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Abstract: Integrity of the cell membrane is a basic requirement for maintaining the biological characteristics of a cell. In this study, changes in the morphology and ultrastructure of HeLa (human cervical carcinoma), HepG2 (human hepatocellular liver carcinoma), and C6 (rat glioma) cells were studied by atomic force microscopy (AFM) both before and after treatment with the anti-cancer drugs, colchicine or cytarabine. In response to both drugs, the microstructure of the cell membrane of all three cell types displayed similar changes; that is, with increases in drug concentration and reaction time, the degree of morphological changes on the surface of cell membrane increased. These changes included increases in the fluctuation of the surface components of the cell membrane, increase in shrinkage, or even the appearance of pores. Cell viability was maintained, as determined by optical microscope observation of gross cell morphology and by MTT assay results. Analysis of the cell membrane root-mean-square (RMS) roughness showed that under the action of colchicine and cytarabine, RMS values for the cell membranes of all three tumor cell types were positively correlated to the drug concentration and reaction time. This research has great significance for the visual diagnosis of early stage apoptosis in tumor cells in response to anti-cancer drugs, as well as in the studies on the interaction between drugs and cells. The use of AFM can be a rapid and sensitive visual method for studying the interaction between cells and drug. [Copyright &y& Elsevier]

Abstract: Rapid assessment of acute myocardial infarction (AMI) was successfully demonstrated using an improved superparamagnetic polymer microsphere-assisted sandwich fluoroimmunoassay to detect two early cardiac markers—myoglobin and human heart-type fatty acid binding protein (H-FABP). This assay used a preparation of superparamagnetic poly(styrene-divinylbenzene-acrylamide) microspheres, glutaraldehyde-coupled capture antibodies (monoclonal anti-myoglobin 7C3 and anti-H-FABP 10E1) grafted onto the polymer microspheres, and a sequential sandwich fluoroimmunoassay using detection antibodies (FITC-labeled anti-myoglobin 4E2 and FITC-labeled anti-H-FABP 9F3). Characterization of the polymer microspheres by TEM, SEM and Fourier transform infrared spectroscopy (FT-IR) showed that the microspheres were uniformly round with an average diameter of 1.12μm, and had a Fe3O4-polymer core–shell structure (shell thickness was about 84nm) with 0.22mmol/g amino groups on their surfaces. The magnetic behavior of the Fe3O4-polymer microspheres was superparamagnetic (M s =13emu/g, H c =13.1Oe). Fluorescence images of the post-immunoassay microspheres recorded using a confocal laser-scanning microscope showed that the average fluorescence intensity was correlated with the concentration of cardiac markers, in agreement with the results obtained by an F-4500 FL spectrophotometer; this indicated that the fluoroimmunoassay could be used to semi-quantitatively detect both myoglobin and H-FABP. The detection limit was 25ng/mL for myoglobin and 1ng/mL for H-FABP. [Copyright &y& Elsevier]

Large-scale single-cell arrays are urgently required for current high-throughput screening of cell function and heterogeneity. However, the rapid and convenient generation of large-scale single-cell array in a multiplex and universal manner is not yet well established. In this paper, we report a simple and reliable method for the generation of a single-cell array by combining pneumatic microvalve arrays (PμVAs) and hydrodynamic single-cell trapping sites in a single microfluidic device. The PμVAs, which can be precisely controlled by actuated pressures, were designed to guide multiple types of cells being trapped in the corresponding single-cell trapping sites located in the fluidic channel. According to the theoretical demonstration and computational simulation, we successfully realized a multiplex single-cell array with three different types of cells by a step-by-step protocol. Furthermore, the analysis of cellular esterase heterogeneity of the three types of cells was concurrently implemented in the device as a proof-of-concept experiment. All the results demonstrated that the method developed in the current study could be applied for the generation of large-scale single-cell array with multiple cell types, which would be also promising and helpful for single-cell-based high-throughput drug test, multipurpose immunosensor and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: The present study presents a new microfluidic device integrated with pneumatic microvalves and a membrane mixer for enzyme-based immunoassay of acute myocardial infarction (AMI) biomarkers, namely, myoglobin, and heart-type fatty acid binding protein (H-FABP). Superparamagnetic microspheres with carboxyl groups on their surfaces were used as antibody solid carriers. A membrane mixer consisting of four ψ-type membrane valves was assembled under the reaction chamber for on-chip performing microsphere trapping and reagent mixing. The entire immunoassay process, including microsphere capture, reagent input, mixing, and subsequent reaction, was accomplished on the device either automatically or manually. The post-reaction substrate resultant was analyzed using a microplate reader. The results show that the average absorbance value is correlated with the concentration of cardiac markers, in agreement with the results obtained using a conventional microsphere-based immunoassay; this indicated that the proposed on-chip immunoassay protocol could be used to detect both myoglobin and H-FABP. The minimum detectable concentration is 5ng/mL for myoglobin and 1ng/mL for H-FABP. [Copyright &y& Elsevier]

Abstract: Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin–biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen–antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2IU/mL, 0.3ng/mL, and 0.5ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general protocol in other bacterial and viral marker analysis. [Copyright &y& Elsevier]

Abstract: In this study, a high-throughput microfluidic system is presented. The system is comprised of seven parallel channels. Each channel contains 32 square-shaped microchambers. After simulation studies on samples loaded into the microchambers, and the solute exchange between the microchambers and channels, the long-term culture of Escherichia coli (E. coli) HB101 in the microchambers is realized. Using the principle that l-arabinose (l-Ara) can induce recombinant E. coli HB101 pGLO to synthesize green fluorescent protein (GFP), the real-time analysis of GFP expression in different initial bacterial densities is performed. The results demonstrate that higher initial loading densities of the bacterial colony cause bacterial cell to enter log-phase proliferation sooner. High or low initial loading densities of the bacterial cell suspension induce the same maximum growth rates during the log-phase. Quantitative on-chip analysis of tetracycline and erythromycin inhibition on bacterial cell growth is also conducted. Bacterial morphology changes during antibiotic treatment are observed. The results show that tetracycline and erythromycin exhibit different inhibition activities in E. coli cells. Concentrations of 3μg/mL tetracycline can facilitate the formation of long filamentous bacteria with the average length of more than 50μm. This study provides an on-chip framework for bacteriological research in a high-throughput manner and the development of recombinant bacteria-based biosensors for the detection of specific substances. [ABSTRACT FROM AUTHOR]