silica-lignin system to prepare an amperometric glucose biosensor

Accepted Manuscript Title: Carbon paste electrode based on functional GOx/silica-lignin system to prepare an amperometric glucose biosensor Authors: Artur J˛edrzak, Tomasz R˛ebi´s, Łukasz Klapiszewski, Jakub Zdarta, Grzegorz Milczarek, Teofil Jesionowski PII: DOI: Reference:

S0925-4005(17)31981-0 https://doi.org/10.1016/j.snb.2017.10.079 SNB 23383

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

13-6-2017 11-10-2017 14-10-2017

Please cite this article as: Artur J˛edrzak, Tomasz R˛ebi´s, Łukasz Klapiszewski, Jakub Zdarta, Grzegorz Milczarek, Teofil Jesionowski, Carbon paste electrode based on functional GOx/silica-lignin system to prepare an amperometric glucose biosensor, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.10.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Carbon paste electrode based on functional GOx/silica-lignin system to prepare an amperometric glucose biosensor

Artur Jędrzak1, a, Tomasz Rębiś2, a, Łukasz Klapiszewski1, Jakub Zdarta1, Grzegorz Milczarek2, Teofil Jesionowski1,*

1

Poznan University of Technology, Faculty of Chemical Technology, Institute of Chemical Technology and Engineering, Berdychowo 4, PL-61965 Poznan, Poland

2

Poznan University of Technology, Faculty of Chemical Technology, Institute of Chemistry and Technical Electrochemistry, Berdychowo 4, PL-61965 Poznan, Poland

*

Corresponding authors: [email protected] (Teofil Jesionowski)

a

These authors contributed equally to the work presented here and should therefore be

regarded as equivalent authors.

Graphical Abstract

Highlights



Biosensor with promising electrochemical parameters for glucose determination



Immobilization of GOx and the development of stable glucose biosensors



Carbon paste electrode based on functional GOx/silica-lignin system is prepare

Abstract

In this paper, a novel procedure for receiving enzyme biosensor based on the cheap and functional silica/lignin (SiO2/Lig) hybrid material is presented. In this study, a functional biohybrid SiO2/Lig was selected to conduct the immobilization of glucose oxidase (GOx) by adsorption on its surface. The immobilized amount of GOx at SiO2/Lig was 25.28 mg g-1, twice as much compared to its amount at non-functionalized SiO2. The system of GOxSiO2/Lig was combined with single-walled carbon nanotubes/platinum nanoparticles support

to prepare a I generation glucose biosensor. Moreover, the GOx-SiO2/Lig based carbon paste electrode with ferrocene redox mediator was evaluated as an active material in II generation glucose biosensor. The GOx-SiO2/Lig/CPE was subjected to an examination in glucose solution

by

electrochemical

techniques

such

as

cyclic

voltammetry

(CV)

and

chronoamperometry. The obtained results suggest that GOx-SiO2/Lig can be a material of choice for preparation of an efficient and low-cost biosensor working in various electrode configurations. The newly obtained glucose biosensor presents promising electrochemical parameters for glucose determination. The glucose-sensing sensitivity amounted 0.78 µA mM-1. The biosensor showed a linear response range of 0.5–9 mM with a detection limit (LOD) of 145 µM.

Keywords:

Glucose

biosensor;

Silica/lignin

hybrid;

Enzyme

immobilization;

Chronoamperometry

1. Introduction Since 1962, when Clark and Lyons created the first biosensor for glucose measurement based on oxygen electrode and glucose oxidase (GOx), a rapid and potent period has started for research on biosensors to be conducted and published [1,2]. The performance of an enzyme electrode is significantly affected by the materials used for immobilization and its assembly mechanism on electrode surface. Techniques such as dropcasting, covalent coupling, encapsulation and physical adsorption are commonly employed to immobilize the enzyme on electrode surface [3–5]. The choice of a new material as a support to immobilize a GOx has been determined on non-toxic influence of heavy metals on GOx, its satisfactory immobilization prosperity, and its high conductivity in the

whole set. Good-working biosensors are characterized by 3S, selectivity, stability and sensivity. 3S is important to create a novel biosensing system for transfer between an immobilized enzyme and an electrode [3,6–8]. In recent years, metal oxides such as (ZnO, CeO2, SiO2) have been intensively studied as a basic surface for enzymes to be immobilized due to their biocompatibility, biostability, nontoxicity or high surface area. For instance, Wang and co-authors have applied ZnO nanocomb structures for glucose detection [9]. Novel sensing material, in view of metal oxides influence, causes better electron conductivity, higher isoelectric point, biocompatibility or bioactivity. The scientific team of Ansari has confined GOx on sol-gel nanostructured CeO2 carrier, which has been deposited on indium-tin-oxide (ITO) glass plate for glucose determination [10]. Kumar and co-workers have developed a ZnO-nanomaterial for biosensing application to detect monosaccharide, glucose, which has been successfully applied for development in the novel sensing system [11]. In their research, Pandey and coworkers used ferrocene encapsulated ORMOSIL (organically modified silica or silicates) to construct a glucose biosensor using immobilized GOx [12]. Lignin is an inexpensive amorphous aromatic biopolymer obtainable in great quantities as a by-product of the pulping process of wood. It is the second most abundant natural polymer, after cellulose, in biomass. In recent years, lignins have been evaluated for their versatile use as electrochemically active electrode materials. Particularly, lignin has been widely used in the development of electrochemical sensors. Various electrodes modified by lignin exhibited enhanced redox activity and electrocatalysis towards reduced nicotinamide adenine dinucleotide (NADH) [13] and hydrazine oxidation [14] as well as nitrate reduction [15]. It has been confirmed that the aromatic character of lignin is responsible for its great affinity for nano-structured carbon materials and causes that they can be subject to strong adsorption on sp2-hybridized carbon surface. Some very interesting examples of obtainment

and characterization of materials such as lignin/carbon nanotubes are described in the literature [16–18]. Furthermore, the use of lignin in nanotechnology can be extended to the preparation of silver and gold nanoparticles having potential application in sensors [19]. In our previous teamwork, the SiO2/Lig hybrid material, had been successfully linked and comprehensively examined among other materials by using Non-Invasive Back Scatter (NIBS), Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS) and other techniques [20,21]. This novel matrix provides new opportunities for its wider application, for example, it can be used as an immobilization surface or in biosensors. The SiO2/Lig hybrid material was tested in electrochemistry. This makes an excellent opportunity and interesting properties for this material, which contributed to being used in building a novel electrochemical system. The SiO2/Lig hybrid material is presented in this work as an efficient material for the immobilization of glucose oxidase. Herein, we also demonstrate the feasibility of the application of hybrid GOx-SiO2/Lig material as a glucose biosensor (see Scheme 1). The electrochemical performance of the glucose biosensor was investigated by cyclic voltammetry and chronoamperometry.



2. Experimental

2.1. Materials and Chemicals Kraft lignin (Lig), glucose oxidase (from Aspergillus niger; contains protein 65–85%; molecular weight: 160 kDa, catalytic activity according to producer specification is 100–250 U/mg) and α-D-Glucose (≥99.5%, MW: 180.16 g mol-1) were purchased from Sigma-Aldrich.

Ferrocene (Fc) was provided by Fluka. Graphite (Drop Sens), single-walled carbon nanotubes (SWCNT, Sigma-Aldrich) conductive aqueous ink 1 mg mL-1 and platinum nanoparticles (PtNP, 3 nm particle size, 1000 ppm in H2O) were provided by Sigma-Aldrich. To determine an analytical performance of the presented biosensor real samples were used – Liquid Glucose 1WW - strawberry taste (Medmess, Warsaw) and Glucose infusion solution 5% (Hasco-Lek, Wroclaw). All reagents and solvents were of reagent-grade quality and were used without further purification. Carbon paste electrode (CPE) with a hole (d = 3 mm, 6 mm deep) and glassy carbon electrode (GC) with d = 3 mm were purchased from BASi.

2.2. Synthesis of SiO2/Lig hybrid material

First, the SiO2 was synthesized by the modified Stöber method, by the hydrolysis of tetraethoxysilane precursor and condensation of TEOS; 50 mL ethyl alcohol (95%); 11 mL aqua ammonia (25%); and 17 mL tetraethoxysilane [22,23]. Second, the SiO2 surface was modified by (3-aminopropyl)triethoxysilane (APTES). Further, the commercial kraft lignin was oxidized by NaIO4 and modified SiO2 was added to the reactor [20,21,24]. The whole system was then mixed by a high-speed stirrer for 1 h. Finally, the hybrid SiO2/Lig material was dried and sieved (sieve size 80 µm) [20,21].

2.3. GOx immobilization of SiO2/Lig and quantification of the amount of immobilized enzyme

Freshly prepared GOx solution in acetate buffer, pH = 5.0 in volume of 1 mL consisting of 5 mg GOx was added to 50 mg of SiO2/Lig hybrid. An immobilization process

was conducted by adsorption. The immobilization process lasted 24 h. Precipitate was thoroughly examined, in similar way to SiO2/Lig, filtrate was also subjected to Bradford analysis in order to quantify the amount of immobilized enzyme on the surface of SiO2/Lig material [25]. The procedure consists in measuring an initial and a final amount of glucose oxidase in the immobilization medium before and after the immobilization process, based on spectrophotometric measurements (λ = 595 nm). A calibration curve was prepared by using the BSA solutions at a known concentration. The resultant solutions were used to calculate the amount of proteins bounded to the support (milligram of the immobilized enzyme per gram of the support).

2.4. Morphology characterization

To determine the morphology characterization was carried out analysis using the Scanning Electron Microscope with Energy Dispersive Spectroscopy (SEM-EDS) and the Atomic Force Microscope (AFM) for our products. The SEM method records on the Jeol 7001TTLS with maximum 30 kV acceleration was used for analysis, with 1.5 nm resolution. The AFM measurements were made using Agilent's 5500 atomic force microscope in intermittent contact mode in ambient conditions. The test material was applied to the mica surface. Substrate surface was previously cleaned by mechanical stripping. The test material was applied to the substrate by spin coating. All in One lever type of resonance frequency of about 150 kHz made by BudgetSensors company ware used for scanning.

2.5. FTIR analysis

To characterize the presence of functional groups in the structure of precursors, the obtained matrix, immobilized product, and samples were subjected to Fourier transform infrared spectroscopy (FTIR). FTIR spectra were made using a Vertex 70 spectrometer (Bruker, Germany). Materials were analyzed in the form of potassium bromide tablets, made by mixing 250 mg of anhydrous KBr and 1 mg of the analyzed substance under pressure of 10 MPa. The FTIR analysis was performed at a resolution of 0.5 cm-1 in a wave number range of 4000–400 cm-1.

2.6. Electrochemical methods

Electrochemical measurements were performed using a μ-Autolab III (ECO Chemie, Netherlands) potentiostat/galvanostat. A three-electrode configuration was used with a Ag/AgCl/(3 M KCl) as a reference electrode, a Pt wire as a counter electrode and a GC or CPE as a working electrode. High purity nitrogen had been used for deoxygenation of the supporting electrolytes before all experiments.

2.7. Fabrication of GOx-SiO2/Lig/Fc based carbon paste electrode

GOx-SiO2/Lig based carbon paste electrode was prepared in a conventional manner by a thorough hand-mixing of GOx-SiO2/Lig (20 mg), graphite powder (20 mg), ferrocene (2 mg) and paraffin oil (2 µL) in an agate mortar with a pestle. The modified carbon paste (CPE/GOx-SiO2/Lig/Fc) was inserted into the hole in the electrode body and smoothed off with filter paper (Whatman). CPE characteristics a special hole in its own construction, which allows GOx-SiO2/Lig to be placed inside.

2.8. Fabrication of GC/SWCNT/PtNP/GOx-SiO2/Lig electrode

Prior to surface modification, the GC was carefully polished with 0.05 μm alumina slurry (Buehler) to obtain a mirror-like surface. After having been sonicated in water/acetone 1:1, v/v mixture for 5 min, the electrode was rinsed with water and dried in an oven at 60 ºC. Before use SWCNT conductive aqueous ink 1 mg mL-1 had been sonicated for 1 h. SWNT/PtNP hybrid materials with different mass ratio were prepared as follows. SWCNT solution (1 mg mL-1) was added to the PtNP solution (1 mg mL-1) in five volume ratios: 90 : 10, 75 : 25, 50 : 50, 25 : 75 and 10 : 90 respectively to prepare the solution with 10 wt%, 25 wt%, 50 wt%, 75 wt% and 90 wt% of PtNP. All the mixed solutions were sonicated for 30 min for homogeneous dispersion. The preliminary experiments on the electrocatalytic activity towards H2O2 suggested that the electrodes with 50 wt% – 75 wt% performed the best activity (Fig. S1, please see Supplementary Materials). Hence, the 50 wt% composition was uded for further study (taking into account also the price of PtNP). Next, 1 mL of SWCNT/PtNP was thoroughly mixed with 1 mg of GOx-SiO2/Lig by magnetic stirr bar in a small vial for 1 h. Finally, 1 µL of the homogenous dispersion was dropped onto the GC surface and allowed to dry in ambient temperature. The resulting electrode was then rinsed with double distilled water to remove any unbound particles.

3. Results and discussion 3.1. Morphological characterization The determination of the morphological structure of the hybrid materials and the resulting carriers was carried out using scanning electron microscopy. For this purpose, SEM

images

were

made

for

GOx-graphite/Fc;

GOx-SiO2/graphite/Fc;

GOx-

SiO2/Lig/Fc/graphite/Fc, which are shown in Fig. 1. A-C respectively. AFM studies were carried out to confirm a change of matrix before and after immobilization of the enzyme GOx. Obtained results are presented in Fig. 1. D-E, respectively. SEM-EDS results of SWCNT (A), (B) and SWCNT/PtNP (C), (D) are shown in Fig. S2 (see Supplementary Material).



The SEM images shown in Fig. 1A show a samples of GOx-graphite/Fc characterized by the occurrence of irregularly shaped particles. In addition, Fig. 1B also presented similar primary particles of graphite, reinforced with silica particles, capable to form aggregates (> 200 nm) and agglomerates (> 1000 nm) are visible. Fig. 3C shows the SEM image of GOxSiO2/Lig/Fc/graphite/Fc, which is one of the components obtained in a further phase of testing hybrid materials. Micrographs represent spherical particles of varying size and regular shape. SEM image analysis of lignin shows the particles that tend to form aggregates and in a further agglomerate stage. The AFM results show changes in morphology before and after the enzyme immobilization process. The thicker layer observed after the immobilization process is due to adsorption of GOx. The AFM images also shows the more smooth edges of the material, which confirm the successful process of immobilization.

3.2. FTIR analysis To investigate the type of functional groups presented in the structure of precursors, and to confirm effective enzyme immobilization FTIR spectroscopy, a glucose oxidase from

Aspergillus niger as well as obtained silica/lignin were used. Obtained results are presented in Fig. 2.

Spectrum of the silica consists of signals with maxima at 3600–3440 cm-1 (–OH), 1100 cm-1 (Si–O–Si), 968 cm-1 (Si–OH), 813 cm-1 (Si–O–Si) and 580–520 cm-1 (Si–O) [26–29]. In the spectrum of lignin, bands at 2940 and 2850 cm-1 (C–H), 1720 cm-1 (C=O), 1605, 1525 and 1435 cm-1 (CAr–CAr and CAr–CAr), as well as around 1100 cm-1 (C–O) can be seen. In the spectrum of synthesized materials signals characteristic for both, silica and lignin precursors, are presented. The presence of these signals is an indirect proof of an effective synthesis of silica/lignin biohybrid matrix [21,24]. The presence of peaks at wave numbers 3410–3270 cm-1 (N–H), 2820 cm-1 (C–H), 1625 cm-1 ( amide I), 1525 cm-1 ( amide II), 1030 cm-1 (C–O) and around 700 cm-1 ( C–O) is observed in the spectrum of glucose oxidase. After GOx attachment to the silicalignin support, in the FTIR spectra of an immobilized enzyme, the intensity of the peak characteristic for the –OH bonds decreased due to the reduction of hydroxyl groups after enzyme attachment [30]. Moreover, maxima of the signals generated by amide groups of GOx are slightly shifted, which proves that the immobilized enzyme retains its catalytic properties. Shifts on the FTIR spectra indicate also changes in the chemical environment of the immobilized oxidase that might have influenced activity of the immobilized enzyme [26–29]. The analysis of the FTIR spectra of a product following immobilization proved the effective enzyme immobilization onto the surface of a biocomposite carrier, which is a key step for the proper preparation of the glucose biosensor.

3.3. Efficiency and stability of the enzyme immobilization process

In order to determine the amount of enzyme immobilized on the SiO2/Lig hybrid and their components, a Bradford analysis was applied [25]. Tests were performed on three samples; silica, graphite (G) and SiO2/Lig. Verification of origin kraft lignin, as a component of this material, was impossible by Bradford method, due to the fact that the kraft lignin is well-soluble in water. Hence, the color of dissolved kraft lignin prevented measurement on UV-Vis spectrophotometer. The highest amount of immobilized GOx (Table 1) was noticed on hybrid material (25.28 mg g-1), which caused excellent properties compared to origin components like silica (12.88 mg g-1) and graphite (7.58 mg g-1). Higher efficiency of GOx immobilization on this hybrid material can be hypothetically related to the formation of hydrogen bonds between enzyme molecules and hydroxyl, carbonyl, quinone as well as amino groups present in support [31]. These functional groups came directly from silica, lignin or a surface modifying agent (APTES), giving a synergy effect occurring in the SiO2/Lig matrix [20,21,26]. The results have confirmed our assumptions, giving us the possibility to use GOx-SiO2/Lig to build the biosensor. Besides evaluation, the amount of the immobilized enzyme and catalytic activity of the GOx immobilized onto silica, graphite and silica/lignin hybrid material was examined. As it can be seen, catalytic activity of the immobilized GOx increases when amount of the immobilized enzyme also increases. However, it should be noticed, that the amount of the immobilized biomolecules increases two times for silica/lignin hybrid. In comparison to silica, catalytic activity does not increase two times, proving that not all molecules of the immobilized catalysts are available for the molecules of substrate. That fact is most probably related to the more complicated structure of the used hybrid in contrast to silica particles which caused steric hindrances.

Operational stability of glucose oxidase immobilized onto silica/lignin hybrid material was presented in Fig. S3 (see Supplementary Material). Figure S3 shows that activity of GOx immobilized onto silica/lignin hybrid slightly decreases with the number of consecutive catalytic cycle. However, after 15 catalytic cycles, immobilized enzyme retains over 60% of its initial properties, which proved that immobilization significantly increases stability of the GOx. Decrease of the catalytic properties of linked biomolecules might be explained mainly by two factors. First enzyme is being inactivated in the presence of H2O2. Moreover, the decrease in catalytic activity might also be related to the partial leakage of the enzyme from the support material during repeated catalytic cycles.

3.4. Electrochemical study on GC/SWCNT/PtNP/GOx-SiO2/Lig modified electrode (I generation biosensor)

In order to investigate the feasibility of GC/SWCNT/PtNP/GOx-SiO2/Lig bioelectrode acting as a first generation glucose biosensor, we initially studied the electrochemical behavior of electrode in the presence of O2 and H2O2 (Fig. 3). As seen in Fig. 3A, the cyclic voltammogram of GC/SWCNT/PtNP/GOx-SiO2/Lig electrode recorded in PBS solution (saturated with O2), suggests that the electrode possesses strong electrocatalytic properties towards reduction of oxygen, as the CV clearly shows a well-defined single reduction peak at -0.1 V. This significant current response can be ascribed to the existence of PtNP that is able to accelerate the slow electron transfer kinetics of O2 at the GC electrode surface [32,33].



As is well known, electrochemical reduction of H2O2 at common solid electrodes occurs at high overpotentials, which is the most important limitation for the selective determination of H2O2 [34,35]. Cyclic voltammograms of GC/SWCNT/PtNP/GOx-SiO2/Lig in the absence and presence of 2 mM hydrogen peroxide are shown in Fig. 3B. When the hydrogen peroxide is present, the anodic and cathodic currents related to the electrooxidation and electroreduction of hydrogen peroxide are observed (Fig. 3B, blue line). An appreciable oxidation current begins at +0.3 V, which is substantially lower than at bare GC [36,37]. This catalytic effect is attributed to the presence of Pt, which has substantially lowers the overvoltage H2O2 oxidation. These results indicate that GC/SWCNT/PtNP/GOx-SiO2/Lig is a suitable active material to determine H2O2 at low overpotential.



In order to confirm the enzymatic activity of immobilized GOx on the electrode, the electrocatalysis of glucose was examined in the presence of oxygen. The electrocatalytic reaction between GOx active center (FAD) and glucose can be represented by Equations (1) and (2) [3]:

glucose + O2 → gluconic acid + H2O2

(1)

O2 + 4H+ + 4e- → 2H2O

(2)

Cyclic voltammetry studies on a GC/SWCNT/PtNP/GOx-SiO2/Lig modified electrode were carried out in PBS (pH=7.0) by increasing the glucose concentration (from 0 to 40 mM). With the addition of each aliquot of glucose, an increase in oxidation current was observed

from 0.4 V up to 0.7 V (Fig. 3). This increase is attributed to H2O2 oxidation, which is released because of an enzymatic reaction of glucose oxidase with glucose (Eq.1). The shape of CV and peak positions for H2O2 oxidation on the GC/SWCNT/PtNP/GOx-SiO2/Lig electrode matches well with the CVs in Fig. 3B, evidence that hydrogen peroxide was enzymatically generated. Interestingly, the decrease of cathodic peak current at -0.1 V is simultaneously observed in Fig. 4 when the glucose concentration increases. This behavior corresponds to the successive consumption of oxygen by GOx allowing the FAD/FADH2 redox couple to regenerate [38]. Figure S4 (see Supplementary Material) shows typical chronoamperometric responses of GC/SWCNT/PtNP/GOx-SiO2/Lig bioelectrode with the successive addition of 5 mM glucose at an applied potential of +0.6 V. When an aliquot of glucose solution was added into the buffer solution, the oxidation current increased steeply to reach a steady-state value. The above-mentioned results show tentatively that the proposed sensor represents promising abilities for sensitive enzymatic detection of glucose at moderate potential. At high concentrations of glucose (typically higher than 10 mM), the GOx catalytic response leveled off, likely due to Michaelis–Menten kinetics. The same behavior was observed for many different enzymatic electrodes and glucose biosensors, exhibiting linearity in initial stages and approaching saturation for higher glucose concentration [39,40].

3.5. Electrochemical study on CPE/GOx-SiO2/Lig/Fc electrode (II generation biosensor)

The application of GOx-SiO2/Lig as an electrode material in the development of II generation glucose biosensors was also investigated. For this purpose a carbon paste electrode with ferrocene as a redox mediator and GOx-SiO2/Lig as a biocatalyst was fabricated. The electrochemical properties of CPE/GOx-SiO2/Lig/Fc were initially studied by CV in PBS (pH

= 7.0) in absence of glucose. According to Fig. 5A, the CPE/GOx-SiO2/Lig/Fc electrode shows a well-defined quasi-reversible redox couple at ca. 0.25 V. These peaks correspond to electroactivity of ferrocene incorporated in the electrode [39]. The CVs of CPE/GOxSiO2/Lig/Fc at different scan rates (5–100 mV s-1) in phosphate buffer are also presented in Fig. 5A. The anodic and cathodic peak currents of the CPE/GOx-SiO2/Lig/Fc redox system increased linearly with an increase of scan rates. As shown in Fig. 5B and C, the peak currents are linear to the square root of scan rates, indicating that diffusion limitations occur through the thick electrode layer, giving contribution to the surface process of CPE/GOx-SiO2/Lig/Fc. Similar electrochemical behavior was also observed for ferrocene redox polymer nanobeads applied in glucose biosensing [39].



When a positive scan approaching and beyond the E0’ value of the mediator is applied to the investigated electrode, the Fc is transferred into its catalytically active Fc+ oxidized form. As can be seen in Fig. 6, the magnitude of the electrochemical response current increases with an increasing concentration of glucose in anodic range (at potential more positive than E0’ of Fc+/Fc couple). This is attributed to the enzymatic catalytic action of GOx immobilized on the SiO2/Lig matrix. Such behavior demonstrates that the CPE/GOxSiO2/Lig/Fc electrode can electrocatalyze the oxidation of glucose by taking Fc+/Fc as a mediator in saturated solutions with N2 according to Equations 3-5 [3]:

glucose + FAD(ox) → gluconolactone + FADH2(red)

(3)

FADH2(red) +2Fc+→ FAD(ox) +2Fc + 2H+

(4)

2Fc → 2Fc+ + 2e−

(5)

Electrochemical impedance spectra recorded at 0.3 V (PBS pH = 7.0) for CPE/GOxSiO2/Lig/Fc and CPE/SiO2/Lig/Fc electrodes are shown in Fig. S5 (see Supplementary Material). The spectra of both electrodes present a semicircle in the high frequency region, corresponding to the electron transfer process, and a linear part at low frequency region, corresponding to diffusion. A model consisting of the total cell resistance (RΩ) in series with a charge transfer resistance (Rct), connected in parallel with a constant phase element CPE and Warburg impedance (W) was used for fitting the spectra. Calculated values of the circuit parameters are shown in Table S1 (see Supplementary Material). From comparing the RΩ values, the immobilization of GOx causes an increase of ohmic resistance from 708 to 987 Ω. This could be attributed to resistive nature enzyme. On the other hand, CPE/GOx-SiO2/Lig/Fc electrode shows a much smaller diameter of the semicircle than the electrode without GOx, that indicates the significantly lower charge transfer resistance. This result suggests that GOx is easily deposited on SiO2/Lig hybrid and promotes fast electron transfer between ferrocene redox couple.



The characteristics of the biosensor are investigated by chronoamperometric measurement under the stirring conditions. The variation of amperometric currents with different pH (from 5.2 to 8.3) was investigated at a fixed concentration of glucose (2 mM). The results are presented in Fig S6 A and B (see Supplementary Material). According to the obtained data, the anodic current increased continuously with the function of pH and reached

a maximum value at pH 7.0. Then, a slight current decrease can be observed. Therefore, pH 7.0 was selected as optimal for the biosensor operation. Temperature influences electrocatalytic performance of the biosensor (Fig. S6 C and D, see Supplementary Material). At low temperature (5 ºC), the kinetics is considerably slowed down and the current response is relatively low. The current was observed to increase with increasing temperature up to room temperature (25 ºC). Further increase of temperature to 35 ºC and 45 ºC caused a drop of current, probably due to faster deactivation of the enzyme. In order to verify the influence of lignin on the biosensor response, we have performed the amperometric oxidation of glucose at the electrode consisting of GOx immobilized on SiO2 (CPE/GOx-SiO2/Fc). Moreover, the measurements were carried out at the electrode consisting of GOx immobilized on graphite (CPE/GOx/Fc). Figure 7A shows a typical current–time response of the CPE/GOx-SiO2/Lig/Fc for successive adding of aliquots of 0.5, 1 and 2 mM of glucose into 10 mL of PBS, in comparison with those at CPE/GOx-SiO2/Fc and CPE/GOx/Fc. As expected, the results presented in Fig. 7A (curve c) indicate a significant improvement of sensing performance, with GOx immobilized in the SiO2/Lig hybrid material. A well-defined catalytic current is measured at CPE/GOx-SiO2/Lig/Fc after each glucose addition. The electrode response deviates from linearity at higher concentration (Fig. 7B) representing a typical characteristic of Michaelis–Menten kinetics. This behavior confirms that immobilized GOx is involved in glucose oxidation [6,40]. The apparent Michaelis-Menten constant (Km) can be calculated from the Lineweaver-Burk equation, 1/Iss = 1/Imax + Km/ImaxC, where Iss is steady state current of glucose oxidation, C and Imax are saturation concentration and corresponding maximum current. According to the data in Fig. S7 (see Supplementary Material) the Km is equal to 62 mM.



A linear relationship between the magnitude of current and glucose concentration has been observed for low glucose concentrations. The calibration curve in Fig. 7B with linear range spans the concentration of glucose from 0.5 up to 9 mM. The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated according to the formula: LOD (LOQ)=(κ · SDa)/b, where κ is 3.3 for LOD, 10 for LOQ and SDa is the standard deviation of the intercept and b is the slope. The determined values of LOD and LOQ amounted to 145 μM and 440 μM, respectively. The analytical performance was compared to the literature data covering the electrochemical detection of glucose at various modified electrodes (Table 2). It appears that the sensitivity, LOD and linear range are comparable to some values obtained for other systems. The precision of the method was estimated by performing twelve replicate determinations of 4 mM Glu on CPE/GOx-SiO2/Lig/Fc electrode. The method is precise because the relative standard deviation (RSD) was 4.72%. Inter day assay precision was also verified. The RSD of repeatability (RSDr) was 0.49% indicating a suitable repeatability of the detection method. The obtained data indicate that CPE/GOx/SiO2/Lig combined with an artificial redox mediator (Fc) can be applied for enzymatic determination of glucose at relatively low concentrations.



3.6. Interference study and stability of the biosensor

Common interference species such as L-ascorbic, uric acid and L-cysteine were studied on the detection of glucose. As seen in Fig. S8 (see Supplementary Material), uric acid (1 mM) and 1 mM L-cysteine give negligible interference (2% increase of current) up to 5 mM glucose for the CPE/GOx-SiO2/Lig/Fc electrode. However, 1 mM l-ascorbic acid

significantly affects the signal response to glucose due to the fact that both species can be oxidized at applied potential of +0.6 V. Such a problem can be avoided by using a permselective layer against anions. Nafion thin films are often used as an interferent rejection coating [8,46,47]. Thus, casting a thin film of Nafion polymer onto the top of the CPE/GOxSiO2/Lig/Fc electrode greatly diminishes the interfering AA species and enhances selectivity of the electrode (Fig. S9, see Supplementary Material).



The operational stability of the CPE/GOx-SiO2/Lig/Fc electrode was tested by continuous CV cycles between -0.4 and 0.6 V (50 mV s-1). It was found that peak currents retained 96% of their initial value after 150 scans, indicating good cycling stability. The storage stability revealed that the peak current of the biosensor decreased by approx. 25% within 3 weeks (Fig. S10, see Supplementary Material). The stability of the resulting biosensor toward glucose oxidation was also investigated for 3 weeks. When not in use, it was stored at 4 ºC in a refrigerator. The amperometric response signal of 5 mM glucose decreased after one week by 8%. The response current of the proposed electrode was reduced to 82% and 73% of its initial value after two and three weeks, respectively (Fig. 8).

3.7. Determination of glucose in real samples

To identify the feasibility of the proposed biosensor, the content of glucose in Liquid Glucose 1WW - strawberry taste (sample 1) and 5% Glucose infusion solution (sample 2) has been determined. Samples were purchased from local pharmacy. The amperometric assays were carried out by standard addition method (three additions of 2 mM glucose). Before

determination, the original samples were diluted with PBS to 100 mL (100-fold) in order to fit in the concentration range of the biosensor. The measurement results were shown in Table 3. The mean content of glucose was determined to be 3.36±0.082 (sample 1) and 2.90±0.084 mM (sample 2) indicating that the method is suitable for the determination of glucose in real samples.

4. Conclusion

The procedures presented here are promising for the immobilization of glucose oxidase and the development of stable glucose biosensors. The demonstrated SiO2/Lig presented satisfactory enzyme loading properties (25.28 mg g-1) with retained electroactivity of FAD/FADH2 redox center. GOx-SiO2/Lig has been revealed as a new biosensor platform for sensitive glucose determination and it may be anticipated that other enzymes and various mediators can be incorporated into the SiO2/Lig hybrid. The glucose biosensor presented high sensitivity of 0.78 µA mM-1. The linear response range of 0.5–9 mM has a detection limit (LOD) 145 µM. The CPE/GOx-SiO2/Lig/Fc can be an attractive alternative for the construction of efficient glucose biosensors. It has practical relevance to the usage of industrial pulp and paper by-product characterized by high accessibility and low cost. The approach described in this work also opens us up to the possibility of further extended studies on various lignin derivatives for the development of biosensors and biofuel cells.

Acknowledgments

The study was financed within the National Science Centre Poland funds according to decision no. DEC-2013/09/B/ST8/00159. The authors acknowledge to Dr. Marek Nowicki for the AFM investigations, Dr. Krzysztof Tadyszak for SEM observations as well as to Dr. Adam Piasecki for the EDS measurements.

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AUTHORS BIOGRAPHIES

Artur Jędrzak received the M.Sc., Eng. degree in Organic Chemistry in 2016 at Poznan University of Technology. Since 2016 he is a Ph.D. student of Chemical Technology at Poznan University of Technology and he is also a member of NanoBioMedical Centre in Poznan. His research interests are biosensors, enzymatic and catalytic systems, synthesis of hybrid/composite materials and receiving magnetic nanoparticles for medical application.

Tomasz Rębiś received a Ph.D. at Poznan University of Technology in 2015. Currently, he is holding a lecturer position in the Institute of Chemistry and Technical Electrochemistry at Poznan University of Technology. His research interests include the development of electrochemical sensors and new electrode materials for sensing.

Łukasz Klapiszewski received the Ph.D. at the Faculty of Chemical Technology, Poznan University of Technology in 2014. Now he is an professor assistant of the Division of Chemical Technology PUT. His research and scientific areas are: biopolymers, inorganic-organic hybrid materials, polymer

processing and environmental pollution protection. He has published over 50 peer-review papers, 3 book chapters and additionally holding over 10 patents and patent applications. He was awarded by Ministry of Science and Higher Education (2014).

Jakub Zdarta received the Ph.D. at the Faculty of Chemical Technology, Poznan University of Technology in 2017. Now he is a PostDoc Researcher at Center for BioProcess Engineering at DTU Chemical Engineering, Technical University of Denmark. His research focuses on enzyme immobilization including utilization of various types of novel support materials and different groups of biocatalysts for practical application. During his doctoral studies, he participated in numerous programs that supported his scientific carriers and mobility including Preludium and Etiuda financed by National Science Center Poland. During last 5 years he published over 20 peer-reviewed articles.

Grzegorz Milczarek received his Ph.D. in Chemical Technology at the Faculty of Chemical Technology, Poznan University of Technology in 1999. Then he moved to Tohoku University (Japan) to work in

Center for Interdisciplinary Research first as a postdoctoral fellow and then as a visiting professor. He also spent couple of months at Faculty of Physics, Chemistry and Biology at Linkoping University (Sweden). Now he is associate professor and head of the Division of General and Analytical Chemistry at the Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology. In years 2008 to 2016 he served as a vice-Dean for Student Affairs at the same faculty, and since 2016 he is the chair of the Institute of Chemistry and Technical Electrochemistry at Poznan University of Technology. His research and scientific interests include: electrochemistry, analytical chemistry, synthesis of nanoparticles, nanostructured and hybrid materials for energy storage as chemical sensors. He has published over 60 peer-review papers in prestigious scientific journals including Science.

Teofil Jesionowski was born in 1970. He received the Ph.D. in Chemical Technology at the Faculty of Chemical Technology, Poznan University of Technology in 1999. Now he is a full professor and head of the Division of Chemical Technology there. From 2008 to 2016 he served as a vice-Dean of the Faculty of Chemical Technology, and since 2016 he is a vice-Rector for Long Life Learning & International Education at Poznan University of Technology. His research and scientific areas are: surface and colloids science, dyes and pigments, biomimetic and hybrid materials, biopolymers, enzyme immobilization, functional fillers, polymer processing, nanocomposites and environmental pollution protection as well as chemical sensors. During his scientific carrier years, he has published over 260 peer-review papers, 5 book chapters and additionally holding over 45 patents and patent

applications. He was awarded by Foundation of Polish Science (1999) as well as Polish Academy of Science (2008). Moreover, he was appointed as Editor/Editorial Board Member of Scientific Reports journal as well as member of Editorial Board of Dyes and Pigments, Physicochemical Problems of Mineral Processing (former Editor) journals, all indexed by Thomson Reuters JCR.

Scheme and figure captions

Scheme 1. Scheme presents acting of GOx-SiO2/Lig into CPE for β-D-glucose. Fig. 1. (A) GOx-graphite/Fc; (B) GOx-SiO2/graphite/Fc; (C) GOx-SiO2/Lig/graphite/Fc; (D) SiO2/Lig before immobilization of GOx; (E) SiO2/Lig after immobilization of GOx. Fig. 2. FTIR spectra of: (A) activated lignin, modified silica, silica-lignin material and (B) glucose oxidase from Aspergillus niger and product after enzyme immobilization. Fig. 3. (A) Cyclic voltammograms of GC/SWCNT/PtNP/GOx-SiO2/Lig in absence (black line)

and

in

the

presence

(red

line)

of

O 2.

(B)

Cyclic

voltammograms

of

GC/SWCNT/PtNP/GOx-SiO2/Lig in the absence (black line) and in presence (blue line) of 2 mM H2O2. Supporting electrolyte: PBS (pH=7.0), scan rate 10 mV s-1. Fig. 4. CV of GC/SWCNT/PtNP/GOx-SiO2/Lig biosensor in PBS (pH=7.0) with increasing glucose concentration. Scan rate: 10 mV s-1. Fig. 5. (A) CV curves of CPE/GOx-SiO2/Lig/Fc at different scan rates (inner to outer curves correspond to 5, 10, 20, 40, 60, 80, 100 mV s-1). (B) and (C) correspond to the relationship between I vs v and I vs v1/2, respectively. Fig. 6. CV spectra of CPE/GOx-SiO2/Lig/Fc in PBS (pH=7.0) with increasing glucose concentration. Scan rate 10 mV s-1.

Fig. 7. (A) Amperometric response of (a) CPE/GOx/Fc, (b) CPE/GOx-SiO2/Fc and (c) CPE/GOx-SiO2/Lig/Fc towards successive addition of 0.5, 1 and 2 mM glucose in pH=7.0 (PBS) at 0.6 V under stirring conditions. (B) Calibration curve between response current of CPE/GOx-SiO2/Lig/Fc and glucose concentration. Fig. 8. Normalized amperometric I−t response of the 5 mM glucose recorded initially (for fresh electrode) and after a period of one, two and three weeks. Potential constant +0.6 V, pH=7.0 (PBS).

Scheme 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Table 1. The amount of GOx immobilized on the silica, graphite and silica/lignin hybrid material. Sample

Immobilized GOx /mg g-1

Catalytic activity /Umg-1 of the support)

Silica

12.88

7.34

Graphite

7.58

3.26

SiO2/Lig) hybrid material

25.28

11.75

Table 2. Comparison of parameters of selected modified electrodes for electrochemical sensing of glucose. Sensitivity Electrode

a

Linear range

/µM

Ref. /mM

graphene/AuNPs/GOD/chitosan

99.5*

180

0.2–4.2

[41]

BPEI-Fc/PEDOT:PSS/GOx/SPCE

66*

-

0.5–4.5

[39]

GCE/Chi-Py/Au/GOx

0.58

68

1.0–20

[42]

Nafion®/GOD/nanoCoPc–Gr

-

15

0.016–1.6

[35]

Gox-PoPD/PtNPs/PVF+ClO4−/Pt

17.4*

18

0.06–9.64

[33]

0.38

38

0.1–1.0

[4]

Nafion/GOD–OMCs/GE

0.053

156

0.5–15

[43]

Nafion/GOx/Aunano/Ptnano/CNT/Au

-

400

0.5–16.5

[44]

GOx/TiO2–SWCNT/ITO

5.32*

10

0.01–1.4

[45]

CPE/GOx-SiO2/Lig/Fc

0.78

145

0.5–9.0

This work

b

c d e

f

GOD/TCT/AP/OMC/GCE g

h

/µA mM-1 or * /µA mM-1 cm-2

Limit of detection

i

*

This means that the units in such cases are (µA mM-1 cm-2) instead of (µA mM-1)

a

Chitosan containing nanocomposites of graphene and gold nanoparticles with glucose oxidase at gold electrode

b

Redox polymer nanobeads of branched polyethylenimine binding with ferrocene with PEDOT:PSS and glucose

oxidase dropped on a screen-printed carbon electrode c

Film of chitosan–polypyrrole–gold nanoparticles with immobilized glucose oxidase

d

Cobalt phthalocyanine nanorods on graphene with nafion and glucose oxidase

e

Immobilized glucose oxidase in electropolymerized poly(o-phenylenediamine) film on a platinum

nanoparticles-polyvinylferrocenium modified electrode

f

Glassy carbon electrode (GCE) with covalently modified ordered mesoporous carbon (OMC) with glucose

oxidase (GOD) g

Highly ordered mesoporous carbons with nafion and glucose oxidase

h

Glucose oxidase (GOx) at the gold and platinum nanoparticles-modified carbon nanotube (CNT) electrode with

nafion i

Indium thin oxide (ITO) electrode with glucose oxidase (GOx) entrapped at single-walled carbon nanotubestitanium dioxide composite

Table 3. Determination of glucose in real samples. The results obtained from three repetitive measurements of two samples.

Number of measurement

Determined Glu concentration (mM) Sample 1

Sample 2

1

3.43

2.93

2

3.27

2.81

3

3.38

2.97

mean

3.36

2.90

standard deviation

0.082 (2.4%)

0.084 (2.9%)

- supplier specification

3.29

2.78