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* These authors contributed equally
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.
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.
Single cells are dynamic, viscoelastic materials1. A multitude of internal and external processes, (e.g., onset of mitosis or remodeling of the extracellular matrix [ECM]), influence their structure and composition2,3,4, often resulting in distinct biophysical properties that complement their current state. In particular, mechanical properties have been shown to be important biomarkers of cellular development, physiology, and pathology, yielding valuable quantitative information that can supplement canonical molecular and genetic approaches
1. Design device geometry
The mechanophenotyping platform presented here is a simple and versatile approach for measuring the biophysical properties of single cells with moderate throughput. Cells are flowed through the microfluidic channel (Figure 1A) using constant pressure-driven flow. As the cells transit, the length of the microfluidic channel and the current pulses produced are recorded using the data acquisition hardware. The acquired signal (Figure 1B,C) is then .......
Measuring the mechanical properties of single cells using this mechanophenotyping technique consists of three stages: device fabrication, data acquisition, and data analysis. Within each stage, there are notable aspects that may significantly impact the experimental results. During device fabrication, consistent channel geometries and device-to-device uniformity are essential for accurate and repeatable results. Specifically, the sidewalls of each device should be relatively smooth (Figure 4Ai
This research was supported by grants from NIBIB 1R01EB024989-01 and NCI 1R01CA190843-01. A. L. and R. R. were supported by an H2H8 Association Graduate Research Fellowship. K. L. C. was supported by a National Science Foundation Graduate Research Fellowship and a Siebel Scholar Fellowship.
....Name | Company | Catalog Number | Comments |
Acetone | J.T. Baker | 5356-05 | Purity (GC) ≥ 99.5% (https://us.vwr.com/store/product/6057739/acetone-99-5-vlsi-j-t-baker) |
Aluminum Foil | n/a | n/a | |
Analog Low-Pass Filter | ThorLabs | EF504 | ≤240 kHz Passband, Coaxial BNC Feedthrough (https://www.thorlabs.com/thorproduct.cfm?partnumber=EF504#ad-image-0) |
Biopsy Punch | Integra Miltex | 33-31AA-P/25 | 1mm, Disposable, with Plunger (https://mms.mckesson.com/product/573313/Miltex-33-31AA-P25) |
Blade | n/a | n/a | |
BNC Cable | Pomona Electronics | 2249-C-12 | https://www.digikey.com/en/products/detail/pomona-electronics/2249-C-12/603323?utm_adgroup=Coaxial%20Cables%20%28RF%29&utm_source=google&utm_ medium=cpc&utm_campaign= Shopping_Product_Cable%20Assemblies_NEW&utm_term= &utm_content=Coaxial%20Cables%20%28RF%29&gclid=Cj0KCQjwlK-WBhDjARIsAO2sErQqnVJ pj5OXVObuTI8ZUf1ZeIn7zvzGnx mCWdePrG6SdEJMF3X6ubUaAs w-EALw_wcB |
Cleanroom Polyester Swab | Thermo Fisher Scientific | 18383 | https://www.fishersci.com/shop/products/texwipe-cleantip-alpha-polyester-series-swabs-6/18383 |
Current Preamplifier | DL Instruments | 1211 | https://www.brltest.com/index.php?main_page=product_info&products_ id=1419 |
Custom PCB (w/ components) | n/a | n/a | see Supplemental files 4 and 5 |
DAQ Terminal Block | National Instruments | BNC-2120 | https://www.ni.com/en-in/support/model.bnc-2120.html |
DAQ to BNC-2110 cable | National Instruments | SHC68-68-EPM | https://www.ni.com/en-in/support/model.shc68-68-epm.html |
Data Acquisition Board (DAQ) | National Instruments | PCI-6251 | https://www.ni.com/docs/en-US/bundle/pci-6251-feature/page/overview.html |
Dessicator | Thermo Fisher Scientific | 5311-0250 | https://www.thermofisher.com/order/catalog/product/5311-0250 |
Female BNC To Banana Plug Adapter | Pomona Electronics | 72909 | https://www.digikey.com/en/products/detail/pomona-electronics/72909/1196318 |
Fetal Bovine Serum (FBS) | VWR | 89510-186 | https://us.vwr.com/store/product/18706419/avantor-seradigm-select-grade-usda-approved-origin-fetal-bovine-serum-fbs |
Glass Cutter | Chemglass | CG-1179-21 | https://chemglass.com/plate-glass-cutters-diamond-tips |
Gold Etchant TFA | Transene | NC0977944 | https://www.fishersci.com/shop/products/NC0977944/NC0977944 |
Hot Plate | Thermo Fisher Scientific | SP131825Â | |
Isopropyl Alcohol | Spectrum Chemical | I1056-4LTPL | Purity (GC) ≥99.5% (https://www.spectrumchemical.com/isopropyl-alcohol-99-percent-fcc-i1056) |
Metal Hardware Enclosure | Hammond Manufacturing | EJ12126 | https://www.digikey.com/en/products/detail/hammond-manufacturing/EJ12126/2423415 |
Methanol | Sigma-Aldrich | 34860 | Purity (GC) ≥99.8% (https://www.sigmaaldrich.com/IN/en/substance/methanol320467561) |
MF-321 Developer | Kayaku Advanced Materials | n/a | https://kayakuam.com/products/mf-321/ |
MICROPOSIT S1813 Positive Photoresist | DuPont | n/a | https://kayakuam.com/products/microposit-s1800-g2-series-photoresists/ |
Phosphate Buffered Saline (PBS) | Thermo Fisher Scientific | 10010049 | https://www.thermofisher.com/order/catalog/product/10010049?SID=srch-hj-10010049 |
Photomask | Fineline Imaging | n/a | Photomask are custom ordered from our CAD designs (https://www.fineline-imaging.com/) |
Plain Glass Microscope Slide | Fisher Scientific | 12-553-5B | Material: Soda Lime, L75 x W50 mm, Thickness: 0.90–1.10 mm |
Plasma Cleaner | Harrick Plasma | PDC-001 | https://harrickplasma.com/plasma-cleaners/expanded-plasma-cleaner/ |
Plastic Petri Dish | Thermo Fisher Scientific | FB0875712 | 100 mm (https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-raised-ridge-100-x-15mm/FB0875712) |
Pressure Controller | Fluigent | MFCS-EZ | https://www.fluigent.com/research/instruments/pressure-flow-controllers/mfcs-series/ |
Pressure Controller Software | Fluigent | MAESFLO | |
Programming & Computation Software | MATLAB | R2021b | for data acquisition and analysis (https://www.mathworks.com/products/matlab.html) |
PTFE Tubing | Cole Parmer | 06417-31 | 0.032" ID x 0.056"Â (https://www.coleparmer.com/i/masterflex-transfer-tubing-microbore-ptfe-0-032-id-x-0-056-od-100-ft-roll/0641731) |
Scepter 2.0 Handheld Automatic Cell Counter | Millapore Sigma | PHCC20060 | https://www.sigmaaldrich.com/IN/en/product/mm/phcc20060 |
Silicon Wafer | Wafer World | 2885 | 76.2 mm, Single Side Polished (https://www.waferworld.com/product/2885) |
Spin Coater | n/a | n/a | |
SU-8 3025 Negative Photoresist | Kayaku Advanced Materials | n/a | https://kayakuam.com/products/su-8-2000/ |
SU8 Developer | Kayaku Advanced Materials | n/a | https://kayakuam.com/products/su-8-developer/ |
Sygard 184 Polydimethlysiloxane | Dow Chemical | 4019862 | https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/ |
Tape | Scotch | 810-341296 | https://www.staples.com/Scotch-Magic-Tape-810-3-4-x-36-yds-1-Core/product_130567?cid=PS:GS:SBD:PLA:OS&gclid= Cj0KCQjwlK-WBhDjARIsAO 2sErRwzrrgjU0NjFkDkne1xm vT7ekS3tdzvAgiMDwPoxocgH VTQZi7vJgaAvQZEALw_wcB |
Titanium, Platinum, Gold | n/a | n/a | |
Triple Output Power Supply | Keysight | E36311A | https://www.newark.com/keysight-technologies/e36311a/dc-power-supply-3o-p-6v-5a-prog/dp/15AC9653 |
UV Mask Aligner | Karl Suss America | MJB3 Mask Aligner |
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