Poster Y15 - An optical aptamer-based cytokine nanosensor detects macrophage activation by bacterial toxins

Introduction: Overactive or dysregulated cytokine expression is hallmark of many acute and chronic inflammatory diseases. This is true for acute or chronic infection, neurodegenerative diseases, autoimmune diseases, cardiovascular disease, cancer, and others. Cytokines such as interleukin-6 (IL-6) are known therapeutic targets and biomarkers for such inflammatory diseases. Platforms for cytokine detection are therefore desirable tools for both research and clinical applications. Single-walled carbon nanotubes (SWCNT) are versatile nanomaterials with near-infrared fluorescence that can serve as transducers for optical sensors. When functionalized with an analyte-specific recognition element, SWCNT emission may become sensitive and selective towards the desired target. SWCNT-aptamer sensors are easily assembled, inexpensive, and biocompatible.

Materials and

Methods: In this work, we introduced a nanosensor design based on SWCNT and a DNA aptamer specific to IL-6. We first evaluated several SWCNT-aptamer constructs based on this simple direct complexation method, wherein the aptamer both solubilizes the SWCNT and confers sensitivity to IL-6. We tested the sensor against a range of IL-6 concentrations to determine its limit of detection. Continuous acquisition of fluorescence spectra during nanosensor deployment allowed us to investigate sensor kinetics. Further, we studied the stability of the SWCNT-aptamer construct over a period of two weeks. To test the sensor’s ability to detect endogenous IL-6, we treated Raw 264.7 cells with lipopolysaccharide (LPS). These activated macrophages expressed elevated levels of IL-6, as confirmed by ELISA. We deployed our nanosensor in conditioned media from the activated macrophages and compared its response to the cytokine levels measured with ELISA.

Results, Conclusions, and Discussions: We identified a 31-nucleotide DNA aptamer that enabled sensitive and specific detection of IL-6 in a variety of testing conditions. The sensor limit of detection, 105 ng/mL, lies in the relevant range for pathological IL-6 levels. Upon investigation of sensor kinetics, we found rapid response within seconds of antigen addition which continued over the course of three hours. We found that this sensor construct is stable, and the aptamer is not displaced from the nanotube surface during IL-6 detection. Finally, we confirmed the ability of this sensor construct to detect macrophage activation caused by LPS in an in vitro model of disease, finding rapid and sensitive detection of macrophage-expressed IL-6. We are confident further development of this sensor will have novel implications for diagnosis of acute and chronic inflammatory diseases, in addition to contributing to the understanding of the role of cytokines in these diseases.

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