AP acknowledges the support of Prof T Venkatesan, Director, NUS Nanoscience and Nanotechnology Initiative and Office of the Deputy President (National University of Singapore) (R-398-000-084-646). CLD acknowledges the support of the Singapore Ministry of Health National Medical Research Council under its clinician scientist funding scheme, NMRC/CSA/035/2012, and the National University of Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Fabrication of the Sensor and Acquisition of the Data
<ol>
	<li>Preparation of the nickel mold
	<ol>
		<li>Coat a 220 nm-thick layer of negative electron beam resist onto a 600 &#181;m-thick 4-in silicon wafer. Write the designed nanohole array on this wafer using an electron beam lithography system.
		<ol>
			<li>To accelerate the e-beam writing, write the patterns with a low dotmap (N) of 20k for each 300 &#181;m field size (A) (i.e., there are 0.4 billion dots mapped on each 300 &#181;m2 area, and each dot will either be exposed by the e-beam or not, depending on the pattern design). Set the e-beam resist exposure dose for the resist to 110 &#181;C cm&#8722;2 and write at a current (I) of 800 pA. 
			NOTE: In e-beam writing, the exposure dose (D) is controlled by the exposure time for each dot (Tdot), calculated by <img alt="Equation 1" src="/files/ftp_upload/55597/55597eq1.jpg" />. For the exposure dose at 110 &#181;C cm&#8722;2, the e-beam dwelling time on each exposed dot is 0.5 &#181;s12. Since the array captures an area of 1.8 mm2, there are a total of 36 patches of 300-&#181;m2 field areas stitched together to form one large, gold nanohole array.</li>
		</ol>
		</li>
		<li>Develop the resist by immersing the 4 inch silicon wafer in the developer solution for 10 s and letting the wafer dry in air.</li>
		<li>Deposit a seed layer of a metal, such as nickel, copper, or aluminum, on the silicon wafer.</li>
		<li>Electroplate the wafer in a plating system in a nickel sulfamate bath. Carry out the electroplating in two steps. In the first step, lasting 95 min, use a current density of 0.7 A dm&#8722;2; this completely fills the nanopatterns with nickel. In the second step, lasting 125 min, use 12 A dm&#8722;2 to reach 300 &#181;m as the final nickel mold thickness (20 nm). Ensure that the pH value is at 3.5 - 3.8 and that the temperature is at 52 - 54 &#176;C.</li>
		<li>Separate the nickel mold from the silicon substrate by applying gentle mechanical force. Soak the nickel mold in about 100 mL of positive photoresist removal reagent overnight to wash away the residue from the e-beam resist.</li>
		<li>Feed the nickel mold into an oven and dry it at 100 &#176;C for 3 h. Clean it in a plasma etching system with O2 gas at 10 sccm and 100 W for 3 min.</li>
	</ol>
	</li>
	<li>Fabrication of the gold nanostructure
	<ol>
		<li>Coat 150 &#181;L of heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane (FDTS) on the nickel mold in a self-assembly monolayer (SAM) coating machine at 80 &#176;C. 
		NOTE: This will form an anti-adhesive layer, which will enable the separation of the mold from the photoresist (&#34;demolding&#34;) after the completion of the nanoimprinting step. The vaporizing time should be 180 s, and the reaction time should be 900 s.</li>
		<li>Imprint the nanopatterns on a 4-in glass wafer that has been coated with a 300 nm-thick layer of photo-curable NIL resist using a nano-imprinter at a pressure of 10 bar and a temperature of 40 &#176;C for 10 min.</li>
		<li>Transfer the mold, the photoresist, and the glass wafer to a UV light curing system and photocure with 75 mW cm-2 of UV exposure for 30 s. 
		NOTE: If all steps have been followed correctly, the nickel mold should easily be demolded from the photoresist.</li>
		<li>In a reactive ion etching (RIE) system, perform a blank etch of the photoresist on the glass substrate, with an O2 gas flow of 10 sccm, at 50 W for 2 s to expose the glass on the indented areas.</li>
		<li>Deposit a 5 nm-thick layer of chromium (Cr) for metal adhesion and a 100-nm layer of gold (Au) for the plasmonic sensor on the glass wafer in an electron-beam deposition machine. Use a deposition rate of 1 &#197; s&#8722;1 for Cr and 2 &#197; s&#8722;1 for Au.</li>
		<li>Perform lift-off of the photoresist by O2 plasma etching for 3 min followed by a 15-s sonification step in acetone.</li>
		<li>Dice the sample into 5 mm &#215; 5 mm chips. The nanohole array will occupy the central 2 mm &#215; 2 mm of the chip.</li>
	</ol>
	</li>
	<li>Acquisition of the data
	<ol>
		<li>Set up the apparatus to make the optical measurements such that a beam of white light exiting through the end of the transmitter optical fiber is collimated and is incident on the sensor surface (nanohole array) at 90&#176;. 
		NOTE: Light is transmitted through the whole nanohole array.</li>
		<li>Collect the transmitted signal with the receiver optical fiber and record it with a UV-visible spectrometer operating within the range of 300 to 1,000 nm.</li>
		<li>Set the acquisition time for each frame to 20 ms. Average 100 frames to obtain the final spectrum to lower the noise in the measurements.</li>
		<li>Use plotting software to analyze the data based on the previously identified transmission peaks (using a Lorentz-based method).</li>
	</ol>
	</li>
</ol>
2. Sensor Bulk Sensitivity Test
<ol>
	<li>Deposit the standard RI liquid into the liquid cell, with the RI varying from 1.31 to 1.39.</li>
	<li>Immerse the sensor chip in the standard RI liquid and align it with the beam of white light. Obtain the transmission spectrum.</li>
	<li>Clean the sensor chip after each measurement with a surface-active cleaning reagent and dry it with nitrogen gas.</li>
</ol>
3. Sensor Surface Modification
<ol>
	<li>Prior to any chemical modifications, clean the sensor chips by sequential immersion in isopropanol, acetone, and deionized water. Dry at room temperature in a stream of dry nitrogen gas.</li>
	<li>Incubate the sensor chips in an ethanolic solution of 0.4 mM 10-carboxy-1-decanethiol and 1.6 mM 1-octanethiol for 12 h at room temperature. 
	NOTE: This will form an amine-reactive self-assembly monolayer (SAM).</li>
	<li>Use ethanol to rinse thoroughly and dry at room temperature.</li>
	<li>Make a mixture of 75 mM sulfo-N-hydroxysuccinimide (sulfo-NHS) and 15 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Immerse the chips in this mixture for 15 min. 
	NOTE: This will activate the carboxylic group of the SAM.</li>
	<li>Spot 50 &#181;L of 200 &#181;g/mL anti-troponin antibody solution made in a pH 4.5 acetate buffer on the sensor surface and incubate for 30 min.</li>
	<li>Deactivate the unreacted esters by immersing the sensor chip in 1 M ethanolamine-HCl solution for 15 min.</li>
	<li>Rinse the chip with deionized water and dry it in a stream of dry nitrogen gas at room temperature.</li>
</ol>
4. cTnI Assay
<ol>
	<li>Block any non-specific binding by spotting 100 &#181;L of 1% bovine serum albumin (BSA) solution onto the surface. Incubate for 15 min.</li>
	<li>Rinse the sensor chips three times in phosphate-buffered saline (PBS) solution. Insert the chip into the measurement cell to record the transmission spectrum. 
	NOTE: This is the reference spectrum.</li>
	<li>Spot 50 &#181;L of cTnI standard onto the chip surface and incubate in a moist environment for 30 min.</li>
	<li>Rinse the sensor chips three times in PBS solution and insert it into the measurement cell to record the transmission spectrum. 
	NOTE: This is the after-binding spectrum.</li>
	<li>Submerge the chips in 50 mM glycine-HCl (pH 2) for 1 min and then rinse in PBS solution three times to regenerate the chip surface. Measure the transmission spectrum in PBS to verify the success of the regeneration step.</li>
</ol>
5. Surface Plasmon Resonance (SPR) Measurement
<ol>
	<li>Run the multiplexed SPR sensor chip on the SPR system with PBS-T buffer. 
	NOTE: The composition of PBS-T buffer is 20 mM Na-phosphate, 150 mM NaCl, and 0.05% Tween 20. The pH is 7.4.</li>
	<li>Use cTnI standard and the antibody, as described in step 4.</li>
	<li>Activate 3 out of the 6 available channels with a mixture of EDC (0.2 M) and sulfo-NHS (0.05 M) for 5 min. Perform a 5 min injection of 50 &#181;g/mL antibody 560 and a 5-min injection of 1 M ethanolamine-HCl solution.</li>
	<li>Rotate the sensor chip by 90&#176; and inject the cTnI standards at different concentrations (75, 30, 7.5, and 2.5 ng/mL).</li>
	<li>Observe the conjugation to the antibody at spots of interaction on the chip in real time through the SPR readout.</li>
	<li>Regenerate the chip by injecting 50 mM glycine-HCl (pH 2) for 1 min.</li>
</ol>

<ol><li>Ebbesen, T. W., Lezec, H. J., Ghaemi, H., Thio, T., Wolff, P. <a target="_blank" href="http://www.nature.com/nature/journal/v391/n6668/abs/391667a0.html">Extraordinary optical transmission through sub-wavelength hole arrays.</a> Nature. 391 (6668), 667-669 (1998).</li>
<li>Krishnan, A., et al. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0030401801015589">Evanescently coupled resonance in surface plasmon enhanced transmission.</a> Optics Comm. 200 (1), 1-7 (2001).</li>
<li>Yang, J. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+optical+transmission+mediated+by+localized+plasmons+in+anisotropic%2C+three-dimensional+nanohole+arrays%5BTitle%5D)+AND+%22Nano+letters%22%5BJournal%5D)">Enhanced optical transmission mediated by localized plasmons in anisotropic, three-dimensional nanohole arrays.</a> Nano letters. 10 (8), 3173-3178 (2010).</li>
<li>Kim, J. H., Moyer, P. J. <a target="_blank" href="http://aip.scitation.org/doi/abs/10.1063/1.2355468?journalCode=apl">Transmission characteristics of metallic equilateral triangular nanohole arrays.</a> Appl Phys Lett. 89 (12), 121106(2006).</li>
<li>Liu, H., Lalanne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscopic+theory+of+the+extraordinary+optical+transmission%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Microscopic theory of the extraordinary optical transmission.</a> Nature. 452 (7188), 728-731 (2008).</li>
<li>Shon, Y. -S., Choi, H. Y., Guerrero, M. S., Kwon, C. <a target="_blank" href="http://link.springer.com/article/10.1007/s11468-009-9079-1">Preparation of nanostructured film arrays for transmission localized surface plasmon sensing.</a> Plasmonics. 4 (2), 95-105 (2009).</li>
<li>Xiang, G., Zhang, N., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localized+surface+plasmon+resonance+biosensing+with+large+area+of+gold+nanoholes+fabricated+by+nanosphere+lithography%5BTitle%5D)+AND+%22Nanoscale+Res+Lett%22%5BJournal%5D)">Localized surface plasmon resonance biosensing with large area of gold nanoholes fabricated by nanosphere lithography.</a> Nanoscale Res Lett. 5 (5), 818(2010).</li>
<li>Valsecchi, C., Brolo, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Periodic+metallic+nanostructures+as+plasmonic+chemical+sensors%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Periodic metallic nanostructures as plasmonic chemical sensors.</a> Langmuir. 29 (19), 5638-5649 (2013).</li>
<li>Gates, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+approaches+to+nanofabrication%3A+molding%2C+printing%2C+and+other+techniques%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">New approaches to nanofabrication: molding, printing, and other techniques.</a> Chem Rev. 105 (4), 1171-1196 (2005).</li>
<li>Guo, L. J. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/adma.200600882/abstract">Nanoimprint lithography: methods and material requirements.</a> Adv Mater. 19 (4), 495-513 (2007).</li>
<li>Wong, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+and+high+yield+nanofabrication+of+precisely+designed+gold+nanohole+arrays+for+fluorescence+enhanced+detection+of+biomarkers%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">High throughput and high yield nanofabrication of precisely designed gold nanohole arrays for fluorescence enhanced detection of biomarkers.</a> Lab on a Chip. 13 (12), 2405-2413 (2013).</li>
<li>Deng, J., Wong, T. I., Sun, L. L., Quan, C., Zhou, X. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0167931716303975">Acceleration of e-beam lithography by minimized resist exposure for large scale nanofabrication.</a> Microelect Eng. 166, 31-38 (2016).</li>
<li>Wu, L., Bai, P., Li, E. P. <a target="_blank" href="https://www.osapublishing.org/josab/abstract.cfm?uri=josab-29-4-521">Designing surface plasmon resonance of subwavelength hole arrays by studying absorption.</a> JOSA B. 29 (4), 521-528 (2012).</li>
<li>Ding, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+a+cardiac+biomarker+in+human+serum+using+extraordinary+optical+transmission+%28EOT%29%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Quantification of a cardiac biomarker in human serum using extraordinary optical transmission (EOT).</a> PloS one. 10 (3), 0120974(2015).</li>
<li>Im, H., Sutherland, J. N., Maynard, J. A., Oh, S. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanohole-based+surface+plasmon+resonance+instruments+with+improved+spectral+resolution+quantify+a+broad+range+of+antibody-ligand+binding+kinetics%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Nanohole-based surface plasmon resonance instruments with improved spectral resolution quantify a broad range of antibody-ligand binding kinetics.</a> Anal Chem. 84 (4), 1941-1947 (2012).</li>
<li>Bhagawati, M., You, C., Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+real-time+imaging+of+protein-protein+interactions+by+LSPR+detection+with+micropatterned+gold+nanoparticles%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Quantitative real-time imaging of protein-protein interactions by LSPR detection with micropatterned gold nanoparticles.</a> Anal Chem. 85 (20), 9564-9571 (2013).</li>
</ol>

The authors have no competing financial interests.

Simulating the interaction between incident light and the nanostructures makes it possible to identify the appropriate peak (in the transmission spectrum), whose shift can be recorded as a function of the concentration of the analyte. It is important to note that the localization of the bands with respect to the structure of the sensor is crucial to the choice of the right band, whose shift can be tracked to sense the analyte. The visualization can be achieved through simulations. This is also critical to the design of a...

Chemical sensors based on nanohole arrays have been a subject of numerous investigations since the first report on extraordinary optical transmission (EOT) was published by Ebbesen et al. in 19981. When light impinges on periodic arrays of nanohole structures of sub-wavelength dimensions, enhanced transmission occurs at specific wavelengths. This occurs when the incident light couples with Bloch-wave surface polariton (BW-SPP) and/or localized surface plasmons (LSP)2.
The underlying physical principle exploited when biosensing with such periodic arrays is simple. Adsorption of molecules onto or near the interface of metal changes the dielectric constant of the medium in contact with the metal, in turn shifting the location of the transmission bands in the spectrum. The spectrum itself can be adjusted by nano-engineering the shape, size, and separation distance3,4,5. By design, sensors based on EOT have characteristic bands in their spectra that facilitate specific assignments6,7,8 during the investigation of molecular binding events. This is a crucial advantage over commercially available surface plasmon resonance (SPR) platforms.
Sensors using EOT typically involve a light source optically aligned such that a collimated beam is incident on the sensing surface. Techniques to generate large nanohole surfaces, such as co-polymer templates and interference and nanosphere lithography, have poor reproducibility9. Due to these limitations in accurately fabricating large surfaces that show the EOT phenomenon, an optical microscope was required to correctly position the light source and detector. To simplify the technique, high-quality nanoimprinting lithography (NIL)10 was employed. This enabled the production of large sensor surface areas11 (mm-scale), removing the need for a microscope to look for the sensing surface on a chip. Instead, this sensor could be easily interfaced with a standard fiber optic cable.
Since the transmission peaks for this nanohole array are contained in the visible to near-infrared region (NIR), it is perfectly suited to sensing binding events for biomolecules in an aqueous environment. The expected optical behavior of the nanohole array was simulated. The result was then verified through studies with liquids of standard refractive indexes (RI). This array was then used to measure the concentration of cardiac troponin I (cTnI) in the complex background of human serum. cTnI is the clinical gold standard for the diagnosis of acute myocardial infarction.
Using this sensor, it is possible to detect and quantify cTnI in human serum at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. The detection is much quicker than the most commonly used technology in this domain, enzyme-linked immunosorbent assay (ELISA). Furthermore, the sensing surface can easily be regenerated and therefore reused. Hence, this work demonstrates the promise of nanohole arrays as a viable point-of-care (POC) technology for biosensing within complex biofluids.

<table><tbody><tr><td>Electron Beam Lithography setup</td><td>Elionix ELS 700</td><td></td><td></td></tr><tr><td>o-Xylene</td><td>Sigma Aldrich</td><td>95662</td><td></td></tr><tr><td>EB resist</td><td>Sumitomo</td><td>NEB-22A2</td><td></td></tr><tr><td>Developer reagent</td><td>Shipley Company</td><td>Microposit MF 321</td><td></td></tr><tr><td>Electroplating machine</td><td>Technotrans AG RD 50</td><td></td><td></td></tr><tr><td>Photoresist stripper&amp;nbsp;</td><td>Rohm and Haas Electronic Materials&amp;nbsp;LLC</td><td>Microposit Remover 1165</td><td></td></tr><tr><td>Etching System</td><td>Trion Phantom</td><td></td><td></td></tr><tr><td>Heptadecafluoro-1,1,2,2-tetrahydrodecyl)trichlorosilane&amp;nbsp;</td><td>Gelest (PA, USA)</td><td>78560-44-8</td><td></td></tr><tr><td>SAM coater&amp;nbsp;</td><td>Sorona Inc.</td><td>AVC 150M</td><td></td></tr><tr><td>Photo-curable NIL resist</td><td>micro resist technology GmbH</td><td>mr-UVCur21-300nm</td><td></td></tr><tr><td>Light Curing System</td><td>Dymax&amp;nbsp;</td><td>Model 2000 Flood</td><td></td></tr><tr><td>E-beam deposition machine</td><td>Denton Explorer</td><td></td><td></td></tr><tr><td>UV-visible spectrometer&amp;nbsp;</td><td>Ocean optic HR2000+ (Dunedin, FL, USA)</td><td></td><td></td></tr><tr><td>Standard refractive index liquids&amp;nbsp;</td><td>Cargill Inc (Cedar Grove, USA)</td><td>18032</td><td></td></tr><tr><td>Plotting software</td><td>Origin</td><td>Origin Pro 9</td><td></td></tr><tr><td>10-carboxy-1-decanethiol&amp;nbsp;</td><td>Dojindo Laboratories (Japan)</td><td>C385-10</td><td></td></tr><tr><td>1-octanethiol&amp;nbsp;</td><td>Sigma-Aldrich, MO, USA</td><td>471386</td><td></td></tr><tr><td>Sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide&amp;nbsp;</td><td>BioRad (CA, USA)</td><td>1762410</td><td></td></tr><tr><td>Anti-troponin antibody 560</td><td>Hytest (Finland)</td><td>4T21</td><td></td></tr><tr><td>Ethanolamine-HCl solution</td><td>BioRad (CA, USA)</td><td>1762450</td><td></td></tr><tr><td>Surface Plasmon Resonance setup</td><td>BioRad XPR36 (Haifa, Israel)</td><td></td><td></td></tr><tr><td>Multiplexed SPR chip</td><td>BioRad</td><td>GLC</td><td></td></tr><tr><td>Human cTnI standard</td><td>Phoenix Pharmaceuticals</td><td>EK -311-05</td><td></td></tr><tr><td>Glycine-HCl</td><td>BioRad (CA, USA)</td><td>1762221</td><td></td></tr></tbody></table>

using extraordinary optical transmission to quantify cardiac biomarkers in human serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

The optical setup for taking measurements is shown in Figure 1A. An image of the actual nanohole array is given in Figure 1B. To understand the physics driving the sensing process, the COMSOL simulation software was used to simulate the distribution of the plasmonic field in an aqueous environment. The results from the simulation were then related to the actual measurement. A previously published study contains details of the ass...

Watch this Scientific Journal Video about Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum at JoVE.com

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

using-extraordinary-optical-transmission-to-quantify-cardiac

Research

JoVE Journal

Bioengineering

6.2K Views.  National University of Singapore. Dit werk beschrijft een nanoimprinting lithografie methode om kwalitatief hoogwaardige sensing matrices die op het principe van buitengewone optische transmissie werken. De biosensor is lage kosten, robuust, makkelijk te gebruiken, en kan detecteren cardiale troponine ik in serum op klinisch relevante concentraties (99th percentiel cutoff ∼10-400 pg/mL, afhankelijk van de test).

Video: Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Medicine

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Biology

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

Samenvatting

Meer video's verkennen