Immobilization Techniques for DNA

biosensor

LECTURE OF SUBJECT:

Dr. sharafaldin Al-musawi

College of Biotecholgy

LECTURE: 6SUBJECT: Biosensors & Biochips

LEVEL: 4

Immobilization Techniques for DNA biosensor

A functional group is a specific group of atoms within a molecule that is responsible for characteristic chemical reactions of that molecule.

There are different covalent attachment chemistries involving various functional groups on the surface of functionalization chip for DNA immobilization.

Among this factor we can refer to:

Carboxyl

Aldehyde

Epoxy

Isothiocyanate

Maleimide

Mercaptosilane

Functional group for DNA Immobilization

Carboxyl group

The carboxyl group is an organic functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group.

Another way to view it is as a carbonyl group (C=O) that has a hydroxyl group (O-H) attached to the carbon atom.

The carboxyl group is commonly written as -C(=O)OH or -COOH.

Reaction of carboxyl group with amine

The aminated DNA can Interact with immobilized carboxylate via

EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] OR

Sulfo-NHS (N-hydroxysulfosuccinimide).

The advantages and disadvantages of the reaction of carboxy functionalized surface with

aminated DNA

Advantages:

Simple method of immobilization High surface coverage of DNA’s Easy coupling reaction

Disadvantages:

Efficiency of immobilization depends on:

i. pHii. Concentrationiii. Ionic strengthiv. Reaction time.

Aldehyde group

Aldehyde: any of a class of organic compounds, in which a carbon atom shares a double bond with an oxygen atom, a single bond with a hydrogen atom, and a single bond with another atom or group of atoms.

The double bond between carbon and oxygen is characteristic of all aldehydes and is known as the carbonyl group.

The aminated DNA can Interact with immobilized

Aldehyde group in bio reception surfaces.

DNA DNA

Aminated DNA

Aldehyde group

Chemical bonding with aminated-DNA

+ H2O

Advantages:

Good stability Long hybridization time High binding strength

Disadvantages:

Limits the absolute signal intensity Stable enough for long term use High hybridization temperature

The advantages and disadvantages of the reaction of aldehyde functionalized surface with

aminated DNA

Epoxy group

An organic chemical that contains a group consisting of an oxygen atom bound to two already connected atoms, usually carbon.

A  functional group that containing an oxygen atom joined to two different groups that are themselves joined to other groups.

Double-strand DNA can be immobilized on the epoxy surface Because guanine bonds easily react with epoxy group.

Epoxy groups can intract with DNA via chemical (covalent) bonding with hydroxyl, amine and sulfhydryl groups.

Advantages:

Easy protocol for immobilization Good stability High binding strength Stable enough for long term use

Disadvantages:

Reactions between DNA and epoxy are extremely slow.

Low Immobilization density

The advantages and disadvantages of the reaction of epoxy functionalized surface with

aminated DNA

Isothiocyanate

Isothiocyanate is the chemical group –N=C=S, formed by substituting the oxygen in the isocyanate group with a sulfur.

DNA DNA

Isothiocyanate groups can interact with DNA via chemical (covalent) bonding with its aminated form.

H2

Advantages:

Well-ordered surface Re-usability High density DNA/area Stable enough for long term use

Disadvantages:

High non-specific hybridizations Long hybridization time

The advantages and disadvantages of the reaction of Isothiocyanate functionalized

surface with aminated DNA

Maleimide group

Maleimide is a chemical compound with the formula H2C2(CO)2NH.

Maleimide group can interact with DNA via chemical (covalent) bonding with its sulfhydrylated form.

DNADNA

Advantages:

Faster immobilization reaction Re-usability Good stability High binding strength

Disadvantages:

High non-specific interaction Degradation in aqueous solutions

The advantages and disadvantages of the reaction of maleimide functionalized surface

with sulfated DNA

Mercaptosilane group

• any of a class of odiferous sulfur containing compounds having the type formula RSH, where R is any hydrocarbon group.

Also called thiol.

Mercaptan

DNA SH

DNA S S

Mercaptosilane can perform chemical (covalent) bonding with DNA-SH.

H2

Advantages:

Stable enough for long term use Re-usability Good stability High binding strength

Disadvantages:

High non-specific interaction High hybridization temperature

The advantages and disadvantages of the reaction of mercaptosilane functionalized

surface with sulfated DNA