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Tuning Polymer-Protein Interaction with Salt
Tuesday, February 14, 2017 Diabetes Mellitus (DM) is caused by the pancreas insufficient produce of insulin or the body cells not responding properly to the blood insulin, which leads to disorder of carbohydrate metabolism. When the body has deficient insulin, tissues cannot take glucose in from blood stream. As an alternative metabolic pathway, burning fatty acids produces ketones (b-hydroxybutyric acid: 3-HB) into bloodstream as well as create energy. Too much concentration of blood ketone body is called ketoacidosis, and it can cause serious disturbance of consciousness, or result in death at the worst case. Therefore it is important to measure blood ketone body concentration and control it. Standard method of measuring blood ketone is venous blood test. Therefore, there is many problems such as 1) difficulty of periodic measurement, 2) risks of infection, 3) pain of injection, 4) waiting time for a measurement result. In this study, we developed a novel enzymatic reactor and H2O2 electrode aiming at the application to measure urine/sweat ketone body non-invasively. To evaluate properties of trial product sensors, we prepared test flow circuit which consisted of continuous flow pump, manual injector, and incubator (30/37 C). Within phosphate buffer solution, different concentration (0.5-113.83 [mM]) of ketone body which mixed in 1.0mL/min of continuous flow path could be detected with high accuracy. 2246-Pos Board B566 Microvolume Dielectric Spectroscopy and Molecular Dynamics of Amino Acids Daniel Havelka, Ondrej Krivosudsky, Jiri Prusa, Michal Cifra. Institute of Photonics and Electronics, Czech Academy of Sciences, Prague, Czech Republic. Accurate knowledge of electromagnetic properties of biosystems is essential for the novel physical methods for therapy and diagnostics in medicine and biotechnology. To study the dynamics and electrical properties of biomolecules, we have designed, fabricated, and tested novel grounded coplanar waveguide-based chip for dielectric spectroscopy of liquid samples in 0.5 - 40 GHz band made on RO4350B substrate and we implemented a method for the permittivity extraction based purely on the extraction from measured scattering parameters. To our knowledge, the proposed bio sensing method represents an advantage over the current known sensing chips because the dielectric function of sample is obtained purely by using precision calibration techniques (NIST multi-line TRL) and computational optimization (CST Microwave Studio) without any a priori assumption about the dielectric model of measured sample. This chip is designed with well-defined active area to achieve the high sensitivity to dielectric change, enables perfect repeatability of sample position without the need of any microfluidics. Furthermore, due to its sensing size, the chip permits to work with only 250 microliter sample volume. In the pioneering experiments, we focused on dielectric (i.e. polarization) properties of solutions of amino acids, as building blocks of proteins. Alanine has been selected. The polar nature of amino acids determines their behavior in aqueous solutions and due to fact that at least one relaxation process is anticipated in microwave band, it allows us to study their dynamics and structure which employ using broadband dielectric spectroscopy. Data extracted from experiment are in an exact agreement in comparison with widely used commercial reflection method by coaxial probe (85070E Dielectric Probe Kit). Data show the trend of rising static permittivity at low frequencies with rising concentration of amino acids and also show the shift of relaxation times. We also performed molecular dynamics simulations to predict the complex permittivity of amino acid solution and obtained good agreement with experimental data. The presented procedure enables simple experimental verification of molecular dynamics of biomolecules by the dielectric spectroscopy using our microvolume chip. We acknowledge support by the Czech Science Foundation, project no. P102/ 15-17102S. Authors participate in COST Action BM1309 and bilateral exchange project between Czech and Slovak Academies of Sciences, no. SAV15-22. 2247-Pos Board B567 Tuning Polymer-Protein Interaction with Salt Monasadat Talarimoghari1, Aleksandra Dylewska1, Marcel Hoffmann1, Gerhard Baaken2, Dalila Chouikhi3, Jean-Francois Lutz3, Jan C. Behrends1. 1 Physiology, University of Freiburg, Freiburg, Germany, 2Physiology, Ionera Technologies GmbH, Freiburg, Germany, 3Institut Charles Sadron, University of Strasbourg, Strasbourg, France. Biological nanopores are known to interact with synthetic and biological polymers, enabling their use in label-free single-molecule analytical tasks such as sequencing and/or mass discrimination. The latter, called nanopore-based single molecule mass spectrometry (Np-SMMS) has, to date, only been shown for one synthetic polymer, poly(ethyleneglycol) (PEG). It is based on the fact that
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binding of PEG inside the pore gives rise to blocks of ionic current, with the degree of block being detectably different for PEG molecules differing in size by only one monomer1,2. In order to extend the range of application of Np-SMMS, and to obtain information on the mechanism underying mass sensitivity of polymer-induced pore block, we have begun to test the interaction of other neutral polymers with biological nanoores. Here, we report on poly(dimethylacrylamide) (PDMA), a water-soluble neutral polymer. Under conditions used for Np-SMMS of PEG with alpha-hemolysine (4M KCl, þ40 mV), we obtained current blocks with polydisperse PDMA (Mn 1500 g/mol by MALDI) that were low in frequency (<0.1 Hz/ mM) short (tau <100 ms) and noisy, resulting in little mass resolution compared to PEG. We reasoned that we might take advantage of a specific salt effect of fluoride anion reported for the aHL pore3 in order to increase blocking frequency and dwell time. Using electrolyte solutions consiting of 4M Kþ as cation and various proportions of Cl- and F- as anions (1:1, 2:1, 3:1, 4:1) we were able to obtain longer events (tau up to 400 ms) and higher frequencies (up to 0.15 Hz/mM), allowing significantly better mass resolution for PDMA than in 4 M KCl. However, the large noise component in the blocked current levels for PDMA as opposed to PEG remained, still compromising the peakto-valley ratio of histograms. These findings suggest that the specific salt effect of F- on polymer-protein interaction is independent of the polymer and may be useful in tuning polymer pore interaction for a range of analytes. (1) Robertson et al. Single-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211. (2) Baaken et al. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore. ACS Nano 2015, 9, 6443-6449. (3) Rodrigues et al. Hofmeister Effect in Confined Spaces: Halogen Ions and Single Molecule Detection. Biophys. J. 2011, 100, 2929-2935. 2248-Pos Board B568 A Novel Capacitive Biosensor for the Detection of Small Molecule S-Nitrosothiols Nikki M. Meyer1, Spencer Burton1, James N. Bates2, Benjamin Gaston1, Stephen J. Lewis1, James M. Seckler1. 1 Pediatrics, Case Western Reserve University, Cleveland, OH, USA, 2 Anesthesiology, Univsersity of Iowa, Iowa City, IA, USA. Small molecule S-nitrosothiols are a class of endogenous chemicals which are produced by various forms of nitric oxide synthase. The regulation of these molecules has been shown to play a role in control of a variety of bodily processes and disease models including breathing, blood pressure, pulmonary hypertension, and asthma. However, these molecules are extremely labile, making in vivo detection extremely challenging as most small molecule S-nitrosothiols exists at very low concentrations in the body. To overcome this challenge, we have developed a capacitive biosensor which employs an organic semiconductor that readily covalently crosslinks to all free amines, free thiols, and S-nitrosylated thiols in solution. Samples were treated with formaldehyde to block all free amines and free thiols, leaving only S-nitrosylated thiols. S- nitrosothiol bonding to the semiconducting surface of the sensing electrode changes its capacitance, allowing for extremely sensitive detection of S-nitrosothiols in biological samples. We will present evidence of attomolar detection of S-nitrosocysteine which can be abolished by the addition of Mercury to the fixing buffer, or by exposing the sample to UV light during fixing, both methods of degrading S-nitrosothiols. We will also present evidence of the presence of small molecule S-nitrosothiols in blood, saliva, and other biological samples. 2249-Pos Board B569 Multiparametric Characterization of Single, Unlabeled Proteins in Solution Jared Houghtaling1,2, Michael Mayer2. 1 Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 2Adolphe Merkle Institute, Universite´ de Fribourg, Fribourg, Switzerland. With large diversity in structure and function, as well as clinical relevance, proteins represent an important target for biophysical characterization. Resistive pulse-based nanopore sensing is a compelling platform for this task, as it provides information about individual proteins during their translocation through the zeptoliter sensing volume inside of a nanopore. Previous work in our group used lipid-coated synthetic nanopores to extract five distinct protein descriptors by analyzing modulations in ionic current, DI, resulting from the translocation and rotation of individual lipid-tethered proteins [1]. However, while coating of nanopore walls with fluid lipid bilayers and tethering proteins with lipid anchors is useful for preventing non-specific protein adsorption, extending translocation times, and increasing specificity, it is technically demanding and its
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binding of PEG inside the pore gives rise to blocks of ionic current, with the degree of block being detectably different for PEG molecules differing in size by only one monomer1,2. In order to extend the range of application of Np-SMMS, and to obtain information on the mechanism underying mass sensitivity of polymer-induced pore block, we have begun to test the interaction of other neutral polymers with biological nanoores. Here, we report on poly(dimethylacrylamide) (PDMA), a water-soluble neutral polymer. Under conditions used for Np-SMMS of PEG with alpha-hemolysine (4M KCl, þ40 mV), we obtained current blocks with polydisperse PDMA (Mn 1500 g/mol by MALDI) that were low in frequency (<0.1 Hz/ mM) short (tau <100 ms) and noisy, resulting in little mass resolution compared to PEG. We reasoned that we might take advantage of a specific salt effect of fluoride anion reported for the aHL pore3 in order to increase blocking frequency and dwell time. Using electrolyte solutions consiting of 4M Kþ as cation and various proportions of Cl- and F- as anions (1:1, 2:1, 3:1, 4:1) we were able to obtain longer events (tau up to 400 ms) and higher frequencies (up to 0.15 Hz/mM), allowing significantly better mass resolution for PDMA than in 4 M KCl. However, the large noise component in the blocked current levels for PDMA as opposed to PEG remained, still compromising the peakto-valley ratio of histograms. These findings suggest that the specific salt effect of F- on polymer-protein interaction is independent of the polymer and may be useful in tuning polymer pore interaction for a range of analytes. (1) Robertson et al. Single-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211. (2) Baaken et al. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore. ACS Nano 2015, 9, 6443-6449. (3) Rodrigues et al. Hofmeister Effect in Confined Spaces: Halogen Ions and Single Molecule Detection. Biophys. J. 2011, 100, 2929-2935. 2248-Pos Board B568 A Novel Capacitive Biosensor for the Detection of Small Molecule S-Nitrosothiols Nikki M. Meyer1, Spencer Burton1, James N. Bates2, Benjamin Gaston1, Stephen J. Lewis1, James M. Seckler1. 1 Pediatrics, Case Western Reserve University, Cleveland, OH, USA, 2 Anesthesiology, Univsersity of Iowa, Iowa City, IA, USA. Small molecule S-nitrosothiols are a class of endogenous chemicals which are produced by various forms of nitric oxide synthase. The regulation of these molecules has been shown to play a role in control of a variety of bodily processes and disease models including breathing, blood pressure, pulmonary hypertension, and asthma. However, these molecules are extremely labile, making in vivo detection extremely challenging as most small molecule S-nitrosothiols exists at very low concentrations in the body. To overcome this challenge, we have developed a capacitive biosensor which employs an organic semiconductor that readily covalently crosslinks to all free amines, free thiols, and S-nitrosylated thiols in solution. Samples were treated with formaldehyde to block all free amines and free thiols, leaving only S-nitrosylated thiols. S- nitrosothiol bonding to the semiconducting surface of the sensing electrode changes its capacitance, allowing for extremely sensitive detection of S-nitrosothiols in biological samples. We will present evidence of attomolar detection of S-nitrosocysteine which can be abolished by the addition of Mercury to the fixing buffer, or by exposing the sample to UV light during fixing, both methods of degrading S-nitrosothiols. We will also present evidence of the presence of small molecule S-nitrosothiols in blood, saliva, and other biological samples. 2249-Pos Board B569 Multiparametric Characterization of Single, Unlabeled Proteins in Solution Jared Houghtaling1,2, Michael Mayer2. 1 Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 2Adolphe Merkle Institute, Universite´ de Fribourg, Fribourg, Switzerland. With large diversity in structure and function, as well as clinical relevance, proteins represent an important target for biophysical characterization. Resistive pulse-based nanopore sensing is a compelling platform for this task, as it provides information about individual proteins during their translocation through the zeptoliter sensing volume inside of a nanopore. Previous work in our group used lipid-coated synthetic nanopores to extract five distinct protein descriptors by analyzing modulations in ionic current, DI, resulting from the translocation and rotation of individual lipid-tethered proteins [1]. However, while coating of nanopore walls with fluid lipid bilayers and tethering proteins with lipid anchors is useful for preventing non-specific protein adsorption, extending translocation times, and increasing specificity, it is technically demanding and its