Method for Measuring Carbon Nanotubes Taken-Up by a Plurality of Living Cells
Carbon nanomaterials (CNM), including carbon nanotubes (CNT) and graphene, are undergoing extensive research due to their diverse commercial applications and potential for biomedical uses. There are many reports indicating the toxicity of various CNMs, which is a potential hazard for biomedical applications as well as the environment. Furthermore, recent studies have shown that CNTs from anthropogenic sources reach human lung cells on a routine basis.1 As such, it has become increasingly critical to be able to accurately analyze the potential risks to environmental health and safety. Despite the numerous analytical methods available, the need remains for an accessible method of analyzing CNM products in aqueous solutions. The presented method for CNM quantification in aqueous solutions, including biological extracts, can determine the concentration between detection limits of 5ng and 575ng and only requires a gel electrophoresis unit, a flatbed scanner, and image analysis software. Figure 1: Flow diagram for method of measuring CNTs taken-up by living cells. Problem: There is a need for rapid, sensitive, and easily-accessible methods for quantification of small amounts of CNMs in liquid samples, especially in biological applications.Solution: A label-free method to quantify CNMs from populations of cells has been presented. This method involves the use of polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), a flatbed scanner, and image analysis software to quantify nanogram amounts of CNMs. Technical Summary:This method of quantifying CNT powders consists of SDS-PAGE followed by white light scanning to create a standard curve (for every batch of CNT powder) that may then be used to quantify CNM concentrations. White light scanning enables the detection of all CNM structures in a sample regardless of whether the CNMs are pristine, oxidized, and/or covalently functionalized. The choice of surfactant was confirmed to have no effect on the quantification of CNMs in the PAGE analysis. Raman microprobe spectroscopy was used to identify presence of CNMs at the gel interface. CNMs of various dimensions can be selectively concentrated at the gel interface by adjusting the porosity of the gel; the smallest graphene and graphene oxide nanoflakes may be analyzed in a 15% polyacrylamide gel (rather than 4% used for SWNTs). Value Proposition:This method allows for rapid, sensitive, and inexpensive label-free detection and quantification of carbon nanomaterials (CNM) in aqueous solutions, including biological extracts. Applications:May be used to study:CNMs in blood & tissue extracts. (Biodistribution & pharmokinetic studies of CNMs in intact animals)ADMET profile - How CNM constructs interact with living systemsHow CNM parameters are involved in the (cell) accumulation process (e.g., CNM dimensions, concentrations, and surface chemistries)Parameters required to assess energy dependence of accumulation (cell incubation times & temperatures) Key Benefits:Inexpensive – Requires only a gel electrophoresis unit, a flatbed scanner, and image analysis software – commonly-available lab equipmentSmall samples – Only 1 microliter aliquots needed for quantificationCustomizable – CNMs of various dimensions can be measured by adjusting porosity of polyacrylamide gelSensitive – Quantifies concentration of all CNT types between with nanogram-scale sensitivity Publication:Wang, Ruhung, et al. “Gel Electrophoresis Method to Measure the Concentration of Single-Walled Carbon Nanotubes Extracted from Biological Tissue.” ACS Publications, Analytical Chemistry, 18 Mar. 2009, pubs.acs.org/doi/abs/10.1021/ac802485n. Related Link: Sigma Aldrich Technology Spotlight – Carbon Nanomaterials: Elemental Analyses and Quantification of Their Accumulation in Living Cells ( Link ) IP Status: United States patent 8,632,671 B2 issued on January 21, 2014.Licensing Opportunity: This technology is available for exclusive or non-exclusive licensing.ID Number: MP 08-021Contact: otc@utdallas.edu 1 Kolosnjaj-Tabi, Jelena, et al. “Anthropogenic Carbon Nanotubes Found in the Airways of Parisian Children.” EBioMedicine, vol. 2, no. 11, Oct. 2015, pp. 1697–1704., doi:10.1016/j.ebiom.2015.10.012.
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