Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

Lack of accurate yet inexpensive diagnostics remains a critical bottleneck in the control and elimination of tuberculosis (TB). Interferon-gamma release assay (IGRA), which involve measurement of the release of the cytokine, interferon-gamma (IFN-γ), in blood samples stimulated with <i>Mycobacterium tuberculosis</i> antigens, is used to detect latent TB infection (LTBI). Use of IGRA in resource-poor settings, in which TB is endemic, is however hindered by the need for specialized equipment for sensitive detection of low amounts of cytokine released. Additionally, recent research has shown the advantage of multiplexed detection of other non-IFN-γ cytokines in improving diagnostic accuracy. However, this requires even more expensive instrumentation and there are no inexpensive or point-of-care compatible multiplexed cytokine detection tools available yet. Here we develop and demonstrate a low-cost, high-throughput, multiplexed cytokine detection platform based on amplified enzymatic silver metallization on a plastic substrate. The assay is performed in microwells formed on a commonly available plastic petri dish and the dry readout of the deposited silver is obtained using a cellphone camera, thus significantly reducing overall cost and complexity of multiplexed cytokine detection. We demonstrate the ability to measure clinically relevant sub-picomolar levels of multiple cytokines, including IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) from a small volume (<5µL) of the same blood sample used in an IGRA. Furthermore, we demonstrate the use of this assay to distinguish IGRA+ and IGRA-participant samples from a TB endemic setting.