PI: Prof. Alexander Revzin Lead Institution: University of California, Davis
The Intellectual Merit
The goal of the proposal will be the development of novel microsystems for analysis and manipulation of immune cells. Immune cells serve as sentinels of infections, malignancies and autoimmune disorders afflicting an individual. Therefore, these cells may be used to gain diagnostic information as well as to enhance understanding of mechanism of immune disease progression. Analysis of immune cells (leukocytes) represents a singular challenge because there is multiple cell subsets present in the body that are often distinguishable only based on the secreted products. This is true for T-lymphocytes that can be categorized into T-helper 1, T-helper 2 or T-helper 17 phenotype based on the secretion of specific proteins (cytokines). Similarly, B-cells represent a heterogeneous population of cells that are distinguished solely based on the production of specific antibodies. Single cell-level, real-time analysis of products secreted by live T-cells and B-cells is not currently possible due to the lack of appropriate bioanalytical tools. To address this shortcoming, this proposal will develop novel microsystems allowing to arrange immune cells in high density single cell arrays and then monitor production of secreted proteins at a single cell level. In addition, they will develop a "sense-and-release" electrode array system for identifying immune cells based on the secreted product and subsequently sorting/releasing these cells. The microsystems developed in this proposal will be used for the analysis of T-cells from normal and autistic children with the goal of helping to identify correlates between immune function and behavioral aberrations.
Broader Impact
An individual cell is the smallest living building block of tissues and organs. Therefore, analysis of single living cells has been at the frontier of biological/life science research for the past decade and half. The use of green fluorescence protein (GFP) and other fluorescent proteins to report on dynamics of how and when genes get turned on in individual cells has revolutionized the field of biological and medical sciences. However, the requirement of having to get GFP-encoding DNA into cells limits application of reporter gene technology to the most robust and easy to transfect cells. In addition, reporter gene technology monitors gene expression as opposed to protein production. The vision of this proposal is to develop novel and transformative bio-microsystems for non-invasive, dynamic monitoring of protein production in difficult-to-transfect primary cells. This proposal will focus on the analysis of leukocytes and will develop biosensors and microsystems for monitoring cytokine production of individual T-lymphocytes. The interdisciplinary team of investigators will provide surface engineering and microfabrication-based solutions to challenges that currently confound single cell analysis, including: 1) creating high-density single cell arrays, 2) integrating multi-analyte sensors with single cells, 3) co-localizing sensing elements with single cells to ensure high local concentration of secreted metabolite. The novel biosensors to be developed in this proposal will be translatable to other cellular systems (e.g. circulating as well as anchorage-dependent cells) and will be broadly applicable in bioengineering, biotechnology and life sciences fields.
The interdisciplinary collaboration of researchers with a diverse expertise in this project provides a unique opportunity and framework for interdisciplinary education and training of secondary school through postdoctoral students at the frontiers of engineering and the life sciences.
The goal of this project was to develop novel biosensors for immune cell analysis. Immune cells frequently reside in blood and serve as first line of defense against infections. Immune cells also serve as sentinels of infections. Our goal was to query immune cells using novel biosensors that allow for blood analysis to be done faster and simpler than current state of art techniques. We also wanted to have the ability to analyze and sort single cells. The focus of our biosensors was detection of small proteins, called cytokines, produced by the immune cells. Cytokine production by the immune cells may be correlated to specific diseases that in the past invaded the immune system. The best example of this can be found in screening for Tuberculosis (TB), where one cytokine called gamma interferon is used as a marker of patient's exposure to TB. Our team developed novel biosensors for gamma interferon and other inflammatory cytokines. These biosensors are based on class of nucleic acid molecules called aptamers. Aptamers are DNA or RNA molecules that can be designed to emit electrical or optical signal upon binding of the biomarker beign detected. We designed aptamers for gamma interferon as well as other cytokines, and created electrodes that changed electrical signal upon exposure to these cytokines. Importantly these electrodes worked well in complex samples such as blood. We have also demonstrated that cytokines may be detected at the level of single cells as well as from a larger group of cells. One important outcome of our project is that the cytokine sensing technology has been patented and is being licensed by a company. Therefore, we hope to translate our research findings into products/devices for monitoring of infectious diseases such as TB.
