The spatiotemporal accuracy of microscale magnetophoresis has improved significantly over the course of several decades of development. However, most of the studies so far were using magnetic microbead composed of nanosphere particle for magnetophoretic actuation purpose. Here, we developed an in-house method for magnetic sample analysis called quadrupole magnetic steering control (QMSC). QMSC was used to study the magnetophoretic behavior of polystyrene microbeads decorated with iron oxide nanospheres-coated polystyrene microbeads (IONSs-PS) and iron oxide nanorods-coated polystyrene microbeads (IONRs-PS) under the influence of a quadrupole low field gradient. During a 4-s QMSC experiment, the IONSs-PS and IONRs-PS were navigated to perform 180° flip and 90° turn formations, and their kinematic results (2 s before and 2 s after the flip/turn) were measured and compared. The results showed that the IONRs-PS suffered from significant kinematic disproportion, translating a highly uneven amount of kinetic energy from the same magnitude of magnetic control. Combining the kinematic analysis, transmission electron microscopy micrographs, and vibrating sample magnetometry measurements, it was found that the IONRs-PS experienced higher fluid drag force and had lower consistency than the IONSs-PS due to its extensive open fractal nanorod structure on the bead surface and uneven magnetization, which was attributed to its ferrimagnetic nature.
Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.
This study aims to identify cancer-associated proteins in the secretome of oral cancer cell lines. We have successfully established four primary cell cultures of normal cells with a limited lifespan without human telomerase reverse transcriptase (hTERT) immortalization. The secretome of these primary cell cultures were compared with that of oral cancer cell lines using 2DE. Thirty five protein spots were found to have changed in abundance. Unambiguous identification of these proteins was achieved by MALDI TOF/TOF. In silico analysis predicted that 24 of these proteins were secreted via classical or nonclassical mechanisms. The mRNA expression of six genes was found to correlate with the corresponding protein abundance. Ingenuity Pathway Analysis (IPA) core analysis revealed that the identified proteins were relevant in, and related to, cancer development with likely involvements in tumor growth, metastasis, hyperproliferation, tumorigenesis, neoplasia, hyperplasia, and cell transformation. In conclusion, we have demonstrated that a comparative study of the secretome of cancer versus normal cell lines can be used to identify cancer-associated proteins.
This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 Vpp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field.
One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biennial reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods in capillaries and microchips, covering the period July 2014-June 2016. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.
One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.
A combination of a modified Feret' (Silvae Genet. 1971, 20, 46-50) extraction buffer and two types of electrophoresis with acrylamide and starch gels were used to characterize allozymes in mature vegetative tissue of a commercially high value species of rattans (Calamus subinermis). From the analysis of allelic segregation from single maternal rattans and their offspring, genetic control of the 16 observed banding zones, which were consistently scorable, was assumed. Seventeen gene loci were identified. The percentage of polymorphic loci within Calamus subinermis was much higher (70.5%) than expected levels of genetic diversity for tropical woody and non-woody species. It is thought that the protocol described may be applied to the analysis of the genetic diversity of all the endangered Calamus species.
The Glasgow Coma Scale (GCS), which classifies patients into mild, moderate or severe traumatic brain injury (TBI), is a system used to prioritize treatment and prognosticate the severity of head injury. In this study, sera of patients with various stages of TBI, as well as control subjects, were analyzed to screen for proteins that may be used to complement the GCS system. By subjecting pooled serum samples to iTRAQ analysis for quantitative comparison of protein abundance, and attesting their altered levels using ELISA, we have detected increased levels of serum amyloid A, C-reactive protein, leucine-rich alpha-2-glycoprotein, lipopolysaccharide-binding protein, fibronectin, vitronectin and alpha-1-antichymotrypsin in patients across all strata of TBI relative to the controls. However, kininogen was decreased only in moderate and severe TBI, whereas apolipoprotein E and zinc-alpha-2-glycoprotein were only increased in severe TBI. Hence, we propose a panel of serum biomarkers, which if analyzed within 24 h of the injury, can be used to diagnose patients with TBI into mild, moderate or severe stratification objectively, thus complementing the traditional GCS.
Mushrooms are considered an important food for their traditionally famous nutritional and medicinal values, although much information about their potential at the molecular level is unfortunately unknown. Edible mushrooms include fungi that are either collected wild or cultivated. Many important species are difficult to cultivate but attempts have been made with varying degrees of success, with the results showing unsatisfactory economical cultivation methods. Recently, proteomic analysis has been developed as a powerful tool to study the protein content of fungi, particularly basidiomycetes. This mini-review article highlights the contribution of proteomics platforms to the study of edible mushrooms, focusing on the molecular mechanisms involved in developmental stages. This includes extracellular and cytoplasmic effector proteins that have potential or are involved in the synthesis of anticancer, antidiabetic, antioxidant, and antibiotic, in blood pressure control, in the supply of vitamins and minerals, and in other responses to environmental changes. The contribution of different proteomics techniques including classical and more advanced techniques is also highlighted.
Binding constants for the enantiomers of modafinil with the negatively charged chiral selector sulfated-β-CD (S-β-CD) using CE technique is presented. The calculations of the binding constants employing three different linearization plots (double reciprocal, X-reciprocal and Y-reciprocal) were performed from the electrophoretic mobility values of modafinil enantiomers at different concentrations of S-β-CD in the BGE. The highest inclusion affinity of the modafinil enantiomers were observed for the S-enantiomer-S-β-CD complex, in agreement with the computational calculations performed previously. Binding constants for each enantiomer-S-β-CD complex at different temperatures, as well as thermodynamic parameters for binding, were calculated. Host-guest binding constants using the double reciprocal fit showed better linearity (r(2)>0.99) at all temperatures studied (15-30°C) and compared with the other two fit methods. The linear van't Hoff (15-30°C) plot obtained indicated that the thermodynamic parameters of complexation were temperature dependent for the enantiomers.
The assembly of carbon nanotubes (CNTs) across planner electrodes using dielectrophoresis (DEP) is one of the standard methods used to fabricate CNT-based devices such as sensors. The medium drag velocity caused by electrokinetic phenomena such as electrothermal and electroosmotic might drive CNTs away from the deposition area. This problem becomes critical at large-scale electrode structures due to the high attenuation of the DEP force. Herein, we simulated and experimentally validated a novel DEP setup that uses a top glass cover to minimize the medium drag velocity. The simulation results showed that the drag velocity can be reduced by 2-3 orders of magnitude compared with the basic DEP setup. The simulation also showed that the optimum channel height to result in a significant drag velocity reduction was between 100 μm and 240 μm. We experimentally report, for the first time, the assembly and alignment of CNT bridges across indium tin oxide (ITO) electrodes with spacing up to 125 μm. We also derived an equation to optimize the CNT's concentration in suspensions based on the electrode gap width and channel height. The deposition of long CNTs across ITO electrodes has potential use in transparent electronics and microfluidic systems.
The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.
Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2-nitrophenol (NP) and 4-chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS-CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at -3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40 s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100- to 737-fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53 μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid-microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07-2.39% (migration time) and 8.28-14.0% (peak area).
Replicative senescence and stress-induced premature senescence (SIPS) cells are known to share certain traits. However, whether these cells are different at the protein level is unclear. Thus, this study has utilized proteomics to identify differences in the proteomes of replicative senescence and SIPS cells compared to normal cells. Replicative senescence was induced by serial passage of normal cells in culture. SIPS was established by exposure to H2 O2 at a subcytotoxic concentration of 20 μM for two weeks. Following 2DE, protein profiles were compared and protein spots that changed in abundance were identified by MALDI-TOF MS. Quantitative real-time RT-PCR was then performed to evaluate the transcript expression of selected altered proteins. A total of 24 and 10 proteins were found to have changed in abundance in replicative senescence and SIPS cells, respectively, when compared to young cells. Quantitative RT-PCR revealed that nine genes showed the same direction of change as observed in the proteomics analysis. Very little overlap was observed between proteins that changed in replicative senescence and SIPS cells, suggesting that although both SIPS and replicative senescence cells share hallmarks of cellular senescence, they were different in terms of proteins that changed in abundance.