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Introduction to Laboratory

exercises for the TFTB34

course

2015

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Outline

• Safety in the laboratory

• Electrochemical exercise

– Introduction to electrochemical techniques and to electrochemical

device and electrochemical cell (potentiostat).

• Biacore exercise

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• Always ware provided protection equipment.

– Laboratory coat.

– Safety google.

– Gloves.

– Contact lens are not allowed in the laboratory.

• Do not drink or eat in the laboratory.– This can only be done outside the laboratory: before leaving the laboratory remove

all protection equipment and wash your hands.

• In case of accident use the appropriate emergency equipment.– Safety shower

– Eye washer

– First aid kit

– INFORM IMMEDIATLY THE DEMONSTRATORS AND OTHER STUDENTS

– CONTACT EMERGENCY SERVICE IF NEEDED

Safety in the Laboratory

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Safety in the laboratory

First aid kitEyes washer

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• Avoid any unsafe behaviour.

– Playing in laboratory is not allowed.

– Performing experiments not specified in the exercise guideline or not authorised by the demonstrators is not allowed.

– Always follow the instruction of the demonstrators.

• Always follow instructions from demonstrators or specified in the exercise guidelines

• Read Risk assessment before entering in the laboratory

IN CASE OF DOUBTS DO NOT HESITATE TO ASK TO THE DEMONSTRATORS.

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In case of anaccident:

1) Call 112

2) Describe what happened, state who you

are…..(see instruction)

3) Guide emergency to you:

• Fysikhuset

• Entrance 57

• First Floor

• Corridor Heisenberg

• Room Number H302/H304

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Exercise 1

• Development of 2 electrochemical enzyme biosensors for

the detection of glucose.

– Sensing approach: Enzymatic biosensor

• Enzyme: Glucose oxidase

– D-glucose + H2O + O2 gluconic acid + H2O2

• Catalitic enzyme biosensor: Use a catalyst Prussian blue to facilitates the

electrochemical detection of an electroactive molecule involved in the

enzymatic reaction.

• Mediated enzyme biosensor: Use an organic molecule (Ferrocene) to

improve electron transfer between enzyme and working electrode.

– Transduction: Electrochemical transduction via amperometric

measurements.

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Hardware

Electrochemical cellMeasurement device (potentiostat)

WARKING ELECTRODE: Is the electrode at which the electrochemical reaction takes place.

REFERENCE ELECTRODE: Is an electrode having a well defined and stable equilibrium

potential. It is used to set the potential of the working electrode.

COUNTER ELECTRODE: It is used in the three electrodes cell setup to allow current flowing and

electroneutrality in the electrolyte solution.

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• Cyclic Voltammetry:

– Cyclic voltammetry consist of measuring the current generated as the

working electrode as a function of the potential applied to it. Potential is

changed between a starting and a final value with a constant speed (scan

rate). The potential scan is performed in the forward and backward

(cycle).

Electrochemical Techniques

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• Application of cyclic Voltammetry:

– Cleaning/activation of surfaces: consecutive oxidation/reduction

processes can allow the removal of impurities from sensor surface

improving in this way the available active area and its conductivity.

– Studying the electrochemical properties of

molecules/surfaces/reactions; By Cyclic voltammetry it is possible to

define if a reaction take place or to identify the most suitable potential for

amperometric detection.

– Functionalise sensor surfaces: Electrodeposition of polymers, grafting

molecules or catalysts is an easy way to provide new functionalities to the

surface.

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Electrochemical Techniques

• Amperometry (measurement):

– This measures the current, as a function of the time, dues to an

electrochemical reaction taking place at the working electrode, and

driven by the application of a constant potential.

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Short description of the glucosensors

preparation• Electrode activation:

– Working electrode is activated (oxidative cleaning) by applying a constant

potential.

• Deposition and characterisation of Catalyst:

– Prussian blue is electrochemically deposited onto the working electrode.

Ferrocene is drop casted onto the sensor surface. Presence of catalyst and

meditator onto the electrode will detected by cyclic voltammetry.

• Enzyme immobilisation:

– Enzyme is drop-casted onto the electrodes and trapped onto it by the formation of

a membrane (Nafion).

• Glucosensor evaluation:

– Amperometric measurements are performed using solution with different

concentrations of glucose.

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Exercise 2 (Sponsored by;

Biacore, General Electric Life Science)

Measurement of antibody/antigen interaction by SPR

measurements.

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• Interactive training:

– An interactive course on how SPR work followed by interactive questioner will be undertaken by the

students.

Experimental Demonstration

• Immobilisation of biorecognition molecules:

– Monoclonal mouse-anti-human β2 microglobulin will be immobilised, via EDC/NHS chemistry onto

Dextran based SPR chip (CM5 chip).

• Capture of antigen:

– A solution containing the antigen (Human β2 microglobulin) will be flowed over the modified and

unmodified channels of the CM5 chip.

• Signal amplification:

– Amplification of the recognition event will be performed by the use of amplification antibodies

(Polyclonal mouse-anti-human β2 microglobulin) that have affinity for the antigen.

• Surface regeneration:

– Regeneration of sensor surface will be performed with glycerol solution.

Short description of the affinity assay

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Timetable

Exercise 1A2015/01/28

Exercise 1B2015/02/4

Exercise 2A2015/02/11

Exercise 2B2014/03/18

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

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Demonstrators

Alina SekretaryovaOnur Parlak