Metallothionein (MT) is a low-molecular-weight protein with a number of roles to play in cellular homeostasis. MT is synthesized as a consequence of a variety of cellular stressors, and has been found in both intracellular compartments and in extracellular spaces. The intracellular pool of this cysteine-rich protein can act as a reservoir of essential heavy metals, as a scavenger of reactive oxygen and nitrogen species, as an antagonist of toxic metals and organic molecules, and as a regulator of transcription factor activity. The presence of MT outside of cells due to the influence of stressors suggests that this protein may make important contributions as a "danger signal" that influences the management of responses to cellular damage. While conventional wisdom has held that extracellular MT is the result of cell death or leakage from stressed cells, there are numerous examples of selective release of proteins by nontraditional mechanisms, including stress response proteins. This suggests that MT may similarly be selectively released, and that the pool of extracellular MT represents an important regulator of various cellular functions. For example, extracellular MT has effects both on the severity of autoimmune disease, and on the development of adaptive immune functions. Extracellular MT may operate as a chemotactic factor that governs the trafficking of inflammatory cells that move to resolve damaged tissues, as a counter to extracellular oxidant-mediated damage, and as a signal that influences the functional behavior of wounded cells. A thorough understanding of the mechanisms of MT release from cells, the conditions under which MT is released to the extracellular environment, and the ways in which MT interacts with sensitive cells may both illuminate our understanding of an important control mechanism that operates in stressful conditions, and should indicate new opportunities for therapeutic management via the manipulation of this pool of extracellular MT.

ProblemQuantitative measurement of proteins in bodily fluids or cellular preparations is critical for the evaluation of biomarkers or the study of complex cellular processes. While immunoassays are the most common quantitative approach used so far, they are not practical for the evaluation of multiple proteins. Microfluidic technology allows a fine spatial control in immobilizing proteins and biomolecules inside microchannels, eliminating cross‐reactivity between competing analytes, and allowing rapid and sensitive detection of targeted antigens for multiple applications. We report the characterization and validation of the Simple Plex™ platform for the detection and quantification of cytokines and chemokines from human and mouse samples.MethodCytokine and chemokine expression levels were determined using Simple Plex cartridges from ProteinSimple. Serum samples were obtained from the Yale Biorepository.ResultsOur data demonstrate an excellent correlation between the results obtained with Simple Plex and conventional immunoassays such as ELISA and Luminex.ConclusionWe describe the characterization and validation of Simple Plex, a novel multi‐analyte, automated microfluidic platform that allows the evaluation of cytokines and chemokines from human and mice biological samples. Simple Plex showed significant advantages over traditional approaches in terms of low sample volume requirements, sensitivity and dynamic range, coefficient of variation, and reproducibility.

Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, spores, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a non-toxic form of Pseudomonas aeruginosa exotoxin A (ntPE), Bacillus globigii, Mycoplasma hyopneumoniae, Listeria monocytogenes, Escherichia coli, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free and highly multiplexed assay requiring nanoliter amounts of capture reagents.

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