# IGEM:IMPERIAL/2006/project/Bio elec interface

(Difference between revisions)
 Revision as of 13:47, 24 August 2006 (view source) (→Range of pH values we are expecting to see from different concentrations of AHL)← Previous diff Revision as of 13:50, 24 August 2006 (view source) (→Range of pH values we are expecting to see from different concentrations of AHL)Next diff → Line 270: Line 270: *The acylase breaks down AHL into fatty acid (carboxylic acid) and HSL: AHL + H20 -> Fatty Acid + HSL *The acylase breaks down AHL into fatty acid (carboxylic acid) and HSL: AHL + H20 -> Fatty Acid + HSL *Since carboxylic acid is a weak acid, we need to find pKa values and calculate H+ concentrations for different concentrations of the acid. *Since carboxylic acid is a weak acid, we need to find pKa values and calculate H+ concentrations for different concentrations of the acid. - *pKa value found from [http://daecr1.harvard.edu/pdf/evans_pKa_table.pdf Harvard University: Evans Group pKa Table]: Carboxylic Acid with X=C6H5    ->  ''' pKa=4.2''' + *pKa value found from [http://daecr1.harvard.edu/pdf/evans_pKa_table.pdf Harvard University: Evans Group pKa Table]: Carboxylic Acid with X=C6H5    ->  ''' pKa = 4.2''' *Working out the Ka: *Working out the Ka: **HA(aq) <-> H+ + A- **HA(aq) <-> H+ + A- **Ka = [H+][A-]/[HA] **Ka = [H+][A-]/[HA] - **pKa = -log(Ka) = 4.2  -> '''Ka=10E-4.2''' + **pKa = -log(Ka) = 4.2  -> '''Ka = 10E-4.2''' + *For [AHL] = 10E-9 M  ->  '''[H+] = 9.99*10E-10 M''' + *For [AHL] = 10E-3 M  ->  '''[H+] = 2.216 M'''

## Biological to Electrical Interface

System level diagram of biological to electrical interface

## Specifications

Bio Reporter Signal Transducer
Expressed by E.coli Detect Bio reporter
Degradation in medium afterwards. Time needed for degradation should be equal to/less than that of GFP Specific response to bio reporter only
No other interaction with E.coli processes Real-time readout
Sufficient expression and secretion of bio reporter in medium for detection by signal transducer Sufficient sensitivity to detect changes in bio reporter
Commercially available

## Possible Implementation

1. Signal transduction could involve (protein) redox-reactions
2. Antigen/Antibody sensors?
3. Protein sensors
4. Hormone/Neurotransmitter sensors (e.g. serotonin)

Scenario 1:
• E.coli is in a liquid medium
• Some bioprocess triggers production of a bio reporter (protein) in E.coli
• The bio reporter is expelled from the cell into the medium
• A sensor will measure the level of bio reporter (protein) in the medium

Scenario 2:
• E.coli is in a liquid medium
• Some bioprocess triggers productio of a bioreporter (protein) in E.coli
• The bio reporter is expelled from the cell into the medium
• There, the bio reporter (protein/enzyme) breaks down a certain chemical present in the medium
• A commercially available sensor then measures the
a) increase in concentration of the breakdown products or,
b) the decrease in concentratio of the initial chemical

## Enzymatic pH sensors: Email-correspondance with Anna

Sent: 01 August 2006 16:34
To: Vohra, Farah
Subject: RE: LB & M9
__HIDER__ <hide> Dear All,

Considering the media compositions that you have just sent to me I would presumed there is a chance for the biosensors I have described to work (with the polymeric membrane pH electrodes as a internal sensors for the enzyme sensor).

The diffusion models of enzymatic pH sensors are the following: model Carasa-Janaty model Moynihana_Wanga model Eddowes model Varanasiego I am sure you can fine in the literature their description and all necessarily equations. However I can provide you also some reference if you want to.

Anna

</hide></showhide>

Sent: 01 August 2006 18:30
To: Sander, Christin Y M
Subject: RE: Models for enzymatic pH sensors
__HIDER__ <hide> I am so sorry but probably some of names in the particular models were misspelled so therefore you could not find them in the literature. It is a crazy day today. Any way I am sending you some references to the models given earlier. Good luck.

1. model Carasa-Janaty

S.D. Caras at all, Anal. Chem., 57 (1985) 1920 S.D. Caras at all, Anal. Chem., 57 (1985) 1924 S.D. Caras at all, Anal. Chem., 57 (1985) 1917

2. model Moynihan-Wanga H.J. Moynihan, N.H.L. Wang, Biotechnol. Prog., 3 (1987) 90

3. model Eddowes M.J. Eddowes at all, Sens. Actuators, 7 (1985) 15 M.J. Eddowes, Sens. Actuators, 7 (1985) 97 M.J. Eddowes, Sens. Actuators, 11 (1987) 256 G.K. Chandler, M.J. Eddowes, Sens. Actuators, 13 (1988) 223

4. model Varanasi S. Varanasi at all, AIChE J., 33 (1987) 558 S. Varanasi at all, Biosens. Bioelectron., 3 (1988) 269 S.O. Ogundiran at all, Biotechnol. Bioeng., 37 (1991) 160

</hide></showhide>

Sent: 08 August 2006 10:50
To: Sander, Christin Y M
Subject: RE: Models for enzymatic pH sensors
__HIDER__ <hide>

Hi Christin,

To be honest I do not think so it is possible to reach the sensitivity range you want (nM). Potentiometric methods are known to have the detection limit around 10-6M (especially for the ion-selective electrodes). In some of the cases of ion-selective electrodes you could go up to 10-9M (look in the literature for Pretsch E or Sokalski T) but personally I do not think you can do that for pH-selective sensors. The only one thing that comes into my mind at the moment is e.g. tri-enzyme sensitive electrode. It would be amperometric set-up and so you could go much lower in the limit of detection. The path of determination would be e.g.

choline esterase converts butyryl choline into butyric acid and choline in presence of water. Then choline is transformed in the presence of oxygen and choline oxidase into betaine and hydrogen peroxide. Then H2O2 is converted using peroxidase into water. Of course H2O2 is a electrochemically detectable.

I send you an example of paper were researches used that pathway. You would have to adjust it to your own expectations. It is just of an idea but maybe it could be useful for your purpose.

Anna </hide></showhide> Paper on tri-enzyme electrode

Sent: 09 August 2006 17:08
To: Sander, Christin Y M
Subject:
__HIDER__ <hide>

Hi Christin,

I am sorry for a late answer but I have been a bit busy. Any way back to our business...As far as I remember the way to obtain the tri-enzymatic sensor was not that complicated (it is doable). Of course there is a danger that procedure could not work as more enzyme incorporated into the biosensors means more complications to get all the enzymes working also sensitivity will be diminish comparing to single enzyme biosensors. You should be aware of these problems. However personally I think that if you strictly follow the procedure of getting tri-enzyme sensor which was presented in the paper I have sent to you have a great chance to obtain positive results. Of course 4 weeks time it is plenty of time to build that type of sensor (probably you can do that over couple of hours only). The enzymes you need should be ordered as soon as possible so you could get them by mail in few days time. As for enzymes ordering please be carefull and check for the references in the recommended paper as well as it is very important to order exactly what they were using in their research work.

Good luck

Anna </hide></showhide>

Sent: 11 August 2006 18:07
To: Vohra, Farah
Subject: RE: iGEM
__HIDER__ <hide>

Hi Farah,

To be honest I think that the enzyme L-gulunolactone oxidase will not cause the enzymatic reaction for HSL . The alternative could be this reaction L-galactono-1,4-lactone + O2 = L-ascorbate + H2O2 where the structure of substrate is more similar to HSL and the chance for the reaction to work would be better. However I think this enzyme is also specific only for a given product. Check out that side: http://www.brenda.uni-koeln.de. Maybe there you could find some useful information. I will think about this problem over the weekend. There is a possibility that this electrochemical detection may not to work at all...There maybe you should reconsider the way of AHL detection (probably optical detection would be here the most safe option).

Anna </hide> </showhide>

Sent: 16 August 2006 15:02
To: Vohra, Farah
Subject: RE:
__HIDER__ <hide>

Hello Farah,

According to my knowledge the lowest in the limit of detection you could go for potentiometric sensors is around 10-7M (there are reported some sensors for which detection limit is around 10-9M or 10-11M but it is not for the case of hydrogen ions). For the detection using optical fibres (with fluorescence dye) the detection limit is similar. If I was you I would probably start from simple glass electrode.

Anna

PS I had one thought more that maybe you could use the amperometric pH measurements. In such a case you could go with LOD a bit lower. I attached some papers to my email so you can have a look and decide what would be better suited for you. </hide></showhide> Urea biosensor based on amperometric pH-sensing with hematein as a pH-sensitive redox mediator
Amperometric pH-sensing biosensors for urea, penicillin, and oxalacetate

## Commercially Available Enzymes

Sites for commercially available enzymes:

The biosensor we're looking at now requires three different enzymes: an esterase, oxidase and peroxidase.

A Japanese company called Amano Enzyme can provide us with acylase and peroxidase and we can maybe use L-gulonolactone oxidase as an oxidase for the sensor.

Various types of acylases can be ordered at Sigma-Aldrich. We now have to decide which one is best to use for our purposes.

## Meeting with Anna on 16/08/06

Discussion with Anna on the probability of the above enzymes working for our system led to the suggestion of using the following reaction: L-galactono-1,4-lactone + O2 = L-ascorbate + H2O2, instead of L-gulonolactone oxidase. L-galactono-1,4-lactone is similar in structure to HSL and has a higher probability of breaking down HSL in our reaction. However, the reaction may still be too specific to use. The acylase, however, should work.

The failure to find appropriate enzymes to make the tri-enzyme approach work means we'll have to use a different approach. Originally, we hoped to use a sensor that could directly measure [AHL] within the liquid medium containing our oscillator system. There have been various method discussed for achieving this such as a bio-luminescent sensor or a tri-enzyme electrode. Unfortunately they all seem a little too complicated considering our time constraints.

As a practical alternative, we discussed transferring samples of [AHL] from our medium into another medium containing a certain concentration of enzyme and measuring the resulting pH changes using a standard pH sensor such as a glass or optical electrode. These sensors are more likely to be able to detect within our desired pH range (pH 5-10).

Anna's offered to compare the two electrodes to give us a better idea as to which would be better to use. Watch this space for further updates!

## Meeting with Pierre Mielot about pH sensor (17/8/2006)

We can build a pH sensor on our own - as described in the paper Fabrication of Anodically Electrodeposited Iridium Oxide Film pH Microelectrodes for Microenvironmental Studies

The sensitivity of the sensor is in the pH range of 4-9.

## Range of pH values we are expecting to see from different concentrations of AHL

• Part C0061 produces AHL(N-acyl homoserine lactone), specifically it produces 3-oxohexanoyl-homoserine lactone (3OC6HSL)
• We ordered the following enzyme, a generic acylase: Acylase I from porcine kidney
• The acylase breaks down AHL into fatty acid (carboxylic acid) and HSL: AHL + H20 -> Fatty Acid + HSL
• Since carboxylic acid is a weak acid, we need to find pKa values and calculate H+ concentrations for different concentrations of the acid.
• pKa value found from Harvard University: Evans Group pKa Table: Carboxylic Acid with X=C6H5 -> pKa = 4.2
• Working out the Ka:
• HA(aq) <-> H+ + A-
• Ka = [H+][A-]/[HA]
• pKa = -log(Ka) = 4.2 -> Ka = 10E-4.2
• For [AHL] = 10E-9 M -> [H+] = 9.99*10E-10 M
• For [AHL] = 10E-3 M -> [H+] = 2.216 M