Difference between revisions of "Arsenic Prototype 4.0"

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==Principles==
 
 
The Arsenic prototype v4.0 is the [http://wiki.biodesign.cc/wiki/Winter_School Winter School] 2016 kit version of [http://wiki.biodesign.cc/wiki/Arsenic-Prototype-3-0 prototype 3.0].
 
The Arsenic prototype v4.0 is the [http://wiki.biodesign.cc/wiki/Winter_School Winter School] 2016 kit version of [http://wiki.biodesign.cc/wiki/Arsenic-Prototype-3-0 prototype 3.0].
  
The basis of the prototype remains the same - the bioreporter, a GMO bacteria expressing eGFP (Green Fluorescent Protein) is incubated with a water sample, where the fluorescence is detected optically and can be quantified in order to measure the concentration of Arsenic in the water
+
==Principles==
 +
The basis of the prototype remains the same as the [http://wiki.biodesign.cc/wiki/Arsenic-Prototype-3-0 prototype 3.0]- the bioreporter, a GMO bacteria expressing eGFP (Green Fluorescent Protein) is incubated with a water sample, where the fluorescence is detected optically and can be quantified in order to measure the concentration of Arsenic in the water
 
<br>
 
<br>
 
* A vial containing the water sample we want to test is positioned on a socket through which a fluorescent excitation LED (blue 488nm for eGFP) passes.  
 
* A vial containing the water sample we want to test is positioned on a socket through which a fluorescent excitation LED (blue 488nm for eGFP) passes.  
* GFP absorbs this blue light (λ=475 nm) and emits green light (λ=504 nm) which is detected by a photosensor on which the light is concentrated with the help of two lenses that avoid loss of intensity
+
* GFP absorbs this blue light (λ=475 nm) and emits green light (λ=504 nm) which is detected by a photosensor on which the light is concentrated with the help of two lenses that avoid loss of intensity.
* A long-pass filter allow only the signal eGFP fluorescence to reach the photosensor
+
* A long-pass filter allows only the eGFP fluorescence signal to reach the photosensor.
 +
* The intensity of the fluorescence is a function of the number of bacterias in the sample. This can be quantified undirectly by measuring the [https://en.wikipedia.org/wiki/Turbidity Turbidity] of the sample.
 +
* A red LED is placed in-line with the photosensor to measure the transmittance, which can be converted into turbidity. The measurement of turbidity will allow us to normalize our results with respect the density of bacterias.
 +
 
 
<br>
 
<br>
  
Moreover, a red LED was added to measure the transmittance, which can be converted into turbidity. The measurement of turbidity will allow us to normalize our results.
+
[[File:Proto4 principle all.png|600px]]
* A red LED is placed in-line with the photosensor
+
 
 +
 
 
<br>
 
<br>
  
With these measures, compared against a standard curve of water containing known arsenic concentrations, one can determine the concentration of arsenic in the sample and so know if the water is drinkable or not.
+
Such a system is easily implemented using a [https://en.wikipedia.org/wiki/Microcontroller microcontroller], a minimalist computer.
 +
A microcontroller has a number of GPIO (general purpose input output). When turned as an output, a GPIO is a terminal that can be
 +
programmatically set to a voltage of 5V (high state, logical 1), or 0V (low state, logical 0). This can be used to turn on or off
 +
external components such as LEDs. When used as an input, we can read the light sensor from them for example. The microcontroller is
 +
programmed in C code. It incorporates also timers that allow to keep track of time.
 +
 
 +
We use an [https://en.wikipedia.org/wiki/Arduino Arduino] board to control this device. The microcontroller on the Arduino board is programmed to carry out the following operations.
 +
# Wait for button to be pushed.
 +
# Turn on the blue LED for a predetermined amount of time.
 +
# Readout the value of the light sensor.
 +
# Turn off the blue LED.
 +
# Turn on the red LED for a predetermined amount of time.
 +
# Readout the value of the light sensor.
 +
# Display the two values on the screen of the device.
 +
 
 +
<br>
 +
 
 +
[[File:Proto4 electronics diagram.png|600px]]
 +
 
 +
<br>
 +
 
 +
The main electronic components of the device are as follows.
 +
* '''Arduino board''': the brain of the device.
 +
* '''Transistors''': the GPIO cannot provide enough current to drive LEDs directly. We use instead transistors. Transistors are like electrically controllable switches. It is a 3-terminal device. When provided with 0V or 5V on one terminal, the two remaining terminals are connected or disconnected, respectively (or vice versa depending on transistor type). The transistor size is proportional to how much current it must let through. We need a large one for the blue LED as it is very bright and has a high current.
 +
* '''LED at 600 nm''': to measure optical density.
 +
* '''LED at 485 nm''': to excite eGFP.
 +
* '''A light sensor''': the sensor we use is a ''light-to-frequency'' sensor. It means that it outputs a square signal with a frequency proportional to the light intensity on the photosensitive part of the sensor.
 +
* '''A screen''': to display results.
 +
* '''Buttons and potentiometer''': for the user to interact with the device.
 +
 
 +
With the measures taken by the device, compared against a standard curve of water containing known arsenic concentrations, one can determine the concentration of arsenic in the sample and so know if the water is drinkable or not.
  
 
<br>
 
<br>
Line 23: Line 57:
 
     blue LED for eGFP fluorescence excitation at 90 degrees from detector
 
     blue LED for eGFP fluorescence excitation at 90 degrees from detector
 
     red LED for transmittance measurements in line with detector
 
     red LED for transmittance measurements in line with detector
     vial holder matching the vial approved by the Swiss authorities
+
     vial holder matching the [http://biodesign.cc/2014/08/29/field-testing-approved/ vial approved by the Swiss authorities]
 
     light to frequency detector
 
     light to frequency detector
 
     a filter to block excitation light
 
     a filter to block excitation light
Line 32: Line 66:
  
 
<br>
 
<br>
 +
 +
==Tools==
 +
 +
* Soldering iron
 +
* Tin
 +
* Knippers
 +
* Plier
 +
* Screwdriver phillips #1
 +
* Vice (for crimping the ribbon cable)
 +
 +
==Safety==
 +
 +
* Don't burn the wires on the table with the soldering iron
 +
* Mind your fingers
 +
* When knipping the legs of the components, hold the loose end so that it doesn't fly into someone's eye.
 +
* Apply common sense
  
 
==Materials==
 
==Materials==
 +
The '''firmware''' is in our github repository for the [https://github.com/BioDesignRealWorld/FluoMeter FluoMeter].<br>
 +
For the full '''Bill of Materials (BOM)''' including reference numbers see this [https://docs.google.com/spreadsheets/d/1YvrxXKR7t9VFFLNv7FZZpDFvK6zu98gKysxAoj49X2o/edit?usp=sharing|google sheet].<br><br>
 +
 +
LCD Shield
 +
 +
* 1x LCD 8x2 HDM08216L-3-L30S
 +
* 1x resistor 1K (R1)
 +
* 1x Trimpot 10K
 +
* 2x Tactile switch 17mm (S1, S2)
 +
* 1x Potentiometer
 +
* 1x Micromatch connector 10 pin SMD female 8-338069-0
 +
* 1x 1x40 pin header straight long (17mm)
 +
* 1x 2x8 pin header straight
 +
 +
LED Driver board
 +
 +
* 1x LED 485nm (blue) SMD XPEBBL-L1-0000-00Z01
 +
* 1x P-channel MOSFET TO-220
 +
* 1x P-channel MOSFET TO-92
 +
* 1x Micromatch connector 10 pin SMD female 8-338069-0
 +
* 1x Resistor 5 ohm 3W
 +
* 1x Resistor 150 Ohms 1/4W
 +
* 1x 1x3 pin header 90 degree bent
 +
* 1x 1x2 pin header 90 degree bent
 +
 +
Optical density (red LED)
 +
 +
* 1x red LED 5mm
 +
* 1x 1x2 pin header straight
 +
 +
Light sensor
 +
 +
* 1x Light To Frequency Converter – TSL235R
 +
* 1x 1x3 pin header straight
 +
 +
Optics
 +
 +
* 2x [[https://www.knightoptical.com/stock/optical-components/uvvisnir-optics/lenses/plastic-lenses/plastic-lenses-singlets/plastic-lens-biconvex-103mmflx10mmdia/ Plastic Biconvex Lenses] LPP1009
 +
* 1x 1.5 x 1cm [http://www.knightoptical.com/stock/optical-components/uvvisnir-optics/filters/long-pass-filters/acrylic-longpass-filters/colour-filter-acrylic-type-50x50mm-510nm-long-pass/ 510nm long pass acrylic filter] 510FAP5050
 +
 +
Miscellanea
 +
 +
* 1x Arduino UNO Rev 3
 +
* 1x 2.1mm DC barrel jack
 +
* 1x 9V battery snap and contact connector
 +
* 5x Jumper cables female-to-female 10 cm
 +
* 1x 9V Battery
 +
* 20cm Ribbon cables 1.27 10 wires
 +
* 2x Micromatch connector 10 pin male 8-215083-0
 +
  
*[https://www.knightoptical.com/stock/optical-components/uvvisnir-optics/lenses/plastic-lenses/plastic-lenses-singlets/plastic-lens-biconvex-103mmflx10mmdia/ Plastic Lens Biconvex] (Knight Opticals)
+
Mechanical
*[http://www.knightoptical.com/stock/optical-components/uvvisnir-optics/filters/long-pass-filters/acrylic-longpass-filters/colour-filter-acrylic-type-50x50mm-510nm-long-pass/ 510nm long pass acrylic filter] (Knight Opticals)
+
* 12x M3 screws 10mm
<br>
+
* 12x M3 nuts
You can find the bill of materials here:
+
* 10x M3 washers
 
<br>
 
<br>
 +
 +
==Before Starting==
 +
[http://mightyohm.com/files/soldercomic/FullSolderComic_EN.pdf Soldering is easy!] Learn it! <br>
 +
 +
 +
[http://www.cableorganizer.com/articles/how-avoid-solder-related-health-hazards.html More soldering safety!]
  
 
==Step by Step Buildling==
 
==Step by Step Buildling==
All files can be found here: <br>
+
 
===Laser Cutting Parameters===
+
===Laser Cutting the Inner Scaffold Pieces===
at chez hackuarium - laser cutter model: Keyland KQG-1060 120W CO2 laser cutter
+
----
 +
at chez [http://hackuarium.strikingly.com/ hackuarium] neighboring @make space of [http://www.univercite.ch/ univercité] - laser cutter model: Keyland KQG-1060 120W CO2 laser cutter
 
<br>
 
<br>
 
<br>
 
<br>
 
{|class="wikitable"
 
{|class="wikitable"
|+Material: particle board HDF (MDF)
+
|+Cutting Parameters for Material: HDF (MDF)
 
|  
 
|  
 
|mode
 
|mode
Line 65: Line 172:
 
|3mm board
 
|3mm board
 
|cut
 
|cut
|15
+
|20
 
|100
 
|100
 
|
 
|
Line 94: Line 201:
 
|}
 
|}
 
<br>
 
<br>
Results<br>
+
With these parameters, the 3mm thickness pieces take 5 min to cut, and the 2mm thickness pieces take min.<br><br>
 
+
Results
[[File:Particleboard_A.JPG|thumb|none|180px|HDF]]
+
<gallery widths=200px heights=200px>
[[File:Particleboard_B.JPG|thumb|none|180px|MDF]]
+
File:Particleboard_A.JPG| high-density fiberboard (HDF)
<br>
+
File:Particleboard_B.JPG| medium-density fiberboard (MDF)
 +
</gallery>
 +
<br><br>
  
 
===Assembling the Electronics===
 
===Assembling the Electronics===
 +
----
 
There are four PCBs for:
 
There are four PCBs for:
  
 
# Blue LED (for the eGFP excitation)
 
# Blue LED (for the eGFP excitation)
# LCD screen
+
# LCD screen  
 
# Red LED (for the transmittance)
 
# Red LED (for the transmittance)
 
# Light to frequency meter (to detect the light)
 
# Light to frequency meter (to detect the light)
 +
 +
'''TIP:''' if this is your first time soldering, we suggest to start with the LCD screen<br>
 +
Each of the PCBs except the LCD screen PCB are fixed onto the lasercut fiber boards using M3 screws.
 +
<br><br>
 +
  
 
====Soldering and Mounting====
 
====Soldering and Mounting====
 
=====Blue LED=====
 
=====Blue LED=====
[[File:Blue_LED_PCB_and_parts.png|400px]]<br>
+
[[File:Blue_LED_PCB_and_parts.png|400px]]<br><br>
 +
''Soldering the pieces:''<br>
 +
<gallery mode="packed"|left>
 +
File:Blue_LED_01.jpg|1. the super bright blue surface mount LED (SMD) piece is manually soldered onto the "backside" of the PCB
 +
File:Blue_LED_02.jpg|2. on the "front side" of the PCB, first solder the resistor
 +
File:Blue_LED_03.jpg|3. clip the legs of the resistor from the back
 +
File:Blue_LED_04.jpg|4. solder the other components (the connectors for the Red LED and the light to frequency meter)
 +
File:Blue_LED_05.jpg|5. Solder in the large MOSFET transistor. Try to bend it so that it stays flat on the PCB, hole aligned.
 +
File:Blue_LED_06.jpg|6. the other MOSFET is soldered into place - the flat side facing the resistor.
 +
File:Smd connector led board.jpg|7. pay attention to the orientation of the ribbon cable connector (the connector is red in this picture, but black in subsequent, it is the same connector)
 +
File:Blue_LED_07.jpg|8. to place the ribbon cable connector, first apply solder on one pad of the PCB (e.g. the circled pad in previous picture)
 +
File:Blue_LED_08.jpg|9. solder the first foot of the ribbon cable connector, ater soldering remove the sticker atop the connector, then proceed with the rest
 +
File:Blue_LED_09.jpg|10. all except the resistor for the super bright LED is soldered
 +
</gallery>
 +
[[File:Blue_LED_11.jpg|400px]]<br>
 +
10. ready for mounting<br><br>
 +
 
 +
''Now mounting onto the particle board:''<br>
 +
<gallery mode="packed"|left>
 +
File:Blue_LED_PCB_mounting_1.jpg|1. M3 screws are placed
 +
File:Blue_LED_PCB_mounting_2.jpg|2. Two washers are placed onto the screws before the PCB
 +
File:Blue_LED_PCB_mounting_3.jpg|3. the SMD Blue LED should face the particle board, and the PCB is bolted in
 +
</gallery>
 +
<br>
 
<br>
 
<br>
  
 
=====LCD screen=====
 
=====LCD screen=====
[[File:LCD_PCB_and_parts.png|400px]]
+
This LCD screen / arduino shield will connect to the blue LED PCB with a ribbon cable<br>
 +
[[File:LCD_PCB_and_parts.png|400px]]<br><br>
 +
 
 +
''Soldering the pieces:''<br>
 +
<gallery mode="packed"|left>
 +
File:Arduino usb tape.jpg|1. take out the Arduino board from its package. Cut a piece of electrical tape and cover the USB connector as shown on the picture.
 +
File:Lcd board headers.jpg|2. take out the '''long''' pin header, break it down into 2 pieces of 6 pins, and 2 pieces of 8 pins. Place all the pins in the female connectors of the Arduino board as shown. The 6-pins go on the side with the analog inputs (A0, A1, etc), and the 8-pins on the digital IO side. You should now be able to place the PCB on top with all pins sticking out
 +
File:LCD_03.jpg|3. place the green PCB board on top of it with the pin headers sticking out at the correct place. Solder all the pin headers in place (in this picture, the resistor is already there, but it is actually easier to do it next)
 +
File:Smd connector screen board.jpg|4. we will now solder the ribbon cable connector just as we did in the previous step (Blue LED steps 8 and 9), again pay attention to orient it as shown here
 +
File:LCD_01.jpg|5. solder resistor 1 on the logo side
 +
File:LCD_05.jpg|6. back to the logo side, place the trimpot on the left, two small buttons S1, S2 on the right, and the large potentiometer on the right. Solder into place from the back
 +
File:LCD_06.jpg|7. solder the pins onto the LCD screen, as the others, solder one, and make sure the placement is good before soldering the other pins
 +
File:LCD_07.jpg|8. LCD screen with view of the pins from the back
 +
File:LCD_08.jpg|9. view of the back with the big button soldered in
 +
</gallery>
 +
[[File:LCD_09.jpg|400px]]<br>
 +
9. done soldering this board, onto the next<br><br>
 +
<br>
 
<br>
 
<br>
  
Line 120: Line 275:
 
[[File:Red_LED_PCB_and_parts.png|400px]]<br>
 
[[File:Red_LED_PCB_and_parts.png|400px]]<br>
 
<gallery mode="packed"|left>
 
<gallery mode="packed"|left>
File:RED_LED_1.JPG|1. first mount the red LED - short leg = negative
+
File:RED_LED_1.JPG|1. first mount the red LED - short leg / flat plastic rim = negative, then solder
File:RED_LED_2.JPG|2. next, the pins, long pins same side as the LED
+
File:RED_LED_2.JPG|2. next, the pins, long pins same side as the LED, then solder
 
File:RED_LED_3.JPG|3. ready to mount on the board
 
File:RED_LED_3.JPG|3. ready to mount on the board
 +
File:RED_LED_4.JPG|4. mounted, view from the PCB side - washers not necessary for the screws
 +
File:RED_LED_5.JPG|5. mounted, view from the LED side
 
</gallery>
 
</gallery>
 
<br>
 
<br>
  
 
=====light to frequency meter=====
 
=====light to frequency meter=====
[[File:LFD_PCB_and_parts.png|400px]]
+
[[File:LFD_PCB_and_parts.png|400px]]<br>
 +
<gallery mode="packed"|left>
 +
File:LFD_1.jpg|1. place the legs of the light to frequency detector (LFD) in the PCB without bending or soldering, the goal will be to align it with the picture on the PCB
 +
File:LFD_2.JPG|2. use the particle board to align the 'eye' of the LFD, fix already the particle board to the PCB by using two M3 screws and bolts, place a washer between the two boards, verify that the light sensor lies flat on the PCB, if not, push it with a plier or a screwdriver
 +
File:LFD_3.JPG|3. solder the LFD and the long pins same side as the LFD
 +
File:LFD_4.JPG|4. back of the soldered PCB aligned with the particle board
 +
File:LFD_5.jpg|5. place a washer to account of the LFD thickness
 +
File:LFD_6.jpg|6. mounted, view from the PCB side
 +
File:LFD_7.JPG|7. mounted, view from the LFD side
 +
File:filter_not_glued.jpg|8. take out the yellow filter and prepare to fix it over the LFD - remove the clear film that is protecting the filter plastic
 +
File:filter_hot_glue.jpg|9. use the glue gun to put a dot of glue on both side of the filter, be careful to not put any glue where the other particle boards will come during assembly
 +
File:filter_glued.jpg|10. the filter after being fixed
 +
</gallery>
 
<br>
 
<br>
  
====Final Assembly====
+
====Battery connector====
 +
<gallery mode="packed"|left>
 +
File:battery_connector_parts.jpg|1. get the DC barrel jack, unscrew the plastic part and drive the wires through it '''before''' soldering anything
 +
File:battery_connector_wires.jpg|2. drive the red wire through the central hoop from inside out, and the black wire to the larger outer rim also from inside out, bend back the wires
 +
File:battery_connector_3rd_hand.jpg|3. with the help of a 3rd hand, immobilize the connector and solder the wires into place
 +
File:Adding_switch.jpg|4. cut one cable from the battery adaptor and solder it on one outer and one middle leg of the switch
 +
File:Switch_stabilization.jpg|5. use a glue gun to stabilize the switch connection
 +
</gallery>
 +
<br>
 +
 
 +
===Lens assembly===
 +
----
 +
# Place the two sides next to each other. They should be both marked with the *same* letter (A/A or B/B).
 +
# Place the lens in the groove on one side.
 +
# Place the second side on top, with letters towards the inside for both sides.
 +
# Fix together with 2 screws and 2 bolts.
 +
<br>
 +
===Vial Stabilizing Ring===
 +
----
 +
<gallery mode="packed"|left>
 +
File:Ring.jpg|1. Either etch a groove on the '''middle''' board or place a ring cut from MDF
 +
File:Ring_glued.jpg|2. Use wood glue or glue gun to glue onto the middle board - make sure the gluing is clean in the inside so that the vial can sit snugly
 +
</gallery>
 +
<br>
 +
 
 +
===Final Assembly===
 +
----
 
Finally, the Red LED and light to frequency meter will be plugged into the Blue LED board with connectors.<br>
 
Finally, the Red LED and light to frequency meter will be plugged into the Blue LED board with connectors.<br>
 +
[[File:Final_assembly_1.JPG|400px]]<br><br>
 +
 +
Then the LCD screen is plugged into the Blue LED board with a ribbon cable.<br>
 +
[[File:Final_assembly_2.JPG|400px]]<br>
  
Then the LCD screen is plugged into the Blue LED board with a ribbon cable.
 
 
The LCD screen board is ready to be mounted on the arduino.<br>
 
The LCD screen board is ready to be mounted on the arduino.<br>
  
Line 140: Line 338:
 
<br>
 
<br>
  
===Testing the Prototype===
+
===Testing the Electronics===
 +
----
 +
====Uploading the arduino code====
 +
If you have not done so already, download the [https://www.arduino.cc/en/Main/Software arduino open software].<br>
 +
The firmware is in our github repository for the [https://github.com/BioDesignRealWorld/FluoMeter FluoMeter].<br>
  
 +
====Reading the measurements====
 +
First test the firmware and the hardware without sample.
 +
* When you push the start button, does the blue LED go on?
 +
* Do you see the measurement on the LCD screen?
 +
* Do you then see the red LED go on?
 +
<br>
 +
Then try it with sample with fluorescence
 +
* Do you see changes in the measurement?
 +
<br>
 +
Now we are ready for the actual bacterial samples. Sample set-up protocol here.
 +
<br>
 +
 +
====Transmittance and absorbance====
 +
What we actually measure with the red LED is the transmittance - how much of the red LED light goes through the sample and reaches the other side.<br>
 +
Absorbance (optical density) is then calculated.<br>
 +
 +
<math>A = -\log_{10} \left(\frac{P}{P_0}\right)</math>
 +
 +
where <math>P/P_0 =</math> (Red LED captured by the light to frequency meter with sample / with buffers (no bacteria))<br>
 +
<br>
 +
 +
 +
====Checking with Bead Standards====
 +
Suggested [[calibration polystyrene bead standards]].<br>
 +
Take the readings to calibrate for scattering/transmittance, and fluorescence.<br>
 +
 +
 +
====Normalizing the Fluorescence Data====
 +
As the fluorescence intensity depends also on the number of bacteria present in the vial, it is necessary to normalize for this - especially when comparing between time points, or if toxicity to the bacteria are seen.<br>
 +
Normalized Fluorescence Units = NFU
 +
 +
<math>NFU = B/A</math>
 +
 +
where <math>B</math> = reading from the blue LED, and <math>A</math> is the absorbance calculated from above.<br>
 +
 +
These are the values that should be used to plot the standard curves, and your samples.<br><br>
 +
 +
===Making the Outer Housing===
 +
----
 +
What we have built so far leaves the samples exposed to ambient light. <br>
 +
So we need to make outer housing that can mount the LCD screen, house the battery, enclose the scaffold inside, and have easy access to put the samples in and out while keeping the light and optics dark. You can find the template files on [https://github.com/BioDesignRealWorld/FluoMeter github].
 +
<br>
 +
 +
<gallery mode="packed-hover">
 +
File:Boxing.jpg
 +
File:switch.jpg
 +
File:Box.jpg
 +
File:Box_LCD.jpg
 +
File:Box_sample_access.jpg
 +
File:Box_allopen.jpg
 +
</gallery>
 +
<br><br>
 +
 +
==Issues and Observations==
 +
During the Winter Workshop of February 1-5 2016, here is the list of issues to be addressed on this workshop<br>
 +
===BOX===
 +
* test for "light proofness" for outer box
 +
* more robust casing
 +
* make a USB cable hole in the outer box - make these holes accessible directly from the outside without having to open the box
 +
* make the access (sample) lid on the opposite side
 +
* LCD on top
 +
<br>
 +
===LIGHT===
 +
* when the battery is first turned on, the first fluorescent reading is much higher than the rest of the readings
 +
::Problem due to interrupt flag not properly cleared. Fix in github.
 +
* battery drains quickly >>> ''alternatives: use a USB battery charger''
 +
[[File:blank_pwr.png|600px]]
 +
* 2ml in the 4ml vial seems to improve eGFP folding, and early detection of the eGFP fluorescence (needs to be confirmed)
 +
* improve signal to noise ratio by [https://en.wikipedia.org/wiki/Lock-in_amplifier signal lock-in-amplification]
 +
* Optimize the blue light for shortening the bleaching of eGFP (shorten time for the blue LED ON, also modulate blue LED intensity)
 +
<br>
 +
===DATA===
 +
* Direct write to file through USB cable (rather than taking manual notes)
 
<br>
 
<br>
  
 
==Links other References==
 
==Links other References==
 +
====Documentation Videos====
 +
* [https://vimeo.com/156707865 Soldering and mounting] time lapse video
 +
* [https://vimeo.com/156707861 Box assembly] time lapse video
 
<br><br>
 
<br><br>
 +
====Biosafety====
 +
* [[GMO Containment in the Field]] discusses how we can bring the bioreporter together with the device in the field in Switzerland -
 +
<br>
 +
[[Category:Ongoing Projects]][[Category:Hardware]]

Latest revision as of 10:43, 13 May 2016

The Arsenic prototype v4.0 is the Winter School 2016 kit version of prototype 3.0.

Principles

The basis of the prototype remains the same as the prototype 3.0- the bioreporter, a GMO bacteria expressing eGFP (Green Fluorescent Protein) is incubated with a water sample, where the fluorescence is detected optically and can be quantified in order to measure the concentration of Arsenic in the water

  • A vial containing the water sample we want to test is positioned on a socket through which a fluorescent excitation LED (blue 488nm for eGFP) passes.
  • GFP absorbs this blue light (λ=475 nm) and emits green light (λ=504 nm) which is detected by a photosensor on which the light is concentrated with the help of two lenses that avoid loss of intensity.
  • A long-pass filter allows only the eGFP fluorescence signal to reach the photosensor.
  • The intensity of the fluorescence is a function of the number of bacterias in the sample. This can be quantified undirectly by measuring the Turbidity of the sample.
  • A red LED is placed in-line with the photosensor to measure the transmittance, which can be converted into turbidity. The measurement of turbidity will allow us to normalize our results with respect the density of bacterias.


Proto4 principle all.png



Such a system is easily implemented using a microcontroller, a minimalist computer. A microcontroller has a number of GPIO (general purpose input output). When turned as an output, a GPIO is a terminal that can be programmatically set to a voltage of 5V (high state, logical 1), or 0V (low state, logical 0). This can be used to turn on or off external components such as LEDs. When used as an input, we can read the light sensor from them for example. The microcontroller is programmed in C code. It incorporates also timers that allow to keep track of time.

We use an Arduino board to control this device. The microcontroller on the Arduino board is programmed to carry out the following operations.

  1. Wait for button to be pushed.
  2. Turn on the blue LED for a predetermined amount of time.
  3. Readout the value of the light sensor.
  4. Turn off the blue LED.
  5. Turn on the red LED for a predetermined amount of time.
  6. Readout the value of the light sensor.
  7. Display the two values on the screen of the device.


Proto4 electronics diagram.png


The main electronic components of the device are as follows.

  • Arduino board: the brain of the device.
  • Transistors: the GPIO cannot provide enough current to drive LEDs directly. We use instead transistors. Transistors are like electrically controllable switches. It is a 3-terminal device. When provided with 0V or 5V on one terminal, the two remaining terminals are connected or disconnected, respectively (or vice versa depending on transistor type). The transistor size is proportional to how much current it must let through. We need a large one for the blue LED as it is very bright and has a high current.
  • LED at 600 nm: to measure optical density.
  • LED at 485 nm: to excite eGFP.
  • A light sensor: the sensor we use is a light-to-frequency sensor. It means that it outputs a square signal with a frequency proportional to the light intensity on the photosensitive part of the sensor.
  • A screen: to display results.
  • Buttons and potentiometer: for the user to interact with the device.

With the measures taken by the device, compared against a standard curve of water containing known arsenic concentrations, one can determine the concentration of arsenic in the sample and so know if the water is drinkable or not.



In summary, the most current version consists of:

   lasercut chassis
   blue LED for eGFP fluorescence excitation at 90 degrees from detector
   red LED for transmittance measurements in line with detector
   vial holder matching the vial approved by the Swiss authorities
   light to frequency detector
   a filter to block excitation light
   LCD screen read out
   based on the arduino



Tools

  • Soldering iron
  • Tin
  • Knippers
  • Plier
  • Screwdriver phillips #1
  • Vice (for crimping the ribbon cable)

Safety

  • Don't burn the wires on the table with the soldering iron
  • Mind your fingers
  • When knipping the legs of the components, hold the loose end so that it doesn't fly into someone's eye.
  • Apply common sense

Materials

The firmware is in our github repository for the FluoMeter.
For the full Bill of Materials (BOM) including reference numbers see this sheet.

LCD Shield

  • 1x LCD 8x2 HDM08216L-3-L30S
  • 1x resistor 1K (R1)
  • 1x Trimpot 10K
  • 2x Tactile switch 17mm (S1, S2)
  • 1x Potentiometer
  • 1x Micromatch connector 10 pin SMD female 8-338069-0
  • 1x 1x40 pin header straight long (17mm)
  • 1x 2x8 pin header straight

LED Driver board

  • 1x LED 485nm (blue) SMD XPEBBL-L1-0000-00Z01
  • 1x P-channel MOSFET TO-220
  • 1x P-channel MOSFET TO-92
  • 1x Micromatch connector 10 pin SMD female 8-338069-0
  • 1x Resistor 5 ohm 3W
  • 1x Resistor 150 Ohms 1/4W
  • 1x 1x3 pin header 90 degree bent
  • 1x 1x2 pin header 90 degree bent

Optical density (red LED)

  • 1x red LED 5mm
  • 1x 1x2 pin header straight

Light sensor

  • 1x Light To Frequency Converter – TSL235R
  • 1x 1x3 pin header straight

Optics

Miscellanea

  • 1x Arduino UNO Rev 3
  • 1x 2.1mm DC barrel jack
  • 1x 9V battery snap and contact connector
  • 5x Jumper cables female-to-female 10 cm
  • 1x 9V Battery
  • 20cm Ribbon cables 1.27 10 wires
  • 2x Micromatch connector 10 pin male 8-215083-0


Mechanical

  • 12x M3 screws 10mm
  • 12x M3 nuts
  • 10x M3 washers


Before Starting

Soldering is easy! Learn it!


More soldering safety!

Step by Step Buildling

Laser Cutting the Inner Scaffold Pieces


at chez hackuarium neighboring @make space of univercité - laser cutter model: Keyland KQG-1060 120W CO2 laser cutter

Cutting Parameters for Material: HDF (MDF)
mode speed power scan mode interval
Groove LED (fluo) scan 50 (100) 35 x_unilat 0.1
3mm board cut 20 100
Text and Logo scan 250 (100) 25 x_unilat 0.3
Groove Lens scan 50 12 x_unilat 0.1
2mm board cut 35 100


With these parameters, the 3mm thickness pieces take 5 min to cut, and the 2mm thickness pieces take min.

Results



Assembling the Electronics


There are four PCBs for:

  1. Blue LED (for the eGFP excitation)
  2. LCD screen
  3. Red LED (for the transmittance)
  4. Light to frequency meter (to detect the light)

TIP: if this is your first time soldering, we suggest to start with the LCD screen
Each of the PCBs except the LCD screen PCB are fixed onto the lasercut fiber boards using M3 screws.


Soldering and Mounting

Blue LED

Blue LED PCB and parts.png

Soldering the pieces:

Blue LED 11.jpg
10. ready for mounting

Now mounting onto the particle board:



LCD screen

This LCD screen / arduino shield will connect to the blue LED PCB with a ribbon cable
LCD PCB and parts.png

Soldering the pieces:

LCD 09.jpg
9. done soldering this board, onto the next



Red LED

Red LED PCB and parts.png


light to frequency meter

LFD PCB and parts.png


Battery connector


Lens assembly


  1. Place the two sides next to each other. They should be both marked with the *same* letter (A/A or B/B).
  2. Place the lens in the groove on one side.
  3. Place the second side on top, with letters towards the inside for both sides.
  4. Fix together with 2 screws and 2 bolts.


Vial Stabilizing Ring



Final Assembly


Finally, the Red LED and light to frequency meter will be plugged into the Blue LED board with connectors.
Final assembly 1.JPG

Then the LCD screen is plugged into the Blue LED board with a ribbon cable.
Final assembly 2.JPG

The LCD screen board is ready to be mounted on the arduino.

We are now ready to test it out.


Testing the Electronics


Uploading the arduino code

If you have not done so already, download the arduino open software.
The firmware is in our github repository for the FluoMeter.

Reading the measurements

First test the firmware and the hardware without sample.

  • When you push the start button, does the blue LED go on?
  • Do you see the measurement on the LCD screen?
  • Do you then see the red LED go on?


Then try it with sample with fluorescence

  • Do you see changes in the measurement?


Now we are ready for the actual bacterial samples. Sample set-up protocol here.

Transmittance and absorbance

What we actually measure with the red LED is the transmittance - how much of the red LED light goes through the sample and reaches the other side.
Absorbance (optical density) is then calculated.

where (Red LED captured by the light to frequency meter with sample / with buffers (no bacteria))


Checking with Bead Standards

Suggested calibration polystyrene bead standards.
Take the readings to calibrate for scattering/transmittance, and fluorescence.


Normalizing the Fluorescence Data

As the fluorescence intensity depends also on the number of bacteria present in the vial, it is necessary to normalize for this - especially when comparing between time points, or if toxicity to the bacteria are seen.
Normalized Fluorescence Units = NFU

where = reading from the blue LED, and is the absorbance calculated from above.

These are the values that should be used to plot the standard curves, and your samples.

Making the Outer Housing


What we have built so far leaves the samples exposed to ambient light.
So we need to make outer housing that can mount the LCD screen, house the battery, enclose the scaffold inside, and have easy access to put the samples in and out while keeping the light and optics dark. You can find the template files on github.



Issues and Observations

During the Winter Workshop of February 1-5 2016, here is the list of issues to be addressed on this workshop

BOX

  • test for "light proofness" for outer box
  • more robust casing
  • make a USB cable hole in the outer box - make these holes accessible directly from the outside without having to open the box
  • make the access (sample) lid on the opposite side
  • LCD on top


LIGHT

  • when the battery is first turned on, the first fluorescent reading is much higher than the rest of the readings
Problem due to interrupt flag not properly cleared. Fix in github.
  • battery drains quickly >>> alternatives: use a USB battery charger

Blank pwr.png

  • 2ml in the 4ml vial seems to improve eGFP folding, and early detection of the eGFP fluorescence (needs to be confirmed)
  • improve signal to noise ratio by signal lock-in-amplification
  • Optimize the blue light for shortening the bleaching of eGFP (shorten time for the blue LED ON, also modulate blue LED intensity)


DATA

  • Direct write to file through USB cable (rather than taking manual notes)


Links other References

Documentation Videos



Biosafety