Determining extracellular analyte concentration with nanoplasmonic sensors

Description (excerpt)

This application is a divisional application of U.S. patent application Ser. No. 15/186,742, filed on Jun. 20, 2016 by Marc P. Raphael et al., entitled IMAGING EXTRACELLULAR PROTEIN CONCENTRATION WITH NANOPLASMONIC SENSORS, which claimed the benefit of U.S. Provisional Application No. 62/181,939, filed on Jun. 19, 2015 by Marc P. Raphael et al., entitled IMAGING EXTRACELLULAR PROTEIN CONCENTRATION WITH NANOPLASMONIC SENSORS, the disclosures of both are incorporated herein by reference, in their entireties. BACKGROUND Aspects of the exemplary embodiment relate to detection of analytes in a fluid and find particular application in connection with the detection of extracellular proteins using nanoplasmonic sensors. From bacterium to eukaryote, a cell's fate is directly tied to its local chemical environment. The measurement of external protein concentrations and gradients by membrane bound receptors is useful in the study of cell differentiation, motility and proliferation. Such dependencies have been deduced by introducing artificial gradients to cell cultures. However, direct measurements of the spatio-temporal concentrations of analytes, which cells themselves produce via secretion, have remained elusive. One roadblock has been the lack of an assay that can measure extracellular protein concentrations in real time without disrupting the signaling pathways of interest. This real time, non-invasive requirement severely limits the techniques that can be employed, including common fluorescent labeling methods. For example, while fluorescent fusion proteins have been useful in the study of of intracellular protein measurements, the technique does not lend itself to extracellular signaling. A tag, such as green fluorescent protein (GFP) tag of about 27 kDa, for example, may compromise the labeled protein's ability to navigate the complexities of the secretory pathway (Wiedenmann et al., “Fluorescent Proteins for Live Cell Imaging: Opportunities, Limitations, and Challenges,” lubmb Life 61 (11):1029-1042 (2009); Costantini, et al., “Fluorescent Proteins in Cellular Organelles: Serious Pitfalls and Some Solutions,” DNA Cell Biol. 32 (11):622-627 (2013)). Even if the proteins are successfully secreted, the result is a diffuse fluorescent glow outside the cell which is difficult to quantify. Fluorescently-labeled antibodies used for immunosandwich assays have been successfully introduced outside of live cells to measure secretions (Bailey, et al., “DNA-encoded antibody libraries: A unified platform for multiplexed cell sorting and detection of genes and proteins,” J. Am. Chem. Soc. 129 (7):1959-1967 (2007); Han, et al., “Polyfunctional responses by human T cells result from sequential release of cytokines,” Proc. Natl. Acad. Sci. U.S.A. 109 (5):1607-1612 (2012); Shirasaki, et al., “Real-time single-cell imaging of protein secretion,” Scientific Reports 4 (2014)). However, the addition of these relatively large probes (typically 150 kDa) is an impediment to downstream signaling and the techniques typically involve isolating individual cells. In both examples, the ability to establish causal relationships between secreted protein concentrations and cell fate, whether the signaling be autocrine, paracrine or endocrine in nature, is hampered by the probes themselves. Solid-state nanosensors have the potential to overcome this impasse. Probes such as nanodiamonds and metallic nanostructures are biocompatible, do not suffer from photobleaching and, advantageous from the protein secretion perspective, are label-free techniques. Nanodiamond sensors are highly sensitive magnetic field detectors resulting from nitrogen vacancies, which makes the technique particularly applicable to detecting metalloproteins (Horowitz, et al., “Electron spin resonance of nitrogen-vacancy centers in optically trapped nanodiamonds,” Proc. Natl. Acad. Sci. U.S.A. 109 (34):13493-13497 (2012); Ermakova, et al., “Detection of a Few Metallo-Protein Molecules Using Color Centers in Nanodiamonds,” Nano Lett. 13 (7):3305-3309 (2013)). Metallic nanoparticles exhibit a localized surface plasmon resonance (LSPR) which is sensitive to changes in the local refractive index of the surrounding medium. Their surfaces can be biofunctionalized for the detection of proteins, lipids, and DNA in cell-free environments (Sepulveda, et al., “LSPR-based nanobiosensors,” Nano Today 4 (3):244-251 (2009); Mayer, et al., “A single molecule immunoassay by localized surface plasmon resonance,” Nanotechnology 21 (25) (2010); Haes, et al., “A nanoscale optical biosensor: Sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles,” J. Am. Chem. Soc. 124 (35):10596-10604 (2002); Nusz, et al., “Label-free plasmonic detection of biomolecular binding by a single gold nanorod,” Anal. Chem. 80 (4):984-989 (2008); Jonsson et al., “Supported lipid bilayer formation and lipid-membrane-mediated biorecognition reactions studied with a new nanoplasmonic sensor template,” Nano Lett. 7 (11):3462-3468 (2007); Dahlin, et al., “Specific self-assembly of single lipid vesicles in nanoplasmonic apertures in gold,” Adv. Mater. 20 (8):1436-1422 (2008); Endo, et al., “Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor,