אנו מודים לקבוצת קניס מאוניברסיטת אילינוי באורבנה-Champaign לדיונים מועיל בנדיבות מתן שימוש במתקנים cleanroom. עבודה זו מומנה על ידי מסלול NIH כדי פרס לצרכן העצמאות, תחת גרנט לא 4R00HG004183-03 (Charles M. שרדר Melikhan Tanyeri). עבודה זו נתמכה על ידי הקרן הלאומית למדע באמצעות מלגת מחקר לתארים מתקדמים על אריק ג&#39;ונסון מ-Chavarria.

<ol>
	<li>Tanyeri, M., Johnson-Chavarria, E. M., Schroeder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Trap+for+Single+Particles+and+Cells.">Hydrodynamic Trap for Single Particles and Cells.</a> Applied Physics Letters. 96, 224101-224101 (2010).</li><li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Optics Letters. 11, 288-290 (1986).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Review of Scientific Instruments. 75, 2787-2809 (2004).</li><li>Gosse, C., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+tweezers:+Micromanipulation+and+force+measurement+at+the+molecular+level.">Magnetic tweezers: Micromanipulation and force measurement at the molecular level.</a> Biophysical Journal. 82, 3314-3329 (2002).</li><li>Chiou, P. Y., Ohta, A. T., Wu, M. C., C, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+manipulation+of+single+cells+and+microparticles+using+optical+images.">Massively parallel manipulation of single cells and microparticles using optical images.</a> Nature. 436, 370-372 (2005).</li><li>Cohen, A. E., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+trapping+and+manipulating+nanoscale+objects+in+solution.">Method for trapping and manipulating nanoscale objects in solution.</a> Applied Physics Letters. 86, 093109-09 (2005).</li><li>Evander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+acoustic+cell+trapping+in+a+microfluidic+perfusion+system+for+online+bioassays&amp;quot;,+Analytical+Chemistry+79.">Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays&amp;quot;, Analytical Chemistry 79.</a> , 2984-2991 (2007).</li><li>Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolithic+microfabricated+valves+and+pumps+by+multilayer+soft+lithography.">Monolithic microfabricated valves and pumps by multilayer soft lithography.</a> Science. 288, 113-116 (2000).</li><li>Kim, M. C., Wang, Z. H., Lam, R. H. W., Thorsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+better+cell+trap:+Applying+Lagrangian+modeling+to+the+design+of+microfluidic+devices+for+cell+biology.">Building a better cell trap: Applying Lagrangian modeling to the design of microfluidic devices for cell biology.</a> Journal of Applied Physics. 103, (2008).</li><li>Lutz, B. R., Chen, J., Schwartz, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+tweezers:+1.+Noncontact+trapping+of+single+cells+using+steady+streaming+microeddies.">Hydrodynamic tweezers: 1. Noncontact trapping of single cells using steady streaming microeddies.</a> Analytical Chemistry. 78, 5429-5435 (2006).</li></ol>

אין ניגודי אינטרסים הכריז.

שיטות microfluidic נוכחי מניפולציה החלקיקים מבוסס על זרימה הידרודינמית ניתן לאפיין שיטות מגע מבוסס או ללא מגע. צור בשיטות מבוססות להשתמש זרימת הנוזל פיזית להגביל ו לשתק חלקיקים בקירות microfabricated ערוץ 9, ואילו ללא מגע שיטות להסתמך על מחזורי זרימה או microeddies 10. בעבוד...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>21 gauge blunt needle</td>
<td>&nbsp;</td>
<td> Zephyrtronics</td>
<td> ZT-5-021-1-L</td>
<td>For punching port holes in PDMS</td>
</tr>
<tr>
<td>3 ml plastic syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309585</td>
<td>For filling valve with oil</td>
</tr>
<tr>
<td>Si wafers</td>
<td>&nbsp;</td>
<td>University Wafer</td>
<td>&nbsp;</td>
<td> 3&#x201D; P(100) single side polished 380 &mu;m test grade</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48404-428</td>
<td>24 x 40 mm #1.5</td>
</tr>
<tr>
<td>DAQ card</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI 6229</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent beads</td>
<td>&nbsp;</td>
<td>Spherotech</td>
<td>FP-2056-2</td>
<td>2.2 &mu;m Nile red</td>
</tr>
<tr>
<td>Fluorinert </td>
<td>&nbsp;</td>
<td>3M</td>
<td>FC 40</td>
<td>Fluorinated carrier oil</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>IX-71</td>
<td>&nbsp;</td>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Version 9.0f3 (32bit)</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>MZ6</td>
<td>For aligning PDMS control layer to fluidic layer.</td>
</tr>
<tr>
<td>Mechanical Convection Oven</td>
<td>&nbsp;</td>
<td> VWR</td>
<td>1300U</td>
<td>For baking devices to create monolithic PDMS slabs with two layers.</td>
</tr>
<tr>
<td>Microfluidic tubing and connectors</td>
<td>&nbsp;</td>
<td>Upchurch Scientific</td>
<td>&nbsp;</td>
<td>1/16 x .020 PFA tubing and super flangeless fittings</td>
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<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>GE Silicones</td>
<td>RTV 615 A&amp;B</td>
<td>&nbsp;</td>
<tr>
<td>Plasma Chamber</td>
<td>&nbsp;</td>
<td>Harrick</td>
<td>PDC-001</td>
<td>&nbsp;</td>
<tr>
<td>Pressure Transducer</td>
<td>&nbsp;</td>
<td>Proportion Air</td>
<td>DQPV1</td>
<td>&nbsp;</td>
<tr>
<td>Spin Coater</td>
<td>&nbsp;</td>
<td>Specialty Coating Systems</td>
<td>G3P-8 Spin Coat</td>
<td>&nbsp;</td>
<tr>
<td>Photoresist</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU 8 2050</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000 Programmable</td>
<td>&nbsp;</td>
<tr>
<td>Terminal Block</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>BNC 2110</td>
<td>For analog output to pressure regulator and read out.</td>
</tr>
<tr>
<td>UV Collimated Light Source and Exposure System</td>
<td>&nbsp;</td>
<td>OAI</td>
<td>Model 30 Enhanced Light Source</td>
<td>&nbsp;</td>		</tbody>
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a microfluidic-based hydrodynamic trap for single particles

היכולת להגביל ולטפל חלקיקים יחיד פתרון חופשית היא טכנולוגיה המאפשרת מפתח עבור המדע הבסיסי והיישומי. שיטות לכידה של חלקיקים המבוססת על טכניקות אופטי, מגנטי, electrokinetic, ואקוסטי הובילו התקדמות מרכזי פיזיקה וביולוגיה מולקולרית החל ברמה התאית. במאמר זה, אנו מציגים שיטה חדשה microfluidic מבוסס על השמנה החלקיקים מניפולציה מבוססת אך ורק על זרימת הנוזל הידרודינמית. באמצעות שיטה זו, אנו מדגימים לכידה של חלקיקים מיקרו בקנה מידה ננו תמיסות מימיות של קשקשים זמן רב. מלכודת הידרודינמית מורכב מכשיר microfluidic משולב עם גאומטריה צולבות חריץ ערוץ שבו שני זרמים מנוגדים להתכנס למינרית, ובכך לייצר זרימת extensional מישוריים עם נקודת קיפאון נוזל (מהירות אפס נקודה). במכשיר זה, חלקיקים כלואים במרכז מלכודת בשלט פעיל של שדה הזרימה לשמור על המיקום החלקיק בנקודת קיפאון הנוזל. באופן זה, חלקיקים לכודים ביעילות פתרון חינם באמצעות בקרת משוב האלגוריתם מיושם עם קוד LabVIEW שהותקן. אלגוריתם הבקרה כוללת רכישת התמונה חלקיק במכשיר microfluidic, ואחריו מעקב החלקיקים, קביעת מיקום החלקיקים centroid, והתאמת הפעילות של זרימת הנוזל על ידי ויסות הלחץ המופעל על שבב שסתום פנאומטי באמצעות ווסת לחץ. בדרך זו, על שבב דינמי שסתום פונקציות מדידה להסדיר את ספיקות יחסית ערוצי לשקע, ובכך לאפשר קנס בהיקף של משרה מלאה קיפאון נקודת לכידה החלקיקים. מלכודת microfluidic מבוססי הידרודינמית מוצגים מספר יתרונות כשיטה השמנה החלקיקים. השמנה הידרודינמית אפשרי עבור כל חלקיק שרירותי ללא דרישות ספציפיות על המאפיינים פיזי או כימי של האובייקט לכודים. בנוסף, השמנה הידרודינמית מאפשרת הכליאה של אובייקט &quot;יחיד&quot; היעד השעיות חלקיק מרוכז או צפוף, וזה קשה באמצעות כוח חלופי בתחום מבוססי שיטות לכידה. מלכודת הידרודינמית הוא ידידותי למשתמש, פשוט ליישם ניתן להוסיף מכשירים microfluidic הקיימים כדי להקל על ניתוח השמנה זמן רב של חלקיקים. בסך הכל, מלכודת הידרודינמית היא פלטפורמה חדשה הכליאה, המיקרומניפולציה, ותצפית של חלקיקי ללא immobilization השטח מבטלת את הצורך שדות אופטיים, מגנטיים, חשמליים perturbative פוטנציאל השמנה פתרון חופשי של חלקיקים קטנים.

Watch this Scientific Journal Video about A Microfluidic-based Hydrodynamic Trap for Single Particles at JoVE.com

A Microfluidic-based Hydrodynamic Trap for Single Particles

במאמר זה, אנו מציגים שיטה microfluidic מבוסס על הכליאה החלקיקים מבוסס על זרימה הידרודינמית. אנו מדגימים לכידה חלקיק יציב בנקודה נוזל הקיפאון באמצעות מנגנון בקרה ומשוב, ובכך מאפשרים כליאה ו המיקרומניפולציה של חלקיקים שרירותי microdevice משולבת.

Microfluidic מבוססי הידרודינמית מלכודת חלקיקים יחיד

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

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16.6K Views.  University of Illinois at Urbana-Champaign. במאמר זה, אנו מציגים שיטה microfluidic מבוסס על הכליאה החלקיקים מבוסס על זרימה הידרודינמית. אנו מדגימים לכידה חלקיק יציב בנקודה נוזל הקיפאון באמצעות מנגנון בקרה ומשוב, ובכך מאפשרים כליאה ו המיקרומניפולציה של חלקיקים שרירותי microdevice משולבת.

Video: A Microfluidic-based Hydrodynamic Trap for Single Particles

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis מבוסס microfluidic לניתוח כמותי על פי דרישה של<em&gt; Elegans Caenorhabditis</em&gt; &#39;נידות

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

פרוטוקול להערכה הידרודינמית יחסית של הפולימר שסתום Tri-עלון

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

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;

שבב microfluidic לניתוח הכימי תכליתי של תאים בודדים

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

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

חוקר מערכות קולטן ליגנד של Cellulosome עם המולקולה בודדת מבוססת AFM חיל ספקטרוסקופיה

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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.

Biochip אלקטרוכימי מבוסס Microfluidic לניתוח הכלאה DNA ללא תווית

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

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

הזמנת תאים בודדים עוברים יחיד מאסר 3D: התקן חדש עבור הקרנת תוכן גבוהה

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

טיפוח של תאי יונקים באמצעות מערכת פניאומטיים Bioreactor יחיד שימוש

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

טכניקה ללא תווית להדמית החלל-הזמן של פרשות תא בודדת

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

פלטפורמת Microfluidic עבור בידוד תפוקה גבוהה מתא בודד ותרבות

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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.

פלטפורמת microfluidic עם איתור Multiplexed אלקטרוני למערכות מעקב מרחבית של חלקיקים

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