Real application of our device

In this section of the wiki, we present how we consider the design of our device and discuss realistic application. To learn more about the market expectations, we met a safety official of ARKEMA. Here are also some information about mecury toxicity.

Mercury poisoning.

Design of the device.

Meeting with ARKEMA.

Mercury poisoning and pollution.

Mercury is a metal, liquid at room temperature. It easily become odorless and colorless vapor at room temperature. The exposure to mercury vapor can be really harmfull since mercury can easily reach the bloodstream by the lung, causing severe damage to kidney and brain. The body can not get rid of the mercury, it accumulates, provoking damages.

Mercury pollution is an issue in air, soil and water. Here we focus on water pollution. Releases of mercury in the environment are mainly caused by the chemical and gold industry and combustion of fossile fuel.

Mercury exists in water at different oxydation levels, mainly $Hg$ and $Hg^{2+}$. It also form inorganic compound with others ions, for example HgCl₂. Some bacteria are able to metabolize $Hg^{2+}$, forming methyl-mercury. This is the reason why mercury is so poisonous, the organometalic compounds are eaten by fishes, which ones are subsequently eaten by humans.

The most known example of mercury poisoning is called the Minamata disease. In Japan the Chisso Corporation, a chemical industry, release large amount of methyl-mercury for more than 30 years in the see. Tousands of people died from eating contaminated fish and shellfish.

Design and use of Mercuro-Coli device

Design

Our objective is to design a device for the detection and quantification of heavy metal pollution in a given sample. Our final device is intended to sense mercury, but for practical reasons, we mainly work with a harmless molecule as a proof of concept: anhydrotetracyclin. One should notice that most of the elements in the design of our device and genetic network would not change whatever the chemical that we could want to quantify.

The device presented here take into account the information provided by modelling results. The device we conceived is rectangular shape, thin, light and very cheap. It is made of machined polycarbonate. Vertical channels are engraved in the plastic.

Each channel form a specific chemical environnement: they contain distincts concentration of IPTG, such that a gradient is obtained on the whole plate.

A graduation is etched below the channels to read the concentration corresponding to the channel where the red stripe appear. The graduation is not shown on the picture above. Experiments are made at the moment to know how to draw the graduation. Even if we have not all the data needed to get an experimental graduation, our modelling work gave us that answer.

Use: prepare the sample, read the result

Packaging

The device is packaged with the channels half-filled with suspension of our engineered bacteria in nutritive media with the IPTG gradient. The post-transcriptionnal system maintains the two pathways of the toggle switch on OFF position. The plate is hermetically covered by an adhesive film. In this state the device can be stored at 4°C in a fridge for several weeks. The shipping package has to maintain this cool temperature. Before use, just let the device at room temperature for some times.

Prepare the sample

A preparation phase is needed before the sample can be introduce in the device. The first step consists in ionization of the mercury, it is crucial because we want to quantify the total amount of mercury in the sample. Many other form are found in nature: mercury is often complexed to others ions or forms organometallic compounds.

Once all the mercury of the sample is in the $Hg^{2+}$ chemical form, the second step is to tetracyclin in water. The tetracyclin, when the sample and the solutions in the channelswill mix, will activate the system by inducing production of rsmY.

Read the result

The water pollution test is performed by introducing the prepared sample in the device and waiting for the result (about one hour, according to the modelling). The introduction of the sample is made through a main channel that supply the other. Check valves will prevent the solutions in the different channels from mixing.

As shown in the illustration below, the red coloration of a channel indicates the presence of pollutant in the sample. The position of the colored channel on the plate gives us the amount of mercury in the water.

Disposal of the device

The device is disposable. Once the test is performed, autoclave the device to sterilize it.

Meeting at ARKEMA and visit of their laboratory

On the 24^th october we met the person in charge of the safety at the ARKEMA's chemical factory in Jarrie. To produce chlorine they use electrochemical techniques that involve mercury. They have to keep the mercury discharges as low as possible. The European laws imposes a concentration of mercury in the discharges inferior to 50 µg/L. But a prefectural order force them to reduce the total mercury concentrationin their discharge to about 5µg/L.

To manage to comply the law, they have to monitor cautiously their mercury emission. To do so, they collect sample of water all day long at several place around the factory so that they can measure mean value of the mercury emitted in a day.

The collected water sample are prepared before being tested. To measure total mercury concentration, all the mercury is reduced in Hg⁽⁰⁾ form. Then the amount of Hg is measured by fluorescence after atomic absorption.

They explained to us why they measure mercury concentration in water and how they proceed. It is very important for them to have reliable system to measure the mercury. The device has to be precise and accurate. According to modelling results, the limit of quantification (for aTc) is 0.1 µM. If we assume that it is the same for mercury, it gives us a limit of detection of 20 µg/L. So our device is precise enough to meet the requirements of European council laws on mercury emission.

According to the World Health Organization, the maximum concentration of mercury in drinkable water is 1 µg/L. According to the directive of the European Council it is 0.05 µg/L. In both case the concentration is below the limit of detection of our system. Several possibility exists to overcome this limitation, we can proceed to a more complex sample preparation by concentrating the mercury (for example 500 times), we might also tune the parameters of our system by mutating the DNA.

References

• PhD thesis: Faïdjiba LOE-MIE, Développement de sels d'onium chélatants et fluorogéniques pour la détection des ions mercuriques en microsystèmes.
• Directive of The European Parliament And The Council Of The European Union
• The French Senate (it refers to european laws)
• www.epa.gov/hg/effects.htm