Este trabajo fue apoyado por el Programa de Descubrimiento del Consejo Australiano de Investigación (ARC) (DP150101065) y el Centro de Investigación ARC para Dispositivos Integrados para análisis de usuario final en niveles bajos (IDEAL) (IH150100028).

1. Preparación de electrodos tBLM
<ol><li>Preparación del primer recubrimiento monocapa<ol>
<li>Sumergir un portaobjetos de microscopio de electrodo con patrón de oro recién pulverizado en una solución etanólica compuesta por una proporción de 3 mM 1: 9 de moléculas "espaciadoras" de bencil-disulfuro-tetra-etilenglicol-OH (el disulfuro de bencilo comprendía un espaciador de cuatro oxígeno-etilenglicol, terminado con un grupo OH) y bencil-disulfuro (tetra-etilenglicol) n = 2 moléculas "atadas" de C20-fitetil. Esto crea el recubrimiento de la primera capa a la que se puede anclar una bicapa. NOTA: El electrodo de oro se fabrica evaporando 100 nm, 99.9995% de oro (5n5 de oro) en diapositivas de policarbonato personalizadas de 25 mm x 75 mm20.</li>
		<li>Incubar electrodos con la primera capa a temperatura ambiente durante al menos 1 h.</li>
		<li>Enjuague los electrodos de oro sumergiéndose en copiosas cantidades de etanol puro durante 30 s.</li>
		<li>Use la corredera de electrodo de oro con la primera monocapa directamente para el siguiente paso o guárdelo en un frasco lleno de etanol puro.</li>
		<li>NOTA: Para garantizar la integridad de la primera capa, minimice cualquier contacto directo con las partes doradas de la diapositiva</li>
	</ol></li>
	<li>Montaje de la primera corredera recubierta monocapa<ol>
<li>Retire con cuidado un portaobjetos de electrodo de oro coplanar de su recipiente con pinzas, asegurándose de no hacer contacto con las áreas estampadas donde se formarán los tBLM. NOTA: Tenga en cuenta identificar el lado de la diapositiva en el que se deposita el oro.</li>
		<li>Deslizamiento seco al aire durante 1 - 2 minutos para eliminar cualquier etanol residual.</li>
		<li>Coloque el electrodo de oro sobre una superficie seca, asegúrese de que el electrodo de oro esté correctamente orientado con la superficie de oro estampada hacia arriba.</li>
		<li>Pelar la cubierta de la capa adhesiva transparente de un laminado delgado y colocar sobre los 6 canales para definir cada pozo.</li>
		<li>Utilice un rodillo de presión para liberar aire entre la corredera y la capa adhesiva transparente, como se muestra en la Figura 1A. NOTA: El tiempo requerido para este paso deberá ser optimizado por el investigador. En este protocolo, los tiempos oscilan entre 2-3 min.</li>
		<li>Introduzca tan pronto como sea posible (dentro de 1-2 minutos) la segunda bicapa lipídica en el primer electrodo recubierto monocapa ensamblado para autoensamblaje para evitar dañar la primera capa.</li>
	</ol></li>
	<li>Preparación de la segunda bicapa lipídica<ol>
<li>Agregue 6 μL de lípidos de 3 mM de interés al primer pozo de la diapositiva de seis pozos. No deje que el borde de la punta de la micropipeta toque la superficie de oro, lo que puede dañar las químicas atadas en el electrodo. NOTA: La mezcla lipídica utilizada en este trabajo consistió en 3 mM 70% de lípidos zwitteriónicos C20 difirtanil-éter-glicero-fosfatidilcolina (DPEPC) y 30% de lípidos éter de difitilicéridos C20 (GDPE) mezclados con 3 mM de colesterol-PEG-Biotina en proporción molar 50:1.</li>
		<li>Introducir 6 μL de la mezcla lipídica en los otros pozos con un espacio de 10 s entre cada adición.</li>
		<li>Incubar cada pozo durante exactamente 2 minutos a temperatura ambiente antes de intercambiar la mezcla de lípidos sobre los electrodos con un tampón como PBS. Espaciar los tiempos para la adición y el intercambio de tampón a 10 s de distancia para que cada pozo se incube con el lípido durante exactamente 2 minutos cada uno.</li>
		<li>Lavar 3 veces más con 50 μL de tampón PBS (pH 7.0). Asegúrese de dejar 50 μL de tampón sobre los electrodos en todo momento. No permita que los electrodos se sequen. NOTA: El desplazamiento del disolvente de etanol con la solución acuosa de esta manera (el método de intercambio de disolvente)permite la rápida formación de una sola bicapa lipídica anclada al electrodo de oro a través de las químicas atadas.</li>
	</ol></li>
	<li>Prueba de la formación de tBLM mediante mediciones de espectroscopia de impedancia eléctrica (EIS)<ol>
<li>Inserte la diapositiva de electrodo preparada en un espectrómetro de impedancia de CA (por ejemplo, Tethapod). Asegúrese de que el espectrómetro esté conectado a través de un puerto USB a una computadora que ejecute el software.</li>
		<li>Abra el software, haga clic en Configuración y abra Hardware.</li>
		<li>Establezca la configuración de hardware para usar la excitación de CA de pico a pico de 25 mV.</li>
		<li>Establezca frecuencias entre 0.1 y 10,000 Hz con dos pasos por década para medidas de impedancia rápida presione ok.</li>
		<li>Haga clic en el menú Configuración y abra Modelo.</li>
		<li>Utilice un modelo de circuito equivalente que describa el electrodo de oro de anclaje como un elemento de fase constante en serie con una resistencia que describa el tampón electrolítico y una red paralelo resistencia-condensador para describir la bicapa lipídica, y presione OK.</li>
		<li>Pulse el botón Inicio para iniciar una medición en tiempo real de la capacitancia de membrana (Cm)y la conducción de membrana (Gm). Los valores de Cm de los tBLM típicos deben estar en el rango de 12,5 nF a 15,5 nF para el 10% de químicas atadas21,22.</li>
		<li>Después de ejecutar el protocolo y finalizar el experimento, guarde los datos.</li>
		<li>Repita la medición con el siguiente pozo.</li>
	</ol></li>
</ol>2. Irradiación láser
<ol><li>Configuración experimental NOTA: El sistema a medida está configurado para cada pozo tBLM individualmente.<ol>
<li>Realice experimentos en una caja a prueba de luz para minimizar el láser peligrosamente.</li>
		<li>Utilice una mesa de óptica para configurar el experimento para reducir las vibraciones no deseadas.</li>
		<li>Coloque el lector de impedancia, donde está conectada la diapositiva de oro, en un escenario XYZ y eléblelo de tal manera que se asiente en el camino de la fuente láser.</li>
		<li>Utilice engranajes microscópicos de enfoque grueso y fino para controlar la altura de la fuente láser para lograr la precisión adecuada.</li>
		<li>Apunte a la trayectoria del láser a lo largo del eje longitudinal de la corredera del electrodo. PRECAUCIÓN: Siempre use gafas de seguridad láser adecuadas y mantenga buenos protocolos de seguridad láser.</li>
		<li>Permita que el láser sintonizado seleccionado se estabilice antes de comenzar el experimento. NOTA: En la figura 2Ase ilustra un esquema de la configuración experimental.</li>
	</ol>
</li>
	<li>Alineación de electrodos láser y oro NOTA: Antes de comenzar, siempre evalúe la potencia de salida del láser utilizando un medidor de potencia para asegurarse de que solo se entreguen vatios muy bajos a los tBLM.<ol>
<li>Ajuste la trayectoria del láser o el ángulo del electrodo de modo que el láser pase a través del líquido que cubre el electrodo y sea visible, de manera uniforme, en la superficie de oro.</li>
		<li>Ajuste la posición de la luz del rayo láser para cada experimento elevando o bajando la fuente del rayo láser utilizando el ajuste fino mientras observa los cambios en la conductancia de la membrana.</li>
		<li>Bloquee la perilla para asegurar la posición de la trayectoria del láser cuando no se observen cambios de conductancia. NOTA: Se generarán valores de conductancia de membrana aumentados cuando el láser interactúe con el electrodo de oro subyacente. Por lo tanto, es importante ajustar la ruta del láser de tal manera que no sean posibles tales interacciones.</li>
	</ol>
</li>
	<li>Preparación de muestras<ol>
<li>Prepare la alineación de la luz del rayo láser (donde no haya cambios en la conductancia de la membrana), como se muestra en la Figura 2,posición 3.</li>
		<li>Agregue GNP de interés (funcionalizados o desnudos) al búfer PBS en el que se sumergen los tBLM mientras el láser está apagado.</li>
		<li>Mezcle el tampón PBS que rodea los tBLM suavemente tres veces, teniendo cuidado de no tocar el electrodo.</li>
		<li>Incubar durante 5-10 min a temperatura ambiente.</li>
		<li>Encienda el láser para irradiar la muestra, utilizando la posición correcta de la luz de rayo láser alineada como se ve en la Figura 2,posición 3.</li>
		<li>Utilice la combinación adecuada de tamaño, forma y concentración de GNP con la longitud de onda de la luz láser. NOTA: El rayo láser de longitud de onda establecida debe acoplarse a la frecuencia de resonancia de plasmón GNP correspondiente.</li>
		<li>Registre la corriente medida continuamente (mediciones en tiempo real).</li>
		<li>Realice los pasos 2.2.1 a 2.3.7, omitiendo la adición de PNB para los experimentos de control.</li>
	</ol>
</li>
</ol>3. Análisis y presentación de datos estadísticos
<ol><li>Exporta los datos a una hoja de cálculo.</li>
	<li>Extraiga el parámetro de conductancia de la membrana frente al tiempo.</li>
	<li>Utilice los datos grabados después de configurar una luz de rayo láser con la posición correcta y antes de la introducción de GNP.</li>
	<li>Normalice los datos dividiendo la conductancia de membrana medida sobre la conductancia de membrana basal. NOTA: Esto confirma que los cambios relativos en los valores de conducción de la membrana provocados por los GNP irradiados introducidos.</li>
	<li>Presentar los datos como gráficos de tiempo (eje x) versus conducción de membrana normalizada (eje y).</li>
</ol>4. Predecir la cantidad de calor localizado generado en los tBLMs a partir de nanopartículas irradiadas (modelo predictivo térmico)
<ol><li>Resolver el problema de la transferencia de radiación de acuerdo con Dombrovsky23, con el fin de calcular la potencia de radiación absorbida en soluciones de nanopartículas irradiadas.</li>
	<li>Calcule la generación de calor incorporando la fuente de calor debida a la radiación absorbida en la ecuación de energía. NOTA: Para una explicación detallada del análisis numérico de la generación de calor en los tBLM a partir de nanopartículas irradiadas y la interfaz nanomaterial-electrodo, consulte 19.</li>
</ol>

<ol>
	<li>Her, S., Jaffray, D. A., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+for+applications+in+cancer+radiotherapy:+Mechanisms+and+recent+advancements.">Gold nanoparticles for applications in cancer radiotherapy: Mechanisms and recent advancements.</a> Advanced Drug Delivery Reviews. 109, 84-101 (2017).</li><li>Pissuwan, D., Valenzuela, S. M., Killingsworth, M. C., Xu, X., Cortie, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+destruction+of+murine+macrophage+cells+with+bioconjugated+gold+nanorods.">Targeted destruction of murine macrophage cells with bioconjugated gold nanorods.</a> Journal of Nanoparticle Research. 9 (6), 1109-1124 (2007).</li><li>Pissuwan, D., Valenzuela, S. M., Miller, C. M., Cortie, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+golden+bullet?+Selective+targeting+of+Toxoplasma+gondii+tachyzoites+using+antibody-functionalized+gold+nanorods.">A golden bullet? Selective targeting of Toxoplasma gondii tachyzoites using antibody-functionalized gold nanorods.</a> Nano Letters. 7 (12), 3808-3812 (2007).</li><li>Zhang, H. -. G., Mehta, K., Cohen, P., Guha, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermia+on+immune+regulation:+a+temperature's+story.">Hyperthermia on immune regulation: a temperature's story.</a> Cancer Letters. 271 (2), 191-204 (2008).</li><li>Gobin, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+resonant+nanoshells+for+combined+optical+imaging+and+photothermal+cancer+therapy.">Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy.</a> Nano Letters. 7 (7), 1929-1934 (2007).</li><li>Jackson, J. B., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering+on+tunable+plasmonic+nanoparticle+substrates.">Surface-enhanced Raman scattering on tunable plasmonic nanoparticle substrates.</a> Proceedings of the National Academy of Sciences. 101 (52), 17930-17935 (2004).</li><li>Emelianov, S. Y., Li, P. -. C., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustics+for+molecular+imaging+and+therapy.">Photoacoustics for molecular imaging and therapy.</a> Physics Today. 62 (8), 34 (2009).</li><li>Dimitriou, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles,+radiations+and+the+immune+system:+Current+insights+into+the+physical+mechanisms+and+the+biological+interactions+of+this+new+alliance+towards+cancer+therapy.">Gold nanoparticles, radiations and the immune system: Current insights into the physical mechanisms and the biological interactions of this new alliance towards cancer therapy.</a> Pharmacology &amp; Therapeutics. 178, 1-17 (2017).</li><li>Mueller, P., Rudin, D. O., Tien, H. T., Wescott, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cell+membrane+structure+in+vitro+and+its+transformation+into+an+excitable+system.">Reconstitution of cell membrane structure in vitro and its transformation into an excitable system.</a> Nature. 194 (4832), 979 (1962).</li><li>Tamm, L. K., McConnell, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+phospholipid+bilayers.">Supported phospholipid bilayers.</a> Biophysical Journal. 47 (1), 105-113 (1985).</li><li>Plant, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+hybrid+bilayer+membranes+as+rugged+cell+membrane+mimics.">Supported hybrid bilayer membranes as rugged cell membrane mimics.</a> Langmuir. 15 (15), 5128-5135 (1999).</li><li>Sackmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+membranes:+scientific+and+practical+applications.">Supported membranes: scientific and practical applications.</a> Science. 271 (5245), 43-48 (1996).</li><li>Alghalayini, A., Garcia, A., Berry, T., Cranfield, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Tethered+Bilayer+Lipid+Membranes+to+Identify+the+Mechanisms+of+Antimicrobial+Peptide+Interactions+with+Lipid+Bilayers.">The Use of Tethered Bilayer Lipid Membranes to Identify the Mechanisms of Antimicrobial Peptide Interactions with Lipid Bilayers.</a> Antibiotics. 8 (1), 12 (2019).</li><li>Khan, M. S., Dosoky, N. S., Williams, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+lipid+bilayer+membranes+for+protein+studies.">Engineering lipid bilayer membranes for protein studies.</a> International Journal of Molecular Sciences. 14 (11), 21561-21597 (2013).</li><li>Urban, P., Kirchner, S. R., M&#252;hlbauer, C., Lohm&#252;ller, T., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+control+of+current+across+lipid+membranes+by+local+heating.">Reversible control of current across lipid membranes by local heating.</a> Scientific Reports. 6, 22686 (2016).</li><li>Palankar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoplasmonically-induced+defects+in+lipid+membrane+monitored+by+ion+current:+transient+nanopores+versus+membrane+rupture.">Nanoplasmonically-induced defects in lipid membrane monitored by ion current: transient nanopores versus membrane rupture.</a> Nano Letters. 14 (8), 4273-4279 (2014).</li><li>Bendix, P. M., Reihani, S. N. S., Oddershede, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurements+of+heating+by+electromagnetically+trapped+gold+nanoparticles+on+supported+lipid+bilayers.">Direct measurements of heating by electromagnetically trapped gold nanoparticles on supported lipid bilayers.</a> ACS Nano. 4 (4), 2256-2262 (2010).</li><li>Plaksin, M., Shapira, E., Kimmel, E., Shoham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+transients+excite+neurons+through+universal+intramembrane+mechanoelectrical+effects.">Thermal transients excite neurons through universal intramembrane mechanoelectrical effects.</a> Physical Review X. 8 (1), 011043 (2018).</li><li>Alghalayini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+heat+transfer+between+gold+nanoparticles+and+tethered+bilayer+lipid+membranes.">Real-time monitoring of heat transfer between gold nanoparticles and tethered bilayer lipid membranes.</a> Biochimica et Biophysica Acta (BBA)-Biomembranes. , 183334 (2020).</li><li>Moradi-Monfared, S., Krishnamurthy, V., Cornell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+machine+biosensor:+construction,+predictive+models+and+experimental+studies.">A molecular machine biosensor: construction, predictive models and experimental studies.</a> Biosensors and Bioelectronics. 34 (1), 261-266 (2012).</li><li>Hoiles, W., Gupta, R., Cornell, B., Cranfield, C., Krishnamurthy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+tethers+on+artificial+cell+membranes:+A+coarse-grained+molecular+dynamics+study.">The effect of tethers on artificial cell membranes: A coarse-grained molecular dynamics study.</a> PloS One. 11 (10), 0162790 (2016).</li><li>Cranfield, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+potential+gradients+and+impedance+measures+of+tethered+bilayer+lipid+membranes:+pore-forming+peptide+insertion+and+the+effect+of+electroporation.">Transient potential gradients and impedance measures of tethered bilayer lipid membranes: pore-forming peptide insertion and the effect of electroporation.</a> Biophysical Journal. 106 (1), 182-189 (2014).</li><li>Dombrovsky, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Radiation heat transfer in disperse systems. , (1996).</li><li>Cornell, B. A., Braach-Maksvytis, V., King, L., Osman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biosensor+that+uses+ion-channel+switches.">A biosensor that uses ion-channel switches.</a> Nature. 387 (6633), 580 (1997).</li><li>Maccarini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructural+determination+of+a+lipid+bilayer+tethered+to+a+gold+substrate.">Nanostructural determination of a lipid bilayer tethered to a gold substrate.</a> The European Physical Journal E. 39 (12), 123 (2016).</li><li>Beugin-Deroo, S., Ollivon, M., Lesieur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayer+stability+and+impermeability+of+nonionic+surfactant+vesicles+sterically+stabilized+by+PEG-cholesterol+conjugates.">Bilayer stability and impermeability of nonionic surfactant vesicles sterically stabilized by PEG-cholesterol conjugates.</a> Journal of Colloid and Interface Science. 202 (2), 324-333 (1998).</li><li>Kendall, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+Structure+of+Cholesterol-Based+Tethered+Bilayer+Lipid+Membranes+on+Ionophore+Activity.">Effect of the Structure of Cholesterol-Based Tethered Bilayer Lipid Membranes on Ionophore Activity.</a> ChemPhysChem. 11 (10), 2191-2198 (2010).</li><li>Jiang, W., Kim, B. Y., Rutka, J. T., Chan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+cellular+response+is+size-dependent.">Nanoparticle-mediated cellular response is size-dependent.</a> Nature Nanotechnology. 3 (3), 145 (2008).</li><li>He, C., Hu, Y., Yin, L., Tang, C., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+particle+size+and+surface+charge+on+cellular+uptake+and+biodistribution+of+polymeric+nanoparticles.">Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.</a> Biomaterials. 31 (13), 3657-3666 (2010).</li><li>Wang, F. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+and+bulk+contributions+to+the+second-order+nonlinear+optical+response+of+a+gold+film.">Surface and bulk contributions to the second-order nonlinear optical response of a gold film.</a> Physical Review B. 80 (23), 233402 (2009).</li></ol>

Los autores declaran los siguientes intereses financieros / relaciones personales, que pueden considerarse como posibles intereses en competencia: El profesor Bruce Cornell es Director de Ciencia y Tecnología en Surgical Diagnostics SDx membranas atadas Pty. Ltd.

Este protocolo describe el uso del modelo tBLM con un sustrato de electrodo coplanar junto con una configuración de alineación láser horizontal que permite el registro de impedancia eléctrica en tiempo real en respuesta a la irradiación láser de nanopartículas de oro. El método de registro EIS presentado aquí construye una lista mínima de experimentos necesarios para proporcionar el registro de los cambios de corriente iónica a través de la membrana, que corresponde al calor g...

El rendimiento hipertérmico de los nanomateriales de oro irradiados ofrece una nueva clase de tratamiento mínimamente invasivo, selectivo y dirigido para infecciones y tumores1. El empleo de nanopartículas que pueden ser calentadas por un láser se ha utilizado para destruir selectivamente las células enfermas, así como para proporcionar un medio para la administración selectiva de fármacos2,3. Una consecuencia del fenómeno de fototermólisis de las nanopartículas plasmónicas calentadas es el daño a las membranas celulares. La membrana bicapa lipídica fluida se considera un sitio particularmente vulnerable para las células que se someten a tales tratamientos porque la desnaturalización de las proteínas intrínsecas de la membrana, así como el daño de la membrana también pueden conducir a la muerte celular4,ya que muchas proteínas están allí para mantener el gradiente de potencial iónico a través de las membranas celulares. Si bien la capacidad de determinar y monitorear la transferencia de calor a nanoescala es de interés clave para el estudio y la aplicación de los GNP irradiados1,5,6,7, la evaluación y comprensión de las interacciones moleculares entre los GNP y las biomembranas, así como las consecuencias directas de los fenómenos de calentamiento inducidos por láser de los GNP incrustados en los tejidos biológicos, aún no se han dilucidado completamente8. Por lo tanto, una comprensión profunda del proceso de hipertermia de los GNP irradiados sigue siendo un desafío. Como tal, el desarrollo de una interfaz nanomaterial-electrodo que imite el entorno natural de las células podría proporcionar un medio para llevar a cabo una investigación en profundidad de las características de transferencia de calor de las nanopartículas de oro irradiadas dentro de los sistemas biológicos.
La complejidad de las membranas celulares nativas es uno de los desafíos significativos en la comprensión de las interacciones de los GNP irradiados en las células. Se han desarrollado varias plataformas de membrana artificial para proporcionar versiones bio-miméticas simples cercanas de la arquitectura y funcionalidad de la membrana lipídica natural, incluidas, entre otras, membranas lipídicas negras9,membranas bicapa planas compatibles10,membranas bicapa híbridas11,membranas bicapa lipídicas amortiguadas con polímeros12 y membranas lipídicas bicapa atadas13. Cada modelo de membrana lipídica artificial tiene claras ventajas y limitaciones con respecto a imitar las membranas lipídicas naturales14.
Este estudio describe el empleo de electrodos recubiertos de membrana lipídica como sensor para evaluar las interacciones de nanopartículas de oro y membrana lipídica, utilizando el modelo tBLM. El esquema de detección de biosensores basado en tBLM proporciona estabilidad y sensibilidad inherentes13 ya que las membranas atadas pueden autorrepararse, a diferencia de otros sistemas (como las membranas formadas por patch-clamp o liposomas) en los que solo una pequeña cantidad de daño a la membrana resulta en su colapso15,16,17,18. Además, debido a que los tBLM son de dimensiones mm2, la impedancia de fondo es órdenes de magnitud más baja que las técnicas de registro de pinza de parche, lo que permite un registro de los cambios en el flujo iónico de la membrana basal debido a las interacciones de nanopartículas. Como resultado de esto, el presente protocolo puede contrastar los cambios en la conductancia de la membrana por GNP unidos que son excitados por láseres cuyas potencias son tan bajas como 135 nW / μm2.
El sistema presentado aquí proporciona un método sensible y reproducible para determinar parámetros láser precisos, tamaño de partícula, recubrimientos de partículas y composición necesarios para diseñar y desarrollar terapias térmicas. Esto es fundamental para el refinamiento de las terapias fototérmicas emergentes, así como para ofrecer información valiosa para mecanismos detallados de transferencia de calor dentro de los sistemas biológicos. El protocolo presentado se basa en el trabajo publicado anteriormente19. Un esquema del protocolo es el siguiente: la primera sección define la formación de tBLM; la segunda sección describe cómo construir la configuración y alinear la fuente láser de excitación; la sección final ilustra cómo extraer información de los datos de espectroscopia de impedancia eléctrica.

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	<tbody>
		<tr>
			<td>30 nm diameter streptavidin-conjugated gold nanoparticles</td>
			<td>Cytodiagnostics</td>
			<td>AC-30-04-05</td>
			<td>This is a streptavidin-conjugated GNPs product ready for use</td>
		</tr>
		<tr>
			<td>30 nm diameter bare gold nanoparticles</td>
			<td>Sigma-Aldrich</td>
			<td>753629</td>
			<td>This is a bare GNPs product ready for use</td>
		</tr>
		<tr>
			<td>Cholesterol-PEG-Biotin (MW1000)</td>
			<td>NANOCS</td>
			<td>PG2-BNCS-10k</td>
			<td>Dissolved in highly pure ethanol</td>
		</tr>
		<tr>
			<td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S1</td>
			<td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Spacer molecules</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&#160;</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Tethered molecules</td>
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			<td>532 nm green laser continuous light</td>
			<td>OBIS LS/OBIS CORE LS, China</td>
			<td>ND-1000</td>
			<td>The power of this laser was ~135&#160;mW&#160;</td>
		</tr>
		<tr>
			<td>tethaPod EIS reader</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-R1</td>
			<td>A reader of conductance and capacitance on six channels simultaneously</td>
		</tr>
		<tr>
			<td>tethaPlate cartridge assembly</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-BG</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Clamp and slide assembly jig</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-A1</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Lipid coated coplanar gold electrodes</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-T10</td>
			<td>Coplanar&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&#160;</td>
		</tr>
		<tr>
			<td>tethaQuick software</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-B1</td>
			<td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td>
		</tr>
		<tr>
			<td>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 99.9%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>pH 7</td>
		</tr>
	</tbody>
</table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

Aquí informamos de un protocolo para investigar la transferencia de calor entre las nanopartículas de oro irradiadas (GNPs) y las membranas lipídicas bicapa por electroquímica utilizando membranas lipídicas bicapa atadas (tBLMs) ensambladas en electrodos de oro. Los GNP modificados irradiados, como los GNP conjugados con estreptavidina, están incrustados en tBLM que contienen moléculas diana, como la biotina. Mediante el uso de este enfoque, los procesos de transferencia de calor entre los GNP irradiados y la membrana lipídica bicapa modelo con entidades de interés están mediados por un rayo láser enfocado horizontalmente. El modelo computacional predictivo térmico se utiliza para confirmar los cambios de conductancia inducidos electroquímicamente en los tBLM. Bajo las condiciones específicas utilizadas, la detección de pulsos de calor requirió la unión específica de las nanopartículas de oro a la superficie de la membrana, mientras que las nanopartículas de oro no unidas no lograron provocar una respuesta medible. Esta técnica sirve como un potente biosensor de detección que se puede utilizar directamente para el diseño y desarrollo de estrategias para terapias térmicas que permiten la optimización de los parámetros del láser, el tamaño de partícula, los recubrimientos de partículas y la composición.

El sustrato de oro sobre el que se pueden crear tBLM se muestra en la Figura 1. En la Figura 2se presenta un esquema de la configuración experimental.
Los electrodos de oro coplanar, como se muestra en la Figura 1A,están hechos de sustrato base de policarbonato de 25 mm x 75 mm x 1 mm con matrices de oro estampadas. Una capa adhesiva transparente define las seis cámaras de medición individuales. El el...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Este trabajo describe un protocolo para lograr un monitoreo dinámico y no invasivo de la transferencia de calor de las nanopartículas de oro irradiadas por láser a las tBLM. El sistema combina la espectroscopia de impedancia para la medición en tiempo real de los cambios de conductancia a través de los tBLM, con un rayo láser enfocado horizontalmente que impulsa la iluminación de nanopartículas de oro, para la producción de calor.

Membranas lipídicas bicapa atadas para monitorear la transferencia de calor entre las nanopartículas de oro y las membranas lipídicas

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

tethered-bilayer-lipid-membranes-to-monitor-heat-transfer-between

Research

JoVE Journal

Biology

2.6K vistas.  University of Technology Sydney. Este trabajo describe un protocolo para lograr un monitoreo dinámico y no invasivo de la transferencia de calor de las nanopartículas de oro irradiadas por láser a las tBLM. El sistema combina la espectroscopia de impedancia para la medición en tiempo real de los cambios de conductancia a través de los tBLM, con un rayo láser enfocado horizontalmente que impulsa la iluminación de nanopartículas de oro, para la producción de calor.

Video: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Método para el aislamiento de Francisella tularensis Membranas externas

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Investigación

Biología

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidina basado en ensayo de fluorescencia, para la determinación de moléculas pequeñas posibles para modificar las propiedades bicapa lipídica

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

Modelado de membranas biológicas con placas de circuitos y medición de señales eléctricas en los axones: Ejercicios de laboratorio para estudiantes

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

La reconstitución de un canal Kv en las membranas lipídicas de Estudios estructurales y funcionales

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

El uso de células Caco-2 para el Estudio de lípidos Transporte por el Intestino

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Aislamiento y composicional Análisis de Planta cutícula lípidos poliéster Monómeros

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Cuantificación de lípidos abundancia y evaluación de la distribución de lípidos en Caenorhabditis elegans por Nilo rojo y aceite rojo O manchas

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Medición del metabolismo energético en el tejido retiniano Explanted utilizando el análisis de flujo extracelular

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Disección y tinción de gotas de lípidos de los ovenocitos en larvas de Drosophila

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...