The project is funded by the Engineering and Physical Sciences Research Council EPSRC (EP/K503745/1), National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) (BRC272/HI/JG/101440) and the UCL change makers.

The protocol follows the guidelines of the University College London&#39;s human research ethics committee.
CAUTION: This protocol contains an electrical hazard and a burn hazard (soldering iron); read both before attempting this protocol. This protocol includes connecting a device to skin. Ensure that at no time there is a path between the skin and electricity mains. Never touch the element of the soldering iron. Hold wires to be heated with tweezers or clamps. Keep the cleaning sponge wet during use. Always return the soldering iron to its stand when not in use. Never put it down on the workbench. Turn unit off and unplug it when not in use.
1. Assembling the Base Components
NOTE: Figure 3 gives a high-level overview of the protocol steps.
<ol>
	<li>To build the Bionic Clicker MK I, plug the bio-signals sensor shield into the microcontroller and screw the EMG cables into the E, M and GND screw terminals of the shield (see Figure 4). Continue to step 1.6.</li>
	<li>To build the MK II, place a row of three header pins into the muscle sensor in the +, - and SIG holes (see Figure 5) from above, and solder underneath.
	<ol>
		<li>Bend the header pins 90 &#176; with a pair of plyers halfway up the pins. This places the pins in the correct position for the case.</li>
		<li>If using the abductor indicis muscle as the input, continue to step 1.3, if not move to step 1.4.</li>
	</ol>
	</li>
	<li>Remove the short black reference cable from the muscle sensor.
	<ol>
		<li>Cut the three EMG cables with a wire cutter to run from the wrist to the back of the hand. Strip the end of the three EMG cables with a wire stripper.</li>
		<li>Place the stripped end of the black wire into the R hole, the blue wire into the E hole, and the red wire into the M hole of the muscle sensor (Figure 5). Solder the wires in place on the underside of the muscle sensor. Move to step 1.5.</li>
	</ol>
	</li>
	<li>Clip two electrode pads into the underside of the muscle sensor and one electrode into the connector of the black reference cable.
	<ol>
		<li>Stick the muscle sensor onto the selected muscle with the electrode pads and place the black reference electrode in an appropriate place.</li>
	</ol>
	</li>
	<li>Cut 8 single-core multi-thread wires to length and strip each end: 5 short (7 cm) wires to run from the microcontroller to the control board (red, black, green, white and blue) and 3 longer (approximately 12 cm but dependent on wrist size) wires (red, black and green) to run from one side of the wrist to the other. 
	NOTE: If placing the muscle sensor on a different muscle make sure that the longer wires will run from the muscle sensor site to the wireless microcontroller site.
	<ol>
		<li>Place the wires into the microcontroller ready for soldering: 2 red wires into the 3V hole, 2 black wires into the GND hole, the long green wire into the A0 hole, the short blue wire into the 2 hole, the long white wire into the 3 hole, and the short green wire into the 5 hole. Solder the wires in place on the underside of the microcontroller.</li>
		<li>Solder the other end of the 3 long wires to 3 header sockets in the order: red, black, green. See Figure 5. If not using the abductor indicis muscle proceed on to step 2.</li>
	</ol>
	</li>
	<li>Place the EMG sensor pads on the hand, as shown in Figure 6, with two of the electrodes at either end of the abductor indicis muscle and one EMG sensor pad on the middle of the back of the hand.
	<ol>
		<li>Clip the electrode pads into the connector end of the muscle sensor cables (push fit). The blue and red electrodes clip above the muscle, the black electrode clips on the back of the hand.</li>
	</ol>
	</li>
</ol>
2. Test EMG Output
<ol>
	<li>Download the library for the bio-signals shield following the link14 from the reference section. Unzip it and place it in the Integrated Development (IDE) libraries folder (usually found in documents/Arduino/libraries). Continue to step 2.3. If building the MK II, proceed to step 2.2.</li>
	<li>Add the microcontroller boards to the IDE, following the instructions15.</li>
	<li>Download &#39;ThresholdTest.ino&#39; for the MK I or &#39;BLEThresholdTest.ino&#39; and &#39;BluefruitConfig.h&#39; for the MK II and open in the IDE software (Supplemental files).</li>
	<li>Unplug the laptop from the mains and then, and only then, plug the microcontroller into the laptop via a Universal Serial Bus (USB) cable.</li>
	<li>Upload the relevant version of the threshold test to the microcontroller and then open the serial monitor (Tools&#62;Serial Monitor). The output of the EMG will now be displayed.</li>
	<li>Move the index finger from side to side and move the hand around without moving the index finger. Write down the values displayed in each case. 
	NOTE: When using the MK II make sure that the cables do not move as it is extremely sensitive to noise generated this way.</li>
	<li>Select a value that is above what is seen when the hand is moved around, but below what is seen when the finger is moved from side to side. Write down this value. 
	NOTE: The value is selected so that the device will only be activated by a purposeful movement of the finger. This is the Threshold Trigger Value, the value at which the device will be activated. The muscle sensor has a gain setting that can be manually altered if the threshold value is hard to find. The electrodes might need to be replaced. If using the abductor indicis muscle, set the gain to minimum as the starting point. The gain setting is altered by the potentiometer on the muscle sensor marked by GAIN, and this can be changed by a small flat-head screwdriver.</li>
</ol>
3. Test Threshold
<ol>
	<li>Download &#39;BoomTest.ino&#39; for the MK I or &#39;BLEBoomTest.ino&#39; and BluefruitConfig.h for the MK II and open it in the IDE software.</li>
	<li>Edit the provided code by replacing &#39;PLACE_YOUR_THRESHOLD_TRIGGER_VALUE_HERE&#39; with the Threshold Trigger Value previously determined in step 2.8. This is line 37 of the code for the MK I and line 47 of the code for the MK II.</li>
	<li>Upload the correct version of BoomTest to the microcontroller and then open the serial monitor (Tools&#62;Serial Monitor).</li>
	<li>Move the hand around (not moving the index finger from side to side); nothing is seen on the serial output.</li>
	<li>Move the index finger from side to side; the word &#39;BOOM&#39; appears. 
	NOTE: If &#39;BOOM&#39; appears at the wrong time or not at all, check the connections and move back to step 2.7.</li>
</ol>
4. 3D Print the MK II Case
<ol>
	<li>If building the MK II, download the stl files for all 5 components of the case (see Figure 7 for all 5 parts). Print the parts of the case by any preferred method. Proceed to step 5.2. If building the MK I, move on to section 5. 
	NOTE: The case has been successfully printed by both fused deposition modelling16 (FDM) and photolithography printers17.</li>
</ol>
5. Solder the Control Board
NOTE: If building the MK II, proceed to step 5.2.
<ol>
	<li>Place a row of two header pins, five 10 K&#937; resistors, a sliding switch and two push button switches for the components as shown in Figure 8A; then solder them in place on the underside of the board.
	<ol>
		<li>Break the copper tracks on the strip board by slicing through with a craft knife, following the grey lines on Figure 8A. This allows for individual tracks to have multiple functions across the board.</li>
		<li>Cut 7 wires (black, red, blue, orange, white, brown and yellow) of the correct length with a wire cutter so that they will run from the forearm to the upper arm (around 30 cm). Cut a red wire of 7 cm, a black wire of 3 cm and an orange and a blue wire of 4 cm.</li>
		<li>Strip both ends of the wires with a wire stripper.</li>
		<li>Place the wires in the control board, following the circuit diagram shown in Figure 9; solder the wires in place on the underside.</li>
		<li>Solder the long red and black wires to a pair of header pins, and then solder the other long wires to a strip of header pins in the order: blue, orange, white, brown, yellow.</li>
		<li>Solder the 5V and GND pins of the wireless module to the header pins on the control board.</li>
		<li>Solder the short orange wire to pin 2 of the wireless communication module and the short blue wire to pin 3.</li>
	</ol>
	</li>
	<li>Place three 10 K&#937; resistors, a sliding switch and two push button switches as shown in Figure 10A and solder them into place on the underside of the board.
	<ol>
		<li>Break the copper tracks on the strip board by slicing through with a craft knife, following the grey lines on Figure 10A. This allows for the track to have multiple functions on the board.</li>
		<li>Cut the wires that were previously soldered to the microcontroller with a wire cutter so that they can run through the mid layer of the microcontroller case to the control board without stopping the case from closing (Figure 10B).</li>
		<li>Place the wires in the control board, following the circuit diagram (Figure 11). Solder the wires in place. Proceed to step 6.2.</li>
	</ol>
	</li>
</ol>
6. Assemble Clicker and Update Microcontroller
<ol>
	<li>Re-assemble the Bionic Clicker, connecting the header connectors from the control board wires to the microcontroller and bio-signals shield (5V and GND on the MK I, pin 22-30 on the MKII). Connect the battery to the microcontroller. See Figure 12. Move on to step 6.3.</li>
	<li>Re-assemble the Bionic Clicker, connecting the header connector from the microcontroller to the muscle sensor (green wire to SIG). See Figure 13.</li>
	<li>Connect the microcontroller to the laptop via USB cable.</li>
	<li>Download &#39;BionicClicker.ino&#39; or &#39;BLEBionicClicker.ino and BluefruitConfig.h and open it in the IDE software.</li>
	<li>Edit the code and replace &#39;PLACE_YOUR_THRESHOLD_TRIGGER_VALUE_HERE&#39; with the Threshold Trigger Value determined in step 2.7 (on line 59 of the code for the MK I, line 83 of the code for the MK II). 
	NOTE: The name that the MK II device appears as when connecting over wireless can be changed by editing line 47 of the code. Replace &#39;Bionic Clicker MK II&#39; with an alternative title.</li>
	<li>Disconnect the microcontroller from the laptop by removing the USB cable.</li>
</ol>
7. Connect the Device to a Computer
<ol>
	<li>If using the MK I, follow the instructions to pair the wireless module to the device by following the manufacturer&#39;s guide18. If using the MK II, connect to the device wirelessly following the procedure to connect a wireless keyboard to the computer being used.</li>
</ol>
8. Test the Clicker
<ol>
	<li>Open some typing software and enter some text, such as &#39;Lorem ipsum dolor sit amet&#39;. This allows the presses to be perceived to test whether these commands are sent and received. 
	NOTE: If the battery is low the device may give erratic behavior; always use a fresh battery.
	<ol>
		<li>Press the manual forward button to see the cursor move forward and the manual backwards button to see the cursor move backwards. Raise the index finger to also move forward.</li>
	</ol>
	</li>
	<li>To test the clicker with the presentation software, raise the index finger to progress the slides. 
	NOTE: The override switch turns the EMG function on and off, and the manual forward and backwards buttons progress and retreat the slides in both scenarios.</li>
</ol>
9. Mount the Clicker
NOTE: If building the MK II move to step 9.2.
<ol>
	<li>If building the MK I, cut the double-sided hook and loop material with scissors, so that it fits comfortably around the wrist. Make sure the loops are facing inwards to not scratch the wrist.
	<ol>
		<li>Cut the double-sided hook and loop material so that it fits comfortably around the upper arm, again make sure the loops face inwards.</li>
		<li>Cut the double-sided hook and loop strips to the size of the microcontroller (10 cm x 5 cm) and the control board (2.5 cm x 6.4 cm). Cut a strip that will fit tightly around the battery (4 cm x 12 cm).</li>
		<li>Using the glue gun, glue the loop side of the strips to the bottom of the microcontroller and the bottom of the control board.</li>
		<li>Attach the control board to the wrist strap. Attach the microcontroller and battery to the upper arm strap.</li>
		<li>Plug everything in: The 9 V battery plugs into the microcontroller with the PP3 connector. The microcontroller and e-health shield connect to the control board via the soldered wires. 
		NOTE: The MK I is now finished.</li>
	</ol>
	</li>
	<li>If building the MK II, cut double-sided hook and loop material 35 mm wide and long enough to wrap around the wrist (around 22 cm for smaller wrists).
	<ol>
		<li>Slide the hook and loop material through the clips at the bottom of the case. Make sure that the loops are facing inwards to not scratch the wrist.</li>
		<li>Plug the wires soldered to the microcontroller terminating in the female header into the male header pins on the muscle sensor, and clip the electrodes into the EMG cables by pushing them on. 
		NOTE: The MK II is now finished. See Figure 14.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Navarro, X., Krueger, T. B., Lago, N., Micera, S., Stieglitz, T., Dario, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+interfaces+with+the+peripheral+nervous+system+for+the+control+of+neuroprostheses+and+hybrid+bionic+systems.">A critical review of interfaces with the peripheral nervous system for the control of neuroprostheses and hybrid bionic systems.</a> J Periph Nerv Syst. 10 (3), 229-258 (2005).</li><li>Yang, D. P., 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=An+anthropomorphic+robot+hand+developed+based+on+underactuated+mechanism+and+controlled+by+EMG+signals.">An anthropomorphic robot hand developed based on underactuated mechanism and controlled by EMG signals.</a> J Bionic Eng. 6 (3), 255-263 (2009).</li><li>Chu, J. U., Moon, I., Lee, Y. J., Kim, S. K., Mun, M. 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+supervised+feature-projection-based+real-time+EMG+pattern+recognition+for+multifunction+myoelectric+hand+control.">A supervised feature-projection-based real-time EMG pattern recognition for multifunction myoelectric hand control.</a> IEEE/ASME Trans Mechatronics. 12 (12), 282-290 (2007).</li><li>Bitzer, S., Van Der Smagt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+EMG+control+of+a+robotic+hand:+towards+active+prostheses.">Learning EMG control of a robotic hand: towards active prostheses.</a> Proceedings 2006 IEEE International Conference on Robotics and Automation. , (2006).</li><li>Cipriani, C., Zaccone, F., Micera, S., Carrozza, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+shared+control+of+an+EMG-controlled+prosthetic+hand:+analysis+of+user-prosthesis+interaction.">On the shared control of an EMG-controlled prosthetic hand: analysis of user-prosthesis interaction.</a> IEEE Trans Rob. 24 (1), 170-184 (2008).</li><li>Tenore, F., Ramos, A., Fahmy, A., Acharya, S., Etienne-Cummings, R., Thakor, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+control+of+individual+fingers+of+a+prosthetic+hand+using+surface+EMG+signals.">Towards the control of individual fingers of a prosthetic hand using surface EMG signals.</a> IEEE EMBS. 2007, 6145-6148 (2007).</li><li>Reinvee, M., P&#228;&#228;suke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+Contemporary+Low-cost+sEMG+Hardware+for+Applications+in+Human+Factors+and+Ergonomics.">Overview of Contemporary Low-cost sEMG Hardware for Applications in Human Factors and Ergonomics.</a> In Proc Hum Fact Ergon Soc Annu Meet. 60 (1), 408-412 (2016).</li><li>George, P. K., Minas, V. L., Agisilaos, G. Z., Christoforos, I. M., Kostas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-Source,+Anthropomorphic,+Underactuated+Robot+Hands+with+a+Selectively+Lockable+Differential+Mechanism:+Towards+Affordable+Prostheses.">Open-Source, Anthropomorphic, Underactuated Robot Hands with a Selectively Lockable Differential Mechanism: Towards Affordable Prostheses.</a> 2015 IEEE/RSJ International Conference of Intelligent Robots and Systems. , (2015).</li><li>Agisilaos, G. Z., Minas, V. L., Christoforos, I. M., Kostas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-Source,+Affordable,+Modular,+Light-Weight,+Underactuated+Robot+Hands.">Open-Source, Affordable, Modular, Light-Weight, Underactuated Robot Hands.</a> IEEE/RSJ International Conference of Intelligent Robots and Systems. , (2014).</li><li>Minas, V. L., Agisilaos, G. Z., Melina, N. B., Kostas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-Source,+Low-Cost,+Compliant,+Modular,+Underactuated+Fingers:+Towards+Affordable+Prostheses+for+Partial+Hand+Amputations.">Open-Source, Low-Cost, Compliant, Modular, Underactuated Fingers: Towards Affordable Prostheses for Partial Hand Amputations.</a> 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. , (2014).</li><li>Chatterjee, H. J., Hannan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Engaging the senses: object-based learning in higher education. , (2015).</li><li>Zainee, N. M., Chellappan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergency+clinic+multi-sensor+continuous+monitoring+prototype+using+e-Health+platform.">Emergency clinic multi-sensor continuous monitoring prototype using e-Health platform.</a> 2014 IEEE Conference on Biomedical Engineering and Sciences (IECBES). , (2014).</li><li>Paul, P., Motskin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engaging+the+Public+with+Your+Research.">Engaging the Public with Your Research.</a> Trends Immunol. 37 (4), 268-271 (2016).</li><li>. e-Health Sensor Platform V2.0 for Arduino and Raspberry Pi Available from: <a target="_blank" href="https://www.cooking-hacks.com/documentation/tutorials/ehealth-biometric-sensor-platform-arduino-raspberry-pi-medical#step3_1">https://www.cooking-hacks.com/documentation/tutorials/ehealth-biometric-sensor-platform-arduino-raspberry-pi-medical#step3_1</a> (2017)</li><li>. Arduino IDE Setup Available from: <a target="_blank" href="https://learn.adafruit.com/add-boards-arduino-v164/setup">https://learn.adafruit.com/add-boards-arduino-v164/setup</a> (2017)</li><li>. Ultimaker 2+ Available from: <a target="_blank" href="https://ultimaker.com/en/products/ultimaker-2-plus">https://ultimaker.com/en/products/ultimaker-2-plus</a> (2017)</li><li>. Form 2 Available from: <a target="_blank" href="https://formlabs.com/3d-printers/form-2/">https://formlabs.com/3d-printers/form-2/</a> (2017)</li><li>. Pairing to Bluefruit Available from: <a target="_blank" href="https://learn.adafruit.com/introducing-bluefruit-ez-key-diy-bluetooth-hid-keyboard/pairing-to-bluefruit">https://learn.adafruit.com/introducing-bluefruit-ez-key-diy-bluetooth-hid-keyboard/pairing-to-bluefruit</a> (2017)</li></ol>

The authors have nothing to disclose.

The saturation of the MK II when used on the abductor indicis is less of an issue than it may first appear. Careful placement of the electrodes and correct gain setting stops this from being an issue when the device is used as a clicker. Unless interested in accurately recording the activity of the abductor indices, this is unlikely to be a problem at all. No over-saturation has been seen on any other muscle after the gain has been set. The false negatives with the MK II are due to the difficulty of selecting the proper threshold value when using the abductor indicis. With larger muscles the difference between the magnitude of non-purposeful activation of the muscle and purposeful tensing of the muscle is greater, allowing for the selection of a threshold point that is further from both the false-positive and false-negative points. On particularly small hands the abductor indicis muscle may be too small for the electrodes to be correctly placed (though with smaller electrode pads this could potentially be solved).
The considerably longer battery life for the MK II is useful for a variety of reasons. Firstly, the MK I device started to act erratically after 45 minutes of use, so it cannot be used for longer demonstrations. Secondly with a multi-hour battery life, the MK II can be considered as a control input for a useful device, and with only a small increase in physical battery size, it could be used as an all-day monitoring device. The wireless microcontroller has 6 analogue inputs and 13 digital inputs; this means that the device could accept signals from multiple muscle sensors to create a device with more degrees of freedom in the control inputs. It should also be noted that the muscle sensor could be replaced by any biosensor with an analogue output to create a device that uses other biological signals as the input. The code of the device can also be easily modified to change its functionality. Changes to the software and hardware of the device allow for simple and varied modifications to the device.
One limitation of the device as it currently stands is that the EMG output cannot be sent wirelessly at a high data rate as this can overload the wireless microcontroller buffer. Another limitation is that the technique uses the abductor indices as the input, and as the muscle is very small, the spacing of the electrodes on the hand almost overlap; if someone has particularly small hands, it may be impossible to place the electrodes correctly over this muscle. 
The device has several advantages over the more expensive devices when it comes to flexibility in potential research projects. It is low cost: the device costs 80 USD and additional EMG channels only cost 35 USD, making it ideal for smaller or student projects. It is easy to customize, the software can be easily edited, and the inputs changed for other hardware. It has a small size, so a person wearing it does not need to carry heavy or bulky equipment. It also appears as a wireless keyboard to other devices, so it can be easily integrated with any compatible wireless device. The device has already been incorporated into an assistive device that will be published in the near-future.
Due to the size and ease of customization of the MK II, it is already being considered for incorporation into several research projects as a wireless EMG module and as a wireless trigger mechanism. It is also being used as the foundation of one of the lab sessions on a master student's course. The main improvement we would like to make to the device is to increase the wireless transmission rate; the goal is to achieve 10 Hz, and whether this will be done through hardware or software is yet to be determined. 
The most critical steps within the protocol are steps 2.6 and 2.7: the selection of the Threshold Trigger Value. In step 2.6, special attention needs to be paid to the movement of the EMG cables, as these can act as antenna and generate motion artifacts; however, if these are kept stationary this is not an issue. In step 2.7, if the selected value is too high, this results in false negatives. If this value is too low, this results in false positives. In the case of the abductor indicis, it is very difficult to find a value that does not result in the occasional false negative, although with larger muscles this does not appear to be an issue. If finding the correct value is still an issue, the gain can be corrected by setting it to the minimum value and increasing it until a large difference between non-activation and activation is seen through the serial readout, with the values staying below the point of saturation.
Overall the MK II is a considerable improvement over the MK I as a potential research device, although because the MK I has a stronger visual impact, it is likely to still be used in future public engagement events.

The Aspire Centre for Rehabilitation Engineering and Assistive Technology investigates techniques that are applicable and transferable between different domains in related areas of interest, including but not limited to, stroke, muscular dystrophy, amputation, the ageing population and training of specialized skills. One area of research that the center is involved in is neuroprosthesis. Of the many techniques used for control of neuroprosthetic arms, EMG is one of the most common inputs for the control systems1,2,3,4,5,6. This is in a large part due to its ease of use and affordability when compared to other control systems7. Recently developed 3D-printed prostheses such as the Ada hand can cost only 1,000 USD when using this type of control8,9,10. However, when attempting to demonstrate such systems to the public there is no easy way of doing so without the aid of an amputee.
To raise the awareness of the research activities in this field to members of the public, a bionic clicker demo device was developed. It is very important to use object-based demonstration as it attracts attention and accelerates the learning and understanding of the subject being taught11. Our device not only helps to teach the concept of EMG but also to increase the knowledge of the current development of modern technologies. Moreover, it inspires younger generations to choose studies within Science, Technology, Engineering and Math (STEM) areas.
The Bionic Clicker MK I was made using readily available parts that were already in use within the lab. It consisted of a microcontroller, a bio-signals shield12, electrodes, a control board, a wireless communication board and a 9 V battery. The device functioned by picking up the activity of the abductor indicis muscle located between the index finger and thumb. It triggers a slide change by mimicking a keyboard and sending a &#39;right keypress&#39; whenever a preselected threshold was reached. The control board allowed for the manual sending of &#39;right&#39; and &#39;left&#39; keypresses (progress slides and retreat slides) and could also be used to override the EMG input if things went wrong during a live demonstration.
As part of the Medical Physics and Biomedical Engineering department public engagement activities, we demonstrated the Bionic Clicker to members of the public. It received an enthusiastic response from the audience and generated interest in starting several collaborations. After the success of the initial device a second version of the device was designed.
The goal for the design of the second device was to produce a device that was cheaper, less cumbersome and more customizable than the first device. The purpose of this device was to design something that could easily be modified for student projects and be cheaply incorporated into existing projects. The main advantage of this device over others available1,2,3,4,5,6 is its ease of use, small size, and low cost. Although the bionic clicker devices may not have the resolution of other research devices, such as trigger devices, they are more than good enough. The MK II would be an ideal basis for any system that uses an EMG threshold to trigger a device, such as a prosthetic controller or assistive device.
The design was based around a wireless-capable microcontroller and a muscle sensor. It also included a 3.7 V 150 mAh lithium polymer battery, a manual control board and a 3D-printed case. Figure 3 shows an overview of the differences between the designs. The MK II design has the same basic functionality as the original device but has significantly more potential functionality for new applications such as wireless EMG monitoring.

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">For the Mark I</td>
			<td style="width:77px;"></td>
			<td style="width:308px;"></td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laptop</td>
			<td></td>
			<td></td>
			<td>Any laptop with USB</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">USB B cable</td>
			<td></td>
			<td></td>
			<td>From laptop to USB-B connection on Arduino</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soldering Station</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot glue gun</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot glue gun glue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Items</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Small Single-Core Multi-thread Wires</td>
			<td></td>
			<td></td>
			<td>Black, Red, Yellow, Brown, Orange White, Blue,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arduino MEGA 2560</td>
			<td>Arduino</td>
			<td>Arduino MEGA 2560</td>
			<td>(Geniuno MEGA 2560 outside US)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-Health Shield v2.0</td>
			<td>Cooking Hacks</td>
			<td>e-Health Sensor Shield V2.0 for Arduino, Raspberry Pi and Intel Galileo [Biometric / Medical Applications]</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMG cables</td>
			<td>Cooking Hacks</td>
			<td>Electromyography Sensor (EMG) for e-Health Platform [Biometric / Medical Applications]</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMG Electrodes</td>
			<td>Sparkfun</td>
			<td>SEN-12969</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">9V battery</td>
			<td></td>
			<td></td>
			<td>Any</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Power cable</td>
			<td></td>
			<td></td>
			<td>PP3 9v connector with jack</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bluefruit EZ-KEY HID</td>
			<td>Adafruit</td>
			<td align="right">1535</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">strip board</td>
			<td>Amazon.co.uk</td>
			<td>Small Stripboard 25 X 64mm Pack of 3</td>
			<td>any similiar stripboard 2.54mm pitch 7x25</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">push button switch</td>
			<td></td>
			<td>COM-00097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">slide switch</td>
			<td>amazon.com</td>
			<td>20 Pcs On/Off/On DPDT 2P2T 6 Pin Vertical DIP Slide Switch 9x4x3.5mm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">resistors</td>
			<td></td>
			<td>COM-11508</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double sided Velcro</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Break Away Headers - Straight</td>
			<td>Sparkfun</td>
			<td>PRT-00116</td>
			<td>2, 2 and 5 needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">For the Mark II</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laptop</td>
			<td></td>
			<td></td>
			<td>Any laptop with USB connection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">USB micro cable</td>
			<td></td>
			<td></td>
			<td>From laptop to USB micro (standard phone connector style)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soldering Station</td>
			<td></td>
			<td></td>
			<td>Any</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Items</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Small Single-Core Multi-thread Wires</td>
			<td></td>
			<td></td>
			<td>Black, Red, Green, White, Blue,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Feather BLE 32U4</td>
			<td>Adafruit</td>
			<td align="right">2829</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MyoWare</td>
			<td>Sparkfun</td>
			<td>SEN-13723</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMG cables</td>
			<td>Sparkfun</td>
			<td>CAB-12970</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMG electrodes</td>
			<td>Sparkfun</td>
			<td>SEN-12969</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3.7 V LiPo</td>
			<td>Adafruit</td>
			<td align="right">1317</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Strip Board</td>
			<td>amazon.co.uk</td>
			<td>Small Stripboard 25 X 64mm Pack of 3</td>
			<td>2.54 pitch 7x9 rows</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Push Button switch</td>
			<td></td>
			<td>COM-00097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">slide switch</td>
			<td>amazon.com</td>
			<td>20 Pcs On/Off/On DPDT 2P2T 6 Pin Vertical DIP Slide Switch 9x4x3.5mm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">resistors</td>
			<td></td>
			<td>COM-11508</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3D printed parts</td>
			<td></td>
			<td></td>
			<td>Can be 3D printed yourself or printed from a website</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double sided Velcro</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Break Away Headers - Straight</td>
			<td>Sparkfun</td>
			<td>PRT-00116</td>
			<td>3 pins needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Female Headers</td>
			<td>sparkfun</td>
			<td>PRT-00115</td>
			<td>3 pins needed</td>
		</tr>
	</tbody>
</table>

the bionic clicker mark i &amp; ii

In this manuscript, we present two 'Bionic Clicker' systems, the first designed to demonstrate electromyography (EMG) based control systems for educational purposes and the second for research purposes. EMG based control systems pick up electrical signals generated by muscle activation and use these as inputs for controllers. EMG controllers are widely used in prosthetics to control limbs.
The Mark I (MK I) clicker allows the wearer to change the slide of a presentation by raising their index finger. It is built around a microcontroller and a bio-signals shield. It generated a lot of interest from both the public and research community.
The Mark II (MK II) device presented here was designed to be a cheaper, sleeker, and more customizable system that can be easily modified and directly transmit EMG data. It is built using a wireless capable microcontroller and a muscle sensor.

The MK II is more affordable, customizable and less cumbersome than the MK I device. The entire MK II costs only slightly more than the bio-signals shield alone (75 USD). The device is significantly smaller sitting on the wrist rather than the arm and the wireless microcontroller could potentially simultaneously support inputs from 6 muscle sensors. The functional battery life of the MK I device is just under an hour using a 9 V 550 mAh battery and the functional battery life of the MK II device (when used as a clicker) is around 8 hours using a 3.7 V 150 mAh battery; see Table 1 for a comparison of the devices.
The Bionic Clicker MK II can have an issue when used on the abductor indicis: the amplifier can saturate and take more than a second to discharge (see Figure 15). Careful placement of the electrodes and correctly setting the gain can overcome this issue. This does not happen with the Bionic Clicker MK I or on any other commonly used muscles for EMG.
Whilst calibrating the devices to find the Threshold Trigger Value, many different values can be observed. They fall into three ranges: the values when the hand is stationary, the values when the hand is moving, and the values when the finger is moved. Table 2 shows recorded values in each range; for the stationary and hand moving ranges, the maximum values are shown and for the finger tensing range the minimum value is shown. The threshold value is selected to lie above the hand moving value and below the finger tensing value. A value nearer to the hand moving range increases the chance of false positive and reduces the chance of false negatives, whilst a value closer to the finger tensing range has the opposite effect.
Both devices where tested for false negatives and false positives when tensing the abductor indices muscle. A false negative was recorded when the device did not trigger a change of slide upon tensing of the muscle and a false positive was recorded if the slide changed when no tensing occurred. Neither device had an issue with false positives, though the MK II device experienced the occasional false negative (less than 5% of the time). The MK I device experiences no false positives or negatives during the first 45 minutes of operation, though the number of false negatives increases rapidly until total device failure between 50 minutes and an hour (see Table 3).
These results show that the device succeeded in its stated aims. Table 1 shows that the MK II is cheaper and has more flexibility than the MK I. Table 2 and Table 3 show that the device functions as intended and can be used as an EMG-based trigger device. Figure 15 shows the issues that can occur if using the abductor indices muscle: this is not a problem that occurs with most muscles and can be fixed by altering the gain. Although the devices have some issues, they are sufficient for the intended use.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55705/55705fig1.jpg" /> 
Figure 1: The Bionic Clicker MK I. This shows the Bionic Clicker MK I and all of its components mounted on the left arm. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55705/55705fig2.jpg" /> 
Figure 2: Block diagram of the devices. Each box represents a separate section of the device; within each box is the functionality that section has as a part of the device. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55705/55705fig3.jpg" /> 
Figure 3: Steps to build the device. A flow diagram containing a high-level overview of each step of the protocol. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55705/55705fig4.jpg" /> 
Figure 4: Initial MK I assembly. Microcontroller with the bio-signals shield and electrode cables. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55705/55705fig5.jpg" /> 
Figure 5: Initial MK II assembly. Microcontroller with the muscle sensor and soldered connections. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55705/55705fig6.jpg" /> 
Figure 6: Electrode Placement. This figure shows the correct placement of the electrodes on the hand when using the abductor indicis. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55705/55705fig7.jpg" /> 
Figure 7: The MK II case parts. The parts of the MK II case ready for printing in a photolithography printer. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55705/55705fig8.jpg" /> 
Figure 8: The MK I control circuit. (a) Circuit board from above (gray marks where the strip board had contacts broken on the underside). (b) Completed Circuit Board. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55705/55705fig9.jpg" /> 
Figure 9: The MK I control board circuit diagram. The circuit diagram for the MK I control board showing the connections between the resistors, switches and wires. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/55705/55705fig10v2.jpg" /> 
Figure 10: The MK I control circuit. (a) Control Board from above (gray mark where the strip board had contact broken on the underside). (b) Completed Circuit Board <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig10v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/55705/55705fig11.jpg" /> 
Figure 11: The MK II control board circuit diagram. The circuit diagram for the MK I control board showing the connections between the resistors, switches and wires. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/55705/55705fig12.jpg" /> 
Figure 12: The assembled MK I. This shows all the components of the MK I device before they have been mounted on the arm. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/55705/55705fig13.jpg" /> 
Figure 13: Assembling the Clicker MK II. (a) Place the microcontroller in the bottom of the case. (b) Place the battery in the mid-section and put on the lid. (c) Place the muscle sensor in its case and put on the lid. (d) Connect the microcontroller to the muscle sensor and connect the battery to the microcontroller. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/55705/55705fig14.jpg" /> 
Figure 14: The completed Bionic Clicker MK II. (a) On the hook and loop strap. (b) On the wrist. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/55705/55705fig15.jpg" /> 
Figure 15: Oversaturation of the muscle sensor. This figure shows what happens when the muscle sensor is oversaturated; the plateaus are when muscle activation was too strong for the current gain setting on the device. <a href="http://cloudflare.jove.com/files/ftp_upload/55705/55705fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>MK I</td>
			<td>MK II</td>
		</tr>
		<tr>
			<td>EMG sensor</td>
			<td>General Bio-sensor</td>
			<td>Dedicated Muscle Sensor</td>
		</tr>
		<tr>
			<td>Wireless</td>
			<td>Separate wireless module</td>
			<td>On the microcontroller board</td>
		</tr>
		<tr>
			<td>EMG over wireless?</td>
			<td>No</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>9 V PP3</td>
			<td>150 mAh LiPo</td>
		</tr>
		<tr>
			<td>Operational Time</td>
			<td>1 h</td>
			<td>8 h</td>
		</tr>
		<tr>
			<td>Build Time</td>
			<td>5 h</td>
			<td>4 h</td>
		</tr>
		<tr>
			<td>Total cost</td>
			<td>$150</td>
			<td>$80</td>
		</tr>
		<tr>
			<td>False Positives (%)</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>False Negatives (%)</td>
			<td>0</td>
			<td>4.7</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of the devices. This table compares several aspects of the devices, from design to functionality.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Stationary (maximum)</td>
			<td>Hand Moving (maximum)</td>
			<td>Finger Tensing (minimum)</td>
			<td>Threshold Value</td>
		</tr>
		<tr>
			<td>MK I</td>
			<td>25</td>
			<td>35</td>
			<td>215</td>
			<td>200</td>
		</tr>
		<tr>
			<td>MK II</td>
			<td>40</td>
			<td>280</td>
			<td>460</td>
			<td>400</td>
		</tr>
	</tbody>
</table>
Table 2: Calibration Results. This table shows the values obtained whilst keeping the hand stationary, moving the hand and finger tensing, as well as the threshold value selected.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Number of false negatives (Tested every 30 s)</td>
			<td colspan="2">Number of false positives (Spontaneous activations)</td>
		</tr>
		<tr>
			<td></td>
			<td>First 45 min</td>
			<td>45 min-1 h</td>
			<td>1-8 h</td>
			<td>First h</td>
			<td>1-8 h</td>
		</tr>
		<tr>
			<td>MK I</td>
			<td>0</td>
			<td>35</td>
			<td>N/A</td>
			<td>0</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>MK II</td>
			<td>4</td>
			<td>1</td>
			<td>40</td>
			<td>0</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 3: Testing of the devices. Comparison of false positives and false negatives between the two devices.
Supplemental code files for MK I and MK II: 
<a href="https://www.jove.com/files/ftp_upload/55705/BionicClicker.ino" target="_blank">Please click here to download &#34;BionicClicker.ino&#34;</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/BLEBionicClicker.ino" target="_blank">Please click here to download &#34;BLEBionicClicker.ino&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/BLEBoomTest.ino" target="_blank">Please click here to download &#34;BLEBoomTest.ino&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/BLEThresholdTest.ino" target="_blank">Please click here to download &#34;BLEThresholdTest.ino&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/BoomTest.ino" target="_blank">Please click here to download &#34;BoomTest.ino&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/ThresholdTest.ino" target="_blank">Please click here to download &#34;ThresholdTest.ino&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/Feather-Featherbase.stl" target="_blank">Please click here to download &#34;Feather-Featherbase.stl&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/Feather-Feathermid.stl" target="_blank">Please click here to download &#34;Feather-Feathermid.stl&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/Feather-Feathertop.stl" target="_blank">Please click here to download &#34;Feather-Feathertop.stl&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/Myo-Myobase.stl" target="_blank">Please click here to download &#34;Myo-Myobase.stl&#34;.</a> 
<a href="https://www.jove.com/files/ftp_upload/55705/Myo-Myolid.stl" target="_blank">Please click here to download &#34;Myo-Myolid.stl&#34;.</a> 
&#160;

Watch this Scientific Journal Video about The Bionic Clicker Mark I & II at JoVE.com

The Bionic Clicker Mark I & II

A device was created to demonstrate electromyography-based control to a lay audience. After the success of the initial device, a second device was made with greater flexibility in functionality for demonstration and research purposes. This protocol describes the process of building and calibrating both devices.

The Bionic Clicker Mark I &amp; II

In this manuscript, we present two 'Bionic Clicker' systems, the first designed to demonstrate electromyography (EMG) based control systems for ...

Research

JoVE Journal

Bioengineering

16.3K Views.  University College London. A device was created to demonstrate electromyography-based control to a lay audience. After the success of the initial device, a second device was made with greater flexibility in functionality for demonstration and research purposes. This protocol describes the process of building and calibrating both devices.

Video: The Bionic Clicker Mark I & II

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont&#8211;free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock&#8217;s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...