Immobilization of HRP in Mesoporous Silica and Its Application for the Construction of Polyaniline Modified Hydrogen Peroxide Biosensor

Polyaniline (PANI), an attractive conductive polymer, has been successfully applied in fabricating various types of enzyme-based biosensors. In this study, we have employed mesoporous silica SBA-15 to stably entrap horseradish peroxidase (HRP), and then deposited the loaded SBA-15 on the PANI modified platinum electrode to construct a GA/SBA-15(HRP)/PANI/Pt biosensor. The mesoporous structures and morphologies of SBA-15 with or without HRP were characterized. Enzymatic protein assays were employed to evaluate HRP immobilization efficiency. Our results demonstrated that the constructed biosensor displayed a fine linear correlation between cathodic response and H2O2 concentration in the range of 0.02 to 18.5 mM, with enhanced sensitivity. In particular, the current approach provided the PANI modified biosensor with improved stability for multiple measurements.


Introduction
Polyaniline (PANI) has been used in the fabrication of various types of enzyme-based biosensors because of its porous structure, as well as its adequate conductivity and thermal stability [1][2][3][4][5][6]. To stabilize the immobilized enzyme in the matrix of PANI film, glutaraldehyde (GA) is usually employed as a bifunctional agent to crosslink enzyme molecules [7][8][9][10], but the crosslinking efficiency under standard conditions is not always satisfactory [10,11], which results in the lower sensitivity and poor stability of the resulting biosensor. Previously, we have electrochemically synthesized the PANI film on a Pt electrode in the presence of bovine serum albumin (BSA), a lysine-rich enzyme, which provides extra free ε-amino groups for the further crosslinking of HRP with glutaraldehyde and has significantly improved the effectiveness of a PANI modified biosensor [1]. Nevertheless, to enhance the efficiency and stability of enzyme immobilization on an electrode is still the major issue of fabricating enzyme-based biosensors.
Over the past few years, immobilizations of enzymes in well-defined mesoporous silica materials have been proven to be promising for enhancing the thermal stabilities and maintaining the catalytic activities of enzymes [12][13][14]. Enzymes entrapped inside the silica mesopores are less susceptible to pH and temperature alternations and organic solvents as well [12,15,16]. Among them,  which is synthesized in the presence of nonionic triblock copolymer P123 as a template under acidic conditions, exhibits well-ordered hexagonal pore arrays of uniform pore size [17,18]. Meanwhile, SBA-15 possesses a large surface area and internal silanol hydroxyls that have affinities for physical adsorption of enzyme molecules [19][20][21][22][23]. Recently, SBA-15 has been successfully employed to entrap glucose oxidase (GOD) to construct a glucose biosensor, achieving with enhanced sensitivity, long-term stability and reproducibility [24,25]. In addition, monoclonal antibodies were immobilized in SBA-15 for the detection of antigen (cTnI) in the serum of patients, which is more convenient and superior to the conventional enzyme-linked immunoadsorbent assay (ELISA) [26]. For the detection of hydrogen peroxide (H 2 O 2 ), SBA-15 loaded with hemoglobin (Hb) has shown a fast amperometric response, a low detection limit, and good stability [27].
In this study, we further exploited the application of mesoporous SBA-15 in entrapping HRP and constructed an amperometric GA/SBA-15(HRP)/PANI/Pt biosensor by immobilizing the SBA-15(HRP) on the electrochemically synthesized PANI film on a Pt electrode. The composite biosensor was then characterized and evaluated for the detection of H 2 O 2 with cyclic voltammetry. In addition, its linear correlation, sensitivity and stability were investigated.
The pore diameter was approximately 100 Å, which was close to the center of the pore size distribution (ca. 92 Å) shown in the inset of Figure 2. The pore size distribution indicated the major pore diameter of mesopores ranged between 80 and 110 Å, which permitted the easier access of HRP molecules because the dimension of the native HRP (MW: ~ 44 kDa) in a neutral buffer solution was predicted to be 62 × 43 × 12 (Å) 3 by a scanning tunneling microscopy (STE) study [28]. Meanwhile, the average pore diameter of SBA-15 estimated by the BJH method was ca. 76 Å (Table 1), which was below the major pore size distribution (80~110 Å), suggesting the presence of micro-channels in the interior of SBA-15.

Immobilization of HRP in SBA-15 Mesopores
When compared with SBA-15, the total specific surface area and the total pore volume of SBA-15(HRP), decreased modestly by about 11% and 8%, respectively, indicating the successful entrapment of HRP within the pores of SBA-15 ( Figure 2 and Table 1). The loading of HRP in SBA-15 was also confirmed with ABTS enzymatic assay, which nearly 395 units of HRP was stably shown in the inset of Figure 2, implied that the retained HRP did not block the entrances of mesopores, but rather resided in the inner space of mesopores. This conclusion was further supported by the identical BJH pore diameters before and after loading of HRP (Table 1).
Furthermore, we utilized the Coomasie Brilliant Blue R-250, which is commonly employed to stain proteins in SDS-PAGE gel analysis, to stain SBA-15 and SBA-15(HRP), respectively. Both were then washed with de-staining solution several times. Our result showed that the blue dye was retained by SBA-15(HRP), but not by SBA-15, suggesting HRP was stably immobilized in SBA-15(HRP). In order to investigate whether the immobilized HRP in SBA-15(HRP) was entrapped inside the pore of SBA-15 or was adsorbed on the surface of SBA-15, we examined another mesoporous silica, MCM-41, which was synthesized by a similar approach as that used for SBA-15, but its pore diameter was estimated as 25 Å, therefore, HRP was supposed to be completely excluded by MCM-41. We found that the adsorption of HRP on the surface of silica MCM-41 was relatively unstable and most of the blue stain was washed away. Therefore, the stably immobilized HRP in SBA-15(HRP) was most likely entrapped inside the pores of SBA-15.

The Surface Morphology of Electrodes
After depositing the SBA-15 on the electrochemically synthesized PANI/Pt electrode, SEM was employed to illustrate the surface morphology of the constructed electrodes. As shown in Figure Table 1, the mesopores of SBA-15 provided the electro-active proteins with a unique environment that might prevent protein aggregation during immobilization. A recent publication has demonstrated that the SBA-15 mesoporous materials accelerated the electron transfer between the entrapped enzyme and electrode [29]. The similar conclusions were also made for other mesoporous silica [30][31][32] and the electron hopping mechanism was proposed to facilitate the electron transfer inside silica mesopores [29,[33][34][35].

The Stability of Constructed Electrode
It has been reported that the electrochemical synthesized PANI film on the Pt electrode may be unstable during the sequential cyclic voltammetric measurements, in which can be improved by the implanted bovine serum albumin [1]. The same results are shown in Figure 7, where the cathodic    loaded with HRP provided the PANI/Pt electrode with not only an enhanced sensitivity but also an improved stability, in which could be further improved by employing glutaraldehyde.

Characterizations of SBA-15 Mesoporous Silica
The

Assay of Protein Activity
The HRP activity assay was performed by the 1-Step™ ABTS protocol (PIERCE Chemical Co., Rockford, IL, USA) according to manufacturer's procedure. In brief, one milliliter of assay mixture

Immobilization of HRP
Prior to immobilization, enzyme stock solution was prepared by dissolving 16.8 mg of HRP (5,000 units) in 1 mL of 0.1 M phosphate buffer (pH 6.2), and then was aliquoted and stored at -80 °C. To perform enzyme immobilization, 20 μL of HRP stock solution (100 units) was first diluted to 200 μL by 0.1 M phosphate buffer (pH 6.2) and subsequently 10 mg of SBA-15 was suspended in the enzyme solution for 1 h at 4 °C on a rotator. The loaded SBA-15 (SBA-15(HRP)) was recovered by centrifugation with 7,000 rpm for 5 min and the supernatant was also collected for protein analysis.

Fabrication of the Biosensor
The PANI/Pt electrode was prepared following a procedure similar to that described our previous publication [1]. The Pt/ceramic electrode with a desired pattern (area: 0.28 cm 2 ) was constructed by sputtering platinum to a ceramic plate (area: 2 cm 2 ) with a shadow mask for 600 sec on a sputter instrument (JFC-1200, JEOL, Tokyo, Japan), then washed with 3M NaOH and 3M HCl, rinsed with water, and finally dried under 50 °C for one hour. A certain amount of aniline was then

Electrochemical Measurement
A PC-controlled CHI621B electrochemical analyzer (CH Instruments, Austin, TX, USA) was employed to run cyclic voltammetric experiments for electrode preparation and hydrogen peroxide measurement. All experiments were performed in a miniature electrochemical cell using a modified PANI/Pt electrode (area: 0.28 cm 2 ) as the working electrode, a platinum wire as the auxiliary electrode, and an Ag/AgCl (3M NaCl) electrode as the reference electrode. The reductions of H 2 O 2 on electrodes were quantified with cyclic voltammetry in 0.1 M phosphate buffer (pH 6.2). The buffer had undergone deoxygenation with highly pure nitrogen for 20 min before a certain amount of H 2 O 2 was added. During the calibration, pure nitrogen gas was gentle purged on the surface of the sample solution to create an anaerobic atmosphere.

Conclusions
We have presented a new strategy for the fabrication of hydrogen peroxide biosensor based on entrapping HRP in mesoporous SBA-15 and depositing on a PANI modified Pt electrode. Our results further indicated that the synthetic SBA-15 particle possessed well-defined pore geometry and high internal surface area that was able to enhance the physical adsorption of enzyme molecules. The proper pore size distribution of SBA-15 was suitable for the entrapment of HRP and maintaining its bioactivity.
Meanwhile, the novel GA/SBA-15(HRP)/PANI/Pt biosensor exhibited enhanced sensitivity and a fine linear correlation between the cathodic response and the concentration of H 2 O 2 in the range of 0.02 to 18.5 mM (R 2 = 0.997). In particular, the current approach by utilizing SBA-15 to entrap HRP provided the biosensor with improved stability for multiple measurements. As shown in Figure 5, the lost of current response was mainly occurred during the initial cyclic voltammetric measurement, suggesting a few of HRP molecules were excluded by the applied potential.
Furthermore, the pore size of SBA-15 can be easily adjusted by controlling the synthesis conditions in the presence of pore expanding reagents, such as 1,3,5-trimethylbenzene (TMB) [21,[37][38][39], and biomolecules with different molecular mass can be entrapped in SBA-15 of proper pore size accordingly. Meanwhile, the internal surface of SBA-15 has been successfully modified by various organic functional groups, such as amine, thiol, and carboxylic acid, thereby providing additional improvement to minimize the leaching of biomolecules [20]. Nevertheless, the entrapment of biomolecules in SAB-15 may provide new aspects of enhancing the performance of enzyme-based biosensors, in particular offering better stability and multiple usages.