real-time-cytotoxicity-assays-in-human-whole-blood

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Adheren, Inc

DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells.

An integrated microdevice is developed for the analysis of gene expression in single cells. The system captures a single cell, transcribes and amplifies the mRNA, and quantitatively analyzes the products of interest. The key components of the microdevice include integrated nanoliter metering pumps, a 200-nL RT-PCR reactor with a single-cell capture pad, and an affinity capture matrix for the purification and concentration of products that is coupled to a microfabricated capillary electrophoresis separation channel for product analysis. Efficient microchip integration of these processes enables the sensitive and quantitative examination of gene expression variation at the single-cell level. This microdevice is used to measure siRNA knockdown of the GAPDH gene in individual Jurkat cells. Single-cell measurements suggests the presence of 2 distinct populations of cells with moderate (approximately 50%) or complete (approximately 0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.

Integrated microfluidic bioprocessor for single-cell gene expression analysis.

Previously, we reported a method for the attachment of living cells to surfaces through the hybridization of synthetic DNA strands attached to their plasma membrane. The oligonucleotides were introduced using metabolic carbohydrate engineering, which allowed reactive tailoring of the cell surface glycans for chemoselective bioconjugation. While this method is highly effective for cultured mammalian cells, we report here a significant improvement of this technique that allows the direct modification of cell surfaces with NHS-DNA conjugates. This method is rapid and efficient, allowing virtually any mammalian cell to be patterned on surfaces bearing complementary DNA in under 1 h. We demonstrate this technique using several types of cells that are generally incompatible with integrin-targeting approaches, including red blood cells and primary T-cells. Cardiac myoblasts were also captured. The immobilization procedure itself was found not to activate primary T-cells, in contrast to previously reported antibody- and lectin-based methods. Myoblast cells were patterned with high efficiency and remained undifferentiated after surface attachment. Upon changing to differentiation media, myotubes formed in the center of the patterned areas with an excellent degree of edge alignment. The availability of this new protocol greatly expands the applicability of the DNA-based attachment strategy for the generation of artificial tissues and the incorporation of living cells into device settings.

Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns.

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.

Nucleic acid aptamers offer significant potential as convenient and evolvable targeting groups for drug delivery. To attach them to the surface of a genome-free viral capsid carrier, an efficient oxidative coupling strategy has been developed. The method involves the periodate-mediated reaction of phenylene diamine substituted oligonucleotides with aniline groups installed on the outer surface of the capsid shells. Up to 60 DNA strands can be attached to each viral capsid with no apparent loss of base-pairing capabilities or protein stability. The ability of the capsids to bind specific cellular targets was demonstrated through the attachment of a 41-nucleotide sequence that targets a tyrosine kinase receptor on Jurkat T cells. After the installation of a fluorescent dye on the capsid interior, capsids bearing the cell-targeting sequence showed significant levels of binding to the cells relative to those of control samples. Colocalization experiments using confocal microscopy indicated that the capsids were endocytosed and trafficked to lysosomes for degradation. These observations suggest that aptamer-labeled capsids could be used for the targeted drug delivery of acid-labile prodrugs that would be preferentially released upon lysosomal acidification.

Viral capsid DNA aptamer conjugates as multivalent cell-targeting vehicles.

Genome-free viral capsids as multivalent carriers for taxol delivery.

Bacteriophage MS2 was used to construct a targeted, multivalent photodynamic therapy vehicle for the treatment of Jurkat leukemia T cells. The self-assembling spherical virus capsid was modified on the interior surface with up to 180 porphyrins capable of generating cytotoxic singlet oxygen upon illumination. The exterior of the capsid was modified with ∼20 copies of a Jurkat-specific aptamer using an oxidative coupling reaction targeting an unnatural amino acid. The capsids were able to target and selectively kill more than 76% of the Jurkat cells after only 20 min of illumination. Capsids modified with a control DNA strand did not target Jurkat cells, and capsids modified with the aptamer were found to be specific for Jurkat cells over U266 cells (a control B cell line). The doubly modified capsids were also able to kill Jurkat cells selectively even when mixed with erythrocytes, suggesting the possibility of using our system to target blood-borne cancers or other pathogens in the blood supply.

Dual-surface modified virus capsids for targeted delivery of photodynamic agents to cancer cells.

A new technique is reported for the attachment of synthetic DNA strands to the surfaces of microbial organisms. This gives algal, bacterial, and fungal cells the ability to bind to complementary strands extending from patterned surfaces that can be produced on platforms such as microfluidic devices. The ability of this method to establish complex 2- and 3-dimensional cocultures comprising multiple organism types is also presented.

Direct attachment of microbial organisms to material surfaces through sequence-specific DNA hybridization.

&#x4EE5;&#x524D;&#x7684;&#x62A5;&#x544A;&#x8868;&#x660E;&#x5408;&#x6210; DNA &#x94FE;&#x53EF;&#x4EE5;&#x9644;&#x52A0;&#x5230;&#x7EC6;&#x80DE;&#x819C;&#x7684;&#x6D3B;&#x7EC6;&#x80DE;&#xFF0C;&#x4F7F;&#x4ED6;&#x4EEC;&#x5177;&#x5907;&#x4EBA;&#x5DE5;&#x7C98;&#x9644;&quot;&#x53D7;&#x4F53;&quot;&#xFF0C;&#x5C06;&#x7ED1;&#x5B9A;&#x5230;&#x4ECE;&#x6750;&#x6599;&#x8868;&#x9762;&#x5EF6;&#x4F38;&#x7684;&#x4E92;&#x8865;&#x94FE;&#x3002;&#x8FD9;&#x79CD;&#x505A;&#x6CD5;&#x662F;&#x7B26;&#x5408;&#x5E7F;&#x6CDB;&#x7684;&#x5355;&#x5143;&#x683C;&#x7C7B;&#x578B;&#x3001; &#x63D0;&#x4F9B;&#x4F18;&#x79C0;&#x6355;&#x83B7;&#x6548;&#x7387;&#xFF0C;&#x5E76;&#x6709;&#x53EF;&#x80FD;&#x53EF;&#x4EE5;&#x7528;&#x6765;&#x521B;&#x5EFA;&#x590D;&#x6742;&#x7684;&#x591A;&#x7EC6;&#x80DE;&#x5B89;&#x6392;&#xFF0C;&#x901A;&#x8FC7;&#x4F7F;&#x7528;&#x591A;&#x4E2A;&#x6355;&#x83B7;&#x5E8F;&#x5217;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x5DE5;&#x4F5C;&#xFF0C;&#x6211;&#x4EEC;&#x5E94;&#x7528;&#x94DD;&quot;&#x6251;&#x817E;&quot;&#x5149;&#x523B;&#x6280;&#x672F;&#x65B9;&#x6CD5;&#x4EE5;&#x5141;&#x8BB8;&#x7EC4;&#x6210;&#x4E0D;&#x540C;&#x7684; DNA &#x5E8F;&#x5217;&#x7684;&#x590D;&#x6742;&#x56FE;&#x6848;&#x7684;&#x9AD8;&#x6548;&#x751F;&#x6210;&#x3002;&#x7531;&#x6B64;&#x4EA7;&#x751F;&#x7684;&#x66F2;&#x9762;&#x7136;&#x540E;&#x6F14;&#x793A;&#x4E86;&#x80FD;&#x591F;&#x6355;&#x83B7;&#x6700;&#x591A;&#x4E09;&#x4E2A;&#x4E0D;&#x540C;&#x7C7B;&#x578B;&#x7684;&#x6D3B;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x7279;&#x5B9A;&#x4F4D;&#x7F6E;&#x3002;&#x8FD9;&#x79CD;&#x65B9;&#x6CD5;&#x7684;&#x5B9E;&#x7528;&#x7A0B;&#x5E8F;&#x88AB;&#x8868;&#x660E;&#x901A;&#x8FC7;&#x89C2;&#x5BDF;&#x56FE;&#x6848;&#x5355;&#x5143;&#x683C;&#x4F5C;&#x4E3A;&#x4ED6;&#x4EEC;&#x901A;&#x8FC7;&#x57FA;&#x4E8E;&#x6269;&#x6563;&#x7684;&#x65C1;&#x5206;&#x6CCC;&#x4FE1;&#x53F7;&#x8FDB;&#x884C;&#x901A;&#x4FE1;&#x3002;&#x9884;&#x8BA1;&#x8FD9;&#x9879;&#x6280;&#x672F;&#x80FD;&#x591F;&#x521B;&#x5EFA;&#x51E0;&#x4E4E;&#x4EFB;&#x4F55;&#x7C7B;&#x578B;&#x7684; 2D &#x5F02;&#x6784;&#x5355;&#x5143;&#x683C;&#x56FE;&#x6848;&#x5E94;&#x8BC1;&#x660E;&#x975E;&#x5E38;&#x6709;&#x7528;&#x7684;&#x5173;&#x952E;&#x95EE;&#x9898;&#x5728;&#x7EC6;&#x80DE;&#x5185;&#x4FE1;&#x53F7;&#x4F20;&#x9012;&#xFF0C;&#x8003;&#x8BD5;&#x5305;&#x62EC;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x548C;&#x80BF;&#x7624;&#x8F6C;&#x79FB;&#x3002;

&#x8702;&#x7A9D;&#x5FAE;&#x7EC6;&#x52A0;&#x5DE5;&#xFF1A; &#x89C2;&#x6D4B;&#x4F7F;&#x7528; lithographically &#x5B9A;&#x4E49;&#x7684; DNA &#x7684;&#x7EC6;&#x80DE;&#x95F4;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x6355;&#x83B7;&#x5E8F;&#x5217;&#x3002;

Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion "receptors" that bind to complementary strands extending from material surfaces. This approach is compatible with a wide range of cell types, offers excellent capture efficiency, and can potentially be used to create complex multicellular arrangements through the use of multiple capture sequences. In this work, we apply an aluminum "lift off" lithography method to allow the efficient generation of complex patterns comprising different DNA sequences. The resulting surfaces are then demonstrated to be able to capture up to three distinct types of living cells in specific locations. The utility of this approach is demonstrated through the observation of patterned cells as they communicate by diffusion-based paracrine signaling. It is anticipated that the ability of this technique to create virtually any type of 2D heterogeneous cell pattern should prove highly useful for the examination of key questions in cell signaling, including stem cell differentiation and cancer metastasis.

Cellular microfabrication: observing intercellular interactions using lithographically-defined DNA capture sequences.

Les rapports pr&eacute;c&eacute;dents ont montr&eacute; que les brins d&#39;ADN synth&eacute;tiques peuvent &ecirc;tre attach&eacute;s &agrave; la membrane plasmique des cellules vivantes pour les &eacute;quiper d&#39;une adh&eacute;rence artificiel &laquo; r&eacute;cepteurs &raquo; qui se lient &agrave; des brins compl&eacute;mentaires qui s&#39;&eacute;tend de surfaces de mat&eacute;riaux. Cette approche est compatible avec un large &eacute;ventail de types de cellules, offres excellentes efficacit&eacute; de capture et peuvent potentiellement &ecirc;tre utilis&eacute;s pour cr&eacute;er des arrangements multicellulaires complexes gr&acirc;ce &agrave; l&#39;utilisation de plusieurs s&eacute;quences de capture. Dans ce travail, nous appliquons une m&eacute;thode de lithographie &laquo; d&eacute;coller &raquo; en aluminium pour permettre la g&eacute;n&eacute;ration efficace de mod&egrave;les complexes comprenant des s&eacute;quences d&#39;ADN diff&eacute;rentes. Les surfaces ainsi obtenues sont alors d&eacute;montr&eacute;s pour pouvoir capturer jusqu&#39;&agrave; trois types distincts de cellules vivantes dans des endroits sp&eacute;cifiques. L&#39;utilit&eacute; de cette approche est d&eacute;montr&eacute;e par l&#39;observation des cellules &agrave; motifs qu&#39;ils communiquent par la signalisation paracrine ax&eacute;e sur la diffusion. Il est pr&eacute;vu que la capacit&eacute; de cette technique de cr&eacute;er pratiquement n&#39;importe quel type de structure cellulaire h&eacute;t&eacute;rog&egrave;ne 2D devrait s&#39;av&eacute;rer tr&egrave;s utile pour l&#39;examen des questions cl&eacute;s dans la signalisation cellulaire, y compris la diff&eacute;renciation des cellules souches et des m&eacute;tastases canc&eacute;reuses.

Microfabrication cellulaire : observation des interactions intercellulaires en utilisant l&#39;ADN d&eacute;finie par lithographically capturent des s&eacute;quences.

Fr&uuml;here Berichten zufolge synthetischen DNA-Str&auml;nge angeschlossen werden k&ouml;nnen, um die Plasmamembran von lebenden Zellen, sie mit k&uuml;nstlichen Adh&auml;sion &quot;Rezeptoren&quot;, die komplement&auml;ren Str&auml;nge von Materialoberfl&auml;chen binden, auszustatten. Dieser Ansatz ist kompatibel mit einer Vielzahl von Zelltypen, bietet hervorragende Effizienz zu erfassen und k&ouml;nnen potenziell zum komplexen Vielzeller Arrangements durch den Einsatz von mehreren Aufnahme-Sequenzen erstellen. In dieser Arbeit wenden wir eine Aluminium &quot;abheben&quot; Lithografie Methode um die effiziente Generierung von komplexen Mustern, bestehend aus unterschiedliche DNA-Sequenzen zu erm&ouml;glichen. Die resultierenden Oberfl&auml;chen werden so gezeigt, um bis zu drei verschiedene Arten von lebenden Zellen an bestimmten Orten zu erfassen zu k&ouml;nnen. Das Dienstprogramm dieses Ansatzes wird durch die Beobachtung der gemusterten Zellen demonstriert, wie sie durch Diffusion basierenden parakrine Signale kommunizieren. Es wird erwartet, dass die M&ouml;glichkeit dieser Technik, praktisch jede Art von 2D heterogene Zellmuster erstellen sehr n&uuml;tzlich f&uuml;r die Pr&uuml;fung der Schl&uuml;sselfragen in Zelle signalisieren, beweisen sollte einschlie&szlig;lich Differenzierung von Stammzellen und Krebsmetastase.

Zellul&auml;re Microfabrication: beobachtende interzellul&auml;re Wechselwirkungen mit lithographically definiert DNA Sequenzen zu erfassen.

Rapporti precedenti hanno mostrato che i filamenti di DNA sintetici possono essere associati a membrana del plasma delle cellule viventi per dotarli di aderenza artificiale &quot;recettori&quot; che si legano ai filamenti complementari che si estende da superfici di materiali. Questo approccio &egrave; compatibile con una vasta gamma di tipi di cellule, offre un&#39;eccellente efficienza di cattura e possono essere potenzialmente utilizzati per creare arrangiamenti pluricellulari complesse attraverso l&#39;uso di pi&ugrave; sequenze di acquisizione. In questo lavoro, si applica un metodo litografia &quot;lift off&quot; in alluminio per consentire la generazione efficiente di modelli complessi comprendenti diverse sequenze di DNA. Le superfici risultanti sono poi dimostrate di essere in grado di catturare fino a tre tipi distinti di cellule viventi in posizioni specifiche. L&#39;utilit&agrave; di questo approccio &egrave; dimostrato attraverso l&#39;osservazione delle cellule fantasia come comunicano di segnalazione paracrina basata sulla diffusione. Si prevede che la capacit&agrave; di questa tecnica di creare praticamente qualsiasi tipo di pattern celle eterogenee 2D dovrebbe rivelarsi molto utile per l&#39;esame delle questioni chiave nella segnalazione delle cellule, tra cui la differenziazione delle cellule staminali e cancro metastasi.

Cellulare microfabbricazione: osservando le interazioni intercellulari mediante DNA litograficamente definite catturano sequenze.

&#x4EE5;&#x524D;&#x306E;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x306F;&#x3001;&#x5F7C;&#x3089;&#x300C;&#x304B;&#x3089;&#x306E;&#x6750;&#x6599;&#x306E;&#x8868;&#x9762;&#x3092;&#x62E1;&#x5F35;&#x3059;&#x308B;&#x88DC;&#x8DB3;&#x306E;&#x7E4A;&#x7DAD;&#x3092;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x53D7;&#x5BB9;&#x4F53;&#x300D;&#x3068;&#x4EBA;&#x5DE5;&#x306E;&#x5BC6;&#x7740;&#x6027;&#x3092;&#x88C5;&#x5099;&#x3059;&#x308B;&#x751F;&#x304D;&#x305F;&#x7D30;&#x80DE;&#x306E;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306B;&#x5408;&#x6210; DNA &#x9396;&#x3092;&#x4ED8;&#x52A0;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x7D30;&#x80DE;&#x306E;&#x7A2E;&#x985E;&#x306E;&#x5E83;&#x3044;&#x7BC4;&#x56F2;&#x3068;&#x4E92;&#x63DB;&#x6027;&#x306E;&#x3042;&#x308B;&#x3001;&#x30AA;&#x30D5;&#x30A1;&#x30FC;&#x512A;&#x308C;&#x305F;&#x52B9;&#x7387;&#x3092;&#x30AD;&#x30E3;&#x30D7;&#x30C1;&#x30E3;&#x3057;&#x3001;&#x53EF;&#x80FD;&#x6027;&#x306E;&#x3042;&#x308B;&#x8907;&#x6570;&#x306E;&#x30AD;&#x30E3;&#x30D7;&#x30C1;&#x30E3; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x8907;&#x96D1;&#x306A;&#x591A;&#x7D30;&#x80DE;&#x30A2;&#x30EC;&#x30F3;&#x30B8;&#x3092;&#x4F5C;&#x6210;&#x3059;&#x308B;&#x306E;&#x306B;&#x4F7F;&#x3048;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x4F5C;&#x696D;&#x3067;&#x306F;&#x3001;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A; DNA &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x69CB;&#x6210;&#x3059;&#x308B;&#x8907;&#x96D1;&#x306A;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306E;&#x52B9;&#x7387;&#x7684;&#x306A;&#x751F;&#x6210;&#x3067;&#x304D;&#x308B;&#x3088;&#x3046;&#x306B;&#x3001;&#x30A2;&#x30EB;&#x30DF;&#x30CB;&#x30A6;&#x30E0;&#x300C;&#x3092;&#x6301;&#x3061;&#x4E0A;&#x3052;&#x308B;&#x300D;&#x30EA;&#x30BD;&#x30B0;&#x30E9;&#x30D5;&#x30A3;&#x6CD5;&#x3092;&#x9069;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x5F97;&#x3089;&#x308C;&#x305F;&#x8868;&#x9762;&#x3001;&#x6700;&#x5927; 3 &#x3064;&#x306E;&#x7570;&#x306A;&#x308B;&#x7A2E;&#x985E;&#x306E;&#x751F;&#x304D;&#x3066;&#x3044;&#x308B;&#x7D30;&#x80DE;&#x306E;&#x7279;&#x5B9A;&#x306E;&#x5834;&#x6240;&#x3092;&#x30AD;&#x30E3;&#x30D7;&#x30C1;&#x30E3;&#x3067;&#x304D;&#x308B;&#x3088;&#x3046;&#x306B;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x5F7C;&#x3089;&#x306F;&#x62E1;&#x6563;&#x306B;&#x57FA;&#x3065;&#x304F;&#x30D1;&#x30E9;&#x30AF;&#x30EA;&#x30F3;&#x3092;&#x5408;&#x56F3;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x901A;&#x4FE1;&#x3059;&#x308B;&#x305F;&#x3081;&#x3001;&#x3053;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306E;&#x30E6;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30C6;&#x30A3; &#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x5316;&#x7D30;&#x80DE;&#x306E;&#x89B3;&#x5BDF;&#x3092;&#x901A;&#x3057;&#x3066;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305D;&#x308C;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3068;&#x764C;&#x8EE2;&#x79FB;&#x3092;&#x542B;&#x3080;&#x4E8B;&#x5B9F;&#x4E0A;&#x3042;&#x3089;&#x3086;&#x308B;&#x30BF;&#x30A4;&#x30D7;&#x306E; 2D &#x7570;&#x8CEA;&#x7D30;&#x80DE;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x4F5C;&#x6210;&#x3059;&#x308B;&#x3053;&#x306E;&#x624B;&#x6CD5;&#x306E;&#x80FD;&#x529B;&#x304C;&#x975E;&#x5E38;&#x306B;&#x6709;&#x7528;&#x306A;&#x7D30;&#x80DE;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x3001;&#x91CD;&#x8981;&#x306A;&#x8CEA;&#x554F;&#x306E;&#x5BE9;&#x67FB;&#x8A3C;&#x660E;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x4E88;&#x60F3;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;

&#x7D30;&#x80DE;&#x5FAE;&#x7D30;&#x52A0;&#x5DE5;: &#x30D1;&#x30BF;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3055;&#x308C;&#x305F;&#x5B9A;&#x7FA9; DNA &#x3092;&#x7528;&#x3044;&#x305F;&#x89B3;&#x5BDF;&#x7D30;&#x80DE;&#x9593;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x30AD;&#x30E3;&#x30D7;&#x30C1;&#x30E3;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC774;&#xC804; &#xBCF4;&#xACE0;&#xC11C; &quot;&#xC218;&#xC6A9; &#xCCB4;&quot; &#xC18C;&#xC7AC; &#xD45C;&#xBA74;&#xC5D0;&#xC11C; &#xD655;&#xC7A5; &#xBCF4;&#xC644; &#xAC00;&#xB2E5; &#xBC14;&#xC778;&#xB529;&#xD560; &#xC778;&#xACF5; &#xC811;&#xCC29; &#xB825;&#xACFC; &#xC7A5;&#xBE44;&#xB97C; &#xC0B4;&#xC544;&#xC788;&#xB294; &#xC138;&#xD3EC;&#xC758; &#xC6D0;&#xD615;&#xC9C8; &#xB9C9;&#xC5D0; &#xD569;&#xC131; DNA &#xAC00;&#xB2E5;&#xC744; &#xCCA8;&#xBD80;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC73C;&#xB85C; &#xB098;&#xD0C0;&#xB0AC;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xBC29;&#xBC95;&#xC740; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC885;&#xB958;&#xC758; &#xC138;&#xD3EC;&#xC640; &#xD638;&#xD658;, &#xC81C;&#xACF5; &#xC6B0;&#xC218;&#xD55C; &#xD6A8;&#xC728;&#xC131;&#xC744; &#xCEA1;&#xCC98;&#xD558;&#xACE0; &#xC5EC;&#xB7EC; &#xCEA1;&#xCC98; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xBCF5;&#xC7A1; &#xD55C; &#xB2E4;&#xC138;&#xD3EC; &#xC870;&#xCE58;&#xB97C; &#xB9CC;&#xB4E4;&#xB97C; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC791;&#xD488;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xC54C;&#xB8E8;&#xBBF8;&#xB284; &quot;&#xB9AC;&#xD504;&#xD2B8;&quot; &#xB9AC;&#xC18C; &#xADF8;&#xB798;&#xD53C; &#xBC29;&#xBC95;&#xC744; &#xC801;&#xC6A9; &#xB2E4;&#xB978; DNA &#xC2DC;&#xD000;&#xC2A4; &#xAD6C;&#xC131; &#xB41C; &#xBCF5;&#xC7A1; &#xD55C; &#xD328;&#xD134;&#xC758; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xC0DD;&#xC131;&#xC744; &#xD5C8;&#xC6A9; &#xD558;&#xB3C4;&#xB85D;. &#xACB0;&#xACFC; &#xC11C;&#xD53C;&#xC2A4;&#xB294; &#xB2E4;&#xC74C; &#xD2B9;&#xC815; &#xC704;&#xCE58;&#xC5D0; &#xC0B4;&#xC544;&#xC788;&#xB294; &#xC138;&#xD3EC;&#xC758; 3 &#xAC00;&#xC9C0; &#xC885;&#xB958;&#xB97C; &#xCEA1;&#xCC98;&#xD560; &#xC218; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xADF8;&#xB4E4;&#xC740; &#xBCF4;&#xAE09; &#xAE30;&#xBC18; paracrine &#xC2E0;&#xD638;&#xC5D0; &#xC758;&#xD574; &#xC758;&#xC0AC; &#xC18C;&#xD1B5;&#xC73C;&#xB85C;&#xC774; &#xBC29;&#xC2DD;&#xC758; &#xC720;&#xD2F8;&#xB9AC;&#xD2F0; &#xD328;&#xD134;&#xD654; &#xB41C; &#xC138;&#xD3EC;&#xC758; &#xAD00;&#xCC30;&#xC744; &#xD1B5;&#xD574; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xADF8;&#xAC83;&#xC740;&#xC774; &#xAE30;&#xC220;&#xC758; &#xB2A5;&#xB825;&#xC744; 2D &#xC774;&#xAE30;&#xC885; &#xC140; &#xD328;&#xD134;&#xC758; &#xAC70;&#xC758; &#xBAA8;&#xB4E0; &#xC885;&#xB958;&#xB97C; &#xB9CC;&#xB4E4; &#xC138;&#xD3EC; &#xC2E0;&#xD638;&#xC5D0;&#xC11C; &#xC8FC;&#xC694; &#xC9C8;&#xBB38;&#xC758; &#xC2DC;&#xD5D8;&#xC5D0; &#xB300; &#xD55C; &#xB9E4;&#xC6B0; &#xC720;&#xC6A9;&#xD55C; &#xC99D;&#xBA85; &#xD574;&#xC57C; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654;, &#xC554; &#xC804;&#xC774; &#xB4F1; &#xC608;&#xC0C1; &#xB429;&#xB2C8;&#xB2E4;.

&#xC140;&#xB8F0;&#xB7EC; &#xC81C;&#xC791;: lithographically &#xC815;&#xC758; &#xB41C; DNA&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAD00;&#xCC30; &#xC138;&#xD3EC; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xCEA1;&#xCC98;&#xD569;&#xB2C8;&#xB2E4;.

Informes anteriores han demostrado que los filamentos de ADN sint&eacute;ticos pueden acoplarse a la membrana plasm&aacute;tica de las c&eacute;lulas vivas para equiparlos con adherencia artificial &quot;receptores&quot; que se unen a los filamentos complementarios que se extiende desde las superficies de materiales. Este enfoque es compatible con una amplia gama de tipos celulares, ofrece excelente eficiencia de captura y potencialmente puede utilizarse para crear arreglos multicelulares complejos mediante el uso de m&uacute;ltiples secuencias de captura. En este trabajo, hemos de aplicar un m&eacute;todo de litograf&iacute;a &quot;Levante&quot; de aluminio para permitir la generaci&oacute;n eficiente de patrones complejos que comprende diferentes secuencias de ADN. Las superficies resultantes son luego demostr&oacute; ser capaz de capturar a hasta tres tipos distintos de c&eacute;lulas vivas en lugares espec&iacute;ficos. La utilidad de este enfoque se demuestra a trav&eacute;s de la observaci&oacute;n de c&eacute;lulas con motivos como se comunican mediante se&ntilde;alizaci&oacute;n paracrina basadas en difusi&oacute;n. Se prev&eacute; que la capacidad de esta t&eacute;cnica para crear pr&aacute;cticamente cualquier tipo de patr&oacute;n heterog&eacute;neo 2D de la c&eacute;lula puede resultar muy &uacute;til para el examen de las cuestiones clave en la se&ntilde;alizaci&oacute;n celular, diferenciaci&oacute;n de c&eacute;lulas madre y met&aacute;stasis del c&aacute;ncer.

Microfabricaci&oacute;n celular: interacciones intercelulares observaci&oacute;n usando ADN definida por litogr&aacute;fico capturan secuencias.

We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3000 molecules of 2 kDa poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.

N-Terminal labeling of filamentous phage to create cancer marker imaging agents.

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

Article	Year
DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells. Angewandte Chemie (International ed. in English)\| PubMed ID: 18798192	2008
Integrated microfluidic bioprocessor for single-cell gene expression analysis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19075237	2008
Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 19505164	2009
DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array. Lab on a chip\| PubMed ID: 19568668	2009
Viral capsid DNA aptamer conjugates as multivalent cell-targeting vehicles. Journal of the American Chemical Society\| PubMed ID: 19603808	2009
Genome-free viral capsids as multivalent carriers for taxol delivery. Angewandte Chemie (International ed. in English)\| PubMed ID: 19921725	2009
Dual-surface modified virus capsids for targeted delivery of photodynamic agents to cancer cells. ACS nano\| PubMed ID: 20863095	2010
Direct attachment of microbial organisms to material surfaces through sequence-specific DNA hybridization. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 22431245	2012
Cellular microfabrication: observing intercellular interactions using lithographically-defined DNA capture sequences. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 22512362	2012
N-Terminal labeling of filamentous phage to create cancer marker imaging agents. ACS nano\| PubMed ID: 22830952	2012
Real time assays for quantifying cytotoxicity with single cell resolution. PloS one\| PubMed ID: 23826123	2013

Sonny C. Hsiao

Biography

JoVE Journal Publications

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