by Radha Gupta and Prof. Ashok Kumar
Over the years sol-gel technology has been explored in order to develop biosensing devices, due to its ambient processing conditions, physical rigidity, chemical inertness and high photochemical and thermal stability. The combination of sol-gel technology with a molecular imprinting technique allows the development of biosensors with the greater sensitivity and selectivity necessary for sensing applications. However, the sol-gel glassy porous matrix limits the diffusion of larger analytes such as high molecular weight proteins and whole cells, due to shrinkage of the gels. As an alternative, cryo-gels (supermacroporous hydrogels) are attractive matrices that allow unhindered diffusion of analytes due to their interconnected systems of macropores (up to 200µm), and their spongy and elastic morphology. These features, in combination with a molecular imprinting technique, provide a new approach for various biotechnological and environmental applications.
The field of biosensor development is fast becoming one of the most exciting areas of research. This is because of the large number of potential applications for these important devices in a variety of fields, including clinical diagnostics, defence, environmental control, drug discovery and the agricultural industries. In addition exciting opportunities for new applications for biosensors are continuously emerging. Compared to conventional analytical methods, biosensors have the potential advantages of specificity, speed, low cost, portability and high sensitivity.
What is a biosensor?
A biosensor consists of an immobilised biological sensing material coupled to a transducer producing a signal proportional to the concentration of the analyte. Essential components include the biological recognition element (bioselector/biomediator), physical transducer (transductor), signal amplifier (e.g., electrical amplifier) and a data processing system. A bioselector is any biologically active substance such as an enzyme, protein, antibody, DNA or a microorganism, which is capable of recognising specific analytes. The processes of recognising specific analytes leads to changes in physical and chemical properties that can be monitored by a suitable transducer system, giving measurable signals. Thus, the transducers can convert the biological and chemical changes into useful electronic data. Depending upon the detection techniques, different types of transducers viz. electrochemical (potentiometric/amperometric), optical, piezoelectrical and calorimetrical are reported in the literature [1].
Despite the enormous number of biosensors under development, few practical systems are commercially available. The two aspects that are the most problematic in developing biosensensors are the incorporation of the bioselector into a suitable matrix and quantitating the interactions between the analytes and the sensing molecules. Immobilisation is a key step in the development of biosensors. It not only helps to achieve the required close proximity between the bioselector and the transducer, but also helps to stabilise the bioselector for reuse. Various immobilisation techniques such as adsorption to solid supports, covalent attachment, cross-linking and entrapment have been applied in different polymeric matrices. No single method or material has emerged as the standard for successful applications.
Sol-gel technology
Sol-gel entrapment has been explored over the years in an attempt to achieve a reproducible and robust immobilisation technique for developing biosensors. This inorganic material is particularly attractive for optical sensing applications due to its simple, ambient processing conditions and the possibility of tailoring it for specific requirements. Sol-gels are inorganic-based, polymeric materials formed by acid- or base-catalysed hydrolysis and condensation of metal alkoxides with a mutual cosolvent. Usually alkoxide precursors such as tetramethyl-orthosilicate (TMOS) and tetraethyl-orthosilicate (TEOS) are used for the preparation of a glassy matrix. The dynamics of the sol-gel process are dependent on various physical and chemical properties of the composition of the sol-gel viz., water to precursor ratio, types of catalyst, choice of precursors, pH, temperature and solvent. The physico-chemical properties of the internal environment change as a result of the initial conditions as well as storage (aging). The variation in these properties over time needs to be understood and tailored to specific biosensor applictions. Recently the use of various organically modified silane precursors (ORMOSILS) viz. methyltriethoxysilane (MTES), propyltrimethoxysilane (PTMS), or dimethyldimethoxysilane (DMDMS) etc. in sol-gel preparation has lead to organically modified sol-gel glasses with tailored features. Sol-gels have been used to develop a number of chemical sensors and biosensors because they can entrap recognition elements, such as pH indicators, proteins, enzymes, antibodies and whole cells. Such biosensors however, have limitations of long-term stability and reusability [2,3]. Molecular imprinting is a versatile method for creating macromolecular matrices that display selective molecular recognition behaviour. Compared with entrapment of biological molecules in sol-gel, molecular imprinting in sol-gel results in biosensors with the greater sensitivity and selectivity necessary for sensing applications.
Molecular imprinting and sol-gel
In molecular imprinting, functional monomers are associated with a template, which is fixed into the polymeric matrix by polymerisation. After removal of the template, the functional groups of the polymeric matrix can then rebind the same template or its analogue. The association between the imprint molecule and the monomers is based on two types of interactions viz. non-covalent and covalent interactions. The non-covalent interactions include hydrogen bonds, ionic bonds, hydrophobic interactions and Van der Waals forces and this approach has been pioneered by Mosbach [4]. The molecular imprinting technique can be applied to different kinds of target molecules, ranging from small organic molecules (eg. pharmaceuticals, pesticides, amino acids and peptides, nucleotide bases, steroids and sugars) to polypeptides, high molecular proteins and even whole cells. Molecularly imprinted materials possess inherent advantages, including robustness, low cost and potential utility in situations where no bioselector molecule is available, as well as an extremely long shelf life without any need for special storage conditions. Sol-gel based materials are ideal for designing and synthesising imprinted materials. Due to a high degree of cross-linking in the (SiO2)n network as well as their rigidity, sol-gel based materials retain the size and shape of the cavities created by the template after its removal. The sol-gel method also provides an efficient way to incorporate organic components into inorganic polymeric materials under mild thermal conditions, and to cast different configurations such as monolith, thin films and powder etc. The availability of a range of pure functional monomers makes their use in molecular imprinting more attractive [5]. The use of imprinted films in sensor technology is advantageous compared to use in bulk imprinted polymers. The progress in the development of sol-gel based molecular imprinted polymers and their applications can be seen from the growing number of publications. Few reports are available on molecular imprinting of proteins viz. human serum albumin in sol-gel; limitation of diffusion due to shrinkage of the gels is a problem [6]. Imprinting of larger biomolecules such as proteins and cells is still a challenge as it requires a macroporous matrix and the unhindered diffusion of analytes within the polymeric matrix.
Cryo-gels: supermacroporous polymeric matrices
An alternative is cryo-gels, supermacroporous polymeric matrices that are synthesised at subzero temperatures. An interconnected systems of macropores (up to 200µm) and the spongy and elastic morphology of cryo-gels allow unhindered diffusion of analytes. These features, in combination with osmotic, chemical and mechanical stability, result in matrices which are eminently suitable for large entities such as protein aggregates, membrane fragments, cell organelles and even whole cells. Applications include cell separations, cell culture and cell-biomaterials. Cryo-gels can be produced from hydrophilic and hydrophobic monomers or polymeric precursors, and can be designed in different sizes and formats (monoliths, sheets, discs, micro-titre plate formats, etc) depending upon the application and scale of the operation [7,8].
Many biomedical applications require viable cells isolated from complex mixtures. The elasticity of cryo-gel along with the continuous porous structure make cryo-gel monoliths highly suitable for applications in affinity cell separation. Under mild conditions high recovery of captured cells with high viability is ensured [9]. Specific ligand coupling for immobilisation as well as matrix architecture also affect the affinity binding of cells to cryo-gel adsorbents. The binding of cells to monolithic cryo-gel was reported to be higher (90–95%) than with the traditional bead methods used for cell separations (76%) [10]. A new concept for the preparation of selective sorbents with high flow path properties has been reported recently [11], where molecular imprinted polymers (MIPs) were embedded into various macroporous gels (MGs). The macroporous composite systems, consisting of MG monoliths embedded with 17β-estradiol MIP particles, were prepared at subzero temperature and tested for trace concentrations of 17β-estradiol (considered as the most important source of endocrine compound interference) from water and from waste-water.
In conclusion, cryo-gels can be more efficiently prepared by applying molecular imprinting techniques, and are useful for various biotechnological applications. Cryo-gels are now becoming commercially available from Protista Biotechnology AB, Lund, Sweden (http://www.protista.se/). We can thus look forward to using molecular imprinting in sol-gel and cryo-gel for various biomedical, bioengineering and environmental applications.
References
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3. Gupta R, Chaudhury NK. Biosens Bioelectron 2007; 22: 2387-99.
4. Mosbach K. Trends Biochem Sci 1994; 19:9-14.
5. Dai S, Shin YS, Barnes CE, Toth LM. Chem Mater 1997; 9: 2521-25.
6. Zhang Z, Long Y, Nie L, Yao S. Biosens Bioelectron 2006; 21: 1244-51.
7. Kumar A, Bansal V, Nandakumar KS, Galaev IY, Roychoudhury PK, Holmdahl R, Mattiasson B. Biotech Bioengg 2006; 93: 636-46.
8. Lozinsky VI, Galaev IY, Plieva FM, Savina IN, Jungvid H, Mattiasson B. Trends in Biotech 2003; 21: 445-51.
9. Dainiak MB, Kumar A, Galaev IY, Mattiasson B. PNAS 2006; 103: 849–54.
10. Kumar A, Rodrıguez-Caballero A, Plieva FM, Galaev IY, Nandakumar KS, Kamihira M, Holmdahl R, Orfao A, Mattiasson B. J Mol Recognit 2005; 18: 84–93.
11. Le Noir M, Plieva F, Hey T, Guieysse B, Mattiasson B. J Chromatogr A 2007; 1154: 158-64.
The authors
Dr Radha Gupta,
Professor Ashok Kumar,
Department of Biological Sciences
& Bioengineering,
Indian Institute of Technology-Kanpur,
Kanpur-208016, Uttar Pradesh
India
e-mail: ashokkum@iitk.ac.in
