by Dr Soichi Yabuki
Immobilisation of biomolecules is a key technology for using these molecules in industry. High-performance biosensors can be obtained by employing suitable immobilisation methods. Two types of immobilisation method for obtaining high-performance biosensors are introduced in this article. One involves biomolecule immobilisation in a novel matrix, a polyelectrolyte complex membrane, and the other involves protein engineered biomolecules immobilised on substrate plates with the same orientation.
High-performance biosensors
To overcome the limitations of using biosensors for research applications, improvements in their performance are necessary. These improvements relate to properties such as sensitivity, selectivity, response time and stability. Sensitivity and selectivity are especially important, and require most attention as biosensors are developed further. This article describes various methods that can be used to enhance the selectivity and sensitivity of electrochemical biosensors.
There are usually two ways to address these issues of selectivity and sensitivity: one is to improve the matrix (i.e., addition of new function to the matrix), and another is to improve the biomolecules involved by technologies such as protein engineering. This article outlines these two ways whereby such improvements can be made.
Matrices for selective and sensitive biosensors
Changing the physicochemical property of the immobilisation matrix for biomolecules is effective for constructing high-performance biosensors. In particular biomolecules that are buried in the matrix are more affected than those on the matrix. For example, if electro-conductive properties can be added to the polymer matrix, biomolecules can be controlled electrochemically [1]. For instance, a biomolecule, glucose oxidase was immobilised in an electro-conductive polymer, polypyrrole, and the activity of the enzyme was controlled electrochemically.
In order to develop effective high-selective biosensors, a perm-selective material can be incorporated between enzyme and solution. Similarly, the production of high-sensitive biosensors can be accomplished with high-loading biomolecules in the matrix. To satisfy both requirements, a novel immobilisation matrix, polyelectrolyte complex membrane, is a suitable material [2].
Polyelectrolyte complex membrane as the immobilisation matrix
Polyelectrolyte complex membrane consists of two polyelectrolytes, a polyanion (negatively-charged polyelectrolyte in neutral solution) and a polycation (positively-charged polyelectrolyte). In the presence of both polyelectrolytes, polyanion and polycation associate spontaneously due to a electrostatic binding force, and form inter-polymer complexes [Figure 1a]. Once the complex forms, the complex cannot dissociate. Biomolecules will be immobilised if the biomolecules co-exist during the complex formation [Figure 1b].
For the preparation of the complex, some polyelectrolytes such as weak poly-acids (polyanion) and poly-bases (polycation) can be used. Poly(allylamine), poly-L-lysine (PLL), poly-L-arginine and polyethylene imine are usually used as polycations, whereas poly(styrenesulphonate) (PSS), poly(vinyl sulphonate), polyglutamate and deoxyribonucleic acid are used as polyanions. There are two main methods for the preparation polyelectrolyte complex membranes. The first method employs the formation of a multilayer, layer by layer, on a solid surface. The second method is a simple preparation method: simultaneous mixing of polyanion and polycation solutions. For this method, the membrane obtained is termed a polyion complex membrane (PIC). The PIC can be formed on any surface of electrodes such as platinum, gold and carbon.
If biomolecules co-exist when polyelectrolytes are simultaneously mixed, the PIC membrane will contain the biomolecules. The immobilised biomolecules retain their activity, i.e., immobilised enzymes demonstrate enzyme activity in PIC. Biomolecules can also be immobilised on the surface of PIC, because the amino group of the polycation (or the carboxyl group of the polyanion) can be used as the covalent bonding site between the polyelectrolyte and biomolecules.
It was found that a PIC membrane that consists of PLL and PSS showed perm-selectivity of solutes [3]; small solutes, with molecular weights lower than 100, can penetrate the PIC membrane easily, whereas larger solutes are excluded by the PIC. In consequence, larger solutes cannot reach the immobilised enzyme and base electrode. This permeation property can enhance the selectivity of solutes and the signal-to-noise ratio (S/N) of the biosensors.
A biomolecule-immobilised PIC membrane can be obtained when a solution of the biomolecules is added during the polyelectrolyte mixing process. In this process, the quantity of biomolecules added influences the degree of immobilisation, i.e., the enzyme activity of the membrane is influenced by the amount of enzyme added. If a high-sensitive enzyme biosensor is needed, the amount of enzyme added can be increased.
As described above, high-performance (high-selective and sensitive) biosensors can be obtained by enzyme-immobilised PIC membranes. The method used to prepare enzyme-immobilised PIC membranes is the simultaneous mixing of polyelectrolytes and biomolecules; actually, a polyelectrolyte solution and enzyme solution are mixed on a supporting plate, and then a counter polyelectrolyte solution is added to the mixture. If the enzyme solution contains other chemical substances, the substances will be immobilised in the PIC. For the (electron) mediators of enzymes, the method can be applied for immobilising the mediators, and these immobilised mediators can even function in the PIC, because the degree of mobility of the mediators is similarly retained in the membrane.
Some electrochemical enzyme biosensors can be constructed using the method for preparing PIC on electrodes, including biosensors for measuring L-lactate, ethanol, amino acids and hydrogen peroxide. If the enzyme substrates are larger than can penetrate the PIC, the problem can be solved by the immobilisation of the enzyme on the surface of the PIC. By immobilising the enzyme in this way, several sensors, such as for glucose, fructose and amino acids, can be obtained. As the resulting biosensors demonstrate less signal for electrochemical interferants such as L-ascorbate, urate and acetaminophen, S/N is improved by using a PIC membrane as the matrix. Moreover, there are several advantages to using PIC; response time and long-term stability are improved.
Unique biosensors based on perm-selective properties
As describe above, PIC exhibits perm-selective properties with solutes. For an amino acid biosensor using PIC, because the penetration of solute would be different from the penetration of amino acids, the activity (sensor response) depends on the size of amino acids. Different sizes of amino acids distribute around the inflection point due to the permeation property, i.e., small amino acids will penetrate through the PIC membrane, whereas large amino acids reach the surface of the PIC, but do not penetrate it [Figure 2a]. In contrast, the sensor activity is not influenced by the penetration of the PIC, which uses an enzyme attached on the surface of the membrane. Differentiation between small D-amino acids and large D-amino acids can be achieved by two enzyme (D-amino acid oxidase)-immobilised PIC membranes [Figure 2b]; the response signals of the two sensors are not different with small D-amino acids such as D-alanine and D-serine, whereas there are large differences between the signals of the two sensors with large D-amino acids such as D-methionine and D-phenylalanine. Using two biosensors, the size of the substrate can be distinguished.
Progress of immobilisation for high-performance biosensors using protein engineering techniques
There are some potential methods for attaining high-performance biosensors that have high selectivity and high sensitivity. Protein engineering, via the substitution of some amino acid residues in enzymes combined with genetic engineering is being used to modify or change biomolecules’ properties. This method is effective in some cases, however, it is difficult to find which mutation improves a biomolecule's sensitivity and selectivity. At the current stage, because the mutation site differs for different biomolecules, the method is not widely applicable, but some research is being initiated in this field.
Immobilised biomolecules with the same orientation and monolayer formation are effective and completely functional. To achieve this formation of biomolecules, binding between base plate and biomolecules should be initiated at the specific amino acid residue of the biomolecules; the replacement of amino acid residues with a new function could be accomplished by enzyme engineering. Fortunately the amino acid cysteine is known to be a suitable binding reagent with gold; the addition of cysteine to the N- or C-terminals is effective for the binding of biomolecules to gold plate [4]. Nowadays, some peptides that bind to various base plates have been reported. By connecting the peptide sequence with the biomolecules, the modified biomolecules can be immobilised on the correspond base plate [5]. Using these methods, immobilised biomolecules with the same orientation and monolayer formation can be obtained.
Future perspectives
To obtain high-performance biosensors, it is important that novel functions of materials are applied to biosensor construction. Along with the progress of material chemistry, novel materials, which can be applied for the biosensor matrix, can be found. In the case of a PIC, different properties of PIC membranes can be obtained by changing the components of the membranes. In fact, the permeability of charged species through the PIC membrane can be designed by changing the component ratio of the membrane. Permeation properties such as the permeation profile and perm-selective property could be changed by using novel polyelectrolytes which are designed for a new function. By using these new PIC membranes, higher-performance biosensors will be constructed.
For protein engineering, currently it is difficult to find which mutation improves a biomolecule's sensitivity and selectivity. In the near future, the appropriate biomolecule modifications for obtaining high-sensitive and selective properties will be established as a result of the accumulation of knowledge on the structure-function relationship of biomolecules. By using modified biomolecules, higher-performance biosensors will be obtained.
References
1. Yabuki S, Shinohara H, Aizawa M. Electro-conductive enzyme membrane. J Chem Soc Chem Commun 1989; 945-946.
2. Yabuki S. Polyelectrolyte complex membranes for immobilizing biomolecules, and their applications to bio-analysis. Anal Sci 2011; 27:695-702.
3. Mizutani F, Yabuki S, Hirata Y. Amperometric biosensors using poly-L-lysine, poly(styrenesulfonate) membranes with immobilized enzyme. Denki Kagaku (presently Electrochemistry) 1995; 63:1100-1105.
4. Vigmond SJ, Iwakura M, Mizutani F, Katsura T. Site-specific immobilization of molecularly engineered dihydrofolate reductase to gold surfaces. Langmuir 1994; 10:2860-2862.
5. Hayashi T, Sano K, Shiba K, Iwahori K, Yamashita I, Hara M. Critical amino acid residues for the specific binding of the Ti-recognizing recombinant ferritin with oxide surfaces of Titanium and Silicon. Langmuir 2009; 25:10901-10906.
The author
Dr Soichi Yabuki
Biomedical Research Dept.
National Institute of Advanced Industrial Science and Technology (AIST)
Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
e-mail: s.yabuki@aist.go.jp
