Alexander Revzin

Biosensor technology: recent advances in threat agent detection and medicine

Biosensors are of great significance because of their capability to resolve a potentially large number of analytical problems and challenges in very diverse areas such as defense, homeland security, agriculture and food safety, environmental monitoring, medicine, pharmacology, industry, etc. The expanding role of biosensing in society and a real-world environment has led to an exponential growth of the R&D efforts around the world. The world market for biosensor devices, according to Global Industry Analysts, Inc., is expected to reach

Aptamer-Based Electrochemical Biosensor for Interferon Gamma Detection

12 billion by 2015. Such expedient growth is driven by several factors including medical and health problems, such as a growing population with a high risk of diabetes and obesity, and the rising incidence of chronic diseases such as heart disease, stroke, cancer, chronic respiratory diseases, tuberculosis, etc.; significant problems with environmental monitoring; and of course serious challenges in security and military applications and agriculture/food safety. A review paper in the biosensor technology area may be structured based on (i) the principles of detection, such as the type of transducer platform, bioanalytical principles (affinity or kinetic), and biorecognition elements origin/properties (i.e. antibodies, enzymes, cells, aptamers, etc.), and (ii) the application area. This review follows the latter strategy and focuses on the applications. This allows discussion on how different sensing strategies are brought to bear on the same problem and highlights advantages/disadvantages of these sensing strategies. Given the broad range of biosensor related applications, several particularly relevant areas of application were selected for review: biological threat agents, chemical threat agents, and medicine.

Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes

In this paper, we describe the development of an electrochemical DNA aptamer-based biosensor for detection of interferon (IFN)-γ. A DNA hairpin containing IFN-γ-binding aptamer was thiolated, conjugated with methylene blue (MB) redox tag, and immobilized on a gold electrode by self-assembly. Binding of IFN-γ caused the aptamer hairpin to unfold, pushing MB redox molecules away from the electrode and decreasing electron-transfer efficiency. The change in redox current was quantified using square wave voltammetry (SWV) and was found to be highly sensitive to IFN-γ concentration. The limit of detection for optimized biosensor was 0.06 nM with linear response extending to 10 nM. This aptasensor was specific to IFN-γ in the presence of overabundant serum proteins. Importantly, the same aptasensor could be regenerated by disrupting aptamer-IFN-γ complex in urea buffer and reused multiple times. Unlike standard sandwich immunoassays, the aptasensor described here allowed one to detect IFN-γ binding directly without the need for multiple washing steps and reagents. An electrochemical biosensor for simple and sensitive detection of IFN-γ demonstrated in this paper will have future applications in immunology, cancer research, and infectious disease monitoring.

Simultaneous detection of cell-secreted TNF-α and IFN-γ using micropatterned aptamer-modified electrodes

Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25 degrees to 37 degrees C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF)-an important liver signaling molecule - into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.

A microdevice for multiplexed detection of T-cell-secreted cytokines

Cellular production of such cytokines as interferon (IFN)-γ and tumor necrosis factor (TNF)-α is used to determine disease-specific immune responses and may be used to diagnose infectious diseases such as tuberculosis. In this paper, we describe the development of micropatterned electrodes functionalized with electroactive aptamers for multiplexed detection of immune-cell-produced cytokines. A sequence of electrode deprotection and aptamer incubation steps were used to assemble anti-IFN-γ DNA aptamers and anti-TNF-α RNA aptamers on individually addressable half-ring electrodes. Aptamer molecules were thiolated for assembly on gold and were functionalized with methylene blue redox reporter for electrochemical signal transduction. Specificity of individual sensors to the correct cytokine species was confirmed by exposure to recombinant cytokines. For cell detection experiments, electrode arrays were integrated into microfluidic devices and incubated with immune cells. Design of the surface was such that a small group of ~400 cells attached in the circular adhesion sites surrounded by half-ring electrodes sensing IFN-γ and TNF-α. The microdevice consisted of two parallel microfluidic channels, each channel containing four cell capture/sensing sites. Upon mitogenic activation, secreted IFN-γ and TNF-α molecules were monitored by performing square wave voltammetry (SWV) at different time points at individually addressable electrodes. This biosensing platform was used to analyze the quantity and rate of cytokine release from primary T cells and a monocyte cell line. Upon further development of this platform may be enhanced to enable detection of larger number of cytokines and used to correlate the levels and dynamics of cytokine release in immune cells to diagnosis and treatment of infectious diseases.

Lensfree Holographic Imaging of Antibody Microarrays for High-Throughput Detection of Leukocyte Numbers and Function

Cytokines are produced by immune cells in response to viral or bacterial pathogens and therefore have significant diagnostic value. The goal of the present study was to develop a miniature device for detection of interleukin (IL)-2 and interferon (IFN)-gamma cytokines secreted by a small population of CD4 and CD8 T-cells. Microarrays of T-cell- and cytokine-specific Ab spots were printed onto poly(ethylene glycol) (PEG) hydrogel-coated glass slides and enclosed inside a microfluidic device, creating a miniature ( approximately 3 microL) immunoreaction chamber. Introduction of the red blood cell (RBC) depleted whole human blood into the microfluidic device followed by washing at a pre-defined shear stress resulted in isolation of pure CD4 and CD8 T-cells on their respective Ab spots. Importantly, the cells became localized next to anti-IL-2 and -IFN-gamma Ab spots. Mitogenic activation of the captured T-cells was followed by immunofluorescent staining (all steps carried out inside a microfluidic device), revealing concentration gradients of surface-bound cytokine molecules. A microarray scanner was then used to quantify the concentration of IFN-gamma and IL-2 near CD4 and CD8 T-cells. This study represents one of the first demonstrations of a microdevice for capturing desired T-cell subsets from a small blood volume and determining, on-chip, cytokine profiles of the isolated cells. Such a microdevice is envisioned as an immunology tool for multi-parametric analysis of T-cell function with direct applications in diagnosis/monitoring of HIV and other infectious diseases.

Control of Mammalian Cell and Bacteria Adhesion on Substrates Micropatterned with Poly(ethylene glycol) Hydrogels

Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.

Detecting Cytokine Release from Single T-cells

A simple method for controlling the spatial positioning of mammalian cells and bacteria on substrates using patterned poly(ethylene glycol) (PEG) hydrogel microstructures is described. These microstructures were fabricated using photolithography on silicon, glass or poly (dimethylsiloxane) (PDMS) surfaces modified with a 3-(trichlorosilyl) propyl methacrylate (TPM) monolayer. During the photogelation reaction, the resulting hydrogel microstructures were covalently bound to the substrate via the TPM monolayer and did not detached from the substrate upon hydration. For mammalian cell patterning, microwell arrays of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding hydrophobic TPM floors inside the microwells. Murine 3T3 fibroblasts and transformed hepatocytes were shown to selectively adhere to the TPM monolayer inside the microwells, maintaining their viability, while adherent cells were not present on the hydrogel walls. The number of cells inside one microwell could be controled by changing the lateral dimension of the microwells, thus allowing only a single cell per microwell if desired. In the case of 30×30 μm microwells, as many as 400 microwells were fabricated in 1 mm2. In addition, PEG hydrogel microstructures were also shown to effectively resist the adhesion of bacteria such as Escherichia coli.

Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion

The cytokine production by leukocytes correlates with bodys ability to mount an immune response and therefore has high diagnostic value. In the present study we employed microfabricated surfaces to capture T-cells from minimally processed human blood, arrange these cells into a single cell array, and then detect interferon (IFN)-gamma released from individual cells. The fabrication of cell capture surfaces started with coating a silane-modified glass slide with a uniform layer of poly(ethylene glycol) (PEG) hydrogel. The hydrogel-coated slide was lyophilized and then incubated with a mixture of monoclonal anti-IFN-gamma and anti-CD4 antibodies (Abs). To define sites for single cell attachment, PEG hydrogel microwells (20 microm diameter) were photolithographically patterned on top of the Ab-containing hydrogel layer. This micropatterning process resulted in fabrication of PEG hydrogel microwells with Ab-decorated bottom and nonfouling walls. To minimize the blood volume requirement and to precisely define shear stress conditions, the engineered surface was enclosed inside a PDMS-based microfluidic device. Introduction of red blood cell (RBC) depleted whole human blood followed by controlled washing led to the isolation of individual CD4 T-cells within PEG microwells. Mitogenic activation and immunofluorescent staining performed inside the microfluidic chamber revealed IFN-gamma cytokine signal colocalized with specific T-cells. The device and process presented here will be expanded in the future to enable multiparametric functional analysis of immune cells organized into high density single cell arrays.

Using growth factor arrays and micropatterned co-cultures to induce hepatic differentiation of embryonic stem cells

Healthy guts exclude oxygen Normally, the lumen of the colon lacks oxygen. Fastidiously anaerobic butyrate-producing bacteria thrive in the colon; by ablating these organisms, antibiotic treatment removes butyrate. Byndloss et al. discovered that loss of butyrate deranges metabolic signaling in gut cells (see the Perspective by Cani). This induces nitric oxidase to generate nitrate in the lumen and disables β-oxidation in epithelial cells that would otherwise mop up stray oxygen before it enters the colon. Simultaneously, regulatory T cells retreat, and inflammation is unchecked, which contributes yet more oxygen species to the colon. Then, facultative aerobic pathogens, such as Escherichia coli and Salmonella enterica, can take advantage of the altered environment and outgrow any antibiotic-crippled and benign anaerobes. Science, this issue p. 570; see also p. 548 Butyrate-producing microbes contribute to synergism between epithelial cell metabolism and immune response regulation to maintain gut heath. Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator–activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward β-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.