Name: mechano-node-pore sensing: a rapid, label-free platform for multi-parameter single-cell viscoelastic measurements
Uploaded: 2022-12-02T14:20:00
Description: Watch this Scientific Journal Video about Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements at JoVE.com

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.

1. Design device geometry
<ol>
	<li>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.</li>
	<li>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...

<ol>
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(PIBM 2014). 92300, (2014).</li><li>Alcaraz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrheology+of+human+lung+epithelial+cells+measured+by+atomic+force+microscopy.">Microrheology of human lung epithelial cells measured by atomic force microscopy.</a> Biophysical Journal. 84 (3), 2071-2079 (2003).</li><li>Li, M., Dang, D., Liu, L., Xi, N., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+in+characterizing+cell+mechanics+for+biomedical+applications:+A+review.">Atomic force microscopy in characterizing cell mechanics for biomedical applications: A review.</a> IEEE Transactions on Nanobioscience. 16 (6), 523-540 (2017).</li><li>Urbanska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+microfluidic+methods+for+high-throughput+cell+deformability+measurements.">A comparison of microfluidic methods for high-throughput cell deformability measurements.</a> Nature Methods. 17, 587-593 (2020).</li><li>Hill, R. 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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</stron...

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 approaches5,6,7. For example, Li et al. recently described the mechanical differences between drug-resistant and drug-responsive acute promyelocytic leukemia cells, while also using RNA-seq to uncover differentially-expressed cytoskeleton-associated genes8. By understanding the complex interplay between single-cell mechanics and cellular function, mechanophenotyping has broader applications in transforming basic science and clinical diagnostics9.
The most widely adopted tool for measuring single-cell mechanics is atomic force microscopy (AFM). While AFM enables a high-resolution, localized measurement of cellular mechanical properties, it remains limited to a throughput of &#60;0.01 cells/s10. Alternatively, optical stretchers, which use two divergent laser beams to trap and deform suspended single cells11, are limited to marginally higher throughputs of &#60;1 cell/s12. Recent advances in microfluidic technologies have enabled a new generation of devices for rapid, single-cell, mechanical assessment12,13. These techniques employ narrow constriction channels14,15, shear flow16, or hydrodynamic stretching17 to deform cells quickly at throughputs of 10-1,000 cells/s18. While the measurement rate of these approaches is considerably faster than conventional techniques, they often trade high-throughput capabilities for limited mechanical readouts (Supplementary&#160;Table 1).&#160;All the aforementioned rapid microfluidic methods focus on basic, single-parameter metrics, such as transit time or deformability ratios, that only reflect a cell&#39;s elastic properties. However, given the intrinsic viscoelastic nature of single cells, a robust and thorough mechanical characterization of cells requires consideration of not only elastic components but also viscous responses.
Mechano-node-pore sensing (mechano-NPS)2,8 (Figure 1A) is a microfluidic platform that addresses existing limitations with single-cell mechanophenotyping. This method enables the measurement of multiple biophysical parameters simultaneously, including cell diameter, relative deformability, and recovery time from deformation, with a moderate throughput of 1-10 cells/s. This technique is based on node-pore sensing (NPS)19,20,21,22,23,24, which involves using a four-point probe measurement to measure the modulated current pulse produced by a cell transiting a microfluidic channel that has been segmented by wider regions, referred to as &#34;nodes&#34;. The modulated current pulse is a result of the cell partially blocking the flow of current in the segments (i.e., &#34;pores&#34;) and nodes, with more current blocked in the former than in the latter. In mechano-NPS, one segment, the &#34;contraction channel&#34;, is narrower than a cell diameter; consequently, a cell must deform to transit the entire channel (Figure 1B). Cell diameter can be determined by the magnitude of the subpulse produced when the cell transits the node-pores prior to the contraction channel (Figures 1B,C). Here, |&#916;Inp|, the current drop when the cell is in the pore,&#160;is proportional to the volume ratio of the cell to the pore, Vcell/Vpore2,8,19. Cell stiffness can be determined by &#916;Tc, the duration of the dramatically larger subpulse produced when the cell transits the contraction channel (Figures 1B,C). A stiffer cell will take longer to transit the channel than a softer one2,8. Finally, cell &#34;recovery&#34;, the cell&#39;s ability to return to its original size and shape post deformation, can be determined by the series of subpulses produced as the cell transits the node-pores after the contraction channel (Figures 1B,C). The recovery time, &#916;Tr, is the time it takes for the current subpulses to return to the magnitude of the previous subpulses, prior to the cell being squeezed. Overall, the modulated current pulses produced as a cell transits the microfluidic channel are recorded and analyzed to extract the relevant single-cell mechanical parameters (Figure 1D)2,8.
The reproducibility and ease of use of this electronics-based microfluidic platform have been previously demonstrated25. Additionally, the platform presents a low barrier to entry for single-cell mechanophenotyping. Standard soft lithography is employed to fabricate microfluidic devices. The measurement hardware consists of inexpensive components, including a simple printed circuit board (PCB), power supply, preamplifier, data acquisition board (DAQ), and computer. Finally, user-friendly code is available for data acquisition and analysis, enabling straightforward implementation. This mechanophenotyping technique can distinguish populations of non-malignant and malignant breast and lung epithelial cell lines, discriminate between sublineages in primary human mammary epithelial cells, and characterize the effects of cytoskeletal perturbations and other pharmacological agents2,8. Overall, this platform is an effective approach for the mechanophenotyping of single cells.

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			<td>Acetone</td>
			<td>J.T. Baker</td>
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			<td>&#8804;240 kHz Passband, Coaxial BNC Feedthrough&#160;(https://www.thorlabs.com/thorproduct.cfm?partnumber=EF504#ad-image-0)</td>
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			<td>Biopsy Punch</td>
			<td>Integra Miltex</td>
			<td>33-31AA-P/25</td>
			<td>1mm, Disposable, with Plunger (https://mms.mckesson.com/product/573313/Miltex-33-31AA-P25)</td>
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			<td>BNC Cable</td>
			<td>Pomona Electronics</td>
			<td>2249-C-12</td>
			<td>https://www.digikey.com/en/products/detail/pomona-electronics/2249-C-12/603323?utm_adgroup=Coaxial%20Cables%20%28RF%29&#38;utm_source=google&#38;utm_ 
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			<td>Cleanroom Polyester Swab</td>
			<td>Thermo Fisher Scientific</td>
			<td>18383</td>
			<td>https://www.fishersci.com/shop/products/texwipe-cleantip-alpha-polyester-series-swabs-6/18383</td>
		</tr>
		<tr>
			<td>Current Preamplifier</td>
			<td>DL Instruments</td>
			<td>1211</td>
			<td>https://www.brltest.com/index.php?main_page=product_info&#38;products_ 
			id=1419</td>
		</tr>
		<tr>
			<td>Custom PCB (w/ components)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>see Supplemental files 4 and 5</td>
		</tr>
		<tr>
			<td>DAQ Terminal Block</td>
			<td>National Instruments</td>
			<td>BNC-2120</td>
			<td>https://www.ni.com/en-in/support/model.bnc-2120.html</td>
		</tr>
		<tr>
			<td>DAQ to BNC-2110 cable&#160;</td>
			<td>National Instruments</td>
			<td>SHC68-68-EPM</td>
			<td>https://www.ni.com/en-in/support/model.shc68-68-epm.html</td>
		</tr>
		<tr>
			<td>Data Acquisition Board (DAQ)</td>
			<td>National Instruments</td>
			<td>PCI-6251</td>
			<td>https://www.ni.com/docs/en-US/bundle/pci-6251-feature/page/overview.html</td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td>Thermo Fisher Scientific</td>
			<td>5311-0250</td>
			<td>https://www.thermofisher.com/order/catalog/product/5311-0250</td>
		</tr>
		<tr>
			<td>Female BNC To Banana Plug Adapter</td>
			<td>Pomona Electronics</td>
			<td>72909</td>
			<td>https://www.digikey.com/en/products/detail/pomona-electronics/72909/1196318</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td>https://us.vwr.com/store/product/18706419/avantor-seradigm-select-grade-usda-approved-origin-fetal-bovine-serum-fbs</td>
		</tr>
		<tr>
			<td>Glass Cutter</td>
			<td>Chemglass</td>
			<td>CG-1179-21</td>
			<td>https://chemglass.com/plate-glass-cutters-diamond-tips</td>
		</tr>
		<tr>
			<td>Gold Etchant TFA</td>
			<td>Transene</td>
			<td>NC0977944</td>
			<td>https://www.fishersci.com/shop/products/NC0977944/NC0977944</td>
		</tr>
		<tr>
			<td>Hot Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>SP131825&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Spectrum Chemical</td>
			<td>I1056-4LTPL</td>
			<td>Purity (GC)&#160; &#8805;99.5% (https://www.spectrumchemical.com/isopropyl-alcohol-99-percent-fcc-i1056)</td>
		</tr>
		<tr>
			<td>Metal Hardware Enclosure</td>
			<td>Hammond Manufacturing</td>
			<td>EJ12126</td>
			<td>https://www.digikey.com/en/products/detail/hammond-manufacturing/EJ12126/2423415</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Purity (GC)&#160; &#8805;99.8% (https://www.sigmaaldrich.com/IN/en/substance/methanol320467561)</td>
		</tr>
		<tr>
			<td>MF-321 Developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/mf-321/</td>
		</tr>
		<tr>
			<td>MICROPOSIT S1813 Positive Photoresist</td>
			<td>DuPont</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/microposit-s1800-g2-series-photoresists/</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>https://www.thermofisher.com/order/catalog/product/10010049?SID=srch-hj-10010049</td>
		</tr>
		<tr>
			<td>Photomask</td>
			<td>Fineline Imaging</td>
			<td>n/a</td>
			<td>Photomask are custom ordered from our CAD designs (https://www.fineline-imaging.com/)</td>
		</tr>
		<tr>
			<td>Plain Glass Microscope Slide</td>
			<td>Fisher Scientific</td>
			<td>12-553-5B</td>
			<td>Material: Soda Lime, L75 x W50 mm, Thickness: 0.90&#8211;1.10 mm&#160;</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>https://harrickplasma.com/plasma-cleaners/expanded-plasma-cleaner/</td>
		</tr>
		<tr>
			<td>Plastic Petri Dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875712</td>
			<td>100 mm (https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-raised-ridge-100-x-15mm/FB0875712)</td>
		</tr>
		<tr>
			<td>Pressure Controller</td>
			<td>Fluigent</td>
			<td>MFCS-EZ</td>
			<td>https://www.fluigent.com/research/instruments/pressure-flow-controllers/mfcs-series/</td>
		</tr>
		<tr>
			<td>Pressure Controller Software</td>
			<td>Fluigent</td>
			<td>MAESFLO</td>
			<td></td>
		</tr>
		<tr>
			<td>Programming &#38; Computation Software</td>
			<td>MATLAB</td>
			<td>R2021b</td>
			<td>for data acquisition and analysis (https://www.mathworks.com/products/matlab.html)</td>
		</tr>
		<tr>
			<td>PTFE Tubing</td>
			<td>Cole Parmer</td>
			<td>06417-31</td>
			<td>0.032&#34; ID x 0.056&#34;&#160;(https://www.coleparmer.com/i/masterflex-transfer-tubing-microbore-ptfe-0-032-id-x-0-056-od-100-ft-roll/0641731)</td>
		</tr>
		<tr>
			<td>Scepter 2.0 Handheld Automatic Cell Counter</td>
			<td>Millapore Sigma</td>
			<td>PHCC20060</td>
			<td>https://www.sigmaaldrich.com/IN/en/product/mm/phcc20060</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>Wafer World</td>
			<td>2885</td>
			<td>76.2 mm, Single Side Polished (https://www.waferworld.com/product/2885)</td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025 Negative Photoresist</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/su-8-2000/</td>
		</tr>
		<tr>
			<td>SU8 Developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/su-8-developer/</td>
		</tr>
		<tr>
			<td>Sygard 184 Polydimethlysiloxane</td>
			<td>Dow Chemical</td>
			<td>4019862</td>
			<td>https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Scotch</td>
			<td>810-341296</td>
			<td>https://www.staples.com/Scotch-Magic-Tape-810-3-4-x-36-yds-1-Core/product_130567?cid=PS:GS:SBD:PLA:OS&#38;gclid= 
			Cj0KCQjwlK-WBhDjARIsAO 
			2sErRwzrrgjU0NjFkDkne1xm 
			vT7ekS3tdzvAgiMDwPoxocgH 
			VTQZi7vJgaAvQZEALw_wcB</td>
		</tr>
		<tr>
			<td>Titanium, Platinum, Gold</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Output Power Supply</td>
			<td>Keysight</td>
			<td>E36311A</td>
			<td>https://www.newark.com/keysight-technologies/e36311a/dc-power-supply-3o-p-6v-5a-prog/dp/15AC9653</td>
		</tr>
		<tr>
			<td>UV Mask Aligner</td>
			<td>Karl Suss America</td>
			<td>MJB3 Mask Aligner&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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 ...

Watch this Scientific Journal Video about Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements at JoVE.com

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

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 ...

We present an electronics-based microfluidic platform for mechanical phenotyping of single cells. Specifically, we measure single cell elastic and viscous properties at a throughput of up to 10 cells per second. Our method requires minimal sample preparation, utilizes a straightforward electronic measurement, replacing expensive optical hardware and complex image analysis, and is non-destructive, meaning our approach is compatible with downstream analyses.Mechano-NPS has been applied to many cell types, including primary samples, and has measured the effect of subcellular components on single cell viscoelasticity. It could be used for understanding cell behavior, determining disease progression, or monitoring drug response. To begin, remove the plasma treated components from the plasma chamber.Pipet a two to one solution of methanol and deionized water on the glass substrate with pre-fabricated electrodes. Then place the PDMS mold with the feature side down on top of the glass substrate. Place the device under the stereoscope to adjust alignment.Bake the aligned device to complete device fabrication. Prepare the pressure source, PCB, benchtop hardware, and data acquisition software. Next, align the clamps header pins with the holes on the PCB and align the clamps spring loaded pins with the electrode contact pads on the microfluidic device.Then insert the clamps header pins into the PCB holes, making sure the spring loaded pins stay aligned with the electrode contact pads. Set up and connect the electronic hardware. Next, set the values to initialize the data acquisition session and set the sample rate for acquisition.Culture and prepared the cells according to the appropriate cell culture protocol of the cell line of choice. Then suspend the cells in a prepared solution of 2%FBS and 1X PBS at a concentration between 100, 000 and 500, 000 cells per milliliter. Keep the cells on ice for the duration of the experiments.To load cells into the microfluidic device, cut 30 centimeters of polytetrafluoroethylene tubing with a razor blade. Use a syringe to drop the cell sample into the end of the tubing and connect the same end into the device inlet. Finally, connect the opposite end of the tubing to the microfluidic pressure controller.To run the experiment, set the desired constant driving pressure on the pressure controller software and allow the sample to fill the device. If bubbles form in the microfluidic channels, use dead-end filling. Plug the device outlet and apply a low pressure to the inlet to force air out through the gas permeable PDMS.Then set the desired voltage by rotating the voltage knob on the power supply and enable the voltage by pressing the on button. Turn on the current preamplifier and set the sensitivity as low as possible. Alternatively, set the gain as high as possible without overloading the preamplifier or exceeding the maximum analog input voltage of the DAQ.Press the green Run button in the MATLAB ribbon menu to begin the data acquisition script NPS. m, and start sampling and saving the data. To end the experiment, press the Stop button in the lower left corner of the live plot window to end the data acquisition script.Then disable the power supply output by pressing the On button. Finally, set the pressure source to zero pressure in the pressure controller software. For data analysis, the raw signal should have a sufficient signal-to-noise ratio to filter out the noise and extract the significant components.Critically, the current signal rise from each node should be robust enough such that subpulses can be easily identified from the difference signal. Malignant MCF-7 cells have a greater wCDI distribution than non-malignant MCF-10A cells, indicating that the malignant MCF-7 cells are softer than their nonmalignant MCF-10A counterparts. MCF-10A and MCF-7 cells treated with latrunculin show an increase in wCDI.A distinct wCDI distribution differentiating the two primary cell types indicates that LEP cells are softer than MEP cells. Nonmalignant MCF-10As and untreated MCF-10A and MCF-7s have a greater proportion of cells that recover instantly, indicating lower viscosity than their malignant or latrunculin treated counterpart. If the live current readout appears abnormal, stop your experiment and inspect the microfluidic channel.Ensure that there are no air bubbles and no clogs such that the cells are flowing freely from the inlet to the outlet. Because our method doesn't harm cells, downstream analyses like RNA-seq or immunofluorescence can be performed. This could help uncover some underlying reasons why cells have distinct mechanical phenotypes.

mechano-node-pore-sensing-rapid-label-free-platform-for-multi

Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

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.

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

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.

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

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

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.

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 ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...