designing-microfluidic-devices-for-studying-cellular-responses-under

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Fu-Jen Catholic University

We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in. x 3 in. (2.54 cm x 7.62 cm) microscope slide.

Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format.

&#x4F20;&#x7EDF;&#x7684;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x7ECF;&#x5E38;&#x88AB;&#x7528;&#x6765;&#x63A2;&#x6D4B;&#x901A;&#x8FC7;&#x5171;&#x8F6D;&#x8367;&#x5149;&#x6297;&#x4F53;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6807;&#x5FD7;&#x7269;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x8367;&#x5149;&#x5171;&#x8F6D;&#x7684;&#x6297;&#x4F53;&#x4F1A;&#x7ED1;&#x5B9A;&#x5C5E;&#x6027;&#xFF0C;&#x5982;&#x5F3A;&#x5EA6;&#x548C;&#x7279;&#x5F02;&#x6027;&#x6297;&#x4F53;&#x7684;&#x6539;&#x53D8;&#x5F80;&#x5F80;&#x65E0;&#x65B9;&#x5F0F;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x4E00;&#x79CD;&#x5229;&#x7528;&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#x5F02; (&#x53CB;&#x7231;&#x8DEF;) &#x663E;&#x5FAE;&#x955C;&#x7684;&#x65E0;&#x6807;&#x7B7E;&#x7684;&#x65B9;&#x6CD5;&#x5B9E;&#x65F6;&#x68C0;&#x6D4B;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6807;&#x8BB0;&#xFF0C;&#x5E76;&#x5C06;&#x5176;&#x5E94;&#x7528;&#x5230;&#x7279;&#x5B9A;&#x9636;&#x6BB5;&#x7684;&#x80DA;&#x80CE;&#x6297;&#x539F; 1 (SSEA1) &#x5BF9;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x5206;&#x6790;&#x3002;&#x5C0F;&#x9F20;&#x5E72;&#x7EC6;&#x80DE;&#x5176;&#x8868;&#x9762;&#x548C; SSEA1 &#x8DCC;&#x5E45;&#x7684;&#x6C34;&#x5E73;&#x8868;&#x793A; SSEA1&#xFF0C;&#x5F53;&#x7EC6;&#x80DE;&#x5F00;&#x59CB;&#x5206;&#x5316;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#xFF0C;&#x6211;&#x4EEC;&#x56FA;&#x5B9A;&#x5316;&#x5C0F;&#x9F20;&#x5E72;&#x7EC6;&#x80DE;&#x548C;&#x975E;-&#x5E72;&#x7EC6;&#x80DE; &#xFF08;&#x63A7;&#x5236;&#xFF09; &#x4F5C;&#x4E3A;&#x82AF;&#x7247;&#x7684;&#x73BB;&#x7483;&#x8868;&#x9762;&#x548C;&#x7EC6;&#x80DE;&#x82AF;&#x7247;&#x4E0E;&#x672A;&#x6807;&#x8BB0; SSEA1 &#x6297;&#x4F53;&#x7684;&#x53CD;&#x5E94;&#x3002;&#x901A;&#x8FC7;&#x4E0E;&#x4EC1;&#x7231;&#x8DEF;&#x663E;&#x5FAE;&#x955C;&#x5728;&#x5B9E;&#x65F6;&#x76D1;&#x89C6;&#x53CD;&#x5E94;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x5B9E; SSEA1 &#x6297;&#x4F53;&#x7ED1;&#x5B9A;&#x53EA;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x548C;&#x975E;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x8868;&#x9762;&#x3002;&#x4ECE;&#x7ED1;&#x5B9A;&#x7684;&#x66F2;&#x7EBF;&#xFF0C;&#x6211;&#x4EEC;&#x51B3;&#x5FC3;&#x4E0E;&#x5E72;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x4E0A;&#x7684; SSEA1 &#x6807;&#x8BB0;&#x6297;&#x4F53;&#x7684;&#x5E73;&#x8861;&#x89E3;&#x79BB;&#x5E38;&#x6570; &#xFF08;Kd&#xFF09;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x5728;&#x4EC1;&#x7231;&#x8DEF;&#x663E;&#x5FAE;&#x955C;&#x53EF;&#x4EE5;&#x7528;&#x4E8E;&#x68C0;&#x6D4B;&#x7EA6;&#x675F;&#x529B;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6807;&#x5FD7;&#x7269;&#x548C;&#x672A;&#x6807;&#x8BB0;&#x7684;&#x6297;&#x4F53;&#x4E4B;&#x95F4;&#x7684;&#x4EB2;&#x7F18;&#x5173;&#x7CFB;&#x7ED1;&#x5B9A;&#x5230;&#x5355;&#x5143;&#x683C; &#xFF1B;&#x6B64;&#x4FE1;&#x606F;&#x53EF;&#x7528;&#x4E8E;&#x6D4B;&#x5B9A;&#x7684;&#x5E72;&#x7EC6;&#x80DE;&#x9636;&#x6BB5;&#x3002;

&#x65E0;&#x6807;&#x7B7E;&#x7684;&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#x5F02;&#x955C;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x8868;&#x9762;&#x6807;&#x5FD7;&#x7684;&#x68C0;&#x6D4B;&#x3002;

Conventional fluorescence microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in often uncharacterized ways. Here we present a method using an oblique-incidence reflectivity difference (OI-RD) microscope for label-free, real-time detection of cell surface markers, and apply it to analysis of stage-specific embryonic antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces, and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies bind only to the surface of the stem cells and not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. Thus, the OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells; this information could be useful for determination of stem cell stages.

Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy.

Conventionnel microscopie &#xE0; fluorescence est couramment utilis&#xE9;e pour d&#xE9;tecter les marqueurs de surface cellulaire par le biais fluorophore anticorps conjugu&#xE9;s. Toutefois, un fluorophore conjugaison d&#x26;#39;anticorps alt&#xE8;re les propri&#xE9;t&#xE9;s de liaison tels que la force et la sp&#xE9;cificit&#xE9; de l&#x26;#39;anticorps de mani&#xE8;re souvent non caract&#xE9;ris&#xE9;s. Nous pr&#xE9;sentons ici une m&#xE9;thode utilisant une barre oblique d&#x26;#39;incidence diff&#xE9;rence de r&#xE9;flectivit&#xE9; (OI-RD) microscope pour sans &#xE9;tiquette, en temps r&#xE9;el de d&#xE9;tection de marqueurs de surface cellulaire, et l&#x26;#39;appliquer &#xE0; l&#x26;#39;analyse de l&#x26;#39;&#xE9;tape de l&#x26;#39;antig&#xE8;ne sp&#xE9;cifique embryonnaire 1 (SSEA1) sur les cellules souches . Les cellules souches de souris express SSEA1 sur leurs surfaces, et le niveau de SSEA1 diminue lorsque les cellules commencent &#xE0; se diff&#xE9;rencier. Dans cette &#xE9;tude, nous avons immobilis&#xE9; cellules souches de souris et non de ces cellules souches (de contr&#xF4;le) sur une surface de verre comme une puce et a r&#xE9;agi le micror&#xE9;seau de cellules non marqu&#xE9;es avec des anticorps SSEA1. En surveillant la r&#xE9;action avec un microscope PO-RD en temps r&#xE9;el, on confirme que les anticorps se lient SSEA1 seulement &#xE0; la surface des cellules souches et non &#xE0; la surface de cellules souches non. &#xC0; partir des courbes de liaison, on d&#xE9;termine la constante de dissociation &#xE0; l&#x26;#39;&#xE9;quilibre (Kd) de l&#x26;#39;anticorps avec les SSEA1 marqueurs sur la surface de cellules souches. Ainsi, le microscope OI-RD peut &#xEA;tre utilis&#xE9; pour d&#xE9;tecter des affinit&#xE9;s de liaison entre les marqueurs de surface cellulaire et des anticorps non marqu&#xE9;s li&#xE9;s aux cellules; cette information pourrait &#xEA;tre utile pour la d&#xE9;termination des stades de cellules souches.

Sans &#xE9;tiquette de d&#xE9;tection de marqueurs de surface sur les cellules souches par obliques incidence Microscopie diff&#xE9;rence de r&#xE9;flectivit&#xE9;

Konventionelle Fluoreszenzmikroskopie wird routinem&auml;&szlig;ig zur Zelle Oberfl&auml;che Marker durch Fluorophor-konjugierten Antik&ouml;rpern erkennen. Jedoch &auml;ndert Fluorophor-Konjugation von Antik&ouml;rpern Bindungseigenschaften wie Festigkeit und Spezifit&auml;t des Antik&ouml;rpers oft uncharakterisiertes M&ouml;glichkeiten. Hier pr&auml;sentieren wir Ihnen eine Methode mit einem schr&auml;g-Inzidenz Reflexionsverm&ouml;gen-Unterschied (OI-RD)-Mikroskop f&uuml;r Etikett-frei, Echtzeit-Erkennung von Zelle Oberfl&auml;che Marker, und wenden sie auf Analyse der embryonalen Phase-spezifisches-Antigen 1 (SSEA1) auf Stammzellen. Maus-Stammzellen schnelles SSEA1 auf ihrer Oberfl&auml;che, und das Niveau der SSEA1 sinkt, wenn die Zellen beginnen zu unterscheiden. In dieser Studie haben wir immobilisiert Maus-Stammzellen und nicht-Stammzellen (Kontrolle) auf einer Glasoberfl&auml;che als ein Microarray und reagiert die Zelle Microarray unbeschriftete SSEA1-Antik&ouml;rper. &Uuml;berwachen Sie die Reaktion mit einem OI-RD-Mikroskop in Echtzeit, best&auml;tigt wir, dass die SSEA1 Antik&ouml;rper nur an der Oberfl&auml;che der Stammzellen und nicht an die Oberfl&auml;che des nicht-Stammzellen binden. Der Bindung-Kurven haben wir die Dissoziation Gleichgewichtskonstante (Kd) des Antik&ouml;rpers mit dem SSEA1-Marker auf der Stammzell-Oberfl&auml;che festgestellt. So k&ouml;nnen Sie das OI-RD-Mikroskop zum erkennen Affinit&auml;ten zwischen Zelle Oberfl&auml;che Marker und unbeschriftete Antik&ouml;rper binden an die Zellen gebunden; Diese Informationen k&ouml;nnten f&uuml;r Bestimmung der Stammzelle Stufen n&uuml;tzlich sein.

Etikett-freie Abfragung der Oberfl&auml;che Marker auf Stammzellen durch schr&auml;g-Inzidenz Reflexionsverm&ouml;gen Unterschied Mikroskopie.

Microscopia di fluorescenza convenzionale viene utilizzata regolarmente per rilevare i marcatori di superficie cella attraverso anticorpi coniugati fluorophore. Tuttavia, fluorophore-coniugazione degli anticorpi altera le propriet&agrave; di associazione come forza e specificit&agrave; dell&#39;anticorpo in modi spesso atipici. Qui vi presentiamo un metodo utilizzando un microscopio di differenza (OI-RD) Incidenza obliqua riflettivit&agrave; per privo di etichetta, rilevazione in tempo reale della cella i marcatori di superficie e si applicano all&#39;analisi della specifica fase embrionale antigene 1 (SSEA1) sulle cellule staminali. Cellule staminali di topo express SSEA1 sulle loro superfici e il livello di SSEA1 diminuisce quando le cellule iniziano a differenziarsi. In questo studio, abbiamo immobilizzato cellule staminali di topo e non-cellule staminali (controllo) su una superficie di vetro come un microarray e ha reagito il microarray delle cellule con anticorpi SSEA1 adenoidi. Monitorando la reazione con un microscopio di OI-RD in tempo reale, abbiamo confermato che i SSEA1 gli anticorpi si legano solo alla superficie delle cellule staminali e non alla superficie delle cellule staminali. Dalle curve associazione, abbiamo determinato la costante di dissociazione di equilibrio (Kd) dell&#39;anticorpo con i marcatori SSEA1 sulla superficie delle cellule staminali. Cos&igrave;, il microscopio OI-RD pu&ograve; essere utilizzato per rilevare l&#39;associazione affinit&agrave; tra i marcatori di superficie delle cellule e degli anticorpi senza etichetta associata alle cellule; Questa informazione potrebbe essere utile per la determinazione delle fasi di cellule staminali.

Rilevamento di marcatori di superficie sulle cellule staminali da microscopia differenza di incidenza obliqua riflettivit&agrave; senza etichetta.

&#x5F93;&#x6765;&#x306E;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1;&#x306F;&#x3001;&#x65E5;&#x5E38;&#x7684;&#x306B;&#x30D5;&#x30EB;&#x30AA;&#x30ED;&#x30D5;&#x30A9;&#x30A2;&#x6A19;&#x8B58;&#x6297;&#x4F53;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x6297;&#x4F53;&#x306E;&#x86CD;&#x5149;&#x4F53;&#x5171;&#x5F79;&#x306F;&#x3001;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x5F37;&#x3055;&#x3068;&#x3001;&#x3057;&#x3070;&#x3057;&#x3070;&#x672A;&#x77E5;&#x306E;&#x65B9;&#x6CD5;&#x3067;&#x6297;&#x4F53;&#x306E;&#x7279;&#x7570;&#x6027;&#x306A;&#x3069;&#x306E;&#x7D50;&#x5408;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306E;&#x30E9;&#x30D9;&#x30EB;&#x30D5;&#x30EA;&#x30FC;&#x3001;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x306E;&#x691C;&#x51FA;&#x306E;&#x305F;&#x3081;&#x306E;&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#xFF08;OI-RD&#xFF09;&#x9855;&#x5FAE;&#x93E1;&#x3092;&#x7528;&#x3044;&#x305F;&#x65B9;&#x6CD5;&#x3092;&#x63D0;&#x793A;&#x3057;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306B;&#x30B9;&#x30C6;&#x30FC;&#x30B8;&#x56FA;&#x6709;&#x306E;&#x80CE;&#x5150;&#x6027;&#x6297;&#x539F;1&#xFF08;SSEA1&#xFF09;&#x306E;&#x5206;&#x6790;&#x306B;&#x9069;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x304C;&#x5206;&#x5316;&#x3092;&#x958B;&#x59CB;&#x3057;&#x305F;&#x3068;&#x304D;&#x306B;&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5E79;&#x7D30;&#x80DE;&#x306F;&#x305D;&#x306E;&#x8868;&#x9762;&#x306B;SSEA1&#x3092;&#x8868;&#x73FE;&#x3057;&#x3001;SSEA1&#x6E1B;&#x5C11;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30A2;&#x30EC;&#x30A4;&#x306E;&#x3088;&#x3046;&#x306B;&#x30AC;&#x30E9;&#x30B9;&#x8868;&#x9762;&#x4E0A;&#x3067;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5E79;&#x7D30;&#x80DE;&#x3068;&#x975E;&#x5E79;&#x7D30;&#x80DE;&#xFF08;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#xFF09;&#x56FA;&#x5B9A;&#x5316;&#x3068;&#x975E;&#x6A19;&#x8B58;SSEA1&#x6297;&#x4F53;&#x3068;&#x7D30;&#x80DE;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30A2;&#x30EC;&#x30A4;&#x3092;&#x53CD;&#x5FDC;&#x3055;&#x305B;&#x305F;&#x3002;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x3067;OI-RD&#x9855;&#x5FAE;&#x93E1;&#x3068;&#x306E;&#x53CD;&#x5FDC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;SSEA1&#x6297;&#x4F53;&#x304C;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x306B;&#x3067;&#x306F;&#x306A;&#x304F;&#x3001;&#x975E;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x306B;&#x306E;&#x307F;&#x7D50;&#x5408;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x305F;&#x3002;&#x7D50;&#x5408;&#x66F2;&#x7DDA;&#x304B;&#x3089;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x4E0A;SSEA1&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x6297;&#x4F53;&#x306E;&#x5E73;&#x8861;&#x89E3;&#x96E2;&#x5B9A;&#x6570;&#xFF08;Kd&#xFF09;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;OI-RD&#x9855;&#x5FAE;&#x93E1;&#x306F;&#x7D30;&#x80DE;&#x306B;&#x7D50;&#x5408;&#x3057;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x672A;&#x6A19;&#x8B58;&#x6297;&#x4F53;&#x3068;&#x306E;&#x7D50;&#x5408;&#x89AA;&#x548C;&#x6027;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x60C5;&#x5831;&#x306F;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x6BB5;&#x968E;&#x306E;&#x6C7A;&#x5B9A;&#x306B;&#x6709;&#x7528;&#x3067;&#x3042;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#x9855;&#x5FAE;&#x93E1;&#x306B;&#x3088;&#x308B;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306E;&#x30E9;&#x30D9;&#x30EB;&#x30D5;&#x30EA;&#x30FC;&#x691C;&#x51FA;

&#xAE30;&#xC874;&#xC758; &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; fluorophore conjugated &#xD56D; &#xCCB4;&#xB97C; &#xD1B5;&#xD574; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4;&#xB97C; &#xAC10;&#xC9C0; &#xD558; &#xC77C;&#xC0C1;&#xC801;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xD56D; &#xCCB4;&#xC758; fluorophore-&#xD65C;&#xC6A9; &#xC885;&#xC885; &#xC0C8;&#xB86D;&#xAC70;&#xB098; &#xBC29;&#xBC95;&#xC73C;&#xB85C; &#xAC15;&#xB3C4; &#xBC0F; &#xD56D; &#xCCB4;&#xC758; &#xD2B9;&#xC774;&#xC131; &#xB4F1;&#xC758; &#xBC14;&#xC778;&#xB529; &#xC18D;&#xC131;&#xC744; &#xBCC0;&#xACBD;&#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0; &#xC6B0;&#xB9AC;&#xAC00; &#xD604;&#xC7AC; &#xB808;&#xC774;&#xBE14; &#xC5C6;&#xB294; &#xB300; &#xD55C; &#xAC04;&#xC811; &#xBC1C;&#xC0DD;&#xB960; &#xBC18;&#xC0AC;&#xB3C4; &#xCC28;&#xC774; (&#xD558;&#xC774;-RD) &#xD604;&#xBBF8;&#xACBD;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xBA54;&#xC11C;&#xB4DC; &#xC140;&#xC758; &#xC2E4;&#xC2DC;&#xAC04; &#xD0D0;&#xC9C0; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4;, &#xADF8;&#xB9AC;&#xACE0; &#xBB34;&#xB300; &#xAD00;&#xB828; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBC30;&#xC544; &#xD56D; &#xC6D0; 1 (SSEA1)&#xC758; &#xBD84;&#xC11D;&#xC5D0; &#xC801;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC6B0;&#xC2A4; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC140;&#xC744; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; &#xB370; &#xC2DC;&#xC791;&#xD560; &#xB54C; &#xADF8;&#xB4E4;&#xC758; &#xD45C;&#xBA74;&#xC5D0; SSEA1 &#xAC10;&#xC18C; &#xC218;&#xC900;&#xC744; SSEA1 &#xC775;&#xC2A4;&#xD504;&#xB808;&#xC2A4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xB9C8;&#xC6B0;&#xC2A4; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBC0F; &#xBE44;-&#xC904;&#xAE30; &#xC138;&#xD3EC; (&#xC81C;&#xC5B4;)&#xB97C; microarray&#xB85C; &#xC720;&#xB9AC; &#xD45C;&#xBA74;&#xC5D0; &#xC6C0;&#xC9C1;&#xC77C; &#xD558; &#xACE0; &#xC140; microarray &#xB808;&#xC774;&#xBE14;&#xC774; &#xC5C6;&#xB294; SSEA1 &#xD56D; &#xCCB4;&#xC640; &#xBC18;&#xC751;. &#xC2E4;&#xC2DC;&#xAC04;&#xC73C;&#xB85C; &#xC624;&#xC774; RD &#xD604;&#xBBF8;&#xACBD;&#xC73C;&#xB85C; &#xBC18;&#xC751;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xC6B0;&#xB9AC;&#xB294; SSEA1 &#xD56D; &#xCCB4; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xD45C;&#xBA74; &#xC544;&#xB2C8;&#xB77C; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xD45C;&#xBA74;&#xC5D0;&#xB9CC; &#xBC14;&#xC778;&#xB529;&#xD560; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xBC14;&#xC778;&#xB529; &#xCEE4;&#xBE0C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; SSEA1 &#xD45C;&#xC2DD;&#xC774; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xC788;&#xB294; &#xD56D; &#xCCB4;&#xC758; &#xD3C9;&#xD615; &#xBD84;&#xB9AC; &#xC0C1;&#xC218; (Kd) &#xACB0;&#xC815;. &#xC624;&#xC774; RD &#xD604;&#xBBF8;&#xACBD;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC140;;&#xC5D0; &#xBC14;&#xC778;&#xB529;&#xB41C; &#xBC14;&#xC778;&#xB529; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4; &#xBC0F; &#xB808;&#xC774;&#xBE14; &#xC5C6;&#xB294; &#xD56D; &#xCCB4; &#xC0AC;&#xC774;&#xC758; &#xC120;&#xD638;&#xB3C4; &#xAC10;&#xC9C0; &#xD558;&#xB294; &#xB530;&#xB77C;&#xC11C;, &#xC774; &#xC815;&#xBCF4;&#xB294; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB2E8;&#xACC4; &#xACB0;&#xC815;&#xC5D0; &#xC720;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB808;&#xC774;&#xBE14; &#xC5C6;&#xB294; &#xAC04;&#xC811; &#xBC1C;&#xC0DD;&#xB960; &#xBC18;&#xC0AC;&#xB3C4; &#xCC28;&#xC774; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95;&#xC5D0; &#xC758;&#xD574; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4; &#xAC80;&#xCD9C;.

Microscop&#xED;a de fluorescencia convencional se utiliza rutinariamente para detectar marcadores de superficie celular a trav&#xE9;s fluor&#xF3;foro anticuerpos conjugados. Sin embargo, fluor&#xF3;foro-conjugaci&#xF3;n de anticuerpos altera las propiedades de uni&#xF3;n tales como la resistencia y la especificidad del anticuerpo en formas a menudo no caracterizados. Aqu&#xED; presentamos un m&#xE9;todo que utiliza una oblicua de incidencia diferencia de reflectividad (OI-RD) microscopio libre de marca, en tiempo real la detecci&#xF3;n de marcadores de superficie celular, y aplicarlo al an&#xE1;lisis de ant&#xED;geno espec&#xED;fico de la fase embrionaria 1 (SSEA1) sobre las c&#xE9;lulas madre . C&#xE9;lulas madre de ratones expresan SSEA1 en su superficie, y el nivel de SSEA1 disminuye cuando las c&#xE9;lulas comienzan a diferenciarse. En este estudio, que inmoviliz&#xF3; c&#xE9;lulas madre de ratones y las c&#xE9;lulas madre no (control) sobre una superficie de vidrio como los microarrays y reaccionaron los microarrays de c&#xE9;lulas con anticuerpos no marcados SSEA1. Mediante la supervisi&#xF3;n de la reacci&#xF3;n con un microscopio de OI-DR en tiempo real, se confirm&#xF3; que los anticuerpos se unen SSEA1 s&#xF3;lo a la superficie de las c&#xE9;lulas madre y no a la superficie de las c&#xE9;lulas madre no. De las curvas de uni&#xF3;n, se determin&#xF3; la constante de equilibrio de disociaci&#xF3;n (Kd) de los anticuerpos con los marcadores SSEA1 en la superficie de c&#xE9;lulas madre. As&#xED;, el microscopio de OI-RD puede ser utilizado para detectar afinidades de uni&#xF3;n entre los marcadores de la superficie celular y los anticuerpos no marcados unidos a las c&#xE9;lulas; esta informaci&#xF3;n podr&#xED;a ser &#xFA;til para la determinaci&#xF3;n de las etapas de c&#xE9;lulas madre.

Libre de marca de detecci&#xF3;n de marcadores de superficie de las c&#xE9;lulas madre por Microscop&#xED;a de incidencia oblicua reflectividad Diferencia

Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four β1-4-linked galactosides, three β1-3-linked galactosides, one β-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin-glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of protein-ligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein-glycan interactions.

Fluorescent labeling agents change binding profiles of glycan-binding proteins.

In this paper, we report a new method to incorporate 3D scaffold with electrotaxis measurement in the microfluidic device. The electrotactic response of lung cancer cells in the 3D foam scaffolds which resemble the in vivo pulmonary alveoli may give more insight on cellular behaviors in vivo. The 3D scaffold consists of ordered arrays of uniform spherical pores in gelatin. We found that cell morphology in the 3D scaffold was different from that in 2D substrate. Next, we applied a direct current electric field (EF) of 338 mV/mm through the scaffold for the study of cells' migration within. We measured the migration directedness and speed of different lung cancer cell lines, CL1-0, CL1-5, and A549, and compared with those examined in 2D gelatin-coated and bare substrates. The migration direction is the same for all conditions but there are clear differences in cell morphology, directedness, and migration speed under EF. Our results demonstrate cell migration under EF is different in 2D and 3D environments and possibly due to different cell morphology and/or substrate stiffness.

Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds.

The wound-healing assay is an easy and economical way to quantify cell migration under diverse stimuli. Traditional assays such as scratch assays and barrier assays are widely and commonly used, but neither of them can represent the complicated condition when a wound occurs. It has been suggested that wound-healing is related to electric fields, which were found to regulate wound re-epithelialization. As a wound occurs, the disruption of epithelial barrier short-circuits the trans-epithelial potential and then a lateral endogenous electric field is created. This field has been proved invitro as an important cue for guiding the migration of fibroblasts, macrophages, and keratinocytes, a phenomenon termed electrotaxis or galvanotaxis. In this paper, we report a microfluidic electrical-stimulated wound-healing chip (ESWHC) integrating electric field with a modified barrier assay. This chip was used to study the migration of fibroblasts under different conditions such as serum, electric field, and wound-healing-promoting drugs. We successfully demonstrate the feasibility of ESWHC to effectively and quantitatively study cell migration during wound-healing process, and therefore this chip could be useful in drug discovery and drug safety tests.

In vitro electrical-stimulated wound-healing chip for studying electric field-assisted wound-healing process.

Reactive oxygen species (ROS) are known to be a key factor in the development of cancer, and many exogenous sources are supposed to be related to the formation of ROS. In this paper, a microfluidic chip was developed for studying the production of ROS in lung cancer cells under different chemical and physical stimuli. This chip has two unique features: (1) five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 are achieved in the culture regions; (2) a shear stress gradient is produced inside each of the five culture areas. Lung cancer cells were seeded inside this biocompatible chip for investigating their response to different concentrations of H2O2, a chemical stimulus known to increase the production of ROS. Then the effect of shear stress, a physical stimulus, on lung cancer cells was examined, showing that the production of ROS was increased in response to a larger shear stress. Finally, two antioxidants, α-tocopherol and ferulic acid, were used to study their effects on reducing ROS. It was found that high-dose α-tocopherol was not able to effectively eliminate the ROS produced inside cells. This counter effect was not observed in cells cultured in a traditional chamber slide, where no shear stress was present. This result suggests that the current microfluidic chip provides an in vitro platform best mimicking the physiological condition where cells are under circulating conditions.

Effects of shear stresses and antioxidant concentrations on the production of reactive oxygen species in lung cancer cells.

One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD) technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR). The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.

Real-time, label-free detection of biomolecular interactions in sandwich assays by the oblique-incidence reflectivity difference technique.

In recent years, various label-free biosensing technologies have been developed for studying the real-time kinetics of diverse biomolecular interactions. These biosensors partially take the place of fluorescence-based methods by providing a comparable sensitivity as well as retaining the conformational and functional integrality of biomolecules to be investigated. However, to completely eliminate the need of fluorescence, throughput is the next big consideration. Microarrays provide a high-throughput platform for screening tens of thousands of biomolecular interactions simultaneously, and many compatible fluorescent scanners have been commercially available. The combination of microarrays and label-free biosensors will be of great interest to researchers in related fields. Microarrays are fabricated by spotting, imprinting, or directly synthesizing biomolecules on solid supports such as glasses, silicon wafers, and other functionalized substrates, and they have been applied to detect DNAs, proteins, toxins, and so on in surface plasmon resonance (SPR) imaging systems and oblique-incidence reflectivity difference (OI-RD) microscopes. Current challenges include increasing sensitivity, reducing sampling time, improving surface chemistry, identifying captured molecules, and minimizing reagent consumption. Future research directions are to improve the instruments themselves, modify the microarray surface for more efficient analyte capture, and combine the systems with mass spectrometry and microfluidics.

Use of Microarrays as a High-Throughput Platform for Label-Free Biosensing.

Microarrays provide a platform for high-throughput characterization of biomolecular interactions. To increase the sensitivity and specificity of microarrays, surface blocking is required to minimize the nonspecific interactions between analytes and unprinted yet functionalized surfaces. To block amine- or epoxy-functionalized substrates, bovine serum albumin (BSA) is one of the most commonly used blocking reagents because it is cheap and easy to use. Based on standard protocols from microarray manufactories, a BSA concentration of 1% (10 mg/mL or 200 μM) and reaction time of at least 30 min are required to efficiently block epoxy-coated slides. In this paper, we used both fluorescent and label-free methods to characterize the BSA blocking efficiency on epoxy-functionalized substrates. The blocking efficiency of BSA was characterized using a fluorescent scanner and a label-free oblique-incidence reflectivity difference (OI-RD) microscope. We found that (1) a BSA concentration of 0.05% (0.5 mg/mL or 10 μM) could give a blocking efficiency of 98%, and (2) the BSA blocking step took only about 5 min to be complete. Also, from real-time and in situ measurements, we were able to calculate the conformational properties (thickness, mass density, and number density) of BSA molecules deposited on the epoxy surface.

Characterization of Bovine Serum Albumin Blocking Efficiency on Epoxy-Functionalized Substrates for Microarray Applications.

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

Cell migration is an essential process involved in the development and maintenance of multicellular organisms. Electric fields (EFs) are one of the many physical and chemical factors known to affect cell migration, a phenomenon termed electrotaxis or galvanotaxis. In this paper, a microfluidics chip was developed to study the migration of cells under different electrical and chemical stimuli. This chip is capable of providing four different strengths of EFs in combination with two different chemicals via one simple set of agar salt bridges and Ag/AgCl electrodes. NIH 3T3 fibroblasts were seeded inside this chip to study their migration and reactive oxygen species (ROS) production in response to different EF strengths and the presence of β-lapachone. We found that both the EF and β-lapachone level increased the cell migration rate and the production of ROS in an EF-strength-dependent manner. A strong linear correlation between the cell migration rate and the amount of intracellular ROS suggests that ROS are an intermediate product by which EF and β-lapachone enhance cell migration. Moreover, an anti-oxidant, α-tocopherol, was found to quench the production of ROS, resulting in a decrease in the migration rate.

Correlation between cell migration and reactive oxygen species under electric field stimulation.

Collective cell migration plays important roles in many physiological processes such as embryonic development, tissue repair, and angiogenesis. A "wound" occurs when epithelial cells are lost and/or damaged due to some external factors, and collective cell migration takes place in the following wound-healing process. To study this cellular behavior, various kinds of wound-healing assays are developed. In these assays, a "wound," or a "cell-free region," is created in a cell monolayer mechanically, chemically, optically, or electrically. These assays are useful tools in studying the effects of certain physical or chemical stimuli on the wound-healing process. Most of these methods have disadvantages such as creating wounds of different sizes or shapes, yielding batch-to-batch variation, and damaging the coating of the cell culture surface. In this study, we used ultraviolet (UV) lights to selectively kill cells and create a wound out of a cell monolayer. A comparison between the current assay and the traditional scratch assay was made, indicating that these two methods resulted in similar wound-healing rates. The advantages of this UV-created wound-healing assay include fast and easy procedure, high throughput, and no direct contact to cells.

A Wound-Healing Assay Based on Ultraviolet Light Ablation.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies best mimicking the in vivo micro-environment. These devices are capable of creating precise and controllable surroundings of pH value, temperature, salt concentration, and other physical or chemical stimuli. Various cell studies such as chemotaxis and electrotaxis can be performed by using such devices. Moreover, microfluidic chips are designed and fabricated for applications in cell separations such as circulating tumor cell (CTC) chips. Usually, there are two most commonly used inlets in connecting the microfluidic chip to sample/reagent loading tubes: the vertical (top-loading) inlet and the parallel (in-line) inlet. Designing this macro-to-micro interface is believed to play an important role in device performance. In this study, by using the commercial COMSOL Multiphysics software, we compared the cell capture behavior in microfluidic devices with different inlet types and sample flow velocities. Three different inlets were constructed: the vertical inlet, the parallel inlet, and the vertically parallel inlet. We investigated the velocity field, the flow streamline, the cell capture rate, and the laminar shear stress in these inlets. It was concluded that the inlet should be designed depending on the experimental purpose, i.e., one wants to maximize or minimize cell capture. Also, although increasing the flow velocity could reduce cell sedimentation, too high shear stresses are thought harmful to cells. Our findings indicate that the inlet design and flow velocity are crucial and should be well considered in fabricating microfluidic devices for cell studies.

Article	Year
Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format. Applied optics\| PubMed ID: 17356635	2007
Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy. BioTechniques\| PubMed ID: 21781038	2011
Fluorescent labeling agents change binding profiles of glycan-binding proteins. Molecular bioSystems\| PubMed ID: 22009201	2011
Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds. Biomicrofluidics\| PubMed ID: 22288000	2012
In vitro electrical-stimulated wound-healing chip for studying electric field-assisted wound-healing process. Biomicrofluidics\| PubMed ID: 24009651	2012
Effects of shear stresses and antioxidant concentrations on the production of reactive oxygen species in lung cancer cells. Biomicrofluidics\| PubMed ID: 24396542	2013
Real-time, label-free detection of biomolecular interactions in sandwich assays by the oblique-incidence reflectivity difference technique. Sensors (Basel, Switzerland)\| PubMed ID: 25490591	2014
Use of Microarrays as a High-Throughput Platform for Label-Free Biosensing. Journal of laboratory automation\| PubMed ID: 25812567	2015
Characterization of Bovine Serum Albumin Blocking Efficiency on Epoxy-Functionalized Substrates for Microarray Applications. Journal of laboratory automation\| PubMed ID: 25976408	2015
Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform. Biomolecules\| PubMed ID: 26193329	2015
Correlation between cell migration and reactive oxygen species under electric field stimulation. Biomicrofluidics\| PubMed ID: 26487906	2015
A Wound-Healing Assay Based on Ultraviolet Light Ablation. Journal of laboratory automation\| PubMed ID: 27139694	2016
Comparison of Chip Inlet Geometry in Microfluidic Devices for Cell Studies. Molecules (Basel, Switzerland)\| PubMed ID: 27314318	2016

Yung-Shin Sun

Biography

JoVE Journal Publications

Other Publications