Immobilization Techniques for DNA biosensor LECTURE OF SUBJECT: Dr. sharafaldin Al-musawi College of Biotecholgy LECTURE: 6 SUBJECT: Biosensors & Biochips LEVEL: 4 Immobilization Techniques for DNA biosensor

Immobilization Techniques for DNA biosensor

Functional group for DNA Immobilization 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

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).

Simple method of immobilization High surface coverage of DNA’s 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: pH Concentration Ionic strength 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.

Chemical bonding with aminated-DNA The aminated DNA can Interact with immobilized Aldehyde group in bio reception surfaces. DNA Aminated DNA Aldehyde group Chemical bonding with aminated-DNA + H2O

Long hybridization time High binding strength The advantages and disadvantages of the reaction of aldehyde functionalized surface with aminated DNA Advantages: Good stability Long hybridization time High binding strength Disadvantages: Limits the absolute signal intensity Stable enough for long term use High hybridization temperature

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.

Epoxy groups can intract with DNA via chemical (covalent) bonding with hydroxyl, amine and sulfhydryl groups. Double-strand DNA can be immobilized on the epoxy surface Because guanine bonds easily react with epoxy group.

Easy protocol for immobilization Good stability High binding strength The advantages and disadvantages of the reaction of epoxy functionalized surface with aminated DNA 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

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

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

Stable enough for long term use Disadvantages: The advantages and disadvantages of the reaction of Isothiocyanate functionalized surface with aminated DNA Advantages: Well-ordered surface Re-usability High density DNA/area Stable enough for long term use Disadvantages: High non-specific hybridizations Long hybridization time

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.

Faster immobilization reaction Re-usability Good stability The advantages and disadvantages of the reaction of maleimide functionalized surface with sulfated DNA Advantages: Faster immobilization reaction Re-usability Good stability High binding strength Disadvantages: High non-specific interaction Degradation in aqueous solutions

Mercaptosilane group Mercaptan any of a class of odiferous sulfur containing compounds having the type formula RSH, where R is any hydrocarbon group. Also called thiol.

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

Stable enough for long term use Re-usability Good stability The advantages and disadvantages of the reaction of mercaptosilane functionalized surface with sulfated DNA Advantages: Stable enough for long term use Re-usability Good stability High binding strength Disadvantages: High non-specific interaction High hybridization temperature