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.

A new green approach to L-histidine and ß-alanine analysis in dietary supplements using rapid and simple contactless conductivity detection integrated with high-resolution glass-microchip electrophoresis.

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 ß-alanine. A new microfluidic method for the determination of L-histidine and ß-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 ß-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.

Recent advances in microscale separation techniques for glycome analysis.

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.

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.

Quantification of multiple microRNAs by microchip electrophoresis assisted by strand displacement amplification.

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.

High-throughput relative quantification of fatty acids by 12-plex isobaric labeling and microchip capillary electrophoresis - Mass spectrometry.

BACKGROUND: 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. RESULTS: Here, we developed a novel method for relative quantification of FAs by combining 12-plex isobaric N,N-dimethyl leucine-derivatized ethylenediamine (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. SIGNIFICANCE: 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.

Isothermal strand displacement polymerase reaction (ISDPR)-assisted microchip electrophoresis for highly sensitive detection of cancer associated microRNAs.

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.

Microchip isotachophoresis for green and sustainable pharmaceutical quality control: Method validation and application to complex pharmaceutical formulations.

Universal microchip isotachophoresis (µITP) methods were developed for the determination of cationic and anionic macrocomponents (active pharmaceutical ingredients and counterions) in cardiovascular drugs marketed in salt form, amlodipine besylate and perindopril erbumine. The developed methods are characterized by low reagent and sample consumption, waste production and energy consumption, require only minimal sample preparation and provide fast analysis. The greenness of the proposed methods was assessed using AGREE. An internal standard addition was used to improve the quantitative parameters of µITP. The proposed methods were validated according to the ICH guideline. Linearity, precision, accuracy and specificity were evaluated for each of the studied analytes and all set validation criteria were met. Good linearity was observed in the presence of matrix and in the absence of matrix, with a correlation coefficient of at least 0.9993. The developed methods allowed precise and accurate determination of the studied analytes, the RSD of the quantitative and qualitative parameters were less than 1.5% and the recoveries ranged from 98 to 102%. The developed µITP methods were successfully applied to the determination of cationic and anionic macrocomponents in six commercially available pharmaceutical formulations.

Bacterial handling under the influence of non-uniform electric fields: dielectrophoretic and electrohydrodynamic effects

This paper describes the modeling and experimental verification of a castellated microelectrode array intended tohandle biocells, based on common dielectrophoresis. The proposed microsystem was developed employing platinumelectrodes deposited by lift-off, silicon micromachining, and photoresin patterning techniques. Having fabricated the microdevice it was tested employing Escherichia coli as bioparticle model. Positive dielectrophoresis could be verified with the selected cells for frequencies above 100 kHz, and electrohydrodynamic effects were observed as the dominant phenomena when working at lower frequencies. As a result, negative dielectrophoresis could not be observed because its occurrence overlaps with electrohydrodynamic effects; i.e. the viscous drag force acting on the particles is greater than the dielectrophoretic force at frequencies where negative dielectrophoresis should occur. The experiments illustrate the convenience of this kind of microdevices to micro handling biological objects, opening the possibility for using these microarrays with other bioparticles. Additionally, liquid motion as a result of electrohydrodynamic effects must be taken into account when designing bioparticle micromanipulators, and could be used as mechanism to clean the electrode surfaces, that is one of the most important problems related to this kind of devices.

Este artigo descreve a modelagem e teste experimental de uma rede de microeletrodos em cremalheira cujo objetivo é o manuseio de células biológicas, com base em dieletroforese comum. O microsistema proposto foi desenvolvido empregando eletrodos de platina depositados por técnicas de 'lift-off', micro-usinagem em silício e litografia com foto-resina. Uma vez fabricado o microdispositivo, este foi testado utilizando a Escherichia coli como modelo de biopartículas. Dieletroforese positiva pode ser observada com as células selecionadas para freqüências acima de 100kHz, e efeitos eletro-hidrodinâmicos foram observados como o fenômeno dominante para menores freqüências. Como resultado, a dieletroforese negativa não pode ser observada pois sua ocorrência se sobrepõe a efeitos eletro-hidrodinâmicos; i.e. a força de arraste viscoso atuando sobre as partículas é superior à força dieletroforética para freqüências em que a dieletroforese negativa deveria ocorrer. Os experimentos ilustram a conveniência deste tipo de micro-dispositivo para o micromanuseio de objetos biológicos, abrindo a possibilidade de uso destas micro-redes com outras partículas biológicas. Além disto, o movimento líquido como resultado dos efeitos eletro-hidrodinâmicos deve ser levado em conta ao se desenhar micromanipuladores de partículas biológicas, e pode ser utilizado como mecanismo para limpar as superfícies dos eletrodos, que é um dos problemas mais importantes relacionados a este tipo de dispositivo.

The relation between Escherichia coli surface functional groups' composition and their physicochemical properties

Escherichia coli surface characteristics including hydrophobicity, electrophoretic mobility and surface functional groups' composition were investigated. These characteristics were determined respectively by water contact angle measurements, microelectrophoresis and x-ray photoelectron spectroscopy (XPS). The relation between the physicochemical properties and functional groups' composition was also examined. The electrophoretic mobility at pH 7 appeared to be governed on the cell surface by the (O=C) functional groups. The cell surface's hydrophilicity was associated with high levels of (C-(O.N)) and (OH-(C-O-C)) functional groups, whereas the cell surface's hydrophobicity was associated with (C-(C,H)) functional groups.

Características de superfície de Escherichia coli, tais como hidrofobicidade, mobilidade eletroforética e composição dos grupos funcionais de superfície, foram estudadas. Essas características foram determinadas por medidas de angulo de contato com água, microeletroforese e espectroscopia fotoeletrônica de raio-X (XPS), respectivamente. A relação entre as propriedades fisicoquímicas e a composição dos grupos funcionais foi também examinada. Aparentemente, a mobilidade eletroforética em pH 7 é controlada na superfície celular pelos grupos funcionais (O=C). A hidrofilicidade da superfície celular estava associada com altos níveis dos grupos funcionais [C-(O.N)] e [OH-(C-O-C)], enquanto a hidrofobicidade estava associada com os grupos funcionais [C-(C,H)].