Immobilization Method for the Preparation of Biosensors Based on pH Shift-Induced Deposition of Biomolecule-Containing Polymer Films

Abstract

Miniaturization of amperometric biosensors is crucially dependent on the availability of methods for the nonmanual immobilization of biological recognition elements on the transducer surface. From an aqueous polymer suspension, the precipitation of a polymer film with entrapped biological recognition elements is initiated by electrochemically induced oxidation of H20 at the electrode surface. Using the locally generated H+ gradient, acidic side chains of the polymer are titrated, leading to a change in the polymer solubility and hence to the controlled deposition of a polymer film. To investigate the properties and limitations of this immobilization technology, the specific features of a glucose biosensor based on polymer-entrapped glucose oxidase and amperometric detection of enzymatically generated H202 were investigated. Besides the reproducibility of the immobilization procedure, the sensitivity (14.59 mA cm(-2) M(-1) at pH 7), long-term stability (up to 5000 measurements in a sequential-injection analyzer), dependence on enzyme concentration, polymer thickness, and possibilities to fabricate multilayer sensor architectures were exploited. In addition, the miniaturization potential of this nonmanual immobilization technology was evaluated by investigating the modification of microband electrode arrays and cross talk between the neighboring microsensors.

Theory of polymer entrapped enzyme ultramicroelectrodes: Fundamentals

“…4 also confirm experimental observations that an optimal size of the entrapment domain exists [24,45,46]. The existence of a Z corresponding to a maximum electrode effectiveness K′ is evident in Fig.…”

“…(20) with C A = C p,A , this allows one to solved for the uniform concentration of analyte in the polymer: (22) where (22) can be used to evaluate S R , which is then substituted into an expression for electrode current obtained by combining Eqs. (10) and (14) ( 23) The results can also be described in terms of a measure of enzyme electrode effectiveness, K ′ ≡ |I|/(4nFD A aC ∞,A ): (24) 4.1.2. Heterogeneous Reaction Approximation-In the opposite limit of consumption of the analyte by the redox-species-producing reaction very close to the polymer surface, Eq.…”

“…Current state-of-the-art immobilization strategies include (i) crosslinking (e.g., attachment of the biological recognition element based on biotin/avidin chemistry [15] or antigen-antibody binding [16]), (ii) covalent attachment to self assembled monolayers [17,18], and (iii) polymer entrapment. Enzyme entrapment in polymer matrices has been demonstrated in conducting polymers [19][20][21], sol gels [22], and hydrogels [23], including electrodeposited electrophoretic paints (EDPs) [24], which are polymer suspensions deposited via a pH shift induced desolubilization. Electrophoretic paint entrapment exhibits favorable characteristics, including ease of implementation, retention of enzyme activity, and extended sensor stability [13,25].…”

“…Electrophoretic paint entrapment exhibits favorable characteristics, including ease of implementation, retention of enzyme activity, and extended sensor stability [13,25]. The model presented in this work was developed to describe the response assuming a probable geometry and likely dominant physics for micro-disk electrodes and polymeric matrices formed via pH shift induced polymer deposition (see section 2.1) [13,24,25].…”

“…The formulation is particularly suitable for micro-disk electrodes and polymeric matrices formed via pH shift induced polymer deposition [24]; however, the model may be extended to biosensors based on different enzyme entrapment methods. Experimental validation of the theory and demonstration of its application are provided in a companion paper [41].…”