Final Presentation

BIODESIGN FOR REAL WORLD

3rd of June 2013

Jasmina Rubattel Emilie Mussard Nicolas Krischer Romain Equey

Plan �  Introduction

�  Aim �  Motivation

�  Background researches �  Coliform bacteria �  Arsenic �  Fluorescence �  Legal framework �  Decision

�  Design criteria �  Prototype I: presentation and demo �  Prototype II: presentation and demo �  Data and analysis �  Conclusions �  Future directions

Introduction •  Bachelor project

•  Team

Aim of the project �  Build a sensor either for Arsenic or for Coliform bacteria detection

�  Sense a pathogen in water �  Process information with a device � Real world application à go out of the lab �  Build something accessible

�  More than just building a biodevice! � Open-source informations � Raise awareness about water quality �  Learn how to work

�  As a group �  With people from different backgrounds

�  Integrate different domains of studies

Motivation The aims have been defined by our motivation as much as our motivation was dependent of the aims! �  Dispose of a new way to learn

� No “knowledge feeding” �  Participation, initiative, try, search, solve problems… � Connect different subject around one goal

�  Real world aspect �  Do with what we have in terms of:

� Knowledge � Materials �  Possibilities

Background Researches •  Coliform bacteria •  Arsenic •  Fluorescence •  Legal framework •  à Decision

Coliform bacteria: General informations �  Gram-negative bacteria, ferment lactose �  Found in nature and in feces of warm-blooded animals �  Used for fecal contamination determination �  Easy to culture, especially E.Coli

•  Most studied coliform •  Found in the intestinal tract of animals •  Mostly harmless, but some strains are

toxic •  E.g. STEC that produces shiga-toxin,

found in ruminants gut

Coliform bacteria: intoxication & detection � Symptoms of coliform intoxication:

�  Bloody diarrhea, vomiting � Complication: Hemolytic uremic syndrome (HUS)

�  HUS consists in clot formation, leading to: �  blocked arteries à Ischemia �  And destroyed red blood cells

� E.Coli detection: � Heterotrophic plate count (HPC) is commonly used, with varying

conditions (incubation, temperature, nutrient) in addition to other tests

� Water considered safe under 100 cfu/ml �  It has some inconvenient, so other detection techniques are being

researched

Arsenic � 33rd element of the periodic table, As �  AsO3, arsenic trioxyde/arsenite: most common

form in the environment. � Soluble àWater can be contaminated by Arsenic

�  Industrial origin � Geological origin

Arsenic poisoning �  Interfer with Krebs cycle (inhibits pyruvate conversion to

acetyl-coA) �  As a slow poison, causes diseases

�  Skin diseases �  Intestinal tract problems � Cancers

�  Maximum concentration advised by WHO: 10μg/L �  Letal dose: 1mg/kg/day

�  Problem in Bangladesh and some Asian countries

Arsenic detection �  Interest in detection:

�  Industrial devices � Academic research

�  Bacteria have a constitutive arsenite and arsenate detection mechanism. �  Expression of a specific membrane protein complex which

serves to pump the arsenite residues only when they are present.

� Use of this mechanism to engineer a biosensor

Legal framework �  International framework

�  Precaution principle �  Substantial equivalence principle

�  Switzerland: Protection of the environment and public health �  Antibiotic resistance gene à Confinement à restrictions

�  Sample-holder: �  Transport

�  3 layers �  Waste gestion

�  It makes us aware of our responsibilities �  It forces us to communicate

àWe were looking what we are allowed to do and we discover that the legal demands forces us to think HOW to continue our research and build our prototype.

Fluorescence �  Emission of light by a substance that has previously been excited

by light at a specific wavelenght or by other electromagnetic radiation.

�  Green fluorescent protein: excited at 395nm, emitting green light at 509nm. �  From jellyfish Aequorea Victoria �  Used in biology for tracking �  Expressed in the reporter bacteria after having sensed Arsenic

�  Measure: light intensity at a specific wavelenght

Decision: the choice of fluorescence �  Do with what already exists, where the most informations are available. �  Work with Bangalore: students, responsive. �  Use fluorescence to detect Arsenic via the bioreporter

�  Fluorescence can also be used to detect E.Coli �  Based on intrisic fluorescence of bacteria components (in the UV range)

�  Amino-acids �  Nucleic acids

Arsenic presence

E.Coli sense As

Production of green fluorescence: measurable, proportional to Arsenic concentration

Activation of GFP gene

Design criteria General: •  Portability •  Low-cost •  Replicability

Fluorescence kit: •  Light-source •  Filter •  Sample-holder •  Receptor •  Data analysis

Prototype I •  Presentation •  Demonstration

Typical Fluorometer

http://openwetware.org/wiki/Citizen_Science/Open_Fluorometer_Project/Resources

Detector

Light Source

Our fluorometer LED

Emitting at a specific wavelength

Sample

Detector Detecting a specific

wavelength

Camera as a detector �  Canon Powershot A530 �  CHDK (Canon hackers development kit)

Image Processing �  ImageJ, an open-source image processing software

Script �  Fiji is similar to ImageJ, but allows to write scripts �  Permits automation of image analysis

Tests of our device �  FITC Dextran �  Constitutively expressing-eGFP E.Coli �  Arsenic biosensor

http://apb.tbzmed.ac.ir/Portals/0/Archive/Vol2No1/Pics/2/2.Fig2.jpg

Demonstration

Improvements �  Addition of a battery and a switch

�  To avoid using an Arduino as a simple battery �  The LED can be individually turned on/off

�  Fixation of the camera in the device �  Pictures more precise

�  Vertical position of the sample �  To allow an easiest change between

different samples

Prototype II •  Presentation •  Demonstration

General Mechanisms

Quantification with the Photoresistance

•  More the light increases, more the resitance decreases so more Vout tends to equal Vin •  Inversely, more the light decreases, more the resistance increases so more Vout tends to be null.

•  Problem: The photoreistance isn’t enough sensitive.

Quantification with the light-to-frequency device

330 ΩLight to frequency device

5 V Sample holderArduino Analogic pin 5

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•  The mechanisms are the same, but only the quantifier is different

Calibration •  Take measures with known arsenic concentrations. •  Plot them into a graph.

•  Light = slope * concentration + const •  Concentration = (light - const) / slope

Data and analysis

What we have done: Prototype 1 with dextran Prototype 1 with eGFP Prototype 2 with dextran Prototype 1 with arsenic biosensor Prototype 2 with arsenic biosensor

Prototype 1 with dextran

0

50

100

150

200

250

0 0.02 0.04 0.06 0.08 0.1 0.12

Gre

en li

ght i

nten

sity

[au]

Concentration of Dextran [g/L]

Prototype 1 with eGFP

y = 102.3x + 3.5759 R² = 0.99917

y = 1E+07x + 415889 R² = 0.99942

1

10

100

1000

10000

100000

1000000

10000000

0.000001 0.00001 0.0001 0.001 0.01 0.1 1

Gre

en L

ight

inte

nsit

y [a

u]

Sample dilution MEAN mean x area

Linear (MEAN) Linear (mean x area)

Prototype 2 with dextran

0

2

4

6

8

10

12

14

16

0 10 20 30 40 50 60

Sign

al

dextran solution [ml]

Prototype 1 with arsenic biosensor

0

2

4

6

8

10

12

14

16

18

20

0 10 20 30 40 50 60 70 80 90 100

Gre

en L

ight

Inte

nsit

y [a

u]

Arsenite [µg]

Prototype 2 with arsenic biosensor �  Failure!

Conclusion

Future directions �  Experiment more our prototypes with arsenic biosensor

�  Learn how the different aspects interact �  Improve the devices

� Test LEDs � Test filters � Add lenses �  Improve reception

�  Improve our prototypes �  Improve CHDK to do the analysis � Redesign the box to be used with a smartphone, create an app � Many samples at the same time

Future directions: General reflexions �  Sample holder

�  Size �  Environment for bacteria activity

�  Change the fluorescent protein �  Bigger difference between excitation and emission �  Longer wavelenght = cheaper LEDs

�  Use another reporter than GFP? �  Shorten reaction time

�  Another arsenic measuring way? �  Living matter = many parameters to manage:

Bacteria number, temperature, phase, oxygen and nutrients, …

Thanks �  Sachiko Hirosue �  Robin Scheibler �  Prof. Michaël Bensimon �  Nina Buffi �  José Artacho �  Sabrina Leuenberger, Heinz Straessle, Charles Joye �  Prof. Martial Geiser, Frederic Truffer, Jean Iwanovski �  Prof. Jan Roelof Van der Meer, Siham Beggah, Davide

Merulla