This work was supported by the startup funding from the School of Engineering, Virginia Commonwealth University and some of electronic devices were prepared at the microfabrication facilities in the Wright Virginia Microelectronics Center. We acknowledge researchers who made contributions for the device and clinical data (Figures 3 and 5 in this paper), acquired from the published article10. W.-H.Y thanks Yoshiaki Hattori for the custom-made, data recording software.

The experiments for device fabrication, skin lamination, and characterization shown in Figures 1, 2, and 4 involved two volunteers, all performed in the Bio-interfaced NanoEngineering Laboratory at Virginia Commonwealth University (VCU), Richmond, VA, USA. This study was approved by the VCU Institutional Review Board (protocol number: HM20001454) and followed the research guidelines from the VCU Human Research. The device and clinical data shown in Figures 3 and 5 were acquired from the published article10 where the experiments on patients were conducted under the protocol (number: STU69718) approved by the Institutional Review Board, Northwestern University, Chicago, IL, USA.
1. Device Fabrication &#160;
NOTE: Figure 2 presents schematic illustrations for the overall fabrication process.
<ol>
	<li>Prepare a carrier substrate
	<ol>
		<li>Cut a bare 3 in&#160;silicon (Si) wafer into the desired size of the electronics by using a diamond blade. 
		NOTE: About half Si wafer gives an ideal size for the wound device.</li>
		<li>Degrease Si wafer with acetone and isopropyl alcohol (IPA). Rinse the wafer with deionized (DI) water and then dry with nitrogen and dehydrate on a hotplate at 110 &#176;C for 3 min.</li>
		<li>Prepare 11 g of polydimethylsiloxane (PDMS) mixture with 10:1 volume ratio of base and curing agent and degas the mixture in a vacuum chamber for an hour. 
		NOTE: PDMS is used for a dry pattern retrieval and transfer printing after the microfabrication, which is preferable to the wet chemical (acetone)-based approach from the previous study7.</li>
		<li>Spin coat 5 g of mixed PDMS solution on the wafer at 3,000 rpm for 1 min&#160;and fully cure on a hotplate at 150 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Deposit materials and pattern electronics
	<ol>
		<li>Treat the PDMS-coated wafer with ultraviolet (UV)/ozone by using a UV lamp (8.9 mW/cm2) for 3 min&#160;to make the surface hydrophilic. 
		NOTE: The hydrophilic surface offers uniform coating of additional layers on the PDMS.</li>
		<li>Spincoat polyimide (PI; 2 ml) on the PDMS coated wafer, by pipetting, at 4,000 rpm for 1 min&#160;to form a 1.2-&#181;m-thick layer, pre-bake on a hotplate at 150 &#176;C for 5 min, and post-bake at 250 &#176;C for 2 hr.</li>
		<li>Deposit chromium (Cr) to form a 20 nm-thick layer and then deposit copper (Cu) to form a 3 &#181;m-thick layer by using electron-beam (e-beam) evaporation (base pressure: ~1&#215;10-7 Torr, deposition pressure: ~1&#215;10-6 Torr, deposition rate: 1 &#8211; 5 &#197;/s). Monitor the film thickness by the deposition controller interface embedded in the evaporator. 
		NOTE: The thick layer of Cu provides enough levels of electrical conductivity on the microscale resistors of the device and thin Cr layer is used to promote adhesion between PI and Cu.</li>
		<li>Spincoat a photoresist (2 ml) with three steps at 900 rpm for 10 sec, 1,100 rpm for 60 sec, and 4,000 rpm for 20 sec&#160;and then cure it on a hotplate at 75 &#176;C for 30 min. 
		NOTE: The sequential steps described above were used to deposit a thick (&#62; 10 &#181;m) photoresist.</li>
		<li>Align Cu electronic patterns (sensors; fractal &#8216;Peano&#8217; design with 35 &#181;m in width and interconnects; serpentine open mesh design with 50 &#181;m in width) at the center of the Si wafer by using a UV aligner (power: 10 mW/sec) with exposure time 25 s. 
		NOTE: The fractal structures are used to provide superior mechanical stretchability, compared to merely meandering features14.</li>
		<li>Develop the photoresist in a diluted base developer (1:2 ratio of developer and DI water) for a minute, rinse with DI water, and dry with nitrogen. Inspect the patterns (Cu fractals and interconnects) using a microscope to confirm the feature size and find any defects from particulates. 
		NOTE: If there are any unwanted defects, then remove photoresist by rinsing with acetone/IPA/DI water. After drying with nitrogen, repeat the steps from 1.2.4 to 1.2.6.</li>
		<li>Etch the Cu layer on the Si wafer by immersing in a wet chemical etchant for ~6 min&#160;(10 ml; mixture of ammonium persulfate and water in ratio of 1:4; etch rate of 8 nm/sec&#160;in 40 &#176;C), rinse with DI water, and dry with nitrogen. Inspect the patterns using a microscope for any over-etched patterns. 
		NOTE: If the patterns are over-etched, it may cause unwanted sharp edges of the features, which could lead to mechanical fracture during device handling and washing process. The prior testing results showed that more than ~20% over-etching of the original patterns caused the aforementioned issues.</li>
		<li>Etch the Cr layer with reactive ion etching (RIE; pressure: 300 mTorr, power: 200 W, CF4 gas: 5 sccm, O2 gas: 10 sccm) for 5 min. Inspect the patterns. 
		NOTE: For etching of the Cr layer, the RIE process is preferable to wet chemical etching that causes unfavorable reaction with the Cu layer.</li>
		<li>Remove the remained photoresist on the metal layers by immersing the wafer in acetone (10 ml), IPA (10 ml), and DI water (20 ml), respectively. Then, dry it with nitrogen.</li>
		<li>Spincoat PI (2 ml) on the metal deposited wafer, by pipetting, at 4000 rpm for 1 min&#160;to form a 1.2-&#181;m-thick layer, pre-bake on a hotplate at 150 &#176;C for 5 min, and post-bake at 250 &#176;C for 2 hr.</li>
		<li>Spincoat a photoresist (2 ml) with three steps at 900 rpm for 10 sec, 1,100 rpm for 60 sec, and 4,000 rpm for 20 sec&#160;and then cure it on a hotplate at 75 &#176;C for 30 min.</li>
		<li>Align PI patterns to encapsulate the Cu electronics (sensors; fractal &#8216;Peano&#8217; design with 35 &#181;m in width and interconnects; serpentine open mesh design with 250 &#181;m in width) with the pre-defined Cu fractals and interconnects by using a UV aligner (power: 10 mW/sec) with exposure time 25 sec.</li>
		<li>Develop the photoresist with a diluted developer (1:2 ratio of developer and DI water) for a minute, rinse with DI water, and dry with nitrogen. Inspect the patterns using a microscope to confirm the feature size and find any defects from particulates. 
		NOTE: If there are any unwanted defects, then remove photoresist by rinsing with acetone/IPA/DI water. After drying with nitrogen, repeat the steps from 1.2.10 to 1.2.13.</li>
		<li>Etch the PI layer with RIE (pressure: 170 mTorr, power: 150 W, O2 gas: 20 sccm) for 25 min. Inspect the patterns.</li>
		<li>Remove the remained photoresist by immersing the wafer in acetone (10 ml), IPA (10 ml), and DI water (20 ml), respectively. Then, dry it with nitrogen.</li>
	</ol>
	</li>
	<li>Prepare an elastomeric membrane
	<ol>
		<li>Prepare a 10 g of encapsulating silicone mixture (1:1 volume ratio of base and curing agent) and add a black ink15 with one to one volume ratio, which is to facilitate control measurements of temperature variation on the skin using an infrared camera. 
		NOTE: The utilized silicone (clear encapsulating rubber) provides unique characteristics of low viscosity, optical clarity, and electrical isolation/protection to the device16.</li>
		<li>Spincoat 8 g of the mixture in a petri dish at 150 rpm for 1 min&#160;to form a 500 &#181;m thick elastomeric membrane and cure at RT for O/N. 
		NOTE: The material needs to be placed on a flat surface for uniform thickness.</li>
		<li>Cut the membrane into the desired size of 70 mm x 30 mm by using a sharp razor blade and gently detach it from the petri dish.</li>
	</ol>
	</li>
	<li>Retrieve and transfer electronics
	<ol>
		<li>Cut a water soluble tape (25 mm x 80 mm) and gently laminate onto the fabricated electronic patterns and place them on a hotplate at 130 &#176;C for 3 min. 
		NOTE: Temperature elevation expands the PDMS layer on the Si wafer to help dissociation of the electronic patterns from the surface.</li>
		<li>Detach the tape rapidly from the PDMS/Si wafer to retrieve the electronic patterns.</li>
		<li>Deposit a 20 nm thick Cr (for adhesion) followed by a 50 nm thick silicon dioxide (SiO2) on retrieved patterns by e-beam evaporation.</li>
		<li>Treat UV/ozone by using UV lamp (365 nm, 8.9 mW/cm2) on the targeted silicone membrane for 2 min&#160;to activate the surface.</li>
		<li>Transfer the patterns to the silicone membrane by placing the retrieved patterns on the tape to the desired location and evenly adding pressure from top side of the patterns down to the substrate. Apply water to dissolve the tape for 5 min. 
		NOTE: The described process of materials transfer is facilitated by covalent bonding (Si-O-Si) between the deposited silicon dioxide&#160;and the UV-activated silicone substrate17.</li>
		<li>Peel off the tape, rinse with DI water, and dry on a hotplate at 90 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Encapsulate the device using a silicone membrane
	<ol>
		<li>Prepare an 10 g of encapsulating silicone mixture (1:1 volume ratio of base and curing agent).</li>
		<li>Cover the cable contact pads with a rectangular PDMS piece (22 &#215; 6 &#215; 1 mm3) by van der Waals bonding with the bottom silicone membrane, to avoid silicone coating the pads.</li>
		<li>Spincoat the 5 g of silicone mixture at 4,000 rpm for 1 min&#160;to form a 5 &#181;m thick layer on the transferred electronics and then cure at RT for O/N.</li>
	</ol>
	</li>
	<li>Connect a flexible ribbon cable for data acquisition
	<ol>
		<li>Apply liquid steel flux (0.5 ml), by pipetting, on the connector pads for 3 sec&#160;to make clean surface.</li>
		<li>Bond a thin, flexible ribbon cable on the contact points with pressure at high temperature (&#62; 60 &#176;C). A typical hair straightener offers easy handling and bonding. 
		NOTE: The micro-film cable is preferable to the conventional hard-wire soldering to avoid any mechanical fracture of the transferred metal membranes on a silicone.</li>
		<li>Check the electrical connection using a digital multimeter. The resistance value is expected less than 1 Ohm between the sensor pad with one end and the other end of the film cable (distance: ~1 cm apart).</li>
		<li>Bond the other end of the ribbon cable to a customized printed circuit board with the same strategy described in the step 1.6.2.</li>
		<li>Check the electrical connection using a digital multimeter.</li>
		<li>Connect the device with the data acquisition hardware by soldering conventional wires on PCB.</li>
	</ol>
	</li>
</ol>
2. Clinical Testing &#160;
<ol>
	<li>Clean the device using a disinfectant solution
	<ol>
		<li>Prepare 205 g of a diluted disinfectant solution (40:1 volume ratio of water and solution).</li>
		<li>Spray the solution (10 g) on the device and soak it for 10 min. 
		NOTE: The diluted disinfectant cleaner can be stored at RT.</li>
		<li>Rinse with water three times and dry it with clean tissues.</li>
	</ol>
	</li>
	<li>Set up a series of equipment for device testing
	<ol>
		<li>Prepare and connect a lock-in amplifier with a current source, a multiplexer, and the custom software installed on a laptop computer for data recording.</li>
		<li>Place an infrared camera on a tripod and focus on a target object for thermography as a reference.</li>
		<li>Set up system parameters of a lock-in amplifier to measure thermal conductivity (frequency: 1 &#38; 3 Hz; time constant: 3 and 1 sec; sensitivity: 1 mV; dynamic reserve: high reserve) and temperature (frequency: 997 Hz; time constant: 300 msec; sensitivity: 2 mV; dynamic reserve: low noise) with the applied constant current (2 mA).</li>
		<li>Connect two wound devices, prepared by microfabrication and transfer printing and mounted on wound and contralateral sites, to the multiplexer right before recording data from a patient.</li>
	</ol>
	</li>
	<li>Record temperature and thermal conductivity 
	NOTE: The data acquisition software is custom-made, which can remote control the lock-in amplifier for real-time data monitoring and saving. In the temperature measurement, each data point is measured every 300 msec for 20 sec. The set of data for the first 10 sec&#160;and next 10 sec&#160;are used to calculate the average temperature value and standard deviation, respectively. The recorded data are then saved as a comma-separated value file, which is used to plot a graph for comparison with data from infrared thermography. In the measurement of thermal conductivity, the 3&#937; signals are directly read from the hardware screen (amplifier), which is then utilized to calculate the thermal conductivity analytically.
	<ol>
		<li>Gently rub the device application sites on the skin using antiseptic alcohol wipes10.</li>
		<li>2.3.2) Laminate two devices on the desired skin locations by gently pressing the device to the skin with fingers to facilitate the soft bonding: one on the surgical wound site and the other on the contralateral location as a reference.</li>
		<li>Measure the electrical voltage (3&#937;), related to thermal conductivity, of the device by starting the data acquisition.</li>
		<li>Evaluate the obtained data to verify the conformal contact of the device to the skin; abnormal value (&#60; 0.1 W/mK) shows bad contact of the device.</li>
		<li>Measure the electrical resistance to determine temperature distribution and record data through the custom software.</li>
		<li>Take optical and IR images of two devices on the skin.</li>
		<li>Compare temperature values from the IR images with the data recorded from the wound device (2.3.5). Add both values to separate columns in a customized spreadsheet.</li>
	</ol>
	</li>
	<li>Analyze the recorded data
	<ol>
		<li>Export the recorded data to the customized template to automatically calculate temperature and thermal conductivity from an array of sensors in the device.</li>
		<li>Plot the data (temperature and thermal conductivity according to the sensor location on a different time scale) for comparison over the course of one month (four sets of data on day 1, 3, 15, and 30).</li>
		<li>Analyze the data by comparing a series of temperature and thermal conductivity data according to time; Values with sudden elevation or drop tell the change of wound healing phase and/or unexpected abnormality on wound sites.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Dargaville, T. R., 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=Sensors+and+imaging+for+wound+healing:+A+review.">Sensors and imaging for wound healing: A review.</a> Biosens Bioelectron. 41, 30-42 (2013).</li><li>Panuncialman, J., Hammerman, S., Carson, P., Falanga, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+edge+biopsy+sites+in+chronic+wounds+heal+rapidly+and+do+not+result+in+delayed+wound+healing.">Wound edge biopsy sites in chronic wounds heal rapidly and do not result in delayed wound healing.</a> J Invest Dermatol. 129, S47-S47 (2009).</li><li>Hess, C. T., Kirsner, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Orchestrating Wound Healing: Assessing and Preparing the Wound Bed. Adv Skin Wound Care. 16 (5), 246-257 (2003).</li><li>Lange-Asschenfeldt, S., 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=Applicability+of+confocal+laser+scanning+microscopy+for+evaluation+and+monitoring+of+cutaneous+wound+healing.">Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing.</a> J Biomed Opt. 17 (7), (2012).</li><li>Crane, N. J., Elster, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrational+spectroscopy:+a+tool+being+developed+for+the+noninvasive+monitoring+of+wound+healing.">Vibrational spectroscopy: a tool being developed for the noninvasive monitoring of wound healing.</a> J Biomed Opt. 17 (7), (2012).</li><li>Jeong, J. W., 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=Materials+and+Optimized+Designs+for+Human-Machine+Interfaces+Via+Epidermal+Electronics.">Materials and Optimized Designs for Human-Machine Interfaces Via Epidermal Electronics.</a> Adv Mater. 25 (47), 6839-6846 (2013).</li><li>Kim, D. H., 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=Epidermal+Electronics.">Epidermal Electronics.</a> Science. 333 (6044), 838-843 (2011).</li><li>Webb, R. C., 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=Ultrathin+conformal+devices+for+precise+and+continuous+thermal+characterization+of+human+skin+(vol+12,+pg+938).">Ultrathin conformal devices for precise and continuous thermal characterization of human skin (vol 12, pg 938).</a> Nat Mater. 12, 1078-1078 (2013).</li><li>Yeo, W. H., 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=Multifunctional+Epidermal+Electronics+Printed+Directly+Onto+the+Skin.">Multifunctional Epidermal Electronics Printed Directly Onto the Skin.</a> Adv Mater. 25 (20), 2773-2778 (2013).</li><li>Hattori, Y., 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=Multifunctional+skin-like+electronics+for+quantitative,+clinical+monitoring+of+cutaneous+wound+healing.">Multifunctional skin-like electronics for quantitative, clinical monitoring of cutaneous wound healing.</a> Adv Healthc Mater. 3 (10), 1597-1607 (2014).</li><li>Guo, S., DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+Affecting+Wound+Healing.">Factors Affecting Wound Healing.</a> J Dent Res. 89 (3), 219-229 (2010).</li><li>Matzeu, G., 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=Skin+temperature+monitoring+by+a+wireless+sensor.">Skin temperature monitoring by a wireless sensor.</a> Ieee Ind Elec. , 3533-3535 (2011).</li><li>Cahill, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal-Conductivity+Measurement+from+30-K+to+750-K+-+the+3-Omega+Method.">Thermal-Conductivity Measurement from 30-K to 750-K - the 3-Omega Method.</a> Rev Sci Instrum. 61 (2), 802-808 (1990).</li><li>Fan, J. A., 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=Fractal+Design+Concepts+for+Stretchable+Electronics.">Fractal Design Concepts for Stretchable Electronics.</a> Nature Commun. 5 (3266), (2013).</li><li>Song, Y. 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=Digital+cameras+with+designs+inspired+by+the+arthropod+eye.">Digital cameras with designs inspired by the arthropod eye.</a> Nature. 497 (7447), 95-99 (2013).</li><li>Jeong, J. W., 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=Capacitive+Epidermal+Electronics+for+Electrically+Safe,+Long-Term+Electrophysiological+Measurements.">Capacitive Epidermal Electronics for Electrically Safe, Long-Term Electrophysiological Measurements.</a> Adv Healthc Mater. 3 (5), 642-648 (2013).</li><li>Zhang, Y. H., 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=Experimental+and+Theoretical+Studies+of+Serpentine+Microstructures+Bonded+To+Prestrained+Elastomers+for+Stretchable+Electronics.">Experimental and Theoretical Studies of Serpentine Microstructures Bonded To Prestrained Elastomers for Stretchable Electronics.</a> Adv Funct Mater. 24 (14), 2028-2037 (2014).</li><li>Carlson, A., Bowen, A. M., Huang, Y. G., Nuzzo, R. G., Rogers, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+Printing+Techniques+for+Materials+Assembly+and+Micro/Nanodevice+Fabrication.">Transfer Printing Techniques for Materials Assembly and Micro/Nanodevice Fabrication.</a> Adv Mater. 24 (39), 5284-5318 (2012).</li><li>Yeo, W. H., Webb, R. C., Lee, W., Jung, S., Rogers, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-integrated+electronics+and+sensor+systems.">Bio-integrated electronics and sensor systems.</a> Proc Spie. 8725, (2013).</li><li>Chung, H. 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=Fabrication+of+Releasable+Single-Crystal+Silicon&#8211;Metal+Oxide+Field-Effect+Devices+and+Their+Deterministic+Assembly+on+Foreign+Substrates.">Fabrication of Releasable Single-Crystal Silicon&#8211;Metal Oxide Field-Effect Devices and Their Deterministic Assembly on Foreign Substrates.</a> Adv Funct Mater. 21 (16), 3029-3036 (2011).</li><li>Kim, H. S., 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=Unusual+strategies+for+using+indium+gallium+nitride+grown+on+silicon+(111)+for+solid-state+lighting.">Unusual strategies for using indium gallium nitride grown on silicon (111) for solid-state lighting.</a> Proc Natl Acad Sci U.S.A. 108 (25), 10072-10077 (2011).</li><li>Padilla-Medina, J. A., 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=Assessment+technique+for+acne+treatments+based+on+statistical+parameters+of+skin+thermal+images.">Assessment technique for acne treatments based on statistical parameters of skin thermal images.</a> J Biomed Opt. 19 (4), 046019-046019 (2014).</li></ol>

The authors declare that they have no competing financial interests.

This article highlights the methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination near the wound tissues, which offers quantitative measurement of skin temperature and thermal conductivity mapping on the skin. 
The key features include the utilization of novel techniques of materials transfer printing and hard-soft materials integration to design and develop the flexible/stretchable, soft electronic device. The use of biocompatible, elec...

In clinical study and biomedical research, monitoring of wound healing has focused on an invasive method that is based on the histological evaluation of tissue morphologic change in wounds1,2. Recently, rapid advancements in electronic technologies enable the development of high-precision imaging and analysis tools that can visually inspect the wound healing process via digital imaging3,4 or confocal scanning microscopy and spectroscopy4,5. However, those imaging approaches require high cost, complicated optical tools and operations, and more importantly, patients need to be immobilized during testing. Therefore, there exists a need for new devices and systems that are quantitative, non-invasive, easy-to-use, inexpensive, and multifunctional to offer more accurate wound management.
Here, we introduce a skin-like electronic system that provides precise, real-time mapping of temperature and thermal conductivity and delivers a precise level of heating at wound sites via conformal lamination of the device non-invasively. This device presents a class of technology, skin-mounted epidermal electronic systems that are designed to match to mechanical and material properties (total thickness, bending stiffness, effective moduli, and mass density) of the epidermis6-9.
The device is designed in a biocompatible, skin-friendly, water-proof, and reusable form that can be washed and disinfected for clinical applications on patients10. The conformal electronic device mounted near the wound tissues captures the inflammation phase (one of wound healing process), caused by increased blood flow and enzymatic reactions to the wound11,12, through quantitative recording of temperature8 and thermal conductivity13, correlated to hydration. Experimental and computational studies determine an optimal mechanics design to accommodate natural motions and applied strains without mechanical fracture and capture the underlying physics of stretching mechanics of the skin-like electronics that laminates conformally on the skin surface, which offers acquisition of high fidelity signals.
The protocols described in this article present the methods of microfabrication for skin-like electronic systems, testing preparation including device cleaning, equipment setup in a clinical setting, and clinical applications for quantitative monitoring of temperature and thermal conductivity on cutaneous wounds.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3&#34; Silicon wafer</td>
			<td>University Wafer, USA</td>
			<td>N/A</td>
			<td>Use as carrier to fabricate the device</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific, USA</td>
			<td>A18-1</td>
			<td>Use to clean a wafer and to remove photoresist</td>
		</tr>
		<tr>
			<td>Isopropanol (IPA)</td>
			<td>Fisher Scientific, USA</td>
			<td>A459-1</td>
			<td>Use to clean a wafer</td>
		</tr>
		<tr>
			<td>AZ4620 photoresist</td>
			<td>AZ Electrionic Materials, USA</td>
			<td>N/A</td>
			<td>Use to make patterns on metals and polymers</td>
		</tr>
		<tr>
			<td>AZ400K developer</td>
			<td>AZ Electrionic Materials, USA</td>
			<td>N/A</td>
			<td>Use to develop AZ4620 photoresist</td>
		</tr>
		<tr>
			<td>Chromium etchant</td>
			<td>Transene, USA</td>
			<td>1020AC</td>
			<td>Use to etch Cr layer of device</td>
		</tr>
		<tr>
			<td>Copper etchant</td>
			<td>Transene, USA</td>
			<td>ASP-100</td>
			<td>Use to etch Cu layer of device</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit (PDMS)</td>
			<td>Dow Corning, USA</td>
			<td>39100000</td>
			<td>Use as a substrate for &#39;dry&#39; retrieval</td>
		</tr>
		<tr>
			<td>PI2545 polyimide</td>
			<td>HD MicroSystem, USA</td>
			<td>N/A</td>
			<td>Use to encapsulate metal layer</td>
		</tr>
		<tr>
			<td>Solaris</td>
			<td>Smooth-On, USA</td>
			<td>N/A</td>
			<td>Use as substrate and to encapsulate device</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Carolina, USA</td>
			<td>741255</td>
			<td>Use as mold to make substrate</td>
		</tr>
		<tr>
			<td>Water-Soluble Wave Solder Tape 5414</td>
			<td>3M, USA</td>
			<td>AM000000217</td>
			<td>Use to retrive device from PDMS layer</td>
		</tr>
		<tr>
			<td>High Activity Liquid Stainless Steel Flux</td>
			<td>Worthington, USA</td>
			<td>331929</td>
			<td>Use to remove oxidation layer on Cu</td>
		</tr>
		<tr>
			<td>Flexible, micro-film cable</td>
			<td>Elform, USA</td>
			<td>N/A (customized)</td>
			<td>Use to make the electrical connection between the electronic device and the data acquisition system</td>
		</tr>
		<tr>
			<td>pH Neutral Cleaner</td>
			<td>Australian Gold, USA</td>
			<td>N/A</td>
			<td>Use as disinfectant solution to clean device in clinical testing</td>
		</tr>
		<tr>
			<td>Solder</td>
			<td>Kester, USA</td>
			<td>24-6337-9703</td>
			<td>Use as material to solder hard wires</td>
		</tr>
		<tr>
			<td>Ultraviolet lamp</td>
			<td>Cole-Parmer, USA</td>
			<td>97600-00</td>
			<td>Use to activate PDMS layer as hydrophilic surface</td>
		</tr>
		<tr>
			<td>Multiplexer</td>
			<td>FixYourBoard, USA</td>
			<td>U802</td>
			<td>Use to acquire measurements from six sensing components&#160;</td>
		</tr>
		<tr>
			<td>DC/AC current source</td>
			<td>Keithley, USA</td>
			<td>6221</td>
			<td>Use to supply current</td>
		</tr>
		<tr>
			<td>SMD Digital Hot Air Rework Station</td>
			<td>Aoyue, China</td>
			<td>968A+</td>
			<td>Use to solder hard wires, to electrically connect between the device and external instruments</td>
		</tr>
		<tr>
			<td>Infrared camera</td>
			<td>FLIR, USA</td>
			<td>435-0001-01-00</td>
			<td>Use to take infrared images in experiment</td>
		</tr>
		<tr>
			<td>Digital multimeter</td>
			<td>Fluke, USA</td>
			<td>117</td>
			<td>Use to check electrical connection</td>
		</tr>
		<tr>
			<td>Lock-in amplifier</td>
			<td>Stanford Research System, USA</td>
			<td>SR830</td>
			<td>Use to perform four-point-probe-measurement</td>
		</tr>
		<tr>
			<td>Electron beam evaporator</td>
			<td>9 scale Vacuum Products, USA</td>
			<td>N/A (customized)</td>
			<td>Use to deposit thin films (Cu and SiO2)</td>
		</tr>
	</tbody>
</table>

fabrication and characterization of a conformal skin-like electronic system for quantitative, cutaneous wound management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

Figure 1 presents an overview of the characteristics of the conformal, skin-like electronic system, designed for quantitative, cutaneous wound management on patients. The multifunctional electronic device consists of microscale fractal structures3,14 and filamentary serpentine traces9,17 on a thin elastomeric membrane that offers exceptional mechanical stretchability and bendability. The compliant device that is completely enclosed by silicone layers enables gentle, reversible lamin...

Watch this Scientific Journal Video about Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management at JoVE.com

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

fabrication-characterization-conformal-skin-like-electronic-system

Research

JoVE Journal

Bioengineering

8.8K Görüntüleme.  Virginia Commonwealth University. This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management. 

Video: Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...