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>

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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, electrically safe, silicone layers that completely enclose the device allows the electronics to be used for the first time in a realistic clinical setting, cutaneous wound management on patients. 
In the protocol of device preparation, the following steps are critical to enhance the fabrication yield. Wet chemical etching of the metal layer (Cu) in the device should involve extreme time control since the etching rate is faster than other metals. The major drawback of Cu over-etching is the change of the combined electrical resistance of the device, which requires additional optimization and adjustment of equipment parameters in the data acquisition system. It should be noted that multiple layers of a water soluble tape needs to be used to avoid fracture during retrieval process of the electronics dissociated from a carrying PDMS. After the transfer printing of electronics with a water soluble tape, it is very important to completely remove any residue from the dissolvable material (polyvinyl alcohol) using multiple rinsing with DI water. In the process of cable bonding to the device, extra caution is required since the device is on a highly compliant and bendable material (low-modulus silicone), while the cable is thin plastic. Thus, careless handling of the device may lead to cause fracture or plastic deformation of metal resistors.
The device characterization and evaluation reveals some limitations in the clinical use. The data acquisition process using a thin cable still requires the connection with a series of equipment, which hinders continuous, long-term recording of skin parameters, relevant to wound management. The measured electrical signals accompany the downstream analysis for diagnosis, which limits on-site/point-of-care use of the device and system. 
Further improvements of the electronic system include the development of the integration of a wireless power supply and telecommunication system with the device and smart-app interface that monitors the skin parameters, collects the data, and diagnoses automatically for alerting to medical personnel. 
In conclusion, new strategies and methods of mechanical analysis and materials processing present a class of technology to develop a conformal, skin-like electronic system for quantitative, cutaneous wound management on patients. The biocompatible, waterproof, easy-to-use, mechanically compliant device provides the functions and characteristics for the use in the clinical settings. This type of electronic system has a potential to address important issues in chronic wound management.

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><tbody><tr><td>3&quot; Silicon wafer</td><td>University Wafer, USA</td><td></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></td><td>Use to make patterns on metals and polymers</td></tr><tr><td>AZ400K developer</td><td>AZ Electrionic Materials, USA</td><td></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 &#039;dry&#039; retrieval</td></tr><tr><td>PI2545 polyimide</td><td>HD MicroSystem, USA</td><td></td><td>Use to encapsulate metal layer</td></tr><tr><td>Solaris</td><td>Smooth-On, USA</td><td></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></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></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&amp;nbsp;</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></td><td>Use to deposit thin films (Cu and SiO&lt;sub&gt;2&lt;/sub&gt;)</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 lamination on the skin through van der Waals interactions alone. The unique characteristics of the device include biocompatibility, waterproofness, ease-of-use, and mechanical flexibility for the use in realistic clinical settings.
The integration of hybrid materials such as polymers and a metal (silicone, polyimide, and copper) yields an electrically safe, waterproof, and biocompatible device (Figure 2A). An array of fractal (copper; Cu) resistors (35 &#181;m in width and 3 &#181;m in thickness) is placed at the neutral mechanical plane, by enclosed polyimide (PI, 1.2 &#181;m in thickness) layers, to minimize applied bending strains on the core material (Cu) in clinical applications.
The total thickness of the device on a silicone membrane is only ~600 &#181;m by offering extreme bendability. The schematic illustrations in Figure 2B describe the microfabrication process of the skin-like electronic system. The fabrication method combines the conventional microfabrication techniques (metallization, photolithography, and etching) with the newly developed transfer-printing techniques (retrieval, transfer, and bonding)9,14,18,19. This type of a device can be scaled up by using large scale transfer printing with an automated printing equipment20,21.
Figure 3 summarizes the mechanical stretchability and electrical functionality of the skin-like electronics, reported in the prior work10. Mechanics and materials study by the finite element method (FEM) offers the optimal system design to accommodate natural motions and applied strains, involved in the clinical use, without mechanical fracture (Figure 3A, top). The experimental study that presents mechanical behavior of the fractal structure with tensile strains up to 30% (Figure 3Aa, bottom) shows a good agreement with the FEM results. The device with microscale resistors is used for quantitative measurement of temperature and thermal conductivity and delivering precise, localized heating (Figures 3B &#8211; 3D). The calibration curve of electrical resistance according to temperature change was obtained by using an infrared camera and a high-sensitivity hot plate (Figure 3B). The evaluation method of the measured thermal conductivity was adapted from the 3 omega technique13 that uses 3 omega voltage signals at two different alternating current frequencies (Figure 3c). Applied electrical current (35 mA with 10 mW) to the fractal resistors occurs Joule heating, which offers controllable temperature actuation in a therapeutic mode (Figure 3D).
For practical, clinical applications, the suggested cleaning process of the hand-held device involves disinfection prior to use on patients. Spraying of a disinfectant solution on the waterproof device and following rinsing in water three times prepares the device for clinical testing (Figures 4A and 4B). The assessment of qualitative biocompatibility of the device utilizes a digital contact microscope to visually inspect the skin surface (Figure 4C), which investigates the change of the skin color and texture over multiple cycles of use on patients. An infrared (IR) thermography can make a quantitative assessment of the skin conditions for about two weeks (Figure 4D) since the side effects such as erythema causes temperature elevation22. The examined devices are laminated near the wound tissues and the contralateral location (as a reference). Recording of relevant parameters of temperature and thermal conductivity is conducted using a data acquisition system and IR imaging in an exam room (Figures 4E and 4F).
Figure 5 presents representative data of quantitative measurement of cutaneous wound healing on a patient from a prior study10. A series of photos in Figure 5A shows the monitoring process of wound healing with the skin-mounted device over the course of one month. The wound device dyed with a black ink was laminated near the surgical wound. Pen marks on the skin guided the mounting of the device on the same location for quantitative data comparison from day 1 to day 30. Measurement of temperature and thermal conductivity variation using an array of sensors in the device and comparison between the wound and reference sites captures the wound healing phase, inflammation (Figures 5B &#8211; 5E). The highly sensitive, six sensors in the wound device were able to capture minimal change of the body temperature and point intense inflammation on Day 3 (Figures 5B &#8211; 5C) and record variation of thermal conductivity (Figures 5D &#8211; 5E). A set of reference data was measured from the contralateral side as a control.
<img alt="Figure 1" src="/files/ftp_upload/53037/53037fig1.jpg" /> 
Figure 1.&#160;Overview of characteristics of the skin-like, wound monitoring device on a patient. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53037/53037fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53037/53037fig2.jpg" /> 
Figure 2.&#160;Device fabrication. (A) Schematic illustration of the device layouts (left; layer 1: transparent silicone at the top, layer 2: PI, layer 3: Cu, layer 4: PI, and layer 5: black silicone at the bottom) and the completed, flexible/stretchable electronics (right). (B) Illustration of the step-by-step fabrication process (cross-sectional view). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53037/53037fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53037/53037fig3.jpg" /> 
Figure 3.&#160;Device characteristics (reproduced with permission from Advanced Healthcare Materials, 3 (10), 1597-1607, 2014)10. (A) Finite element method (FEM) results (top) and the corresponding experimental results (bottom) of a fractal structure under uniaxial tensile strains up to 30 %. (B) Measurement of temperature using six sensors for device calibration. (C) Measurement of thermal conductivity using three sensors for device calibration. (D) Infrared thermography of the device that was used as a micro-heater with localized Joule heating. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53037/53037fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53037/53037fig4.jpg" /> 
Figure 4.&#160;Clinical testing process. (A) Disinfection of the device using a cleaning solution. (B) Rinsing with water to clean the surface for clinical testing. (C) Skin assessment using a digital contact microscope (left) and magnified view of the skin (right). (D) Infrared thermography of the skin for quantitative assessment of temperature variation. (E) Clinical setting for wound management in an exam room. (F) Magnified photo of the laminated devices near the wound (right leg) and contralateral site (left leg) tissues. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53037/53037fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53037/53037fig5.jpg" /> 
Figure 5.&#160;Representative data of quantitative management of wound healing with the device (reproduced with permission from Advanced Healthcare Materials, 3 (10), 1597-1607, 2014)10. (A) Photos of the wound with the device over the course of a month. (B) Recording of temperature distribution near the wound for one month with six sensors in the device (inset). (C) Recording of temperature distribution on a contralateral location as a reference. (D) Recording of thermal conductivity near the wound for one month with three sensors in the device (inset). (E) Recording of thermal conductivity on a contralateral location as a reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53037/53037fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K Views.  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

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

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

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

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

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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 ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Developmental Biology

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering the growing interest in telemedicine and personalized care driven by the rise of the Internet of Things era, wearable sensors have expanded their field of application into healthcare. To ensure the collection of clinically relevant data, these devices need to establish conformable interfaces with the human body to provide high-signal-quality recordings and long-term operation. To this end, this paper presents a method to easily fabricate conformable thin tattoo- and soft textile-based sensors for their application as wearable organic electronic devices in a broad spectrum of surface electrophysiological recordings. 
The sensors are developed through a cost-effective and scalable process of cutaneous electrode patterning using poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), the most popular conductive polymer in bioelectronics, on off-the-shelf, wearable substrates. This paper presents key steps in electrode characterization through impedance spectroscopy to investigate their performance in signal transduction when coupled with the skin. Comparative studies are required to position the performance of novel sensors with respect to the clinical gold standard. To validate the fabricated sensors' performance, this protocol shows how to perform various biosignal recordings from different configurations through a user-friendly and portable electronic setup in a laboratory environment. This methods paper will allow multiple experimental initiatives to advance the current state of the art in wearable sensors for human body health monitoring.

Two recent technologies-tattoo and textiles-have demonstrated promising results in cutaneous sensing. Here, we present the fabrication and evaluation methods of tattoo and textile electrodes for cutaneous electrophysiological sensing. These electronic interfaces made of conductive polymers outperform the existing standards in terms of comfort and sensitivity.

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering ...

Development of a Benchtop Model for Evaluating the Compatibility of Wound Dressing Materials with Negative Pressure Wound Therapy Systems

Negative pressure wound therapy (NPWT) systems facilitate wound healing by applying sub-atmospheric pressure to the wound bed, which promotes granulation tissue formation and reduces inflammation. Wound dressings can be used with these systems to enhance healing; however, the effects of dressings on NPWT device performance are challenging to assess. The purpose of this study was to develop a benchtop flesh analog model for testing the compatibility of wound dressing materials with NPWT devices. In this study, a chitosan-based advanced wound care device was evaluated for its effects on NPWT performance under maximum and minimum therapy pressures. The goal was to use the model to compare pressure readings and fluid collection for samples with and without the chitosan wound care device. The benchtop model was constructed using a plastic box connected to multiple pressure gauges. A circular defect was created on a piece of pork belly, used as the flesh analog, and inserted into the box. The defect was filled with standard NPWT foam or foam combined with the wound dressing. Simulated body fluid containing bovine serum was added to the box, which was then tested at either maximum (-200 mmHg) or minimum (-25 mmHg) pressures for 72 h. Pressure and fluid collection were recorded every 12 h. The NPWT system successfully maintained pressure over the 72 h test period, both with and without the test dressings. The addition of the wound dressings did not impact fluid collection. The test box proved effective as a benchtop model, as it could be sealed and maintained vacuum conditions over the 72 h testing period. This model successfully demonstrated its utility in evaluating the compatibility of wound dressing materials with NPWT systems.

This study presents a benchtop model designed to evaluate the compatibility of wound dressing materials with negative pressure wound therapy systems by assessing pressure and fluid collection over 72 h under continuous and intermittent pressure settings.

Negative pressure wound therapy (NPWT) systems facilitate wound healing by applying sub-atmospheric pressure to the wound bed, which promotes granulation ...

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

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