The immobilization of the biorecognition system on the transducer is a critical step in biosensor development, as it enhances the ease of manipulation, operation, and reuse of the biosensor, leading to longer use times and potential cost savings.
Methods of Immobilization
Several methods have been developed since the 1960s, involving both physical and chemical processes:
Physical Methods
- Adsorption: This is one of the simplest methods, where the biorecognition element is physically adsorbed onto the transducer surface. However, it can be less stable and may lead to desorption over time[1][5].
- Entrapment: The biorecognition element can be entrapped within inorganic or organic gels, such as polyacrylamide or alginate. This method helps in retaining the biological activity of the element while preventing it from leaching out[1][5].
Chemical Methods
- Covalent Bonding: This involves forming covalent bonds between the biorecognition element and the transducer surface. Techniques such as cross-linking with agents like glutaraldehyde or using functional groups to attach the biological molecules are common. This method provides a stable and durable immobilization[1][5].
- Confinement within Semipermeable Membranes: The biorecognition element can be confined within semipermeable membranes, allowing the analyte to pass through while keeping the biological component in place[1].
Advanced Immobilization Techniques
Electro-Deposition
- This method involves the deposition of electronically conducting polymers, such as polypyrrole or poly(dicarbazole), onto microelectrodes. Enzymes like glucose oxidase (GOx) can be deposited in defined regions, making it suitable for multiplexed or array systems[4].
Photo-Polymerization
- Photoimmobilization using p-nitrophenylazides or photodeprotection is used for the fabrication of oligonucleotide and protein arrays. This method is precise and can be used to create complex patterns[4].
Soft Patterning Approaches
- Soft lithography techniques, such as microcontact printing or micromolding, are used for patterning proteins and cells. These methods allow for the precise placement of biological molecules on the transducer surface[4].
Advantages and Applications
- XY-Dimensionality: Techniques like electro-deposition, photo-polymerization, and soft patterning allow for immobilization with XY-dimensionality, making them applicable to multiplexed or array systems. This is particularly useful for high-throughput analysis and multi-analyte detection[4].
- Stability and Reusability: Chemical immobilization methods, such as covalent bonding, enhance the stability and reusability of the biosensor, reducing the need for frequent replacement of the biorecognition element[1][5].
- Minimized Interferences: Electropolymerized films can prevent interferences and electrode fouling, improving the overall performance of the biosensor[4].
Examples and Applications
- Enzyme-Based Biosensors: Enzymes like GOx have been immobilized using electro-deposition on microamperometric or microconductimetric electrodes, enhancing the sensitivity and selectivity of glucose biosensors[4].
- Protein and Oligonucleotide Arrays: Photoimmobilization has been extensively used for the fabrication of protein and oligonucleotide arrays, which are crucial in genomics, proteomics, and diagnostics[4].
- Cell and Tissue-Based Biosensors: Soft lithography techniques have been used to pattern cells and tissues, enabling the development of biosensors for monitoring complex biological interactions[4].
In summary, the choice of immobilization technique depends on the specific requirements of the biosensor, including the type of biorecognition element, the desired stability and reusability, and the need for multiplexed analysis. Advanced techniques such as electro-deposition, photo-polymerization, and soft patterning offer significant advantages in terms of precision, stability, and applicability to array systems.
