Arsenic Prototype 4.0

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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