Immobilization of Enzymes through One-Pot Chemical Preoxidation and Electropolymerization of Dithiols in Enzyme-Containing Aqueous Suspensions To Develop Biosensors with Improved Performance

Abstract

A protocol of one-pot chemical preoxidation and electropolymerization of monomers (CPEM) in enzyme-containing aqueous suspensions (or solutions) was proposed as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors, which was proven to be effective for the monomer of 1,4-benzenedithiol (BDT), 1,6-hexanedithiol, o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. As a case examined in detail, a well-dispersed aqueous suspension of the poorly soluble BDT was obtained through its dispersion assisted by ultrasonication and coexisting GOx, which was then subject to chemical preoxidation through adding K3Fe(CN)6, yielding many composites of insoluble BDT oligomers with lots of high-activity enzyme molecules entrapped. Some insoluble composites were then electrochemically codeposited with poly(1,4-benzenedithiol) on an Au electrode, yielding an enzyme film with high-load and high-activity enzyme immobilized. The glucose biosensor prepared here from the CPEM protocol showed much better performance than that from the preoxidant-free conventional electropolymerization (CEP) protocol, with a detection sensitivity increase by a factor of 32 in this case. The GOx-based and AP-based first-generation biosensors developed from the present CPEM protocol all exhibited notably improved performance compared with the analogues from the preoxidant-free CEP protocol. The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate various electrode modification processes. The values of quantity and enzymatic specific activity (ESA) of the immobilized enzymes were evaluated through the EQCM and the conventional UV-vis spectrophotometric method, given that the CPEM protocol notably improved the quantity and the ESA of immobilized enzymes as compared with the preoxidant-free CEP protocol. The proposed CPEM protocol may be interesting in a number of fields, including biosensing, biocatalysis, biofuel cells, bioaffinity chromatography, and biomaterials, and the successful electropolymerization of dithiols in aqueous suspensions (two-phase electropolymerization) may open a new avenue for many monomers that are poorly soluble in neutral aqueous solutions to in situ immobilize biomolecules for bioapplications.

Contributions by a Novel Edge Effect to the Permselectivity of an Electrosynthesized Polymer for Microbiosensor Applications

“…These implantable biosensors must also be able to reject electrochemical interference by reducing agents present in the tissue, and a common strategy is the incorporation of a permselective membrane on the biosensor surface, such as electropolymerized PPD. 43,[75][76][77][78][79][80] This polymer is unusual 81 in that it displays very high permeability to the biosensor signal transduction molecule (H 2 O 2 , see…”

Electropolymerization of catecholamines after laccase-catalyzed preoxidation to efficiently immobilize glucose oxidase for sensitive amperometric biosensing

“…An enzyme film electrode can be readily prepared by the electropolymerization protocol, but the chemical polymerization protocol often requires an additional and special step for modification of the chemical polymerization in solution and the electropolymerization on the electrode. Recently, the chemical and electrochemical polymerization of dopamine (DA) and dithiols have been proposed in our laboratory as a new and effective protocol to immobilize several enzymes for highly sensitive biosensing [13][14][15][16].…”

High‐Performance Amperometric Sensors Using Catalytic Platinum Nanoparticles‐Thionine‐Multiwalled Carbon Nanotubes Nanocomposite

“…Figure 3d displays the amperometric responses on GOx/PtNPs-TH-MWCNTs (r TH-Pt = 0.10 : 1)/ Au at 0.5 V to successive additions of glucose, and the time required to reach 95 % of each steady-state response is within 5 s. The GOx/PtNPs-TH-MWCNTs (r TH-Pt = 0.10 : 1)/Au electrode exhibits a linear response to glucose in the concentration range from 1.65 mM to 11.1 mM (r 2 = 0.999), with a sensitivity of 0.14 A M À1 cm À2 and a detection limit of 0.05 mM (S/N = 3). The sensitivity here is probably the greatest one for first-generation GOx-based glucose biosensing to date [35][36][37][38].…”