BiosensorM. Fatih Abasıyanık

•Biosensors are actively used in science•Self-Monitoring of Blood Glucose with

Glucose Sensor has a market (8.8 Billion Dollars in 2008)

•Biosensor :

Aim:

•Understand the parts of biosensors•How they work•To be able to discriminate biosensors

according to their quality•To be able to design new biosensors•To be able to discuss the papers about

biosensors•To be able to improve biosensors•To be able to classify results of sensors.

Contents

•Introduction to Sensors•Transduction Elements•Sensing Elements•Performance Factors•Electrochemical Sensors and Biosensors •Photometric Applications•Mass-Sensitive and Thermal Sensors•Specific Applications

Book

•“CHEMICAL SENSORS AND BIOSENSORS”

Sensor?

•A device or organ To detects certain external stimuli and responds in a distinctive manner

Basic sensors

•An acid turns blue litmus paper red and a base turns red litmus paper blue

pH meter

Typesa) Physical sensors: measure

▫ Distance▫ Mas▫ Temperature▫ Pressure

b) Chemical sensors: measure▫ Chemical substrances by chemical or physical response

c) Biosensors: measure▫ Chemical substrances by using a biological sensing

element.

• All of them connected to a transducer visible response occurs

•Chemical Sensors and biosensors detect chemicals called analyte.•If analyte is a biomolecule, the device can be called biosensor

Recognation element

Types of transducer• Electrochemical biosensor: mostly used

▫ Potentiometric: measure potential▫ Voltammetric : an increasing (decreasing)

potential is applied to the cell until Ox. (re.) of the substrance to be analysed occure and a sharp rise (fall) in the current to give a peak.

▫ Conductometric▫ FET based

• Optical • Piezo electric• Thermal

İmmobilization

Performance factors

•Selectivity•Sensitivity•Accuracy•Response time•Recovery time•Lifetime

Biosensor: Biorecognation Unit (Sensing Element; biologic detection unit)

Sensing Elements•It is concerned with various ways in which

a sensor can recognize an analyte. •specific for that analyte alone,

▫Selective ;responding to the required analyte more than to other species.

Types of Sensing Elements▫İonic (chemical sensors)▫Molecular (chemical sensor and biosensor)▫Biological (biosensor)

A- Ionic Recognation: Ion-Selective Electrodes

• Possible to be used in biosensors.▫ Example: urea ammonia

• They are based on the principle of the emf of a concentration cell.

• A potentiometric device in which the change in emf is proportional to the logarithm of the analyte concentration.

• The selectivity by the membrane separating the analyte solution from the internal reference solution.

0.00.5

• Interferences▫ Sensors respond to one ion more than to others,

although there is often a small response to unwanted ions. This is known as interference.

• For example, ▫ a fluoride ISE responds to hydroxide at one

tenth of the response level (to fluoride) for equal concentrations of the ions.

• The level of interference is measured by the selectivity coefficient▫ kij=the selectivity coefficient.

0.00.50.1

ISAB (ionic-strength adjustment buffer): [OH] declined to 10-9M pOH:9 . it is lower than detection limit for OH

•ionsconductor for electreicty▫quantity measurement▫resistance (1/conductivity)▫conductance depends on…..;

degree of ionization (storng or weak) charge of ion number of ions mobility of ions (related to size,

smallfastermore conductive) conductivity not used alone

ion chromography (extensivily)

A- Ionic Recognation: Conducting Devices

• selectivityimportant• electrodes not selective alone• to gain a selectivitymodify them• two ways

▫ modify themselves▫ modify its surface coated with polymers

•modify themselves▫best exampleCPE(Carbon paste electrode)

suitable electrodegraphite powder with Nugola stiff paste mix with a modified component (electroactive;such as ferrocene) or a complexing agent extracting electroactive analyte into the surface of the paste

A- Ionic Recognation: Modified electrodes

•modify electrode surface by coating with different types of polymers

•Mainly 3 typesa) conducting polymersb) ion-exchange polymersc) redox polymers

•a) Conducting polymers most studied ones: polyacetylene, polypyrrole, polyaniline and

polythiophene easy to polymerize: electrochemically oxidizing the substrade on

the electrode. solvents and counter ions effect on the polymer

A- Ionic Recognation: Modified electrodes

Thermodynamiclyrxn to be controlled

M= analyte L=Recognition AgentM-L complex, or M or L respond to a particular transducer (optical, electrochemical etc)Optical response: an absorption or fluorescence change to the analyte PVC (polyvintylchloride) successfully used for such biosensor (ion-selective electrodes also)Potentiometic e. (used)

B- Molecular Recognition: 1. Chemical Recognition Agents

Some Application with such recognitin agents

•Analyte: Iron (II)•Recognation Agent: 2,2’-bipyridyl in a poly

(vinyl pyridine) (PVP) membrane.•Method: iron(II) oxidized by linear-sweep

voltametry.•Complexation rxn (M-L) accumulation of iron

•İndicators can be used to analyze the analytes.

•Optodes: Optical biosensors (colorimeter or spectrophotometer)…. Used to detect the colour change

•Molecular Size:▫If the analyte is smaller than the others in

the sample, size is selective and molecular sieves are used as recognition agent.

Examples:

•The antibiotic, valinomycin is a neutral ionophore•Making complex with potassium ions selectively•K fits into its cavity•Valionmycin used as a recognition reagent to detect K

• Biological systemsthe major selective elements• They must attach themselves to one particular

substrate, but not to others.

1. Enzymes2. Antibodies3. Nucleic acids4. Receptors

Affinity biosensors: Sensors with antibodies of receptors as Recognition Elements

B- Molecular Recognition: 2. Biological Recognition Agents

1. Enzymes

A. Free (purified) EnzmyesB. Tissue MaterialsC. Micro-organismsD. Organelles (Mitochondria)

1. EnzymesA. Purified Enzyme

Many enzymesoxidation-reduction (electrochemically)

S:substrate ; E: Enzyme; ES:enzyme-substrate complex; P:product

Michaelis-Menten Equation

enzymes

enzymes

Double-reciprocal plot

enzymes

Advantages and Disadvantages of using Enzymes in sensors

ADVANTAGES DISADVANTAGES

•Highly selective

•Catalytically active

improve sensitivity

•Fairly fast-acting

•One of the most known

biological components

•Expensive

•A loss activity when

immobilized on a

transducer

•Tending to lose activity

after a relatively short

period time

B. Tissue Materials:•Tissue multiplicity of enzymes

▫Thus, not selective as purified enzymes ▫But, longer life protected (in its environ.)

•Response slow (diffusion)•Cheaper

▫Example: Banana tissue sensor: [Eggins (1997)] Banana biosensor

enzymes

• For dopamine, a catcholamine found in brain containing a complex of polyphenolases which catalyse the oxidation of polyphenolic compounds

• İts derivatives: flavanols found in beers and wine

• Polyphenol oxidase catalyse the rxn.

• İt is found in banana

enzymes

•Advantages:▫Enzyme in natural environment▫Activity stable▫Less expensive▫They sometimes work when purified enzymes fail

•Disadvantages:▫Loss selectivity▫Diffusion problems

enzymes

C. Microorganisms:•They play important role in

Brewing Pharmaceutical synthesis Food manufacture Waste-water treatment Energy production

•MO immobilized to transducer directly•MO can …..

Assimilate organic compounds Produce electroactive metabolites

enzymes

•Advantages: Cheaper Less sensitive to inhibiton by solutes, more

tolerant to pH & temperature Longer lifetimes

•Disadvantages: Longer response time Longer recovery activity times Many enzymes and less selectivity

enzymes

D. Mitochondria (organelles)

•Sub-cellular multi-enzyme particelscatalyze an analyte

enzymes

2. Antibodies:•Versitile (large spectrum)•Possible to develope for any antigen•İts function: to bind an invading antigen

and remove it from harm

antibodies

Heavy chain

light chainhingesFab: antigen-binding fragment

Gerald edelman & Rondey porter

Fc: crystalizable F

•Labelling Ab Radioisotopes Enzymes Red cells (RBC) Fluorescent probes Chemiluminescent probes Metal tags

▫Mass change etc also can be seen

antibodies

•Advantages:▫High selectivity▫Ultra sensitive▫Bind very powerfull▫TNT also can be detected by antibodiesR.D. Shankaran, K.V. Gobi, K. Matsumoto, T. Imato, K.

Toko and N. Miura, Highly sensitive surface plasmon resonance immunosensor for parts-per-trillion level detection of 2,4,6-trinitrophenol, Sens. Actuators B:

Chem. 100 (2004), pp. 425–430.•Disadvantages.

No catalytical

antibodies

3. Nucleic Acids:•like antibodies•DNA probesused to detect genetic

diseases, cancers and viral infections•DNA assays can involve the addition of

labelled DNA to the system Radioactive Photometric Enzymic Electroactive

nucleic acids

•Examples to NA biosensornucleic acids

4. Receptors:•A receptor is a structure in a cell, which

can trigger an amplified physiological response when it is bound to a particular ligand (an agonist). ▫ An agonist a chemical binding to a receptor and

triggers a response by the cell. An agonist often mimics the action of a naturally occurring substance. An agonist produces an action. An antagonist blocks an action of an agonist.

The response may be either …a. ion-channel opening, b. production of second messenger systems,

or c. activation of enzymes.

• Recepors tend to have an affinity for a range of structural related compounds.

• Like others, also they are labelled• Examples:

▫Biosensor for Anaestherics

Transducers

Types of transducers

1. Electrochemical Transducersa) Potentiometricb) Voltammetry and Amperometricc) Conductivity

2. Field Effective Transistors3. Photometric Sensors4. Others

1. Electrochemical Transducers

metal (such as silver) is placed in a solution containing ions(such as silver ions), there is a charge separation across the boundary between the metal and the solution. an electron pressure, usually termed a porential.It cannot be measured directly, and requirestwo such electrode-electrolyte combinations. Each of these is called a half-cell. Such a combination is called an electrochemical cell

• Half cells conntected by two ways▫ internally by means of an

electrically conducting bridge or membrane.

▫ Externally by a potential measuring device, such as a digital voltmeter (DVM).

• Wire has a very high internal impedance, such that very little current will flow through it. The electrical circuit is now complete and the emf of the cell can be measured.

• This value is the difference between the electrode potentials of the two half-cells.

• Depends on a number of factors, • (i) the nature of the electrodes, • (ii) the nature and concentrations of

the solutions in each half-cell, • (iii) the liquid junction potential

across the membrane (or salt bridge).

Other Reference Electrodes• SHE is a reference electrode (RE) to which

other electrodes may be referred. • While it is not difficult to set up an SHE in

the laboratory, it is not very convenient for routine measurements as such an electrode involves flowing hydrogen gas, which is potentially explosive.

• Other secondary reference electrodes are therefore used▫Easy set up▫Non-polarizable▫Reproducable electrode potential▫Many varieties, 2 are in common

• Silver chloride soluble

• İt consists of a silver wire coated with silver chloride dipping into a solution of sodium chloride

Calomel means mercurous chloride (Hg2Cl2).

A mercury pool in contact with a paste made by mixing mercury chloride powder and saturated potassium chloride

• ion-selective electrodes (ISE)• left test solution (a reference

electrode dipped into). • The ion-selective membrane is in the

middle (dividing the test solution from the standard solution, with a fixed concentration of the ions being measured. A second reference electrode is dipped into standard solution is placed.

• The two reference electrodes are connected through a high impedance voltmeter - usually a digital voltmeter.

• The electrode system is then usually calibrated with standard solutions in one of a number of ways, as to be described..

• An ISE is an ion-selective electrode, designed to respond to one particular ion more than others.

• A potentiometric device: the potential of the electrode (measured against an appropriate reference electrode) is proportional to the logarithm of the activity (or concentration) of the ion being tested.

• Such a device usually responds rapidly, with a linear range of about 10-6 to 1 M for most ISEs.

• It operates on the principle of a concentration cell, in that it contains a selective membrane which develops a potential if there is a concentration difference across the membrane of the ion being tested.

•Precautions for ISE▫İonic strengtconstant▫pH controlled▫Add components to Minimize or eliminate

interfering ions.

▫ISA (ionic strength adjusters) or TISABs (total-ionic-strength adjustment buffers) present

Measurement and Calibration

1. Calibration Graphs and Direct Read▫ Preapare a series of standards▫ Read them▫ Read the sample▫ Make a plot (log[concentration] versus

potential)▫ Estimate

Measurement and Calibration2. Standard Addition:•Prepare a standard whose concentreation is probabily 10 times higher than sample•Read sample•Add stanadrd to sample and read it•Use the following formula to estimate the concentration

Measurement and Calibration3. Gran plot: •Similar to standard addition method•Series of standard prepared•Add them to sample and read

Electrochemical Transducers2. Amperometric

2. Linear Sweep VoltammetryVoltammetry:information about an analyte is obtained by measuring the current as the potential is varied.

1) linearly varying potential between a working electrode and a reference electrode in 2) an electrochemical cell containing a high concentration of an indifferent electrolyte to make the solution conduct - called the supporting electrolyte and 3) an oxidizable or reducible species - the electroactive species.

Current monitored

Potential against currentploted =voltammogramThis techniqulinear sweep voltammetry (LSV) LSV  is a voltammetric method where the current at a working electrode is measured while the potential between the working electrode and a reference electrode is swept linearly in time.

Auxillary (supporting) electrode

Reference electrode

working electrode

A :current very smallA-B: becouse of impurities (background current) B: potenital approaches the reduction potential of the oxidized species.B-C: higher potential electrons from the electrode to the Ox at increasing rate

The increase in reduction rate cell current to increase

inet (cell current) ic (cathodic-reduction- current) İa (anodic-oxidation current)

•E ↑ic ↑ but ia ↓▫Rise in voltammetric wave▫At C:

[Ox] is limited İt is depleted by reduction Rate of fresh Ox from bulk

of solution A peak in current Id: diffusion limited current

-IL

•But we will need high matematical calculations

•We have another straightforward solution•We have current at the point C in our

hand•How we can find this to calculate

[analyte]

3. Cyclic Voltammetry▫Why: to understand the mechanism of

Redox rxn▫How works: again 3 electrodes like

voltametry

▫What the difference is Potential reversed so reverse everything (if

rxnreversible

• While Ox at the electrode surface depleted by the reduction replaced by the Red. (diffuses into the solution)

• Hence, if we reverse the potential sweep from the positive side of the peak, we shall observe the reverse effect.

• As the potential sweeps back towards the redox potential, the reduced species will start to be re-oxidized to Ox. The current will now increase in the negative (oxidizing) direction until an oxidation peak is reached.

• two peaks: 1st: the reduction of the original substrate 2nd: reoxidation of the product back to the original substrate.

• peak currents almost identical heights.• The average of the two peak potentials is equal to the standard

redox potential, regardless of the concentration of substrate or its diffusion coefficients or rates of electron transfer.

4. Chronoamperometry• Instead of sweeping the potential, the latter is

stepped in a square-wave fashion to a potential just past where the peak would appear in linear-sweep voltammetry.

• The current is then monitored as a function of time. Decay occurs because of the collapse (or spreading out) of the diffusion layer

•decay is proportional to the reciprocal of the square root of time, as shown by the Cottrell equation:

•Chronoamperometry can be used to determine any of the variables in the above equation, providing that the others are known. A,C,D or n

5. Amperometryusual name for the analytical application of the chronoamperometric technique. With certain cell and electrode configurations, the decaying current reaches an approximately steady state after a certain time. This is shown by the shaded part of the curve in the figure left The current has become effectively independent of time, as indicated by the following equation:

Constant related to the diffusion layer

thickness

6. Conductivity• inverse of resistance• A measure of the ease of passage of electric

current through a solution.• Ohm’s law

▫ E=I/R ; L=1/R and therefore ▫ E=I/L

The conductivity varies according to • the charge on the ion, • the mobility of the ion • the degree of dissociation of the

ion.

•impedance: conductance, capacitance and inductance attantion

• In principle, a change in conductance can be used to follow any reaction that produces a change in the number of ions, the charge on the ions, the dissociation of the ions or the mobility of the ions. Usually a differential type of cell is used, as shown in the following Figure.

7. Piezo-Electric EffectPrinciples• In 1880, The Curie brothers discovered “

1. anisotropic crystals give out an electrical signal when mechanically stressed.” 2. if an electrical signal is applied to such crystals, they will deform mechanically (With the application of an oscillating electrical potential, the crystal will vibrate).

• Every crystal has its own natural resonant frequency of oscillation, which can be modulated by its environment. The usual value of this frequency is in the 10 MHz region, i.e. radiofrequency.

• The actual frequency is dependent on the mass of the crystal, (any other material coated on it)

• The change in resonant frequency (Af) resulting from the adsorption of an analyte on its surface can be measured with high sensitivity (500-2500 Hz g-’),

• when applied in sensors can thus result in devices with pg detection limits.• The relationship between the surface mass change, Δm, and the change in

resonant frequency, Δf, is given by the Sauerbrey equation, as follows:

• Δm:the mass in grams of the adsorbed material on an area A (cm2) of the sensing region,

• Δ f : the overall resonant frequency. For a 15 kHz crystal, a resolution of 2500 Hz kg-l is likely, so that a detection

limit of g (1 pg) is achievable.

• Materials showing the piezo-electric effect ceramic materials such as barium and lead titanates

• Some organic polymers, such as poly (vinylidene fluoride) (PVDF) (-CF2-CH2-CF2-),, also form crystals with piezo-electric properties.

The frequency at which an oscillator works is usually determined by a quartz crystal. When a direct current is applied to such a crystal, it vibrates at a frequency that depends on its thickness, and on the manner in which it is cut from the original mineral rock. Some oscillators employ combinations of inductors, resistors, and/or capacitors to determine the frequency. However, the best stability (constancy of frequency) is obtained in oscillators that use quartz crystals

3. Photometric SensorsAbasıyanık M. F.

•Most bioassays photometric type•How workChanges in species with

photometric properties.•Example:

• The problem: how to make a sensor by using an optical technique.

• The basic optical response is based on the Beer-Lambert law (usually referred to as Beer’s law), as follows:

• IO: the intensity of the incident light; • I: the intensity of the transmitted light;• A: the absorbance (usually measured directly by an

instrument), • ε: extinction coefficient, • C : the concentration of the analyte, • L: the pathlength of light through the solution. (result in

limitation of size of sensors, which does not occur in electrochemical devices)

What are the advantages of optical sensors?i. No ‘reference electrode’ is needed, although a reference source is

often useful.ii. There is no electrical interference.iii. An immobilized reagent does not have to be in contact with any

optical fibres, and can easily be replaced.iv. There are no electrical safety hazards.v. Some analytes, such as oxygen, can be detected in equilibrium.vi. They are highly stable with respect to calibration, especially if the

ratio of the intensities at two different wavelengths can be measured.

vii. They can respond simultaneously to more than one analyte by using multiple immobilized reagents with different wavelengths for response, e.g. 02 and CO.

viii. Multi-wavelength measurements can be made to monitor changes in the state of the reagent.

ix. They have potential for higher-information content than electrical transducers.

What are the disadvantages of optical sensors?i. They will only work if appropriate reagent phases can be

developed.ii. They are subject to background ambient light interference. This

may be excluded either directly or by using a modulation technique.

iii. They have a limited dynamic range when compared to electrical sensors – typically 102 compared with 106 – 1012 for ion-selective electrodes.

iv. They are extensive devices, and dependent on the amount of reagent, and hence difficult to miniaturize.

v. There are problems with the long-term stability of the reagents under incident light.

vi. Response times may be slow because of the time of mass transfer of analytes to the reagent phase.

Optical Techniques

Main types of photometric behaviour in biosensor applications:

1. Ultraviolet-visible absorption2. Fluorescence (and phosphorescence)

emission3. Bioluminescence4. Chemiluminescence5. Internal reflection spectroscopy (IRS)6. Laser light scattering methods

1. Ultraviolet and Visible Absorption SpectroscopyThe absorbance is measured by passing

incident radiation through the monochromator of a spectrometer and then measuring its intensity with a photomultiplier or photodiode, thus producing an electrical signal which is proportional to the absorbance at a particular wavelength.

The measured absorbance is linearly proportional to the concentration.

However, the apparatus ▫relatively cumbersome ▫Expensive,

2. Fluorescence Spectroscopy (animation)• Species absorb light, • the excited species will decay in one of a variety

of ways. • decay to the lowest excited singlet state, • re-emit radiation, usually at a lower wavelength

than the original excitation. • This phenomenon is known as fuorescence,• Emitted light can be measured

•Turn off light

•Quenching refers to any process which decreases the fluorescence intensity of a given substance. 

•The excited molecule transfer its energy to the new one (quenching)

LuminescenceEmission of lightChemiluminescence : certain chemical

RXN emission of light without heat – Bioluminescence: The similar emission of

light from biological systems.

3. Chemiluminescence,As a result of the oxidation (with O2 or

H2O2) of certain substances (example:luminol), visible light 'in the cold‘ and in the absence of any exciting illumination.

• Luminol is normally used as a label and employed in any assay involving;▫ oxygen, ▫ hydrogen peroxide or ▫ peroxidase.

• particularly useful with immunoassays▫ But sensitivity is limited because the quantum yield is only 1%.

• Example: ▫ A particularly interesting approach, combining both luminescence and

fluorescence▫ An antigen labelled with the luminol, while the corresponding

antibody labelled with a fluorescent compound.▫ the emission from the luminol will excite the fluorescence. ▫ Ag-Ab: luminol emits light of 460 nm, which excites the fluorescor, ▫ İt emits fluorescence in turn at 525 nm, ▫ thus results in an increased quantum yield. ▫ At the same time, unlabelled antigen combines with labelled

antibody, ▫ in which case there is no fluorescence but emission▫ luminol at 460 nm permits analysis of both bound and unbound.

• Their usage in Biosensors • Examples:

▫ luminol with hydrogen peroxide and peroxidase. ▫A fibre-optic sensor for H202 : peroxidase

immobilized on PAGE (polyacrylamide gel) + luminol at the end of the fibre.

▫The luminescence is detected in situ no external light source is needed. The sensor can be connected directly to the

photodiode. It will detect 1-10 mM H202, with a response time of

2 min. One obvious application is to 'connect' the

sensor to a glucose-glucose oxidase reaction system to

determine glucose, for which a linear concentration range of 0 15- 1.5 mM can be obtained.

A photodiode is a type of photodetector capable of converting light into either current or voltage, depending upon the mode of operation

• A recent example:• adamantyl dioxetine phosphate adamantyl

dioxetine anion (by alkaline phosphatase ), unstable fluoresces. fluorescence lifetime is several minutes, unlike

conventional luminescence • Such behaviour could be used in many types of

assay which involve phosphate ester hydrolysis using alkaline phosphatase.

• adamantyl dioxetane phosphate compete with other organic phosphates for the phosphatase.

4. BioluminescenceCertain biological species (firefly) emit

luminescence. This originates in a group of substances of varied structures known as luciferins.

The enzyme-catalysed oxidation of luciferin results in luminescence.

•Rxnvery sensitive down to femtomole [10-15 ]•example:

▫determination of creatine kinase related to myocardial infarction and muscle disorders

•Bacterial luciferases do not use luciferins but ..

• Most analytical reactions NADH for FMN to FMNH2

• Example:

•NADH oxidationfor ethanol determination

alcohol dehydrogenase

Advantages of luminescence methods when compared with other photometric methods

•In most photometric methods: ▫comparison of light absorbed in the presence

of the analyte with the corresponding light in the absence of the analyte,(which will not usually be zero).

•Luminescence (chemiluminescence-bioluminescence) is measured against a background of complete absence of light.

•Much more sensitive detectors much lower levels of light detecteddetection limits very much lower

Optice transducers

•Fiber optic glasses (optic fibers) key components. An optical fiber is a glass or plastic fiber that carries light along its length.

•Light is kept in the core of the optical fiber by total internal reflection. 

•Total internal reflection is an optical phenomenon that occurs when a ray of light strikes a medium boundary at an angle larger than a particular critical angle with respect to the normal to the surface.

• When light crosses a boundary between materials with

different refractive indices, the light beam will be partially

refracted at the boundary surface, and partially reflected.

However, if the angle of incidence is greater (i.e. the ray

is closer to being parallel to the boundary) than the critical

angle – the angle of incidence at which light is refracted

such that it travels along the boundary – then the light

will stop crossing the boundary altogether and instead be

totally reflected back internally. This can only occur

where light travels from a medium with a higher

refractive index to one with a lower refractive index. For

example, it will occur when passing from glass to air, but

not when passing from air to glass

glass

air

• Optical fibers regarded as ‘light conductors’ or ‘light wires’.

• metal electrical wires conduct electricity (often over very long distances)

• Optical fibres does the same for light. ▫ optical fibres are even for telephone transmission.

• Optical fibres behave as waveguides for light. • Original fibres are made of glass, • but now polymeric materials used

▫ much cheaper than glass and the metal wires • The light waves are propagated along the fibre by

total internal rejection (TIR).

• TIR depends on the angle of incidence and the refractive indices of the media:

• 2.7.7 Device Construction• system works better with a ‘reference blank’, with the light

source then being split between the sample and the reference. • Detection may be effected at different analytical and

reference wavelengths and one then obtains the ratio of the signals at the two wavelengths.

• This eliminates scatter and source fluctuations.• Waveguides for different forms of light may need to be made

of different• materials, as follows:• λ > 450 nm, plastic (such as polyacrylamide)• λ > 350 nm, glass• λ < 350 nm, fused silica• λ > 1000 nm, germanium crystal• In a photometric sensor, the reagent has to be immobilized so

that it can interact with the analyte, probably to form a complex with distinctive optical properties which can then be monitored by the sensor.

• The criteria for the application of these are as follows:

Choice of immobilization support Immobilization of indicators, thus

retaining activity in the desired rays Immobilization of biorecognition

molecules with retention of activity Cell geometry Choice of source and detector

components• portable device low-power

components (desirable) such as LED (light emitting diode)sources and photodiode detectors.

• LEDs have only a limited range of wavelengths

• Solid-Phase Absorption Label Sensors

Performance Factors

Chapter IIIIV. Week

1.Selectivity: •the ability to discriminate between different substances. It is a function of the selective component (biodetection element), although sometimes the operation of the transducer contributes to the selectivity.

i)Ion-selective Electrodes:•Respond to particular ions

▫BUT, nearly all can respond to some other ions (interference)▫Interference can be measured and for commercial sensors, it should be given.

Selectivity

• The interference is expressed in the Nicolskii-Eisenman equation

• ki,j:Electrode potential and a selectivity coefficient,

ai : activity of the primary analyte of charge naj: activity of the interfering analyte of charge zE: potantial of the system ; K and S: konstant

Selectivity

• ki,j determined by a “two-point, mixed solution method”

• Example: ▫ The cell potantial primary analyte (E1) (0.001M)

▫ 0.001M (primary analyte)+0.1 or 0.01M potentially interfering ion (E2)

Selectivity

Question: Transducer: A calcium ISES: +29.6 mV/decade in a 0.001 M solution. E1: -20.1 mV (in a 0.001 M calcium chloride solution)E2: - 19.8 mV (mixed sol. 0.00 1 M calcium chloride and 0.1 M sodium chloride).

Selectivity

Calculate the selectivity coefficient for calcium ions in the presence of sodium.

2. Sensitivity Sensitivity

2. SensitivityRange, Linear Range and Detection Limits

Sensitivity

it is important to know ..•what concentration range is covered and• over what section of this range the response is linear. •At the lower level is the detection limit.

DL is the concentration of analyte at which the extrapolated linear portion of the calibration graph intersects the baseline – a horizontal line corresponding to zero change in response for several decades of concentration change.

How to calculate DETECTION LIMIT: There are many definitions of DL.

•Linear Range: should include effective constentration of the target

•For example: for determination of glucose, biosensor’s linear range cover (0.2-20mM, possible concentration of glucose in blood, normal-diabetic respectively)

•Potentiometric sensor’s linear range larger

pH (H+)-selective electrode’s linear range from ph 0-12 (100-1012)

•Amperometric sensors and biosensors do not ranges have ranges of much more than two or three powers of ten

Sensitivity

•Statistical anaylsis important for bioanalytical chemistry▫Mean, standart deviation, CV (Coefficient

of Variation), ▫CV = (Standard Deviation / Mean) * 100▫Example

Sensitivity

CV ( )

3. Time Factors3.1. Responce TimesSome definations•the time that elapses between a stimulus

and the response to it.•The amount of time it takes for a device to

react to an input signal.•time to allow the system to come to

equilibrium, the response time.

Response Time

•Chemical sensors: their response time short▫ For example: nitrate electrodes the most reproducible

results were obtained after stirring the solution in contact with the electrode for ~30 s.

•Biosensors:▫Response times from a few sec.- to a few

min.▫Up to 5 min acceptable▫>10 min too long.

•if the time becomes too long it can affect the usefulness of the method for repetitive routine analyses.

Response Time

3.2. Recovery Times: the time that elapses before a sensor is ready to be used for another sample measurement.

It may be ▫immediate ▫after one measurement the sensor system

has to rest to resume its base equilibrium before it can be used with the next sample.

•In many publications, these times (recovery and response) are combined

Recovery Time

3.3. Lifetimes:Biosensors and chemical sensor’s lifetimes

can be different•The most robust pH electrodes tend to

deteriorate after months of use.•What does lifetime mean? Types?1. The response during continous use•the sensor is in constant contact with an

analyte solution and successive readings are made over a period of, e.g. hours.

•This lifetime in use can be defined as the time after which the response has declined by a given percentage (say 5%).

lifetimes

2. Shelf life stability: the time over which the assembled sensor is stored, perhaps in a buffer solution or an ISAB (ionic-strength adjustment buffer)

3. Storage stability: the period when the sensor is stored ▫ dry in its packing, for an ion-selective electrode, ▫ when a biological material is stored separately, perhaps

refrigerated.• All organic material deteriorates with time,

especially when taken out of its natural environment. • Biosensor studies to show how the response of the

biosensor to a standard sample changes with time over hours, days and even months.

• Generally pure enzymes have the lowest stability, whereas tissue preparations have the highest.

lifetimes

Examples to Techniques to improve lifetime of biomaterials of biosensor:

•Gibson et all in stabilizing a range of enzymes by using a mixture of the followings as additives (stabilizers)

a polyelectrolyte (DEAE-dextrin) A sugar alcohol (lacticol)

▫Enhanced the retention of enzyme activity during.. Desiccation Thermal stress

▫They optimized this method on .. Alcohol dehydrogenase Horseradish peroxidase

▫Other scientist perform on 12 other enzymes

lifetimes

Examples:• Alcohol oxidase (alcohol determination; with membrane

immobilization and• horseradish peroxidase (amperometric oxidation of

hydrogen peroxide ) with a mediated coupled reaction and N-methylphenothiazine- tetracyanoquino- dimethane (NMP-TCNQ) on a graphite electrode. ▫ In both cases, stabilizers promoted a considerable increase

in storage stability in the shelf life (dried form; 37°C)

Similarly; • L-glutamate biosensor (NMP-TCNQ-modified graphite

electrodes) • L-glutamate oxidase

▫ an increase in shelf life

Other workers, (just the DEAE-dextran polyelectrolyte) • glucose oxidase biosensors, were less successful.

▫ In fact, lyophilized glucose oxidase alone is extremely stable, (for 2 years at 0°C and for 8 years at - 15°C)

▫ In solution, it is most stable at pH 5, while below pH 2 and above pH 8 the catalytic activity is rapidly lost.

lifetimes

3.4. Precision and Accuracy•accuracy of a measurement: the degree of closeness of measurements of a quantity  to its actual (true) value •precision (reproductibilty) of a measurement system: the degree to which repeated measurements under unchanged conditions show the same results

Precision and Accuracy

High accuracy Low precision

Low accuracy High precision

•Standard addition method▫The method of standard addition is used

in instrumental analysis to determine concentration of a substance (analyte) in an unknown sample by comparison to a set of samples of known concentration, similar to using a calibration curve.

▫Standard addition can be applied to most analytical techniques and is used instead of a calibration curve to solve the matrix effect problem.

Precision and Accuracy

Different Transducer

•Performance ▫Types of analyte1. Urea biosensor2. Amino acid biosensor3. Glucose biosensor4. Uric acid▫affected by the amount of enzyme▫Type of immobilization▫Some examples

1. Urea Biosensorİn table forward, urea biosensor compared

Cationic (NH4+, pH and gas (NH3 and CO2 )

pH sensor more enzyme needed Below 5mM linear Longest response time

Ammonia Gas electrode Best one 4 months stabek Use little enzyme Short response time

Cationic Best rsponse time Excellent response range 3 weeks stable

1. Urea BiosensorA urea biosensor on an ENFET (urease in sensing

gate) pH ▫ 1.3-16.7 mM urea▫ 2 weeks storage at 4 degree C▫ Response time 2 min.▫ No interfering with glucosei creatinine and albumine

Ir-Pd MOS (metal oxide semiconductor) deviceRead the abstract

Determination of urea with an ammonia gas-sensitive semiconductor device in combination with ureaseF. Winquista, A. Spetza, I. Lundströma and B. Danielssonb

aLaboratory of Applied Physics, Linköping Institute of Technology, S-581 83 Linköping SwedenbDepartment of Pure and Applied Biochemistry, Chemical Centre, University of Lund, S-220 07 Lund SwedenReceived 3 April 1984.  Available online 18 January 2002. 

AbstractAn ammonia gas-sensitive Ir/Pd MOS capacitor is used for urea determinations with the aid of urease in two different systems. One combination utilizes a reaction column with immobilized urease in a flow-injection system. The lower limit of urea detection for 150-μl samples was 0.2 μM. Urea in whole blood and blood serum was determined after a 500-fold dilution, and 15 samples per hour could be assayed. The relative standard deviation was 4.6% (n=10). Recovery tests were satisfactory. Values obtained for urea in serum correlated well with those from a spectrophotometric method. The other combination is based on a small flow cell with free urease enclosed between a dialysis membrane and a gas-permeable membrane. Urea was determined in the concentration range 0.01–50 mM. The enzyme probe could be used for up to four days without changes of behaviour.

2. Glucose Biosensor

2

7 2)

Health Care

• Mesurements of blood, gases ions and metabolites regularly analyzed.

• Convential methods slow• Chemisensors and biosensorsfast• Latest ExacTech® biosensor: in 12 second

• Problem: sensors for different analytes needs specialist scientist (Nurse) to operate it.

• BUT, new smart biosensors because of FET technology (field effect transistors) that can combine several measurements in a sensor unit. ▫ Sodium, potassium, calcium and pH by one

sensor▫ Glucose, lactate and urea

Health Care

Health Care

Dream for scientist to produce....

•An implanted biosensor for continuous monitoring of a metabolite▫HOW

Via a microprocessor to a controlled drug-delivery system throug the skin. Diabetes

Automatit delivery system gives insulin (artifical pancrease)

Health Care

Control of Industrial Process

•2. Control of Industrial Process •Environmental Monitoring:

Health Care

• Mesurements of blood, gases ions and metabolites regularly analyzed.

• Convential methods slow• Chemisensors and biosensorsfast• Latest ExacTech® biosensor: in 12 second

• Problem: sensors for different analytes needs specialist scientist (Nurse) to operate it.

• BUT, new smart biosensors because of FET technology (field effect transistors) that can combine several measurements in a sensor unit. ▫ Sodium, potassium, calcium and pH by one

sensor▫ Glucose, lactate and urea

Health Care

Dream for scientist to produce....

•An implanted biosensor for continuous monitoring of a metabolite▫HOW

Via a microprocessor to a controlled drug-delivery system throug the skin. Diabetes

Automatit delivery system gives insulin (artifical pancrease)

Health Care

Control of Industrial Process

•2. Control of Industrial Process •Environmental Monitoring: