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This paper describes the use of PolyJet 3D printing to fabricate microchip electrophoresis devices with integrated microwire electrodes for amperometric detection. The fabrication process involves 3D printing of two separate pieces, a channel layer and an electrode layer. The channel layer is created by 3D printing on a pre-fabricated mold with a T-intersection. For the electrode layer, a stencil design is printed directly on the printing tray and covered with a piece of transparent glass. Microwire electrodes are adhered over the glass piece (guided by underlaying stencil) and a CAD design of the electrode layer is then printed on top of the microwire electrode. After delamination from the glass after printing, the microwire is embedded in the printed piece, with the stencil design ensuring that alignment and positioning of the electrode is reproducible for each print. After a thermal bonding step between the channel layer and electrode layer, a complete electrophoresis device with integrated microelectrodes for amperometric detection results. It is shown that this approach enables different microwire electrodes (gold or platinum) and sizes (100 or 50 µm) to be integrated in an end-channel configuration with no gap between the electrode and the separation channel. These devices were used to separate a mixture of catecholamines and the effect of separation voltage on the potential voltage applied on the working electrode was also investigated. In addition, the effect of electrode size on the number of theoretical plates and limit of detection was studied. Finally, a device that contains different channel heights and a detection electrode was 3D-printed to integrate continuous flow sampling with microchip electrophoresis and amperometric detection. [ABSTRACT FROM AUTHOR]

The abundance of potentially habitable hypersaline environments in our solar system compels us to understand the impacts of high-salt matrices and brine dynamics on biosignature detection efforts. We identified and quantified organic compounds in brines from South Bay Salt Works (SBSW), where evapoconcentration of ocean water enables exploration of the impact of NaCl- and MgCl2-dominated brines on the detection of potential biosignature molecules. In SBSW, organic biosignature abundance and distribution are likely influenced by evapoconcentration, osmolyte accumulation, and preservation effects. Bioluminescence assays show that adenosine triphosphate (ATP) concentrations are higher in NaCl-rich, low water activity (aw) samples (<0.85) from SBSW. This is consistent with the accumulation and preservation of ATP at low aw as described in past laboratory studies. The water-soluble small organic molecule inventory was determined by using microchip capillary electrophoresis paired with high-resolution mass spectrometry (µCE-HRMS). We analyzed the relative distribution of proteinogenic amino acids with a recently developed quantitative method using CE-separation and laser-induced fluorescence (LIF) detection of amino acids in hypersaline brines. Salinity trends for dissolved free amino acids were consistent with amino acid residue abundance determined from the proteome of the microbial community predicted from metagenomic data. This highlights a tangible connection up and down the "-omics" ladder across changing geochemical conditions. The detection of water-soluble organic compounds, specifically proteinogenic amino acids at high abundance (>7 mM) in concentrated brines, demonstrates that potential organic biomarkers accumulate at hypersaline sites and suggests the possibility of long-term preservation. The detection of such molecules in high abundance when using diverse analytical tools appropriate for spacecraft suggests that life detection within hypersaline environments, such as evaporates on Mars and the surface or subsurface brines of ocean world Europa, is plausible and argues such environments should be a high priority for future exploration. Key Words: Salts—Analytical chemistry—Amino acids—Biosignatures—Capillary electrophoresis—Preservation. Astrobiology 24, 795–812. [ABSTRACT FROM AUTHOR]

The growing consumption of plant-based milk substitutes raises important questions about their composition. The various additives used by manufacturers, including those employed as flavor enhancers, protein additives, and stabilizers, may contain both protein and non-protein nitrogen components. In our study, we examined not only popular milk alternatives but also other milk substitutes made from specific plants. We present a reproducible and rapid method for the simultaneous qualitative and quantitative determination of the total nitrogen content in milk alternatives, focusing on applicability. Using the microchip gel electrophoretic method, we determined that the total nitrogen content differed from the protein content indicated on the packaging. Our results, along with statistical evaluations, supported the hypothesis that different brands of products, derived from the same plant source, resulted in different microfluidic profiles, likely due to the presence of additives. As expected, the microfluidic profiles of additive-free products differed from those of fortified products made from the same plant-based milk replacer. Total nitrogen content provides crucial information for individuals with kidney disease, as is essential to reduce the burden on the kidneys to slow deterioration, alleviate symptoms and avoid complications. [ABSTRACT FROM AUTHOR]

The analysis of dietary supplements is far less regulated than pharmaceuticals, leading to potential quality issues. Considering their positive effect, many athletes consume supplements containing l-histidine and β-alanine. A new microfluidic method for the determination of l-histidine and β-alanine in dietary supplement formulations has been developed. For the first time, capacitively coupled contactless conductivity detection was employed for the microchip electrophoresis of amino acids in real samples. A linear relationship between detector response and concentration was observed in the range of 10–100 µmol L–1 for l-histidine (R2 = 0.9968) and β-alanine (R2 = 0.9954), while achieved limits of detection (3 × S/N ratio) were 4.2 µmol L–1 and 5.2 µmol L–1, respectively. The accuracy of the method was confirmed using recovery experiments as well as CE-UV-VIS and HPLC-UV-VIS techniques. The developed method allows unambiguous identification of amino acids in native form without chemical derivatization and with the possibility of simultaneous analysis of amino acids with metal cations. [ABSTRACT FROM AUTHOR]

The glycomic analysis holds significant appeal due to the diverse roles that glycans and glycoconjugates play, acting as modulators and mediators in cellular interactions, cell/organism structure, drugs, energy sources, glyconanomaterials, and more. The glycomic analysis relies on liquid‐phase separation technologies for molecular purification, separation, and identification. As a miniaturized form of liquid‐phase separation technology, microscale separation technologies offer various advantages such as environmental friendliness, high resolution, sensitivity, fast speed, and integration capabilities. For glycan analysis, microscale separation technologies are continuously evolving to address the increasing challenges in their unique manners. This review discusses the fundamentals and applications of microscale separation technologies for glycomic analysis. It covers liquid‐phase separation technologies operating at scales generally less than 100 µm, including capillary electrophoresis, nanoflow liquid chromatography, and microchip electrophoresis. We will provide a brief overview of glycomic analysis and describe new strategies in microscale separation and their applications in glycan analysis from 2014 to 2023. [ABSTRACT FROM AUTHOR]

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in‐gel probing and separation resolution. Here, a highly sensitive temperature‐controlled single‐cell western blotting (tc‐scWB) method is introduced, which is based on a thermo/photo‐dualistic‐sensitive polyacrylamide hydrogel, namely acrylic acid‐functionalized graphene oxide (AFGO) assisted, N‐isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high‐temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low‐temperature for better antibody penetration into the hydrogel. The tc‐scWB method enables the separation and profiling of small‐molecule‐weight proteins with highly crosslinked gel (12% T) in SDS‐PAGE. The tc‐scWB is demonstrated on three metabolic and ER stress‐specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc‐scWB is a general toolbox for the analysis of low‐abundance small‐molecular functional proteins at the single‐cell level. [ABSTRACT FROM AUTHOR]

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. Methods: In the first step, the microchip capillary electrophoresis technique was used to serve as a separation and detection system. Analytes were injected in the 40 mm long microchannel with 10 mm sidearms. Then, they were separated by applying a direct electrical field (+1800 V) between the buffer and detection reservoirs. 2-(n-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 5) was used as a 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-reference 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 to extract 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 optimal extraction cycles were found to be 8 with adding 1% NaCl to the aqueous phase. Analyzing time of the mentioned analytes was less than 100s, the precision range was 3.3 - 8.2 with a 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 systems for detecting fenitrothion and parathion insecticides. [ABSTRACT FROM AUTHOR]

The world is facing challenges in terms of environmental sustainability and rising costs. In this context, miniaturized equipment such as microfluidic devices can be used to manipulate small volumes of fluids in micrometer-sized channels incorporating multiple components such as pumps, valves and mixers. Due to their high flexibility, these devices can provide sustainable alternatives to traditional platforms. The present study focuses on the design of a user-friendly and reliable microfluidic capillary electrophoresis chip for pharmaceutical applications. To meet pharmaceutical requirements in terms of quantification performance and overcome the injection variability usually observed with such microfluidic systems, a reliable and reproducible design for hydrodynamic injection using passive valves has been developed. It has been successfully used for synthetic cathinone analysis.

The present study involved the fabrication and testing of a Microchip electrophoresis (MCE) device for pulse amperometry based detection of pesticides from their mixture. We were able to separate and then quantify three distinct types of insecticides, namely Chlorpyrifos, Imidacloprid, and Fipronil using on chip MCE followed by pulsed amperometric detection. All these results were obtained with an inhouse developed potentiostat cum controller unit with a detection time of only 15 min, employing a minimal sample size of 2 μL without any preconcentration or extraction procedure. The limit of detection (LOD) was calculated as 42.69 μM, 62.61 μM, and 71.14 μM or 14.96, 16.0 and 31.09 ppm, respectively for Chlorpyrifos, Imidacloprid, and Fipronil and their respective migration times as 536 ± 6.3 s, 484 ± 1.7 s, and 604 ± 3.5 s (n = 14). The sensitivity of detection was determined as 0.03 nA/μM for Chlorpyrifos, 0.0265 nA/μM for Imidacloprid, and 0.035 nA/μM for Fipronil. In addition, the efficacy of the produced microchip was confirmed by analysing soil extract spiked with known pesticides concentrations while the recovery percentage, representing a ratio of calculated concentration to spiked concentration multiplied by hundred was found as 84.3% (±9.4%) (n = 9). Thus, integrating microchip technology with the developed analytical instruments presents significant promise for practical field applications and the analysis of diverse analytes by way of creating a library where the migration coefficient and peak detection current are needed for any analyte which can be made cationic or anionic using a suitable buffer.

Hemoglobin (Hb) disorders are among the most common monogenic diseases affecting nearly 7% of the world population. Among various Hb disorders, approximately 1.5% of the world population carries β-thalassemia (β-Thal), affecting 40,000 newborns every year. Early screening and a timely diagnosis are essential for β-thalassemia patients for the prevention and management of later clinical complications. However, in Africa, Southern Europe, the Middle East, and Southeast Asia, where β-thalassemia is most prevalent, the diagnosis and screening for β-thalassemia are still challenging due to the cost and logistical burden of laboratory diagnostic tests. Here, we present Gazelle, which is a paper-based microchip electrophoresis platform that enables the first point-of-care diagnostic test for β-thalassemia. We evaluated the accuracy of Gazelle for the β-Thal screening across 372 subjects in the age range of 4–63 years at Apple Diagnostics lab in Mumbai, India. Additionally, 30 blood samples were prepared to mimic β-Thal intermediate and β-Thal major samples. Gazelle-detected levels of Hb A, Hb F, and Hb A2 demonstrated high levels of correlation with the results reported through laboratory gold standard high-performance liquid chromatography (HPLC), yielding a Pearson correlation coefficient = 0.99. This ability to obtain rapid and accurate results suggests that Gazelle may be suitable for the large-scale screening and diagnosis of β-Thal. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 20 mmol/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 1.9 kV. 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 (0.085 μg·mL−1 and 0.077 μg·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. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb) disorders are among the most common monogenic diseases affecting nearly 7% of the world population. Among various Hb disorders, approximately 1.5% of the world population carries β-thalassemia (β-Thal), affecting 40,000 newborns every year. Early screening and a timely diagnosis are essential for β-thalassemia patients for the prevention and management of later clinical complications. However, in Africa, Southern Europe, the Middle East, and Southeast Asia, where β-thalassemia is most prevalent, the diagnosis and screening for β-thalassemia are still challenging due to the cost and logistical burden of laboratory diagnostic tests. Here, we present Gazelle, which is a paper-based microchip electrophoresis platform that enables the first point-of-care diagnostic test for β-thalassemia. We evaluated the accuracy of Gazelle for the β-Thal screening across 372 subjects in the age range of 4–63 years at Apple Diagnostics lab in Mumbai, India. Additionally, 30 blood samples were prepared to mimic β-Thal intermediate and β-Thal major samples. Gazelle-detected levels of Hb A, Hb F, and Hb A2 demonstrated high levels of correlation with the results reported through laboratory gold standard high-performance liquid chromatography (HPLC), yielding a Pearson correlation coefficient = 0.99. This ability to obtain rapid and accurate results suggests that Gazelle may be suitable for the large-scale screening and diagnosis of β-Thal.

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. [ABSTRACT FROM AUTHOR]

The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches. There are limitations with current protein sensing methods. Here the authors report DigitISA, a digital immunosensor assay based on microchip electrophoretic separation and single-molecule detection that enables quantitation of protein biomarkers in a single, solution-phase step. [ABSTRACT FROM AUTHOR]

A method for the determination of selected aromatic amino acid biomarkers of oxidative stress using microchip electrophoresis with electrochemical detection is described. The separation of the major reaction products of phenylalanine and tyrosine with reactive nitrogen and oxygen species was accomplished using ligand exchange micellar electrokinetic chromatography with a PDMS/glass hybrid chip. Electrochemical detection was achieved using a pyrolyzed photoresist film working electrode. The system was evaluated for the analysis of the products of the Fenton reaction with tyrosine and phenylalanine, and the reaction of peroxynitrite with tyrosine. [ABSTRACT FROM AUTHOR]

The problem of overlapping peaks has been a challenge in microchip electrophoresis (ME) signal analysis. However, traditional peak fitting algorithms have difficulty analyzing overlapping peaks due to the high dependence on the starting point. In this study, we propose a symmetrical peak fitting method named the tent-mapped whale optimization algorithm and Levenberg–Marquardt (TWOALM), which combines a whale optimization algorithm (WOA) improved by one of the most commonly used chaotic maps, the tent map and the Levenberg–Marquardt (LM) algorithm. Specifically, we first derive the fitted model for the overlapping peaks, showing that it is separable nonlinear least squares, significantly reducing the number of parameters to be optimized. Second, we integrate the tent map into the WOA, which improves the convergence speed of the peak fitting algorithm. Finally, we propose an efficient peak-fitting algorithm that combines the improved WOA and LM. The advantage of the proposed algorithm is that it is significantly faster than WOA and significantly more accurate than the LM algorithm. The results of fitting the synthetic peaks and ME signals showed that the combination of the chaotic map-based WOA algorithm and the LM algorithm can significantly improve the peak fitting performance and provide an effective solution for the analysis of overlapping peaks. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) is rapidly growing as a therapeutic modality for vaccination and the treatment of a wide range of diseases. As a result, there is an increased demand for mRNA-based analytical methods capable of assessing purity and stability, which are considered critical quality attributes (CQAs). In recent decades capillary electrophoresis (CE) has emerged alongside liquid chromatography (LC) as an important tool for the assessment of purity and stability of mRNA therapeutics. CE offers a variety of advantages over conventional LC or gel-based analytical methods, including reduced injection volume, increased resolution, and increased separation efficiency. In this study we compared CE-based analytical methods: the Agilent RNA 6000 Nano Kit, the Revvity RNA Reagent Kit, the Sciex RNA 9000 Purity and Integrity Kit, and the Agilent HS RNA Kit. These methods were evaluated on their vendor-recommended instruments: the Bioanalyzer, LabChip GXII, PA800 Plus, and Fragment Analyzer, respectively. We assessed the ability of these methods to measure mRNA integrity, purity, and stability. Furthermore, several parameters for each method were also assessed: selectivity, precision, resolution, analysis time, and ease of use. Based on our results, all four methods are suitable for use in the characterization of in vitro transcribed (IVT) mRNA, depending on the intended application. The Sciex RNA 9000 Purity and Integrity kit method achieved the highest selectivity and resolving power compared with the other methods, making it the most suitable for high-resolution, in-depth sample characterization. In comparison, the Agilent RNA 6000 Nano Kit, Revvity RNA Reagent Kit, and Agilent HS RNA Kit achieved lower selectivity and resolution, but their faster analysis times make them more suitable for high-throughput and screening applications. • 4 CGE-based analytical methods were evaluated. • Methods were comparatively benchmarked using four IVT mRNA samples. • The PA800 Plus-based method demonstrated the highest selectivity and resolution. • The other 3 methods are more suitable for high-throughput applications. [ABSTRACT FROM AUTHOR]

Fatty acids (FAs) are essential cellular components and play important roles in various biological processes. Importantly, FAs produced by microorganisms from renewable sugars are considered sustainable substrates for biodiesels and oleochemicals. Their complex structures and diverse functional roles in biochemical processes necessitate the development of efficient and accurate methods for their quantitative analysis. Here, we developed a novel method for relative quantification of FAs by combining 12-plex isobaric N,N - di methyl leu cine-derivatized e thyle n ediamine (DiLeuEN) labeling and microchip capillary electrophoresis-mass spectrometry (CE-MS). This method enables simultaneous quantification of 12 samples in a single MS analysis. DiLeuEN labeling introduced tertiary amine center structure into FAs, which makes them compatible with the positive mode separation of commercial microchip CE systems and further improves the sensitivity. The CE separation parameters were optimized, and the quantification accuracy was assessed using FA standards. Microchip CE-MS detection exhibited high sensitivity with a femtomole level detection limit and a total analysis time within 8 min. Finally, the applicability of our method to complex biological samples was demonstrated by analyzing FAs produced by four industrially relevant yeast strains (Saccharomyces cerevisiae , Yarrowia lipolytica YB-432 , Yarrowia lipolytica Po1f and Rhodotorula glutinis). The analysis time for each sample is less than 1 min. This work addresses the current challenges in the field by introducing a method that combines microchip-based capillary electrophoresis separation with multiplex isobaric labeling. Our method not only offers remarkable sensitivity and rapid analysis speed but also the capability to quantify fatty acids across multiple samples simultaneously, which holds significant potential for extensive application in FA quantitative studies in diverse research areas, promising an enhanced understanding of FA functions and mechanisms. [Display omitted] • Integrated 12-plex DiLeuEN isobaric labeling with CE-MS to enable rapid and precise fatty acids (FAs) relative quantification. • Enhanced the throughput of FAs quantification with notable precision and accuracy through 12-plex DiLeuEN isobaric labeling. • Achieved fast FAs separation in 8 min with fmol level detection limits via microchip CE-MS. • Successfully applied the developed method for quantitative analysis of FAs across four different yeast strains. [ABSTRACT FROM AUTHOR]

• An ultrasensitive quantification method for multiple miRNAs was established. • Strand displacement amplification was developed to amplify multiple miRNAs. • Three associated miRNAs in A549 cells were successfully detected. MicroRNAs (miRNAs) are increasingly recognized as potential biomarkers for the early diagnosis of cancer. However, the concurrent detection of multiple miRNAs in biological samples presents a significant challenge due to their high homogeneity and low abundance. This study introduced a novel approach combining strand displacement amplification (SDA) with microchip electrophoresis (MCE) for the simultaneous quantitation of trace levels of three miRNAs associated with cancer: miRNA-21, miRNA-145, and miRNA-221. Specifically designed probes were utilized to selectively capture the target miRNAs, thereby initiating the SDA process in a single solution without cross-interference. Under optimized conditions, the SDA-MCE method achieved the limit of detection (LOD) as low as 0.02 fM (S/ N = 3) and the limit of quantitation (LOQ) as low as 0.1 fM across a broad linear range spanning from 0.1 fM to 1 pM. The SDA reaction was completed in approximately 1.5 h, and all target products were separated within 135 s through MCE. Application of this method for the simultaneous detection of these three miRNAs in human lung cancer cell samples yielded satisfactory results. Featuring high sensitivity, rapid analysis, minimal reagent consumption, and straightforward operation, the proposed MCE-SDA strategy holds considerable promise for multi-miRNAs detection applications. [ABSTRACT FROM AUTHOR]

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.

Miniaturized synthesis of positron emission tomography (PET) tracers is poised to offer numerous advantages including reduced tracer production costs and increased availability of diverse tracers. While many steps of the tracer production process have been miniaturized, there has been relatively little development of microscale systems for the quality control (QC) testing process that is required by regulatory agencies to ensure purity, identity, and biological safety of the radiotracer before use in human subjects. Every batch must be tested, and in contrast with ordinary pharmaceuticals, the whole set of tests of radiopharmaceuticals must be completed within a short-period of time to minimize losses due to radioactive decay. By replacing conventional techniques with microscale analytical ones, it may be possible to significantly reduce instrument cost, conserve lab space, shorten analysis times, and streamline this aspect of PET tracer production. We focus in this work on miniaturizing the subset of QC tests for chemical identity and purity. These tests generally require high-resolution chromatographic separation prior to detection to enable the approach to be applied to many different tracers (and their impurities), and have not yet, to the best of our knowledge, been tackled in microfluidic systems. Toward this end, we previously explored the feasibility of using the technique of capillary electrophoresis (CE) as a replacement for the "gold standard" approach of using high-performance liquid chromatography (HPLC) since CE offers similar separating power, flexibility, and sensitivity, but can readily be implemented in a microchip format. Using a conventional CE system, we previously demonstrated the successful separation of non-radioactive version of a clinical PET tracer, 3'-deoxy-3'-fluorothymidine (FLT), from its known by-products, and the separation of the PET tracer 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-cytosine (D-FAC) from its α-isomer, with sensitivity nearly as good as HPLC. Building on this feasibility study, in this paper, we describe the first effort to miniaturize the chemical identity and purity tests by using microchip electrophoresis (MCE). The fully automated proof-of-concept system comprises a chip for sample injection, a separation capillary, and an optical detection chip. Using the same model compound (FLT and its known by-products), we demonstrate that samples can be injected, separated, and detected, and show the potential to match the performance of HPLC. Addition of a radiation detector in the future would enable analysis of radiochemical identity and purity in the same device. We envision that eventually this MCE method could be combined with other miniaturized QC tests into a compact integrated system for automated routine QC testing of radiopharmaceuticals in the future. Graphical abstract Miniaturized quality control (QC) testing of batches of radiopharmaceuticals via microfluidic analysis. The proof-of-concept hybrid microchip electrophoresis (MCE) device demonstrated the feasibility of achieving comparable performance to conventional analytical instruments (HPLC or CE) for chemical purity testing.

Nitrite is considered an important target analyte for environmental monitoring. In water resources, nitrite is the result of the nitrogen cycle and the leaching processes of pesticides based on nitrogenous compounds. A high concentration of nitrite can be associated with intoxication processes and metabolic disorders in humans. The present study describes the development of a portable analytical methodology based on microchip electrophoresis coupled with amperometric detection for the determination of nitrite in environmental water samples. Electrophoretic and detection conditions were optimized, and the best separations were achieved within 60 s by employing a mixture of 30 mmol L−1 lactic acid and 15 mmol L−1 histidine (pH = 3.8) as a running buffer applying 0.7 V to the working electrode (versus Pt) for amperometric measurements. The developed methodology revealed a satisfactory linear behavior in the concentration range between 20 and 80 μmolL−1 (R2 = 0.999) with a limit of detection of 1.3 μmolL−1. The nitrite concentration was determined in five water samples and the achieved values ranged from (28.7 ± 1.6) to (67.1 ± 0.5) µmol L−1. The data showed that using the proposed methodology revealed satisfactory recovery values (83.5–103.8%) and is in good agreement with the reference technique. Due to its low sample consumption, portability potential, high analytical frequency, and instrumental simplicity, the developed methodology may be considered a promising strategy to monitor and quantitatively determine nitrite in environmental samples. [ABSTRACT FROM AUTHOR]

Initial screening of criminal evidence often involves serological testing of stains of unknown composition and/or origin discovered at a crime scene to determine the tissue of origin. This testing is presumptive but critical for contextualizing the scene. Here, we describe a microfluidic approach for body fluid profiling via fluorescent electrophoretic separation of a published mRNA panel that provides unparalleled specificity and sensitivity. This centrifugal microfluidic approach expedites and automates the electrophoresis process by allowing for simple, rotationally driven flow and polymer loading through a 5 cm separation channel; with each disc containing three identical domains, multi-sample analysis is possible with a single disc and multi-sample detection per disc. The centrifugal platform enables a series of sequential unit operations (metering, mixing, aliquoting, heating, storage) to execute automated electrophoretic separation. Results show on-disc fluorescent detection and sizing of amplicons to perform comparably with a commercial 'gold standard' benchtop instrument and permitted sensitive, empirical discrimination between five distinct body fluids in less than 10 min. Notably, our microfluidic platform represents a faster, simpler method for separation of a transcriptomic panel to be used for forensically relevant body fluid identification. [ABSTRACT FROM AUTHOR]

Adulteration of alcoholic beverages is a worldwide concern for authorities, mainly due to risks to human health. This report describes the use of portable analytical platforms combined with chemometrics to trace the authenticity profile of whisky. Rapid analysis by miniaturized methodologies based on separation and colorimetric reactions as well as pH and conductivity measurements is proposed as a strategy for forensic screening at the point of need. Anionic profiles of whisky were obtained using a microchip electrophoresis system with integrated contactless conductivity detection. The indirect detection of caramel and the determination of alcohol content were performed by colorimetric reactions on paper-based analytical devices, which were explored in single microzones and foldable devices, respectively. These methodologies were successfully applied to a total of 50 whisky samples, 45 of which were adulterated and 5 authentic. A correlation study involving these markers and their analytical response through different techniques was then performed. A chemometric tool employing all these parameters was able to distinguish more than 90% of adulterated samples from authentic ones, demonstrating that the proposed methodology is effective for mapping whisky samples. These portable methods have great potential for use at the site of apprehension, due to the low consumption of samples and speed of analysis. [ABSTRACT FROM AUTHOR]

Keywords: lab-on-PCB; printed circuit board; biomedical applications; electronics; sensors; biosensors; actuators; lab-on-chip; microfluidics EN lab-on-PCB printed circuit board biomedical applications electronics sensors biosensors actuators lab-on-chip microfluidics N.PAG N.PAG 4 08/03/22 20220701 NES 220701 The use of Printed Circuit Boards (PCBs) has seen a remarkable growth over the last decade, with applications in engineering, medicine, biology, chemistry, etc. The lab-on-PCB devices are an interesting improvement of lab-on-chip devices due to the inexpensive characteristic of Printed Circuit Boards, and the possibility of including microfluidic circuits, sensors, and actuators, together with electronics in the same substrate. Therefore, the achievement of a robust development of biosensor using PCB substrates is mandatory for a successful future of lab-on-PCB devices. Apart from sensors and biosensors, the lab-on-PCB devices can include actuators, such as microheaters [[4]], and devices for microfluid handling, for example, electrowetting on dielectric devices (EWOD) [[5]]. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • An aptamer-mediated double SDA-MCE method was first explored for rapid and ultrasensitive detection of S. typhimurium. • The proposed method shows high sensitivity and the limit of detection for S. typhimurium down to 6 CFU/mL. • This method has satisfactory expectation for bacterial disease diagnosis. [ABSTRACT FROM AUTHOR]

In the microchip electrophoresis with capacitively coupled contactless conductivity detection, the stray capacitance of the detector causes high background noise, which seriously affects the sensitivity and stability of the detection system. To reduce the effect, a novel design of planar grounded capacitively coupled contactless conductivity detector (PG-C4D) based on printed circuit board (PCB) is proposed. The entire circuit plane except the sensing electrodes is covered by the ground electrode, greatly reducing the stray capacitance. The efficacy of the design has been verified by the electrical field simulation and the electrophoresis detection experiments of inorganic ions. The baseline intensity of the PG-C4D was less than 1/6 of that of the traditional C4D. The PG-C4D with the new design also demonstrated a good repeatability of migration time, peak area, and peak height (n = 5, relative standard deviation, RSD ≤ 0.3%, 3%, and 4%, respectively), and good linear coefficients within the range of 0.05–0.75 mM (R2 ≥ 0.986). The detection sensitivity of K+, Na+, and Li+ reached 0.05, 0.1, and 0.1 mM respectively. Those results prove that the new design is an effective and economical approach which can improve sensitivity and repeatability of a PCB based PG-C4D, which indicate a great application potential in agricultural and environmental monitoring. [ABSTRACT FROM AUTHOR]

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.. [ABSTRACT FROM AUTHOR]

In this work, we demonstrate for the first time the design and fabrication of microchip electrophoresis devices containing cross-shaped channels and spiral electrodes around the separation channel for microchip electrophoresis and capacitively coupled contactless conductivity detection. The whole device was prepared in a digital light processing–based 3D printer in poly(ethylene glycol) diacrylate resin. Outstanding X-Y resolution of the customized 3D printer ensured the fabrication of 40-μm cross section channels. The spiral channels were filled with melted gallium to form conductive electrodes around the separation channel. We demonstrate the applicability of the device on the separation of sodium, potassium, and lithium cations by microchip electrophoresis. [ABSTRACT FROM AUTHOR]

We report for the first time a microfluidic electrophoretic mobility reversal assay (MEMRA) for double-stranded DNA (dsDNA) detection using zinc-finger proteins (ZFPs) and a polyacrylamide-gel (PAG) sieving matrix. Microfluidic DNA analysis was actively studied because of its importance in biology and medicine. Most microfluidic DNA detection techniques rely on time-consuming denaturation and hybridization processes. To address this limitation, ZFP was employed as a novel affinity probe, which directly binds to a specific sequence of dsDNA without denaturation and renaturation. A mildly alkaline electrophoresis buffer (pH 8.6) was used for our MEMRA, instead of a strongly alkaline buffer (pH 10.75) for separating the ZFP–dsDNA complex from interfering species. At pH 8.6, the mobility of ZFP was reversed upon binding with dsDNA (complex pI = ~ 5.33), and unbound ZFP (pI = ~ 9.3) was excluded from loading. Therefore, the ZFP–dsDNA complex was detected without zone interferences. Furthermore, nonspecific interactions and band dispersion, observed in strongly alkaline buffer, were effectively mitigated in the MEMRA. The ZFP–dsDNA complex was fully separated (separation resolution ≥ 2.0) and detected rapidly (12–15 s at a separation distance of 160–240 μm) using on-chip photopatterned 3–16%T discontinuous PAG. The MEMRA performance was excellent, providing a detection limit of 50 pM and a detection range of 100 pM–500 nM for seb (Staphylococcus enterotoxin B) gene dsDNA oligonucleotides. We expect that our ZFP-based MEMRA will find broad utility in biology and medicine where the rapid, specific, and quantitative detection of dsDNA is of paramount importance. [ABSTRACT FROM AUTHOR]

Researches on detection of human papillomavirus (HPV) high-risk samples were carried out by polymerase chain reaction (PCR) coupled with microchip electrophoresis (MCE). Herein, we introduced a simple, rapid, automated method for detecting high-risk samples HPV16 and HPV18. In this research, general primers were initially selected to obtain sufficient detectable yield by PCR to verify feasibility of MCM method for HPV detection, then type-specific primers were further used to evaluate the specificity of MCE method. The results indicated MCE method was capable of specifically detecting high-risk HPV16 and HPV18, and also enabled simultaneous detection of multiplex samples. This MCE method described here has been successfully applied to HPV detection and displayed excellent reliability demonstrating by sequencing results. The inherent capability of MCE facilitated HPV detection conducted in a small chip with automated, high throughput, massive parallelized analysis. We envision that MCE method will definitely pave a way for clinical diagnosis, and even on-site screening of cervical cancer.

The problem of overlapping peaks has been a challenge in microchip electrophoresis (ME) signal analysis. However, traditional peak fitting algorithms have difficulty analyzing overlapping peaks due to the high dependence on the starting point. In this study, we propose a symmetrical peak fitting method named the tent-mapped whale optimization algorithm and Levenberg–Marquardt (TWOALM), which combines a whale optimization algorithm (WOA) improved by one of the most commonly used chaotic maps, the tent map and the Levenberg–Marquardt (LM) algorithm. Specifically, we first derive the fitted model for the overlapping peaks, showing that it is separable nonlinear least squares, significantly reducing the number of parameters to be optimized. Second, we integrate the tent map into the WOA, which improves the convergence speed of the peak fitting algorithm. Finally, we propose an efficient peak-fitting algorithm that combines the improved WOA and LM. The advantage of the proposed algorithm is that it is significantly faster than WOA and significantly more accurate than the LM algorithm. The results of fitting the synthetic peaks and ME signals showed that the combination of the chaotic map-based WOA algorithm and the LM algorithm can significantly improve the peak fitting performance and provide an effective solution for the analysis of overlapping peaks.

Two decades ago, a problem echoed by many individuals and authorities was the complexity of chemical and biochemical analysis that typically necessitated well-equipped laboratories and trained personnel able to undertake complex sample pre-treatment procedures and operate specialized and expensive instrumentation. The first work by Hang Tng et al. reported a saliva antigen rapid test (AP-ART) that was based on multimodal viral capture using both S-specific polyclonal antibodies and recombinant human ACE2 at the test line and signal enhancement using linker-free, signal amplification gold nanoparticles introduced via a parallel flow channel. [Extracted from the article]

We present an experimental and numerical investigation of electrokinetic instability (EKI) in microchannel flow with streamwise conductivity gradients, such as those observed during sample stacking in capillary electrophoresis. A plug of a low-conductivity electrolyte solution is initially sandwiched between two high-conductivity zones in a microchannel. This spatial conductivity gradient is subjected to an external electric field applied along the microchannel axis, and for sufficiently strong electric fields an instability sets in. We have explored the physics of this EKI through experiments and numerical simulations, and supplemented the results using scaling analysis. We performed EKI experiments at different electric field values and visualised the flow using a passive fluorescent tracer. The experimental data were analysed using the proper orthogonal decomposition technique to obtain a quantitative measure of the threshold electric field for the onset of instability, along with the corresponding coherent structures. To elucidate the physical mechanism underlying the instability, we performed high-resolution numerical simulations of ion transport coupled with fluid flow driven by the electric body force. Simulations reveal that the non-uniform electroosmotic flow due to axially varying conductivity field causes a recirculating flow within the low-conductivity region, and creates a new configuration wherein the local conductivity gradients are orthogonal to the applied electric field. This configuration leads to EKI above a threshold electric field. The spatial features of the instability predicted by the simulations and the threshold electric field are in good agreement with the experimental observations and provide useful insight into the underlying mechanism of instability. [ABSTRACT FROM AUTHOR]

Anemia affects over 25% of the world's population with the heaviest burden borne by women and children. Genetic hemoglobin (Hb) variants, such as sickle cell disease, are among the major causes of anemia. Anemia and Hb variant are pathologically interrelated and have an overlapping geographical distribution. We present the first point-of-care (POC) platform to perform both anemia detection and Hb variant identification, using a single paper-based electrophoresis test. Feasibility of this new integrated diagnostic approach is demonstrated via testing individuals with anemia and/or sickle cell disease. Hemoglobin level determination is performed by an artificial neural network (ANN) based machine learning algorithm, which achieves a mean absolute error of 0.55 g dL−1 and a bias of −0.10 g dL−1 against the gold standard (95% limits of agreement: 1.5 g dL−1) from Bland–Altman analysis on the test set. Resultant anemia detection is achieved with 100% sensitivity and 92.3% specificity. With the same tests, subjects with sickle cell disease were identified with 100% sensitivity and specificity. Overall, the presented platform enabled, for the first time, integrated anemia detection and hemoglobin variant identification using a single point-of-care test. [ABSTRACT FROM AUTHOR]

[Display omitted] • Microfluidic devices enable fully automated and integrated at-line monitoring systems. • Microchip capillary electrophoresis is a promising tool for bioprocess monitoring. • Microfluidic affinity assays can be used for detection of bioprocess target proteins. • Miniaturization enables PAT at reduced cost and without need of complex instruments. Monitoring technologies for Process Analytical Technology (PAT) in mammalian cell cultures are often focusing on the same hand full parameters although a deeper knowledge and control of a larger panel of culture components would highly benefit process optimization, control and robustness. This short review highlights key advances in microfluidic affinity assays and microchip capillary electrophoresis (MCE). Aiming at the miniaturization and integration of PAT, these can detect at-line a variety of metabolites, proteins and Critical Quality Attributes (CQA's) in a bioprocess. Furthermore, discrete analytical components, which can potentially support the translation of increasingly mature microfluidic technologies towards this novel application, are also presented as a comprehensive toolbox ranging from sample preparation to signal acquisition. [ABSTRACT FROM AUTHOR]

Purpose: The aim of our study was to investigate the correlation between cfDNA concentration and fragment size fraction with FDG PET/CT- and CT-derived parameters in untreated NSCLC patient. Methods: Fifty-three patients diagnosed of locally advanced or metastatic NSCLC who had undergone FDG PET/CT, CT and cfDNA analysis prior to any treatment were included in this retrospective study. CfDNA concentration was measured by fluorometry and fragment size fractions were determined by microchip electrophoresis. [18F]F-FDG PET/CT was performed and standardised uptake values (SUV), metabolic tumour volume (MTV) and total lesion glycolysis (TLG) were calculated for primary, extrapulmonary and total disease. CT scans were evaluated according to RECIST 1.1 criteria. Results: CfDNA concentration showed a positive correlation with extrapulmonary MTV (r2 = 0.36, P = 0.009), and extrapulmonary TLG (r2 = 0.35, P = 0.009) and their whole-body (wb) ratios. Higher concentrations of total cfDNA were found in patients with liver lesions. Short fragments of cfDNA (100–250 bp) showed a positive correlation with extrapulmonary MTV (r2 = 0.49, P = 0.0005) and extrapulmonary TLG (r2 = 0.39, P = 0.006) and their respective wb ratios, and a negative correlation with SUVmean (r2 = −0.31, P = 0.03) and SUVmean/SUVmax ratio (r2 = −0.34, P = 0.02). A higher fraction of short cfDNA fragments was found in patients with liver and pleural lesions. Conclusions: This study supports the hypothesis that cfDNA concentration and short cfDNA fragment size fraction reflect the tumour burden as well as metabolic activity in advanced NSCLC patients. This suggests their suitability as complementary tests for a more accurate diagnosis of tumour metabolic behaviour and to allow personalised therapies. [ABSTRACT FROM AUTHOR]

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies. [ABSTRACT FROM AUTHOR]

• Elevated cell-free DNA (cfDNA) concentrations were observed in postmortem subjects. • cfDNA level increased with postmortem intervals in subjects who died of asphyxia. • cfDNA concentration was slightly higher in subjects who died from internal cause. • A high concentration of a 150–200 bp fragment characterized postmortem samples. • A fragment larger than 10,000 bp, which originated from necrosis, was found in only two subjects. Cell-free DNA (cfDNA) originates from apoptotic and/or necrotic cells. Few reports are available that examine cfDNA from postmortem samples. Therefore, this study investigated differences between postmortem and biogenic subjects in concentration and fragment distribution of serum cfDNA. We also clarified features of serum cfDNA in postmortem subjects. The results revealed that postmortem subjects had significantly higher cfDNA concentrations than healthy controls and patients with cardiac disease. Serum cfDNA concentrations increased slightly with postmortem interval in subjects who died of asphyxia, and they were slightly higher in subjects who died from internal vs. external causes. Microchip electrophoresis of serum cfDNA revealed a fragment larger than 10,000 bp in only two postmortem subjects; we speculate that the fragment may have originated from necrotic cells. A relatively high concentration of one 150–200 bp fragment was characteristic of postmortem samples. This fragment may have been derived from apoptosis or other processes. We also observed ladder fragments in some subjects who died from external causes. Although additional research is needed for verification, serum cfDNA concentrations and fragment patterns possibly be used as a tool to estimate postmortem intervals and cause of death. [ABSTRACT FROM AUTHOR]

Non-aqueous capillary electrophoresis (NACE) on microfluidic chips is still a comparatively little explored area, despite the inherent advantages of this technique and its application potential for, in particular, lipophilic compounds. A main reason is probably the fact that implementation of NACE on microchips largely precluded the use of polymeric substrate materials. Here, we report non-aqueous electrophoresis on a thiol-ene-based microfluidic chip coupled to mass spectrometry via an on-chip ESI interface. Microchips with an integrated ESI emitter were fabricated using a double-molding approach. The durability of thiol-ene, when exposed to different organic solvents, was investigated with respect to swelling and decomposition of the polymer. Thiol-ene exhibited good stability against organic solvents such as methanol, ethanol, N-methylformamide, and formamide, which allows for a wide range of background electrolyte compositions. The integrated ESI emitter provided a stable spray with RSD% of the ESI signal ≤8%. Separation efficiency of the developed microchip electrophoresis system in different non-aqueous buffer solutions was tested with a mixture of several drugs of abuse. Ethanol- and methanol-based buffers provided comparable high theoretical plate numbers (≈ 6.6 × 104–1.6 × 105 m−1) with ethanol exhibiting the best separation efficiency. Direct coupling of non-aqueous electrophoresis to mass spectrometry allowed for fast analysis of hydrophobic compounds in the range of 0.1–5 μg mL−1 and 0.2–10 μg mL−1 and very good sensitivities (LOD ≈ 0.06–0.28 μg mL−1; LOQ ≈ 0.20–0.90 μg mL−1). The novel combination of non-aqueous CE on a microfluidic thiol-ene device and ESI-MS provides a mass-producible and highly versatile system for the analysis of, in particular, lipophilic compounds in a wide range of organic solvents. This offers promising potential for future applications in forensic, clinical, and environmental analysis. [ABSTRACT FROM AUTHOR]

The long-term consumption of food with pesticide residues has harmful effects on human health and the demand for pesticide detection technology tends to be miniaturized and instant. To this end, we demonstrated the first application of indirectly detecting two carbamate pesticides, metolcarb and carbaryl, by gold nanoparticle–modified indium tin oxide electrode in dual-channel microchip electrophoresis and amperometric detection (ME-AD) system. m-Cresol and α-naphthol were obtained after pesticide hydrolysis in alkaline solution, and then separated and detected by ME-AD. Parameters including the detection potential and running buffer concentration and pH were optimized to improve the detection sensitivity and separation efficiency. Under the optimal conditions, the two analytes were completely separated within 80 s. m-Cresol and α-naphthol presented a wide linear range from 1 to 100 μM, with limits of detection of 0.16 μM and 0.34 μM, respectively (S/N = 3). Moreover, the reliability of this system was demonstrated by analyzing metolcarb and carbaryl in spiked vegetable samples. [ABSTRACT FROM AUTHOR]

More and more studies have found that microRNAs (miRNAs) are markers of cancer, and detection of miRNAs is beneficial for early diagnosis and prognosis of cancer. In this paper, the isothermal strand displacement polymerase reaction (ISDPR), which is an enzyme-assisted nucleic acid amplification method, was studied to combine with microchip electrophoresis (MCE) for a simultaneously detection of two cancer related miRNAs named microRNA-21 (miR-21) and microRNA-221 (miR-221). In the ISDPR amplification, two different DNA hairpins (HPs) were specifically designed, so that miR-21 and miR-221 could respectively bind to HPs and started ISDPR amplification to generate two different products which were ultimately detected by MCE. The optimal conditions of ISDPR were carefully investigated, and the limits of detection (LOD) of miR-21 and miR-221 were as low as 0.35 fM and 0.25 fM (S/N = 3) respectively under these conditions. The human lung tumor cells and serum samples were analyzed by this ISDPR-MCE method and satisfactory results were obtained, which means that this method is of high sensitivity, high efficiency, low reagent consumption and simple operation in miRNAs detection. [Display omitted] • ISDPR-MCE was first used for simultaneous detection of miRNAs. • The LODs of miR-21 and miR-221 are as low as 0.35 fM and 0.25 fM. [ABSTRACT FROM AUTHOR]