fine-tuning-the-size-and-minimizing-the-noise-of-solid-state-nanopores

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

University of Ottawa

The surface stress induced during the formation of alkanethiol self-assembled monolayers (SAMs) on gold from the vapor phase was measured using a micromechanical cantilever-based chemical sensor. Simultaneous in situ thickness measurements were carried out using ellipsometry. Ex situ scanning tunneling microscopy was performed in air to ascertain the final monolayer structure. The evolution of the surface stress induced during coverage-dependent structural phase transitions reveals features not apparent in average ellipsometric thickness measurements. These results show that both the kinetics of SAM formation and the resulting SAM structure are strongly influenced both by the surface structure of the underlying gold substrate and by the impingement rate of the alkanethiol onto the gold surface. In particular, the adsorption onto gold surfaces having large, flat grains produces high-quality self-assembled monolayers. An induced compressive surface stress of 15.9 +/- 0.6 N/m results when a c(4x2) dodecanethiol SAM forms on gold. However, the SAMs formed on small-grained gold are incomplete and an induced surface stress of only 0.51 +/- 0.02 N/m results. The progression to a fully formed SAM whose alkyl chains adopt a vertical (standing-up) orientation is clearly inhibited in the case of a small-grained gold substrate and is promoted in the case of a large-grained gold substrate.

Surface stress, kinetics, and structure of alkanethiol self-assembled monolayers.

We measure the surface stress induced by electrochemical transformations of a thin conducting polymer film. One side of a micromechanical cantilever-based sensor is covered with an electropolymerized dodecyl benzenesulfonate-doped polypyrrole (PPyDBS) film. The microcantilever serves as both the working electrode (in a conventional three-electrode cell configuration) and as the mechanical transducer for simultaneous, in situ, and real-time measurements of the current and interfacial stress changes. A compressive change in surface stress of about -2 N/m is observed when the conducting polymer is electrochemically switched between its oxidized (PPy+) and neutral (PPy0) state by cyclic voltammetry. The surface stress sensor's response during the anomalous first reductive scan is examined. The effect of long-term cycling on the mechanical transformation ability of PPy(DBS) films in both surfactant and halide-based electrolytes is also discussed. We have identified two main competing origins of surface stress acting on the PPy(DBS)/ gold-coated microcantilever: one purely mechanical due to the volume change of the conducting polymer, and a second charge-induced, owing to the interaction of anions of the supporting electrolyte with the gold surface.

Redox-induced surface stress of polypyrrole-based actuators.

A working model has been developed which can be used to significantly increase the accuracy of cantilever deflection measurements using optical beam techniques (used in cantilever-based sensors and atomic force microscopes), while simultaneously simplifying their use. By using elementary geometric optics and standard vector analysis it is possible, without any fitted or adjustable parameters, to completely and accurately describe the relationship between the cantilever deflection and the signal measured by a position sensitive photo-detector. By arranging the geometry of the cantilever/optical beam, it is possible to tailor the detection system to make it more sensitive at different stages of the cantilever deflection or to simply linearize the relationship between the cantilever deflection and the measured detector signal. Supporting material and software has been made available for download at http://www.physics.mun.ca/beauliu_lab/papers/cantilever_analysis.htm so that the reader may take full advantage of the model presented herein with minimal effort.

A complete analysis of the laser beam deflection systems used in cantilever-based systems.

The surface stress response of micromechanical cantilever-based sensors was studied as a function of the morphology, adhesion, and cleanliness of the gold sensing surface. Two model systems were investigated: the adsorption of alkanethiol self-assembled monolayers at the gas-solid interface and the potential-controlled adsorption of anions at the liquid-solid interface. The potential-induced surface stress, on a smooth and continuous polycrystalline Au(111)-textured microcantilever in 0.1 M HClO4, is in excellent agreement with macroscopic Au(111) single-crystal electrode results. It is shown that ambient contaminants on the sensing surface dramatically alter the surface stress-potential response. This observation can be misinterpreted as evidence that for polycrystalline Au(111) microcantilever electrodes, surface stress is dominated by surface energy change. Results for anions adsorption on gold are in contrast to the gas-phase model system. We demonstrate that the average grain size of the gold sensing surface strongly influences the magnitude of the surface stress change induced by the adsorption of octanethiol. A 25-fold amplification of the change in surface stress is observed on increasing the average gold grain size of the sensing surface from 90 to 500 nm.

Microcantilever-based sensors: effect of morphology, adhesion, and cleanliness of the sensing surface on surface stress.

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

Nonexponential kinetics of DNA escape from alpha-hemolysin nanopores.

&#x57FA;&#x4E8E;&#x7EB3;&#x7C73;&#x5B54;&#x7684;&#x8BBE;&#x5907;&#x63D0;&#x4F9B;&#x4E86;&#x5355;&#x5206;&#x5B50;&#x68C0;&#x6D4B;&#x548C;&#x5206;&#x6790;&#x7684;&#x80FD;&#x529B;&#xFF0C;&#x901A;&#x8FC7;&#x7535;&#x6CF3;&#x9A7E;&#x9A76;&#x901A;&#x8FC7;&#x7EB3;&#x7C73;&#x7EA7;&#x5B54;&#x9699;&#x6EB6;&#x6DB2;&#x4E2D;&#x7684;&#x5206;&#x5B50;&#x3002;&#x7EB3;&#x7C73;&#x5B54;&#x63D0;&#x4F9B;&#x9AD8;&#x5EA6;&#x5BC6;&#x95ED;&#x7684;&#x7A7A;&#x95F4;&#x5185;&#x7684;&#x5355;&#x4E00;&#x6838;&#x9178;&#x805A;&#x5408;&#x7269;&#x53EF;&#x4EE5;&#x5206;&#x6790;&#x5728;&#x9AD8;&#x541E;&#x5410;&#x91CF;&#x7531;&#x5404;&#x79CD;&#x5404;&#x6837;&#x7684;&#x624B;&#x6BB5;&#x4E4B;&#x4E00;&#x548C;&#x5B8C;&#x7F8E;&#x7684; processivity &#x53EF;&#x4EE5;&#x5728;&#x7A84;&#x5B54;&#x5F3A;&#x5236;&#x6267;&#x884C;&#xFF0C;&#x786E;&#x4FDD;&#x672C;&#x673A;&#x7684;&#x591A;&#x805A;&#x6838;&#x82F7;&#x9178;&#x4E2D;&#x78B1;&#x57FA;&#x987A;&#x5E8F;&#x53CD;&#x6620;&#x5728;&#x68C0;&#x6D4B;&#x5230;&#x7684;&#x4FE1;&#x53F7;&#x7684;&#x987A;&#x5E8F;&#x3002;Kilobase &#x957F;&#x5EA6;&#x805A;&#x5408;&#x7269; (&#x5355;&#x94FE;&#x57FA;&#x56E0;&#x7EC4; DNA &#x6216; RNA) &#x6216;&#x5C0F;&#x5206;&#x5B50; &#xFF08;&#x4F8B;&#x5982;&#xFF0C;&#x6838;&#x82F7;&#xFF09; &#x53EF;&#x53D1;&#x73B0;&#x5E76;&#x6CA1;&#x6709;&#x653E;&#x5927;&#x6216;&#x8D34;&#x6807;&#x3001; &#x72EC;&#x7279;&#x7684;&#x5206;&#x6790;&#x80FD;&#x529B;&#xFF0C;&#x4F7F;&#x5F97;&#x5EC9;&#x4EF7;&#x3001; &#x5FEB;&#x901F;&#x7684; DNA &#x6D4B;&#x5E8F;&#x7684;&#x53EF;&#x80FD;&#x6027;&#x7684;&#x7279;&#x70B9;&#x3002;&#x8FDB;&#x4E00;&#x6B65;&#x7684;&#x7814;&#x7A76;&#x548C;&#x53D1;&#x5C55;&#xFF0C;&#x4EE5;&#x514B;&#x670D;&#x5F53;&#x524D;&#x9762;&#x4E34;&#x7684;&#x6311;&#x6218;&#x5728; DNA &#x94FE;&#x4E2D;&#x6BCF;&#x4E2A;&#x8FDE;&#x7EED;&#x7684;&#x6838;&#x82F7;&#x9178;&#x7EB3;&#x7C73;&#x5B54;&#x9274;&#x5B9A;&#x63D0;&#x4F9B;&#x4E86;&#x7B2C;&#x4E09;&#x4EE3;&#x7684;&#x524D;&#x666F;&#x5C06;&#x5E8F;&#x5217;&#x4E8C;&#x500D;&#x4F53;&#x7684;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x57FA;&#x56E0;&#x7EC4;&#x4E2D;&#x7EA6; 24 h &#x5927;&#x7EA6; 1000 &#x7F8E;&#x5143;&#x7684;&#x6587;&#x4E66;&#x3002;

&#x6F5C;&#x529B;&#x548C;&#x6311;&#x6218;&#x7684;&#x7EB3;&#x7C73;&#x5B54;&#x6D4B;&#x5E8F;&#x3002;

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.

The potential and challenges of nanopore sequencing.

Un dispositif bas&#xE9; sur nanopore fournit une seule mol&#xE9;cule de d&#xE9;tection et de capacit&#xE9;s d&#x26;#39;analyse qui sont obtenus par &#xE9;lectrophor&#xE8;se de conduire mol&#xE9;cules en solution &#xE0; travers un pore &#xE0; l&#x26;#39;&#xE9;chelle nanom&#xE9;trique. Le nanopore offre un espace tr&#xE8;s confin&#xE9; &#xE0; l&#x26;#39;int&#xE9;rieur qui seul polym&#xE8;res d&#x26;#39;acides nucl&#xE9;iques peuvent &#xEA;tre analys&#xE9;es &#xE0; haut d&#xE9;bit par un d&#x26;#39;une vari&#xE9;t&#xE9; de moyens, et la processivit&#xE9; parfait qui peut &#xEA;tre ex&#xE9;cut&#xE9;e dans un pore &#xE9;troit veille &#xE0; ce que l&#x26;#39;ordre natif des nucl&#xE9;obases dans un polynucl&#xE9;otide est refl&#xE9;t&#xE9; dans la s&#xE9;quence de signaux qui est d&#xE9;tect&#xE9;e. Polym&#xE8;res de longueur kilobases (ADN g&#xE9;nomique simple brin ou d&#x26;#39;ARN) ou de petites mol&#xE9;cules (par exemple, nucl&#xE9;osides) peuvent &#xEA;tre identifi&#xE9;s et caract&#xE9;ris&#xE9;s sans amplification ou l&#x26;#39;&#xE9;tiquetage, une capacit&#xE9; unique d&#x26;#39;analyse qui fait bon march&#xE9;, rapide de l&#x26;#39;ADN de s&#xE9;quen&#xE7;age une possibilit&#xE9;. Des recherches plus pouss&#xE9;es et de d&#xE9;veloppement pour relever les d&#xE9;fis actuels &#xE0; nanopore identification de chaque nucl&#xE9;otide successives dans un brin d&#x26;#39;ADN offre la perspective d&#x26;#39;instruments &#x26;quot;troisi&#xE8;me g&#xE9;n&#xE9;ration&#x26;quot; qui sera s&#xE9;quencer un g&#xE9;nome diplo&#xEF;de mammif&#xE8;re d&#x26;#39;environ $ 1.000 dans environ 24 h.

Le potentiel et les d&#xE9;fis de la Nanopore S&#xE9;quen&#xE7;age

Ein Nanopore-Ger&auml;t bietet Einzelmolek&uuml;l-Erkennung und analytischen F&auml;higkeiten, die erreicht werden, indem man elektrophoretisch Molek&uuml;le in L&ouml;sung durch eine Pore Nanobereich. Die Nanopore bietet sehr engstem Raum innerhalb dessen einzelne Nukleins&auml;ure-Polymere k&ouml;nnen bei hohem Durchsatz durch eine Mischung der Mittel analysiert werden und die perfekte Processivity, die in einem engen Poren erzwungen werden kann sorgt daf&uuml;r, dass die native die Nukleinbasen in einem Polynukleotid-Orden in der Reihenfolge der Signale reflektiert wird, das erkannt wurde. Kilobase L&auml;nge Polymere (einzelstr&auml;ngige genomische DNA oder RNA) oder kleine Molek&uuml;le (z.B. Nucleoside) k&ouml;nnen identifiziert und ohne Verst&auml;rkung oder Etikettierung, eine einzigartige analytische F&auml;higkeit, die preiswerte, schnelle DNA Sequenzierung M&ouml;glichkeit macht charakterisiert werden. Weitere Forschung und Entwicklung zur &Uuml;berwindung der gegenw&auml;rtigen Herausforderungen Nanopore Identifizierung jedes aufeinanderfolgende Nukleotids in einem DNA-Strang bietet die Aussicht auf der &quot;dritten Generation&quot; Instrumente, die ein diploide S&auml;ugetier-Genom-f&uuml;r ca. $1.000 in ca. 24 Stunden Sequenz.

Das Potenzial und die Herausforderungen der Nanopore-Sequenzierung.

Un dispositivo basato su nanopore fornisce rilevazione singola molecola e capacit&agrave; analitiche che vengono raggiunti dalla guida elettroforeticamente molecole in soluzione attraverso un poro di nano-scala. Il nanopore fornisce uno spazio molto limitato entro cui polimeri dell&#39;acido nucleico singolo possono essere analizzati a high throughput da uno di una variet&agrave; di mezzi, e il processivity perfetto che pu&ograve; essere applicato in un poro stretto garantisce che l&#39;ordine nativo delle basi azotate in un polinucleotide si riflette nella sequenza dei segnali che &egrave; rilevata. Polimeri di lunghezza kilobase (single-stranded genomic del DNA o RNA) o piccole molecole (per esempio, nucleosidi) possono essere identificati e caratterizzati senza amplificazione o etichettatura, una singolare capacit&agrave; analitica che rende poco costoso, rapido DNA sequencing una possibilit&agrave;. Ulteriore ricerca e sviluppo per superare le attuali sfide a nanopore identificazione di ogni successiva nucleotidi in un filamento di DNA offre la prospettiva di &#39;terza generazione&#39; strumenti che saranno sequenziare un genoma diploide mammiferi per circa $1.000 in circa 24 h.

Le potenzialit&agrave; e le sfide di nanopore sequenziamento.

&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x306F;&#x3001;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x691C;&#x51FA;&#x3068;&#x96FB;&#x6C17;&#x6CF3;&#x52D5;&#x7684;&#x306B;&#x30CA;&#x30CE;&#x30B9;&#x30B1;&#x30FC;&#x30EB;&#x306E;&#x7D30;&#x5B54;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x6EB6;&#x6DB2;&#x4E2D;&#x306E;&#x5206;&#x5B50;&#x3092;&#x99C6;&#x52D5;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x9054;&#x6210;&#x3055;&#x308C;&#x305F;&#x5206;&#x6790;&#x6A5F;&#x80FD;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x306F;&#x3001;&#x5358;&#x4E00;&#x306E;&#x6838;&#x9178;&#x30DD;&#x30EA;&#x30DE;&#x30FC;&#x306F;&#x3001;&#x69D8;&#x3005;&#x306A;&#x624B;&#x6BB5;&#x306E;&#x4E00;&#x3064;&#x3067;&#x3001;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8;&#x3067;&#x89E3;&#x6790;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x305D;&#x306E;&#x4E2D;&#x306B;&#x975E;&#x5E38;&#x306B;&#x9650;&#x3089;&#x308C;&#x305F;&#x30B9;&#x30DA;&#x30FC;&#x30B9;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3001;&#x72ED;&#x3044;&#x7D30;&#x5B54;&#x306B;&#x9069;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x5B8C;&#x74A7;&#x306A;&#x80FD;&#x529B;&#xFF08;processivity&#xFF09;&#x306F;&#x3001;&#x30DD;&#x30EA;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x306E;&#x5869;&#x57FA;&#x306E;&#x305D;&#x306E;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x306E;&#x9806;&#x5E8F;&#x3092;&#x4FDD;&#x8A3C;&#x691C;&#x51FA;&#x3055;&#x308C;&#x305F;&#x4FE1;&#x53F7;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306B;&#x53CD;&#x6620;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30AD;&#x30ED;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x9577;&#x3055;&#x306E;&#x30DD;&#x30EA;&#x30DE;&#x30FC;&#xFF08;&#x4E00;&#x672C;&#x9396;&#x30B2;&#x30CE;&#x30E0;DNA&#x307E;&#x305F;&#x306F;RNA&#xFF09;&#x307E;&#x305F;&#x306F;&#x5C0F;&#x5206;&#x5B50;&#xFF08;&#x4F8B;&#x3048;&#x3070;&#x3001;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30B7;&#x30C9;&#xFF09;&#x304C;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x30B7;&#x30FC;&#x30AF;&#x30A8;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x3001;&#x5B89;&#x4FA1;&#x3001;&#x8FC5;&#x901F;&#x306A;DNA&#x3092;&#x4F5C;&#x308B;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x5206;&#x6790;&#x80FD;&#x529B;&#x3001;&#x5897;&#x5E45;&#x307E;&#x305F;&#x306F;&#x6A19;&#x8B58;&#x305B;&#x305A;&#x306B;&#x540C;&#x5B9A;&#x3057;&#x3001;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3002; DNA&#x9396;&#x306E;&#x305D;&#x308C;&#x305E;&#x308C;&#x306E;&#x9023;&#x7D9A;&#x3057;&#x305F;&#x200B;&#x200B;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x306E;&#x540C;&#x5B9A;&#x3092;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x306B;&#x73FE;&#x5728;&#x306E;&#x8AB2;&#x984C;&#x3092;&#x514B;&#x670D;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x3055;&#x3089;&#x306A;&#x308B;&#x7814;&#x7A76;&#x3068;&#x958B;&#x767A;&#x306F;&#x3001;&#x7D04;24&#x6642;&#x9593;&#x3067;&#x7D04;&#x5343;&#x30C9;&#x30EB;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x500D;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x3092;&#x52D9;&#x3081;&#x308B; &#x26;quot;&#x7B2C;&#x4E09;&#x4E16;&#x4EE3;&#x26;quot;&#x306E;&#x697D;&#x5668;&#x306E;&#x898B;&#x901A;&#x3057;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3068;&#x8AB2;&#x984C;

Nanopore &#xAE30;&#xBC18; &#xC7A5;&#xCE58; &#xB2E8;&#xC77C; &#xBD84;&#xC790; &#xAC80;&#xCD9C; &#xBC0F; electrophoretically &#xB098;&#xB178; &#xAE30; &#xACF5; &#xD1B5;&#xD574; &#xC194;&#xB8E8;&#xC158;&#xC5D0;&#xC11C; &#xBD84;&#xC790;&#xB97C; &#xC6B4;&#xC804; &#xD558; &#xC5EC; &#xB2EC;&#xC131; &#xD558;&#xB294; &#xBD84;&#xC11D; &#xAE30;&#xB2A5;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. Nanopore&#xB294; &#xB2E8;&#xC77C; &#xD575; &#xC0B0; &#xD3F4;&#xB9AC;&#xBA38;&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xC218;&#xB2E8;&#xC5D0; &#xC758;&#xD574; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9;&#xC5D0;&#xC11C; &#xBD84;&#xC11D; &#xD558; &#xACE0; &#xC881;&#xC740; &#xAE30; &#xACF5;&#xC5D0; &#xC801;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xC644;&#xBCBD; &#xD55C; processivity &#xBCF4;&#xC7A5; &#xAC10;&#xC9C0; &#xC2E0;&#xD638; &#xC21C;&#xC11C;&#xC5D0;&#xB294; polynucleotide&#xC5D0;&#xC11C; nucleobases&#xC758; &#xAE30;&#xBCF8; &#xC21C;&#xC11C; &#xBC18;&#xC601; &#xB429;&#xB2C8;&#xB2E4; &#xB9E4;&#xC6B0; &#xD55C;&#xC815; &#xB41C; &#xACF5;&#xAC04;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. Kilobase &#xAE38;&#xC774; &#xACE0;&#xBD84;&#xC790; (&#xB2E8;&#xC77C; &#xAC00;&#xB2E5; genomic DNA &#xB610;&#xB294; RNA) &#xB610;&#xB294; &#xC791;&#xC740; &#xBD84;&#xC790; (&#xC608;&#xB97C; &#xB4E4;&#xC5B4;, nucleosides)&#xC744; &#xC2DD;&#xBCC4; &#xD558; &#xACE0; &#xC99D;&#xD3ED; &#xB610;&#xB294; &#xB77C;&#xBCA8;, &#xC800;&#xB834;, &#xBE60;&#xB978; DNA &#xC2DC;&#xD000;&#xC2F1; &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xB9CC;&#xB4DC;&#xB294; &#xB3C5;&#xD2B9;&#xD55C; &#xBD84;&#xC11D; &#xAE30;&#xB2A5; &#xC5C6;&#xC774; &#xD2B9;&#xC9D5; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCD94;&#xAC00; &#xC5F0;&#xAD6C;&#xC640; &#xAC1C;&#xBC1C; DNA &#xAC00;&#xB2E5;&#xC5D0; &#xC788;&#xB294; &#xAC01; &#xC5F0;&#xC18D; &#xB41C; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC;&#xC758; nanopore &#xC2DD;&#xBCC4;&#xC5D0; &#xD604;&#xC7AC;&#xC758; &#xC5B4;&#xB824;&#xC6C0;&#xC744; &#xADF9;&#xBCF5; &#xD558;&#xAE30; &#39;&#xC81C; 3 &#xC138;&#xB300;&#39;&#xC758; &#xC804;&#xB9DD;&#xC774; &#xC81C;&#xACF5; &#xC57D; 24 &#xC2DC;&#xAC04;&#xC5D0; &#xC57D; $ 1000 diploid &#xD3EC;&#xC720;&#xB958; &#xAC8C;&#xB188;&#xC744; &#xC2DC;&#xD000;&#xC2F1; &#xD558;&#xB294; &#xC545;&#xAE30;.

&#xC7A0;&#xC7AC;&#xB825;&#xACFC; nanopore &#xC2DC;&#xD000;&#xC2F1;&#xC758; &#xB3C4;&#xC804;&#xC785;&#xB2C8;&#xB2E4;.

Un dispositivo basado en nanopore ofrece una sola mol&#xE9;cula de detecci&#xF3;n y capacidad de an&#xE1;lisis que se alcanzan por la electroforesis de conducir las mol&#xE9;culas en soluci&#xF3;n a trav&#xE9;s de un poro de nano-escala. El nanoporo proporciona un espacio muy limitado en el que un solo pol&#xED;meros de &#xE1;cidos nucleicos puede ser analizada con un alto rendimiento por una de una variedad de medios, y la procesividad perfecta que puede ser ejecutada en un poro estrecho asegura que el orden natural de las nucleobases en un polinucle&#xF3;tido se refleja en la secuencia de las se&#xF1;ales que se detecta. Pol&#xED;meros kilobases de longitud (de cadena simple de ADN gen&#xF3;mico o ARN) o mol&#xE9;culas peque&#xF1;as (por ejemplo, los nucle&#xF3;sidos) pueden ser identificados y caracterizados sin amplificaci&#xF3;n o etiquetado, una capacidad &#xFA;nica anal&#xED;tica que hace que el ADN de bajo costo, r&#xE1;pida secuenciaci&#xF3;n una posibilidad. Investigaci&#xF3;n y desarrollo para superar los retos actuales para la identificaci&#xF3;n de cada nanopore sucesiva de nucle&#xF3;tidos en una hebra de ADN ofrece la perspectiva de los instrumentos de tercera generaci&#xF3;n que se secuencia un genoma diploide de mam&#xED;feros por aproximadamente $ 1.000 en aproximadamente 24 horas.

El potencial y los desaf&#xED;os de la secuenciaci&#xF3;n Nanopore

Weak molecular interactions drive processes at the core of living systems, such as enzyme-substrate interactions, receptor-ligand binding, and nucleic acid replication. Single-molecule force spectroscopy is a remarkable tool for revealing molecular scale energy landscapes of noncovalent bonds, by exerting a mechanical force directly on an individual molecular complex and tracking its survival as a function of time and applied force. In principle, force spectroscopy methods can also be used for highly specific molecular recognition assays, by directly characterizing the strength of bonds between probe and target molecules. However, complexity and low throughput of conventional force spectroscopy techniques render such biosensing applications impractical. Here we demonstrate a straightforward single-molecule approach, suitable for both biophysical studies and molecular recognition assays, in which a approximately 3 nm silicon nitride nanopore is used to determine the bond lifetime spectrum of the biotin-neutravidin complex. Thousands of individual molecular complexes are captured and dissociated in the solid-state nanopore under constant applied forces, ranging from 400 to 900 mV, allowing us to extract the location of the energy barrier that governs the interaction, mapped at Deltax approximately 0.5 nm. These results highlight the capacity of a solid-state nanopore to detect and characterize intermolecular interactions and demonstrate how this could be applied to rapid, highly specific molecular detection assays.

Single-molecule bonds characterized by solid-state nanopore force spectroscopy.

Many interactions drive the adsorption of molecules on surfaces, all of which can result in a measurable change in surface stress. This article compares the contributions of various possible interactions to the overall induced surface stress for cantilever-based sensing applications. The surface stress resulting from adsorption-induced changes in the electronic density of the underlying surface is up to 2-4 orders of magnitude larger than that resulting from intermolecular electrostatic or Lennard-Jones interactions. We reveal that the surface stress associated with the formation of high quality alkanethiol self-assembled monolayers on gold surfaces is independent of the molecular chain length, supporting our theoretical findings. This provides a foundation for the development of new strategies for increasing the sensitivity of cantilever-based sensors for various applications.

Cantilever-based sensing: the origin of surface stress and optimization strategies.

A detailed understanding of the kinetics of DNA motion though nanometer-scale pores is important for the successful development of many of the proposed next-generation rapid DNA sequencing and analysis methods. Many of these approaches require DNA motion through nanopores to be slowed by several orders of magnitude from its native translocation velocity so that the translocation times for individual nucleotides fall within practical timescales for detection. With the increased dwell time of DNA in the pore, DNA-pore interactions begin to play an increasingly important role in translocation kinetics. In previous work, we and others observed that when the DNA dwell time in the pore is substantial (>1 ms), DNA motion in α-hemolysin (α-HL) pores leads to nonexponential kinetics in the escape of DNA out of the pore. Here we show that a three-state model for DNA escape, involving stochastic binding interactions of DNA with the pore, accurately reproduces the experimental data. In addition, we investigate the sequence dependence of the DNA escape process and show that the interaction strength of adenine with α-HL is substantially lower relative to cytosine. Our results indicate a difference in the process by which DNA moves through an α-HL nanopore when the motion is fast (microsecond timescale) as compared with when it is slow (millisecond timescale) and strongly influenced by DNA-pore interactions of the kind reported here. We also show the ability of wild-type α-HL to detect and distinguish between 5-methylcytosine and cytosine based on differences in the absolute ionic current through the pore in the presence of these two nucleotides. The results we present here regarding sequence-dependent (and dwell-time-dependent) DNA-pore interaction kinetics will have important implications for the design of methods for DNA analysis through reduced-velocity motion in nanopores.

Long dwell-time passage of DNA through nanometer-scale pores: kinetics and sequence dependence of motion.

We report the use of an array of electrically gated ~200 nm solid-state pores as nanofluidic transistors to manipulate the capture and passage of DNA. The devices are capable of reversibly altering the rate of DNA capture by over 3 orders of magnitude using sub-1 V biasing of a gate electrode. This efficient gating originates from the counter-balance of electrophoresis and electroosmosis, as revealed by quantitative numerical simulations. Such a reversible electronically tunable biomolecular switch may be used to manipulate nucleic acid delivery in a fluidic circuit, and its development is an important first step toward active control of DNA motion through solid-state nanopores for sensing applications.

Control of DNA capture by nanofluidic transistors.

We present a methodology for preparing silicon nitride nanopores that provides in situ control of size with sub-nanometer precision while simultaneously reducing electrical noise by up to three orders of magnitude through the cyclic application of high electric fields in an aqueous environment. Over 90% of nanopores treated with this technique display desirable noise characteristics and readily exhibit translocation of double-stranded DNA molecules. Furthermore, previously used nanopores with degraded electrical properties can be rejuvenated and used for further single-molecule experiments.

Article	Year
Surface stress, kinetics, and structure of alkanethiol self-assembled monolayers. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 15301492	2004
Redox-induced surface stress of polypyrrole-based actuators. The journal of physical chemistry. B\| PubMed ID: 16853242	2005
A complete analysis of the laser beam deflection systems used in cantilever-based systems. Ultramicroscopy\| PubMed ID: 17174033	2007
Microcantilever-based sensors: effect of morphology, adhesion, and cleanliness of the sensing surface on surface stress. Analytical chemistry\| PubMed ID: 17914755	2007
Nonexponential kinetics of DNA escape from alpha-hemolysin nanopores. Biophysical journal\| PubMed ID: 18775965	2008
The potential and challenges of nanopore sequencing. Nature biotechnology\| PubMed ID: 18846088	2008
Single-molecule bonds characterized by solid-state nanopore force spectroscopy. ACS nano\| PubMed ID: 19751064	2009
Cantilever-based sensing: the origin of surface stress and optimization strategies. Nanotechnology\| PubMed ID: 20081290	2010
Long dwell-time passage of DNA through nanometer-scale pores: kinetics and sequence dependence of motion. Biophysical journal\| PubMed ID: 21689531	2011
Control of DNA capture by nanofluidic transistors. ACS nano\| PubMed ID: 22762282	2012
Precise control of the size and noise of solid-state nanopores using high electric fields. Nanotechnology\| PubMed ID: 22983670	2012

Vincent Tabard-Cossa

Biography

JoVE Journal Publications

Other Publications