Post-doc Associate Virginia G.Piper Center for Personalized Diagnostics Biodesign Institute, Arizona State University 1001S, McAllister Ave. Phone (Office): (480) 965 7049 E-mail: yuxiaobo_hupo@hotmail.com
I started my Ph.D. research from 2003 in the department of biochemistry, Beijing Institute of Radiation Medicine, China. My research aim was to develop new detection techniques for protein microarrays. During this time, I developed a new label-free detection method for protein array based on Electrochemical Impedance Spectroscopy, which had been successfully demonstrated in the detection of protein-protein and protein-DNA interactions. From 2006 to 2007, I was working in Beijing Proteome Research Center. As the group leader of antibody array, my work was to develop new techniques for proteomics research. During this period, I developed an assembly 96-well protein array system, which had been successfully applied into the screening of antibodies, protein-protein interactions and clinical diagnostics research.
From 2008 to 2009, I obtained the Alexonder von Humboldt Fellowship (Alexander von Humboldt Foundation, Germany) and was working in Natural and Medical Sciences Institute at the University of Tuebingen. My advisor is Dr. Thomas Joos, a world leading expert and opinion leader within the field of protein microarray technologies and applications. My work was to perform the assay development of multiplexed immunoassays using Luminex bead-based array system and screen for biomarkers associated with inflammatory diseases using self-developed and commercial multiplexed cytokine and chemokine assays. During the last two years, I developed four multiplexed immunoassay panels including cytokines, chemokines and soluble receptors. As their important roles in the human immune system, the relationship between the changes of these protein levels with inflammatory diseases was investigated. In addition, I developed a new microfluidic bead-based immunoassay technique (µFBI) which can detect up to 100 different proteins with high-sensitivity using only 1 microliter sample, 1 microliter detetecion antibody and 1 microliter fluorescent molecules. This significant save the reagents and extends the applications of protein microarrays in the study of minute amount of sample materials, like tumor biopsies or tissue sections. I also have collaborations with Dr. Li-Tang Yan, associate professor in Hsinghua University. Our aim is to study drug carrier and cell membranes interactions using simulation and experimental techniques.Our results could provide valuable insights into the mechanism of drug delivery and would be very helpful for the design of new highly-efficient and safety drug carriers.
From the January of 2010, I came to Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute. My advisor is Professor Joshua LaBaer, one of the nation’s foremost investigators in the rapidly expanding field of personalized medicine, formerly director of the Harvard Institute of Proteomics. My research is to study protein functions and discovery of disease related biomarkers using high-density Nucleic Acid Programmable Protein Arrays (NAPPA) Microarrays. To date, I have got 17 publications and 2 Chinese patents. The papers were published in the journals of Clinical Chemistry, Analytical Chemistry, Proteomics, PLos One, Journal of Biomolecular Screening, ACS Nano, NanoScale, Macromolecules, and Analyst, etc.
Research Interests:
1. Develop New Technologies for Protein Microarrays and Molecular Diagnostics
2. Applications of Protein Microarrays in the Study of Protein Functions and Discovery of Disease Related Biomarkers.
3. High-throughput Screening of Antibodies and Small Molecule Drugs
Abstract: BACKGROUND: Over the last 10 years, DNA microarrays have achieved a robust analytical performance, enabling their use for analyzing the whole transcriptome or for screening thousands of single-nucleotide polymorphisms in a single experiment. DNA microarrays allow scientists to correlate gene expression signatures with disease progression, to screen for disease-specific mutations, and to treat patients according to their individual genetic profiles; however, the real key is proteins and their manifold functions. It is necessary to achieve a greater understanding of not only protein function and abundance but also their role in the development of diseases. Protein concentrations have been shown to reflect the physiological and pathologic state of an organ, tissue, or cells far more directly than DNA, and proteins can be profiled effectively with protein microarrays, which require only a small amount of sample material. CONTENT: Protein microarrays have become well-established tools in basic and applied research, and the first products have already entered the in vitro diagnostics market. This review focuses on protein microarray applications for biomarker discovery and validation, disease diagnosis, and use within the area of personalized medicine. SUMMARY: Protein microarrays have proved to be reliable research tools in screening for a multitude of parameters with only a minimal quantity of sample and have enormous potential in applications for diagnostic and personalized medicine.
Abstract: In this work, the authors developed a new screening approach using multiplexed immunization and immunogen array analysis to improve the efficiency of antibody screening for high-throughput antibody generation. The immunogen array is based on a 96-well format in which different immunogens and negative as well as positive controls are immobilized in each well, thus making it possible to screen hundreds of antibody candidates simultaneously. To demonstrate this approach, a model of 4 mixed immunogens immunization was employed. In total, 675 antibody candidates were screened before and after established antibody hybridomas in parallel with immunogen arrays and enzyme-linked immunosorbent assay. The signal intensity, specificity, and cross-reactivity of produced antibody candidates were analyzed using a hierarchical cluster algorithm to track the characteristics of antibody candidates during antibody generation, which might reduce the number of false-positive and false-negative binding of antibodies. Moreover, 4 monoclonal antibodies that were produced successfully recognized their corresponding target antigens.
Abstract: BACKGROUND: Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material. METHODOLOGY/PRINCIPAL FINDINGS: A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30. CONCLUSIONS/SIGNIFICANCE: The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections.
Abstract: Understanding the interactions of dendrimers with biological membranes is of fundamental importance in determining their potential biomedical applications like drug delivery vehicles and gene therapeutic agents. Herein we perform systematically mesoscopic simulations to investigate the interactions and binding structures in complexes comprised of charged dendrimers with lipid bilayer membranes. For these purposes, various interaction strengths between the outer-dendrimer hydrophilic component and lipid heads and those between the inner-dendrimer hydrophobic component and lipid tails are used in the simulations. The external force is also induced into the complexes by stretching the membranes to examine the influence of the dendrimer binding on the stabilization of the lipid bilayer membranes. Our simulations demonstrate that the increasing attraction between outer dendrimer and lipid heads leads to wider spread of dendrimer along the membrane surface, while the attraction between the inner dendrimer and lipid tails has a great effect on the insertion of the dendrimer into the bilayer membrane. It is found that the dendrimer can induce a hole in the tense bilayer membrane at earlier time for a stronger attraction between the hydrophobic dendrimer component and lipid tails, which prompts the failure of the membrane affected by the external forces or surroundings. The findings could provide some guidelines for the design of the dendrimers with defined molecular architectures and prompt the understanding for the stabilization of the tense membranes and the potential cytotoxicity of the charged dendrimers in the dendrimerâlipid bilayer membrane complexes
Abstract: Dendrimers have successfully proved themselves as functional nanodevices for drug delivery because they can render drug molecules a greater water solubility, bioavailability, and biocompatibility. It has recently been suggested that the structural changes of cell membranes (e.g., local lipid density and actual pore or hole) could affect the permeability across them for dendrimers. However, to understand these effects requires direct measurements in a single cell and is thus very difficult and more challenging. Here we use mesoscopic simulations to investigate the tension-mediated complexes comprising charged dendrimers and lipid bilayer membranes. The structures of membranes are alternated by adjusting their surface tensions. Our simulations demonstrate that the permeability of charged dendrimers can be effectively enhanced in the tense membranes, and the permeability in the actual hole is several times higher than that in the lipid-poor section. The possible mechanism of charged dendrimer-induced pore nucleation in the tense membranes is evaluated. The findings have implications in tuning intracellular delivery rates and amounts in nanoscale complex and chemotherapeutics.
Abstract: Inflammation is a defense reaction of an organism against harmful stimuli such as tissue injury or infectious agents. The relationship between the infecting microorganism and the immune, inflammatory, and coagulation responses of the host is intricately intertwined. Due to its complex nature, the molecular mechanisms of inflammation are not yet understood in detail and additional diagnostic tools are required to clarify further aspects. In recent years, protein microarray-based research has moved from being technology-based to application-oriented. Protein microarrays are perfect tools for studying inflammatory diseases. High-density protein arrays enable new classes of autoantibodies, which cause autoimmune diseases, to be discovered. Protein arrays consisting of miniaturized multiplexed sandwich immunoassays allow the simultaneous expression analysis of dozens of signaling molecules such as the cytokines and chemokines involved in the regulation of the immune system. The data enable statements to be made on the status of the disease and its progression as well as support for the clinicians in choosing patient-specific treatment. This chapter reviews the technology and the applications of protein microarrays in diagnosing and monitoring inflammatory diseases.
Abstract: Electrochemical impedance spectroscopy (EIS) combined with a gold electrode array was developed to detect multiple antibody-antigen interactions. Hepatitis B surface antigen (HBsAg), as a model sample, was employed to evaluate the characteristics of the biosensor. The array was fabricated by immobilizing antibodies on the self-assembled molecules surface of the electrodes. The surface characteristics of the array during the binding process including the antibody-antigen conjugation and the sandwich complex with HRP-labeled antibody, as well as the precipitation layer, were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy, respectively. A linear relationship between electron-transfer resistance and the concentrations of HBsAg ranged from 10 pg ml(-1) to 1 ng ml(-1) and the detection limit of 10 pg ml(-1) was obtained. 100 pg ml(-1) antigen samples, such as rat IgG, HBsAg and HBeAg, as well as the antigen mixture, were incubated with the relative antibody-modified electrodes on the array. No obvious cross-talk reaction was observed. All these results confirm the feasibility of applying electrochemical impedance spectroscopy to the electrode array.
Abstract: With the growth of the "-omics" such as functional genomics and proteomics, one of the foremost challenges in biotechnologies has become the development of novel methods to monitor biological process and acquire the information of biomolecular interactions in a systematic manner. To fully understand the roles of newly discovered genes or proteins, it is necessary to elucidate the functions of these molecules in their interaction network. Microarray technology is becoming the method of choice for such a task. Although protein microarray can provide a high throughput analytical platform for protein profiling and protein-protein interaction, most of the current reports are limited to labeled detection using fluorescence or radioisotope techniques. These limitations deflate the potential of the method and prevent the technology from being adapted in a broader range of proteomics applications. In recent years, label-free analytical approaches have gone through intensified development and have been coupled successfully with protein microarray. In many examples of label-free study, the microarray has not only offered the high throughput detection in real time, but also provided kinetics information as well as in situ identification. This article reviews the most significant label-free detection methods for microarray technology, including surface plasmon resonance imaging, atomic force microscope, electrochemical impedance spectroscopy and MS and their applications in proteomics research.
Abstract: An electrochemical impedance biosensor array with protein-modified electrodes was designed and fabricated in this report. To demonstrate its feasibility of the detection of multiple antigen-antibody binding reactions based on a label-free approach, human IgG (hIgG), rat IgG (rIgG), human globin and bovine serum albumin were immobilized, respectively, on the gold electrodes and then the resultant array was incubated with goat anti-hIgG, goat anti-rIgG, anti-human globin antibody and the mixture of three antibodies, respectively. The results indicated that the electron transfer resistance of the electrodes was significantly changed due to formation of the antigen-antibody conjugated layer. In addition, experimental conditions such as the protein concentration for the immobilization and screen were studied and optimized. Furthermore, the surface of various protein-modified electrodes was imaged with atomic force microscopy and the height distribution of protein particles was obtained with the Particle Analysis Software. The relative results were fully in accordance with the ones from the electrochemical impedance spectroscopy.
Abstract: An electrochemical impedance spectroscopy method of detection for aptamer-based array electrodes is reported in which the binding of aptamers immobilized on gold electrodes leads to impedance changes associated with target protein binding events. Human IgE was used as a model target protein and incubated with the aptamer-based array consisting of single-stranded DNA containing a hairpin loop. To increase the binding efficiency for proteins, a hybrid modified layer containing aptamers and cysteamine was fabricated on the photolithographic gold surface through molecular self-assembly. Atomic force microscopy analysis demonstrated that human IgE could be specifically captured by the aptamer and stand well above the self-assembled monolayer (SAM) surface. Compared to immunosensing methods using anti-human IgE antibody as the recognition element, impedance spectroscopy detection could provide higher sensitivity and better selectivity for aptamer-modified electrodes. The results of this method show good correlation for human IgE in the range of 2.5-100 nM. A detection limit of 0.1 nM (5 fmol in a 50-microL sample) was obtained, and an average of the relative standard deviation was <10%. The method herein describes the first label-free detection for arrayed electrodes utilizing electrochemical impedance spectroscopy.
Notes: One of the Most-Accessed Articles, July-September 2005
