Microchip Gel Electrophoresis Research Articles

Detection and separation of proteins using micro/nanofluidics devices.

Microfluidics is the technology or system wherein the behavior of fluids' is studied onto a miniaturized device composed of chambers and tunnels. In biological and biomedical sciences, microfluidic technology/system or device serves as an ultra-high-output approach capable of detecting and separating the biomolecules present even in trace quantities. Given the essential role of protein, the identification and quantification of proteins help understand the various living systems' biological function regulation. Microfluidics has enormous potential to enable biological investigation at the cellular and molecular level and maybe a fair substitution of the sophisticated instruments/equipment used for proteomics, genomics, and metabolomics analysis. The current advancement in microfluidic systems' development is achieving momentum and opening new avenues in developing innovative and hybrid methodologies/technologies. This chapter attempts to expound the micro/nanofluidic systems/devices for their wide-ranging application to detect and separate protein. It covers microfluidic chip electrophoresis, microchip gel electrophoresis, and nanofluidic systems as protein separation systems, while methods such as spectrophotometric, mass spectrometry, electrochemical detection, magneto-resistive sensors and dynamic light scattering (DLS) are discussed as proteins' detection system.

A comparative study of CE-SDS, SDS-PAGE, and Simple Western-Precision, repeatability, and apparent molecular mass shifts by glycosylation.

SDS gel electrophoresis is a commonly used approach for monitoring purity and apparent molecular mass (Mr) of proteins, especially in the field of quality control of biopharmaceutical proteins. The technological installation of CE-SDS as the replacement of the slab gel technique (SDS-PAGE) is still in progress, leading to a continuous improvement of CE-SDS instruments. Various CE-SDS instruments, namely Maurice (CE-SDS/CE-SDS PLUS) and Wes by ProteinSimple as well as the microchip gel electrophoresis system LabChip® GXII Touch™ HT by PerkinElmer were tested for precision and repeatability compared to SDS-PAGE (Bio-Rad). For assessing these quality control parameters, standard model proteins with minor post-translational modifications were used. Overall, it can be concluded that the CE-SDS-based methods are similar to SDS-PAGE with respect to these parameters. Quality characteristics of test systems gain more significance by testing proteins that do not behave like model proteins. Therefore, glycosylated proteins were analyzed to comparatively investigate the influence of glycosylation on Mr determination in the different instruments. In some cases, high deviations were found both among the methods and with regard to reference values. This article provides possible explanations for these findings.

Droplet sample introduction to microchip gel and zone electrophoresis for rapid analysis of protein-protein complexes and enzymatic reactions.

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6s with 1200V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.

Microchip gel electrophoretic analysis of perchloric acid-soluble serum proteins in systemic inflammatory disorders.

Perchloric acid (PCA) precipitation is a well‐known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA‐soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods. After PCA treatment of the serum, the soluble proteins in the supernatant were analyzed by SDS‐PAGE and by microchip gel electrophoresis (MGE). Characteristic changes of the electrophoretic patterns of the PCA‐soluble fractions were observed. Four characteristic bands (at ∼11, ∼65, ∼85, and ∼120 kDa) with varying intensity were detected by MGE. The proportion of the ∼65, ∼85, and ∼120 kDa bands were significantly higher in systemic inflammatory conditions than in healthy individuals (p < 0.001), and characteristic patterns were observed in patients with acute inflammation. The marked differences in the acid‐soluble protein patterns, which were observed in patients with ongoing systemic inflammation, might be a good indicator of inflammation. The MGE analysis is a fast screening and quantification method for the detection of characteristic changes among acid‐soluble serum proteins.

"Microchip Electrophoresis," with Respect to "Profiling of Aβ Peptides in the Cerebrospinal Fluid of Patients with Alzheimer's Disease".

Aggregation of beta-amyloid peptides especially Aβ1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This event is normally accompanied by a relative reduction of the concentration of Aβ1-42 in the cerebrospinal fluid (CSF) of patient developing the signs of Alzheimer's disease. Here, we describe methods for isolation and for microchip gel electrophoresis of Aβ peptides in polydimethylsiloxane (PDMS) microfluidic chip. The method was applied to compare the relative concentration of Aβ1-42 with other Aβ peptides, for example, Aβ 1-40 in CSF. In order to increase the sensitivity of detection, Aβ peptides in the CSF samples were first captured and concentrated using magnetic beads coated with specific anti-Aβ antibodies.

On-line coupling of sample preconcentration by LVSEP with gel electrophoretic separation on T-channel chips.

To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments.

Rapid Identification of Gram-Negative Bacteria with and without CTX-M Extended-Spectrum β-Lactamase from Positive Blood Culture Bottles by PCR Followed by Microchip Gel Electrophoresis

We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum β-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.

A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis

Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve.

Microchip Electrophoresis Profiling of Aβ Peptides in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease

The preferential aggregation of Aβ1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This is accompanied by a relative reduction of the concentration of Aβ1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer's disease. Here, we describe a microchip gel electrophoresis method in polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aβ peptides in cerebrospinal fluid. To control the electroosmotic flow (EOF) in the PDMS channel and also to reduce the adsorption of the peptides to the surface of the channel, a new double coating using poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) and methylcellulose-Tween-20 was developed. With this method, separation of five synthetic Aβ peptides (Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42) was achieved, and relative abundance of Aβ1-42 to Aβ1-37 could be calculated in different standard mixtures. We applied our method for profiling of Aβ peptides in CSF samples from nonAlzheimer patients and patients with Alzheimer's disease. Aβ peptides in the CSF samples were captured and concentrated using a microfluidic system in which magnetic beads coated with anti-Aβ were self-organized into an affinity microcolumn under the a permanent magnetic field. Finally, we could detect two Aβ peptides (Aβ1-40 and Aβ1-42) in the CSF samples.

M-marker 2009, a Marker set for Mapping Medaka Mutants Using PCR Length Polymorphisms with an Automated Microchip Gel Electrophoresis System

We have developed a highly effective marker set for assigning medaka mutants to specific linkage groups (LG) in bulk segregation analysis by PCR product length polymorphisms (PLPs). The marker set permits analysis using an automated microchip electrophoresis system as well as conventional agarose gel electrophoresis.

Exceeding 20 000‐fold concentration of protein by the on‐line isotachophoresis concentration in poly(methyl methacrylate) microchip

In this research, a simple on-line microchip gel electrophoresis with ITP was applied for the concentration and separation of BSA and its immunoassay complex with mAb in a single cross form PMMA microchip. We investigated the ITP concentration effect in PMMA MCE using combination of leading electrolytes, terminating electrolytes and other factors. We realized an ITP-based concentration and separation of BSA and its immunoassay complexes in standard cross-channel microchip gel electrophoresis, which exceeded 2000-fold concentration of BSA immunocomplex using Tris-H3PO4 as a leading electrolyte and Tris-gamma-amino butyric acid as a terminating electrolyte. In addition, we also realized concentration of BSA sample in water, which was more than 20 000-fold and was the result of the concentration effect from combining ITP and the sample stacking techniques.

Impact of Thawing on RNA Integrity and Gene Expression Analysis in Fresh Frozen Tissue

Biobanks of fresh, unfixed human tissue represent a valuable source for gene expression analysis in translational research and molecular pathology. The aim of this study was to evaluate the impact of thawing on RNA integrity and gene expression in fresh frozen tissue specimens. Portions of snap frozen tonsil tissue, unfixed or immersed in RNAlater, were thawed at room temperature for 0 minute, 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours before RNA extraction. Additionally, tonsil tissue underwent repetitive freezing and thawing cycles. RNA integrity was analyzed by microchip gel electrophoresis and gene expression by quantitative real-time polymerase chain reaction for selected genes (FOS, TGFB1, HIF1A, BCL2, and PCNA). Minimal RNA degradation was detected after 30 minutes of thawing in unfixed samples. This degradation was accompanied by relevant changes in gene expression for FOS and BCL2 at 45 minutes. Modified primer design or the use of different housekeeping genes could not rectify the changes for FOS. Repetitive thawing cycles had similar effects on RNA integrity. The incubation of the tissue in RNAlater efficiently prevented RNA degradation. In conclusion, degradation of RNA in frozen tissue occurs first after several minutes of thawing. Already minimal decrease in RNA quality may result in significant changes in gene expression patterns in clinical tissue samples.

Microchip gel electrophoresis with programmed field strength gradients for ultra-fast detection of canine T-cell lymphoma in dogs

This paper describes the applicability of microchip gel electrophoresis using a programmed field strength gradients (MGE-PFSG) method coupled with a polymerase chain reaction (PCR) for the ultra-fast diagnosis of canine T-cell lymphoma. The variable region in the T-cell receptor γ (TCRγ) gene from a T-cell lymphoma was used in PCR amplification. The contributions of the various parameters, including the effects of the molecular weight, concentration of the sieving matrix and field strength in MGE, were examined. 0.5% poly (ethyleneoxide) (PEO, M r 8 000 000) was used as the sieving matrix for the ultra-rapid separation of the amplified-PCR products (90 and 130-bp DNA fragments) from the PFSG at an effective length of 20 mm in a glass microchip. The PCR products (90 and 130-bp DNA) of the T-cell lymphoma were analyzed within 41.7 ± 0.1 s, 15.5 ± 0.2 s and only 7.0 ± 0.1 s using a low-constant field strength, high-constant field strength and the PFSG, respectively. When 11 clinical samples were analyzed using the MGE-PFSG method, there was a 100% correlation with those obtained using conventional slab gel electrophoresis. The ultra-fast detection and rapid separation capabilities of MGE-PFSG make it an efficient tool for diagnosing T-cell lymphoma in clinical samples with high sensitivity.

Catheter-Related Fungemia Due to Fluconazole-Resistant Candida nivariensis

We report on a case of fungemia due to fluconazole-resistant Candida nivariensis (MIC, > or =128 microg/ml). Internal transcribed spacer PCR followed by microchip gel electrophoresis with a blood culture that tested positive revealed a unique pattern different from those of other pathogenic yeasts.

Trace Analysis of DNA: Preconcentration, Separation, and Electrochemical Detection in Microchip Electrophoresis Using Au Nanoparticles

We have developed a simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method for trace analysis of DNA. The microchip comprised of three parallel channels: the first two are for the field-amplified sample stacking and subsequent field-amplified sampled injection steps, while the third one is for the microchip gel electrophoresis (MGE) with ED (MGE-ED). To improve preconcentration and separation performances of the method, the stacking and separation buffers containing the hydroxypropyl cellulose (HPC) matrix were modified with gold nanoparticles (AuNPs). The formation of AuNPs and HPC/AuNP-modified buffers were characterized by UV-visible spectroscopy and TEM experiments. The conducting polymer-modified electrode was also modified with AuNPs to enhance detection performances of the electrode. The conducting polymer/AuNP layers act as electrocatalysts for the direct detection of DNA based on their oxidation in a solution phase. The total sensitivity was improved by approximately 25 000-fold when compared with a conventional MGE-ED analysis. The calibration plots were linear (r2 = 0.9993) within the range of 0.003-1.0 pg/microL for a 20-bp DNA sample. The sensitivity was 0.20 nA/(fg/microL), with a detection limit of 5.7 amol in a 50-microL sample, based on S/N = 3. The applicability of the method for the analysis of 13 fragments present in a 100-bp DNA ladder was successfully demonstrated.

Capillary and microchip gel electrophoresis for simultaneous detection of Salmonella pullorum and Salmonella gallinarum by rfbS allele-specific PCR

We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54 ± 1 °C and an MgCl 2 concentration of 2.8–5.6 mM. Under an electric field of 333.3 V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) ( M r 600 000), the amplified PCR products were analyzed within 6 min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30 s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency.

Rapid Identification of Staphylococcal Strains from Positive-Testing Blood Culture Bottles by Internal Transcribed Spacer PCR Followed by Microchip Gel Electrophoresis

PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis (MGE) was evaluated for its usefulness in identification of staphylococci. Forty ITS PCR patterns were demonstrated among 228 isolated colonies of Staphylococcus aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA; 91 strains), 11 patterns for methicillin-resistant S. aureus (MRSA; 99 strains), and 3 patterns for both MSSA and MRSA (38 strains). Thirty-seven control strains of coagulase-negative staphylococci (CNS) representing 16 species showed unique ITS PCR patterns (24 patterns) at the species and subspecies levels: two patterns for S. caprae, S. cohnii, S. haemolyticus, and S. saprophyticus; three patterns for S. lugdunensis; four patterns for S. capitis; and one pattern for each of the other CNS species. The combined PCR-MGE method was prospectively adapted to the positive blood culture bottles, and this method correctly identified MSSA and MRSA in 102 (89%) of 114 blood cultures positive for S. aureus on the basis of the ITS PCR patterns. Eight ITS PCR patterns were demonstrated from 166 blood culture bottles positive for CNS. The most frequent CNS species isolated from blood cultures were S. epidermidis (76%), S. capitis (11%), and S. hominis (8%). Overall, all 280 blood culture bottles shown to contain a single Staphylococcus species by routine phenotypic methods were correctly identified by the PCR-MGE method at the species level, whereas the organism failed to be identified in 8 culture bottles (3%) with mixed flora. The PCR-MGE method is useful not only for rapid identification ( approximately 1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: Computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.

마이크로칩젤 전기영동에서 충진젤 혼합물을 이용한 ORF 바이러스의 진단

We have developed a novel polymerase chain reaction (PCR)-microchip gel electrophoresis (MGE) method based on the sieving gel mixture of commercially available poly(vinylpyrrolidone) (PVP) and hydroxy ethyl cel- lulose (HEC) for the rapid detection a nd diagnosis of the orf virus (ORFV) fr om Korean indigenous goat. After ampli- fication of 594-bp DNA fragment from the B2L gene of ORF virus, the amplicon was analyzed by the MGE separation. The glass microfluidic chip (64 mm total length (36 mm effective length) × 90 µm width × 20 µm depth) allowed the fast detection and diagnosis of ORFV in the mixture of 1.0% PVP (Mr 360,000) and 1.0% HEC (Mr 250,000) as a sieving matrix with better resolution and reproducibility of DNA frag ments. Under the electric fi eld of 277.8 V/cm, the 594-bp DNA was analyzed within 4 min. Compared to traditional slab gel electrophoresis, the PCR-MGE method was twenty times faster and an effective clinical method for the quantitative analysis of ORFV.

Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchip.

We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection.