Immobilization Techniques in Biosensors

Posted on Mar 3, 2025 in Biology

Immobilization Techniques in Biosensors

Immobilization: The technique used for the physical or chemical fixation of cells, organelles, enzymes, or other proteins (e.g., monoclonal antibodies) onto a solid support, into a solid matrix, or retained by a membrane, in order to increase their stability and make possible their repeated or continued use.

Methods of Immobilization

The selective element must be connected to the transducer. This presents particular problems if the former is biological in nature. Several classes of methods of connection have evolved, as follows:

  • Adsorption: The simplest method is adsorption onto a surface.
  • Microencapsulation: Trapping between membranes – one of the earliest methods to be employed.
  • Entrapment: The selective element is trapped in a matrix of a gel, paste, or polymer – this is a very popular method.
  • Covalent attachment: Covalent chemical bonds are formed between the selective component and the transducer.
  • Cross-linking.

Probe Immobilization Techniques in Biosensors

The general process is as follows:

  1. Cleaning the biosensor substrate.
  2. Activation of the biosensor surface (if necessary for glass and silicon wafers).
  3. Functionalization of the biosensor surface.
  4. Immobilization of the biomolecule as a recognition part.

Preparation of Surface for Biomolecule Immobilization

After cleaning the surface of the biosensor:

  • Modification of the surface to create functional groups by:
    • Polymer Coating
    • Glow Discharge
    • Dipping
    • Spin Coating
    • Electrochemical deposition
    • Langmuir and Langmuir-Blodgett films
    • Chemical Modifications
    • Self-assembled monolayers (SAMs)

Self-Assembled Monolayers (SAMs)

SAMs are formed by simply immersing a substrate into a solution of the surface-active material. The driving force for the spontaneous formation of the 2D assembly includes chemical bond formation of molecules with the surface and intermolecular interactions.

Glass

Glass is one of the most preferred solid supports, due primarily to its low cost, low intrinsic fluorescence, transparency, resistance to high temperature, and a relatively homogeneous chemical surface.

Array Platforms

  • Surface-Based Substrates: Glass, Silicon, Gold, Polymeric, etc.
  • Biochips (DNA Array): Produced using bubble jet printing technology.
  • Protein-Chips

Patterning

  • Soft Lithography
  • Photolithography
  • Robotic printing (Microspotting; Ink-jet printing)
  • Lithography with AFM (Dip-pen lithography; Conductive AFM lithography; Nanoshaving/nanografting)

Lithography

  • UV Lithography
  • X-Ray Lithography
  • Electron Beam Lithography
  • Ion Beam Lithography
  • Nanoimprint Lithography
  • Scanning Probe Lithography

Lithography involves the transfer of a pattern to a photosensitive material by selective exposure to a radiation source such as light.

Atomic Force Microscope (AFM)

The AFM head employs an optical detection system in which the tip is attached to the underside of a reflective cantilever. A diode laser is focused onto the back of a reflective cantilever. As the tip scans the surface of the sample, the laser beam is deflected off the attached cantilever, and the photodetector measures the difference in light intensities between the upper and lower photodetectors, and then converts it to voltage.