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Xueqin Wang,
Qin Tu,
Bin Zhao,
Yanfei An,
Jian-Chun Wang,
Wenming Liu,
Mao-Sen Yuan,
Saeed Mahmoud Ahmed,
Juan Xu,
Rui Liu,
Yanrong Zhang,
Jinyi Wang
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ABSTRACT: Intracellular reactive oxygen species (ROS) have been extensively shown to play an important role in the regulation of cell proliferation and cell cycle progression. The effects of endogenous ROS on the proliferation and differentiation of cancer stem cells (CSCs) have received increasing attention because of the unique properties of these cells that allow them to drive tumor growth and evade conventional cancer therapies. In this study, poly(l-Lysine) (PLL)-modified Fe(3)O(4) nanoparticles were synthesized to label CSCs derived from U251 glioblastoma multiform. A featured peroxidase-like activity within PLL-modified Fe(3)O(4) nanoparticles that could greatly reduce intracellular H(2)O(2) activity was identified. We also found that PLL-modified Fe(3)O(4) nanoparticles could accelerate the progression of CSC cell cycle, probably due to the impaired activity of endogenous ROS in CSCs. These results show that growth and proliferation of CSCs could be promoted by Fe(3)O(4) nanocarriers in an ROS-dependent manner, and Fe(3)O(4) nanocarriers may be suitable for certain tumor therapies as a drug delivery system.
Biomaterials 11/2012; · 7.40 Impact Factor
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ABSTRACT: Cell labeling using magnetic nanoparticles is an increasingly used approach in noninvasive behavior tracking, in vitro separation of cancer stem cells (CSCs), and CSC-based research in cancer therapy. However, the impact of magnetic labeling on the biological properties of targeted CSCs, such as self-renewal, proliferation, multi-differentiation, cell cycle, and apoptosis, remains elusive. The present study sought to explore the potential effects on biological behavior when CSCs are labeled with superparamagnetic iron oxide (SPIO) nanoparticles in vitro. The glioblastoma CSCs derived from U251 glioblastoma multiforme were labeled with poly(L-lysine) (PLL)-modified γ-Fe(2)O(3) nanoparticles. The iron uptake of glioblastoma CSCs was confirmed through prussian blue staining, and was further quantified using atomic absorption spectrometry. The cellular viability of the SPIO-labeled glioblastoma CSCs was assessed using a fluorescein diacetate and propidium iodide double-staining protocol. The expressed specific markers and multi-differentiation of SPIO-labeled glioblastoma CSCs were comparatively assessed by immunocytochemistry and semi-quantitative RT-PCR. The effects of magnetic labeling on cell cycle and apoptosis rate of glioblastoma CSCs and their differentiated progenies were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of glioblastoma CSCs and their differentiated progenies were not affected by SPIO labeling compared with their unlabeled counterparts. Moreover, the magnetically labeled CSCs displayed an intact multi-differentiation potential, and could be sub-cultured to form new tumor spheres, which indicates the CSCs capacity for self-renewal. In addition, cell cycle distribution, apoptosis rate of the magnetically labeled glioblastoma CSCs, and their differentiated progenies were not impaired. Therefore, the SPIO-labeled CSCs could be a feasible approach in conducting further functional analysis of targeted CSCs.
Biomaterials 05/2012; 33(14):3719-32. · 7.40 Impact Factor
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ABSTRACT: Ethanol consumption is associated with the risk of breast cancer progression; however, the mechanism of relationship has not
yet been fully explained. Research on breast cancer cell migration after ethanol stimulation may give hope for a better understanding
of the disease and oncotherapy. Conventional cell migration assays such as Boyden chamber and wound-healing assays are easy
to conduct for this purpose; however, these assays have inherent limitations. In this study, we quantified the effect of ethanol
on MCF-7 hunam breast cancer cells using a microfluidics-based wound-healing assay. Wounds were prepared by partially digesting
a confluent cell sheet using parallel laminar flows in the presence of protease trypsin. The cells at the leading edge of
the wound remained intact. Cell image analysis indicates that all the cells cultured in the microdevice took on a good morphology
and monolayer growth status. Cell viability assay demonstrates that cell viability decreased with the increase in ethanol
concentration and treatment time. For 0, 22, 43, and 65mmol/l of ethanol, cell viability after being cultured for 24h was
100%, 99.6%, 99.4%, and 98.4%, respectively. Studying MCF-7 human breast cancer cell migration when treated with different
ethanol concentrations revealed that the cell migration distance is directly proportional with ethanol concentration. After
being cultured for 24h at 37°C and 5% CO2, the maximal cell migration distance was 231, 283, and 332μm for 22, 43, and 65mmol/l ethanol, respectively; all results
were higher than the blank test (i.e., ethanol-free test, 218μm). These findings will be beneficial in developing microfluidic
device applications for future research on breast tumor therapy in a biomimetic microenvironment and for developing new methods
for breast cancer therapy.
KeywordsMicrofluidics–Laminar flow–Breast cancer cells–Migration–Ethanol
Microfluidics and Nanofluidics 04/2012; 10(6):1333-1341. · 3.37 Impact Factor
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Li Ren,
Jian-Chun Wang,
Wenming Liu,
Qin Tu,
Rui Liu, Xueqin Wang,
Juan Xu,
Yaolei Wang,
Yanrong Zhang,
Li Li,
Jinyi Wang
[show abstract]
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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 5 ng/mL for myoglobin and 1 ng/mL for H-FABP.
Biosensors & bioelectronics 02/2012; 35(1):147-54. · 5.43 Impact Factor
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ABSTRACT: On the basis of the host-guest interactions between azobenzenes and cyclodextrins, a new strategy for the preparation of a dually functionalized poly(dimethylsiloxane) (PDMS) surface was investigated using surface-initiated atom-transfer radical polymerization (SI-ATRP) and click chemistry. The PDMS substrates were first oxidized in a H(2)SO(4)/H(2)O(2) solution to transform the surface Si-CH(3) groups into Si-OH groups. Then, the SI-ATRP initiator 3-(2-bromoisobutyramido)propyl(trime-thoxy)silane was grafted onto the substrates through a silanization reaction. Sequentially, the poly(ethylene glycol) (PEG) units were introduced onto the PDMS-Br surfaces via SI-ATRP reaction using oligo(ethylene glycol) methacrylate. Afterward, the bromide groups on the surface were converted to azido groups via nucleophilic substitution reaction with NaN(3). Finally, the azido-grafted PDMS surfaces were subjected to a click reaction with alkynyl and PEG-modified β-cyclodextrins, resulting in the grafting of cyclodextrins onto the PDMS surfaces. The composition and chemical state of the modified surfaces were characterized via X-ray photoelectron spectroscopy, and the stability and dynamic characteristics of the cyclodextrin-modified PDMS substrates were investigated via attenuated total reflection-Fourier transform infrared spectroscopy and temporal contact angle experiments. The surface morphology of the modified PDMS surfaces was characterized through imaging using a multimode atomic force microscope. A protein adsorption assay using Alexa Fluor594-labeled bovine serum albumin, Alexa Fluor594-labeled chicken egg albumin, and FITC-labeled lysozyme shows that the prepared PDMS surfaces possess good protein-repelling properties. On-surface studies on the interactions between azobenzenes and the cyclodextrin-modified surfaces reveal that the reversible binding of azobenzene to the cyclodextrin-modified PDMS surfaces and its subsequent release can be reversibly controlled using UV irradiation. Sandwich fluoroimmunoassay of the cardiac markers myoglobin and fatty acid-binding protein demonstrates that the cyclodextrin-modified PDMS surfaces can be repeatedly utilized in disease biomarker analysis.
Analytical Chemistry 11/2011; 83(24):9651-9. · 5.86 Impact Factor
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08/2011; , ISBN: 978-953-307-271-5
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ABSTRACT: This work describes chemical heat shock transformation of foreign plasmid DNA into bacterial host Escherichia coli cells using a capillary-composited microfluidic device. Transformation processes of the loading, mixing, heat shock and recovery of the transformation mixture were carried out automatically in a linear fashion. In addition, by utilizing the capillary with a hollow cylindrical chamber as heating source, simple, low cost local heat shock with accurate heat shock time to transformation mixture was obtained on the microdevice. Results demonstrated that plasmid DNA could be effectively transformed into E. coli, and the transformation efficiency and frequency were as the same level or better than conventional tube-based method. This work complements other microfluidic technologies for potential gene cloning and functional genomics studies.
Journal of Bioscience and Bioengineering 07/2011; 112(4):373-8. · 1.79 Impact Factor
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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 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/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.
Biosensors & bioelectronics 03/2011; 26(7):3353-60. · 5.43 Impact Factor
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ABSTRACT: Driven by clinical needs, nerve regeneration studies have recently become the focus of research and area of growth in tissue engineering. Biomimetic polymer synthesis and functional interface construction is a promising solution to induce neuritic sprouting and guide the regenerating nerve. However, few studies have been made on primary hippocampal neurons. In this study, a new type of acetylcholine-like biomimetic polymers for their potential in biomaterial-modulated nerve regeneration application is synthesized using click chemistry and free radical polymerization. The structure of the synthesized polymers includes a "bioactive" unit (acetylcholine-like unit) and a "bioinert" unit [poly(ethylene glycol) unit]. To explore the effects of the bioactive unit and the bioinert unit on neuronal growth, different ratios of the two initial monomers poly(ethylene glycol) monomethyl ether-glycidyl methacrylate (MePEG-GMA) and dimethylaminoethyl methacrylate (DMAEMA) were employed and five different polymers were synthesized. Their chemical structures were characterized using (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their physical properties (including molecular weight, polydispersity, glass transition temperature, and melting point) were determined using gel permeation chromatography and differential scanning calorimetry. Culturing of the primary rat hippocampal neurons on the polymeric surfaces show that the ratio of the two initial monomers utilized for polymer synthesis significantly affects neuronal growth. Rat hippocampal neurons show different growth morphologies on different polymeric surfaces. The polymeric surface prepared with 1:60 (mol/mol) of MePEG-GMA to DMAEMA induces neuronal regenerative responses similar to that on poly-l-lysine, a very common benchmark material for nerve cell cultures. These results suggest that acetylcholine-like biomimetic polymers are potential biomaterials for neural engineering applications, particularly in modulating the growth of hippocampal neurons.
Biomaterials 02/2011; 32(12):3253-64. · 7.40 Impact Factor
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ABSTRACT: An improved approach for the surface modification of poly(dimethylsiloxane) (PDMS) using carboxymethyl cellulose (CMC), carboxymethyl beta-1,3-dextran (CMD), and alginic acid (AA) was investigated. The PDMS substrates were first oxidized in a H(2)SO(4)/H(2)O(2) solution to transform the Si-CH(3) groups on their surfaces into Si-OH groups. Then methacrylate groups were grafted onto the substrates through a silanization reaction using 3-(trimethoxysilyl)propyl methacrylate. Sequentially, cysteamine was conjugated onto the silanized surfaces by the reaction between the thiol and methacrylate groups under 254 nm UV exposure. Afterward, the amino-terminated PDMS substrates were sequentially reacted with CMC, CMD, and AA in the presence of N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, resulting in the grafting of polysaccharides onto PDMS surfaces. The composition and chemical state of the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS). In addition, the stability and dynamic characteristics of the polysaccharide-grafted PDMS substrates were investigated by XPS and temporal contact angle experiments. A protein adsorption assay using bovine serum albumin (BSA), chicken egg albumin, lysozyme, and RNase-A showed that the introduction of CMD and AA can reduce the adsorption of negatively charged BSA and chicken egg albumin, but increase the adsorption of the positively charged lysozyme and RNase-A. However, CMC-modified PDMS surfaces showed protein-repelling properties, regardless of whether the protein was positively or negatively charged. A cell culture and migration study of glioma C6, MKN-45, MCF-7, and HepG-2 cells revealed that the polysaccharide-modified PDMS greatly improved the cytocompatibility of native PDMS.
Analytical Chemistry 08/2010; 82(15):6430-9. · 5.86 Impact Factor
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ABSTRACT: We presented an integrated microfluidic system for dynamical study of cell-microenvironmental interactions. We demonstrated its precisely spatio-temporal control in the flow direction and the multi-site staying of the fluids by groups of monolithic microfabricated valves through digital operation, aside from the regulated communication between two loci based on real-time microenvironment transition. Using this system, a series of functional manipulations, including specific delivery, addressable surface treatment, positional cell loading and co-culture were performed quickly and efficiently for biological applications. Sequentially, we carried out the potential utility of this system in the research of dynamic microenvironmental influence to cells using a patho-physiological interaction during cancer initiation and progression. Our results exhibit the passive role but collaborative response of NIH 3T3 fibroblasts to the soluble signals from hepatocellular carcinoma cells, and also the variable behaviors of carcinoma cells under different environmental stimulation. This system can facilitate the in vitro investigation of cell-microenvironmental interactions occurred in numerous biological and pathogenic processes.
Lab on a Chip 07/2010; 10(13):1717-24. · 5.67 Impact Factor
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Jinyi Wang,
Zongfang Wan,
Wenming Liu,
Li Li,
Li Ren, Xueqin Wang,
Peng Sun,
Lili Ren,
Huiying Zhao,
Qin Tu,
Zhiyun Zhang,
Na Song,
Lei Zhang
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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.
Biosensors & bioelectronics 09/2009; 25(4):721-7. · 5.43 Impact Factor
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Jinyi Wang, Xueqin Wang,
Li Ren,
Qiang Wang,
Li Li,
Wenming Liu,
Zongfang Wan,
Linyan Yang,
Peng Sun,
Lili Ren,
Manlin Li,
Heng Wu,
Jinfeng Wang,
Lei Zhang
[show abstract]
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ABSTRACT: This study demonstrates an improved magnetic protein microsphere-aided sandwich fluoroimmunoassay for the analysis of myoglobin and heart-type fatty acid binding protein (H-FABP), early protein markers associated with acute myocardial infarction. In preparation for the assay we constructed superparamagnetic human serum albumin (HSA)/gamma-Fe(2)O(3) microspheres, and grafted capture antibodies (monoclonal antimyoglobin 7C3 and anti-H-FABP 10E1) onto the protein microspheres using the avidin-biotin system. Then the antibody-carrying microspheres were used in a sequential sandwich fluoroimmunoassay along with detection antibodies (Alexa fluor594-labeled antimyoglobin 4E2 and FITC-labeled anti-H-FABP 9F3). The magnetic HSA/gamma-Fe(2)O(3) microspheres were characterized by scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectrophotometry, atomic absorption spectrophotometry, and vibrating sample magnetometry. Fluorescence images of the post-immunoassay microspheres recorded using an inverted fluorescence 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 semiquantitatively detect both myoglobin and H-FABP. The detection limit was 10 ng/mL for myoglobin and 1 ng/mL for H-FABP.
Analytical Chemistry 07/2009; 81(15):6210-7. · 5.86 Impact Factor
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Jinyi Wang,
Li Ren, Xueqin Wang,
Qiang Wang,
Zongfang Wan,
Li Li,
Wenming Liu,
Xuming Wang,
Manlin Li,
Dewen Tong,
Ajing Liu,
Bingbing Shang
[show abstract]
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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 1.12 microm, and had a Fe(3)O(4)-polymer core-shell structure (shell thickness was about 84 nm) with 0.22 mmol/g amino groups on their surfaces. The magnetic behavior of the Fe(3)O(4)-polymer microspheres was superparamagnetic (M(s)=13 emu/g, H(c)=13.1 Oe). 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 25 ng/mL for myoglobin and 1 ng/mL for H-FABP.
Biosensors & bioelectronics 05/2009; 24(10):3097-102. · 5.43 Impact Factor
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Jinyi Wang,
Qiang Wang,
Li Ren, Xueqin Wang,
Zongfang Wan,
Wenming Liu,
Li Li,
Huiying Zhao,
Manlin Li,
Dewen Tong,
Juan Xu
[show abstract]
[hide abstract]
ABSTRACT: A carboxylated superparamagnetic microbead-assisted sandwich fluoroimmunoassay was successfully demonstrated for the analysis of the early protein markers, myoglobin and human heart-type fatty acid-binding protein (H-FABP), associated with acute myocardial infarction. This assay approach consisted of the preparation of superparamagnetic polymer microbeads using a dispersion polymerization, followed by grafting of capture antibodies (monoclonal anti-H-FABP 10E1 and anti-myoglobin 7C3) onto the polymer microbeads using EDC-NHS protocol, and then a sequential sandwich fluoroimmunoassay using detection antibodies (FITC-labeled anti-H-FABP 9F3 and FITC-labeled anti-myoglobin 4E2). The Fe(3)O(4) nanoparticles and carboxylated Fe(3)O(4)-polymer microbeads were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, and X-ray diffraction. The fluoroimmunoassay images were recorded using a confocal laser-scanning microscope, and the average fluorescence intensity of the microbeads was found to correspond to the concentration of each cardiac marker, in agreement with the results obtained by a spectrofluorophotometer. The carboxylated magnetic microbead-assisted protocol could be utilized to semi-quantitatively detect both myoglobin and H-FABP.
Colloids and surfaces. B, Biointerfaces 05/2009; 72(1):112-20. · 2.60 Impact Factor
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ABSTRACT: In this paper, we obtain the strong consistency and asymptotic distribution of the Theil–Sen estimator in simple linear regression models with arbitrary error distributions. We show that the Theil–Sen estimator is super-efficient when the error distribution is discontinuous and that its asymptotic distribution may or may not be normal when the error distribution is continuous. We give an example in which the Theil–Sen estimator is not asymptotically normal. A small simulation study is conducted to confirm the super-efficiency and the non-normality of the asymptotic distribution.
Journal of Statistical Planning and Inference.
