Difference between revisions of "Team:ZJU-China/InterLab"

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<div class="title">
 
<div class="title">
<div class="tl">INTERLAB&nbsp;<h5>OVERVIEW&nbsp;</h5></div>
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<div class="tl">HARDWARE&nbsp;&nbsp;<h5>ABSTRACT&nbsp;&nbsp;</h5></div>
 
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<p>Reliable and repeatable measurement is important in all science experience, so as the synthetic biology. Aiming to repeat measurements in different labs and make measurement tools more available, the Measurement Committee chose GFP as the measurement marker, providing with a detailed protocol and data analysis form, and invite all the iGEM teams to participate in the Interlab study. This is the Fifth Measurement InterLab and we are honored to take part in the study this year.</p>
+
<p>At the very beginning of hardware construction, all biological products and other auxiliary reagents (like Au nanoparticles and Nafion) have to immobilized on the electrode. To meet the need of large-scale manufacturing and high accuracy, we used a 2D printer to print these materials onto IDEs (<span style="font-weight: bolder;"> I</span>nter<span style="font-weight: bolder;">D</span>igital <span style="font-weight: bolder;">E</span>lectrode) to modify the Au-surface. Then we assembled IDEs, amplifier, convertor and digital interface to an entire circuit. The signal from the circuit is transmitted to an iPhone through Bluetooth. Several blood samples from a qualified Hospital were collected for access our hardwares and enzyme IDEs. </p>
</br>
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<p>The objective of the Interlab this year is to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD. Following the requirements and protocol gave by the Measurement Committee, we have obtained and uploaded the data. We also submitted flow cytometry data for extra credit.</p>
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<img style="margin-left: 25% !important; width: 50% !important;" src="https://static.igem.org/mediawiki/2018/8/85/T--ZJU-China--HDhardware.png" />
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  <p style="text-align: center;"> Fig. 1  Work flow of our hardware </p>
 
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<span class="psg_ttl">Methods and Materials</span>
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<span class="psg_ttl">2D printer</span>
<span class="psg_ttl psg_subtitle">Transformation</span>
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<p>Our 2D printer consists of a constant-flow pump, motors, an Arduino Uno microcontroller. The printer’s user interface is based on a Windows software by Steamduino which support several file formats like CAD scripts or normal pictures. Imported files will be converted to 2D routes and transmitted to the microcontroller. The speed of motors and flow rate of the "ink" are controllable in the panel. Since the space between lead feet of IDEs is about 4 mm, the printer can reach an accuracy of 3 mm. The path-programmable printer offers more possibilities for developers to design on the level of electrodes. For example, separated areas of the chip can be modified with different enzymes. </p>
<p>Transform <i>Escherichia coli</i> DH5&alpha; with these following plasmids (all in pSB1C3):</p>
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<img style="width: 60% !important; margin-top: 1em;" src="https://static.igem.org/mediawiki/2018/b/be/T--ZJU-China--HD02.png" />
<p style="padding-left: 3em; margin-top: .6em;">&bull; Negative control: BBa_R0040</p>
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<h5>Fig. 2 Real picture of a 2D printer and its control panel</h5>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Positive control: BBa_I20270</p>
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<span class="psg_ttl">IDE (interdigital electrode) design</span>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 1: BBa_J364000</p>
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<p>IDE (interdigital electrode) is composed of two interdigital electrodes with two connection tracks, on an insulative substrate. IDE is cheaper than normal electrodes and can work with low volumes of sample. The interdigitated configuration typically enhances sensitivity and detection limits.</p>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 2: BBa_J364001</p>
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                    <p></br>The IDE circuit is planted on a PET (Polyethylene terephthalate) platform. And circuits itself are covered with copper, nickel and gold (in a manner from inside to surface) to enhance the conductivity. The current in side IDEs are led out through a FPC (Flexible Printed Circuit) connector which fits to the size of IDE lead feet.</p>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 3: BBa_J364002</p>
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<span class="psg_ttl psg_subtitle">First generation of IDE</span>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 4: BBa_J364007</p>
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<p style="margin-bottom: 1em !important;"></br>Taking our 2D-printer’s precision into consideration, we designed our first generation of IDE (see Fig. 1). The part with black lines is 6×interdigital working electrodes, the green part is a counter electrode and the red part is a reference electrode. But in the first design, two interdigital electrodes in pair are linked to the same lead foot which significantly lower the space for further DIY. </br></p>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 5: BBa_J364008</p>
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<img style="display: inline; width: 70% !important; padding-left: 23% !important;" src="https://static.igem.org/mediawiki/2018/5/5b/T--ZJU-China--HD03.png" />
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 6: BBa_J364009</p>
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<h5><span style="width: 5% !important; vertical-align: top;">Fig 3.</span><span style="padding-left: 2%; width: 93% !important; text-align: left;">First generation of IDE. The part with black lines is 6×interdigital working electrodes, the green part is a counter electrode and the red part is a reference electrode.</span></h5>
<p></br>Resuspend DNA in selected wells in the Distribution Kit with 10 &micro;L ddH20. Thaw competent cells on ice. Pipette 25 &micro;L of competent cells into 1.5 mL tube per transformation and add 2 &micro;L of resuspended DNA into it. Incubate on ice for 30 min. Heat shock tubes at 42&deg;C for 90 sec. Then incubate on ice for 5 min.</p>
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<span class="psg_ttl psg_subtitle">Second generation of IDE</span>
<p></br>Add 1000 &micro;L LB media with Chloramphenicol (1000&times;) to each transformation. Incubate at 37&deg;C for 1 hours, shaking at 200-300 rpm.</p>
+
<p>To make it easier to print protein solution to the surface and also for more possibility in further desgin, we modified our IDEs and create the second generation of IDEs (see Fig.2). In the new design, lead feet were enlarged to 0.44 mm. Gaps in between reached to 0.48 mm to avoid short circuit. And we separated each pair of interdigital electrode to 2 independent lead feet. </p>
<p></br>Pipette 100 &micro;L of each transformation onto LB plates (Chloramphenicol, 1000&times;). Spread with sterilized spreader. Incubate transformations overnight (14-18 hours) at 37&deg;C.</br></p>
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<img style="width: 70%; padding-left: 10%;" src="https://static.igem.org/mediawiki/2018/2/2b/T--ZJU-China--HD05.png" />
<span class="psg_ttl psg_subtitle">Colonies Selection</span>
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<h5><span style="width: 5% !important; vertical-align: top;">Fig 4.</span><span style="padding-left: 2%; width: 93% !important; text-align: left;">Second generation of IDE. The part with black lines is 6×interdigital working electrodes, the green part is a counter electrode and the red part is a reference electrode.</span></h5>
<p>Pick 2 single colonies from each of plate and inoculate it on 5-10 mL LB medium with Chloramphenicol (1000&times;). Grow the cells overnight (16-18 hours) at 37&deg;C and 220 rpm.</br></p>
+
<span class="psg_ttl">Integration </span>
<span class="psg_ttl psg_subtitle">Calibration</span>
+
<p>As the chemical reaction signal is normally small (10<sup>-7</sup>-10<sup>-5</sup> A), a circuit which reaccepts the current signal must amplify the signal to a large voltage signal for sampling in microcontroller. And due to the characters of degenerative circuit, a convertor is needed for sampling subsequently.</p>
<p>We used the plate reader Synergy Neo2 for all the measurements and we used black 96 well plates with flat, transparent bottom.</p>
+
<span class="psg_ttl psg_subtitle">integrated circuit design</span>
<p style="margin-top: .6em; font-weight: bolder;"></br>&bull; OD600 Reference Point</p>
+
<p>We connect a Transimpedance Amplifier together with a Bluetooth module an expansion on Arduino Nano microcontroller. Following figure showed our main design and workable circuit we tested. This part of hardware can amplify currents produced on IDE and the amplification result is 1×106 times depend on impedance on the amplifier.<sup style="font-size: .4em !important;">[1]</sup></p>
<p style="padding-left: 1em;"></br>Add 100 &micro;l LUDOX into wells A1, B1, C1, D1 and 100 &micro;l of H2O into wells A2, B2, C2, D2. Then measure absorbance at 600 nm of all samples in all standard measurement modes in instrument and turn off the pathlength correction at the same time. The temperature setting was 26.6&deg;C. Record the data.</p>
+
<img style="width: 70%; padding-left: 10%;"src="https://static.igem.org/mediawiki/2018/c/ca/T--ZJU-China--HD04.png" />
<p style="margin-top: .6em; font-weight: bolder;"></br>&bull; Particle Standard Curve</p>
+
<h5 style="line-height: 2em !important;">Fig 5. Integrated circuit design on amplifiers and convertors.</br></h5>
<p style="padding-left: 1em;"></br>Obtain the tube labeled &ldquo;Silica Beads&ldquo; from the InterLab test kit and vortex vigorously for 30 seconds. Then immediately pipet 96 &micro;L microspheres into a 1.5 mL eppendorf tube. Add 904 &micro;L of ddH2O to the microspheres and vortex well.</p>
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<img style="width: 70%; padding-left: 10%;" src="https://static.igem.org/mediawiki/2018/4/4d/T--ZJU-China--HD06.png" />
<p style="padding-left: 1em;"></br>Prepare the serial dilution of microspheres as shown below. Set 4 copies.</p>
+
<h5 style="line-height: 2em !important;">Fig 6. Prototype on board while testing</h5>
<img src="https://static.igem.org/mediawiki/2018/thumb/7/7c/T--ZJU-China--interlab01.png/585px-T--ZJU-China--interlab01.png" />
+
<span class="psg_ttl psg_subtitle">Interface and software</span>
<h5>Fig.1 Dilution of microspheres <sup style="font-family: .8em;">[1]</sup></h5>
+
<p>We printed the "ink" containing modified enzymes on IDEs. IDEs' output feet are connected to the in-port of the second part hardware with a small current and circuits can accordingly form a voltage big enough to be sampled. An Arduino Nano microcontrollor and a blueteeth module are used to transfer data to mobile phones running the WeChat (a well-known social media app) mini-program for visualization. Cell phones receive the voltage signal and transfer them into an actual current value by algorithm, and then send processed user-readable results to the applets. This app is developed in the WeChat Platform. Some screen shoots in the app are showed below.</p>
<p style="padding-left: 1em;"></br>Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6&deg;C. Record the data.</p>
+
<img style="width: 70%; padding-left: 10%;" src="https://static.igem.org/mediawiki/2018/8/84/T--ZJU-China--HD07.png" />
<p style="margin-top: .6em; font-weight: bolder;"></br>&bull; Fluorescence standard curve</p>
+
<h5>Fig 7. Screen shots of prototype display</h5>
<p style="padding-left: 1em;"></br>Spin down fluorescein stock tube. Prepare 10x fluorescein stock solution (100 &micro;M) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 &micro;M.</p>
+
<p></br>The hardware implementation not only solves the need to report wet experiment results, but also is much more user friendly, especially for non-professionals.In order to verify the stability and accuracy, we conducted dozens of tests in the hospital using real blood samples and contacted the experts to evaluate the hardware. In the figure showed below, the hardware model works well on distinguish patient samples and healthy samples. And the IDE chips response very quickly within 10s to plasma spacemen. But there is still limitation on whole blood detection. When applied with whole blood, the detaction time is lengthened and signals become very unstable. The modification is on schedule in the future plan.</p>
<p style="padding-left: 1em;"></br>Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.</p>
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<img src="https://static.igem.org/mediawiki/2018/thumb/f/f4/T--ZJU-China--interlab02.png/519px-T--ZJU-China--interlab02.png" />
+
<h5>Fig.2 Dilution of fluorescein <sup style="font-family: .8em;">[1]</sup></h5>
+
<p style="padding-left: 1em;"></br>Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6&deg;C. Record the data.</p>
+
<span class="psg_ttl psg_subtitle">Cell measurement</span>
+
<p style="padding-left: 1em;">Make a 1:10 dilution of of the overnight cultures prepared after colony selection in LB medium + Chloramphenicol and measure Abs 600 of these 1:10 diluted cultures. Then dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber or covered with foil to block light). Incubate the cultures at 37&deg;C and 220 rpm. Take 500 &micro;L samples of the cultures from each of the 8 devices, two colonies per device, at 0 and 6 hours of incubation and add them into 96 well plates as shown below. Place samples on ice before measurements.</p>
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<img src="https://static.igem.org/mediawiki/2018/3/3d/T--ZJU-China--interlab03.png" />
+
<h5>Fig.3 Loading samples <sup style="font-family: .8em;">[1]</sup></h5>
+
<p style="padding-left: 1em;"></br>Measure the samples (Abs 600 and fluorescence measurement). The cell measurement was under the same condition with the particle standard curve and the fluorescence standard curve, using the same plate.</br></p>
+
<span class="psg_ttl psg_subtitle">Counting colony-forming units (CFUs)</span>
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<p>Measure the OD600 of cell cultures, making sure to dilute to the linear detection range of the plate reader. Then dilute the overnight culture to OD600 = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture and check the OD600 to make sure it is 0.1.</p>
+
<p></br>Do the following serial dilutions as blow.</p>
+
<img src="https://static.igem.org/mediawiki/2018/thumb/e/e3/T--ZJU-China--interlab04.png/585px-T--ZJU-China--interlab04.png" />
+
<h5>Fig.4 Dilutions <sup style="font-family: .8em;">[1]</sup></h5>
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<p></br>Count the colonies on each plate with fewer than 300 colonies. And multiple the colony count by the final dilution factor on each plate to get the colony forming units (CFU) per 1mL of an OD600 = 0.1 culture.</p>
+
+
<span class="psg_ttl">Results</span>
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<span class="psg_ttl psg_subtitle">OD600 Reference point</span>
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<table class="table table-striped">
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<thead><tr>
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<th><p></p></th>
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<th><p>LUDOX CL-X</p></th>
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<th><p>H<sub>2</sub>O</p></th>
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</tr></thead>
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<tbody>
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<tr>
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<td><p>Replicate 1</p></td>
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<td><p>0.063</p></td>
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<td><p>0.039</p></td>
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</tr>
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<tr>
+
<td><p>Replicate 2</p></td>
+
<td><p>0.058</p></td>
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<td><p>0.038</p></td>
+
</tr>
+
<tr>
+
<td><p>Replicate 3</p></td>
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<td><p>0.056</p></td>
+
<td><p>0.036</p></td>
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</tr>
+
<tr>
+
<td><p>Replicate 4</p></td>
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<td><p>0.054</p></td>
+
<td><p>0.037</p></td>
+
</tr>
+
<tr>
+
<td><p>Arith. Mean</p></td>
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<td><p>0.058</p></td>
+
<td><p>0.038</p></td>
+
</tr>
+
<tr>
+
<td><p>Corrected Abs600</p></td>
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<td><p>0.020</p></td>
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<td><p></p></td>
+
</tr>
+
<tr>
+
<td><p>Reference OD600</p></td>
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<td><p>0.063</p></td>
+
<td><p></p></td>
+
</tr>
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<tr>
+
<td><p>OD600/Abs600</p></td>
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<td><p>3.111</p></td>
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<td><p></p></td>
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</tr>
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</tbody>
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</table>
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+
<h5>Tab.1 OD600 reference point</h5>
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<span class="psg_ttl psg_subtitle">Particle Standard Curve</span>
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<img src="https://static.igem.org/mediawiki/2018/b/b3/T--ZJU-China--interlab05.png" style="display:inline-block; width: 45%; margin-left:5%;" />
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<img src="https://static.igem.org/mediawiki/2018/2/27/T--ZJU-China--interlab06.png" style="display:inline-block; width:45%;" />
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<h5>Fig.5 Particle standard curve 1 | Fig.6 Particle standard curve 2</h5>
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<span class="psg_ttl psg_subtitle">Fluorescence standard curve</span>
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<img src="https://static.igem.org/mediawiki/2018/b/bd/T--ZJU-China--interlab07.png" style="display:inline-block; width: 45%; margin-left:5%;" />
+
<img src="https://static.igem.org/mediawiki/2018/d/db/T--ZJU-China--interlab08.png" style="display:inline-block; width:45%;" />
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<h5>Fig.7 Fluorescence standard curve 1 | Fig.8 Fluorescence standard curve 2</h5>
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<span class="psg_ttl psg_subtitle">Cell measurement</span>
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<table class="table table-striped">
+
<thead><tr>
+
<th><p>Hour 0:</p></th>
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<th><p>Neg. Control</p></th>
+
<th><p>Pos. Control</p></th>
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<th><p>Device 1</p></th>
+
<th><p>Device 2</p></th>
+
<th><p>Device 3</p></th>
+
<th><p>Device 4</p></th>
+
<th><p>Device 5</p></th>
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<th><p>Device 6</p></th>
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<th><p>LB + Chlor (blank)</p></th>
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</tr></thead>
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<tbody>
+
<tr>
+
<td><p>Colony 1, Replicate 1</p></td>
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<td><p>5</p></td>
+
<td><p>6</p></td>
+
<td><p>9</p></td>
+
<td><p>8</p></td>
+
<td><p>4</p></td>
+
<td><p>14</p></td>
+
<td><p>7</p></td>
+
<td><p>5</p></td>
+
<td><p>6</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 2</p></td>
+
<td><p>5</p></td>
+
<td><p>5</p></td>
+
<td><p>9</p></td>
+
<td><p>8</p></td>
+
<td><p>4</p></td>
+
<td><p>14</p></td>
+
<td><p>7</p></td>
+
<td><p>6</p></td>
+
<td><p>5</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 3</p></td>
+
<td><p>5</p></td>
+
<td><p>5</p></td>
+
<td><p>9</p></td>
+
<td><p>8</p></td>
+
<td><p>4</p></td>
+
<td><p>14</p></td>
+
<td><p>8</p></td>
+
<td><p>6</p></td>
+
<td><p>6</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 4</p></td>
+
<td><p>5</p></td>
+
<td><p>4</p></td>
+
<td><p>9</p></td>
+
<td><p>8</p></td>
+
<td><p>4</p></td>
+
<td><p>13</p></td>
+
<td><p>6</p></td>
+
<td><p>5</p></td>
+
<td><p>5</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 1</p></td>
+
<td><p>4</p></td>
+
<td><p>5</p></td>
+
<td><p>9</p></td>
+
<td><p>8</p></td>
+
<td><p>5</p></td>
+
<td><p>12</p></td>
+
<td><p>6</p></td>
+
<td><p>5</p></td>
+
<td><p>5</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 2</p></td>
+
<td><p>5</p></td>
+
<td><p>5</p></td>
+
<td><p>9</p></td>
+
<td><p>9</p></td>
+
<td><p>4</p></td>
+
<td><p>12</p></td>
+
<td><p>7</p></td>
+
<td><p>5</p></td>
+
<td><p>4</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 3</p></td>
+
<td><p>5</p></td>
+
<td><p>5</p></td>
+
<td><p>8</p></td>
+
<td><p>8</p></td>
+
<td><p>5</p></td>
+
<td><p>13</p></td>
+
<td><p>7</p></td>
+
<td><p>6</p></td>
+
<td><p>5</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 4</p></td>
+
<td><p>5</p></td>
+
<td><p>4</p></td>
+
<td><p>9</p></td>
+
<td><p>8</p></td>
+
<td><p>5</p></td>
+
<td><p>13</p></td>
+
<td><p>6</p></td>
+
<td><p>5</p></td>
+
<td><p>6</p></td>
+
</tr>
+
</tbody>
+
</table>
+
 
+
<h5>Tab.2 Raw plate reader measurements of fluorescence raw at 0 Hour</h5>
+
+
<table class="table table-striped">
+
<thead><tr>
+
<th><p>Hour 6:</p></th>
+
<th><p>Neg. Control</p></th>
+
<th><p>Pos. Control</p></th>
+
<th><p>Device 1</p></th>
+
<th><p>Device 2</p></th>
+
<th><p>Device 3</p></th>
+
<th><p>Device 4</p></th>
+
<th><p>Device 5</p></th>
+
<th><p>Device 6</p></th>
+
<th><p>LB + Chlor (blank)</p></th>
+
</tr></thead>
+
<tbody>
+
<tr>
+
<td><p>Colony 1, Replicate 1</p></td>
+
<td><p>12</p></td>
+
<td><p>47</p></td>
+
<td><p>80</p></td>
+
<td><p>63</p></td>
+
<td><p>13</p></td>
+
<td><p>180</p></td>
+
<td><p>25</p></td>
+
<td><p>28</p></td>
+
<td><p>6</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 2</p></td>
+
<td><p>13</p></td>
+
<td><p>48</p></td>
+
<td><p>84</p></td>
+
<td><p>67</p></td>
+
<td><p>12</p></td>
+
<td><p>194</p></td>
+
<td><p>26</p></td>
+
<td><p>30</p></td>
+
<td><p>7</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 3</p></td>
+
<td><p>12</p></td>
+
<td><p>46</p></td>
+
<td><p>79</p></td>
+
<td><p>66</p></td>
+
<td><p>12</p></td>
+
<td><p>196</p></td>
+
<td><p>26</p></td>
+
<td><p>30</p></td>
+
<td><p>7</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 4</p></td>
+
<td><p>12</p></td>
+
<td><p>47</p></td>
+
<td><p>87</p></td>
+
<td><p>65</p></td>
+
<td><p>13</p></td>
+
<td><p>192</p></td>
+
<td><p>24</p></td>
+
<td><p>31</p></td>
+
<td><p>8</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 1</p></td>
+
<td><p>13</p></td>
+
<td><p>48</p></td>
+
<td><p>79</p></td>
+
<td><p>55</p></td>
+
<td><p>11</p></td>
+
<td><p>112</p></td>
+
<td><p>29</p></td>
+
<td><p>23</p></td>
+
<td><p>7</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 2</p></td>
+
<td><p>14</p></td>
+
<td><p>50</p></td>
+
<td><p>82</p></td>
+
<td><p>59</p></td>
+
<td><p>12</p></td>
+
<td><p>121</p></td>
+
<td><p>27</p></td>
+
<td><p>23</p></td>
+
<td><p>8</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 3</p></td>
+
<td><p>13</p></td>
+
<td><p>49</p></td>
+
<td><p>81</p></td>
+
<td><p>65</p></td>
+
<td><p>13</p></td>
+
<td><p>126</p></td>
+
<td><p>32</p></td>
+
<td><p>26</p></td>
+
<td><p>6</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 4</p></td>
+
<td><p>15</p></td>
+
<td><p>47</p></td>
+
<td><p>86</p></td>
+
<td><p>65</p></td>
+
<td><p>12</p></td>
+
<td><p>137</p></td>
+
<td><p>34</p></td>
+
<td><p>26</p></td>
+
<td><p>7</p></td>
+
</tr>
+
</tbody>
+
</table>
+
<h5>Tab.3 Raw plate reader measurements of fluorescence raw at 6 Hour</h5>
+
<table class="table table-striped">
+
<thead><tr>
+
<th><p>Hour 0:</p></th>
+
<th><p>Neg. Control</p></th>
+
<th><p>Pos. Control</p></th>
+
<th><p>Device 1</p></th>
+
<th><p>Device 2</p></th>
+
<th><p>Device 3</p></th>
+
<th><p>Device 4</p></th>
+
<th><p>Device 5</p></th>
+
<th><p>Device 6</p></th>
+
<th><p>LB + Chlor (blank)</p></th>
+
</tr></thead>
+
<tbody>
+
<tr>
+
<td><p>Colony 1, Replicate 1</p></td>
+
<td><p>0.080</p></td>
+
<td><p>0.067</p></td>
+
<td><p>0.061</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.070</p></td>
+
<td><p>0.059</p></td>
+
<td><p>0.063</p></td>
+
<td><p>0.069</p></td>
+
<td><p>0.042</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 2</p></td>
+
<td><p>0.081</p></td>
+
<td><p>0.071</p></td>
+
<td><p>0.060</p></td>
+
<td><p>0.074</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.062</p></td>
+
<td><p>0.066</p></td>
+
<td><p>0.067</p></td>
+
<td><p>0.042</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 3</p></td>
+
<td><p>0.077</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.061</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.059</p></td>
+
<td><p>0.069</p></td>
+
<td><p>0.066</p></td>
+
<td><p>0.046</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 4</p></td>
+
<td><p>0.080</p></td>
+
<td><p>0.069</p></td>
+
<td><p>0.061</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.070</p></td>
+
<td><p>0.064</p></td>
+
<td><p>0.067</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.045</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 1</p></td>
+
<td><p>0.075</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.059</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.070</p></td>
+
<td><p>0.064</p></td>
+
<td><p>0.067</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.045</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 2</p></td>
+
<td><p>0.073</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.058</p></td>
+
<td><p>0.071</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.064</p></td>
+
<td><p>0.058</p></td>
+
<td><p>0.068</p></td>
+
<td><p>0.043</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 3</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.056</p></td>
+
<td><p>0.067</p></td>
+
<td><p>0.068</p></td>
+
<td><p>0.066</p></td>
+
<td><p>0.055</p></td>
+
<td><p>0.060</p></td>
+
<td><p>0.042</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 4</p></td>
+
<td><p>0.072</p></td>
+
<td><p>0.069</p></td>
+
<td><p>0.060</p></td>
+
<td><p>0.070</p></td>
+
<td><p>0.065</p></td>
+
<td><p>0.062</p></td>
+
<td><p>0.052</p></td>
+
<td><p>0.060</p></td>
+
<td><p>0.044</p></td>
+
</tr>
+
</tbody>
+
</table>
+
 
+
<h5>Tab.4 Raw data of Abs600 measurement at 0 hour</h5>
+
+
<table class="table table-striped">
+
<thead><tr>
+
<th><p>Hour 6:</p></th>
+
<th><p>Neg. Control</p></th>
+
<th><p>Pos. Control</p></th>
+
<th><p>Device 1</p></th>
+
<th><p>Device 2</p></th>
+
<th><p>Device 3</p></th>
+
<th><p>Device 4</p></th>
+
<th><p>Device 5</p></th>
+
<th><p>Device 6</p></th>
+
<th><p>LB + Chlor (blank)</p></th>
+
</tr></thead>
+
<tbody>
+
<tr>
+
<td><p>Colony 1, Replicate 1</p></td>
+
<td><p>0.437</p></td>
+
<td><p>0.456</p></td>
+
<td><p>0.533</p></td>
+
<td><p>0.513</p></td>
+
<td><p>0.505</p></td>
+
<td><p>0.491</p></td>
+
<td><p>0.538</p></td>
+
<td><p>0.483</p></td>
+
<td><p>0.049</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 2</p></td>
+
<td><p>0.509</p></td>
+
<td><p>0.502</p></td>
+
<td><p>0.516</p></td>
+
<td><p>0.573</p></td>
+
<td><p>0.516</p></td>
+
<td><p>0.522</p></td>
+
<td><p>0.531</p></td>
+
<td><p>0.494</p></td>
+
<td><p>0.054</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 3</p></td>
+
<td><p>0.416</p></td>
+
<td><p>0.477</p></td>
+
<td><p>0.491</p></td>
+
<td><p>0.453</p></td>
+
<td><p>0.442</p></td>
+
<td><p>0.479</p></td>
+
<td><p>0.488</p></td>
+
<td><p>0.467</p></td>
+
<td><p>0.042</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 1, Replicate 4</p></td>
+
<td><p>0.458</p></td>
+
<td><p>0.492</p></td>
+
<td><p>0.489</p></td>
+
<td><p>0.454</p></td>
+
<td><p>0.445</p></td>
+
<td><p>0.428</p></td>
+
<td><p>0.458</p></td>
+
<td><p>0.449</p></td>
+
<td><p>0.041</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 1</p></td>
+
<td><p>0.530</p></td>
+
<td><p>0.446</p></td>
+
<td><p>0.421</p></td>
+
<td><p>0.448</p></td>
+
<td><p>0.467</p></td>
+
<td><p>0.385</p></td>
+
<td><p>0.365</p></td>
+
<td><p>0.451</p></td>
+
<td><p>0.046</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 2</p></td>
+
<td><p>0.556</p></td>
+
<td><p>0.459</p></td>
+
<td><p>0.490</p></td>
+
<td><p>0.518</p></td>
+
<td><p>0.577</p></td>
+
<td><p>0.449</p></td>
+
<td><p>0.344</p></td>
+
<td><p>0.469</p></td>
+
<td><p>0.049</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 3</p></td>
+
<td><p>0.516</p></td>
+
<td><p>0.475</p></td>
+
<td><p>0.510</p></td>
+
<td><p>0.551</p></td>
+
<td><p>0.576</p></td>
+
<td><p>0.490</p></td>
+
<td><p>0.409</p></td>
+
<td><p>0.487</p></td>
+
<td><p>0.051</p></td>
+
</tr>
+
<tr>
+
<td><p>Colony 2, Replicate 4</p></td>
+
<td><p>0.600</p></td>
+
<td><p>0.420</p></td>
+
<td><p>0.481</p></td>
+
<td><p>0.575</p></td>
+
<td><p>0.545</p></td>
+
<td><p>0.496</p></td>
+
<td><p>0.436</p></td>
+
<td><p>0.521</p></td>
+
<td><p>0.045</p></td>
+
</tr>
+
</tbody>
+
</table>
+
<h5>Tab.5 Raw data of Abs600 measurement at 6 hour</h5>
+
<p></br>The results of the CFUs and the flow cytometry data have been submitted in time by online forms and a zip file.</p>
+
<img style="width: 60% !important;" src="https://static.igem.org/mediawiki/2018/4/4c/T--ZJU-China--interlab.png" />
+
 
+
<h5>Fig.9 The results of the CFUs and the flow cytometry data</h5>
+
+
<span class="psg_ttl">Feedback</span>
+
<p></br>Thanks to the detailed protocol, our Interlab experiments went on well through the process. The Excel provided makes it convenient to process data. And the warnings are necessary, such as LUDOX cannot be stored in refrigerator. Besides, the protocol really pays attention to the operation errors and reminds us in advance.</p>
+
<p></br>However, there are some disadvantages. Some of the materials are not sufficient enough, like Silica Beads. More samples and replicates can be involved to avoid mistakes.</p>
+
 
+
<span class="psg_ttl">Summary</span>
+
<p></br>Our experiments have got a great result, showing that the protocol is rather detailed and easy to operate. We are pleased to share our data with other teams around the world and we sincerely hope the Interlab study this year goes well.</p>
+
 
+
 
+
<span class="psg_ttl">References</span>
+
<p>[1] <a style="color: #131124;" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf</a></p>
+
<p>[2] <a style="color: #131124;" href="https://2018.igem.org/Measurement/InterLab">https://2018.igem.org/Measurement/InterLab</a></p>
+
<p>[3] <a style="color: #131124;" href="https://2018.igem.org/Measurement/InterLab/Plate_Reader">https://2018.igem.org/Measurement/InterLab/Plate_Reader</a></p>
+
<p>[4] <a style="color: #131124;" href="https://2018.igem.org/Measurement/InterLab/Flow_Cytometry">https://2018.igem.org/Measurement/InterLab/Flow_Cytometry</a></p>
+
+
+
+
+
 
+
 
+
 
 
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Revision as of 20:46, 17 October 2018

Improve Part
HARDWARE  
ABSTRACT  

At the very beginning of hardware construction, all biological products and other auxiliary reagents (like Au nanoparticles and Nafion) have to immobilized on the electrode. To meet the need of large-scale manufacturing and high accuracy, we used a 2D printer to print these materials onto IDEs ( InterDigital Electrode) to modify the Au-surface. Then we assembled IDEs, amplifier, convertor and digital interface to an entire circuit. The signal from the circuit is transmitted to an iPhone through Bluetooth. Several blood samples from a qualified Hospital were collected for access our hardwares and enzyme IDEs.


Fig. 1 Work flow of our hardware


2D printer

Our 2D printer consists of a constant-flow pump, motors, an Arduino Uno microcontroller. The printer’s user interface is based on a Windows software by Steamduino which support several file formats like CAD scripts or normal pictures. Imported files will be converted to 2D routes and transmitted to the microcontroller. The speed of motors and flow rate of the "ink" are controllable in the panel. Since the space between lead feet of IDEs is about 4 mm, the printer can reach an accuracy of 3 mm. The path-programmable printer offers more possibilities for developers to design on the level of electrodes. For example, separated areas of the chip can be modified with different enzymes.

Fig. 2 Real picture of a 2D printer and its control panel
IDE (interdigital electrode) design

IDE (interdigital electrode) is composed of two interdigital electrodes with two connection tracks, on an insulative substrate. IDE is cheaper than normal electrodes and can work with low volumes of sample. The interdigitated configuration typically enhances sensitivity and detection limits.


The IDE circuit is planted on a PET (Polyethylene terephthalate) platform. And circuits itself are covered with copper, nickel and gold (in a manner from inside to surface) to enhance the conductivity. The current in side IDEs are led out through a FPC (Flexible Printed Circuit) connector which fits to the size of IDE lead feet.

First generation of IDE


Taking our 2D-printer’s precision into consideration, we designed our first generation of IDE (see Fig. 1). The part with black lines is 6×interdigital working electrodes, the green part is a counter electrode and the red part is a reference electrode. But in the first design, two interdigital electrodes in pair are linked to the same lead foot which significantly lower the space for further DIY.

Fig 3.First generation of IDE. The part with black lines is 6×interdigital working electrodes, the green part is a counter electrode and the red part is a reference electrode.
Second generation of IDE

To make it easier to print protein solution to the surface and also for more possibility in further desgin, we modified our IDEs and create the second generation of IDEs (see Fig.2). In the new design, lead feet were enlarged to 0.44 mm. Gaps in between reached to 0.48 mm to avoid short circuit. And we separated each pair of interdigital electrode to 2 independent lead feet.

Fig 4.Second generation of IDE. The part with black lines is 6×interdigital working electrodes, the green part is a counter electrode and the red part is a reference electrode.
Integration

As the chemical reaction signal is normally small (10-7-10-5 A), a circuit which reaccepts the current signal must amplify the signal to a large voltage signal for sampling in microcontroller. And due to the characters of degenerative circuit, a convertor is needed for sampling subsequently.

integrated circuit design

We connect a Transimpedance Amplifier together with a Bluetooth module an expansion on Arduino Nano microcontroller. Following figure showed our main design and workable circuit we tested. This part of hardware can amplify currents produced on IDE and the amplification result is 1×106 times depend on impedance on the amplifier.[1]

Fig 5. Integrated circuit design on amplifiers and convertors.
Fig 6. Prototype on board while testing
Interface and software

We printed the "ink" containing modified enzymes on IDEs. IDEs' output feet are connected to the in-port of the second part hardware with a small current and circuits can accordingly form a voltage big enough to be sampled. An Arduino Nano microcontrollor and a blueteeth module are used to transfer data to mobile phones running the WeChat (a well-known social media app) mini-program for visualization. Cell phones receive the voltage signal and transfer them into an actual current value by algorithm, and then send processed user-readable results to the applets. This app is developed in the WeChat Platform. Some screen shoots in the app are showed below.

Fig 7. Screen shots of prototype display


The hardware implementation not only solves the need to report wet experiment results, but also is much more user friendly, especially for non-professionals.In order to verify the stability and accuracy, we conducted dozens of tests in the hospital using real blood samples and contacted the experts to evaluate the hardware. In the figure showed below, the hardware model works well on distinguish patient samples and healthy samples. And the IDE chips response very quickly within 10s to plasma spacemen. But there is still limitation on whole blood detection. When applied with whole blood, the detaction time is lengthened and signals become very unstable. The modification is on schedule in the future plan.