Introduction

Class III peroxidases (EC 1.11.1.7) constitute a large multigene family found in plants. They are heme-containing enzymes that catalyze the oxidation of a wide range of organic and inorganic substances through the reduction of peroxides such as H2O21,2. These enzymes are extensively used in numerous applications, such as diagnostic kits and biosensors3,4, decolorization and degradation of dyes5, bioremediation of phenolic compounds6, organic synthesis7, and cancer treatment8.

Horseradish Peroxidase (HRP), a member of the class III plant peroxidases, is one of the most well-known and extensively used enzymes in biotechnological, industrial, environmental, and analytical applications9,10,11,12. Recently, Fattahian et al.13 introduced a novel plant peroxidase gene from Lepidium draba, belonging to the Brassica family, and recombinantly expressed in the prokaryotic system. Using the basic local alignment search tool (BLAST), the amino acid sequence of Lepidium draba peroxidase (LDP) shows 93% similarity and 89% identity with HRP C1A. Moreover, due to catalytic activity and refolding yield, it is introduced as a suitable plant peroxidase for industrial applications.

One of the main applications of plant peroxidases is in biosensors and diagnostic kits14. Biosensors are extensively applied in medicine15, the food industry16, and environmental monitoring17. In medicine, biosensors detect biomolecules such as glucose, cholesterol, lactate, and uric acid18,19. Glucose is an essential metabolite for the human body, and its elevated blood glucose levels are associated with diseases including diabetes, heart attack, kidney failure, blindness, and islet cell carcinoma20,21. Consequently, highly sensitive and accurate glucose determination is vital for disease prevention and clinical diagnostics. Additionally, measuring glucose concentration is important in the food industry, biology, chemistry, and environmental protection22,23. In the medical field, numerous analytical techniques have been developed for glucose detection, such as electrochemical24, fluorescence, and colorimetric biosensing methods25. Colorimetric biosensing methods are highly promising due to their selectivity, excellent sensitivity, low cost, and the ability for results to be interpreted visually based on color change. Currently, most colorimetric glucose biosensors are based on the use of glucose oxidase (GOx) and peroxidase. GOx catalyzes the oxidation of β-D-glucose in the presence of O2 to produce gluconic acid and H2O2, which further oxidizes chromogenic substrate agents in the presence of peroxidase to produce colorimetric detectable signals20. However, the primary challenges associated with using soluble enzymes in industrial applications are their lack of reusability and their low thermal and storage stability26. Immobilization is one of the developed ways to overcome these problems5. To date, several immobilization methods have been developed, including physical adsorption, covalent linkage, encapsulation, entrapment, and cross-linking27,28. Among these, covalent linkage of enzymes on carriers provides stable and strong binding, thereby minimizing enzyme leaching from the carrier surface29,30. In this approach, selecting an appropriate carrier (characterized by properties such as being mechanically resistant, reactive groups, and surface area) is one of the most important factors31. The immobilization of enzymes on solid supports is a critical factor that influences the success of enzyme-based biosensors32.

Recently, Fe3O4 nanoparticles (nFe3O4) have attracted significant interest for enzyme immobilization because of their properties, including low toxicity, large surface area, suitable physical properties, and easy recovery and reusability from the medium using external magnetic fields33,34,35. Furthermore, modification of nFe3O4 with biopolymers such as chitosan can protect the nanoparticles from oxidation and provide functional groups (e.g., amino, hydroxymethyl, and hydroxyl), necessary for enzyme immobilization34,36.

Chitosan (CS), poly (1–4)-2-amino-2-deoxy-D-glucose, is a polysaccharide derived from the partial deacetylation of chitin. Due to its excellent biochemical properties, including biocompatibility, biodegradability, availability, low cost, non-toxicity, and antibacterial activity, chitosan has found widespread applications29,37,38,39,40. So far, numerous studies have reported the use of chitosan-iron oxide nanoparticles for enzyme immobilization within the framework of biosensor development41,42,43,44.

In this work, glutaraldehyde-activated, chitosan-coated Fe3O4 nanoparticles (nFe3O4-CS-GDA) were used as an appropriate support for the immobilization of the recombinant LDP enzyme. Immobilization conditions, including GDA concentration, coupling time, and the amount of the support, were optimized to achieve the maximum immobilization efficiency. Subsequently, confirmation of the immobilizing enzyme on the support, physicochemical and kinetic properties, thermostability, and storage stability of the immobilized enzyme were assessed and compared with those found in the free enzyme. The reusability of the IE was evaluated, and its applicability was tested on colorimetric glucose sensing. Covalently linked LDP on the chitosan-coated Fe3O4 NPs (nFe3O4-CS) was confirmed using FT-IR, XRD, and FE-SEM. The data showed that the IE had improved affinity to the substrates (TMB and H2O2) and enhanced catalytic efficiency compared to the native enzyme. Furthermore, the stability of the IE against heat and storage conditions improved significantly. Finally, the applicability of the IE enzyme was tested and compared with the free enzyme in glucose sensing, with results indicating that immobilization meaningfully increased the sensitivity of the glucose detection.

Materials and methods

Materials

Ni-NTA agarose resin was purchased from Agarose Bead Technology (ABT) Company. Hemin, kanamycin, imidazole, monopotassium phosphate (KH2PO4), and dipotassium phosphate (K2HPO4) were obtained from Molekula (Gillingham, Dorset, UK). 3,3′,5,5′-tetramethylbenzidine (TMB), isopropyl-β-d-thiogalactopyranoside (IPTG), and chitosan were obtained from BioBasics (Markham, Canada). Glutaraldehyde 50% (v/v) was obtained from Zaozhuang Kerui Chemicals Co., Ltd (Shandong, China), and β-D-glucose was purchased from Merck. Glucose oxidase from Aspergillus niger was supplied by Sigma Aldrich. Fe3O4 NPs (average size of 60 nm and > 99.2 purity) were provided by NaBond Technology Inc.

LDP expression and purification

The recombinant strain (E. coli T7 SHuffle) containing the pET28a (+)-LDP expression system was cultured in the Luria Bertani (LB) medium according to the one-step production of soluble and active form of the peroxidase, under the Iranian patent number of 91,172. Briefly, 1 mL of overnight culture was inoculated into 100 mL of LB medium containing kanamycin (50 µg/mL) at 37 °C, 180 rpm min−1 until the OD600 reached 0.6–0.8. IPTG was then added to a final concentration of 0.1 mM, and the culture was induced at 18 °C, 180 rpm min−1 for 7 h. Cells were harvested by centrifugation (at 6,000×g) for 10 min. The precipitated cells were suspended in lysis buffer, sonicated for 30s, and centrifuged (at 6,000×g) for 45 min at 4 °C. The recombinant enzyme was purified through a Ni-NTA chromatography column with imidazole as an elution buffer. Purification was confirmed using 12% SDS-PAGE gel electrophoresis according to the Laemmli method45, and the protein content was measured using the Bradford assay46.

Determination of enzyme activity

Peroxidase activity was measured according to Krainer et al.47. One milliliter reaction mixture contains 10 mM H2O2, 0.6 mM TMB in potassium phosphate (50 mM), and 50 µL of free LDP or immobilized LDP. The change in absorbance at 653 nm (the extinction coefficient of TMB was 3.9 × 104 mol−1 cm−1) due to TMB oxidation was recorded at 30s intervals. One unit of LDP activity was defined as the enzyme amount needed for the oxidation of 1 µmol TMB in 1 min at 25 °C and pH 6.0.

Synthesis of chitosan-coated magnetic nanoparticles

Chitosan-coated magnetic nanoparticles were synthesized as reported by Lin et al.48, with minor modifications. In brief, 0.25 g of chitosan (CS) was dissolved in acetic acid (1%, 50 mL). Afterwards, 2 g of magnetic nanoparticles (nFe3O4) were added to this solution, and the mixture was stirred vigorously at room temperature. After 30 min, 50 mL of 1 M NaOH was added via syringe into the mixture to prepare nFe3O4 coated with chitosan (nFe3O4-CS). The particles were washed repeatedly with deionized water until a neutral pH was achieved and then dried at 50 °C in a vacuum oven.

Immobilization of the LDP

In the current study, LDP was covalently anchored on nFe3O4-CS using glutaraldehyde (GDA). To achieve optimal immobilization efficiency, several parameters including GDA concentration (1.25, 2.5, and 5% (v/v)), coupling durations (2, 4, 6, 18, and 20 h), and the quantity of support (0.2, 0.1, and 0.05 g), were evaluated in presence of constant enzyme concentration (1 mg/mL) in the immobilization mixture. Then after, the resulting nanostructures were washed 3 times with deionized water to remove excess unbounded components such as glutaraldehyde. Subsequently, 6 mL of 1 mg/mL LDP solution was added to GDA GDA-activated support. After gentle stirring at 25 °C for 18 h, the product was collected magnetically using an external magnet, before being washed three times with phosphate buffer (50 mM, pH 7.0), and then collected. The resulting nFe3O4-CS-GDA-LDP was resuspended in 2 mL of PBS buffer (50 mM, pH 7) for further use.

The LDP immobilization efficiency was calculated according to Eq. (1)36:

$${\text{Immobilization}}\;{\text{efficiency }}\left[ \% \right] = \frac{{{\text{The}}\;{\text{concentration}}\;{\text{of}}\;{\text{protein}}\;{\text{immobilized}}\;{\text{on}}\;{\text{the}}\;{\text{nFe}}_{{\text{3}}} {\text{O}}_{{\text{4}}} - {\text{CS}}\;{\text{carrier}}}}{{{\text{Total}}\;{\text{protein}}\;{\text{concentration}}\;{\text{of}}\;{\text{free}}\;{\text{LDP}}}} \times 100$$
(1)

The concentration of protein immobilized on the nFe₃O₄–CS carrier was calculated as the total protein concentration minus the concentration of unbound protein in the supernatant.

Characterization of nFe3O4-CS-GDA-LDP

Immobilization of the enzyme on the nanostructure was confirmed using techniques such as FTIR, XRD, FE-SEM, and EDX analyses.

FTIR spectra of nFe3O4, nFe3O4-CS, and nFe3O4-CS-GDA-LDP were recorded in the range of 4000–400 cm− 1 by Thermo Nicolet AVATAR 370 (Courtaboeuf, France). X-ray diffraction (XRD) measurements: crystal structure of the samples was obtained using a XRD Rigaku Ultima IV in the laboratory of the Mahamax, an online characterization platform in Tehran, Iran. FE-SEM images of the samples were studied using MIRA3 LMU (Tescan Company, Czech Republic). The chemical composition analysis was performed using the energy-dispersive X-ray spectroscopy (EDX).

Optimum pH and temperature

To measure optimum pH, the residual activities of both free and IE were evaluated at different pH values at room temperature (25 °C). The buffers used were sodium acetate (pH 3.0–5.6, 50 mM) and potassium phosphate (pH 5.8-8.0, 50 mM)49. To obtain the optimum temperature, residual activities of the free and the IE were investigated in potassium phosphate buffer (50 mM) under the optimum pH with temperatures ranging from 15 °C to 40 °C. The results were presented as residual activity based on the amount of protein present in the free and immobilized samples.

Thermal stability and half-life of the free and the immobilized LDP

Thermostability was measured by incubating free and immobilized LDP at different temperatures (20–50 °C) for 10 min. Following incubation, samples were kept in an ice bath for 5 min, and enzyme activity was measured as described in “Determination of enzyme activity” section.

Deactivation rate constant (kd) was determined from the Arrhenius plot of log residual activity (%) in relation to time (min) using Eq. (2)50:

$${\text{Slope}} = k_{d}$$
(2)

The half-life (t1/2) of the enzymes, determined as the duration required for the enzyme to diminish to 50% of its initial activity51, was calculated using Eq. (3):

$$t_{{1/2}} = Ln~2/k_{d}$$
(3)

wherein t1/2 (min) represents the half-life of the enzyme and kd (min− 1) denotes the thermal inactivation rate constant.

Kinetic parameters

The kinetic parameters, Michaelis-Menten constant (Km) and maximum reaction velocity (Vmax) of the free and the immobilized LDP were determined using different substrate concentrations of TMB and H2O2. The constant concentration of TMB (0.6 mM) was examined with various concentrations of H2O2 (0.01 to 20 mM) in optimum conditions. Furthermore, various concentrations of TMB (0.01 to 1 mM) were evaluated in the presence of a constant concentration of H2O2 at 10 mM. The turnover number (kcat s−1) for both the free and IE was determined using Eq. (4):

$$k_{{cat}} = V_{{max}} /E_{T}$$
(4)

In this context, Vmax (mM.s− 1) represents the maximum reaction velocity, while [ET] pertains to the total concentration (mM) of enzymes present within the reaction medium. The catalytic efficiency constant (s−1·mM−1) for both free and immobilized LDP was derived as the turnover number (kcat s−1), and the Michaelis-Menten constant, as delineated in Eq. (5):

$${\text{Catalytic efficiency}} = k_{{cat}} /k_{m}$$
(5)

Storage stability of the free and immobilized LDP

Storage stability of the free and immobilized LDP was assessed by quantifying their residual enzymatic activities over 60 days at 4 °C in a phosphate buffer solution (pH 6.0, 50 mM). Residual enzymatic activities were quantified at designated intervals (every 7 days) employing the following equation 51:

$${\text{Residual activity}}\left( \% \right) = \left( {E/E_{0} } \right){\text{1}}00$$
(6)

where E and E0 denote the enzymatic activities at time t (min) and at the initial time point, t = 0 min, respectively. The initial activity of the free and IE was considered 100%.

Reusability of the immobilized LDP

To assess the reusability of the immobilized LDP, a singular reaction cycle was conducted in a volume of 1.0 mL of reaction mixture, which comprised 10 mM H2O2, 0.6 mM TMB, and a 50 mM phosphate buffer (pH 6.0). Subsequently, the immobilized LDP was washed three times with phosphate buffer at pH 6.0, using an external magnet to eliminate any residual substrate, and then reintroduced into the subsequent reaction cycle. The residual activity after each cycle was determined as described in “Storage stability of the free and immobilized LDP” section.

Glucose detection concentration

Glucose detection was carried out as follows: 25 µL GOx (1 mg/mL), different concentrations of glucose (0.1, 0.2, 0.4, 0.8, 1, 3, 5, and 10 mM), and 50 mM phosphate buffer (pH 6.0) were mixed in a final volume of 500 µL and incubated at 37 °C for 45 min. Then LDP enzyme (free or immobilized) (0.3 mg/mL), TMB (0.6 mM), and 50 mM phosphate buffer (pH 6.0) were added. After 10 and 30 min incubation at 37 °C, the color intensity of the mixtures was measured at 653 nm using UV-spectroscopy (Termo Scientific Evolution 300). The limit of detection value was calculated using Eq. (7)52:

$${\text{LOD}} = ~\frac{{3\;{\text{Standard}}\;{\text{deviation}}\;{\text{of}}\;{\text{blank}}}}{{{\text{Slop}}\;{\text{of}}\;{\text{calibration}}\;{\text{curve}}}}$$
(7)

Results and discussion

Expression and purification of the Recombinant LDP

LDP expression was carried out using the T7 SHuffle strain in the presence of 0.1 mM IPTG, and the subsequent purification process was achieved using a Ni-NTA-Sepharose affinity chromatography column. The purified proteins were analyzed on 12% SDS-PAGE, and the recombinant LDP was estimated to be approximately 34 KD of a single band as deduced from SDS-PAGE (Fig. 1). The high quality of the purified protein (> 95%) was observed in lanes 3, 4, 5, 6, and 7. These fractions were collected and stored at -20 °C to be applied for further studies.

Fig. 1
Fig. 1
Full size image

SDS-PAGE of the purified recombinant LDP with different concentrations of imidazole, lane “A” is the protein ladder, lane “B” is the before column sample as control, lane “C” is the eluted sample passed from the column with wash buffer, Lane umbers 1, 2, 3, 4, 5, 6, 7, and 8 are related to the protein output from the column with concentrations of 300, 300, 250, 250, 200, 200, 100, 50 mM imidazole, respectively.

Immobilization efficiency

To obtain the highest residual activity and immobilization efficiency of the IE, several factors, including GDA concentration, coupling time, and the amount of support, were tested in phosphate buffer (50 mM, pH 6.0) containing a constant enzyme concentration of 1 mg/mL at 25 °C. Among the tested support concentrations, only 0.05 g yielded results (data not shown). The effect of GDA concentration on the remaining activity and immobilization efficiency of the enzyme is shown in Fig. 2a. As revealed in the figure, the lowest and highest GDA concentrations yielded lower residual activity of the IE than was observed at 2.5% GDA concentration. This can be attributed to the inadequacy of low GDA concentrations to generate sufficient crosslinking for the enzyme; conversely, an excessive crosslinking may lead to a distortion of the enzyme’s tertiary structure53. Such structural distortion may impede the accessibility and accommodation of the substrate, consequently affecting the retention of enzyme activity54. Therefore, 2.5% was chosen as the optimal GDA concentration for enzyme immobilization; under these conditions, the highest immobilization efficiency was obtained (30%).

Fig. 2
Fig. 2
Full size image

(a) Optimization of glutaraldehyde concentrations and (b) time of coupling for LDP immobilization on the support.

In Fig. 2b, the impact of coupling time on residual activity and immobilization efficiency is depicted. As shown in the figure, the residual activity of the IE was gradually elevated by increasing immobilization time, so that the maximum was observed at 18 h, and by increasing coupling time over 18 h, residual activity changed considerably. This may occur because the immobilization of the enzyme on the support reached a saturated duration of 18 h. With the extension of the reaction time, any augmentation in the quantity of IE may significantly enhance the steric hindrance experienced by the IE molecules, resulting in a slight reduction in enzyme activity. By increasing the immobilization time, immobilization efficiency was also improved, so that it reached a maximum of 30% after 18 h.

According to the results, under the experimental conditions in phosphate buffer (50 mM, pH 6.0) containing 0.05 g of nFe3O4-CS, 1.0 mg/mL of LDP, and at 25 °C, the optimal GDA concentration and incubation time were obtained at 2.5% (v/v) and 18 h, respectively. Similar results regarding the GDA concentration and coupling time were reported by several studies which were conducted to immobilize HRP on a nanostructure by the use of GDA. For example, Lu et al.55 documented the immobilization of HRP on glutaraldehyde-activated carbon nanospheres using 2% glutaraldehyde (GDA) (v/v), with a coupling time of 18 h at 25 °C. Zai et al.56 successfully immobilized HRP on chitosan–halloysite hybrid-nanotubes (CTS–HNT) using 2.5% GDA, at 4 °C for 15 h. Keshta et al.27 documented immobilization of HRP on Fe3O4@NH2 NPs at room temperature overnight.

Under optimal immobilization conditions, an immobilization efficiency of 30% was obtained. El-Shishtawy et al.51 immobilized the catalase enzyme on chitosan-coated zinc oxide nanoparticles. They reported an immobilization efficiency of 37%. Jankowska et al.57 reported a 27% immobilization yield for the covalent immobilization of HRP on Electrospun fibers. Gomez et al.58, using glutaraldehyde as a coupling agent to immobilize HRP on glass beads, reported a 40% immobilization efficiency. Chagas et al.59 showed that the immobilization efficacy of turnip peroxidase on chitosan beads was 51%.

Immobilized confirmation

FT-IR spectra analysis

The FT-IR spectra of naked nFe3O4, nFe3O4-CS, and nFe3O4-CS-GDA-LDP are shown in Fig. 3. For naked nFe3O4, an absorption band at approximately 598 cm−1 corresponds to the Fe-O stretching vibrations (Fig. 3a)60. In nFe3O4–CS NPs (Fig. 3b), the band at 1073 cm−1 is attributed to C-O-C stretching vibrations, demonstrating the binding of chitosan to the nFe3O4 NPs61. In Fig. 3c, the band at about 1061 cm−1 (C–N bond) shows the crosslinking of GDA to chitosan62. Furthermore, new bands observed at around 1651 cm−1 and 1541 cm−1 correspond to the –CONH– (amide I) and amide II vibrations of protein34,63, confirming the binding of the enzyme to the support.

Fig. 3
Fig. 3
Full size image

FT-IR spectra of (a) nFe3O4, (b) nFe3O4-CS, and (c) immobilized LDP.

FE-SEM analysis

FE-SEM images (Fig. 4a–c) provide a detailed examination of nFe3O4, nFe3O4-CS, and immobilized LDP. The surface morphology of the chitosan-coated and immobilized nanoparticles exhibited significant changes: nanoparticle surface roughness increased following chitosan treatment, subsequent glutaraldehyde activation, and enzyme immobilization. The coating of the surface with chitosan and the enzyme’s subsequent attachment to the activated surface could be the cause of this phenomenon64. Additionally, the EDX spectrum of Fe3O4 showed the presence of iron and oxygen, and the Fe3O4-CS spectrum revealed the presence of carbon, nitrogen, iron, and oxygen. Finally, the EDX spectrum of the immobilized LDP showed the presence of carbon, nitrogen, iron, oxygen, and sulfur. Therefore, the EDX results confirmed the presence of the essential elements in the respective materials, as shown in Fig. 4a a–c, which confirmed the immobilization of the enzyme on the support.

Fig. 4
Fig. 4
Full size image

FE-SEM images and corresponding EDX spectra of (a) nFe3O4, (b) nFe3O4-CS, and (c) immobilized LDP. E: Elements; W: Weight; A: Atomic. XRD pattern of the samples (d).

XRD analysis

Figure 4d illustrates the XRD patterns corresponding to the naked nFe3O4, nFe3O4-CS, and immobilized LDP, wherein six distinctive peaks associated with nFe3O4 were identified (220, 311, 400, 422, 511, and 440) across all samples. The identified peaks show that the synthesized nanoparticles are comprised of pure nFe3O4 exhibiting a spinel crystallographic structure. These results suggest the nanoparticles preserved the crystalline structure of nFe3O4 after the chitosan coating and enzyme immobilization processes34,38.

Effect of pH and temperature on the free and immobilized LDP activity

The effect of pH on the activity of immobilized LDP and free LDP was studied in the pH range of 3.0–8.0 at 25 °C (Fig. 5a). Both enzyme forms exhibited maximum activity at pH 6.0, which was considered the optimal pH value. This finding is consistent with the findings documented for most plant peroxidases, which typically show optimal enzymatic activity in the pH range of 4.5 to 6.027. In Patel et al.65, the optimal pH values for HRP activity were determined to be 6 and 7 on rGO-Fe3O4-M1 and Fe3O4 particles, respectively. Chang et al.66 reported that immobilization of HRP on a superparamagnetic Fe3O4/graphene oxide nanocomposite yielded similar optimal pH values of approximately 6.4 for both free and immobilized HRP. In another study, Muley et al.67 explained that the immobilization of cellulase on iron oxide magnetic nanoparticles did not change the optimal pH, which remained between 6.0 and 6.5. Compared to the free LDP, the immobilized LDP exhibited higher residual activity across all pH values, indicating improved pH adaptability. This phenomenon may be attributed to the covalent interactions between the enzyme and chitosan-coated magnetic nanoparticles (CMNP), which have potentially constrained the conformational changes, resulting in enhanced stability across an extended pH spectrum68. It should be noted that the IE exhibited the highest residual activity from pH 3 to 7, while for free LDP peak activity was from pH 5 to 7.

Fig. 5
Fig. 5
Full size image

Effect of (a) pH and (b) temperature on residual activity of the free and immobilized LDP. (c) Thermal stability of LDP enzyme. (d) Time course of stability against heat of the free and immobilized LDP at 50 °C. Bars show one standard deviation of the mean (n = three replicates).

The optimum temperature of the free and immobilized LDP was determined by measuring residual activity across a temperature range from 15 °C to 40 °C at optimal pH, as is shown in Fig. 5b. The optimum temperature of the free LDP was recorded at 30 °C, while the immobilized LDP peaked at 25 °C (room temperature). In widely circulated literature, it was documented that the optimum temperature of the IEs shifts towards higher29,49,69 or lower70,71,72 values compared to the related free enzymes. The high residual activity of the immobilized LDP at room temperature facilitates its application in diagnostic kits. Compared to the free LDP, the immobilized LDP demonstrated higher residual activity, demonstrating that immobilized LDP had better activity at a temperature range from 20 to 40 °C. Chattopadhyay & Mazumdar observed that HRP exhibited its highest activity at temperatures of 25–40 °C11. Moreover, Xie et al.73 reported that, compared with free HRP, the stability and enzymatic activity of the immobilized HRP on Fe3O4@PAA-6-armPEG-NH2 showed a significant enhancement across a broad temperature range. Generally, interactions between enzymes and between the enzyme and its support increase with increasing temperature, which may affect the enzyme’s optimal temperature74. However, immobilization of LDP on the Zn-MOF nanostructure70 and surface mutation (N186R and N198R)75 has been reported to decrease the optimum temperature, and this decrease was accompanied by changes in the enzyme structure. Consequently, it may be speculated that the LDP structure was also affected through covalent immobilization on CS-nFe3O4.

Thermal stability and half-life of the free and immobilized LDP

Thermal stability of the free and immobilized LDP was studied at various temperatures ranging from 20 to 50 °C. As shown in Fig. 5c, IE exhibited better thermal stability across all temperatures, approximately 10% higher stability compared to the free one. On the other hand, the free and immobilized LDP displayed maximum activity at temperatures ranging from 30 to 40 °C, a similar pattern that was reported recently by Farhadi et al.70. Furthermore, Huang et al.76 reported that the lipase, which was covalently immobilized on magnetic iron nanoparticles by the carbodiimide, showed enhanced stability in response to variations in temperature.

The time courses of enzyme stability against heat during a 90 min test at 50 °C are shown in Fig. 5d. The residual activity of the free and immobilized LDP was 62.67% and 82.18%, respectively. After 30 min incubation of the enzymes at 50 °C, approximately 7% of the initial activity was lost for IE and more than 30% for the free enzyme. The deactivation rate constant (kd) is an important parameter for advancing economic bioprocesses at an industrial scale30. At 50 °C, the kd for the IE was 0.0023 min−1, which is lower than that of the free enzyme (0.0052 min−1). A lower kd for the immobilized LDP signifies a lower rate of denaturation77. Additionally, the half-life (t1/2) of the enzyme at this temperature increased from 133.30 min (for the free enzyme) to 301.37 min upon immobilization, suggesting that immobilization of the enzyme effectively enhances its thermostability. To date, literature has reported that stability and resistance to temperature variation improve upon enzyme immobilization78. Covalent immobilization of an enzyme to a support often limits its conformation, thereby leading to greater resilience against thermal denaturation79.

Kinetic parameters

As summarized in Table 1, Km values of the immobilized LDP for substrates (TMB and H2O2) decreased, rather than the free LDP. On the other hand, the results demonstrated that the affinity toward TMB and H2O2 of the immobilized LDP was drastically enhanced by a factor of 1.7 and 3.8-fold in comparison with the free counterpart. These findings are consistent with some recently reported data. For example, Farhadi et al.70 elucidated that the immobilization of LDP on the Zn-MOF enhances enzyme affinity towards TMB by a factor of around 1.5. Gao et al.80 reported immobilization of HRP on hierarchically porous magnetic metal-organic frameworks (HP-Zr-MOF@Fe3O4) also increased enzyme affinity towards the substrate by over 2.2 times.

In addition, the Vmax of the immobilized LDP was markedly higher than that observed for the free enzyme. In particular, the Vmax of the immobilized LDP increased by about 2 and 4-fold for TMB and H2O2, respectively, compared to that of the free enzyme. This enhancement may be attributed to the fact that immobilization alters the structural conformation of the enzyme and optimizes the functionality of its catalytic active site, thus facilitating an increase in the reaction rate and promoting the conversion of the substrate to product6.

Besides, the Kcat and kcat/Km for immobilized LDP were higher than those for free LDP. The turnover number (kcat) exhibits a significant magnitude, and the process of product formation within catalysis becomes increasingly advantageous. Furthermore, an elevated catalytic efficiency (kcat/Km) indicates a superior proficiency of the enzyme and facilitates its conversion into products (Table 1)81. Jaiswal et al.82 documented that the catalytic efficiency of the immobilized laccase in chitosan beads increased more than 10-fold in comparison to that of the free form. Also, Vineh et al.83 reported that, following the immobilization of HRP on functionalized reduced graphene oxide by covalent bonding, the kcat/Km demonstrated an approximate 8.5-fold increase. The pronounced affinity exhibited towards the substrate, coupled with the catalytic efficacy of LDP immobilized on nFe3O4-CS-GDA, underscores the proficiency and effectiveness of the employed support and immobilization methodology84.

Table 1 Kinetic parameters of the free and immobilized LDP for TMB and H2O2.
Full size table

Storage stability of the free and immobilized LDP

To assess enzyme storage stability, the residual enzymatic activity was evaluated following the storage for 60 days at 4 °C. The results showed that about 20% and 46% of the initial activity of free and IE was restored after around 2 months, respectively. According to Fig. 6a, 50% of the initial activity for the free and immobilized LDP was reduced after 23 and 48 days, respectively. These results demonstrate that enzyme immobilization improved its storage stability approximately 2-fold compared to the free enzyme. This can be due to the covalent interactions between the enzyme and the support, as reported by Sahu et al.29. They documented that the storage stability of the immobilized HRP on chitosan-Fe2O3 nanoparticle coated with graphene oxide significantly improved following covalent bonding of the HRP to the support matrix.

Fig. 6
Fig. 6
Full size image

(a) Storage stability of the free and immobilized LDP at 4 °C in the period time of 60 days. (b) Reusability of immobilized LDP. Bars show one standard deviation of the mean (n = three replicates). For more details, see the Experimental procedures. Spectrophotometric plot for standard glucose assay with (c) free LDP and (d) immobilized LDP.

Reusability of the immobilized LDP

Because chitosan-coated magnetic nanoparticles possess magnetic properties and biocompatibility, immobilized enzymes can facilitate recovery processes for subsequent reutilization85.

The capacity for enzyme reusability constitutes one of the primary advantages associated with the immobilization of the enzymes. This attribute of permitting enzymes to be utilized across multiple cycles represents the most critical determinant for environmental and industrial applications, owing to its economic efficiency29,84,86. The performance of immobilized LDP across multiple reaction cycles is illustrated in Fig. 6b. The immobilized LDP exhibited a retention of about 73% activity over five cycles of TMB oxidation, facilitated by its rapid and efficient separation through magnetic means, indicating good operational stability of the immobilized LDP. Keshta et al.27 reported that the HRP immobilized on Fe3O4@NH2 with guaiacol as substrate retained 60% of its initial activity after 5 cycles. Mohammadi et al.87 reported a reuse of immobilized enzyme on epoxy-functionalized silica, and showed 61% retention of its enzymatic activity subsequent to 5 cycles of use. The activities of the IE exhibited a gradual decline concomitant with an increase in the number of reuse cycles. These findings may be explained by the inactivation of the enzyme, which is attributed to the phenomena of leakage and denaturation of the enzyme. Also, the decrease may be due to the loss of particle fraction during use and washing as reported by Abdella et al. 50. The capacity for reusability of the IE constitutes a critical determinant for practical applications aimed at minimizing expenses and simplifying procedures36,88. Recently, Farhadi et al.70 reported that the process of centrifugation employed during the application of MOFs for enzyme immobilization results in the agglomeration of materials, which consequently leads to enzyme leakage, thereby adversely impacting their reusability80. In our work, the use of chitosan-coated magnetic nanoparticles effectively addresses this challenge by facilitating the swift separation of immobilized enzymes from the reaction medium when subjected to an external magnetic field.

Glucose detection

Glucose determination was performed based on the combined reaction of GOx and peroxidase (LDP/nFe3O4-CS-GDA-LDP) activity. In this process, glucose was oxidized in the presence of GOx enzyme to produce gluconic acid and hydrogen peroxide. Then, hydrogen peroxide was detected quantitatively in a colorimetric reaction of TMB to produce blue-colored oxidized TMB (ox-TMB), with a characteristic absorption peak at 653 nm89. The produced dye intensity represented the glucose content of the sample. As shown in Fig. 6c, d, in the presence of free LDP and immobilized LDP, the ox-TMB intensities increase with increasing concentrations of glucose in the media. The glucose biosensing process shows linearity as 0.1 to 1 mM (after 30 min) and 0.1 to 10 mM (after 10 min) for glucose solution, and a limit of detection around 0.027 and 0.025, for the free LDP and immobilized LDP, respectively. In Table 2, a comparison of the results obtained in this study and other studies on glucose colorimetric biosensors is presented. Filiz et al. 3 documented the synthesis of electrospun blended chitosan-poly (vinyl alcohol) nanofibers for a colorimetric glucose biosensor of GOx/HRP. The experimental findings showed that the colorimetric reaction was linear for glucose detection, ranging from 2.7 to 13.8 mM, with a limit of detection at 2.7 mM. Zhu et al.21 designed a semi-quantitative approach for the determination of glucose utilizing self-calibration based on GOD/HRP bi-enzyme colorimetry through the application of tree-shaped paper strips. The linear detection range was from 1.0 to 11.0 mM, accompanied by a detection limit of 0.3 mM. Furthermore, Skonta et al.90 presented a colorimetric glucose biosensor based on chitosan films. The linear range was observed from 0.1 to 0.8 mM with a detection limit of 0.033 mM.

Also, Soni & Jha.91 developed a non-invasive glucose biosensor utilizing a paper strip for the analysis of saliva. The designed optical biosensor exhibited a detection range spanning from 9 to 1350 mg/dL of glucose, alongside a notable limit of detection of 22.2 mg/dL. Zhang et al.92 constructed a paper-based device (µPAD) sensor for the colorimetric detection of glucose based on glucose oxidase/horseradish peroxidase (GOx/HRP) bi-enzymatic system using TMB as chromogenic agent. The µPAD could realize the quantitative detection of glucose within 0.5–2.84 mM, and the detection limit was 0.28 mM. Ultimately, Gabriel et al.93 documented a colorimetric approach for the quantification of glucose utilizing GOx and HRP enzymes on a paper device, achieving a linear glucose detection range from 0.1 to 1 mM, with a detection limit of 0.05 mM.

Considering that the physiological blood glucose level ranges from 3.0 to 8.0 mM20. More specifically, the lower detection limit and significantly extended linearity range observed in this study, comparable to that reported in the literature (Table 2), indicate the great potential of immobilized LDP for glucose detection.

Table 2 Comparison of analytical performance of some enzyme-based glucose biosensors.
Full size table

Conclusion

In the current study, recombinant LDP peroxidase was immobilized on a magnetic nanoparticle; the physicochemical properties of LDP-magnetic nanoparticle were investigated, and its potential in glucose detection was examined. Our results demonstrate that covalent immobilization of LDP on nFe3O4-CS greatly enhances its functionality, as revealed by higher residual activity and affinity to its substrates. Additionally, thermostability, storage stability, and reusability of the enzyme were improved. The enhanced residual activity and stability of the IE make it suitable for biomedical and food analysis, particularly for glucose detection and reliable quantification. Surprisingly, the glucose detection concentration range increased by more than 10-fold, and the incubation time was reduced by more than 3-fold for the IE compared to the free enzyme.

Further research is needed to increase immobilization efficiency. Further studies using the IE on the other colorimetric biosensors (e.g., cholesterol, urea, etc.) would be more attractive for research. Moreover, the determination of the exact mechanism underlying the improved stability of LDP upon covalent immobilization on chitosan-coated magnetic nanoparticles could provide valuable insights.

Overall, the enhanced reactivity and catalytic efficiency, coupled with enzyme recyclability and strong binding, improved the operational stability of the LDP immobilized on nFe3O4-CS-GDA, underscoring the proficiency and effectiveness of the employed support and immobilization methodology. In conclusion, the IE can be introduced as a suitable enzyme for biomolecule detection in clinical practice, enabling precise quantification, and can also be used in various industrial applications.