Name: Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics (Video) | JoVE Journal
Uploaded: 2025-05-30T14:20:00.000+00:00
Duration: 6 min 3 s
Description: Explore microfluidics-based methods for investigating microtubule polymer mechanics using PDMS devices and computational modeling. This approach enables high throughput analysis of cytoskeletal dynamics with stable flow control.

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We are grateful for the support and resources provided by the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE), where a portion of this research was conducted. This work was partially funded through an NIH NIGMS grant to M. Zanic (R35 GM1192552) and NSF ID 2018661 grant to M. Zanic. M. Rogers received support from the NIH T32 GM08320 grant and a VINSE pilot funding award. L. Richardson is supported by the NSF GRFP Grant No. 1937963. The authors would also like to thank Dr. Alice Leach, David Schaffer, Dr. Christina McGahan, and the entire&#160;Zanic lab for their assistance and support.

The authors have no conflicts of interest. The authors disclose the use of ChatGPT-4o OpenAI for text revision and proofreading.

The primary goal of this protocol was to design and fabricate a microfluidic device suitable for the investigation of microtubule mechanics in vitro. The design was based on the desire to utilize the intrinsic benefits of PDMS-based microfluidic devices while also including a combination of features that would enable robust and customizable high-throughput experimentation.
This goal has been successfully achieved, resulting in fabrication protocols and general guidelines that can serve as a basis for future users of this system. The inclusion of redundant bubble traps in the device decreases the likelihood of protein denaturation due to the presence of air bubbles. While we still have some unplugging and re-plugging of tubing in the device, these bubble traps reduce the probability of experimental failure. Future improvements to the microfluidic setup could even further reduce the amount of manual tubing manipulations made during an experiment. Moreover, the integration of the microfluidic device with an automated flow control software allows for a significant customization of experimental conditions while reducing the possibility of manual error. We have demonstrated the device&#39;s successful performance by fabricating the device and then growing, stabilizing, and bending microtubule extensions in the device using automatic, controller-regulated flow. Furthermore, by establishing a gradient of distinct fluorescently labeled tubulin solutions within the same device, we showed that multiple conditions can be run simultaneously in a single device. Aided by computational modeling and analysis techniques, our system can probe and determine the biomechanical properties of microtubules, such as flexural rigidity52,56,57,58,59.
Potential future improvements would facilitate an even more robust system and associated experimental analysis. First, the photoresist deposition, exposure, and baking were crucial parameters that demonstrated some variability. The relatively tall feature sizes of the SPR photoresist required very gradual heating and cooling to prevent thermal cracking, which could ruin the devices. While thinner devices were attempted, we found issues with the manipulation of these smaller feature sizes. Attention to detail and patience are crucial for replicating devices of this thickness with SPR photoresist. Different photoresists may be used to solve this issue, depending on availability.
Taken together, the microfluidic device and protocol here allow for a range of experimental setups with more robust, high-throughput testing capabilities than previous flow-cell assays47. Furthermore, experiments can be automated using flow controllers to maintain precise flow profiles or concentration gradients in the device, reducing variability inherent to manual users. Future potential applications of this setup include investigating the effects of microtubule-associated proteins on microtubule flexural rigidity, dynamics, lattice damage, and repair, as well as the biomechanical interactions of microtubules and actin filaments54,60,61,62,63,64,65,66,67,68,69,70. The integration of microfabrication, automated flow control, and computational modeling and analysis techniques creates a versatile system suited for studying the cellular cytoskeleton in vitro.

Microfluidics enables precise control of miniscule fluid volumes, often less than one microliter, by the intricate design and fabrication of fluid-flow channels1,2. The small scale of microfluidic devices gives rise to unique engineering phenomena. Namely, the Reynolds number-a dimensionless measure of the ratio between inertial and viscous forces in fluid flow-is small, typically on the order of O(10) or lower in microfluidics, underscoring the importance of viscous forces in microfluidic devices. Additionally, the Péclet number, which compares convective to diffusive transport, shows that convective transport is generally negligible in microfluidics3,4,5. This diffusion-driven, laminar flow regime in microfluidics is advantageous, as it supports parallel experiments on a single device by maintaining precise fluid gradients.
Photolithography remains the primary method for fabricating microfluidic devices6,7,8. In brief, this process involves creating a 'master' etched template of the microfluidic design (Figure 1). A photosensitive substrate is prepared, and a photomask of the microfluidic design selectively exposes areas of photoresist to ultraviolet radiation. Subsequent etching methods develop the substrate, producing a relief of the design. Polydimethylsiloxane (PDMS) is often cast and cured onto the master. The cured PDMS, which adopts the negative features of the design, is then removed from the master and bonded to a glass coverslip. This entire fabrication process typically takes 1-2 days, enabling quick design iterations and the production of multiple devices. Detailed reviews of soft lithography and microfabrication processes are available in other references1,2,3,10,11,12,13.
<img alt="Photolithography process diagram: UV exposure, photoresist development, PDMS mold fabrication steps." class="xfigimg" src="/files/ftp_upload/68185/68185fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Overviews of the traditional photolithography process and microfabrication process. (A) Traditional photolithography process and (B) microfabrication process. Depending on the application and desired photoresist characteristics, a negative or a positive photoresist can be used, even though they will yield the same design master. Characteristics such as feature height desired or photoresist melting temperature help determine the appropriate photoresist type. This figure has been modified with permission from Rogers (2022)14. <a href="https://www.jove.com/files/ftp_upload/68185/68185fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The use of microfluidics has expanded the possibilities in many research fields, with its most recent impact being in the biological sciences. Given its small scale, microfluidics allows for the precise management of limited, valuable resources such as cells or proteins. Even more impactful is the tunability of microfluidic systems to mimic physiological conditions, such as modifications in substrate stiffness, the exertion of force on a specimen, and even the integration of electrical current. Additionally, the use of microfluidics offers the ability to manipulate multiple reagents in parallel and to rapidly prototype and iteratively refine system designs. These features enable the miniaturization of entire laboratory workflows onto a single device, commonly referred to as a "lab-on-a-chip"1,6,9,15,16,17,18,19.
One of the cell-biological applications of microfluidics is investigation of microtubule polymers. Microtubules are an essential component of the cell's cytoskeleton, playing a vital role in processes such as cell division and intracellular cargo transport20,21. As the most rigid element of the cytoskeleton, microtubules exhibit an elastic modulus comparable to that of Plexiglass22,23. Their robust mechanical properties are crucial for various cellular functions, including, for example, cardiomyocyte contraction, where they cyclically bend and relax during the heart's systolic and diastolic phases24. Microfluidic devices have been previously adopted to investigate the properties of microtubules and their higher-order structures in vitro. Indeed, microfluidics has been used to probe microtubule polymerization dynamics, microtubule-microtubule interactions, and the effects of microtubule-associated proteins on microtubule mechanical properties25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41.
While the introduction of microfluidics into the microtubule field has brought about many exciting discoveries, room for improvement still lies in the adaptation of these devices for microtubule research. In this work, we address two specific limitations that persist in studying microtubules in microfluidic devices: the potential for air bubble formation within the device, typically introduced by manual manipulation of microfluidic devices, and the underutilization of high-throughput assays. First, manual manipulations, such as plugging and unplugging tubing, can introduce bubble formation into channels. Air bubble formation within a flow cell is catastrophic, as air bubbles can denature proteins, shear microtubule polymers, and adversely affect cell cultures42,43. In addition, sharp corners and oblique angles in the device result in non-uniform surface wetting, increasing the possibility of air entrainment. Numerous techniques have been developed to reduce the formation, persistence, and impact of air bubbles; however, the use of bubble mitigation methods is not universal42,43,44,45,46. Furthermore, although one of the major advantages of using microfluidics is the ability of high-throughput experimentation, microfluidics has not yet been used to scale-up microtubule research. Microfluidic devices can be designed to test multiple experimental conditions in parallel on the same device. For example, fluid gradients can be used to direct the flow of different microtubule-associated proteins or drugs, enabling their targeted delivery to specific regions of partitioned microtubules within the same device.
Here, we iteratively designed a microfluidic device that addresses these limitations. We provide the step-by-step protocol of the fabrication of the device, thereby enabling a wider audience to employ microfluidic technology in their microtubule research. This device design incorporates bubble-trapping features and utilizes an automated flow control system to reduce manual intervention while also enabling gradients of solutions in the device for high-throughput analyses. In summary, the development of this microfluidic design can facilitate broader research and understanding of microtubule mechanics while offering valuable improvements to experimental designs across the broader microtubule research field.

<table><tbody><tr><td>0.6 mL microcentrifuge tubes (clear)</td><td>Any brand</td><td></td><td>Low retention type is preferred</td></tr><tr><td>1.5 mL microcentrifuge tubes (clear)</td><td>Any brand</td><td></td><td>Low retention type is preferred</td></tr><tr><td>1.5 mm standard biopsy punch</td><td>Integra LifeSciences</td><td>33-31A-P/25</td><td></td></tr><tr><td>100x/1.49 numerical aperture TIRF objective</td><td>Nikon</td><td></td><td></td></tr><tr><td>22 x 22 mm glass coverslips</td><td>ThorLabs</td><td>CG15CH</td><td></td></tr><tr><td>3&quot; single side polished silicon wafers</td><td>University Wafer</td><td>447</td><td></td></tr><tr><td>4&quot; Petri dish</td><td>Any brand</td><td></td><td></td></tr><tr><td>450 &amp;micro;L, Open-Top Thinwall Ultra-Clear Tube</td><td>Beckman Coulter, Inc.</td><td>345843</td><td>Referred to as &#039;airfuge tube&#039; in the protocol</td></tr><tr><td>488-, 561, and 640-nm solid state lasers</td><td>Nikon</td><td></td><td></td></tr><tr><td>A-95 Fixed-Angle Rotor</td><td>Beckman Coulter, Inc.</td><td>347595</td><td></td></tr><tr><td>Acetone</td><td>Any brand</td><td></td><td></td></tr><tr><td>Airfuge Air-Driven Ultracentrifuge</td><td>Beckman Coulter, Inc.</td><td>347854</td><td>Referred to as &#039;airfuge&#039; in the protocol</td></tr><tr><td>Alexa Fluor 488 Microscale Protein Labeling Kit</td><td>Thermo Fisher Scientific</td><td>A30006</td><td></td></tr><tr><td>Aluminum foil</td><td>Any brand</td><td></td><td></td></tr><tr><td>Andor iXon Ultra EM-CCD</td><td>Nikon</td><td></td><td></td></tr><tr><td>Andor NEO sCMOS</td><td>Nikon</td><td></td><td></td></tr><tr><td>AutoCAD</td><td>Autodesk</td><td></td><td>Generic versions can be used</td></tr><tr><td>Bovine brain unlabeled tubulin (purified)</td><td>N/A</td><td></td><td>Made in house, but can be purchased</td></tr><tr><td>Casein</td><td>MilliporeSigma</td><td>C7078</td><td></td></tr><tr><td>Catalase</td><td>MilliporeSigma</td><td>C9322</td><td></td></tr><tr><td>Clean Dry Air (CDA) (pressurized gas)</td><td>Any brand</td><td></td><td></td></tr><tr><td>Compressed air supply</td><td>Any brand</td><td></td><td>Connects to the microfluidic flow controller</td></tr><tr><td>COMSOL Multiphysics software</td><td>COMSOL, Inc.</td><td></td><td></td></tr><tr><td>Custom brass stage adapter</td><td>N/A</td><td></td><td>Made in house to fit our 22 mm x 22 mm coverslips onto the microscope</td></tr><tr><td>De-ionized water</td><td>Any brand</td><td></td><td></td></tr><tr><td>Dessicator</td><td>Any brand</td><td></td><td></td></tr><tr><td>D-glucose</td><td>MilliporeSigma</td><td>G7528</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>MilliporeSigma</td><td>D0632</td><td></td></tr><tr><td>EGTA</td><td>MilliporeSigma</td><td>324626</td><td></td></tr><tr><td>Elveflow Smart Interface (ESI) software</td><td>Elveflow</td><td></td><td></td></tr><tr><td>Flangeless PFA fittings with ETFE &amp;frac14;&amp;rdquo;-28 to 1/16&amp;rdquo; outer diameter ferrules&amp;nbsp;</td><td>Darwin Microfluidics</td><td>CIL-XP-245X</td><td>Used to connect the tubing from the micrewtube source vials to the flow sensor via the pressurized reservoir rack</td></tr><tr><td>Fluiwell 4-Channel 2 mL Low Pressure</td><td>Fluigent</td><td>14002001</td><td>Used to connect the flow control system to the the micrewtubes. Also refered to as &#039;pressurized reservoir rack&#039;</td></tr><tr><td>Fume hood</td><td>Any brand</td><td></td><td></td></tr><tr><td>Glucose oxidase</td><td>MilliporeSigma</td><td>G6125</td><td></td></tr><tr><td>GMPCPP</td><td>Jena Bioscience</td><td>NU-405L</td><td></td></tr><tr><td>Guanosine triphosphate (GTP)</td><td>MilliporeSigma</td><td>G8877</td><td></td></tr><tr><td>Hot plate</td><td>Any brand</td><td></td><td></td></tr><tr><td>HS-625 high-speed emission filter wheel</td><td>Finger Lakes Instrumentation</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>N/A</td><td></td><td>Open access</td></tr><tr><td>Incubator</td><td>Any brand</td><td></td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Any brand</td><td></td><td></td></tr><tr><td>Karl Suss MA-6 mask aligner</td><td>SUSS MicroTec</td><td></td><td></td></tr><tr><td>Magnesium chloride</td><td>MilliporeSigma</td><td>1.05833</td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks</td><td></td><td></td></tr><tr><td>MEGAPOSIT SPR 220 7.0 photoresist</td><td>Dow, Inc.</td><td></td><td></td></tr><tr><td>Microfluidic Fittings 6-40 to 1/4&quot;-28 Adapters Kit</td><td>Darwin Microfluidics</td><td>LVF-KFI-08</td><td>Used to connect the tubing from the micrewtube source vials to the flow sensor via the pressurized reservoir rack (Fluiwell rack)</td></tr><tr><td>Microfluidic Fittings Female Luer Lock Adapter Kit</td><td>Darwin Microfluidics</td><td>LVF-KFI-04</td><td>Used to connect the syringe to the tubing</td></tr><tr><td>Microfluidic flow controller</td><td>Elveflow</td><td>OB1 MK3+</td><td></td></tr><tr><td>Microfluidic flow sensor</td><td>Elveflow</td><td>MFS3</td><td>This flow sensor range is 0-80 &amp;mu;L&amp;nbsp;</td></tr><tr><td>MICROPOSIT MF-319 developer</td><td>Dow, Inc.</td><td></td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>NIS-Elements software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Nitrogen (pressurized gas)</td><td>Any brand</td><td></td><td></td></tr><tr><td>Objective heater</td><td>Tokai Hit</td><td></td><td></td></tr><tr><td>One-Piece Fingertight 10-32 Coned Fitting for 1/16&quot; OD Tubing&amp;nbsp;</td><td>Darwin Microfluidics</td><td>CIL-F-120X</td><td>Used to connect the syringe to the tubing</td></tr><tr><td>Paclitaxol (Taxol)</td><td>Tocris Bioscience</td><td>1097</td><td></td></tr><tr><td>Photolithography masks</td><td>N/A</td><td></td><td>Made by an external party using our designs</td></tr><tr><td>PIPES</td><td>Thermo Fisher Scientific</td><td>172615000</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Plasma flowmeter system</td><td>Harrick Plasma</td><td>PDC-FMG</td><td>Integrates with plasma cleaner to enable flow control of pressurized gas</td></tr><tr><td>Plastic bulb pipet</td><td>Any brand</td><td></td><td></td></tr><tr><td>Pluoronic F-127</td><td>MilliporeSigma</td><td>P2443</td><td>Referred to as &#039;poloxomer 407&#039; in the protocol</td></tr><tr><td>Potassium chloride</td><td>Research Products International</td><td>P41000</td><td></td></tr><tr><td>Saint Gobain Performance Plastics&amp;nbsp;Tube Tygon .020 ID</td><td>Thermo Fisher Scientific</td><td>50-206-8921</td><td>Refered to as &#039;1.5 mm tubing&#039; and &#039;tubing&#039; in the protocol</td></tr><tr><td>Scalpel</td><td>Any brand</td><td></td><td></td></tr><tr><td>Spin coater</td><td>Cost Effective Equipment, LLC.</td><td>200x</td><td>This model may be discontinued</td></tr><tr><td>Standard pipets and tip sets</td><td>Any brand</td><td></td><td></td></tr><tr><td>Standard plastic syringe</td><td>Any brand</td><td></td><td>We used a 10 mL Luer-slip syringe</td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow, Inc.</td><td></td><td>Referred to as &#039;PDMS&#039; and &#039;curing agent&#039; in the protocol</td></tr><tr><td>T339 Micrewtube with Lip Seal and Flat Screw Cap</td><td>Medline Industried, LP.</td><td>T339</td><td>Referred to as &#039;source vial&#039; in the protocol. We used both 0.5 mL and 1.5 mL sizes</td></tr><tr><td>TAMRA, SE; 5-(and-6)-Carboxytetramethylrhodamine, Succinimidyl Ester</td><td>Invitrogen</td><td>C1171</td><td>Referred to as &#039;TTR&#039; in the protocol</td></tr><tr><td>Trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane</td><td>MilliporeSigma</td><td>448931</td><td></td></tr><tr><td>Trion Phantom RIE ICP&amp;nbsp;</td><td>Trion Technology, Inc.</td><td></td><td>This plasma cleaner is only used in Step 1.1 of the protocol. Another plasma cleaner, like the one used for PDMS bonding, can be used instead; we just prefer the much lower vacuum achievable by this system for cleaning the silicon wafer</td></tr><tr><td>TRITC Polyclonal Antibody</td><td>Thermo Fisher Scientific</td><td>A6397</td><td>Referred to as &#039;anti-rhodamine antibody&#039; in the protocol</td></tr><tr><td>Tweezers</td><td>Any brand</td><td></td><td></td></tr><tr><td>Vacuum pump</td><td>Any brand</td><td></td><td></td></tr></tbody></table>

development of a microfluidics-based approach for investigating microtubule polymer mechanics

Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics

In this protocol, we describe the design and fabrication of a microfluidic device developed for the investigation of microtubule polymer mechanics. The design utilizes the intrinsic benefits of Polydimethylsiloxane (PDMS)-based microfluidic devices and introduces several features to enable a robust and customizable high-throughput experimental approach. The developed device incorporates redundant bubble-trapping capabilities to prevent the occurrence of detrimental air bubbles. Furthermore, the device interfaces with an automated flow control system to reduce manual intervention and enable high-throughput analyses. Commercial simulation software is utilized to better develop and understand the fluid transport using this system. Finally, we demonstrate the capability to conduct multiple experiments simultaneously within a single device by growing microtubule extensions with distinct fluorescent labels in different sections of the device. Overall, this microfluidic flow system can be used to probe microtubule polymer mechanics and provides improvements in experimental design for broader microtubule in vitro studies. The synthesis of microfabrication, automated flow control, and computational modeling approaches enables a flexible system ideally suited for probing the cellular cytoskeleton in vitro.

This protocol details the design and fabrication of a microfluidic device suitable for investigating microtubule polymer mechanics. The synthesis of microfabrication, automated flow control, and computational modeling techniques enables a flexible system ideally suited for probing the cellular cytoskeleton in vitro.

In this protocol, we describe the design and fabrication of a microfluidic device developed for the investigation of microtubule polymer mechanics. The ...

development-microfluidics-based-approach-for-investigating

Research

JoVE Journal

Bioengineering

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Explore microfluidics-based methods for investigating microtubule polymer mechanics using PDMS devices and computational modeling. This approach enables high throughput analysis of cytoskeletal dynamics with stable flow control.

Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics

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