Авторы хотели бы поблагодарить немецкого фонда научных исследований (DFG) для частичного финансирования этой работы в рамках гранта чисел UR 70/10-01 и UR 70/12-01.

<ol><li>Spindel, S., Sapsford, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+optical+detection+platforms+for+multiplexed+detection+of+proteins+and+the+need+for+point-of-care+biosensors+for+clinical+use%5BTitle%5D)+AND+%22Sensors+%28Basel%29%22%5BJournal%5D)">Evaluation of optical detection platforms for multiplexed detection of proteins and the need for point-of-care biosensors for clinical use.</a> Sensors (Basel). 14 (12), 22313-22341 (2014).</li>
<li>Luppa, P. B., Bietenbeck, A., Beaudoin, C., Giannetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinically+relevant+analytical+techniques%2C+organizational+concepts+for+application+and+future+perspectives+of+point-of-care+testing%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Clinically relevant analytical techniques, organizational concepts for application and future perspectives of point-of-care testing.</a> Biotechnol Adv. 34 (3), 139-160 (2016).</li>
<li>Gauglitz, G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25014344">Point-of-Care Platforms.</a> Annu Rev Anal Chem. 7 (1), 297-315 (2014).</li>
<li>Jung, W., Han, J., Choi, J. W., Ahn, C. H. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0167931714004316">Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies.</a> Microelectron Eng. 132, 46-57 (2014).</li>
<li>Yager, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+diagnostic+technologies+for+global+public+health%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Microfluidic diagnostic technologies for global public health.</a> Nature. 442 (7101), 412-418 (2006).</li>
<li>Fu, E., Yager, P., Floriano, P. N., Christodoulides, N., McDevitt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perspective+on+diagnostics+for+global+health%5BTitle%5D)+AND+%22IEEE+Pulse%22%5BJournal%5D)">Perspective on diagnostics for global health.</a> IEEE Pulse. 2 (6), 40-50 (2011).</li>
<li>Yager, P., Domingo, G. J., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Point-of-care+diagnostics+for+global+health%5BTitle%5D)+AND+%22Ann+Rev+Biomed+Eng%22%5BJournal%5D)">Point-of-care diagnostics for global health.</a> Ann Rev Biomed Eng. 10, 107-144 (2008).</li>
<li>Dincer, C., Bruch, R., Kling, A., Dittrich, P. S., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+point-of-care+testing+-+xPOCT%5BTitle%5D)+AND+%22Trends+Biotechnol%22%5BJournal%5D)">Multiplexed point-of-care testing - xPOCT.</a> Trends Biotechnol. 35, (2017).</li>
<li>Horak, J., Dincer, C., Bakirci, H., Urban, G. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0925400513012057">A disposable dry film photoresist-based microcapillary immunosensor chip for rapid detection of Epstein-Barr virus infection.</a> Sens Actuators B Chem. 191, 813-820 (2014).</li>
<li>Method for the fabrication of a &#34;lab on chip&#34; from photoresist material for medical diagnostic applications. US patent. , 7,691,623 (2010).</li>
<li>Kling, A., Dincer, C., Armbrecht, L., Horak, J., Kieninger, J., Urban, G. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1877705815024571">Electrochemical microfluidic platform for simultaneous multi-analyte detection.</a> Procedia Eng. 120, 916-919 (2015).</li>
<li>Armbrecht, L., Dincer, C., Kling, A., Horak, J., Kieninger, J., Urban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-assembled+magnetic+bead+chains+for+sensitivity+enhancement+of+microfluidic+electrochemical+biosensor+platforms%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms.</a> Lab Chip. 15, 4314-4321 (2015).</li>
<li>Dincer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designed+miniaturization+of+microfluidic+biosensor+platforms+using+the+stop-flow+technique%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Designed miniaturization of microfluidic biosensor platforms using the stop-flow technique.</a> Analyst. 141, 6073-6079 (2016).</li>
<li>Weltin, A., Kieninger, J., Enderle, B., Gellner, A. K., Fritsch, B., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymer-based%2C+flexible+glutamate+and+lactate+microsensors+for+in+vivo+applications%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">Polymer-based, flexible glutamate and lactate microsensors for in vivo applications.</a> Biosens Bioelectron. 61, 192-199 (2014).</li>
<li>Kling, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multianalyte+Antibiotic+Detection+on+an+Electrochemical+Microfluidic+Platform%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Multianalyte Antibiotic Detection on an Electrochemical Microfluidic Platform.</a> Anal Chem. 88 (20), 10036-10043 (2016).</li>
<li>Bruch, R., et al. Clinical on-site monitoring of ß-lactam antibiotics for a personalized antibiotherapy. Sci Rep. , (2017).</li>
<li>Horak, J., Dincer, C., Qelibari, E., Bakirci, H., Urban, G. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0925400514015482">Polymer-modified microfluidic immunochip for enhanced electrochemical detection of troponin i.</a> Sens Actuators B Chem. 209, 478-485 (2015).</li>
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<li>Mattox, D. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMattox%2C+DM+%22Handbook+of+Physical+Vapor+Deposition+%28PVD%29+Processing%22">Handbook of Physical Vapor Deposition (PVD) Processing</a>. , Elsevier Inc. (2010).</li>
</ol>

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Представленные здесь для изготовления microfluidic электрохимический биодатчик протокол позволяет развитие лоу кост, компактный и простой в использовании платформа для обнаружения биомолекул. В зависимости от assay, потом используется на биосенсор могут быть обнаружены несколько различных ...

За последние два десятилетия диагностические приложения стали элементарных для углубленных исследований в области глобального общественного здравоохранения. Традиционно лабораторные диагностические инструменты используются для обнаружения заболеваний. Даже несмотря на то, что они по-прежнему играть ключевую роль в диагностике заболеваний, тестирования точки обслуживания (POCT) выполнена у пациента или пациент сам стал более и более распространенным явлением в последние годы. Особенно в таких случаях, которые требуют немедленного лечения, например, острый инфаркт миокарда или диабет мониторинг быстрое подтверждение клинического вывод имеет важное значение. Следовательно существует растущая потребность POCT устройств, которая может эксплуатироваться не экспертами и что одновременно способны выполнять точные в vitro диагностические тесты в короткое время1,2,3,4 .
Уже достигнуты значительные успехи, достигнутые в области POCT. Однако есть еще много проблем, чтобы преодолеть5,6,,78. Для платформы POCT будет успешно запущен на рынок и конкурировать с лабораторной диагностики, устройство должно строго соответствовать следующим требованиям: (i) предоставлять точные и количественные результаты, которые согласуются с лабораторией выводы; (ii) имеют короткий пример к результат раз, позволяя немедленного лечения пациента; (iii) располагают незатрудненного и простой обработки, даже при ведении неподготовленных людей и требуют вмешательства свернутого пользователя; и (iv) состоят из датчика лоу кост подразделение, предназначены для однократного использования приложений. Кроме того оборудование Бесплатная диагностика благоприятны, главным образом в бедных ресурсами среды3,4,6.
Из-за этих серьезных требований только две POCT систем на основе электрохимических обнаружения (например, глюкозы в крови тест полоски) и иммуноанализа бокового потока (например, домашние тесты беременности) были успешно начаты на рынок так далеко. Однако обе системы страдают от недостатков, таких, как низкая производительность (т.е. мониторинга глюкозы крови имеет неточные результаты и анализы бокового потока только предоставляют результаты качественных измерений (положительный или отрицательный))4, 6. Эти недостатки обычных систем POCT привели растущий спрос на изучение новых технологий, которые предлагают быстрый, лоу кост и количественного обнаружения точки зрения ухода за4,5.
Для решения этих задач, стоящих перед POCT устройств, технология DFR недавно использовалась для изготовления одноразовых и лоу кост биодатчики9,10,11,12, 13 , 14. по сравнению с мягких и жидких литографических материалы, такие как PDMS или Су-8, DFRs настоящее время множество преимуществ: они (i) доступны в различных композиций и толщины (от нескольких микрон до нескольких миллиметров); (ii) имеют очень грубо площадь поверхности, которая облегчает адгезии к различным материалам; (iii) функция Потрясающе толщина единообразия; (iv) предлагают дешевые, снисходительный и высок объём производства для массового производства; (v) являются легко резать с различными инструментами лоу кост, как пару простых ножницы; и (vi) позволяют для создания трехмерных структур, таких как microfluidic каналы, путем укладки нескольких DFR слои друг на друга.
С другой стороны DFRs в целом имеют относительно бедных разрешение по сравнению с жидкой фоторезистов, который является главным образом вызваны толщины пленки и увеличение расстояния между DFR благодаря защитной пленке, которая дополнительно позволяет света и маски рассеяние. Тем не менее для изготовления комплексных microfluidic биодатчиков, DFRs очень подходит для лоу кост массового производства.
Таким образом мы представляем в этой работы, изготовление и применение биосенсор на основе DFR электрохимических microfluidic. Подробный протокол описывает каждый шаг производства биосенсор платформы, на чипе иммобилизации модель на основе ДНК анализа, и ее электрохимических индикация с использованием метода stop потока. Это универсальная платформа позволяет выявлять многочисленные виды биомолекул, используя различные пробирного технологий (целломике,например, геномики и протеомики) или пробирного форматов (например, конкурентоспособной, бутерброд или прямой). Основываясь на такой платформе DFR, наша группа ранее успешно продемонстрировала быстрый и чувствительных квантификации различных аналитов, включая антибиотики13,,1516 (тетрациклин, pristinamycin и β лактамные антибиотики), тропонин я17и вещество P-18.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pyralux</td><td>DuPont</td><td>AP8525R</td><td>Used as polyimide substrate</td></tr><tr><td>MA-N 1420</td><td>Micro Resist Technology</td><td>MA-N1420</td><td>Lift-off resit to define the platinum depostion</td></tr><tr><td>Ma-D 533s</td><td>Micro Resist Technology</td><td>MaD533S</td><td>Developer for MA-N1420</td></tr><tr><td>Platinum</td><td>-</td><td>-</td><td>Electrode and contact pad material</td></tr><tr><td>Ma-R 404s</td><td>Micro Resist Technology</td><td>MaR404S</td><td>Remover for MA-N1420</td></tr><tr><td>SU-8 3005</td><td>MicroChem Corp.</td><td>SU-8-3005</td><td>Photoresist to define the electrode area and as insulation</td></tr><tr><td>1-methoxy-2-propanol acetate</td><td>Sigma-Aldrich</td><td>108-65-6</td><td>Developer for SU-8 3005</td></tr><tr><td>2-Propanol</td><td>VWR</td><td>8.18766.2500</td><td>Removing of the SU-8 developer</td></tr><tr><td>1020R</td><td>Ultron Systems Inc.</td><td>1020R</td><td>UV sensitive adhesive tape for protection of contact pads</td></tr><tr><td>Arguna S</td><td>Degussa</td><td>1935</td><td>For Silver depostion on reference electrode</td></tr><tr><td>KCl</td><td>Methrom</td><td>62308.020</td><td>For chloridation of the silver reference electrode</td></tr><tr><td>Pyralux</td><td>DuPont</td><td>PC1025</td><td>Dry film photoresist</td></tr><tr><td>Sodium carbonat</td><td>Fluka</td><td>71352</td><td>Developer for Pyralux PC1025</td></tr><tr><td>Hydrogen chloride</td><td>Sigma-Aldrich</td><td>30720</td><td>To top the development of the DFR</td></tr><tr><td>Teflon AF 1600</td><td>DuPont</td><td>AF1600</td><td>For employing the stopping barrier</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PA104</td><td>Mega Electronics</td><td>-</td><td>Bubble etch tank</td></tr><tr><td>FED 53</td><td>Binder</td><td>9010-0018</td><td>Oven</td></tr><tr><td>SPIN150</td><td>APT</td><td>-</td><td>Spin coater</td></tr><tr><td>Pr&amp;auml;zitherm</td><td>Harry Gestigkeit GmbH</td><td>PZ 28-2</td><td>Hot plate</td></tr><tr><td>Hellas</td><td>Bungard Elektronik</td><td>40000</td><td>Exposure unit</td></tr><tr><td>Tetra30-LF-PC</td><td>Diener</td><td>-</td><td>Plasma unit</td></tr><tr><td>Univex 500</td><td>Leybold</td><td>-</td><td>Physical vapor deposition unit</td></tr><tr><td>Shaker S4</td><td>ELMI</td><td>-</td><td>Orbital shaker</td></tr><tr><td>Sonorex Super 10 P</td><td>Bandelin</td><td>783</td><td>Sonic bath</td></tr><tr><td>6221 DC and AC</td><td>Keithley</td><td>-</td><td>Current source</td></tr><tr><td>HRL 350</td><td>Ozatec</td><td>-</td><td>Laminator unit</td></tr><tr><td>Vaccum pen</td><td>EFD</td><td>-</td><td>Vacuum pen</td></tr></tbody></table>

dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation

В последние годы биомаркер диагностика стала незаменимым инструментом для диагностики заболеваний человека, особенно для диагностики точки обслуживания. Платформы easy-to-use и лоу кост датчик весьма желательно измерить различные типы аналитов (например, биомаркеры, гормоны и наркотики), количественно и конкретно. По этой причине сухой пленки фоторезиста технологии - включение дешево, снисходительный и высок объём изготовления - был использован для изготовления microfluidic биосенсор, представленные здесь. В зависимости от помощи биопроб, используемые впоследствии универсальная платформа способен обнаруживать различные типы биомолекул. Для изготовления устройства платиновые электроды строятся на гибкой полиимидные (PI) фольги в шаг только чистый номер процесса. PI фольги служит субстрат для электродов, которые изолированы на основе эпоксидных фоторезиста. Microfluidic канал впоследствии генерируется путем разработки и ламинирования сухой пленки фоторезиста (DFR) фольги на вафельные PI. С помощью гидрофобные остановки барьер в канал, канал разделяется на две конкретные области: иммобилизации раздел для энзим соединенный assay и электрохимические измерения ячеек для считывания сигнала растворенного в воде.
На чипе биопроб иммобилизации осуществляется путем адсорбции биомолекул на поверхности канала. Фермента глюкоза оксидаза используется как датчик для генерации электрохимических сигнала. Присутствии субстрат, глюкоза, производится перекиси водорода, который обнаружен на Платиновый рабочих электродом. Остановка потока техника применяется для получения усиления сигнала наряду с быстрого обнаружения. Различные биомолекул количественно может быть измерена с помощью системы внедрены microfluidic, давая указание различных видов заболеваний, или, относительно лечебного препарата мониторинг, содействие персонализированной терапии.

Проектирование и изготовление биосенсор Microfluidic платформа:
Изготовление чипов биосенсор microfluidic реализуется на уровне вафельные методами стандартных офсетным, используя несколько слоев DFR. Эта стратегия изготовление опирается на ламин?...

Watch this Scientific Journal Video about Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation at JoVE.com

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Сухой пленки фоторезиста на основе электрохимический биодатчик Microfluidic платформа: Изготовление устройств, на чипе Assay подготовка и работы системы

Биосенсор microfluidic платформа была разработана и изготовлены с использованием технологии лоу кост сухой пленки фоторезиста для быстрого и чувствительной квантификации различных аналитов. Эта система одноразовых позволяет для электрохимических индикация по чип прикол энзим соединенный анализов методике стоп потока.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

dry-film-photoresist-based-electrochemical-microfluidic-biosensor

Research

JoVE Journal

Bioengineering

11.7K Views.  University of Freiburg. Биосенсор microfluidic платформа была разработана и изготовлены с использованием технологии лоу кост сухой пленки фоторезиста для быстрого и чувствительной квантификации различных аналитов. Эта система одноразовых позволяет для электрохимических индикация по ?...

Video: Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Микрофлюидных На чипе Захват-циклоприсоединения Реакция на Обратимо Остановите малых молекул или многокомпонентные структуры для биосенсора приложений

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Chemistry

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Флуоресценция методы обнаружения микрофлюидных платформ капли

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Шелковый Фильм культуры система В пробирке Анализ и Дизайн биоматериала

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Микрофлюидных Чип для Универсальный химического анализа отдельных клеток

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Микрожидкостных основе Электрохимический Биочип для этикеток без анализа ДНК-гибридизации

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Цифровой Microfluidics автоматизированного протеомных Обработка

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

Водные капельки, используется как ферментативные микрореакторы и их электромагнитного привода

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Микрожидкостных Платформа с уплотненными электронным детектором для пространственного слежения частиц

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Электроуттинг на основе цифровой микрофлюидной платформы для автоматизированного фермента связанных Иммуносорбент Ассей

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Механо-узловое измерение пор: быстрая платформа без меток для многопараметрических одноэлементных вязкоупругих измерений

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Сухой пленки фоторезиста на основе электрохимический биодатчик Microfluidic платформа: Изготовление устройств, на чипе Assay подготовка и работы системы

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