A Novel Responsive Sensor for Penicillium italicum Fruit Fungus Based on Mesoporous CaMn4O8-G-SiO2 Nanocomposite

In this study, we present the development of a novel CaMn4O8–G–SiO2 (CaMnGS) sensor to detect the presence of Penicillium italicum mold. CaMnGS as ternary type nanocomposite was synthesized using a self-assembly technique. CaMnGS sample demonstrated outstanding stability, high selectivity, and notable characteristics for Penicillium italicum fungus detection. For Penicillium italicum fungus sensing, the CaMnGS displayed a large linear range of (50–100) μL, and a low detection limit of 0.50 μL. Significantly, the CaMnGS sensor was capable of swiftly detecting the Penicillium italicum fungus in wastewater. The CaMnGS has been proven to improve the selectivity for Penicillium italicum and had tremendous potential for Penicillium italicum fungal sensing. The mesoporous CaMnGS sensor may also detect the presence or absence of the fungus Penicillium italicum. This method might be used to identify the fungus Penicillium italicum. This is also the first attempt to discuss the fabrication of an electrochemical sensor employing a mesoporous CaMnGS nanoparticle composite as a platform for the selective detection of Penicillium italicum. Penicillium italicum sensing


Introduction
Penicillium italicum is one of the most troublesome molds, and accounts for nearly 25% of citrus output globally; during storage and transportation, its infection is widespread [1]. Penicillium italicum, sometimes known as blue mold, is the costliest postharvest issue for citrus crops. It is a wound pathogen that exclusively infects damaged citrus trees. Fruitto-fruit contact may transmit the illness. Penicillium italicum may develop particularly at or below 10 °C, therefore cold storage does not preclude its presence or infection. This is what causes it to be infectious, even when fruit are stored in standard, protected conditions [2]. Numerous research efforts have been conducted to eradicate and inhibit the development of Penicillium italicum in citrus species [3], but no studies on the identification of Penicillium italicum magnetoresistance switching [7] all use manganese oxide compounds based on CaMnO 3 as electrode. Nanomaterials made from graphene are being studied for many new applications. Their vast surface area, electrical and thermal conductivity, mechanical strength, and optical transmittance make them stand out. Easy production, excellent stability in multiple solvents, and surface functionalization make graphene a desirable material for electrochemical sensors [8][9][10][11][12][13][14].
The development of mesoporous materials has grown exponentially in recent decades. Mesoporous silica particles have very interesting applications in sensing. To preserve or modify the optical or binding properties of the pores of the material, sensors are covalently bound to the surface of the material that is acting as a solid support. The sensing compound is loaded into the pores without covalent interaction. The selectivity achieved by interacting the analyte with the layer decorating the material adds to the amplifying impact of high transducer loading into the pores [15,20].
Mycotoxins are often detected in centralized labs using procedures that include HPLC, fluorescence, SPR, chemiluminescence, ELISA, quartz crystal microbalance, and solidphase extraction. These techniques are sensitive and selective, but necessitate heavy equipment, complicated sample preparation and clean-up processes, expert operators, and overall high expenses. Thus, disposable biosensors with point-of-care capabilities are a viable tool for mycotoxin detection. Recently, nanomaterials have been employed to construct biosensors with better electrocatalytic capabilities. The most widely used nanomaterial for biosensors is graphene. Graphene's unique qualities include huge surface area (2630 m 2 /g), excellent electrical and thermal conductivity, good biocompatibility, cheap cost, heterogeneous electron transfer (HET), and high stability (125 Gpa). Graphene-based nanomaterials also offer superior macroscopic conductivity and electrocatalytic activity [21].
To achieve the all of properties, we designed a graphenebased electrode with unique characteristics and an increased redox electrochemical signal that resulted in sensitivity and selectivity for Penicillium italicum fungi. In this work, we used a CaMn 4 O 8 -G-SiO 2 (CaMnGS) modified electrode to detect Penicillium italicum fungi as a biomarker of infections.

Synthesis of Mesoporous CaMn 4 O 8 -G (CaMnG)
After 0.1 g of graphite oxide (GO) was added into 550 mL of water, the reaction mixture was irradiated with a highintensity ultrasonic probe at RT in air for 70 min.

Synthesis of Mesoporous CaMn 4 O 8 -G-SiO 2 (CaMnGS)
The CaMn 4 O 8 -G solution was drop-by-drop added to a beaker containing 0.15 g of mesoporous silica powder, which was synthesized using our previously reported method [21] and stirred at 120 °C for 24 h. Next, the mixture was ultra-sonicated for 30 min. The powder was then filtered, washed with ethanol, and dried at 70 °C overnight. After that, it was calcined at 700 °C at a heating rate of 10 °C/min, and then held at 700 °C for 5 h.

Characterization of Materials
The phase structure and purity of the as-synthesized products were examined by X-ray diffraction (XRD; Rigaku, Japan) with Cu-Kα radiation (λ = 1.

Electrochemical Measurements
Three electrodes were used in this experiment, namely a platinum counter electrode, an Ag/AgCl reference electrode, and an aqueous buffer solution as the electrolyte. The detection limit was calculated using Eq. (1) shown below [22,23]: where, Sv is the standard deviation of the response of the curve, and S the slope of the calibration curve. CV measurements were made from (− 0.3 to + 0.2) V versus Ag/AgCl at 10 mV s −1 . has now been established. Orthorhombic perovskite structure is seen in CaMn 4 O 8 . The (004), (011), (200), (112), (020), (013), (116), and (020) planes in the XRD pattern of mesoporous CaMnGS are connected to various XRD peaks in the XRD pattern. The orthorhombic structure was clearly visible in every pattern in the produced material, with no traces of impurity phases [24]. Figure 1 (b) shows the EDS analysis of CaMnGS that was used to identify its elemental composition. EDS identified a high concentration of C, O, Ca, Mn, and Si elements, but no additional contaminants. The CaMnGS sample quantitative investigation discovered significant amounts of C (3.47%), O (48.44%), Ca (25.50%), Si (5.9%), and Mn (23.53%), in addition to other elements. SEM, TEM, and HRTEM images of CaMn, CaMnG, and CaMnGS samples were collected for electron microscopy studies to examine sample morphology, as shown in Fig. 2.

Characterization of the CaMnGS Sample
According to the results of TEM and HRTEM images, the self-assembly synthesis yields agglomerated CaMn nanoparticles [25], with an average diameter as indicated in Fig. 2a. When combined with graphene and mesoporous SiO 2 , as shown in Figs. 2b and c, SEM images of CaMnG and CaMnGS reveal the material's well-distributed nature. As shown SEM images, the particle size of CaMnG increased when graphene was added to CaMn, and the particle size of CaMnGS increased further when mesoporous SiO 2 was added to CaMnG. CaMnG and CaMnGS are both confirmed by HRTEM images. In Fig. 2f, a HRTEM image representing mesopore is presented. The pore size shows a size of about 18 nm. Figure 3 shows the (a) N 2 adsorption-desorption isotherms, (b) surface area plot, (c) t-plots, and (d) pore distribution. Mesoporous materials are known to exhibit the H2 Type IV isotherm and a Type H2 hysteresis loop in the (0.6-0.9) P/P 0 range. In the adsorbent, mesopores were discovered to have a pore size distribution of 13.97 nm, confirming the existence of mesopores. The type of adsorption isothermal curve is an important factor that indicates the shape of pores. The formation of an isothermal curve hysteresis loop is largely over all range, which indicates the formation of mesopores uniformly. Uniform formation of mesopores will increase the effect of sensor sensitivity and reactivity with an increase in adsorption amounts.
XPS analysis was used to determine the surface chemical compositions of the mesoporous CaMnGS. Figure 4 shows the Ca, Mn, C, O, and Si results. The CaMnGS nanocomposite's chemical states and surface composition were confirmed using an XPS examination, which was carried out in the lab. Figure S1a of the Supplementary information shows the XPS analysis of Ca2p that was used to detect the chemical state of Ca in CaMnGS. To distinguish one peak from the other, the Ca2p spectra has two peaks with binding energies of (348.12 and 351.62) eV for Ca2p 3/2 and Ca2p 1/2 respectively. According to the binding energies of calcium in the CaMnGS [26], the Ca2p peaks in the XPS spectrum are consistent. There are two peaks at (641.85 and 654.02) eV for Mn 2p 3/2 and Mn 2p 1/2 , which are characteristic of Mn 3+ -based materials [27] in Fig. S1b of the SI. This shows that the CaMnGS surface prefers Mn substitution. Figure  S1c of the SI shows the C1s spectra exhibit peaks at (284.08 and 289.08) eV that are thought to represent C − C bonding and O − C = O. Figure S1d of the SI, which depicts O1s spectra, shows C − O bonding at 532.02 eV [20]. The presence of SiO 2 on the CaMnGS surface is seen in Fig. S1e of the SI at 102.03 eV [22]. Figure 5(a) shows the EIS findings. Due to its high electrical conductivity, the CaMnGS has a charge-transfer resistance of 25 Ω cm −2 .
The initial charge-transfer resistance (between 3 ~ 150 Z' regions) of CaMnGS was found to be lower than that of CaMn and CaMnG. This suggests that graphene addition significantly increases the electrical conductivity, and therefore, the electrochemical performance in a CaMnGS  sample. As illustrated in Fig. 5b, Raman spectra may be used to study the graphene oxide D and G bands for structural information. A ratio of ID/IG of CaMn was not shown, which indicates that peaks were not shown because it did not contain graphene. In the case of CaMnG, a relatively large amount of graphene is contained, and the ratio of ID/ IG shows a relatively high peak intensity of 0.92. In addition, the ratio of ID/IG of CaMnGS was close to 1.0, which was relatively small. This shows a small peak intensity due to the reason that the amount of graphene is less than the amount of CaMnG.
Both of these bands are associated with the out-of-plane breathing mode of the Sp 2 atom induced by defects and E 2g phonons near the Brillion zone center. The G band is created by the stretching of carbon sp 2 atom bonds, whereas the D band is caused by structural disorder [27]. The ID/ IG ratio (derived from the intensity of the D and G bands) may be used to describe the graphitic structural disorder in carbon materials. Because of the various functional groups generated during the oxidation of graphite powder, GO has a very disordered structure, as seen in Fig. 5b. The D band is located at 1352 cm −1 , while the G band is located at 1596 cm −1 . The maximum peak current shows 6.6 × 10 −1 mA cm −2 for CaMnGS electrode, while CaMn and CaMnG are found at (2.0 × 10 −3 and 3.0 × 10 −4 ) mA cm −2 , respectively. According to CaMn, CaMnG, and CaMnGS, the peak current improved in CaMnGS due to the CaMnGS electrode electroconductivity, which was discovered to have further increased by adding graphene and SiO 2 to the working electrode surface. It has been shown that graphene and SiO 2 work synergistically to enhance electron transport between the redox probe and electrode surface. The electrochemical performance of the CaMnGS working electrode has been significantly improved because of the addition of graphene and SiO 2 to the porous structure. In part, this is due to the large surface area of the composite material, and the functional group located there [28]. Exposure to Penicillium italicum fungus reduced the current for CaMn and CaMnG to 1.0 × 10 −4 and 5.0 × 10 −5 mA cm −2 , respectively, as shown in Fig. 7(a), but Fig. 7(b) shows that the peak current for CaMnGS electrode decreased to 2.0 × 10 −5 mA cm −2 .

Electrochemical Behavior of CMGS
This is due to the CaMnGS electrode SiO 2 surface modification, which increases the rate at which electrons move from the electrolytic solution to the electrode. There are chemical and biological components on the CaMnGS surface that drastically restrict the pace at which electrons may be transferred. The study confirmed that CaMnGS electrodes for biosensing must be modified with conductive nanomaterials on the surface to collect the tiniest of biological activity signals. A decrease in current density and an increase in insulating quality are achieved by Penicillium italicum covalently adhering to the electrode surface [28]. For further study, it was decided to build a biosensor to measure the quantitative amount of Penicillium italicum to simultaneously identify the fungus, which is discussed in more detail in the next section. Chronoamperometry was also used to measure Penicillium italicum fungus, as seen in Fig. 8.
The CaMnGS electrode was utilized to facilitate a suitable reaction, which was then delivered to the electrochemical detection cell for chronoamperometry, together with the various concentrations of Penicillium italicum. As the Penicillium italicum concentration rises, the observed current diminishes. Covalent connections formed between the Penicillium italicum fungus and the biosensor inhibit the movement of electrons from the solution to electrode surface. In terms of linear detection range, low detection limit, and analysis time, the proposed biosensor possesses all of these features, and more [29]. The best biosensor response was discovered at pH 7.0, which is a critical factor in biosensor response. Lower reaction was seen at higher pH values. Biosensor sensitivity was only about half of what it was when the pH level was higher in the acidic range. In the next part, we study pH dependence to determine the suitable pH value.
Because the pH of the electrolyte might affect the peak potential and peak current of the modified electrode, measuring the proton-to-electron ratio involved in the electrode reaction is more advantageous than determining other parameters. Because a change in pH results in a change in the current density of the electrode, the two are related. As a result, the pH range is also critical for biosensors. Figure 9  shows the results of a pH scan performed at a rate of 10 mVs −1 throughout the pH range (4 to 9).
There is a tiny shift in current density in the pH range (4 − 9), which we can see. During a study of CaMnGS peak currents, it was discovered that pH 7.5 was the most appropriate for biological chemical detection applications.
Additional comparators for evaluating the CaMnGS sensor selectivity included kitchen fungus, bread fungus, and biofilm. An electrode was immersed with a variety of strains of the same concentration, as seen in Fig. 10, to estimate the current density. Penicillium italicum exhibited a lower value when the other two species were injected dropwise, although the value of Penicillium italicum was 5 × 10 −5 mA cm −2 .
The sensor was able to correctly identify Penicillium italicum, as shown by the results. This phenomenon happened due to the hydrogen bonding of Penicillium italicum with mesoporous CaMnGS sensor. Penicillium italicum binds with hydrogen bonding and collapses the electron transfer by blocking the active site. Then any other biological material can't bind. Detection of the selectivity was carried out after the appropriate pH and thermal states had been established. Figure 11 depicts the temperature-dependent sensitivity of CaMnGS electrodes to Penicillium italicum.
For the CaMnGS sensor, the sensor sensitivity was evaluated to ensure that the sensor sensitivity of Penicillium italicum was affected by temperature changes. To demonstrate its capacity to detect temperature, the CaMnGS electrode performed well in temperature ranges of 20 to 70 °C. When the temperature rises over 65 °C, the Penicillium italicum is eliminated, as shown by our sensor, and the CaMnGS sensor may transfer electrons and enhance the conductivity of the sensor, confirming this. In parallel, the temperature dependency test provides the thermal stability determination and confirmation of the absence or presence of Penicillium italicum. While at 37 °C, the current density is 3.0 × 10 −5 mA cm −2 , due to the presence of Penicillium italicum in the sensor, which prevents electron transfer, when the temperature is raised to 65 °C, the Penicillium italicum is destroyed, resulting in an increase in the current density to 1.0 × 10 −4 mA cm −2 . As a result, the ideal sensor temperature to detect Penicillium italicum well is (30 to 50) °C [30]. Electrochemical properties of rare metal also offer superior macroscopic conductivity and electrocatalytic activity [31].  The sensor effect measured at a temperature of 20 °C showed similar results at a temperature condition of 25 °C. In addition, the temperature conditions at 37 °C showed similar results to the sensor effect measured at 40 °C. Therefore, in this study, sensor effects were presented under three typical temperature conditions.

Conclusions
For Penicillium italicum detection and sensing, CaMnGS mesopores were produced. When compared to Penicillium italicum, the CaMnGS produced mesoporous CaMnGS demonstrated high sensitivity and specificity. CaMnGS has been proven to improve the selectivity for Penicillium italicum. Because of its ability to both qualitatively and quantitatively identify Penicillium italicum, it is a viable solution for low-resource regions. Penicillium italicum may be detected in food samples using affordable point-of-use and high-throughput quantitative methods devised by the researchers. Penicillium italicum contamination in fruit may be detected using simple procedures in the field, in storage, and in transit situations. Consequently, the high sensitivity, cheap fabrication costs, and real-time operation of the suggested detection method are very promising, opening the way for a cost-effective and real-time sensor. Penicillium italicum infections may be detected and treated early, preventing the development of multidrug-resistant Penicillium italicum.
Author Contribution W-CO contributed to the study conception and design. Material preparation, data collection, and analysis were performed by KYC, C-HJ, JYC and WKJ. The first draft of the manuscript was written by W-CO and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding The authors have not disclosed any funding.
Data Availability All data generated or analyzed during this study are included in this article.

Conflict of interest
The authors declare no conflict of interest with this work.
Ethical Approval All data generated or analyzed during this study are included in this published article.

Research Involving Human and Animal Rights
This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent
Informed consent was obtained from all individual participants included in the study.