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

Summary

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.

Abstract

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.

Introduction

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 composition^2,3,4, 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

Protocol

1. Design device geometry

1. Choose the width of the sizing and recovery segments so that it is wider than the diameter of the largest cells to be measured but also maintains a sufficient signal-to-noise ratio (SNR). See Supplementary Table 2 for examples of different sizing and recovery segment widths for various cell lines.
2. Choose the contraction segment width to apply a 30%-40% strain to the average size of the cells that are to undergo mechanophenotyping. Strain is defined as , where

Results

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 .......

Discussion

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

Materials

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	SP131825
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" (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