field of microchip electrophoresis. Overall, microchip electrophoresis requires further study to increase its suitability for the separation and analysis of complex biological samples.

Progress in on-line, at-line, and in-line coupling of sample treatment with capillary and microchip electrophoresis over the past 10 years: A review.

The review presents an evaluation of the development of on-line, at-line and in-line sample treatment coupled with capillary and microchip electrophoresis over the last 10 years. In the first part, it describes different types of flow-gating interfaces (FGI) such as cross-FGI, coaxial-FGI, sheet-flow-FGI, and air-assisted-FGI and their fabrication using molding into polydimethylsiloxane and commercially available fittings. The second part deals with the coupling of capillary and microchip electrophoresis with microdialysis, solid-phase, liquid-phase, and membrane based extraction techniques. It mainly focuses on modern techniques such as extraction across supported liquid membrane, electroextraction, single drop microextraction, head space microextraction, and microdialysis with high spatial and temporal resolution. Finally, the design of sequential electrophoretic analysers and fabrication of SPE microcartridges with monolithic and molecularly imprinted polymeric sorbents are discussed. Applications include the monitoring of metabolites, neurotransmitters, peptides and proteins in body fluids and tissues to study processes in living organisms, as well as the monitoring of nutrients, minerals and waste compounds in food, natural and wastewater.

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.

Concepts and recent advances in microchip electrophoresis coupled to mass spectrometry: Technologies and applications.

The online coupling of microchip electrophoresis (ME) as a fast, highly efficient, and low-cost miniaturized separation technique to mass spectrometry (MS) as an information-rich and sensitive characterization technique results in ME-MS an attractive tool for various applications. In this paper, we review the basic concepts and latest advances in technology for ME coupled to MS during the period of 2016-2021, covering microchip materials, structures, fabrication techniques, and interfacing to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization-MS. Two critical issues in coupling ME and ESI-MS include the electrical connection used to define the electrophoretic field strength along the separation channel and the generation of the electrospray for MS detection, as well as, a miniaturized ESI-tip. The recent commercialization of ME-MS in zone electrophoresis and isoelectric focusing modes has led to the widespread application of these techniques in academia and industry. Here we summarize recent applications of ME-MS for the separation and detection of antibodies, proteins, peptides, carbohydrates, metabolites, and so on. Throughout the paper these applications are discussed in the context of benefits and limitations of ME-MS in comparison to alternative techniques.

Nanofluidic devices for the separation of biomolecules.

Over the last 30-years, microchip electrophoresis and its applications have expanded due to the benefits it offers. Nanochip electrophoresis, on the other hand, is viewed as an evolving area of electrophoresis because it offers some unique advantages not associated with microchip electrophoresis. These advantages arise from unique phenomena that occur in the nanometer domain not readily apparent in the microscale domain due to scale-dependent effects. Scale-dependent effects associated with nanochip electrophoresis includes high surface area-to-volume ratio, electrical double layer overlap generating parabolic flow even for electrokinetic pumping, concentration polarization, transverse electromigration, surface charge dominating flow, and surface roughness. Nanochip electrophoresis devices consist of channels with dimensions ranging from 1 to 1000 nm including classical (1-100 nm) and extended (100 nm - 1000 nm) nanoscale devices. In this review, we highlight scale-dependent phenomena associated with nanochip electrophoresis and the utilization of those phenomena to provide unique biomolecular separations that are not possible with microchip electrophoresis. We will also review the range of materials used for nanoscale separations and the implication of material choice for the top-down fabrication and operation of these devices. We will also provide application examples of nanochip electrophoresis for biomolecule separations with an emphasis on nano-electrophoresis (nEP) and nano-electrochromatography (nEC).

Electrokinetic characterization of hybrid NOA 81-glass microchips: Application to protein microchip electrophoresis with indirect fluorescence detection.

A low-cost and straightforward hybrid NOA (Norland optical adhesive) 81-glass microchip electrophoresis device was designed and developed for protein separation using indirect fluorescence detection. This new microchip was first characterized in terms of surface charge density via electroosmotic mobility measurement and stability over time. A systematic determination of the electroosmotic mobility (µeo ) over a wide pH range (2-10) and at various ionic strengths (20-50 mM) was developed for the first time via the neutral marker approach in an original simple frontal methodology. The evolution of µeo was proved consistent with the silanol and thiol functions arising from the glass and the NOA materials, respectively. The repeatability and reproducibility of the measurements on different microchips (RSD < 14%) and within 15 days (less than 5% decrease) were successfully demonstrated. The microchip was then applied for the efficient electrophoretic separation of proteins in a zonal mode coupled with indirect fluorescence detection, which is, to our knowledge, the first proof of concept of capillary zone electrophoresis in this hybrid microsystem.

Monitoring of biologically active substances in clinical samples by capillary and microchip electrophoresis with contactless conductivity detection: A review.

Contactless conductivity detection (C4D) as a universal detection technique plays an important role in combination with efficient electrophoretic separation carried out in capillaries (CE) or on microchips (ME) in the analysis of clinical samples. C4D is particularly sensitive in the quantification of low molecular weight biogenic substances such as inorganic cations and anions, amino acids, amines, low molecular weight organic acids, saccharides and many drugs such as antibiotics, analgesics, anaesthetics or antiepileptics. Biogenic substances are determined in CE/C4D or ME/C4D directly in their native form without derivatization and sample matrix treatment is often based only on dilution or addition of an organic solvent. The limit of detection for most CE/C4D determinations is at the micromolar concentration level, which is sufficient to monitor physiological or therapeutic levels of most of low molecular weight biogenic substances. Therefore, CE/C4D and ME/C4D are widely used for sequential monitoring of nutrients, metabolites and waste products at the level of individual tissues and organs, low-invasive detection of inborn errors of metabolism and cystic fibrosis, pharmacokinetic monitoring and therapeutic drug monitoring. Innovative trends such as electrophoretic stacking, microdialysis, electromembrane extraction, portable and disposable CE instruments and minimally invasive clinical sampling techniques are mentioned. A critical evaluation of the positives and negatives of this technique is presented, covering the main applications published over the last 10 years.

Highly sensitive detection of MUC1 by microchip electrophoresis combining with target recycling amplification and strand displacement amplification.

Mucin 1 (MUC1) is usually overexpressed in a variety of malignant tumors, and quantitative analysis of MUC1 plays an important role in the early diagnosis of cancer. In this work, a highly sensitive MUC1 assay was developed by integrating microchip electrophoresis (MCE) with target recycling amplification (TRA) and strand displacement amplification (SDA). Specifically, the presence of MUC1 can trigger the exposure of the designed hairpin probe (HP) to initiate SDA and an amplified amount of ssDNA is produced finally. The amount of these ssDNA can be detected by MCE, then the concentration of MUC1 can be obtained through the correlation between MUC1 concentration and ssDNA concentration. The experimental results show that the MCE signal had a good linear relationship with MUC1 concentration in the range of 1.0 pg/mL - 1.0 × 103 pg/mL with a low limit of detection of 0.23 pg/mL under the optimal conditions (S/N = 3). Additionally, the assay had been successfully applied to detect MUC1 in biological samples with satisfactory results, providing an alternative assay for the detection of other tumor markers owing to the high sensitivity, high selectivity, simple operation and low sample consumption.

Rapid determination of NSAIDs by capillary and microchip electrophoresis with capacitively coupled contactless conductivity detection in wastewater.

A simple, rapid method using CE and microchip electrophoresis with C4 D has been developed for the separation of four nonsteroidal anti-inflammatory drugs (NSAIDs) in the environmental sample. The investigated compounds were ibuprofen (IB), ketoprofen (KET), acetylsalicylic acid (ASA), and diclofenac sodium (DIC). In the present study, we applied for the first time microchip electrophoresis with C4 D detection to the separation and detection of ASA, IB, DIC, and KET in the wastewater matrix. Under optimum conditions, the four NSAIDs compounds could be well separated in less than 1 min in a BGE composed of 20 mM His/15 mM Tris, pH 8.6, 2 mM hydroxypropyl-beta-cyclodextrin, and 10% methanol (v/v) at a separation voltage of 1000-1200 V. The proposed method showed excellent repeatability, good sensitivity (LODs ranging between 0.156 and 0.6 mg/L), low cost, high sample throughputs, portable instrumentation for mobile deployment, and extremely lower reagent and sample consumption. The developed method was applied to the analysis of pharmaceuticals in wastewater samples with satisfactory recoveries ranging from 62.5% to 118%.

Determination of naphthenic acids in produced water by using microchip electrophoresis with integrated contactless conductivity detection.

This study reports for the first time the use of a microchip electrophoresis (ME) device with integrated capacitively coupled contactless conductivity detection (C4D) to analyze naphthenic acids in produced water. A mixture containing 9-anthracenecarboxylic, 1-naphthoic, and benzoic acids was separated and detected using a running buffer composed of 10 mmol L-1 carbonate buffer (pH = 10.2). The separation was achieved within ca. 140 s with baseline resolution greater than 2 and efficiency values ranging from 1.9 × 105 to 2.4 × 105 plates m-1. The developed methodology provided linear correlation with determination coefficients greater than 0.992 for the concentration ranges between 50 and 250 µmol L-1 for benzoic and 9-anthracenecarboxylic acids, and between 50 and 200 µmol L-1 for 1-naphthoic acid. The achieved limit of detection values varied between 4.7 and 7.7 µmol L-1. The proposed methodology revealed satisfactory repeatability with RSD values for a sequence of eight injections between 5.5 and 7.7% for peak areas and lower than 1% for migration times. In addition, inter-day precision was evaluated for sixteen injections (a sequence of four injections performed during four days), and the RSD values were lower than 11.5 and 4.9% for peak areas and migration time, respectively. Five produced water samples were analyzed, and it was possible to detect and quantify 9-anthracenecarboxylic acid. The concentrations ranged from 1.05 to 2.24 mmol L-1 with recovery values between 90.8 and 96.0%. ME-C4D demonstrated satisfactory analytical performance for determining naphthenic acids in produced water for the first time, which is useful for petroleum or oil industry investigation.

3D printed microfluidic device for automated, pressure-driven, valve-injected microchip electrophoresis of preterm birth biomarkers.

A 3D printed, automated, pressure-driven injection microfluidic system for microchip electrophoresis (µCE) of preterm birth (PTB)-related peptides and proteins has been developed. Functional microvalves were formed, either with a membrane thickness of 5 µm and a layer exposure time of 450 ms or with a membrane thickness of 10 µm and layer exposure times of 300-350 ms. These valves allowed for control of fluid flow in device microchannels during sample injection for µCE separation. Device design and µCE conditions using fluorescently labeled amino acids were optimized. A sample injection time of 0.5 s and a separation voltage of 450 V (460 V/cm) yielded the best separation efficiency and resolution. We demonstrated the first µCE separation with pressure-driven injection in a 3D printed microfluidic device using fluorescently labeled PTB biomarkers and 532 nm laser excitation. Detection limits for two PTB biomarkers, peptide 1 and peptide 2, for an injection time of 1.5 s were 400 pM and 15 nM, respectively, and the linear detection range for peptide 2 was 50-400 nM. This 3D printed microfluidic system holds promise for future integration of on-chip sample preparation processes with µCE, offering promising possibilities for PTB risk assessment.

Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation.

The increased use of biopharmaceuticals calls for improved means of bioprocess monitoring. In this work, capillary electrophoresis (CE) and microchip electrophoresis (MCE) methods were developed and applied for the analysis of amino acids (AAs) in cell culture supernatant. In samples from different days of a Chinese hamster ovary cell cultivation process, all 19 proteinogenic AAs containing primary amine groups could be detected using CE, and 17 out of 19 AAs using MCE. The relative concentration changes in different samples agreed well with those measured by high-performance liquid chromatography (HPLC). Compared to the more commonly employed HPLC analysis, the CE and MCE methods resulted in faster analysis, while significantly lowering both the sample and reagent consumption, and the cost per analysis.

Microchip Free-Flow Electrophoresis for Bioanalysis, Sensing, and Purification.

The separation of complex mixtures is ubiquitous throughout molecular biology, and techniques such as gel-based electrophoresis are common laboratory practice. Such methods are not without their drawbacks, however, which include non-specific interactions between analyte and the separation matrix, poor yields in purification and non-continuous analyte throughput. Microfluidic techniques, which exploit physical phenomena unique to the microscale, promise to improve many aspects of traditional laboratory procedures. These methods offer a quantitative, solution-based alternative to traditional gel electrophoresis, with rapid measurement times enabling the analysis of transient or weak biomolecular interactions that would be challenging to observe with traditional methods. Here, we present a protocol for the lithographic fabrication and operation of microfluidic chips capable of free-flow electrophoretic (FFE) fractionation and analysis of biological analytes. We demonstrate the efficacy of our approach through a protein-sensing methodology based on FFE fractionation of DNA-protein mixtures. In addition, the FFE technique described here can be readily adapted to suit a variety of preparative and analytical applications, providing information on the charge, zeta-potential, and interactions of analytes.

An integrated plastic microchip for enhancing electrophoretic separation using tunable pressure-driven backflows.

Microfluidic CE (MCE) is an effective solution for rapid and sensitive determination of multiple analytes. Herein, a dynamic coated cyclic olefin copolymer microchip was developed having an on-chip micropump for fluid velocity adjusting in electrophoretic separations. This micropump was fabricated by constructing a polyacrylamide gel membrane at one channel terminal. Once applying electric field across the membrane, a pressure-driven flow generated automatically to balance the electroosmotic flow (EOF) mismatch at the channel-membrane interface. The influence of gel precursor concentration and operating voltages on the fluid velocity was carefully evaluated. Moreover, the highly integration of injection, separation, and pumping units of the MCE system minimized the dead volume and provides satisfied column efficiency. Experiments showed that by adjusting of pumping voltage reduced the fluid velocity by a factor of 6, resulting six- and threefold resolving power enhancements of rhodamine dye mixture and amino acid mixture, respectively. Furthermore, the developed MCE method was applied for rhodamines and amino acids quantitation in food and cosmetics, with standard addition recoveries of 87.3-106.9% and 89.9-117.4%, respectively. These results were also confirmed by standard HPLC method, revealing the application potential in fast and onsite analysis of complex samples.

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.