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Biosensors

Biosensor : Any device that uses specific biochemical reactions to detect chemical compounds in biological samples.

A sensor that integrates a biological element with a physicochemical transducer to produce an electronic signal proportional to a single analyte which is then conveyed to a detector.

a) Thin layer of active biological material in contact with electrical transducer b) Transducer converts observed change (physical / chemical) in quantifiable signal

c +d+ e )Electronic signal magnitude proportional to concentration of a specific

compound

Based on specificity and sensitivity of biological systems

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Biocatalysis (Enzyme/metabolic): Substrate consumption/product liberation is measured and converted into quantifiable signal. Bioaffinity: These sensors are based on binding interactions between the immobilised biomolecule and the analyte of interest. These interactions are highly selective. Examples include antibody-antigen interactions, nucleic acid for complementary sequences and lectin for sugar.

Biosensors applications 1. Control of critical metabolites during surgery

2. Ambulatory and Hospital Emergencies:

– Obviously expensive and time consuming analysis in central laboratories

– Accelerates the diagnosis and early treatment

– Less risk of damage to the sample

3. Domestic Diagnosis:

• Pregnancy test

• Glucose Control in Diabetics

4. In vivo applications :

– artificial pancreas

– Correction metabolite levels

– Problems: Miniaturization and Biocompatibility

5. Industrial, military ans environmental applications:

– Food industry

– Cosmetics

– Fermentations control

– Quality control

– Explosives Detection

– Detection of nerve gases and / or biological toxins

– Pollution control

–

– .

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TYPES OF BIOSENSORS

1. Electrochemistry Biosensors

– Amperometric: measurement of electrical currents associated with electrons involved in redox processes

– Potenciometric: ion-selective electrodes

– Conductimetric: measurement of conductivity changes associated with changes in the ionic environment of the solutions

2. Termometric Biosensors

3. Piezoelectric biosensors

4. Optical biosensors

Evanescente wave

Surface plasmon resonance

5. Cell Biosensors

6. Immunosensors

3. Detection: Steady-state or flow injection analysis

An enzyme electrode

1. Thin enzyme layer with high specific activity,

2. Good selection of membranes

Response controlled by diffusion through the permselective membrane

(not by enzyme kinetics)

Enzyme activity low - thick membrane needed to achieve linear response, response slow

Enzyme activity high - thin membrane OK, rapid response

Protective

membrane

Enzyme layer Permselective

membrane

Electrode Sample E

P1 P1

S1

S2

P2 P2

P2*

-

-

-

-

I

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Background noise

Analyte Signal

D S

dS/ dt

Time

S

c

D S/ D c Linear response

Detection limit

Biosensor parameters 1. Sensitivity 2. Linear response 3. Detection limit 4. Background noise 5. Baseline drift 6. Selectivity 7. Response 8. Operating stability 9. Shelf life

S = 3 N

N

Analyte

Assay of the detection limit

Response time and kinetics in biosensors

RESPONSE TIME : Time necessary for having 95% of the response. Recovery Time: Time necessary to recovery the baseline.

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Screen print

Matrix carrier

Mobile wiper

Paste

Screen grid

Screen printing

The sensor body is made from ceramics. A gold working electrode (a) is surrounded by an Ag/AgCl reference electrode (b) and gold auxiliary electrode (c). Silver output contacts (d). The ruler in the bottom is in millimeter scale

https://www.youtube.com/watch?v=

GxThyTTztmQ

A. Surface modified electrodes

Electrode material: graphite, glassy carbon, gold, SnO2

Coupling: carbodiimide, glutaraldehyde, adsorption

B. Polymer-based electrodes

Crosslinking with Os(bpy)23+/2+ redox polymer,

electropolymerized polypyrrole, o-phenylethylamine

C. Bulk modified composite electrodes

Graphite-silicone oil paste, paraffin oil paste,

epoxy composite

D. Tissue-modified carbon paste electrodes

Asparagus tissue, tobacco callus tissue,

horseradish root, kohlrabi skin

Electrode designs

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1. Electrochemistry Biosensores: 1.1 Amperometric

• Enzyme catalyzed redox reactions

• Constant voltage between two electrodes

• Current due to the reaction on the electrodes

Between the central platinum cathode and the surrounding anode of silver applied potential of 0.6 volts. The circuit is closed with saturated KCl solution.

The molecular oxygen dissolved in the cathode is reduced in the platinum catode. Electrons are released and produces electrical current can be measured.

C a t o d e ( P t )

A n o d e ( A g )

Bridge KCl

O 2 + 2 H 2 O + 2 e - H 2 O 2 + 2 O H -

2 H 2 O 2 + 2 e - 2 O H -

4 A g 4 A g + + 4 e -

4 A g + + 4 C l - 4 A g C l

4 e -

- +

0 , 6 - 0 , 7 v

A) Hard epoxy resin (B) platinum cathode in the center of a projection. (C) silver anode in a circular (D) Rubber ring spacer holding a paper soaked in an electrolyte and a polytetrafluoroethylene membrane separating the electrodes from the reaction mixture.

Glucosa-oxidasa Invertase

Ele

ctro

do

Glucose

D-gluconolactone

H2O2

Sacarose

Fructose Glucose

O2

glucose

sacarose

time

response

GOD

Fluxe

Glucose and sacarose determination

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Fish freshness: After death, the nucleotides of fish undergo a series of progressive degradation reactions: ATP > ADP > AMP > IMP > HxR > Hx > Xantine > Uric Acid

The accumulation of inosine and hypoxanthine nucleotides is an indicator of the time after the fish died and its storage conditions.

Biosensor: Xanthine oxidase and nucleoside phosphorylase immobilized in a triacyl cellulose membrane of an oxygen electrode

K < 20 Fresh fish can be eaten raw 20 > K < 40 Fish must be cooked K > 40 Non-eatable fish

The nucleotides could be determined using the same sample and electrode, but adding nucleotidase and adenosine deaminase

Analyte Enzyme Reaction Alcohol Alcohol oxidase Ethanol + O2 Acetaldehyde + H2O2

D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2

Lactose Galactose oxidase Lactose + O2 Galactose dialdehyde der. + H2O2 L-Lactate L-Lactate oxidase L-Lactate + O2 Pyruvate + H2O2 Starch Amyloglucosidase Starch + H2O β-D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2 Sucrose Invertase Sucrose + H2O α-D-Glucose + β-D-Fructose Mutarotase α-D-Glucose β-D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2

First generation biosensors - response to the substrates in solution

1. Reduction of oxygen with a Clark type electrode at -0.6 V (vs SCE) 2. Oxidation of hydrogen peroxide at a Pt electrode at +0.7 V 3. Measuring of pH change

Glucose + O2 Gluconic acid + H2O2

Examples of hydrogen peroxide measuring biosensor

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• More specific oxidase reagent to oxidize

• Fast electron transfer rates

• Ability to be regenerated easily

• Biocatalytic membrane retainable

• Not react with other molecules, including molecular oxygen

Redox mediators in amperometric Biosensors ( 2nd generation)

• Tetracyanoquinodimethane: partial electron acceptor

• Ferrocene, tetrathiofulvalene and N-methyl phenazinium partial electron donor

• Hydroquinone and ferrocyanide soluble mediators

• Device sensing area with single use electrode • Deposition on a plastic strip. Reference electrode consists of Ag / AgCl and

carbon electrode with glucose oxidase and ferrocene mediator • Both electrodes covered with hydrophilic tissue. Passage of molecules of

different size, homogeneous diffusion, prevents uneven evaporation • Duration 6 months • 2-25 mM detection in blood drop • Results in 30 seconds

Glucose biosensor

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Possible by the ability of pyrrole to polymerize by electrochemical oxidation in mild conditions enough to trap enzymes and mediators without denaturation

Second generation biosensors - mediated electron transfer between enzyme and electrode - can be easily miniaturized

Third generation biosensors - direct electron transfer between enzyme and electrode

Cell-based based biosensors - cheaper than purified enzymes, Nocardia erythropolis cells immobilised in polyacrylamide or agar (cholesterol oxidase) Cholesterol + O2 Cholest-4-en-3-one + H2O2

In the third generation biosensor the redox cofactor of the enzyme is covalently (or electrostatically) bound to the working electrode. This facilitates the re-reduction (or re-oxidation) of the enzymes after they have interacted with their substrates and assists in carrying electrons or holes to or from the working electrode.

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1.2 Potentiometric biosensors

Three types of ion-selective electrodes which are of use in biosensors:

• Glass electrodes for cations.

• Glass pH electrodes coated with a gas-permeable

membrane selective for CO 2 3 2

• The iodide electrode is useful for the

determination of I- in the peroxidase reaction.

, NH or H S.

H2O2 + 2H+ + 2I- peroxidase I2 + 2H2O

1.2 POTENTIOMETRIC BIOSENSORS

• The biosensor consists of an immobilised enzyme

membrane surrounding the probe from a pH meter.

D-glucose + O 2 2 2 D-glucono-1,5-lactone+H O

Glucose oxidase

D-gluconate+ H +

H 2 O

Penicillin Penicilloic acid+H + Penicillinase

H 2 2 2 NCONH + 2H O 2NH 3 3 + HCO - + H + Urease (pH 9.5)

Neutral lipids + H 2 O Glycerol + fatty acids +H + Lipase

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Potentiometric biosensors (ENFET) enzyme-linked field effect transistors

Schematic diagram of the section across the width of an ENFET. The actual dimensions of the active area is about 500 mm long by 50 mm wide by 300 mm thick. The main body of the biosensor is a p-type silicon chip with two n-type silicon areas; the negative source and the positive drain. The chip is insulated by a thin layer (0.1 mm thick) of silica (SiO2) which forms the gate of the FET. Above this gate is an equally thin layer of H+-sensitive material (e.g. tantalum oxide), a protective ion selective membrane, the biocatalyst and the analyte solution, which is separated from sensitive parts of the FET by an inert encapsulating polyimide photopolymer.

FET

Drain Gate

Source

-

-

-

-

-

-

Insulator

+

+ + + +

(Electron Channel)

(Not conductive enough)

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FET

Drain Gate

Source

-

Insulator

+

+ + + +

Threshold Voltage

FET

Drain Gate

Source

-

-

-

-

-

-

- -

Insulator

+ + + +

+

+ + + +

- - - - - -

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Urea biosensor

NH2CONH2 +3H2O

2HN 4 + + HCO3

- + OH-

Urease

Immobilized urease Surgery and Renal Dialysis Reaction causes great change in ion concentration

An alternating field between two electrodes allows the determination of changes in conductivity, avoiding unwanted electrochemical processes Electrodes arranged to occupy minimal space 0.1 and 10 mM urea

amidases,

decarboxylases,

esterases,

phosphatases and

nucleases.

1.3 Conductometric Biosensors

2. Thermometric biosensors Enzyme-catalysed reactions exhibit the same enthalpy changes as spontaneous chemical reactions. Considerable heat evolution is noted (5-100kJ/mol). The thermal biosensors constructed have been based on:

• direct attachment of the immobilised enzyme or cell to a thermistor

• Immobilisation of the enzyme in a column in which the thermistor has been embedded.

DT=− DHnp/Cp

Reaction confined insulated devices (a) Accurate proper insulation The analyte stream passes through a heat exchanger (b) The reaction takes place in a small reactor (c) The difference in temperature of the analyte enters and exits the product is determined by interconnected thermistors (d) Differences up to 0.0001 ° C

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Reactions used in thermometric biosensors

Flow injection thermal biosensor array for

simultaneous determination of lactate, glucose, urea

and penicillin.

Bin Xie, Kumaran Ramanathan, Bengt Danielsson

Mini/micro thermal biosensors and other related devices for biochemical/clinical analysis and monitoring

TrAC Trends in Analytical ChemistryVolume 19, Issue 5, May 2000, Pages 340–349

Exemples of thermometric biosensors

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The principle of this sensor type is based on the discovery of there being a linear relationship between the change in the oscillating frequency of a piezoelectric (PZ) crystal and the mass variation on its surface. Sauerbrey discovered in 1959 that the change in mass is inversely proportional to the change in frequency of the resonating crystal (usually at MHz frequencies).

DF = -2.3x106 F2DM/A (Sauerbrey equation) DF = frequency change in oscillating crystal in Hz, F = frequency of piezoelectric quartz crystal in MHz, DM = mass of deposited film in g, and A = area of electrode surface in cm2. The change in mass occurs when the analyte interacts specifically with a biospecific agent immobilised on the crystal surface. The crystal may be coated with antibodies, enzymes or organic materials. Frequency changes smaller than 1MHz may be measured providing nanogram sensitivity.

3. Piezoelectric biosensors

Piezoelectric biosensor: A piezoelectric transducer with biological sensing layer

(AG - AB)

Piezoelectric biosensors Sensors configured as an oscillator can be equipped with an antenna for remote sensing and control. Another advantageous feature of using piezoelectric materials is that the same electromechanical transduction mecha-nism can be used not only for a sensing, but also for actuation. This property is essential for the design of piezoelectric surgical micro-cutters or liquid micro-flow systems.

Piezoelectric immuno-nanosensors are inexpensive, easy-to-use, and feature rapid response, hence they may allow for wide screenings and the development of effective preventive strategies for a broad range of diseases, including bio-agent detection such as anthrax or smallpox, viral infections, and cancer

www.tms.org/pubs/journals/JOM/0010/Kumar/Kumar-0010.html

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A piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7. The immunosensor could detect the target bacteria in a range of 103–108 CFU/ml within 30–50 min. The cleaned crystals were immersed in an ethanol solution of 16- mercaptohexadecanoic acid (MHDA). MHDA-modified crystals were treated with EDC (Carbodiimide)-NHS (Nhydroxysuccinimide) to convert the terminal carboxylic group to an active NHS ester. After rinsing with water and drying, anti-E. coli O157:H7 antibodies were added onto Au electrodes and stored at 4 C overnight (at least 15 h). The excess antibodies were removed by rinsing with PBS.

X.L.Su and Y.Li, Biosensors and Bioelectronics 19 (2004) 563–574

Immobilization of Antibodies in a quartz crystal microbalance (QCM)

4. OPTICAL BIOSENSORS The basis of these systems is that enzymatic reactions alter the optical properties of some substances allowing them to emit light upon illumination. Means of optical detection include fluorescence, phosphorescence, chemi/bioluminescence

• Advantages of optical biosensors include: due to fibre optics, miniaturisation is possible in situ measurements are possible in vivo measurements are possible diode arrays allow for multi-analyte detection signal is not prone to electromagnetic interference

• Disadvantages include:

ambient light is a strong interference fibres are very expensive indicator phases may be released with time

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4.1 Fibre optics (optodes) are a subclass of optical waveguides which operate

using the principle of total internal reflection. Light incident on the interface between two dielectric media will be either reflected or refracted according to Snell’s Law.

If light is entered into a fibre (surrounded by a medium of lower refractive index) at a shallow enough angle, the light will be confined within. Thus, the optical fibres consist of a core of high refractive index surrounded by a cladding of slightly lower refractive index, with the whole fibre protected by a non-optical jacket. Light input, and hence output, is dependent on the diameter of the fibre. As a very small diameter is required for flexible fibres, this size is a limiting factor in the fabrication of the fibres. For this reason, fibres are made from bundles which have the advantage of efficient light collection and flexibility. Fibre bundles of 8, 16 and more fibre strands are available.

Detects changes in concentration of oxygen in determining the reduction in the fluorescence of a fluorophore (quenching)

Absorption at 630 nm Change from yellow to greenish blue of bromocresol green when bound to serum albumin at pH 3.8 Linear response to albumin in a range of from 5 to 35 mg/cm3

Optical fiber Optical cell

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There are basically two different configurations used at the tip of the fibre-optic probe :

-the distal cuvette configuration -waveguide binding

configuration

The distal cuvette configuration involves immobilisation of detection molecules in a porous, transparent medium at the fibre tip. The fluorescence changes when the analyte diffuses and is bound. Excitation comes from out of the fibre and emission is coupled back into the fibre

The waveguide binding tip configuration involves the binding of fluorescent-labelled detector molecules (e.g. antibodies) to covalently attached analyte molecules on the fibre surface. As the label is close to the surface it is excited by the evanescent wave emanating from the fibre and the resulting fluorescence is coupled back into the fibre. Free analyte competes for the binding sites on the recognition molecules, permitting them to diffuse away from the surface with a resultant decrease in fluorescence.

In a radiation sensor that travels along a waveguide by total internal reflection creates a field called evanescent electromagnetic field, which can penetrate a specific distance from the surface depending on the angle of incidence at the interface and the wavelength of the excitation radiation.

The evanescent wave in turn can interact with the medium, causing an electromagnetic field to the medium can return with higher refractive index, resulting in changes in the light continuing along the waveguide. Any molecular interaction occurs in the field (such as an analyte binding to an immobilized receptor on the surface of the waveguide) produces changes in the characteristics of the light propagating through the waveguide that can be measured and related with the concentration of analyte.

Evanescent wave: It is based on a total internal reflection fluorescence, which consists in the absorption and emission of photons.

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(a) The evanescent wave penetrates pass the fiber core surface and excites the labeled antibody–protein G complexes. Only the interaction between the analyte and the immobilized molecular complexes within the evanescent wave region can be sensed. (b) Conceptual schematic of a FRET immunosensor. When the antibody binds with the target antigen, a conformational change in the 3D structure of an antibody will occur, resulting in non-radiative energy transfer from the donor to acceptor fluorophores.

Evanescent wave

Plasmons are collective oscillations of the conduction electrons in a metal. The surface plasmon resonance occurs when a polarized light is directed from a layer of higher refractive index (a prism) to a lower index of refraction, a metallic layer of gold or silver, which lies between the prism and the sample. Light incident on the interface between the metal and the prism causes a surface plasmon excitation to a given angle of incidence called resonance angle. The resonance angle depends strongly on the refractive index of the medium adjacent to the metal sheet, so its variations will be proportional to the concentration. The binding of the analyte to the recognition element is a refractive index change on the surface of the metal and, therefore, a shift of the resonance angle. All biomolecules have refractive properties, so no labeling required

4.2 Surface plasmon resonance (SPR)

5 µm

2.5 µm

0 µm

5 µm

2.5 µm

0 µm

105.16 nm

52.58 nm

0 nm

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The surface is divided into 4 flow cells, which can be used individually or in a number of combinations.

Biomolecular Interaction Analysis BIACORE

Properties: -Aqueous environment (hydrogel, containing 97-98% water) -Mobility (chains are not cross-linked) -Efficient use of the evanescent field (thickness about 100 nm) -Increased sensitivity (more coupling sites than on a flat surface) -Allows covalent coupling (through carboxyl groups)

Sensor Chips

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The ligand, is immobilised on the dextran matrix of a sensor chip. The second interactant, the analyte, is injected in a continuous flow by means of a microfluidic system. Molecular association and dissociation events produce variations in the SPR signal that are recorded as resonance units (RU)as a function of time. The mathematical evaluation of the resulting curves, called sensorgrams, allows to calculate the kinetic parameters and the equilibrium affinity of the ligand-analyte interaction.

Change in SPR angle of 0.1= 1000 RU = 1 ng/mm2

Microbial cells as biocatalysts, possess certain advantages over the purified enzymes when used in biosensors:

cheap Longer half-life Less sensitive to inhibition, the pH and temperature self-healing capacity

5. Cell Biosensors

Disadvantages: Slower response Slower recovery rate Low selectivity easily disintegrating Milder conditions

Very useful when you require multiple steps or the presence of coenzymes Living or dead cells

Receptor-cell-transistor biosensor technology

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B cell lines were genetically engineered to express cytosolic aequorin, a calcium sensitive bioluminiscent protein from jellyfish, as well as membrane-bound antibodies specific for pathogens of interest (Rider et al., 2003) Cross-linking of the antibodies with specific pathogens increases intracellular calcium concentrations within seconds, causing the aequorin to emit light. This sensor was named CANARY (cellular analysis and notification of antigen risks and yields) and it has an excellent speed, sensitivity, and specificity.

•The CANARY Cell-based sensor detected as few as 50 CFU of Y. pestis in a total assay time of 3 minutes.

•The system was also tested in one food sample-E. coli O157:H7 in lettuce.

•500 CFU/g of E. coli O157:H7 in lettuce was detected in less than 5 minutes, including the sample preparation.

•1000 CFU of B. anthracis spores

It can distinguish pathogenic E. coli O157:H7 from nonpathogenic E. coli strains.

A B-lymphocyte cell line was encapsulated in a collagen gel matrix This assay measures alkaline phosphatase or lactate dehydrogenase released by cells infected with pathogens or exposed to different toxins. The system was tested using different strains of Listeria, listeriolysin O, and enterotoxins from Bacillus species.

Banerjee et al., Laboratory Investigation (2007) 1-11.

Biosensor Based on Immobilized Indicator Cells

Very fast (1-6 hours) Good sensitivity (MOI>10:1) Portability-collagen entrapped cells remain viable in 48 well plates for 48 hours.

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The main advantage of cell-based biosensors is that they provide information about the physiological effects of pathogens/toxins. Capable of distinguishing between viable pathogenic strains from nonpathogenic ones or dead cells. Sensitivity and specificity comparable to current methods. Expected to have broad applications in food testing, animal health, biodefense, disease diagnosis etc.