In the emerging world of medical diagnostics and biological discovery, there is a growing need for highly sensitive and reliable protein detection platforms to make rapid and quantitative measurements in a multiplexed fashion. Using a technology developed in our lab called Stop Flow Lithography (SFL), we are able to generate encoded microgel particles containing one or multiple protein sensing regions. SFL couples the patterning of flows afforded by laminar flow in microfluidic devices with the patterning of light provided by UV lithography. In this manner, we can pattern both the spatial location of probe molecules within a particle (e.g. stripes) and the geometric shape of the particles, giving rise to a barcode. Acrylate modified biological probes are added into the monomer stream and are directly conjugated into the gel matrix. We take advantage of a suspension-based array format to leverage near-solution kinetics and the hydrating environment of a hydrogel scaffold to give proteins a favorable environment for capture and detection. We analyze our particles post-assay using a home-built microfluidic scanning system.
Using both aptamer probes and antibody probes, we have demonstrated capability to detect proteins with high sensitivity and specificity. We initially interfaced our system with DNA aptamer probes against a crucial protein in the blood clotting cascade called thrombin. The aptamer probe we used, HTDQ, is a well characterized thrombin binder that interacts with the heparin binding exosite of the protein. In this set of experiments, the HTDQ-thrombin interaction was studied in the context of hydrogel particles to determine affinity between the probe and target and to ascertain the limit of detection of thrombin. Without the use of additional spacers during immobilization or the need to use multiple aptamer probes to achieve an avidity affect, we were able to show direct detection limits comparable to those in existing microarray platforms.
In tandem, we have done work where we functionalize our particles with monoclonal antibody capture probes. We synthesized particles that could detect il-2, TNF-α, IFN-γ and MIP1-β, which are all cytokines that are highly relevant in the immune response to Human Immunodeficiency Virus -1 (HIV-1). Hybridization assays were initially carried out in 90% fetal bovine serum (FBS), and we were able to detect single pg/mL levels of all cytokines in this complex media without any fouling of the particles. Particles performed equally well in multiplexed settings, showing minimal cross reactivity and high specificity when they were in the presence of several other antibodies; we observed the same detection ranges in multiplexed assays. After initial proof of concept experiments in the FBS, we moved on to analysis of cell culture supernatants derived from peripheral blood mononuclear cells (PBMCs) of HIV-1 infected patients. We were able to discriminate cytokine response from different cell lines, allowing us to profile the response across a range of patients at various disease states.
See more of this Group/Topical: Topical 3: 2011 Annual Meeting of the American Electrophoresis Society (AES)