Biosensor: Biorecognition elements

2015-02-03

Valerio.beni@liu.se

Outline

� Nomenclature

� Natural biorecognition elements

� Enzymes

� Antibodies

� Cell

� Semisynthetic biorecognition elements

� Nucleic Acid

� Aptamers

� Synthetic Recognition elements

� Imprinted polymers

Of what we will talk?

Biorecognition elements

� Tissues

� Microorganisms

�Organelles

� Cell receptors

� Enzymes

� Antibodies

� Nucleic acids

� Synthetic receptors

Nomenclature according to biorecognition element

Catalytic biosensors: Use enzymes as biorecognition

element.

Immunosensors: Use antibodies as biorecognition element.

Genosensors: Use Nucleic acid as biorecognition element.

Aptasensor: Use aptamers as biorecognition element.

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 an unique part (epitope) of their specific 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.

• High variability in the Fab region allows more efficient recognition of the foreigner substances.

• Fc portion mediates function, eg. Incorporation in the membrane of B cells and determine the antibody class.

• 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 divided in monoclonal or polyclonaldepending on their recognition abilities.

Monoclonal Antibodies (Mab)

When B-cells are activated toproliferate and produceantibody, the progeny (cellderived from) of a single parentcell will all produce antibodywith the same specificity(recognise a single epitope ofthe antigene). The response ofthese progeny is calledmonoclonal.

Polyclonal Antibodies

antigen

However, different B-cells canproduce antibodies against thesame antigen, but these willbe against different epitopes,with slightly different specificity,and in this respect, the totalresponse is polyclonal (mixtureof 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 can be engineered to react to the presence

of chemicals with the production of an easily quantifiable

marker protein.

This can be achieved by introducing in the microorganism a

specific reporter gene that will be overexpressed in the

presence of the targeted molecule.,

Example of this reporter are: bacterial luciferase, green

fluorescent protein, beta-galactosidase.

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

Cell/microorganism adesion or methabolic response can be used to perform animal free studies on:

• 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 biorecognition elements in analytical devices with ever increasing frequency.

Aptamer interact with their specific target via structural and chemical (hydrophobic hydrophylic interaction, electrostatic interaction, hydrogen bond, van der Waals forces).

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 (molecularly imprinted polymers)

• Molecular imprinted polymers are synthetic polymers formed around a

molecule (target) that acts as a template.

• The imprinting process generate polymers containing spatially organised

functional groups complementary to those of the template.

• Removal of the template results in a polymer able to recognise the analyte.

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 synthesis (macro to nano)

In-situ electrodeposition

Examples of application for MIP

Detection of folic acid via QCM measurements

Adv. Mat. Lett. 2011, 2(4), 264-267

Replacing antibodies in ELISA

Anal. Chem. 2013, 85, 8462−8468

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