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    • In another approach to mediatorless sensors recombinant form

    2018-10-30

    In another approach to mediatorless sensors, recombinant forms of HRP were directly bound to polycrystalline gold to fabricate a hydrogen peroxide sensor [3]. It was shown that gold electrodes with recombinant HRP exhibited good electron transfer and produced sensitive and stable responses to hydrogen peroxide. Carbon nanotubes (CNTs) are also used to mediate electron transfer. Zhu et al. used an electrode coated with CNTs on which GOD and HRP enzymes in an electro-polymerized pyrrole film were co-immobilized [16]. They showed that single wall CNTs can effectively transfer electrons between an enzyme layer and gold electrode. Multi-walled carbon nanotubes functionalized with thionin were used by Shobha Jeykumari et al. [8] to immobilize HRP and GOD, and the sensor showed a good detection range and stability. Some peptide nanotubes (PNTs) are conductive and can be used for electron transfer [4]. PNTs are biocompatible, and therefore can be used as an immobilization agent for mediatorless sensing in vivo. Additionally, PNTs were reported to help an enzyme retain its activity and stability against temperature change [12]. Some peptide monomers and protein fragments have been investigated for use as a bridge to connect electron donors to acceptors for electron transfer purposes, and their intramolecular electron transfer properties have been reported [6]. This generated the idea of using peptide nanotubes for enzyme immobilization and electron transfer without the need for extra mediator molecules. Park et al. used diphenylalanine PNTs for immobilizing enzymes for glucose sensing. However, they used a mediator (HQ) in their system for electron transfer [13]. In this study, we encapsulated HRP inside PNTs that were deposited on a gold electrode to detect hydrogen peroxide without using HQ. Amperometric response was produced with the step change of hydrogen peroxide concentration. The amperometric result of the PNT/HRP system was compared with the sensor consisting of HRP attached to a gold electrode without PNTs and using HQ mediator. Two other systems, one consisting of both HRP encapsulating PNTs and HQ (PNT/HQ/HRP) and the other with HRP with neither PNT nor HQ, were investigated for comparison. For all tested electrodes, 3,3′-dithiodipropionic GDC0941 di(N-hydroxysuccinimide ester) self-assembled monolayer (SAM) was used to immobilize HRP and PNTs on the electrode surface [11,12,14].
    Materials and methods
    Results and discussion Fig. 2 shows the SEM images of PNTs before and after HRP encapsulation. The inside of most PNTs appeared darker after HRP encapsulation which is thought to be because of the accumulation of HRP inside the PNTs. There are spectroscopic methods (FTIR and UV–Vis spectroscopy) that were used to verify the HRP encapsulation inside PNTs (not described in this manuscript) which can be found in Park et al. [12]. Amperometry results for the aforementioned cases are shown in Fig. 3. The concentration of hydrogen peroxide was increased from 0 to 58.8mM by 4.9mM increments. Fig. 3 shows that the system containing both PNT and HQ generated greater current signal throughout the entire tested range than the other systems. The HRP only system (no HQ/no PNT) showed no clear stepwise increase in current signal with hydrogen peroxide addition. Furthermore, for the HRP only system, much more frequent and larger fluctuations were observed in the response. The current generation by the PNT/HRP system was either the same as or a little lower than the HQ/HRP system indicating that electron transfer by PNT was comparable to that of HQ. The current generated by the PNT/HQ/HRP system increased most rapidly with hydrogen peroxide concentration. The increasing rates of PNT/HRP and HQ/HRP systems were almost the same. Fig. 4 presents the current change with hydrogen peroxide concentration. Enzyme kinetics can be described using the Michaelis–Menten type equation [11]:where I is the current which depends on the concentration of hydrogen peroxide, I is the apparent maximum current measured and K are Michaelis–Menten parameters. By taking the reciprocal of Eq. (1), the inverse of current can be related to the inverse of hydrogen peroxide concentration with a linear expression as shown in Fig. 5. By plotting the experimental data in the concentration range of 15–60mM, I and K values were obtained. Table 1 summarizes the Michaelis–Menten parameters for all cases. More electrons were transferred when the value of I was higher. The largest I, 5.8mA, was observed in the PNT/HRP/HQ system. The I values for the system containing only PNT (PNT/HRP) and the system containing HRP and HQ (HQ/HRP) are about the same which means the ability of PNTs to transfer electrons is approximately equivalent to that of HQ. The system without PNT and HQ has the lowest I which shows that there was less electron transfer in the absence of PNT or HQ. K values seem to be lower when the electrons were transferred directly between the enzyme and hydrogen peroxide, so the system without PNT and HQ showed smallest K. A large K usually indicates low affinity, which, in our case, can be explained by more indirect electron transfer in the presence of PNT and HQ. Here, larger K didn\'t negatively affect the sensor performance because the effect of I on the signal seemed to be more significant (as Fig. 4 and Table 1 show).