Zhan-Hui Wang

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.

Phage M13Ko7 Detection With Biosensor Based On Imaging Ellipsometry And Afm Microscopic Confirmation

Abstract A rapid detection and identification of pathogens is important for minimizing transfer and spread of disease. A label-free and multiplex biosensor based on imaging ellipsometry (BIE) had been developed for the detection of phage M13KO7. The surface of silicon wafer is modified with aldehyde, and proteins can be patterned homogeneously and simultaneously on the surface of silicon wafer in an array format by a microfluidic system. Avidin is immobilized on the surface for biotin-anti-M13 immobilization by means of interaction between avidin and biotin, which will serve as ligand against phage M13KO7. Phages M13KO7 are specifically captured by the ligand when phage M13KO7 solution passes over the surface, resulting in a significant increase of mass surface concentration of the anti-M13 binding phage M13KO7 layer, which could be detected by imaging ellipsometry with a sensitivity of 109 pfu/ml. Moreover, atomic force microscopy is also used to confirm the fact that phage M13KO7 has been directly captured by ligands on the surface. It indicates that BIE is competent for direct detection of phage M13KO7 and has potential in the field of virus detection.

Investigation of interaction between two neutralizing monoclonal antibodies and SARS virus using biosensor based on imaging ellipsometry

Two neutralizing human scFv, b1 and h12 were identified initially using ELISA,employing highly purified virus as the coating antigen. The biosensor technique based on imaging ellipsometry was employed directly to detect two neutralizing monoclonal antibodies and serial serum samples from 10 SARS patients and 12 volunteers who had not SARS. Further, the kinetic process of interaction between the antibodies and SARS-CoV was studied using the real-time function of the biosensor. The biosensor is consistent with ELISA that the antibody h12 showed a higher affinity in encountering the virus than antibody b1. The affinity of antibody b1 and antibody h12 was 9.5 × 106 M−1 and 1.36 × 107 M− 1, respectively. As a label free method, the biosensor based on imaging ellipsometry proved to be a more competent mechanism for measuring serum samples from SARS patients and the affinity between these antibodies and the SARS coronavirus.

The development of biosensor with imaging ellipsometry

The concept of biosensor with imaging ellipsometry was proposed about ten years ago. It has become an automatic analysis technique for protein detection with merits of label-free, multi-protein analysis, and real-time analysis for protein interaction process, etc. Its principle, and related technique units, such as micro-array, micro-fluidic and bio-molecule interaction cell, sampling unit and calibration for quantitative detection as well as its applications in biomedicine field are presented here.

Imaging ellipsometry for the visualization of bio-molecular layers

Imaging ellipsometry is presented as a visualization technique to study bio-molecular layers. The layers have become more and more attractive in materials science, especially layers with thickness similar to cellular layers and with physiological activities which are very important in molecular biology and medicine. Normally bio-molecular layers are very thin, with thickness of between sub-nanometer and several tens nanometer. They are transparent in the visible range of light, so that they are recognized as a phase object in physics. Imaging ellipsometry is non-destructive and exhibits a high sensitivity to phase transitions within thin layers. It is capable of imaging local variations in the optical properties such as thickness due to the presence of different surface concentrations of bio-molecule or different deposited molecules. Imaging ellipsometry is based on conventional ellipsometry with charge coupled device (CCD) technique. The images are captured with a computer with image processing technique. It has high sensitivity to thickness variation (resolution in the order of angstrom), and high sampling speed (25 pictures with more than 10/sup 5/ pixels per second).

Application of Optical Protein-chip in Detecting Phage M13KO7

Abstract Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1×1010 pfu/mL. Each variation of the layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1×1010–2.5×1010 pfu/mL, with the sensitivity of 109 pfu/mL. Compared with other methods, the optical protein-chip, requiring only short measurement time, label free, is a quantitative test, and can be visualized. This study could be significant on the interactions between the antibody and the virus, showing potential in the early diagnosis of virosis.

Optical protein chip as microarrays for protein interaction determination

One kind of optical protein chip and a ProteinChip analysis system have been introduced. The basic principle is that one kind of optical imaging technique - imaging ellipsometry is used to visualize optical biochips with the lateral thickness (or surface concentration) distribution of protein layers attached on a patterned surface. The chip is processed with surface patterning, surface modification and ligand immobilization to form a sensing surface with multi-bioactivity. The affinity between proteins is used to realize non-labeling micro-assays for protein identifications and protein interactions. A model of the chip and some demonstration results are presented.

Detection of somatropin and corticosterone with imaging ellipsometry

Imaging ellipsometry has been recently developed to visualize the lateral thickness distribution of biomolecular layers with a high sensitivity for a thickness variation, i.e., the visualization of molecular interaction on biolayer with a measurement of thickness variation. By using this technique, we have made the detection of protein hormone, human somatropin (HGH) and steroid hormone, corticosterone: BSA. In the study, silicon wafers were used as substrates on which the anti-HGH or anti-corticosterone was adsorbed to make a biochip, and then the biochip was incubated in solutions containing HGH or corticosterone: BSA for molecular interaction. Thus, the hormones could be detected by the thickness increase, which caused by the specific affinity between hormones and their anti-bodies. In present study we can detect HGH in concentrations of 0.08, 0.008 and 0.004 IU/ml and corticosterone:BSA in concentrations even till 0.5 /spl mu/g/ml and 0.05 /spl mu/g/ml.

Soluble Angiopoietin Receptor Tie-2 in Patients with Acute Myocardial Infarction and its Detection by Optical Protein-chip

The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P<0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.

Immune-microassay with optical proteinchip for protein detection

An optical proteinchip upon antigen-antibody affinity and the chip analysis system have been used in immune analysis. The chip is processed with chip design, surface patterning, surface modification, ligand immobilization to form a microarray with many immune probes-one kind of antigen (or antibody) occupying one unit in the microarray and many such units in matrix. In microreactor of bio-molecule interaction, when ligand met its receptor, they would bind into bio-specific complex, which resulted in the thickness of the probe surface layer (surface concentration of antigen-antibody) increasing. The variation of protein pattern on the chip surface corresponding to the ligand immobilization and protein interactions was screened with optical imaging ellipsometry. A nonlabeling microassay for protein detection was realized with antigen-antibody affinity and nondisturbance optical sampling, which was available for the direct test of crude samples, such as serum, etc. Some application results in immune analysis were presented also.