Outline
� Nomenclature
� Natural biorecognition elements
� Enzymes
� Antibodies
� Cell
� Semisynthetic biorecognition elements
� Nucleic Acid
� Aptamers
� Synthetic Recognition elements
� Imprinted polymers
Of what we will talk?
Main elements of a biosensor.
(a) Biorecognition element
(b) transducer
(c) amplifier
(d) Signal converter
(e) recorder
Biorecognition elements
� Tissues
� Microorganisms
�Organelles
� Cell receptors
� Enzymes
� Antibodies
� Nucleic acids
� Synthetic receptors
Nomenclature according to biorecognition element
Catalytic biosensors: Use enzymes.
Immunosensors: Use antibodies.
Genosensors: Use Nucleic acid.
Aptasensor: Use aptamers.
Enzymes
Protein or ribonucleproteins which present highly selective catalytic
properties towards specific substrates.
Why enzymatic reaction are important?
How does it work a biocatalyst?
Enzyme have very fast turnover
Glucose oxidase Process up to 900 molecule/s
Nomenclature
Enzymes are divided in families according to their activity.
Oxidoreductases: catalyze oxidation/reduction reactions (ex. Peroxidase, glucose oxidase, glucose dehydrogenase, Alcohol dehydrogenase)
Transferases: transfer a functional group (methylase)
Hydrolases: catalyze the hydrolysis of various bonds (ex. Urease, creatinase)
Lyases: cleave various bonds by means other than hydrolysis and oxidation
Isomerases: catalyze isomerization changes within a single molecule
Ligases: join two molecules with covalent bonds.
Some examples
D-glucose + H2O + O2 gluconic acid + H2O2
UREASE
(NH2)2CO + 2H2O + H+ HCO3- + 2NH4+ 2NH3 + 2H+
ALCOHOL DEHYDROGENASE
C2H5OH + NAD+ C2H5O + NADH
Generation of NADH: NADH NAD+ + 2e- + H+
GLUCOSE OXIDASE
How can I measure enzymatic reaction?
Several parameters can be measured to investigate
Enzyme-based biosensors:
• Consumption of a substrate (loss in O2 for glucose
oxidase),
• Generation of a product (H2O2 for glucose oxidase, NH4+
for the Urease, NADH in Alcohol dehydrogenase)
•Measure the alteration of a molecule (mediator) not
directly involved in the enzymatic reaction.
For example: GLUCOSE OXIDASE
D-glucose + H2O + O2 gluconic acid + H2O2
• Reduction in O2 concentration:
O2 + 4e- + 2H2O 4OH-
• Oxidation of H2O2 :
H2O2 2H+ + O2 + 2e-
• Use of Mediators:
D-glucose + 2 Medox+ gluconic acid + 2 Medred
Electrons flow in mediated and not mediated sensors
Cass, A.E.G., Davis, G., Francis, G.D., Hill, H.A.O., Aston, W.J., Higgins, I.J., Plotkin, E.V., Scott, L.D.L. and
Turner, A.P.F. (1984) Ferrocene-mediated enzyme electrode for amperometric determination of glucose.
Analytical Chemistry 56, 667-671.
Antibodies
Antibodies (also known as immunoglobulin) are proteins produced by B cells (a type of white blood cell) and that are used by the immune system to identify foreign substances (e.g. bacteria, virus)
Antibodies are specifically binding to a unique part (epitope) of a target molecule (antigen).
The binding interaction between antibody and antigen is called “affinity binding”
Basic Structure
1. Variable region (Fab)
2. Constant region (Fc)
3. Heavy chain: contain a
variable domain (VH) and a
constant domain (CH1), a
connection chain, and two more
constant domains (CH2 y CH3).
4. Light chain with a variable
domain (VL) and a constant
domain (CL)
5. Recognition region
6. Connection region.
Cabohydrate
chain
Speceficity of Antibodies
Some important facts
Fab is the portion of the antibody binding the antigen;
Fc portion mediates function, eg. Incorporation in the membrane of B cells
Five main classes or isotypes: IgG, IgA, IgD, IgM, IgE are known.
Andibodies may exist as monomers, dimers (eg. IgA) and pentamers(eg. IgM)
Antibodies can be monoclonal or monoclonal.
Monoclonal Antibodies (Mab)
When B-cells are activated to proliferate and produce antibody, the progeny of a single parent cell will all produce antibody with the same specificity. The response of these progeny is monoclonal (This can be manipulated in vitro).
Polyclonal Antibodies
antigen
However, exist different B-cells can produce antibodies against the same antigen, but against a different epitope, with slightly different specificity, and in this respect, the total response is polyclonal (mixture of antibodies).
Antibodies production
Polyclonal Monoclonal
How to use antibodies in biosensors
Enzyme-Linked ImmunoSorbent Assay
ELONA
Electrochemical Immunosensor
DNA
Genome: The to totality of the genetical information of a cell/organism.
Nucleic Acid: basic constituent of the DNA.
Gene: Segment of the DNA that code the production of a protein.
DNA Structure
The unique chemical and recognition properties of nucleic acid allow the development of biosensors that can provide specific qualitative and semi-quantitative analytical information.
Highly chargedHydrophilic
Hydrogen boundHydrophobic
Purines-Pyrimidines interaction
Purines Pyrimidines
DNA Double Helix
• Double helix structure of
DNA was published by
James Watson and
Francis Crick in 1953
• Two polynucleotide
chains held together by
H bonds between bases
• Antiparallel strands
Use of DNA: hybridisation assay
Whole cell biosensor
Microorganisms engineered to react to the presence of
chemical signals with the production of an easily
quantifiable marker protein.
The regulatory system in the bacterial cell is exploited to
drive expression of a specific reporter gene, such as
bacterial luciferase, green fluorescent protein, beta-
galactosidase.
This is achieved by fusing the DNA for a promoter-less
reporter gene to an extra copy of the selected
regulate-able promoter and introducing this
construction into the bacterial or yeast cell
Examples include heavy metal resistance (heavy metal
sensors), organic compound degradation (organic
compound sensors), cellular stress responses (to
obtain general toxicity sensors) and DNA damage
repair (mutagenicity).
Application of cell based sensors
http://www-analytik.chemie.uni-regensburg.de/wegener/nanoscreen.htm
Toxicology
Drugs screening
Cell stress
Biocompatibility studies
Semisynthetic receptors: Aptamers
Aptamers are artificial nucleic acid (RNA/DNA) ligands that can be generated against amino acids, drugs, proteins and other molecules.
Name derives form the Latin word ‘aptus’, which means ‘to fit’.
They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and amplification, called systematic evolution of ligands by exponential enrichment (SELEX).
Aptamers are proposed as alternatives to antibodies as biorecognitionelements in analytical devices with ever increasing frequency.
James, W. (2000) Encyclopedia of Analytical Chemistry, Ed. R.A. Mayers, pp. 4848-4871.
Systematic Evolution of Ligands by EXponential enrichment (SELEX)
Advantages of Aptamers
Reporter molecules can be adjusted to aptamers at precise locations not involved in binding
Labelling of antibodies can cause loss in affinity
Aptamer generation does not require animals
Requires the use of animals
Aptamers are produced by chemical synthesis resulting in little or no batch to batch variation
Antibodies often suffer from batch to batch variation
Selection conditions can be manipulated to obtain aptamers with properties desirable for in vitro assays
Identification of antibodies that recognize targets under conditions other than physiological is not feasible
Denaturated aptamers can be regenerated within minutes, aptamers are stable to long-term storage and can be transported at ambient temperature
Antibodies have limited shelf life and are sensitive to temperature and may undergo denaturation
Kinetic parameters such as on/off rates can be changed on demand
Kinetic parameters of Ab-Ag interactions cannot be changed on demand
Toxins as well as molecules that do not elicit good immune response can be used to generate high affinity aptamers
Limitations against target representing constituents of the body and toxic substances
AptamersAntibodies
Reporter molecules can be adjusted to aptamers at precise locations not involved in binding
Labelling of antibodies can cause loss in affinity
Aptamer generation does not require animals
Requires the use of animals
Aptamers are produced by chemical synthesis resulting in little or no batch to batch variation
Antibodies often suffer from batch to batch variation
Selection conditions can be manipulated to obtain aptamers with properties desirable for in vitro assays
Identification of antibodies that recognize targets under conditions other than physiological is not feasible
Denaturated aptamers can be regenerated within minutes, aptamers are stable to long-term storage and can be transported at ambient temperature
Antibodies have limited shelf life and are sensitive to temperature and may undergo denaturation
Kinetic parameters such as on/off rates can be changed on demand
Kinetic parameters of Ab-Ag interactions cannot be changed on demand
Toxins as well as molecules that do not elicit good immune response can be used to generate high affinity aptamers
Limitations against target representing constituents of the body and toxic substances
AptamersAntibodies
Applications
Examples of possible targets Aptamer for detection of cancer cells
Synthetic Receptors
Scheme of 3- and 2-Dimensional Imprinting Polymerisation
Cross-linked polymer formed around a molecule that acts as a template, template
subsequently removed. Imprints containing functional groups complementary to those
of template remain in the polymer
Virtual library
2-(DIETHYLAMINO)ETHYL METHACRYLATE(DEAEM)
O
O
N
STYRENE
N
4-VINYLPYRIDINE
p-DIVINYLBENZENE
ALLYLAMINE
NH2
ACRYLONITRILE
C N
ACRYLIC ACID ACRYLAMIDE ACROLEIN
H
O
NH2
O
OH
O
2-HYDROXYETHYL METHACRYLATE
O
O
OH
2-ACRYLAMIDO-2-METHYL-1-PROPANESULFONIC ACID
(AMPSA)
2-VINYLPYRIDINE 1-VINYLIMIDAZOLE
NN N
O
NHSO3H
H
UROCANIC ACID ETHYL ESTER
(UAEE)
ETHYLENE GLYCOL DIMETHACRYLATE
(EGDMA)
OO
O
OO
O
N
N
N,N-METHYLENEBISACRYLAMIDE
m-DIVINYLBENZENE
NH
O
NH
O
ITACONIC ACID(IA)
OH
OHO
O
METHACRYLIC ACID(MAA)
OH
O
UROCANIC ACID(UA)
O
HO
N
N
TRIFLUOROMETHACRYLIC ACID(TFMAA)
OH
OCF3
How to prepare them: bulk preparation
Example of application
Summary
• We have learned which are the possible recognition elements that can be used for biosensors.
• We learned that the specific biological function of biomolecules can be used for performing biorecognition.
� Enzyme/substrate
� DNA/DNA
� Antibody/antigen
• Biomolecules/organisms can be engineered to perform recognitions that are not present in nature
� DNA (Aptamers) /Proteins; DNA (Aptamers)/ small molecules
� Genetically modified cells
• Synthetic materials can be used to mimic biorecognition.
� Molecular imprinted polymers