Ali Rahimian

Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode

Inflammatory cytokines are secreted by immune cells in response to infection or injury. Quantification of multiple cytokines in parallel may help with disease diagnosis by illuminating inflammatory pathways related to disease onset and progression. This paper describes development of an electrochemical aptasensor for simultaneous detection of two important inflammatory cytokines, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To enable multiplexing, IFN-γ and TNF-α aptamers were labeled with anthraquinone (AQ) and methylene blue (MB) redox reporters respectively. Random immobilization of two aptamer on gold exhibited redox peaks at -0.37 V (AQ) and -0.15 V (MB) vs. Ag/AgCl reference. When challenged with either IFN-γ or TNF-α, redox signal of the appropriate reporter changed in concentration dependent manner. To demonstrate one possible application of this sensing approach, electrodes were integrated into microfluidic devices and used to dynamically monitor cytokine release from immune cells. Two cell types, primary human CD4 T-cells and U937 monocytic cells, were used to compare differences in cytokine secretions upon stimulation. These cells were infused into the microfluidic devices and stimulated to commence cytokine production. Release of IFN-γ and TNF-α was monitored concurrently from the same small group of cells over the course of 2h. The strategy of encoding specific aptamer types with unique redox reporters allows sensitive and specific detection of multiple protein biomarkers from the same electrode.

Development of an aptasensor for electrochemical detection of exosomes

Exosomes are small (50-100 nm in diameter) vesicles secreted from various mammalian cells. Exosomes have been correlated with tumor antigens and anti-tumor immune responses and may represent cancer biomarkers. Herein, we report on the development of an aptamer-based electrochemical biosensor for quantitative detection of exosomes. Aptamers specific to exosome transmembrane protein CD63 were immobilized onto gold electrode surfaces and incorporated into a microfluidic system. Probing strands pre-labeled with redox moieties were hybridized onto aptamer molecules anchored on the electrode surface. In the presence of exosomes these beacons released probing strands with redox reporters causing electrochemical signal to decrease. These biosensors could be used to detect as few as 1×10(6) particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays relying on anti-CD63 antibodies. Given the importance of exosome-mediated signal transmission among cells, our study may represent an important step towards development of a simple biosensor that detects exosomes without washing or labeling steps in complex media.

Reconfigurable microfluidics with integrated aptasensors for monitoring intercellular communication

We report the development of a microsystem integrating anti-TNF-α aptasensors with vacuum-actuatable microfluidic devices that may be used to monitor intercellular communications. Actuatable chambers were used to expose to mitogen a group of ~600 cells while not stimulating another group of monocytes only 600 μm away. Co-localizing groups of cells with miniature 300 μm diameter aptamer-modified electrodes enabled monitoring of TNF-α release from each group independently. The microsystem allowed observation of the sequence of events that included 1) mitogenic activation of the first group of monocytes to produce TNF-α, 2) diffusion of TNF-α to the location of the second group of cells and 3) activation of the second group of cells resulting in the production of TNF-α by these cells. Thus, we were able to experimentally verify reciprocal paracrine crosstalk between the two groups of cells secreting the same signalling molecule. Given the prevalence of such cellular communications during injury, cancer or immune response and the dearth of available monitoring techniques, the microsystem described here is envisioned to have significant impact on cell biology.

Microfluidic compartments with sensing microbeads for dynamic monitoring of cytokine and exosome release from single cells.

Monitoring activity of single cells has high significance for basic science and diagnostic applications. Here we describe a reconfigurable microfluidic device for confining single cells along with antibody-modified sensing beads inside 20 picoliter (pL) microcompartments for monitoring cellular secretory activity. An array of ∼7000 microchambers fabricated in the roof of the reconfigurable microfluidic device could be raised or lowered by applying negative pressure. The floor of the device was micropatterned to contain cell attachment sites in registration with the microcompartments. Using this set-up, we demonstrated the detection of inflammatory cytokine IFN-γ and exosomes from single immune cells and cancer cells respectively. The detection scheme was similar in both cases: cells were first captured on the surface inside the microfluidic device, then sensing microbeads were introduced into the device so that, once the microcompartments were lowered, single cells and microbeads became confined together. The liquid bathing the beads and the cells inside the compartments also contained fluorescently-labeled secondary antibodies (Abs). The capture of cell-secreted molecules onto microbeads was followed by binding of secondary antibodies - this caused microbeads to become fluorescent. The fluorescence intensity of the microbeads changed over time, providing dynamics of single cell secretory activity. The microdevice described here may be particularly useful in the cases where panning upstream of sensing is required or to analyze secretory activity of anchorage-dependent cells.

On-chip regeneration of aptasensors for monitoring cell secretion

We report on the use of reconfigurable microfluidics for on-chip regeneration of aptasensors used for continuous monitoring of cell-secreted products.

One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids

3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures. STATEMENT OF SIGNIFICANCE Our paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues.

Detecting cell-secreted growth factors in microfluidic devices using bead-based biosensors

Microfluidic systems provide an interesting alternative to standard macroscale cell cultures due to the decrease in the number of cells and reagents as well as the improved physiology of cells confined to small volumes. However, the tools available for cell-secreted molecules inside microfluidic devices remain limited. In this paper, we describe an integrated microsystem composed of a microfluidic device and a fluorescent microbead-based assay for the detection of the hepatocyte growth factor (HGF) and the transforming growth factor (TGF)-β1 secreted by primary hepatocytes. This microfluidic system is designed to separate a cell culture chamber from sensing chambers using a permeable hydrogel barrier. Cell-secreted HGF and TGF-β1 diffuse through the hydrogel barrier into adjacent sensing channels and are detected using fluorescent microbead-based sensors. The specificity of sensing microbeads is defined by the choice of antibodies; therefore, our microfluidic culture system and sensing microbeads may be applied to a variety of cells and cell-secreted factors.

Nanowire Aptasensors for Electrochemical Detection of Cell-Secreted Cytokines

Cytokines are small proteins secreted by immune cells in response to pathogens/infections; therefore, these proteins can be used in diagnosing infectious diseases. For example, release of a cytokine interferon (IFN)-γ from T-cells is used for blood-based diagnosis of tuberculosis (TB). Our lab has previously developed an atpamer-based electrochemical biosensor for rapid and sensitive detection of IFN-γ. In this study, we explored the use of silicon nanowires (NWs) as a way to create nanostructured electrodes with enhanced sensitivity for IFN-γ. Si NWs were covered with gold and were further functionalized with thiolated aptamers specific for IFN-γ. Aptamer molecules were designed to form a hairpin and in addition to terminal thiol groups contained redox reporter molecules methylene blue. Binding of analyte to aptamer-modified NWs (termed here nanowire aptasensors) inhibited electron transfer from redox reporters to the electrode and caused electrochemical redox signal to decrease. In a series of experiments we demonstrate that NW aptasensors responded 3× faster and were 2× more sensitive to IFN-γ compared to standard flat electrodes. Most significantly, NW aptasensors allowed detection of IFN-γ from as few as 150 T-cells/mL while ELISA did not pick up signal from the same number of cells. One of the challenges faced by ELISA-based TB diagnostics is poor performance in patients whose T-cell numbers are low, typically HIV patients. Therefore, NW aptasensors developed here may be used in the future for more sensitive monitoring of IFN-γ responses in patients coinfected with HIV/TB.

Reconfigurable microfluidic device with integrated antibody arrays for capture, multiplexed stimulation, and cytokine profiling of human monocytes

Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)-transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.