Research Application Biosensor Technology TFTB 34 · Research Application Biosensor Technology TFTB...

Research Application

Biosensor Technology TFTB 34

February 27, 2013 Examiner Fredrik Winquist

Linköpings Tekniska Högskola Jenny Sahlin, Emmy Österberg

Development of an instant drug detection through analysis of saliva

Project summary When giving painkillers to people who themselves may have problems to determine how they feel, such

as children and elderly, it can be useful to have a device that check how much of the active substrate

from previous tablets still circulating the blood. Since children develop at different rate the

recommended dose may not always fit the individual child. By develop a device a sample of saliva from

the child could say if it’s time to take a new dose or not.

According to SvD overdosing of paracetamol is one of the most common way for girls at the age of 15‐16

to try to make suicide. If they fail, liver damage often is one of the complications and transplantation will

be necessary. If the device existed, the nature of the overdose could be determined and the right

treatment could be put in faster and more lives and livers could be saved.

The device will be made of a glass capillary with a layer of MIP on the inside in which a sample of saliva

is sucked up by capillary force. When an active molecule in the sample binds to a binding site on the MIP

a change in colour occurs and will give an indication of the concentration of painkillers in the blood. For

starters we want to develop a stick for each of our four most common painkillers, ibuprofen,

paracetamol, diclofenac and acetylsalicylic acid.

Amount of money applied for: 7 745 532 SEK


Appendix

1. Description of the project The goal is to develop a biosensor for detection of paracetamol and nonsteroidal anti‐inflammatory

drugs (NSAID), such as aspirin, ibuprofen and diclofenac, directly from a saliva sample. By using

molecular imprinted polymers (MIP) the concentration of the active molecule in the saliva could be

detected instantly.

1.1 Advantages with the project

Development of a device that could measure the levels of drugs directly from saliva would facilitate the

dosing of medications. It is especially necessary when treating children since it can be hard to determine

when the effect of the painkillers has gone off. Since children develop at different rate the

recommended dose may not always fit the individual child. By using the stick developed in this project a

sample of saliva from the child could say if it’s time to take a new dose or not.

A second target group is elder people that sometimes can’t remember if they have taken their

painkillers. By using the stick they would quickly and easily see if they had already taken a tablet or not.

It would also be useful at nursing homes since the staff could use it to check if the patients have taken

their pill or not since elderly at nursing homes sometimes can be a bit senile.

According to SvD overdosing of paracetamol is one of the most common way for girls at the age of 15‐16

to try to make suicide. If they fail, liver damage often is one of the complications and transplantation will

be necessary. (1) If there exist a method to quick confirm the nature of the overdose, the right

treatment could be put in faster and more lives and livers can be saved.

1.2 Method

The first challenge in this project will be to determine whether or not the active molecule from ordinary

painkillers are excreted in saliva. For paracetamol it’s already determined that it’s possible (2) but for

the others this will be done with HPLC (high performance liquid chromatography) which is a method

that can detect almost any molecule. When confirmed that the molecules are extracted into the saliva

comparative blood tests will be done. These tests are done to compare the ratio between the

concentration of the molecule in blood respectively in saliva to get the proportional between them.

During the last years the development of MIP:s has increased rapidly and the use of them in biosensors

are big. One of the large benefits is that they are possible to mass produce and are very cheap. That’s

why whey will be used in this project to develop a specific method to measure the concentration of each

drug.


MIPs for ibuprofen, aspirin and paracetamol, separately, has already been developed by three individual

research groups (3)(4)(5). The active substrate were used as a template together with acrylamide as

functional monomer. The MIPs has not been tested in saliva before so it’s necessary to test them in this

new media. There is a MIP developed for diclofenac as well, but it uses 2‐vinylpyridine as functional

monomer instead of polyacrylamide as the others (6). Therefore the MIP for diclofenac must be

modified before it’s possible to use it in this project so it has polyacrylamide as functional monomer

instead. When a molecule binds to a binding site in the MIP, a change in colour must occur. If a enzyme

together with a coloured substrate are mixed in the polyacrylamide they could start a reaction, when a

molecule binds to the binding site, that gives a change in colour as a result. As far as the molecules

reaches in the stick the change in colour will reach the same.

Design of the drug detection stick

To begin with, one stick for each active molecule will be developed. The principle of the drug detection

stick is based on a capillary made of glass. The inside of the glass capillary should be covered with

polyacrylamide in which the MIP are made. A small bud on one side of the stick is used to suck up saliva

directly from the mouth and due to capillary forces the sample is adsorbed into the capillary and

introduced to the MIPs. When the right molecule are attached to the binding site on the MIP, a change

in colour occurs. Due to the concentration of molecules in the saliva sample the change in colour will

reach various length in the capillary. On the outside of the glass stick a gradation will tell how big the

concentration of molecules are in the sample by reading it where the change in colour no longer occurs.

The number of binding sites per gradation step should be proportional to the current concentration of

molecules.

After developing the stick it need to be calibrated so right amount of binding sites per gradation step

exist compared to common concentrations of active substrate in saliva.

The last step in the process is to test the stick on humans to confirm that it really works. Comparison

with HPLC need to be done so the gradient on the stick follows the concentration in the blood.


1.3 Activity plan

Year 1: Investigate if it’s possible to measure the active molecule of the painkillers in saliva.

Determine the ratio between the concentration of molecules in saliva compared to the

concentration in blood

Year 2:

Develop MIP:s specific for the chosen molecules and change them so they all work in

polyacrylamide gel

Modify the MIPs so they change color when right molecule connect to the binding site

Design the stick

Year 3:

Calibrate the stick so it’s the right amount of binding sites per gradation step.

Test the stick on humans and check with HPLC so it’s calibrated correctly.

2. Future prospects In the future we hope that this device is commonly used both at home and in nursing homes as a

facilitating method. If the technology will be easy to use, development opportunities are large. Maybe a

universal stick, measuring more than one substance at a time, could be developed to help in acute

overdoses of indeterminate nature at ER:s. In such a stick the most common substances of the same

kind could be combinated, like the four painkillers this project has been focused on. Later on a glass stick

that measures drugs as heroin, cocaine and LSD could be developed as a useful tool for the police when

they suspect a person for drug abuse.


Another use of this technology could be in veterinary medicine where the patients vary in size and

breed. Even if the dose of painkillers is based on weight today, different individuals of the same weight

may need different dose due to differences in metabolism.


3. Budget Year 1 Year 2 Year 3 Project leader (25 %) 120 000 121 000 122 000

Doctorates (200 %) 624 000 631 200 638 400

Medical doctor (25 %) 120 000 0 120 000

Engineer (100 %) 0 420 000 0

Engineer (25 %) 0 0 105 000

LKP (52 %) 430 560 590 200 492 440

Chemicals 20 000 100 000 50 000

Materials 50 000 200 000 60 000

Electronics, computer 25 000 60 000 25 000

Travels 10 000 10 000 10 000

Person testing 100 000 0 200 000

Summary 1 499 560 2 132 200 1 822 840

Administrative fees (42 %) 629 815 895 524 765 593

Total 2 129 375 3 027 724 2 588 433

Total amount applied for: 7 745 532 SEK


References 1)http://www.svd.se/nyheter/inrikes/varktabletter‐vanligast‐nar‐unga‐tar‐overdoser_5830515.svd,

2013‐02‐21, 08:58

2) Chatterjee K et. al., Effects of trichloroethylene anaesthesia on salivary paracetamol elimination.

Indian Journal of Physiology and pharmacology, 37 (1993) 79‐81

3) Monser L et. al., A Molecularly Imprinted Polymer for the Selective Solid‐Phase Extraction of

Ibuprofen from Urine Samples, The Open Chamical and Biomedical Methods Journal, 4 (2011) 7‐13

4) Huang ZF et. al., Synthesis and Research of Molecule Recognition Capability of Aspirin Molecularly

Imprinted Polymer, Fine Chemicals, 22 (2005) 1‐4

5) He XW et. al., Study of paracetamol molecularly imprinted polymers applied to solid phase extraction,

Analytica Chimica Acta, 62 (2004) 73‐81

6) Knopp D et. al., Selective trace analysis of diclofenac in surface and wastewater samples using solid‐

phase extraction with a new molecularly imprinted polymer, Analytica Chimica Acta, 620 (2008) 73‐81.

http://www.svd.se/nyheter/inrikes/varktabletter-vanligast-nar-unga-tar-overdoser_5830515.svd

TFTB34 – Biosensor Technology Martin Hyvönen Research application Karoline Sverkström Spring 2013 Patrik Tunón

APPLICANTS Martin Hyvönen

Karoline Sverkström

Patrik Tunón

DEVELOPMENT OF A REAL-TIME WATER

QUALITY CONTROL SENSOR

CONCEPT A sensing device dragged by larger fishing vessels to measure physiological conditions in the

Baltic Sea. The system itself is thought to work much like the in-line monitoring system for

lakes as used by J.V. Capella et al. (2013).1 That is having a sensory device connected,

possibly through a fiber optical cable, to a data logger which wirelessly transmits data by

means of the ships internet connection to a data storage server. The data will later be

analyzed, both by looking at single sensor measurement and by means of multi variant

analyses of all sensors. The resulting data is later to be compared with existing geographical

data concerning for instance wildlife habitats of different species and near shore cities,

harbors and facilities, evaluating their effect on the overtime changes of physiological

conditions in the Baltic Sea.

AMOUNT OF MONEY APPLIED FOR: 3 510 183 SEK


BACKGROUND Water is very important, therefore the quality of water and our effects on water are being

monitored, however these testes are usually limited to taking a couple of local samples and sending

those to central labs for testing2. This means that the data gathering process become both personnel

limited and with few data points it gets difficult to evaluate data over time.

In Sweden alone 310 million kronor are spent on water monitoring and improvement3. The

monitoring work is distributed between local and state authorities. Sweden also has the longest

stretch of coast toward the Baltic Sea and is therefore in extra need of refined analytical equipment.

When it comes to oceans four of the most important factors for Eutrophication are nitrate, nitrite,

phosphates and iron4, 5. However other ions are also of significance when it comes to water quality

due to their effect on pH disturbance of natural concentrations and toxicity. 4

TARGET COMPOUNDS There are three major categories of components that affect water quality which each contain

multiple species. Therefore to create a versatile instrument an array with multiple ionophores will

be used.6, 7

Eutrophic agents of importance are as follows phosphates, nitrate, ammonia and nitrite. These

chemicals stem mostly from over fertilized farms and insufficiently processed waste water. It is

important to monitor these chemicals because eutrophication leads to algal blooms that in turn may

lead to dead ocean and sea floors through using up all oxygen on the bottom. 2, 6, 7

Metals and alkali metals are especially interesting for drinking water due to their effects on water

quality such as taste, ability to corrode metals and toxicity. 6, 7

Environmental organic toxins our societies’ reliance on chemicals puts a stress on our environment

however where Eutrophic agents and metals are the same over the world whereas these organic

toxins change. For example Africa still have a problem with DDT and PCB while most western

countries have noticed a clear decline in these substances. Therefore the array will contain multiple

channels where the investigated chemical can be changed to match the geographic position of

investigation and expected toxins.8, 9

SENSOR DEVELOPMENT The device construction can be divided largely in four steps. The first being to construct a prototype

device by using currently developed sensors. The system will involve a large number of different

sensors. Primarily electrochemical sensor devices as proposed by Grady Hanrahan et al (2004) for

measurement of several types of ion concentrations. Detecting markers of over-fertilization such as

phosphates using sensors as described by Christopher Warwick et al (2012). Also an oxygen sensitive

sensor as described by Zhen Wang et al (2013) as well as standard sensors for electric conductance, pH

TFTB34 – Biosensor Technology Martin Hyvönen Research application Karoline Sverkström Spring 2013 Patrik Tunón and pressure, as well as sonars to measure depth of measurement and GPS devices to track the

measurement positions.11-14

Step two is small scale testing, searching for interested collaboration partners both by means of funding

and crews willing to connect the system to their ships. Step three being optimization of the sensor device

and possibly attempting to incorporate the sensory part of the system into a smaller microfluidic device

by means of the circuit printing technique described by Liang Li Wu (2011). The final step is gaining

national acceptance of the system and applying for funds for large scale testing and mapping of the

Baltic Sea. The results from this study are primarily to be compared versus handbook 2007:4 by the

Swedish environmental agency10 and also by using the evaluation grounds used in AlControl’s

brochure regarding water testing in wells 7. Interesting would also be to compare the results versus

wildlife conditions with the help of data from the Swedish environmental protection agency6. The

devices are considered to be powered by means of the ships internal energy supply. 11-14

ACTIVITY PLAN The project will be divided in to four phases; literature study and market survey, prototype

construction, evaluation of prototype and transform of prototype to a ready product.

The project is initiated by a literature study during the first phase. Parallel with the literature study,

a market survey needs to be carried out. The market survey serves to give information on the

available sensors on the market. During the same time, funds will be raised by getting in touch with

companies or authorities of interest. Requirements of the Real-time Water Quality Control sensor

need to be set. The first phase will go on for six months.

During the second phase, a prototype is developed. Ion sensors, pH sensors as well as temperature

sensors available on the marked will be purchased and used in a Real-time Water Quality Control

sensor prototype. This phase will take 12 months.

After the prototype is constructed, the third phase will begin. A vast study were the prototype will

be thoroughly evaluated will prolong for six months. During this phase, some collaboration with

companies or authorities needs to take place.

If the prototype withstands all requirements, the fourth phase can start. The prototype will be

transformed into a ready product that can be manufactured in large quantity. During this step

negotiations with the patent office, future customers and independent financers of the

manufacturing process will take place. This phase will require 12 months.


ADVANTAGES WITH THE PROJECT The major advantages of this concept of environmental monitoring is the possibility to vastly

increase the amount and quality of environmental maritime data by making real time, position

coupled measurements of multiple species. By using the existing fishing industry as a transporting

platform for the sensory device a wide measurement area, reducing the resources needed for water

measurements.

BUDGET Costs Year 1 Year 2 Year 3

Project leader (25%) 120 000 120 000 120 000

Assisstent (100 %) 282 000 298 500 307 500

LKP (52%) 209 040 217 620 222 300

Biosensors 100 000 50 000 10 000

Spare parts 20 000 20 000 20 000

Ionophores 50 000 40 000 20 000

Fieldtesting 15 000 30 000 30 000

Chemicals 15 000 10 000 10 000

Materials 60 000 20 000 10 000

Travels 5 000 20 000 20 000

Sum 876 040 826 120 769 800

Administrative fees (42%) 367 937 346 970 323 316

Total 1 243 977 1 173 090 1 093 116


REFERENCES

1. Capella JV, Bonastre A, Ors R, Peris M. In line river monitoring of nitrate concentration by means of a wireless sensor network with energy harvesting. Sensors Actuators B: Chem 2013 2;177(0):419-27.

2. Sundqvist J. Assessment of organic waste treatment. In: Renewables-based technology. John Wiley & Sons, Ltd; 2006. .

3. Pengar för vattenvård [Internet]havet.nu: Havet.nu [cited 2013 02/25]. Available from: http://www.havet.nu/index.asp?d=190&id=39625.

4. Department: water affairs South Africa. EUTROPHICATION -national eutrophication monitoring programme design. :2013-02-25. Available from http://www.dwa.gov.za/iwqs/eutrophication/NEMP/02Eutrophication.pdf.

5. Silver MW, Bargu S, Coale SL, Benitez-Nelson CR, Garcia AC, Roberts KJ, Sekula-Wood E, Bruland KW, Coale KH. Toxic diatoms and domoic acid in natural and iron enriched waters of the oceanic pacific. Proceedings of the National Academy of Sciences 2010 November 30;107(48):20762-7.

6. http://www.naturvardsverket.se/ [Internet]: Naturvårdsverket; c29 november 2012 [cited 2013 02/21]. Available from: http://www.naturvardsverket.se/.

7. ALcontrol Laboratories. Test av ditt brunnsvatten v6. [revised 2013cited 2013-02-21]Available from http://se.alcontrol.com/sites/default/files/Alcontrol/Documents/PDF/SE/Test_av_ditt_Brunnsvatten_2013_v6.pdf.

8. Euro Chlor. PCBs, DDT and dioxin. [revised 2002:2013-02-25. Available from http://www.eurochlor.org/media/49366/8-11-4-16_marine_ra_pcbs_ddt_dioxin.pdf.

9. In Africa, DDT Makes a comeback to save lives [Internet]Executive Intelligence Review.: Executive Intelligence Review. [cited 2013 02/25]. Available from: http://www.larouchepub.com/other/2004/sci_techs/3124ddt_africa.html.

10. Hanrahan G, Patil DG, Wang J. Electrochemical sensors for environmental monitoring: Design, development and applications. J Environ Monit(8):657.

11. Wang Z, Liu D, Gu H, Zhu A, Tian Y, Shi G. NTA-modified carbon electrode as a general relaying substrate to facilitate electron transfer of SOD: Application to in vivo monitoring of O2− in a rat brain. Biosensors and Bioelectronics 2013 5/15;43(0):101-7.

12. Microfluidic printed circuit boards. Electronic components and technology conference (ECTC), 2011 IEEE 61st; 2011. ID: 1.

13. Warwick C, Guerreiro A, Soares A. Sensing and analysis of soluble phosphates in environmental samples: A review. Biosens Bioelectron 2013 Mar 15;41:1-11.

14. Bilaga A till handbok 2007:4 Bedömningsgrunder för sjöar och vattendrag. Naturvårdsverket 2007:http://www.naturvardsverket.se/Documents/publikationer/620-0148-3.pdf.

http://www.havet.nu/index.asp?d=190&id=39625

http://www.dwa.gov.za/iwqs/eutrophication/NEMP/02Eutrophication.pdf

http://www.naturvardsverket.se/

http://www.naturvardsverket.se/

http://se.alcontrol.com/sites/default/files/Alcontrol/Documents/PDF/SE/Test_av_ditt_Brunnsvatten_2013_v6.pdf

http://se.alcontrol.com/sites/default/files/Alcontrol/Documents/PDF/SE/Test_av_ditt_Brunnsvatten_2013_v6.pdf

http://www.eurochlor.org/media/49366/8-11-4-16_marine_ra_pcbs_ddt_dioxin.pdf

http://www.larouchepub.com/other/2004/sci_techs/3124ddt_africa.html

http://www.naturvardsverket.se/Documents/publikationer/620-0148-3.pdf

25/02/2013 Research application Daniel Falk TFTB34 Group 3 Theodor Nevo

The applicant Theodor Nevo, Daniel Falk

Project title Dental health based on salive analysis

Summary Dental hygiene should always be a priority; a lot of money could be saved in healthcare all over the

world if people could take better care of their teeth. So to help and motivate people an easy‐to‐use

strip test could be used to detect change in the urea and arginine level through the use of molecular

imprinted polymers (MIPs) as they are highly selective. Higher levels correspond to a cleaner, more

resistant mouth. Lower levels on the other hand can correspond to a higher number of acidic

bacteria that in turn will promote caries and periodontitis.

The levels will be detected through signal change from the polymers when the molecule binds to it.

Applied for 3 326 000:‐

1


Goal To create a functioning test strip that can give a close approximation as to the users’ dental health

and risk for dental sickness.

Background There are a lot of factors that comes into dental hygiene. And a lot of them are individual like age,

gender, salvatory levels and more. We have a lot of bacteria in our mouth, but normaly they are in

equilibrium with the host, but there is a fine line to walk between oral health and disease. The same

bacteria can under the right conditions instead promote dental caries and periodontal disease

through metabolic changes. Especially in the caries case there is an acidification. Because of an

increase in acid producing bacteria and that the death of the less acidic bacteria that normally is

associated with dental health. These bacteria protects from acidification by hyrdolysing urea or

arganine into ammonia.1

Periodontitis is an inflammatory disease that affects the tissue surrounding the teeth and can if

untreated cause teeth loosening and teeth loss. Triggering Receptor Expressed on Myeloid cells 1

(TREM‐1) are one of the markers for periodontitis that can be found with greatly elevated levels

during infection.2

Metods

Molecular imprinted polymers MIP is a biosensor that has the ability to function as an artificially receptor or antibody to use for

chemical analysis, separation and catalysis. The MIP has specific binders that bind molecules used as

target binders and then is a polymerization step followed. In essence, on a template gets monomers

first pre‐assembled and after they bind to the template is a cavity created through polymerization. In

the next step, the template is leaving and the resulting polymer and functional groups is left and a

artificially receptor is created. The MIP can be used as a biosensor on where analytic elements are

binding and a chemical or physical signal is created and can be transuded to an electrical signal which

can be measured. The polymer has the advantage of being highly resistant to extreme pH values.

One type of MIP based on the change of conductivity can transduce a measurable signal dependent

on the amount of analyte bounded to the polymer. Two electrodes are separated by a polymer

membrane and the change is translated to an electrical signal which is measurable. It is important

that the molecules to be detected by conductivity are porogentetic through the membrane.

The technique with conductivity based assay for fast and easy measurements of chemical analyt have

shown good results in earlier studies but more research need to be done. Better knowledge of what

kind of polymer to be used in MIP is needed to give a fast and costly efficient biosensor.

An alternative is to use a functional fluorophore in the MIP that will be quenched when the molecule

binds to it. This might be a more cost effective method but less precise as the fluorophore might not

be strong enough to give a reliable color difference.

2


Targets Urea and arganine are two targets that are directly linked to the dental hygiene. They will be

targeted by functionalized monomers through MIP.

Triggering Receptor Expressed on Myeloid cells 1 is a marker for periodontitis that would be good if it

could be quantified. Will be target by functionalsed monomers through MIP as well, but that might

be hard because it might not be small enough.

The system The idea is to create a film with MIPs for each target and through a color change (fluorophore

quenching) or change in conductivity get a signal back. Might just be that the change in conductivity

are measured and then converted to a number.

The sample will be applied in a designated spot, and through capillary force be applied over the films.

This can be couple to a small displace that will give the measured values, or they might be

recalculated to a health index.

Advantages This project might evolve into commercial, easy‐to‐use product that got a part with a limited lifetime.

Thus needs to be renewed. Will be able increase the dental health and awareness of the users, and

might even give a pointer to when to contact a doctor or dentists.

References

1. Toro E, Nascimento MM, Suarez‐Perez E, Burne RA, Elias‐Boneta A, Morou‐Bermudez E. The effect of sucrose on plaque and saliva urease levels in vivo. Arch Oral Biol 2010 3;55(3):249‐54.

2. Bostanci N, Öztürk VO, Emingil G, Belibasakis GN. Elevated oral and systemic levels of soluble triggering receptor expressed on myeloid cells‐1 (sTREM‐1) in periodontitis. J Dent Res 2013;92(2):161‐5.

Appendix

Budget Project Budget Year 1 Year 2 Year 3

Project leader 102 000 103 000 104 000

Assistant 280 000 283 000 285 000

LKPG 198 000 200 000 202 000

Materials, Chemicals 60 000 40 000 40 000

Computers, Electronics 90 000 90 000 60 000

Travels 6 000 6 000 30 000

Others 25 000 29 000 29 000

Unexpected costs 25 000 25 000 30 000

3


4

Sum 786 000 776 000 780 000

Administration fees (42%) 330 000 326 000 328 000

Total 1 116 000 1 102 000 1 108 000

Tina Lindell, Katarina Eken, Erika Johansson TFTB34 Biosensor Technology

2013-02-24

Research Application

Applicant Linköping University Applied Physics IFM

Project leader Fredrik Winqvist Linköping University Applied Physics IFM

Project Title Development of a SPR sensor for detection of pathogenic bacteria in milk

Funds applied for: 3 279 000 SEK

Project summary Two billion tons of food, almost half of all the food produced in the world annually, ends up as waste every year. Unnecessarily strict use-by dates are believed to be one of the reasons behind food waste. The aim of this project is to find a better, more accurate, way of telling when the food has passed its use-by date and should be thrown away. This application focuses on reducing the waste of milk by creating a biosensor which could determine when the milk is inedible. The sensor is based on surface plasmon resonance (SPR) combined with immobilized lectin that binds to carbohydrates on pathogens. It should be placed on the milk carton so that light from the surroundings can reach the sensor through a polarization filter and the reflected light can be observed. When there are no bacteria on the sensor then there will not be any reflected light. When the reflection angle shifts due to bacteria binding to the surface, the incoming light is reflected and can be seen by the observer.


2013-02-24

1 Description of the project Two billion tons of food, almost half of all the food produced in the world annually, ends up as waste

every year1. This means that large amounts of land, energy, fertilizers and water also have been lost

due to the wastage. If we are to succeed in the challenge of sustainably meeting our future food

demand, we cannot continue this wastage.2 Further on, during food production, greenhouse gases

are created that affect the climate and contribute to eutrophication. In other words, the waste of

food is also very bad for the environment.3

Unnecessarily strict use-by dates are believed to be one of the reasons behind food waste 1. These

kinds of dates should be treated as guidelines or suggestions, not hard-and-fast rules. Use-by dates

are not completely useless though, they are simply not as useful as common sense. 4 The idea behind

this research application is to find a better, more accurate, way of telling when the food has passed

its use-by date and should be thrown away. The goal is to reduce the food waste and thereby create

conditions for a sustainable future. This application focuses on reducing the waste of milk by creating

a biosensor which could determine when the milk is inedible. The biosensor is supposed to be placed

on each and every milk package which, potentially, may result in a large profit.

Milk should be kept cold to stay drinkable as long as possible. It is recommended to store the milk in

a temperature of maximum +8 degrees and the best-before date is set on the basis of such storage.

The beverage has significantly longer lifetime if it is kept in +4 degrees or colder. A survey made by

some students at Sveriges Lantbruksuniversitet (SLU) showed that the best-before date is misleading

and that the milk lasts several days after the best-before date.5

2 Method The sensor is based on surface plasmon resonance (SPR) combined with immobilized lectin that binds

to carbohydrates on pathogens. In milk there are several kinds of bacteria that constitutes the

problem, hence the sensor must be sensitive to pathogenic bacteria in general and not to specific

bacteria.

The sensor should be placed on the milk carton so that light from the surroundings can reach the

sensor through a polarization filter and the reflected light can be observed. When there are no

bacteria on the sensor then there will not be any reflected light. When the reflection angle shifts due

to bacteria binding to the surface, the incoming light is reflected and can be seen by the observer.

This change indicates that the milk contains bacteria and therefore no longer is suitable for

consumption.

When designing the sensor, bacteria that are relevant have to be determined to ensure that the

bacteria are recognized by lectin. In unpasteurized milk there are four different bacteria that are of

interest. They are ehec, listeria, yersinia, campylobacter6. The specific bacteria in pasteurized milk

1 (Smithers, 2012)

2 ( Institution of mechanical engineers, 2013)

3 (Tomasson, 2012)

4 (Wolchover, 2011)

5 (Konsumentföreningen Stockholm, 2013)

6 (Livsmedelsverket, 2012)


2013-02-24

that spoils the milk have to be evaluated before sensors for this purpose can be designed so the

bacteria which constitute the problem are targeted.

3 Activity plan

Activity Time (from 1-36 months i.e 3 years)

Description

Project planning 1st month Preparation of project outline.

Evaluation of the different bacteria in unpasteurized and pasteurized milk

Months 2-4 Finding the bacteria useful for the project and evaluating their different properties.

Evaluation of lectin binding to bacteria

Months 2-4 Looking into the lectin-bacteria complex.

Lectin immobilization Months 2-4 Immobilizing the lectin on the SPR surface.

Evaluation of lectin immobilization Months 2-4 Evaluating if the lectins are correctly placed on the surface and if they are binding to the bacteria.

First progress report Month 5 Summary of progress so far

Setup of SPR Months 6-9 Finding a suitable wavelength for the incoming light and a reference for the refracted light

Testing the setup with lectin and bacteria

Months 6-9 Checking that the system works.

Evaluation of detection point Months 6-9 Deciding a suitable reflection angle with respect to the amount of bacteria on the surface.

Optimization of visual detection points

Months 6-9 Testing that the shift is large enough to give a good output.

Second progress report Month 10 Progress this far

Design of visual interface Months 11-13 Construction of a suitable and clear visual interface for the customers.

Constructing a simple control Months 14 and 15 Making it easy for the user to see if there is a problem with the sensor

Integration of the device on to the milk carton

Month 16-18 Practical placement of the sensor


2013-02-24

Third progress report Month 19 Progress so far

Market testing Months 20-23 A group of people will test the product for a few months and give feedback

Evaluation of the market research Months 24-27 Evaluating any problems that might have come up

Final optimization Month 28 Tweaking of the product

Final report Month 29 Final product

Commercialization Months 30-36 Marketing

4 Budget The budget below is calculated in SEK.

2014 2015 2016

Project leader 120 000 122 000 125 000

Assistant 300 000 301 000 302 000

LKP (52%) 218 000 220 000 222 000

Chemicals 50 000 20 000 20 000

Electronics 50 000 30 000 10 000

Travels 10 000 10 000 50 000

Marketing - - 100 000

Other 10 000 10 000 10 000

Sum 758 000 713 000 839 000


Total cost per year 1 076 000 1 013 000 1 191 000

Grand total applied for: 3 279 000 SEK


2013-02-24

References Institution of mechanical engineers, 2013. Global food waste not, want not. [Online]

Available at: http://www.imeche.org/knowledge/themes/environment/global-food

[Accessed 19 02 2013].

Konsumentföreningen Stockholm, 2013. Så länge håller mjölk. [Online]

Available at: http://slangintematen.se/hallbarhet-pa-mjolk/

[Accessed 19 02 2013].

Livsmedelsverket, 2012. Opastöriserad mjölk. [Online]

Available at: http://www.slv.se/sv/Fragor--svar/Fragor-och-svar/Drycker/Opastoriserad-mjolk/

[Accessed 24 02 2013].

Smithers, R., 2012. Almost half of the world's food thrown away, report finds. [Online]

Available at: http://www.guardian.co.uk/environment/2013/jan/10/half-world-food-waste

[Accessed 19 02 2013].

Tomasson, L., 2012. Vetenskap och allmänhet. [Online]

Available at: http://v-a.se/wordpress/wp-content/uploads/SLUTRAPPORT_bastfore2011.pdf

[Accessed 19 02 2013].

Wolchover, N., 2011. Do 'Use-By' Dates Cause Americans to Toss Food Too Soon?. [Online]

Available at: http://www.lifeslittlemysteries.com/1484-use-by-dates-americans-toss-food-too-

soon.html

[Accessed 19 02 2013].

TFTB34 Linköping University Malin Strömqvist 2012‐02‐19 Linnea Brusved Andersson

The ATP dependent bacterial controller BacCheck

A research application

Summary

A lot of healthy and perfectly edible food is wasted only because it has passed it`s ‘best before

date’. This food waste is an increasing problem that we sooner or later have to deal with and if

solved could save the society a fortune. The problem is based on the difficulty to determine

whether the food is healthy or not with just a simple useful method.

The aim of this project is to significantly reduce the food waste by introducing a new way of food

labeling at the market by using an ATP dependent biosensor. The new biosensor called BacCheck

is based on the luciferase catalyzed reaction (conversion of luciferin) which results in light

emission in the presence of bacteria. Thus, BacCheck will help you to determine if the food is

contaminated with bacteria or not with just a quick look at the label.

The development of BacCheck will be pursued at Linköping University in contact with research

groups and the project will span a period of three years.

Funds applied for: 3 123 716 SEK


Background Today many tons of healthy and perfectly edible food are wasted all over the world only because it has passed its ‘best before’ date. This waste of food is escalating and costs a lot of money for the society to no avail, something that should be in everyone's interest to change. Almost all packaged food requires a durability mark such as ‘best before’ date or sometimes ‘use by’ date mark to make sure the customer gets satisfying information about the food quality. As a seller it is your responsibility to ensure that the food stays fresh as long as indicated and that the labeling gets as exact as possible. The today labeling of food seems to be a bit confusing since a lot of people interpret the ‘best before’ date as when the date is passed the food is stale and inedible. The ‘best before’ date indicates the period for which food can reasonably be expected to retain its optimal condition1 and should not be interpreted as ‘rot after’ this date. The quality of a product depends on many factors such as manufacturing, packaging and storage and it often will remain fresh and edible for longer if kept as directed in intact package2.

The development of a new food label will eliminate the problem of the “freshness estimation”

of a product and simplify a lot for the customers and sellers as well. The new BacCheck

biosensor will help you to determine if the food is contaminated or not since it is sensitive for

bacteria.

To get the ability to detect bacteria we would like to take advantage of the natural phenomena

called bioluminescence. Bioluminescence is the creation and emission of light due to a chemical

reaction in a living organism. The light occurs when chemical energy is converted to light

energy, an energy‐dependent reaction involving luciferin and the catalytic enzyme luciferase

according to the following reaction3.

ATP + luciferin + O2 AMP + Oxyluciferin + PPi +CO2 + light

It is the enzyme luciferase that, in presence of the energy molecule adenosine triphosphate

(ATP), converts luciferin to oxyluciferin. Oxyluciferin is a highly unstable, singlet‐excited

compound which emits light upon relaxation to its ground state4. This reaction enables

1 http://www.defra.gov.uk/food‐farm/food/labelling/ 2012‐02‐18 2 http://www.slv.se/sv/grupp1/Markning‐av‐mat/Sa‐marks‐maten/Hur‐lange‐haller‐varan/ 2012‐02‐18 3 http://www.sciencedaily.com/articles/b/bioluminescence.htm 2012‐02‐18 4 http://www.ebi.ac.uk/interpro/potm/2006_6/Page2.htm 2012‐02‐18

http://www.defra.gov.uk/food-farm/food/labelling/

http://www.slv.se/sv/grupp1/Markning-av-mat/Sa-marks-maten/Hur-lange-haller-varan/

http://www.sciencedaily.com/articles/b/bioluminescence.htm

http://www.ebi.ac.uk/interpro/potm/2006_6/Page2.htm


detection of bacteria and would therefore function as a good contamination indicator in the

new biosensor BacCheck.


The aim of this project is to expand the food labeling by developing the BacCheck sensor to get

the customer a more satisfied and straight away information about the product and a direct

indication of its freshness. Of course this will facilitate for the sellers at the market as well and

the reduced food waste will support the whole society. By developing BacCheck we could make

a huge effort and drastically reduce the food waste.

Design of BacCheck

As ATP is necessary for almost all reactions in living organisms this lightening reaction can

function as a bacterial indicator5 and tell us if the food contains bacteria or not. The BacCheck

will consist of an ATP permeable dialysis membrane which gathers luciferin and luciferase. ATP

will be the only compound that transfers through the membrane and luciferase will catalyze the

conversion of luciferin to oxyluciferin only when bacteria (ATP) are present in the food.

Oxyluciferin will send out light as it goes back to the ground state and this light will serve as a

marker for bacterial damage. This means that the label will light up when the food has been

contaminated of bacteria and is inedible.

The dialysis membrane will prevent luciferin and luciferase to come in contact with the food but

permits ATP to pass through. When ATP is generated by living organisms such as bacteria it will

pass through the membrane and enables the catalysis which leads to light emission. Since ATP is

a approximate small molecule (Mw 507,18 u) due to most proteins, including luciferin and

luciferase, it is possible to limit the passage only to ATP by using an appropriate molecular

weight cut off. As a suggestion a polymer based dialysis membrane with a molecular weight cut

5 http://hyperphysics.phy‐astr.gsu.edu/hbase/biology/atp.html 2012‐02‐18

http://hyperphysics.phy-astr.gsu.edu/hbase/biology/atp.html


off around 510 u should do the work.

Budget

Proposed budget

Year 1 Year 2 Year 3 Project leader (25%) 120 000 130 000 130 000

Assistant (100%) 280 000 290 000 290 000

LKP (52%) 208 000 218 400 218 400

Materials 65 000 80 000 150 000

Electronics 0 0 0

Travels 0 5 000 15 000

Sum 673 000 723 400 803 400


Total 955 660 1 027 228 1 140 828

The total cost after three years for this biosensor will be approximately 3 123 716 SEK.

Activity plan

For the project we will need a project leader (25%) and one assistant (100%). During the first

year we will create the dialysis membrane at Linköpings University and therefore no travels will

be needed. We carry on the second year with optimizing the membrane to create the perfect

one in collaboration with specialists around Sweden. The last year we will be focusing on mass

production and commercialization.


References

www.defra.gov.uk/food‐farm/food/labelling/ 2012‐02‐18 www.slv.se/sv/grupp1/Markning‐av‐mat/Sa‐marks‐maten/Hur‐lange‐haller‐varan/ 2012‐02‐18 www.sciencedaily.com/articles/b/bioluminescence.htm 2012‐02‐18 www.ebi.ac.uk/interpro/potm/2006_6/Page2.htm 2012‐02‐18 www.hyperphysics.phy‐astr.gsu.edu/hbase/biology/atp.html 2012‐02‐18

http://www.defra.gov.uk/food-farm/food/labelling/

http://www.slv.se/sv/grupp1/Markning-av-mat/Sa-marks-maten/Hur-lange-haller-varan/

http://www.sciencedaily.com/articles/b/bioluminescence.htm%202012-02-18

http://www.ebi.ac.uk/interpro/potm/2006_6/Page2.htm%202012-02-18

http://hyperphysics.phy-astr.gsu.edu/hbase/biology/atp.html

2013-02-25

Linköpings University, Applied Physics (IFM)

Magnus Bernhardsson, Thomas Österberg

Development of a portable, lightweight QCM biosensor for

rapid detection of allergens in foods

Funds applied for (SEK): year 2014 year 2015

2 710 000 1 370 000 1 340 000

Summary Allergy, both the trivial kind and severe forms that may have potentially lethal consequences,

is an increasing problem in today’s society.

The goal with this project is that people with allergies will have a detector in their possession

which enables detection of dangerous allergens potentially present in food, saving them from

both uncomfortable and even dangerous situations.

Detection of the allergen of interest is carried out by utilizing QCM technology. A disposable

stick is inserted into the sample food and then analyzed for allergen response using a docking

station compatible with a cellular phone for evaluation.

The system focuses on fast detection, no more than a few minutes, and user friendliness.

2013-02-25

Introduction

In developed countries, welfare diseases becomes a greater problem for each year. Our

lifestyles nowadays brings with it not only obvious problems such as obesity, but also more

subtle kinds like that of allergy. Allergy, both at serious and trivial levels, is not an exception

and an increasing number of people throughout the western world are only a single

misfortunate meal from getting hospitalized or worse, each and every day. (Burks 2008, Kim,

Drake-Lee 2003)

Purpose

The purpose with this project is to develop a simple, easy to use and portable detector of

allergens, e.g. from nuts, in foods for people with severe allergies. This sensor might come in

handy is when you aren't really sure if your food contains allergens or not, for instance when

you visit a restaurant.

The idea with the sensor is that you insert the sensor into your food and get a fast reading

whether the food contains allergens or not. This will help to ease the worry of being exposed

to unnecessary and even dangerous circumstances.

In this study quartz crystal microbalance (QCM) technology will be utilized, used in the

development of the sensor and antibodies against antigens that may be found in nuts will be

used in the detection process.

For increased marketing value, simplicity and user friendliness the design of the system

revolves around the ability to integrate it with the user’s cellular phone.

Design

Overview

A disposable teststrip fitted with a ‘needle’ with an integrated QCM device for detection (see

Figure 1) is coupled with a pocket sized docking station with the necessary electronics,

software and display feature. The idea is that the docking station is connected to the user’s

mobile phone. A label free system like QCM is essential for ensuring the simplicity of the

design, user friendliness and easy interpretation.

Principle for detection

Before the needle is inserted into the sample food the protecting outer membrane or film

keeping the sample pad hydrated and keeping it from getting contaminated is removed.

The inner membrane keeps the QCM with the antibodies in a liquid phase which allows the

antigens upon contact with the sample to diffuse towards it. When the antigens reach the

antibodies a sandwich complex is formed which increases the mass associated with the QCM

which induces a change in its oscillation frequency. This change will be detected and

converted to a electric signal sent on to the connected docking station that will give a reading

of the present allergen concentration.

2013-02-25

Since the analyte of interest is a protein, which has a relatively large mass, the mass change

induced by its binding should be sufficient to yield a distinguishable signal even for low

concentrations of antigen. A boarderline value for qualitative evaluation should be around 10

ppm (Poms et al. 2004). Should the detection level however prove to be insufficient, the

system could be modified using gold nanoparticles which increases the surface area and

allows for binding of more antibodies (Chu, Lin et al. 2012).

Figure 1. Schematic over the teststrip. The QCM platform is integrated within the needle and is covered by two

membrane layers; one outer, protective layer keeping the sample pad hydrated and protected from contamination and one inner membrane keeping the QCM with antibodies in a liquid phase.

References

BURKS, A.W., 2008. Peanut allergy. The Lancet, 371(9623), pp. 1538-1546.

CHU, P.-., LIN, C.-., CHEN, W.-., CHEN, C.-. and WEN, H.-., 2012. Detection of gliadin in foods using a quartz crystal microbalance biosensor that incorporates gold nanoparticles. Journal of Agricultural and Food Chemistry, 60(26), pp. 6483-6492.

KIM, D.S. and DRAKE-LEE, A.B., 2003. Infection, allergy and the hygiene hypothesis: Historical perspective. Journal of Laryngology and Otology, 117(12), pp. 946-950.

POMS, R.E., KLEIN, C.L. and ANKLAM, E., 2004. Methods for allergen analysis in

food: A review. Food additives and contaminants, 21(1), pp. 1-31.

2013-02-25

Appendix

Proposed budget

Year 1 Year 2

Project leader (25 %) 96 000,00 97 000,00

Assistant (100 %) 264 000,00 266 000,00

LKP (52 %) 187 200,00 188 760,00

Materials 50 000,00 70 000,00

Electronics 270 000,00 230 000,00

Travels 10 000,00 10 000,00

Sum 877 200,00 861 760,00

Administrative fees (42%) 490 386,21 479 205,52

Total 1 367 586,21 1 340 965,52

Grand total: 2 508 551,73

Linköpings Tekniska Högskola

MIP Sensor for Caffeine Concentration Measurement

Henrik Kleinhans, Mark le Moine, Kanyarat Srichada

Funds applied for: 3 300 000 SEK 2013-02-21

Summary Caffeine is the most consumed psychoactive drug in the world. To regulate the caffeine in-take it would be desirable to have a device that can determine the caffeine content. The aim of this project is therefore to develop a sensor that fulfills this desire. The project can be divided into two parts; constructing the sensor in question respectively designing a holder for the sensor.

Appendix

Background and Methods Caffeine is a psycho active drug, stimulative of the central neural system. Commonly known, it

affects the wakefulness and makes you more alert. It is an antagonist to adenosine, a

neurotransmitter substance which promotes sleepiness. Shortly described, by blocking the substance

one can prevent symptoms of tiredness, thus affecting the concentration level. [1]

The structure of the caffeine molecule is an alkoloid consisting of two cyclic rings with several

nitrogen and oxygen where hydrogen bonding can occur. The structure can be seen below compared

to adenosine. Similarities can be observed; the two cyclic regions are conspicuous and could be of

importance when the both substances bind to the receptor.

As we can see in the picture above, the adenosine receptor recognizes many of the molecules

specific sites. [2] Various binding modes are possible between transmembrane protein and the

substance. When constructing the molecular imprinted polymer (MIP) the information above could

be of interest.

Molecularly imprinted polymers, MIPs The idea behind a MIP is more or less to create a casting on a molecular level which sterically only

fits and binds the analyte of interest. The procedure of casting is as follows:

1. The analyte is surrounded by different molecules with which it has intermolecular

interactions with, e.g. hydrogen bonds, dipole-dipole or dipole-ionic interactions which fits

the analyte sterically.

2. The surrounding molecules have a monomer handle, which are used together with other

monomers (crosslinkers) and an initiator to make a polymer network that contains the

analyte.

3. After polymerization, the analyte is washed away from the polymer, leaving an imprint that

has an affinity for the analyte since the functional groups that had intermolecular

interactions with the analyte is still present. In the polymer, the relative positions of the

molecules that interact with the analyte are kept constant.

Figur 1: Construction of the MIP

When designing the MIP, interactions between monomers and caffeine could be of the same nature

as in the adenosine receptor (e.g. pi-pi interactions, hydrogen bonds and hydrophobic interactions)

as mentioned above, although it is not necessary to use amino acids as there exist many organic

compounds that have similar chemical properties. Using the natural receptor which is desired to

simulate as inspiration to create a man-made receptor for the same simulant has a certain

intellectual charm to it. [3]

Design of the Sensor and Practical Use The sensor would contain a column with MIPs specific to the caffeine molecule. This column is

connected to a PQC (piezoelectric quartz crystal) that can detect the difference in mass when

caffeine binds to the column and the concentration can be calculated. [4] The sensor can be placed

in a cartridge, and a small amount of beverage can be applied on it. The cartridge can then be placed

in a special holder. A simple display could be suitable to show the caffeine level and an estimated

time of degradation of the substance in the body.

Although the sensor is independent and can work on its own, manufacturers could implement the

cartridge as a standard in newer coffee dispenser models. This is just a thought if the sensor would

become successful among customers. In this case all you would need is a compatible coffeemaker

in your home with this simple cartridge.

Figure 2: PCB (Printed Circuit Board) with a PQC (Piezoelectric Quartz Crystal) chip and a processor unit, placed inside a cartridge. The cartridge contains a MIP column. The cartridge can be inserted in a holder.

Activity plan What the project group should work with:

1. Develop a MIP with high affinity and selectivity for caffeine.

Find functional monomers that match caffeine. Find which cross linker that gives the most

available cavities for new caffeine molecules to bind to.

Find out which method that removes the template (caffeine) best.

Make a column with the MIP, through which fluid can pass.

2. Develop a rinsing fluid for the MIP.

The chemical properties of the fluid should not be harmful for the end-user.

The rinsing fluid should be stable for at least 1 month.

3. Develop a PQC for the cartridge.

Either develop a PQC from scratch or test an existing model that is compatible with the MIP.

Find how to couple the column to the PQC.

4. Design the MIP cartridge and the analyzer.

This could either be done by the project group or by an external company.

Goal and Advantages with the Project The main goal is to determine the level of caffeine in caffeine containing beverages. In other words

we want to develop a method to measure the concentration of caffeine molecules, for example in

hot coffee, tea, energy drinks, mate etc. The method and sensor should be independent of the

beverage.

The sensor could be used to determine whether a beverage is strong or not by detecting the caffeine

concentration. Whereas strong coffee would contain larger amounts of the stimulant drug than

weaker coffee. By measuring the strength, people who are uncertain if they should take another cup

in the evening and still be able to sleep at bedtime, would easier be able to decide if they should

take that extra cup or not. It would even be possible to decide a certain volume of coffee, tea etc.

that can be consumed.

In case people would want to quit drinking coffee, the sensor could be useful to minimize the

caffeine intake over time. Similar to when smokers use nicotine patches or gum, by controlling the

intake.

Advantedges:

Gain knowledge to make the production of MIP more efficient.

The final prototype or blue-print can be sold to companies willing to mass-produce the

product.

If the product becomes very popular among costumers, companies may implement the

cartridge as a standard in, for example, coffee machines. Or even make it possible to connect

the cartridge holder with the machine.

By using the sensor, you will be able to measure the concentration of caffeine.

As an individual you will be able to regulate the caffeine intake.

Producers of caffeine containing beverages can utilize the sensor to measure if the caffeine

levels are sufficient.

A lot of work is done in this area and this might facilitate the project.

Budget

Budget Year 1 Year 2 Year 3

Project leader (25%) 95000 95000 95000

Assistant (100%) 280 000 280 000 280 000

LKP (52%) 195000 195000 195000

Material 80000 140000 80000

Equipment 30000 30000 30000

Travels 10000 10000 15000

Sum 690000 750000 695000

Administrative fees (42%)

289800 315000 291900

Total 979800 1065000 986900 3031700

References

[1] http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=2519

[2] Liu, Yuli, et.al 2011; 2011. Computational study of the binding modes of caffeine to the adenosine A2A receptor. The Journal of Physical Chemistry B 115, (47) (10/05; 2013): 13880-90.

[3] Suryanarayanan, Vembu, Cheng-Tar Wu, och Kuo-Chuan Ho. 2010. Molecularly imprinted electr chemical sensors. Electroanalysis 22, (16): 1795-811.

[4] Mohammed Zougagh , Angel Ríos, Miguel Valcárcel. 2005. Automatic selective determination of caf-feine in coffee and tea samples. Analytica Chimica Acta 539, 117–124

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=2519

2013‐02‐18 Tobias Benselfelt Torbjörn Sveds

Research application

Applicant Molecular Imprinting program (MIP) Applied Physics (IFM) Linköping University

Project title Analysis and purification targeting pharmaceuticals in waste water using Molecularly Imprinted Polymers and biosensor technology

Applied amount 8 400 000 SEK

Summary Pharmaceutical residues in waste water are a severe problem for many eco systems and do often end up in our drinking water. With this research we will develop a brand new technology for small molecule water purification. The goal is to develop a system which can be integrated in the current water purification technology and can be developed for many different kind of biomolecules. The system will examine waste water to detect the compound levels, process the information automatic and use it for parallel purification. It will process the most prominent pharmaceuticals at same time for completely pure water. Biosensor technology like SPR or QCM will be used for quantitative analysis of the water and the biomolecules it contains, and will be linked to a purification system for controlled reagent release. Molecularly Imprinted Polymers (MIPs) will be used for both sensing and elimination of compound from water using the high selectivity and affinity properties.


Appendix 1: Project description.

Background

The consumption of pharmaceuticals increase as the population grows and new products are

introduced on the market. This also results in an increasing amount of bioactive components in

waste water through urine and feces. These components have more or less biological effects on

different eco systems and will circulate back into our drinking water. Stockholm vatten recently

published a report in which they identified 78 unique compounds flowing through their purification

network [1]. The current purification techniques lack the ability to identify and remove these

compounds from the waste water and research to increase the effectiveness against pharmaceuticals

is high priority. As the population mean age increase and people tends to live longer, even more

pharmaceuticals are consumed and released into our eco systems and drinking water.

Molecularly imprinted polymers (MIPs) creation is a technique using a target molecule template (in

this application a drug) and cast a solid polymeric material around it. The template is first pre‐

assembled with functional monomers and their specific molecular interactions with the template.

After that the polymerization is initiated creating a cavity for the template. The monomers are

localized in the same way as they were connected to the template with molecular interactions

leading to a high affinity and very specific binding site for the template molecule. MIPs can be used

as artificial antibodies or have properties that mimic an enzyme or receptor. [2] These properties can

also be used as a way of separation and purification when binding small molecules to a larger

structure.

Surface Plasmon Resonance (SPR) spectroscopy is based on the fact that a thin gold film absorbs light

and form a surface plasmon at a certain angle of incidence depending on the mass of the molecules

attached to it [4]. The technique has successfully been combined with molecularly imprinted films for

precise detection of biomolecules [3]. Advantages with this technique include fast measurements

and the fact that it’s possible to implement several different detection films on the same small chip.

This makes it possible to scan for many different pharmaceuticals and get continuously data at real

time. One possible disadvantage though, would be that, as the measurements depend on mass, small

molecules might be hard to detect and get quantitative data on.

Quartz crystal microbalance (QCM) is an extremely sensitive microbalance based on piezoelectric

properties of a quartz crystal. Small changes in mass on the surface of the crystal will change the

resonance frequency of the quartz crystal which can be measured [4].

Purpose and Goal

The purpose of this research is to develop a combined sensor and purification system that targets the

most problematic and prominent pharmaceuticals in waste water. The system will contain a

biosensor for parallel identification of multiple pharmaceutical levels using biosensor technology and

MIPs as ligand. The sensor will be in direct contact to the purification system that uses MIPs to bind

compounds in larger structures for easy sedimentation or filtration. The goal is a functional system

which is easy to integrate in the current purification technology.

2013‐02‐18 Tobias Benselfelt Torbjörn Sveds Method

Research regarding the most prominent pharmaceuticals and their effect on the environment will be

the first phase of the project. Due to the huge variety of compounds it is necessary to select the most

important molecules for further development of the system. MIPs for the selected compounds must

be designed and evaluated. Different affinity for sensing MIPs and purification MIPs will probably be

constructed for easy continuous processing.

The biosensor technology used must be evaluated and chosen. Questions to answer are how low

concentrations can be detected and if some preparation of the waste water is needed. In which

phase of the waste water purification should the system be incorporated, before or after current

purification processes?

The preferable sensor technology must be developed for simultaneous sensing of many

pharmaceuticals with multiple quartz crystals or different “fields” on a large SPR surface. The design

of the flow channels relative to the sensor surfaces should be developed for as easy as possible

detection.

Filtration/sedimentation/column separation of the MIPs must be tested and evaluated. Which

method is fastest and best? Is there any MIPs left after purification? Which method is best for

recycling of the MIPs in order to lower operating cost?

Some kind of automatic control of MIP release for lower operating cost when the compound levels

are low must be created. The software that will generate the MIP release into the purification

process must be programmed and tested.

Advantages with the project

The research will provide a new technique for disposal of pharmaceuticals from waste water which is

a big environmental problem. It will also provide a new method for separation and purification which

is easy to develop in the future and can be used in various areas of industry and for future

contaminations.

MIP technology is a very simple method to create reagents with high selectivity and affinity for

almost any desired molecule.

Future

The global water asset is the single most important resource on our planet. The water scarce and

delicate resource and is increasingly contaminated by industrial waste, pharmaceuticals etc. This

research and product can be widely used in the future, not only for pharmaceuticals, but also other

contaminants in water.


Appendix 2

Activity plan

Activity Month people

Project planning 0‐2 2

Literature research 3‐6 2

MIP construction 7‐16 2

Adapt for practical use 0‐24 1

Sensor design 13‐36 2

Electronic design 25‐48 1

Recycling 49‐60 1

Continous measurement development 25‐48 1

Software design 37‐60 1

Practical testing and calibration 49‐60 4

Final report 55‐60 2


Appendix 3

Budget

Crew 2014 2015 2016 2017 2018

Project leader (25%) 96000 98000 100000 102000 104000

Biosensor engineer (100%) 280000 286000 292000 298000 304000

Analytical chemist (100%) 266000

Electronic engineer (100%) 304000 312000

Waste water expert (25%) 80000 82000

Programmer (50%) 145000 146000

LKP (52%) 375440 242320 361920 445640 288080

Materials and other

Chemicals 100000 100000 50000

Electronics 150000 150000

Equipment 50000 50000 50000 50000 50000

Travels 10000 10000 10000 10000 10000

Other 10000 10000 10000 10000 10000

Sum 1267440 878320 1327920 1522640 912080

Administrative fees (42%) 532325 368894 557726 639509 383074

Total/year 1799765 1247214 1885646 2162149 1295154

Total 8389928 SEK


References

1. Cajsa Wahlberg,Berndt Björlenius, Nicklas Paxéus Läkemedelsrester i Stockholms vattenmiljö: Förekomst, förebyggande åtgärder och rening av avloppsvatten, Stockholm Vatten AB, 2010. Available at: http://www.stockholmvatten.se/Aktuellt/Projekt/Lakemedel‐i‐avloppsvatten/ used: 2013‐02‐19.

2. YE, L. and HAUPT, K., 2004. Molecularly imprinted polymers as antibody and receptor mimics for assays, sensors and drug discovery. Analytical and Bioanalytical Chemistry, 378(8), pp. 1887‐1897.

3. YOSHIKAWA, M., GUIVER, M.D. and ROBERTSON, G.P., 2008. Surface plasmon resonance studies on molecularly imprinted films. Journal of Applied Polymer Science, 110(5), pp. 2826‐2832.

4. Advanced Biosensor Technology, Agora for life science technologies, Linköpings University, course: TFTB34

Research application: Alexander Reissig & Pelle Lundberg

Development of enzyme for specific reaction with THC and Photon detection

Background It is well known that alcohol heavily limits driving ability, and that it is the source of many motor

vehicle accidents. However, the negative effect on driving from other drugs, such as cannabis, has

more recently been established and it is now clear that driving under the influence of cannabis or

other psychoactive drugs is a problem that needs to be addressed in a similar way to alcohol.1

Related to this is the fact that the political climate on cannabis is slowly shifting to a broader

acceptance of use and the increased possibility of legalization. This is for example seen in the United

States, where recent legalization of drug abused was introduced and passed in two states.

With these two factors in mind, the need for an easy to use, reliable, efficient and non-invasive road-

side device for cannabis testing of drivers is evident.

Recent large scale studies of oral fluid tests in traffic situations conclude that all though there are

promising applications, the analysis time and error rates could be lowered, especially for smaller

devices.2

The method used today for THC detection is blood samples, hair samples and saliva. Old tests that

used saliva was Duquenois–Levine reagent but turned out to give false positives by cross reactions

between different vegetable oils and other substances, which made them extremely inaccurate and

could not be used. This makes the laboratory or large stationary equipment still the main source for

this kind of analyses.


Method To be able to make an easy to use biosensor that is adapted for field use the methods used must be

as non-complex as possible. This to minimize errors during usage and to make it as robust as possible

with a long life span. Our solution is based on using old and developed techniques that is extremely

reliable.

The process begins with obtaining a saliva/Oral fluid sample from the person you want to test for

THC. The sample is placed in the apparatus that ideally is handheld. The potential THC presence in

the sample will react with a special enzyme which will react with the THC to release a phosphate

(ppi). From this stage old and well tested technics will be applied to create a detectable signal.

The ppi will be used by the signal generating part of the technic called pyrosequencing.

Adenosine 5´ phosphosulfate (APS) reacts with ppi and dephosphorylates trough sulfurylase to make

an ATP that in turn drives the next reaction that generates a photon.3

The photon that gets released from the reaction will then be quantified by a photomultiplier which

gives a measurement that represents the amount of THC molecules in the sample since we get one

photon for every THC molecule in the collected sample.

By being able to get this sort of specific measurements it will be easy to adapt to the wanted legal

limits for the amount present. Since we can get exact measurements that can be both high and very

low it can be adapted to be very specific. 3

Research

The area that we search funds for is the research around finding and develop the needed enzyme for

the initial reaction to release a ppi needed for signal detection and for development of an early

prototype. This will mean research of the cannabis plant for identification of already present

enzymes that can be used for reaction with THC or its derivats.

For the prototyping part there is need for schematics over the apparatus and needed materials such

as photomultipliers for light detection.

http://en.wikipedia.org/wiki/Adenosine_5%C2%B4_phosphosulfate


Budget

Column1 year 1 year 2 year 3

Project leader (25%) 96 000 100 000 102 000

Assistant (100%) 264 000 268 000 230 000

LKP (52%) 187 200 191 360 172 640

Chemicals 60 000 60 000 30 000

Materials 10 000 10 000 50 000

Electronics computers 60 000 20 000 20 000

Travels 10 000 10 000 20 000

Other

Sum 687 200 659 360 624 640

Administrative fees (42%)

288 624 276 931 262 349

Total 975 824 936 291 886 989

Total 2 799 104 over three years

References

1. Movig KLL, Mathijssen MPM, Nagel PHA, et al. Psychoactive substance use and the risk of motor

vehicle accidents. Accident Analysis and Prevention. 2004;36(4):631-636.

2. Evaluation of oral fluid screening devices by TISPOL to harmonise european police requirements

(ESTHER). . 2006.

3. T. Strachan AR. Human molecular genetics. 4th ed. New York: Wiley-Liss; 2011.

Linköpings Tekniska Högskola Sara Johansson sarjo870 TFTB34 Group 10 Alexander Lundberg alelu559

Gustaf Carlsson gusca415

Development of mould detecting biosensor for commercial use

Picture 1. Mould on walls in an apartment. (1) Funds applied for: 3 000 000 SEK Summary Mould is a known health hazard that can cause allergies and cancer. When mould spread it can do so by leaving spores that travel through air. The research idea is to catch spores in a cryo trap and with use of antibodies in a quartz crystal microbalance detect the spores. This mould detector can be used for early mould detection in restaurants and large-‐scale catering establishments in order to suppress mould before it becomes a problem.

2

Development of mould detecting biosensor for commercial use Background and problem description

Mould is a microscopic fungi that grows through a threadlike system, called mycelium. The mycelium can grow beneath the surface without a sign of mould on the surface. When the mould is “mature” it pops out as spots on the surface that can be seen with the bare eye. The spores it produces cause the colour of the mould. When the spores are dry they spread by the air and if they end up at good conditions they start colonising other surfaces and the process repeats itself. Good conditions for fungi in general are warm and moist surroundings. Some moulds produce mycotoxins and aflatoxin, which are poisonous and cause health issues. Aflatoxin is known to cause cancer. Moulds are also known for causing asthma and allergies. Mould is found on food, but also on the walls and ceilings of buildings. Common mould species in food are penicillium, aspergillus and cladosporium.(2) If mould growth is undetected it can result in severe health hazards, as described above. To prevent this from happening we propose a novel detector for moulds through air using biosensor technology.

Our idea

The idea is to detect mould particles, in this case spores. This could be done particularly in restaurants and large-‐scale catering establishments. It could also be used in homes, especially those of people oversensitive to moulds. Cryo trapping will be used to improve the detection of particles. The spores are condensed on a cold metal bar. The cryo trap will be covered with silicon inside to prevent water particles from adhering. Particles in the trap will be released through a rapid pulse of heat, thus releasing the spores at the same time in order to achieve a higher concentration than in the air.(3) This should allow earlier detection, while the levels of spores are still low. The spores are then collected at a biosensor. Spores may have to be fragmented to be able to bind to the antibodies. One possible method for performing this task is using UV light. The biosensor that we propose for detection of spores will be a quartz crystal microbalance. Antibodies specific for mould spores are to be attached to the quartz crystal. When the spores bind, the mass increases and the resonance frequency changes, thus the difference in mass can be calculated. (4) Further research is needed to determine if antibodies can be specific to a certain genus’ spores and to find antibodies for each type of mould. If genus specific antibodies are found, multiple quartz crystals will be necessary. Each quartz crystal would be covered with antibodies for one genus of mould, thus allowing the sensor to differentiate between different moulds.

3

Research area

Research will be done to find out if the spores can bind to antibodies at their natural size. If they cannot, a method for fractionating the spores in suitable pieces for analysis in the biosensor is needed. Using UV light seems promising. Appropriate antibodies binding to specific genus’ spores antigen need to be investigated. A prototype of the sensor will be designed.

Future prospects

The product can after small modifications be used as a detector for house moulds. The system could have large commercial value, because mould is a major health hazard. Earlier detection of mould growth would increase the health and thus the standard of living for countless of people. High profits can be made if authorities adopt the our new method and legislate so restaurants have to adopt and use the system.

Budget

Budget (numbers in SEK) Year 1 Year 2

Project leader (25%) 120 000 125 000

Microbiologist (50%) 150 000 0

Chemist (100%) 312 000 318 000

LKP 31,42% (5) 182 864 139 191

Materials 50 000 70 000

Antibodies 90 000 50 000

Chemicals 10 000 8 000

Computer and electronics 30 000 20 000

Travels 15 000 10 000

Sum 959 864 740 191

Administrative fees 42% 695 074 536 000

Total 1 654 938 1 276 191

Total project cost is 2 931 129 SEK. Hence the amount applied for is 3 000 000 SEK.

4

Description of budget posts Project leader: 25% of the project leader’s time will be allocated to this project. Microbiologist: The tasks of the microbiologist include research connected to mould spores. Approximately 50% of the time is allocated to the project. During the second year the chemist is assumed to have learned how to conduct this work as well. Chemist: Assistant working full time with the project, taking care of daily tasks and administration. LKP: General payroll tax Computer and electronics: Fee paid to the university for providing equipment. Travels: Trips to different seminars and conventions to promote the concept. Administrative fees: Fees paid to the university

References

1. Pure property care, (latest update 2013-‐02-‐24) [Electronic picture] Available at: http://www.purepropertycare.co.uk/pictures/content/images/mould1.jpg 2. United states department of agriculture: Foods safety and inspections survice, (latest update 2013-‐02-‐22) [Electronic]. Available at: http://www.fsis.usda.gov/FactSheets/Molds_On_Food/#1 3. ATAS GL international, (latest update 2013-‐02-‐22) [Electronic]. Available at: http://www.atasgl.com/html/cryotrap.html 4. Marx KA (2003), Quartz Crystal Microbalance: A Useful Tool for Studying Thin Polymer Films and Complex Biomolecular Systems at the Solution−Surface Interface, Biomacromolecules, 4 (5), 1099–1120 5. Skatteverket (Latest update 2013-‐02-‐22) [Electronic] Available at: http://www.skatteverket.se/foretagorganisationer/forarbetsgivare/socialavgifter/arbetsgivaravgifter.4.233f91f71260075abe8800020817.html

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