Electrophoresis Chip Research Articles

Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis

The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.

Two Benches PDMS Free Flow Electrophoresis Chip

This paper puts forward a kind of two benches structure of the glass-PDMS free flow electrophoresis chip. We use die casting method to produce microstructure, and use PDMS as its structure layer. The mold is made in tape-PMMA by micro cementing technology. During the progress of PDMS curing, gold wires are directly integrated in the electrode groove to form the chips electrodes. PDMS and glass are sealed in natural bonding method. Chips glass surface of separation chamber realizes the modification through sticking polyimide tape. Visual technology is conducted for chip electrophoresis separation. The sample is the mixture of methyl green and Rhodamine B. The separation effect of methyl green and Rhodamine B is obvious when the electric field is 31 V/cm. the "two benches" between the electrode groove and the separation chamber block the electrolytic bubbles into the separation chamber. When the separation chamber height is 70 μm and the electric field is 50 V/cm, the air bubbles can be glidingly discharged from electrode groove. The samples steady separation time is more than 2 hours, and this free flow electrophoresis chip realizes continuous work.

Chip electrophoresis as a novel approach to measure the polyphenols reactivity toward human saliva.

Saliva is a biological fluid with a multifunctional role that makes it interesting in terms of research and diagnostic possibilities. In food research, human saliva represented a useful tool by which we measure the tactile sensation elicited by polyphenol-rich beverages called astringency. A method based on SDS-PAGE analysis of saliva before and after the binding reaction with wine polyphenols has been successfully used in previous studies for measuring wine astringency by means of the saliva precipitation index. In this work, chip electrophoresis was used alternatively to SDS-PAGE and results were compared. Chip electrophoresis provides a very good reproducibility for wine and grape astringency. Moreover, this approach is much faster than the conventional SDS-PAGE method requiring several hours for an analysis. Another advantage over traditional gel is lower sample and reagent volume requirements, as well as the lower and less toxic wastes, contributing benefits to health and environment. The application of this novel method allowed, using the principal component analysis, to distinguish grapes and wines according to the saliva precipitation index and structural characteristics determined by the phoroglucinolysis analysis.

Chip electrophoretic separation of highly homologous ammodytoxin isoforms: Three neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom

Ammodytoxins (Atxs), a group of Ca(2+) -dependent neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom, are mainly responsible for venom toxicity. Within the Atx group, LD50 values between three isoforms, A, B, and C are differing with AtxA exhibiting an LD50 value by an order of magnitude lower (more toxic) than the other two isoforms. This difference in toxicity justifies the necessity to prepare suitable antibodies and thus isoform separation to characterize the Atx content of Vipera ammodytes ammodytes venom is of importance. However, a high homology between the three Atx isoforms (differences in only two, respectively, three residues within the last 18 amino acids at the C-terminus, total length 122 residues) hindered the successful separation of isoforms to date. As the investigated phospholipases A2 were reported to exhibit differences in pI values, we concentrate with the current work on the separation of Atx isoforms after fluorescence labeling via chip electrophoresis on a commercially available instrument to build the basis for a fast and easy to handle screening method. In the course of our work, we were able to show that samples of AtxA, AtxB, and AtxC declared to be homogenous by standard analytical techniques consisted indeed of more than one isoform of which the relative amounts were calculated by using the newly developed method.

Automation Highlights from the Literature

Automation Highlights from the Literature

A novel microfluidic chip electrophoresis strategy for simultaneous, label-free, multi-protein detection based on a graphene energy transfer biosensor

A new type of high-throughput and parallel optical sensing platform with a single-color probe based on microfluidic chip electrophoresis combined with aptamer-carboxyfluorescein/graphene oxide energy transfer is reported here. Label-free protein multi-targets were detected, even in challenging complex samples without any pre-treatment.

Microfluidic chip electrophoresis investigation of major milk proteins: study of buffer effects and quantitative approaching

Results of microfluidic chip technology using SEP buffer were comparable to those of SDS-PAGE and previously reported data.

Sensitive determination of reactive oxygen species in cigarette smoke using microchip electrophoresis–localized surface plasmon resonance enhanced fluorescence detection

A sensitive approach to the determination of reactive oxygen species (ROS) in puffs of cigarette smoke (CS) has been developed. The experimental system consists of a microfluidic chip electrophoresis and a laser induced fluorescence (LIF) device enhanced by localized surface plasmon resonance. Core-shell Ag@SiO2 nanoparticles were prepared and then immobilized on the surface of the microchannel to increase the fluorescence intensity based on localized surface plasmon resonance-enhanced fluorescence (LSPREF) effect. The ROS in puffs of CS were trapped via the oxidation of 2',7'-dichlorodihydrofluorescein (DCHF) that had been loaded on polyacrylonitrile (PAN) nanofibers in a micro-column. Determination of ROS was based on the amount of 2',7'-dichlorofluorescein (DCF), which is the sole product from DCHF oxidation. With the optimization of the trapping efficiency, we detected about 8.0 pmol of ROS per puff in the mainstream CS. This microchip electrophoresis-SPREF system enables sensitive quantitation of ROS in CS with low consumption of reagent, material, and analysis time.

Characterization of cross‐linked gelatin nanoparticles by electrophoretic techniques in the liquid and the gas phase

Biodegradable nanoparticles (NPs) and hence e.g. NPs prepared from glutaraldehyde crosslinked gelatin (gelatin NPs) are lately receiving increased attention in various fields like pharmaceutical technology and nutraceutics as biocompatible carriers for hardly water soluble drugs, molecules intended for sustained release or targeted transport. However, in vivo application of such materials requires a thoroughly characterization of corresponding particles. In a previous manuscript we demonstrated the applicability of chip electrophoresis for the separation of gelatin NPs from NP building blocks. Following our previous results we intensified our efforts in the characterization of gelatin NPs by electrophoresis in the liquid (capillary and chip format) and the gas phase (gas phase electrophoretic mobility molecular analysis, GEMMA). In doing so, we demonstrated differences between nominally comparable (from the concentration of initially employed material for crosslinking) gelatin NP preparation batches concerning (i) the amount of obtained NPs, (ii) the degree of NP crosslinking, (iii) the amount of NP building blocks present within samples and (iv) the electrophoretic mobility diameter of NPs. Differences were even more pronounced when NP preparations from batches with different content of initially employed gelatin were compared. Additionally, we compared three setups for the removal of low molecular weight components from samples after fluorescence labeling of NPs. In overall, the combination of the three employed analytical methods for gelatin NP characterization - CE in the capillary and the chip format as well as GEMMA - allows a more thoroughly characterization of NP containing samples.

Label-Free Fluorescence Detection of Aromatic Compounds in Chip Electrophoresis Applying Two-Photon Excitation and Time-Correlated Single-Photon Counting

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

Chip electrophoresis of gelatin‐based nanoparticles

Recently, biodegradable nanoparticles received increasing attention for pharmaceutical applications as well as applications in the food industry. With the current investigation we demonstrate chip electrophoresis of fluorescently (FL) labeled gelatin nanoparticles (gelatin NPs) on a commercially available instrument. FL labeling included a step for the removal of low molecular mass material (especially excess dye molecules). Nevertheless, for the investigated gelatin NP preparation two analyte peaks, one very homogeneous with an electrophoretic net mobility ofμ = -24.6 ± 0.3 × 10(-9) m(2) /Vs at the peak apex (n = 17) and another more heterogeneous peak withμ between approximately -27.2 ± 0.2 × 10(-9) m(2) /Vs and -36.6 ± 0.2 × 10(-9) m(2) /Vs at the peak beginning and end point (n = 11, respectively) were recorded. Filtration allowed enrichment of particles in the size range of approximately 35nm (pore size employed for concentration of gelatin NPs) to 200nm (pore size employed during FL labeling). This corresponded to the very homogeneous peak linking it to gelatin NPs, whereas the more heterogeneous peak probably corresponds to gelatin not cross-linked to such a high degree (NP building blocks). Several further gelatin NP preparations were analyzed according to the same protocol yielding peaks with electrophoretic net mobilities between -23.3 ± 0.3 × 10(-9) m(2) /Vs and -28.9 ± 0.2 × 10(-9) m(2) /Vs at peak apexes (n = 15 and 6). Chip electrophoresis allows analyte separation in less than two minutes (including electrophoretic sample injection). Together with the high sensitivity of the FL detection - the LOD as derived for the first main peak of the applied dye from the threefold standard deviation of the background noise values 80 pM for determined separation conditions - this leads to a very promising high throughput separation technique especially for the analysis of bionanoparticles. For gelatin NP preparations, chip electrophoresis allows for example the comparison of preparation batches concerning the amount of NPs and gelatin building blocks as well as the indirect assessment of the degree of gelatin cross-linking (from obtained FL signals).

Universal Microfluidic Automaton for Autonomous Sample Processing: Application to the Mars Organic Analyzer

A fully integrated multilayer microfluidic chemical analyzer for automated sample processing and labeling, as well as analysis using capillary zone electrophoresis is developed and characterized. Using lifting gate microfluidic control valve technology, a microfluidic automaton consisting of a two-dimensional microvalve cellular array is fabricated with soft lithography in a format that enables facile integration with a microfluidic capillary electrophoresis device. The programmable sample processor performs precise mixing, metering, and routing operations that can be combined to achieve automation of complex and diverse assay protocols. Sample labeling protocols for amino acid, aldehyde/ketone and carboxylic acid analysis are performed automatically followed by automated transfer and analysis by the integrated microfluidic capillary electrophoresis chip. Equivalent performance to off-chip sample processing is demonstrated for each compound class; the automated analysis resulted in a limit of detection of ~16 nM for amino acids. Our microfluidic automaton provides a fully automated, portable microfluidic analysis system capable of autonomous analysis of diverse compound classes in challenging environments.

Microchip Electrophoretic Analysis of Phaseolin Patterns and Its Comparison with Currently Used SDS-PAGE Techniques

The goal of this work was to compare reproducibility of phaseolin patterns of common bean obtained by two electrophoretic protein separation techniques including the conventional SDS-PAGE and an automated chip electrophoresis system. Five standard cultivars of common bean provided by the United States Department of Agriculture (Beltsville, Maryland) that represented five phaseolin types, T (Tendergreen), C (Contender) and S (Sanilac), B (Boyaca) and P (Pampa), were used in this study. Comparison of the phaseolin patterns revealed that the chip-on-a-lab electrophoresis provided a good reproducibility. The phaseolin polymorphism included four to seven polypeptides typical for the pattern composition of the T, C and S types. The polymorphism of the B and P patterns was also established. Phaseolin polypeptides separated by the microchip electrophoresis exhibited differences with respect to the molecular weights and electrophoretic mobility as compared to the SDS-PAGE technique. This phenomenon could be attributed to the absence of a solid separation phase in the microchip electrophoresis. Moreover, this technique has potential to substantially accelerate screening of large bean germplasm collections since it allows for the accurate analysis of the higher number of individual plants within accessions than the conventional, tedious and time consuming SDS-PAGE method.

High Performance of Cyclic Olefin Copolymer-Based Capillary Electrophoretic Chips

This paper demonstrates a simple, one step, and low cost surface modification technique for producing cyclic olefin copolymer (COC) polymer-based microcapillary electrophoresis chips consisting highly hemocompatible microchannels by UV-photografting with N-vinylpyrrolidone (NVP) monomer. An optimal condition has been identified to achieve the best surface grafting process. It has been found that this surface treatment enables extremely high surface wettability, hemocompatibility, and bond strength to the microchannels. The surface grafting was confirmed by attenuated total reflection Fourier transform-infrared spectroscopic (ATR-FTIR) study. In vitro protein adsorption using fluorescent labeled bovine serum albumin (FITC-BSA) into the COC microchannel results indicates that the modified chips have excellent protein resistance ability because of the increase of surface hydrophilicity. Hence, the modified chips showed fast, reproducible and high efficient separations of proteins (up to 51,000 theoretical plates per meter). Moreover, this surface modification process show no loss in the optical transparency to the modified microchannel surfaces: an important requirement for real capillary electrophoresis since the fluorescent intensity is directly related to the amount of adsorbed protein on the surface. Therefore, we believe that this simple and promising route of surface modification could be very useful for developing high performance COC microfluidic devices for the separation of proteins, amino acids, and other biomolecules.

Simple Boric Acid-Based Fluorescent Focusing for Sensing of Glucose and Glycoprotein via Multipath Moving Supramolecular Boundary Electrophoresis Chip

Boric acid-based fluorescent complex probe of BBV-HPTS (boronic acid-based benzyl viologen (BBV) and hydroxypyrene trisulfonic acid trisodium salt (HPTS)) was rarely used for sensitive sensing of saccharide (especially glycoprotein) via electrophoresis. We proposed a novel model of moving supramolecular boundary (MSB) formed with monosaccharide or glycoprotein in microcolumn and the complex probe of BBV-HPTS in the cathodic injection tube, developed a method of MSB fluorescent focusing for sensitive recognition of monosaccharide and glycoprotein, and designed a special multipath capillary electrophoresis (CE) chip for relative experiments. As a proof of concept, glucose and hemoglobin A1c (HbA1c) were respectively used as the mode saccharide and glycoprotein for the relevant demonstration. The experiments revealed that (i) the complex of BBV-HPTS could interact with free glucose or bound one in glycoprotein; (ii) the fluorescent signal was a function of glucose or glycoprotein content approximately; and (iii) interestingly the fluorescent band motion was dependent on glucose content. The developed method had the following merits: (i) low cost; (ii) low limit of detection (down to 1.39 pg/mL for glucose and 2.0 pg per capillary HbA1c); and (iii) high throughput (up to 12 runs or more per patch) and speed (less than 5 min). The developed method has potential use for sensitive monitoring of monosaccharide and glycoprotein in biomedical samples.

Integrated chip electrophoresis and magnetic particle isolation used for detection of hepatitis B virus oligonucleotides

Rapid and sensitive detection is a key step in the effective and early response to the global hazard of various viral diseases. In this study, an integrated isolation of hepatitis B virus (HBV)-specific DNA fragment by magnetic nanoparticles (MNPs) and its immediate analysis by microchip CGE was performed. Microfluidic CE chip was used to accommodate the complete process of viral DNA isolation by MNPs including hybridization and thermal denaturation followed by CE separation. Beforehand, calibration curves of HBV fragments were constructed. For isolation by MNPs, specific streptavidin-biotin interaction was used to bind complementary HBV fragment to magnetic particles. After analysis of isolated HBV by regular MNPs method, innovative approach was performed. The commercial CE chip (Bio-rad) was successfully used to execute HBV fragment isolation. Detection using LIF with detection limit of 1 ng/mL was accomplished.

Determination of nitrite and nitrate in cerebrospinal fluid by microchip electrophoresis with microsolid phase extraction pre-treatment

A new method for the determination of nitrite and nitrate, indicators of various neurological diseases (meningitis, multiple sclerosis, Parkinson's disease) in cerebrospinal fluid (CSF) on an electrophoresis chip was developed. An on-line combination of isotachophoresis (ITP) with capillary electrophoresis (CE) on a poly(methylmethacrylate) chip assembled with coupled separation channels (CC) and contact conductivity detectors was employed. ITP separations performed at low pH (3.6) in the first separation channel enabled a highly selective transfer of the analytes to the second CE stage working under micellar conditions implemented by zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)-propanesulfonate. The proposed method achieved low limits of detection varied from 0.2 to 0.4μgL(-1) when the sample volume injected onto the chip (9.9μl) was almost the same as the volume of both separation channels. Preferable working conditions on the CC chip (suppressed hydrodynamic and electroosmotic flow) contributed for reproducible migration velocities (intra-day reproducibility up to 2.1% RSD) and determinations of trace concentrations of nitrite and nitrate (intra-day precision up to 3.0% RSD). Huge amount of chloride present in CSF (approx. 4.5gL(-1)) was removed from analyzed CSF samples by microsolid phase extraction performed on silver-form resin prior to the ITP-CE analysis. Developed method provided fast (approx. 20min total analysis time) and reliable determinations of trace nitrite and nitrate and could be fully integrated into the analysis of CSF samples.

Towards an integrated device that utilizes adherent cells in a micro-free-flow electrophoresis chip to achieve separation and biosensing

We immobilized adherent human embryonic kidney (HEK) cells--which are able to trace adenosine triphosphate (ATP)--inside a microfluidic free-flow electrophoresis (μFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 μM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.

Exploring chip-capillary electrophoresis-laser-induced fluorescence field-deployable platform flexibility: Separations of fluorescent dyes by chip-based non-aqueous capillary electrophoresis

Microfluidic chip electrophoresis (chip-CE) is a separation method that is compatible with portable and on-site analysis, however, only few commercial chip-CE systems with laser-induced fluorescence (LIF) and light emitting diode (LED) fluorescence detection are available. They are established for several application tailored methods limited to specific biopolymers (DNA, RNA and proteins), and correspondingly the range of their applications has been limited. In this work we address the lack of commercially available research-type flexible chip-CE platforms by exploring the limits of using an application-tailored system equipped with chips and methods designed for DNA separations as a generic chip-CE platform – this is a very significant issue that has not been widely studied. In the investigated Agilent Bioanalyzer chip-CE system, the fixed components are the Agilent chips and the detection (LIF at 635nm and LEDIF at 470nm), while the chemistry (electrolyte) and the programming of all the high voltages are flexible. Using standard DNA chips, we show that a generic CE function of the system is easily possible and we demonstrate an extension of the applicability to non-aqueous CE (NACE). We studied the chip compatibility with organic solvents (i.e. MeOH, ACN, DMF and DMSO) and demonstrated the chip compatibility with DMSO as a non-volatile and non-hazardous solvent with satisfactory stability of migration times over 50h. The generic CE capability is illustrated with separations of fluorescent basic blue dyes methylene blue (MB), toluidine blue (TB), nile blue (NB) and brilliant cresyl blue (BC). Further, the effects of the composition of the background electrolyte (BGE) on the separation were studied, including the contents of water (0–30%) and buffer composition. In background electrolytes containing typically 80mmol/L ammonium acetate and 870mmol/L acetic acid in 100% DMSO baseline separation of the dyes were achieved in 40s. Linearity was documented in the range of 5–28μmol/L, 10–100μmol/L, 1.56–50nmol/L and 5–75nmol/L (r2 values in the range 0.974–0.999), and limit of detection (LOD) values were 90nmol/L, 1μmol/L 1.4nmol/L, and 2nmol/L for MB, TB, NB and BC, respectively.

Crossing the Border towards Deep UV Time-Resolved Microscopy of Native Fluophores

More than 20 years ago, single photon counting based techniques evolved as one recognized standard in fluorescence detection. In combination with confocal microscopy FLIM (Fluorescence Lifetime Imaging Microscopy) and FCS (Fluorescence Correlation Spectroscopy) became established techniques for investigations down to the single molecule level. Up to date, these experiments typically are carried out in the visible up to the near infrared spectral range.Based on recent advances in fiber amplified laser technology [1] and ultrasensitive detection, we present a novel approach to extend time-correlated single photon counting (TCSPC) into the deep UV using 266 nm excitation. Hereby, direct access is granted to the native fluorescence of biomolecules originating from appropriate chromophoric groups such as the amino acids tryptophan and tyrosine within proteins. As first results, we will present label-free FLIM of cells where the aromatic amino acids within the proteins become visible. As a benchmark, also FCS with organic fluorophores in the deep UV will be shown.Another application of time-resolved fluorescence microscopy in the deep UV includes microfluidics and thus enables label-free detection and identification of various aromatic analytes in chip electrophoresis [2, 3]. Fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for different substances in the electropherogram with the aim to identify aromatic compounds in mixtures. Based on the time-correlated single photon counting the background fluorescence can be discriminated resulting in improved signal-to-noise-ratios. In addition, microchip electrophoretic separations with fluorescence lifetime detection can be performed with protein mixtures emphasizing the potential for biopolymer analysis.