Howard Scholars Program, Cycle for Survival, the Alan and Sandra Gerry Metastasis Research Initiative, Mr
Howard Scholars Program, Cycle for Survival, the Alan and Sandra Gerry Metastasis Research Initiative, Mr. and although proteins diminished sensitivity. Here, we discovered an unexpected phenomenon wherein the carbon nanotube optical response to nucleic acids can be enhanced by K+ Channel inhibitor denatured proteins. Mechanistic studies found that hydrophobic patches of the denatured protein chain interact with the freed nanotube surface after hybridization, resulting in enhanced shifting of the nanotube emission. We employed this mechanism to detect an intact HIV in serum, resulting in specific responses within minutes. This work portends a route towards point-of-care optical detection of viruses or other nucleic acid-based analytes. sensor, therefore the experiment was terminated at this time point. Because the kinetics are similar to the well-established response of the sensor to RNA, and because of the investigation of different protein responses in figure S6, we are confident that the sensor is responding to the viral RNA specifically. Such methods could take place directly in serum samples where point of care detection K+ Channel inhibitor is desirable. Field applications in low-resource settings are also possible, as the DNA-nanotube sensor solution and SDS are both highly stable and can be stored at room temperature. In addition, an inexpensive and robust detection device could be developed using standard photodetectors and LED light sources for deployment in regions lacking critical biomedical infrastructure. Many viruses of interest, such as Ebola, are viruses with single-stranded RNA genomes that may be amenable to detection with this method. While we have shown here an application with a lentivirus model, we believe that SDS could also be applied to detection of single-strand oligonucleotides in other viruses K+ Channel inhibitor and pathogen types, as well as in circulating cancer cells. Materials and Methods DNA-suspension of carbon nanotubes Carbon nanotubes produced by the HiPco process (Unidym, Sunnyvale, CA) were mixed with DNA oligonucleotides (IDT DNA, Coralville, IA) at a 2:1 mass ratio in 1 mL of PBS buffer (Thermo Fisher Scientific Waltham, MA) and ultrasonicated for 30 minutes at 40% amplitude (Sonics & Materials, Inc.). The DNA sequences used for suspension were GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTCAGTTTTGCATAGATTTGCACA (GT15miR19), GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTTTTTTTTTTTTTT ((GT)15-(T)15), and GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAAAAAAAAAAAAAA ((GT)15-(A)15) all purchased from IDT (Coralville, IA). Following ultrasonication, the dispersions were ultracentrifuged (Sorvall Discovery 90SE) for 30 minutes at 280,000 g and the top 80% of the supernatant was collected. Absorbance spectra were acquired using a UV/Vis/nIR spectrophotometer (Jasco V-670, Tokyo, Japan). The concentration for HiPco samples was calculated using the extinction coefficient Abs910 = 0.02554 L mg?1cm?1. Excess DNA was removed via 100 kDa Amicon centrifuge filters (Millipore). The DNA-nanotube complexes were re-suspended in PBS (Thermo Fisher Scientific Waltham, MA). Fluorescence spectroscopy measurements of carbon nanotubes Fluorescence emission spectra from aqueous nanotube solutions were acquired with a home- built spectroscopy system as described19. Briefly, carbon nanotube samples were assayed in a 96 well plate format on an inverted microscope using a tunable white-light laser (NKT Photonics) coupled to a variable bandpass filter. Emission light was directed into a spectrometer with a focal length of 320 mm and aperture ratio of f/4.6 (Princeton Instruments IsoPlane SCT 320) and InGaAs array camera (Princeton Instruments 640 512 pixel NIRvana). For all experiments, K+ Channel inhibitor measurements were taken in triplicate across three wells. Following spectra acquisition, custom code written in Matlab applied spectral corrections and background subtraction as described19. Corrected spectra were used to fit the data with Voigt functions. Error bars and linear fits were computed with GraphPad IL1R2 antibody Prism 6. Hybridization conditions Concentrated sodium dodecyl sulfate (Sigma-Aldrich) was prepared in PBS buffer and added to samples to the final concentration indicated, or 1% wt/vol where not indicated; control samples had an equivalent volume of PBS added. Carbon nanotubes were used at a final concentration of 2 mg/L for all experiments. Fetal bovine serum (FBS) (Life Technologies) and human lipoprotein deficient serum (LPDS) (J65516 Lipoprotein Deficient Serum, human, BT-931 from Alfa Aesar) were used for serum-based experiments, and had concentrated nanotubes or SDS added directly to the whole serum. Bovine serum albumin, bovine Y-globulins, casein, and hydrolyzed casein (all from Sigma-Aldrich) were resuspended in PBS to the indicated concentrations. Concentrated target DNA (TGTGCAAATCTATGCAAAACTGA) complementary to the DNA-nanotube sensor or non-complementary control DNA (TCGGTCAGTGGGTCATTGCTAGT) (both from IDT DNA, Coralville, IA) was added to the indicated final concentration. Non-treated controls received an equivalent volume of PBS. Hybridization was allowed to proceed overnight for equilibrium measurements. Kinetics were measured immediately after addition of target. Assessment of (GT)15-(T)15 and (GT)15-(A)15 used 0.2% SDBS (Sigma-Aldrich) as described19 to verify expected behavior with AAAAAAAAAAAAAAA or TTTTTTTTTTTTTTT (IDT DNA, Coralville, IA). HIV production Lentivirus HIV was produced from transfected product packaging cells (293T) using 100 ng pLX304 plasmid38,.