1. Basic knowledge of sensors and biosensors
Jana Pekarkova, CEITEC BUT

2. What is a biosensor IUPAC Definition:
Biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a electrochemical transducer element.
Biosensors are devices capable of detecting specific biological analytes and convert their presence and concentration to another signal which can be easily analyzed, typically: electrical, temperature, optical.
Biosensors combine a biological component with a physicochemical detector.
Biochemical reactions can be mediated by:
isolated enzymes,
immunosystems,
tissue,
organelles
whole cells.

3. History of biosensors
1916: First report on immobilization of proteins: adsorption of invertase on activated charcoal
1922: First glass pH electrode
1956: Clark published his definitive paper on the oxygen electrode.
1962: First description of a biosensor: an amperometric enzyme electrodre for glucose (Clark)
1969: Guilbault and Montalvo – First potentiometric biosensor urease immobilized on an ammonia electrode to detect urea
1970: Bergveld – ion selective Field Effect Transistor (ISFET)
1975: Lubbers and Opitz described a fibre-optic sensor with immobilised indicator to measure carbon dioxide or oxygen.
1975: First commercial biosensor ( Yellow springs Instruments glucose biosensor)
1975: First microbe based biosensor, First immunosensor
1976: First bedside artificial pancreas (Miles)

4. History of biosensors
1980: First fibre optic pH sensor for in vivo blood gases(Peterson)
1982: First fibre optic-based biosensor for glucose
1983: First surface plasmon resonance (SPR) immunosensor
1984: First mediated amperometric biosensor: ferrocene used with glucose oxidase for glucose detection.
1987: Blood-glucose biosensor launched by MediSense ExacTech. HISTORY OF BIOSENSORS
1990: SPR based biosensor by Pharmacia BIACore
1992: Hand held blood biosensor by i-STAT
1996: Launching of Glucocard 1998: Blood glucose biosensor launch by LifeScan FastTake
1998: Roche Diagnostics by Merger of Roche and Boehringer mannheim
CURRENT: Quantom dots, nanoparicles, nanowire, nanotube, etc.

5. Basic principle of biosensor
Basic principle of biosensors involved three elements:
1) biological recognition element which is highly specific towards biological material analytes, integrated or connected to the physico-chemical transducer
2) transducer – transduces signal from biological target to electrical signal
3) amplification and detection - produce discrete or continuous digital electronic signal that is proportional to a specific analyte or group of similar analytes.

6. Elements of biosensors
Sample: The biological component or analyte which is under study
Transducer: A transducer is more generally defined as a device which converts energy from one form to another. Which is combination of,
Bioreceptor: The sensitive biological element a biologically derived material or biomimetic component that interacts (binds or recognizes) the analyte under study.
Electrical Interfaces: The detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into electrical signal form.
Electronic System: Combination of electronic devices i.e. Amplifier, signal processer and display device that are primarily responsible for the display of the results in a user-friendly way.

7. Elements of biosensor

8. Biological element Component used to bind the target molecule
Specifically interact with a target compound (compound to be detected)
Highly specific, stable under storage conditions and immobilized
Microorganism
Tissue
Cell
Organelle
Nucleic Acid
Enzyme
Enzyme Component
Receptor
Antibody

9. Physicochemical transducer
Acts as an interface, measure the physical change that occurs with the raction at the bioreceptor then transforms signal into measurable electrical output

10. Detector
Signals from the transducer are passed to a microprocessor where they are amplified and analyzed.

11. Basic characteristic of a biosensor
Linearity: Linearity of the sensor should be high for the detection of high substrate concentration
Sensitivity: Value of the electrode response per substrate concentration
Selectivity: Chemicals Interference must be minimized for obtaining the correct result
Response time: Time necessary for having 95% of the response

12. Advantages Highly specific
Independent of factors like stirring, pH, etc.
Linear response, Tiny and Biocompatible
Easy to use, Durable
Require only Small Sample Volume
Rapid, Accurate, Stable and Sterilizable

13. Principle of detection
Biosensors can be divided according to the type transducer into four basic groups:
1) Optical biosensors - can be further divided into
colorimetric,
fluorescent,
luminescent
interferometer;
2) Gravimetric biosensors - can be based on the piezoelectric effect or operate on the principle of acoustic waves;
3) Calorimeters (thermometric) biosensors;
4) Electrochemical biosensors, which includes biosensors:
amperometric,
potenciometric
conductometric,
impedimetric.

14. Optical biosensors These sensors measure:
the intensity of the emitted light (luminescence)
absorbance,
reflectance
change in refractive index
as a result of biological or chemical reactions.
 They exceed in:
sensitivity
selectivity.
Application:
determination of effective dose
of medicament
labeling and imaging

15. Optical biosenors
The most common method of detecting and quantifying biological compounds is still based on the fluorescence activity - fluorescent properties of most organic fluorophores are sensitive to environment changes.
They are proposed for general use - provide the possibility of multiple compounds detection within a single device
Fluorescence intensity is proportional to the intensity of excitation light, sample concentration and quantum yield
For excitation radiation is often used tungsten lamp (VIS), deuterium lamp (UV), xenon lamp (time-stable fluorescence), laser (time-variable fluorescence). Polarized radiation is used in fluorescence anisotropy.

16. Chemiluminiscence
emission of light (luminescence) as the result of a chemical reaction
Biosensor for NO determination using dye-labeled cytochrome c´ as bioreceptor
When the cytochrome c′ binds nitric oxide, there is a blue shift (∼10 nm) in the emission peak of the fluorescence spectrum and an damping of the overall fluorescence intensity
Biosensor for hydrogen peroxide detection
The enzyme horseradish peroxidase (HRP) catalyzes luminol oxidation by hydrogen peroxide and produces a high and durable chemiluminescence intensity compared with other catalyst.
Luminol + 2H2O2 → 3-aminophtalate + N2 + 3H2O + hv
HRP

17. Bioluminiscence Production and emission of light by a living organism
Form of chemiluminescence
The chemical reaction that results in bioluminescence requires two unique chemicals: luciferin and either luciferase or photoprotein.
Luciferin reacts with oxygen to create light:
CO2, adenosine monophosphate (AMP) and phosphate groups (PP) are released as waste products. Luciferase catalyzes the reaction, which may be mediated by cofactors such as calcium or magnesium ions
For some types of luciferin also the energy-carrying molecule adenosine triphosphate (ATP)
Firefly luciferin and luciferase are commercially available for measuring the amount of ATP in biological materials

18. Calorimetric biosensor
Many enzyme catalysed reactions are exothermic (generating heat)
This change in temperature is detected by the transducer
The amount of heat generated is proportional to the analyte concentration
The temperature changes are usually determined by thermistors at the entrance and exit of small packed bed columns containing immobilised enzymes within a constant temperature environment.
Under such closely controlled conditions, up to 80% of the heat generated in the reaction may be registered as a temperature change in the sample stream.
This may be simply calculated from the enthalpy change and the amount reacted.
Advantages: does not require frequent calibration,
Disadvantages: less sensitive to optical and electrochemical properties of the sample.
Application: for analysis of foods, pharmaceuticals, cosmetics.

19. Calorimetric biosensor
The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d).
From there, it flows past the reference thermistor (e) and into the packed bed bioreactor (f, 1ml volume), containing the biocatalyst, where the reaction occurs.
The change in temperature is determined by the thermistor (g) and the solution passed to waste (h).
External electronics (l) determines the difference in the resistance, and hence temperature, between the thermistors.

20. Gravimetric biosensor
Based on piezoelectric effect, use the basic principle of a response to a mass variation
Contains a piezoelectric element: usually quartz crystal coated with gold layer, which performs the function of the electrode
After applied electric field, crystal oscillated at a specific frequency
Quartz Crystal Microbalance (QCM)
On the surface of the gold layer, biologically active matter is deposited, which is capable binding the analyte from the solution. After binding the analyte to the active compound, weight (stiffness) of system increases, while the resonant frequency of oscillation proportionately decreases.

21. Gravimetric biosensor
Surface Acoustic Wave biosensor (SAW)
The sensor transduces an input electrical signal into a mechanical wave which can be easily influenced by physical phenomena.
The device can then transduces this wave back into an electrical signal.
Changes in amplitude, phase, frequency or time delay between input and output electrical signal can be used to measure the presence of the desired phenomenon.
Advantages: easy and cheap
Desadvantages: small specifity, selectivity and sensitivity
Usage: non-invasive technologies in diagnostic

22. Electrochemical biosensor
Measure electrical signal, which is generated during the biochemical interaction between the biologically active part and the substrate.
Potentiometric biosensors have ion selective electrodes that detect a change in voltage depending on the analyte concentration.
Amperometric biosensors measure current generated by applying constant potential between two electrodes.
Working electrode is formed either by noble metal or printed layer covered with biologically active comound.
Conductometric and impedimetric biosensors measuresthe change in electrical conductivity or resistance of the solution depending on the number of ions or electrons produced during biochemical reactions between biologically active component and the analyte.

23. Amperometric biosensor
Amperometric measurement at two- or three-electrode arrangement: working, counter (auxialiary) and reference electrode
A voltage (potential) is applied to the working electrode and between working and counter electrode potenecial difference is determined
The measured current changes as an electroactive analyte is oxidized at the anode or reduced at the cathode.
Current is stoichiometrically proportional to the concentration of the analyte (substrate).
The most common electroactive components: oxygen and hydrogen peroxide
Historically the first transducer in amperometric biosensors: Clark electrode, based on measurement of molecular oxygen unconsumed during the enzymatic reaction.

24. Biosensor with immobilized AChE
Solution with sensor → add substrate ATCh → ATCh is decomposed to form the current response
AChE catalysts decomposition ATCh to thiocholin (TCh):
AChE isolated from the electric organ eel is immobilized on the working electrode of the sensor
Active center of immobilized enzyme is blocked by adding inhibiting pesticide and current response decrease.
This model determines actual toxic effect of inhibitors on the nervous system.

25. Amperometric biosensor
Linear current-concentration characteristics are obtained from the amperometric measurements
Current (μA)
concentration
Concentration (%)
Potential (V)

26. Glucose biosensor Glucose detection:
Nanoporous composite film composed of zirconium oxide and Nafion (perfluorosulfonated ionomer) for capturing glucose oxidase on glassy carbon electrode covered with a thin layer of platinum
Nafion combined with zirconia allows faster response and greater sensitivity of the biosensor in comparison with biosensor composed only from Nafion or zirconia.
Achieve 95% of the current response in less than 5 seconds
Nafion is copolymer of tetrafluoroethylen (Teflon®) and perfluoro-3,6-dioxa-4-methyl-7-octene-sulfonic acid

27. Enzyme amperometric sensor of O2 and H2O2
Biosensors using enzymes oxidases as biorecognition element
Enzymes oxidized molecule of substrate (analyte) in the presence of oxygen, thus hydrogen peroxide or water is formed :
S – substrate
P – product
Fist type: a flavin coenzyme oxidase (usually yellow in color, e.g., glucose oxidase, lactate oxidase)
Second type: predominantly at tyrosinase oxidase (copper-containing enzyme

28. Biosensor with immobilized AChE
to detect toxic effects of some pesticides
Acetylcholinesterase (AChE) at nerve synapses of animal decomposes chemical messenger of impulses acetylthiocholine (ATCh)
organophosphorus and carbamate pesticides inhibit AChE → destruction of pests
active sites of enzyme AChE is inhibited by pesticides
ATCh is accumulated in synapse  continuous transmission of nerve impulses  nervous breakdown of coordination is occured
insect or mammal gets spasm and eventually dies

29. Glucose biosensor
Glucose reacts with glucose oxidase (GOD) to form gluconic acid
Two electrons and two protons are also produced
Glucose mediator reacts with surrounding oxygen to form H2O2 and GOD
Now this GOD can reacts with more glucose
Higher the glucose content, higher the oxygen consumption

30. Potentiometric biosensors
detecting potential on the interface between the metal and solution (liquid, solid)
based on measurements of ion concentration, but also neutral particles
record the change of potential on pH, which is proportional to the concentration of ions (e.g. protons) released during the enzymatic reaction

31. Potentiometric sensor
voltage change depending on the concentration of the analyte is given by the Nernst equation:
Electrode of the second kind: 1 reference and 1 ion selective electrode (ISE)
Potential of the working electrode versus the reference electrode ISE is measured
Current flow is very small, because of high output impedance of electrodes
Impedance matching electrodes: a low impedance connection of measuring circuits = simple impedance converter (amplifier with high input resistance)
kij is selective coefficient (the smaller, the higher concentration of the interfering ion is tolerated)

32. Ion selective electrode (ISE)
electrode with electro-active material → sensitivity to particular ions
sensitive part of the electrode - semipermeable membrane
reference electrode – e.g. Ag/AgCl - is often part of the measurement system (commercial systems - combined electrodes)
potential of the working electrode against reference electrode is measured against - it must be constant over time

33. Ion selective electrode
ISE membranes:
monocrystalline membranes for ISE sensitive to fluorides
polycrystalline membranes for ISE sensitive to cyanide or iodides
polymeric membranes consisting of two components:
Polymer provides mechanical mechanical support
Ionophore or other electroactive component establishes the desired electrochemical properties
PVA based membranes: excellent adhesion to the thick-film electrodes formed by Au or AgPd paste
- contain internal reference electrolyte,
- or are closely contacted directly on the electrode surface without an internal electrolyte solution

34. Immobilization of biorecognition parts on ISE
procedures depend on the type of the detected substances
application: environmental protection (water pollution control), medicine
ISE sensitive to ammonium ions (NH4+): determination of urine or amino acids by the enzyme urease
A) conductive mesh from graphite composite and resin covered with PVC matrix containing nonactin (i.e. Ionophore)
B) microencapsulation urease in sol-gel matrix of tetramethylorthosilicate
C) encapsulation of urease in a polymeric matrix of polyethyleneimine and poly(carbamoyl) sulfonate
In a similar manner it is possible in PVC membrane immobilized together with enzyme urease also creatinase and create bienzyme biosensor suitable for the determination of creatine in human serum.

35. Biosenzors according to the part of recognition layer
Enzyme: the most common, based on the catalytic and binding ability of enzymes enable specific detection (lock and key method).
Advantage: sensitivity
Products of the reaction catalyzed by the enzymes can be detected directly or using marker.
Whole cell: (or Tissue) use bacteria, fungi, yeasts, animal and plant cells.
They are based on the special properties of certain microorganisms such as bacterial metabolism or bioluminiescence.
Affinity: biorecognition parts can be antibodies, nucleic acids, aptamers, lectins or hormones.
RNA aptamer specific for biotin

36. Affinity biosensor Biosensors based on antigen/antibody system
Called immunosensors: use highly specific binding between an antigen and its antibody
This bond can be detected e.g. by fluorescence labeling, non-specific interactions have to be minimalized.
Biosensors with nucleic acid are based on the specific complementarity of DNA bases (A-T and G-C).
Due to this complementarity, biosensors may detect e.g. small amount of bacterial DNA by hybridization reaction that takes place between the searching DNA chain and known DNA chain in biorecognition membrane.
Biosensors based on biomimetic material: artificial or synthetic devices that mimic the function of organic biosensors, e.g. Aptasensors.

37. Modification of electrode surface
Biorecognition part can be directly captured on the electrode : incorporation into the electrode matrix (e.g. carbon paste), or captured in a matrix of conductive and redox polymers
Conductive polymers have a large number of conjugated π electrons responsible for conductivity, low ionization potential and a large electron affinity
polypyrrole, polythiophene, polyacetylene and polyphenylene
electrochemical synthesis of these polymers allows the direct deposition of the polymer on the electrode surface and also the incorporation of the enzyme, including mediators
Alternative: non conductive polymers e.g. polyphenol has isolated several aromatic rings without delocalization π electrons)

38. Modification of electrode surface
Carbon paste is a mixture of carbon powder (graphite) and binder component .
Carbon powder provides function of electrode or biosensor for electrochemical measurements.
Suitable materials have a particle size in micrometers and uniform distribution, high chemical purity and low adsorption capacity.
Type and quality of graphite as well as its quantity influence the resulting properties of the biosensor.
The most frequently: spectroscopic graphite, carbon black, acetylene black, glassy carbon.

39. Modification of electrode surface
Screen printed electrode: various types of inks depending on the applications:
low temperature - carbon based inks
high temperature - inks containing metal particles
The most common forms of electrochemical biosensors include electrode with carbon paste for discrete or continuous measurement.

40. Modification of electrode surface
The advantage of screen printing
Each layers (reference and auxiliary electrode, recognition layer or insulating layer) may be printed on the same surface
entire electrochemical cell is disposed on one sensor strip
sensor has a dimension of only a few centimeters
sensors can be produced in large quantities in identical conditions
need only a minimum amount of analyte (just one drop)

41. Electrochemical detection
Modified WE (CNTs, Cu2O)
printed, spray-coated
direct grown (PECVD, CVD)
Working electrode was modified by CNTs or Cuprous oxide nanoparticles via spray coating. Our colleagues also fabricated direct grown CNTs on silicone substrate using PECVD.
Prasek, J. et al. Nanoscale Research Letter (2011), 6, 385

42. Electrochemical detection of glucose
Cu2O preparation
Working electrode fabrication
Electrochemical detection of glucose
Electrochemical sensor with working electrode modified with Cu2O was used for electrochemical oxidation of glucose. A high blood glucose level may be a sign of prediabetes or diabetes mellitus. In alcaline solution (0,1M NaOH) oxidation of glucose took place. Enediol intermediate is formed, it is further oxidised to gluconalacetone and gluconic acid.
Electrochemical sensor with working electrode modified with CNTs was used for electrochemical of insuline. Insuline is composed of aminoacids like Triptofan and Tyrosin. In this case Tyrosin is oxidised on the surface of electrode.
(0.1M NaOH, 5mM glucose in 0.1M NaOH)

43. Electrochemical detection of insulin
Working electrode fabrication
Electrochemical detection of insulin
Calibration curve for insulin concentrations from 250 nM to 1.6 μM
CV response of MWCNTs/DMF modified electrode to 10 μM insulin solution
Electrochemical sensor with working electrode modified with Cu2O was used for electrochemical oxidation of glucose. A high blood glucose level may be a sign of prediabetes or diabetes mellitus. In alcaline solution (0,1M NaOH) oxidation of glucose took place. Enediol intermediate is formed, it is further oxidised to gluconalacetone and gluconic acid.
Electrochemical sensor with working electrode modified with CNTs was used for electrochemical of insuline. Insuline is composed of aminoacids like Triptofan and Tyrosin. In this case Tyrosin is oxidised on the surface of electrode.

44. Thank you for your attention!
This project is funded by the Norwegian Financial Mechanism. Registration number: NF-CZ07-ICP Name of the project: „Formation of research surrounding for young researchers in the field of advanced materials for catalysis and bioapplications“