Quantification of ochratoxin A in a coffee sample utilizing an electrochemical aptasensor fabricated through encapsulation of toluidine blue within a Zn- based metal-organic framework

doi:10.21203/rs.3.rs-4956429/v1

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Quantification of ochratoxin A in a coffee sample utilizing an electrochemical aptasensor fabricated through encapsulation of toluidine blue within a Zn- based metal-organic framework

https://doi.org/10.21203/rs.3.rs-4956429/v1

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Due to the widespread presence of mycotoxins and their significant impact on both health and the economy, there is a clear need for a fast and cost-effective analytical tool to measure these harmful substances. In response to this, an electrochemical aptasensor has been developed specifically for the sensitive and selective determination of ochratoxin A (OTA), one of the most important mycotoxins. The aptasensor utilizes a glassy carbon electrode that has been modified with toluidine blue (TB) encapsulated in a Zn-based metal organic framework (TB@Zn-MOF). The results demonstrate that in the presence of OTA, the peak current of the differential pulse voltammogram (DPV) related to TB oxidation is notably decreased. The changes in the oxidation peak current of TB encapsulated in Zn-MOF, both in the absence and presence of OTA, serve as an analytical signal for accurately measuring its concentration. With the proposed aptasensor, OTA can be measured within a linear concentration range of 1.0 × 10^− 4 − 200.0 ng mL^-1, with a detection limit of 2.1×10^− 5 ng mL^-1. Furthermore, this aptasensor design is suitable for measuring OTA concentration in coffee powder samples. This represents the first report to utilize TB@Zn-MOF in designing an applicable aptasensor to OTA measurement. The high porosity of the Zn-MOF allows for a large number of TB molecules to be encapsulated in its cavities, while its large surface area enables more OTA aptamers to be immobilized on the electrode surface. These two key features significantly enhance the sensitivity of the aptasensor in measuring OTA concentration.

Electrochemical aptasensor. Toluidine blue. Zn

based metal organic framework. Ochratoxin A

Mycotoxins, including aflatoxins, zearalenone, ochratoxins, fumonisins and trichothecenes, are produced by more than 200 mold species [1]. The growth conditions for these fungi depend on factors such as temperature, humidity, and the type of material. Ochratoxin A (OTA) is a specific type of mycotoxin produced by Penicillium and Aspergillus fungi [2]. OTA can contaminate various commodities, such as dried fruits, cereal grains, chocolate, nuts, coffee, wheat, corn, milk, and animal feed [3, 4]. This mycotoxin is difficult to remove from the food chain due to its thermal and chemical stability [5]. OTA poses potential hazards, including nephrotoxicity, hepatotoxicity, carcinogenicity, neurotoxicity, teratogenicity, and immunotoxicity [6]. Coffee is a widely consumed functional beverage worldwide. However, its complex nature makes its susceptible to contamination at different stages of production, including harvesting, storage, and roasting. Some fungi not only damage the coffee fruit but also lead to the formation of mycotoxins like OTA by affecting the grains [7]. Research has found that coffee beans contain OTA both before and after roasting, indicating that roasting alone cannot significantly decrease OTA content in coffee [8]. The European Union (E.U.) has established a maximum permitted limit for OTA in roasted/ground coffee beans at 5.0 ng/ml^− 1 [9]. Therefore, it is crucial to develop a method for accurately measuring low levels of OTA in coffee. Several conventional methods, including thin–layer chromatography [10], capillary electrophoresis [11], high–performance liquid chromatography [12] and mass spectrometry [13] have been used to determine OTA concentration. However, these methods have limitations, such as the requirement for highly skilled personnel, expensive equipment, and lengthy sample preparation times, which restrict their usage. In recent decades, electrochemical biosensors have emerged as a promising platform for mycotoxin measurement. Aptamer-based electrochemical biosensors, in particular, have gained popularity for OTA detection due to their high selectivity and sensitivity, affordability, ease of use, miniaturization and portability [14–18]. Aptamers are synthetic single-stranded oligonucleotides that exhibit high affinity for target molecules. They can bind to various species, including microorganisms, proteins, cells, metal ions, amino acids, drugs, and vitamins [19].

Using nanomaterials can increase the sensitivity of electrochemical aptasensors. One such class of nanomaterials is metal-organic frameworks (MOFs), which are organic-inorganic hybrid materials made up of organic linkers and metal ions/clusters [20]. MOFs can be controlled and regulated for specific applications due to the interactions between the organic linkers and metal ions [21]. They are often used to develop biosensors because of their well-structured nature, large surface area, are biodegradability, and biocompatibility [22]. The high porosity and large surface area of MOFs make them useful materials for loading electroactive compounds and increasing the sensor sensitivity. Some examples of electroactive materials that have been loaded into MOFs include methylene blue [23], hemin [24], and toluidine blue [25].

In this study, a sensing platform was developed by encapsulating toluidine blue (TB) within a Zn-based metal-organic framework (TB@Zn–MOF). The high porosity of Zn-MOF allows for a large number of TB molecules to be encapsulated inside the cavities. These properties greatly enhance the response and sensitivity of the aptasensor. The TB@Zn–MOF modified electrode was then utilized to fabricate an electrochemical aptasensor for the determination of OTA. The OTA aptamer was immobilized on the TB@Zn–MOF modified electrode and incubated with OTA. To quantitatively measure OTA, the peak current of the differential pulse voltammogram was measured before and after incubating OTA with the immobilized aptamer. The difference between these two currents represents the analytical response of the aptasensor. The proposed aptasensor was also successfully employed to measure OTA coffee powder sample, obtaining satisfactory results. To the best of our knowledge, this is the first report to utilize TB@Zn-MOF in designing an applicable aptasensor to OTA measurement.

Apparatus and methods

Electrochemical measurements were performed with an Autolab potentiostat/galvanostat model PGSTAT 30 (Eco chemic Utrecht, The Netherlands) equipped with the General Purpose Electrochemical System (GPES) and the Frequency Response Analyzer (FRA) software. A three-electrode system was employed consisting of a modified glassy carbon electrode (GCE) with a diameter of 2 mm, a platinum rod, and an Ag/AgCl/KCl (sat’d) electrode which served as the working, auxiliary, and reference electrode, respectively. To adjust the pH values a Metrohm model 827 pH/mV meter was used. A MIRA3 TESCAN field emission scanning electron microscope (FE-SEM) at an acceleration voltage of 15.0 kV was used to study the morphology of TB@Zn-MOF. A Bruker Vector 22 FT-IR spectrophotometer (Bruker, Germany) was used to record the FT-IR spectra in the range of 400 to 4000 cm^− 1.

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed in a phosphate buffer (0.1 M, pH 7.0) containing 5.0 mM [Fe(CN)₆]^3−/4− and 0.1 M KCl. To record cyclic voltammograms, the potential was scanned from − 0.04 V to 0.6 V with a scan rate of 50 mV s^− 1. The frequency range of 10^− 1 to 10⁵ Hz with an amplitude of 5.0 mV and a formal potential of 250 mV were also used to record the EIS data. Differential pulse voltammograms were recorded in a phosphate buffer (0.1 M, pH 7.4) over the potential range of -0.4 V to 0 V, using a step potential of 5 mV and a modulation amplitude of 25 mV.

Materials

Nafion (5% w/v) was prepared from Aldrich Company. Zinc nitrate Zn(NO₃)₂, toluidine blue (TB), 1,3,5-benzenetricarboxylic acid (H₃BTC), potassium ferricyanide (K₃[Fe(CN)₆]), potassium ferrocyanide (K₄[Fe(CN)₆]), tris–hydroxymethylaminomethane hydrochloride (Tris), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro–chloride (EDC), N-hydroxysuccinimide (NHS), hydrochloric acid (HCl), potassium chloride (KCl), hydroxide (NaOH), phosphoric acid (H₃PO₄), methanol (CH₃OH), ethanol (C₂H₅OH), bovine serum albumin (BSA), and the other reagents were purchased from Merck Company. Ochratoxin A (OTA), aflatoxin M1 (AFM1), and aflatoxin B1 (AFB1) were purchased from Libios Co. (France). The oligonucleotide sequence of 5′-NH₂-C₆H₁₂-GATCG GGTGT GGGTG GCGTA AAGGG AGCAT CGGAC ACGCC ACCCA CACA-3' that is related to the OTA aptamer was purchased from Bioneer Co. (South Korea).

Synthesis of TB@Zn-MOF

To prepare TB@Zn-MOF, 0.26 g of Zn(NO₃)₂.6H₂O was dissolved in 5.0 mL of double-distilled water. Then, a solution of 0.1 g H₃BTC in ethanol (5.0 mL) was added to this solution with vigorous stirring. Subsequently, 0.01 g of TB was dissolved in 1.0 mL of double-distilled water and added to the above mixture. The mixture was stirred for 1 hour at room temperature. The prepared solution was heated in a water bath at 85°C for 12 hours. The substance was centrifuged at 8000 rpm for 5 minutes, and the sediment was rinsed several times with double distilled water. The resulting TB@Zn–MOF was dried at 65°C for 12 hours [25].

Fabrication of the aptasensor

To fabricate the aptasensor, initially, a GCE was polished using alumina powder and subsequently rinsed with double distilled water. To modify the GCE, 1.0 mg TB@Zn–MOF and 3.0 µL Nafion (0.1%) were dispersed in 1.0 mL double–distilled water and ultrasonicated for 10 minutes. 3.0 µL of the prepared TB@Zn–MOF suspension was dropped onto the electrode surface and allowed to dry at room temperature. The modified electrode was then immersed in EDC/NHS solution (4:1 ratio) for 20 minutes to activate the carboxylic acid groups of TB@Zn–MOF. In the next step, the OTA aptamer with a concentration of 7.0 µM was immobilized on the modified electrode surface by incubating in a humid chamber for 4 hours (Apt/TB@Zn–MOF/GCE). The electrode surface was then rinsed with a phosphate buffer (0.1 M, pH 7.4). To block possible active sites, 5.0 µL of BSA solution (1.0%) was then applied to the Apt/TB@Zn–MOF/GCE surface for 30 minutes. After that, OTA at different concentrations was incubated on the surface of BSA/Apt/TB@Zn–MOF/GCE for 2 hours. Finally, the differential pulse voltammograms of BSA/Apt/TB@Zn–MOF/GCE were recorded before and after the incubation of OTA. The changes in the oxidation peak current of TB on the surface of these electrodes were considered as the analytical response. Scheme 1 illustrates the details of the preparation process of TB@Zn–MOF and sensing mechanism of the aptasensor.

Preparation of solution from coffee powder

The coffee powder was purchased from a local supermarket and used as a real sample. To prepare the sample, 1.0 g of coffee powder was mixed with 5.0 mL of methanol with vigorous stirring for 30 minutes at room temperature. The supernatant was then filtered via filter paper and diluted ten times with methanol. Subsequently, 0.5, 1.0, and 5.0 ng mL^− 1 OTA was added to the diluted solutions. Eventually, the OTA concentration in the prepared sample was determined using the developed aptasensor.

Characterization of TB@Zn-MOF

The surface morphology of TB@Zn-MOF was investigated using FESEM. As shown in Fig. 1A, the TB@Zn-MOF exhibits a filamentous morphology, indicating a distinct and well-defined 3D structure. Figure 1B displays the FTIR spectrum of TB@Zn-MOF. The absorption bands at 1715 cm^− 1 and 1453 cm^− 1 correspond to the asymmetric and symmetric stretching vibrations of the carboxylate group in TB@Zn-MOF, respectively. The strong absorption band at 3432 cm^− 1 is attributed to the stretching vibration of the primary amine in TB. The absorption peaks at approximately 3084, 1610, and 1228–1387 cm^− 1 correspond to the stretching vibrations of the C–H bond of unsaturated compounds, the stretching vibration of the C = N bond, and the stretching vibration of the C–N bond in aromatic amine, respectively. The stretching vibrations of the C–N and C–S bonds were also observed in the range of 1281 − 894 cm^− 1. Energy dispersive X-ray spectroscopy (EDS) was used to identify the compositions of different elements in TB@Zn-MOF. The EDS spectrum of TB@Zn-MOF is shown in Fig. 1C, which reveals the presence of C, N, Zn, S, O, and Cl elements. The uniform distribution of N and S elements in TB@MOF is likely due to the presence of TB molecules. TB not only controls the deprotonation of ligands, but it also regulates the electrostatic adsorption of the H₃BTC ligand with negatively charged ions and TB ions with positively charged ions. As a result, TB molecules can effectively be enclosed within the cavities of Zn-MOF. The EDS mapping of TB@Zn-MOF is displayed in Fig. 2, showing the homogeneous distribution of the elements C, N, O, S, Zn, and Cl.

Sensing mechanism of OTA using the proposed aptasensor

Since TB is an electroactive species, TB encapsulated in Zn-MOF can generate an electrochemical signal in the aptasensor. To ensure accurate monitoring of the preparation steps, differential pulse voltammograms of the proposed aptasensor were recorded after each stage of the aptasensor fabrication process. Figure 3 shows the differential pulse voltammograms of the bare GCE (BGCE), TB@Zn-MOF/GCE, Apt/TB@Zn-MOF/GCE and OTA/BSA/Apt/TB@Zn-MOF/GCE in a 0.1 M phosphate buffer (pH 7.4). As depicted in this figure, no significant oxidation peak is observed for the BGCE. However, when the GCE was modified by TB@Zn-MOF, a considerable oxidation peak was observed at a potential of approximately − 0.25 V. This sharp oxidation peak can be attributed to the oxidation of encapsulated TB in Zn-MOF. The carboxylic groups of TB@Zn-MOF were activated by EDC/NHS, and the OTA aptamer was then immobilized on the modified electrode surface (Apt/TB@Zn-MOF/GCE). The immobilization of the aptamer leads to a decrease in the oxidation peak current of TB. This phenomenon can be attributed to the blocking effect of the aptamer, which decreases the electron transfer of TB and thus the electrochemical signal. After incubation of BSA and OTA on the Apt/TB@Zn-MOF/GCE, the peak current of TB also decreases. This reduction is caused by the further obstruction of the electrode surface and hindrance to the electron transfer at the TB@Zn-MOF. Therefore, the TB peak current was changed before and after incubating OTA with immobilized aptamer. The difference between these currents served as the analytical response of the aptasensor for the determination of OTA.

Tracking the fabrication steps of the OTA aptasensor

During the fabrication process of the aptasensor, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were employed to monitor its electrochemical behavior. Specifically, Nyquist plots (Fig. S1A) and cyclic voltammograms (Fig. S1B) were recorded for the following setups: bare glassy carbon electrode (BGCE), TB@Zn-MOF modified GCE (TB@Zn-MOF/GCE), aptamer immobilized TB@Zn-MOF modified GCE (Apt/TB@Zn-MOF/GCE), and OTA/BSA/aptamer immobilized TB@Zn-MOF modified GCE (OTA/BSA/Apt/TB@Zn-MOF/GCE), using a redox probe comprising of 5.0 mM [Fe(CN)₆]^3−/4− and 0.1 M KCl in a 0.1 M phosphate buffer solution at pH 7.0. The Nyquist plot reveals two distinct sections: a semicircular region and a linear region. The semicircle’s diameter represents the charge transfer resistance (R_ct), while the linear section indicates a diffusion-controlled process. Comparing different setups, the Nyquist plot of the BGCE exhibits a small semicircle, which increases upon modification with TB@Zn-MOF due to poor conductivity and the presence of carboxyl groups on the TB@Zn-MOF surface, hindering electron transfer between the redox probe and the electrode surface. Subsequently, immobilizing the aptamer on TB@Zn-MOF/GCE further increases the semicircle's diameter and R_ct, primarily due to electrostatic repulsion between the negative charges on the aptamer backbone and [Fe(CN)₆]^3−/4−. Notably, after incubation of OTA at a concentration of 0.5 ng mL^− 1 on BSA/Apt/TB@Zn-MOF/GCE, a significant increase in R_ct is observed. This can be attributed to the interaction between the immobilized aptamer and OTA, generating additional negative charges and effectively blocking the electrode surface. The cyclic voltammograms shown in Fig. S1B perfectly align with the results obtained from the EIS technique. In fact, the CV method further validates the steps involved in constructing the aptasensor.

Optimization of experimental parameters

To ensure optimal performance, several parameters that may influence the aptasensor's response were optimized. These parameters include aptamer concentration, aptamer immobilization time, and OTA incubation time. Throughout all experiments, the OTA concentration remained constant at 0.5 ng mL^− 1. Differential pulse voltammetry was used to record the peak current of Apt/TB@Zn-MOF/GCE before (I_a) and after (I_b) OTA incubation in a 0.1 M phosphate buffer solution at pH 7.4. The difference between I_a and I_b, denoted as ΔI, (ΔI = I_a – I_b) served as the analytical signal. The effect of aptamer concentration on the response of the aptasensor was investigated by incubating different concentrations of the aptamer on the TB@Zn-MOF/GCE surface for 4 hours. After blocking the electrode surface with BSA, OTA (0.5 ng mL^− 1) was incubated for 2 hours. Differential pulse voltammograms were recorded and ΔI values were calculated. The plot in Fig. S2A shows the ΔI values against the various concentrations of aptamer. It is evident that the response is enhanced with increasing aptamer concentration up to 7.0 µM, after which it slightly decreases. This can be attributed to the fact that with increasing aptamer concentration up to 7.0 µM, non-specific adsorption of aptamer molecules is low and the aptamer orientation remains favorable. Beyond this concentration, non-specific adsorption of aptamer molecules increases, leading to a decrease in the response. Thus, 7.0 µM was chosen as the optimal aptamer concentration for subsequent experiments. To optimize the immobilization time of the OTA aptamer, the aptamer was applied to the TB@Zn-MOF/GCE surface at an optimal concentration for 2 to 8 hours. OTA (0.5 ng mL^− 1) was then incubated on the electrode surface for 2 hours. The plot in Fig. S2B shows the ΔI values against the immobilization time of the aptamer. The largest ΔI value is observed at 4 hours, indicating that the active sites on TB@Zn-MOF are saturated after this time. Therefore, 4 hours was chosen as the aptamer immobilization time for further experiments. The optimization of the incubation time of the immobilized aptamer with OTA was performed by applying 0.5 ng mL^− 1 OTA to the BSA/Apt/TB@Zn-MOF/GCE surface for different time intervals (1 to 4 hours). The results in Fig. S2C show that the ΔI value increases with increasing incubation time up to 2 hours. This indicates that the structure of the aptamer-OTA complex is fully formed by this time. Therefore, 2 hours was determined to be a sufficient time for the interaction of OTA with the aptamer in subsequent experiments.

Analytical performance of the designed aptasensor

The analytical performance of the aptasensor was investigated using the differential pulse voltammetry (DPV) method, which is a sensitive electrochemical technique. The differential pulse voltammograms of BSA/Apt/TB@Zn-MOF/GCE were recorded before and after incubation with OTA concentrations ranging from1.0×10^− 4 to 200.0 ng mL^− 1. As shown in Fig. 4, the peak oxidation current of TB decreased with increasing OTA concentration. The ΔI values exhibited a linear relationship with the logarithm of OTA concentration within the range of 1.0×10^− 4 to 200.0 ng mL^− 1 (Inset of Fig. 4). The detection limit of OTA was estimated to be 2.1×10^− 5 ng mL^− 1 using the calibration diagram and the equation y_l.o.d.= y_bl. + 3s_bl [26]. In this equation, s_bl and y_bl refer to the standard deviation and mean response of three replicate measurements in a blank solution. The detection of OTA has been reported in several studies. Table 1 compares the analytical parameters of OTA using the proposed aptasensor with those from other studies [6, 27–29]. The data in this table demonstrates that the designed aptasensor has a superior linear concentration range and excellent detection limit compared to other aptasensors.

Table 1

Comparison of the analytical parameters of OTA determination using the aptasensor developed in this work with values reported in other works
Modified electrode^a	Method^b	Linear range (ng mL^–1)	Detection limit (ng mL^–1)	Real sample	Reference
HRP/AuNPs/aptamer/BSA/cDNA/rGO/PAMAM/GCE	DPV	5.0–5.0×10⁵	31.0×10^–2	Wine	[6]
Apt/SA/Nafion–f–MWCNTs/SPCE	DPV	5.0×10^–3–10.0	1.0×10^–3	Malt	[27]
cDNA–MCH–Apt–modified AuE	EIS	5.0×10^–2–10.0	5.0×10^–2	Malt	[28]
Apt–Fc/BSA/cDNA/Ru@SiO₂–BPQDs/AuNPs–PEI–MWCNTs/GCE	ECL	1.0×10^–1–320.0	3.0×10^–2	Wheat– Oats	[29]
BSA/Apt/TB@Zn–MOF/GCE	DPV	1.0×10^–4–200.0	2.1×10^–5	Coffee	This work

^aApt/SA/Nafion–f–MWCNTs/SPCE: Aptamer/streptavidin/Nafion–stabilized functionalized multi walled carbon nanotubes/screen printed carbon electrode; HRP/AuNPs/aptamer/BSA/cDNA/rGO/PAMAM/GCE: Horseradish peroxidase/modified gold nanoparticles/aptamer/Bovine serum albumin/complementary DNA/graphene oxide/polyamidoamine/modified GCE; cDNA/MCH/Apt/modified AuE: complementary DNA/6–mercapto–1–hexanol/Aptamer/modified Au electrode; Apt–Fc/BSA/cDNA/Ru@SiO₂–BPQDs/AuNPs–PEI–MWCNTs/GCE: Aptamer–ferrocenes/Bovine serum albumin/complementary DNA/Ru(bpy)₃²⁺@silica(SiO₂)–BPQDs/gold nanoparticles–polyethyleneimine–multi–walled carbon nanotubes/glassy carbon electrode; BSA/Apt/TB@Zn–MOF/GCE: Bovine serum albumin/aptamer/Toluidine blue@Zn–based metal–organic frameworks/modified GCE.

^bDPV: Differential pulse voltammetry, EIS: Electrochemical impedance spectroscopy, ECL: Electrochemiluminescence.

Study of the selectivity, repeatability, reproducibility, and stability of the aptasensor

The selectivity of the aptasensor towards other mycotoxins is a crucial consideration in its development. Therefore, the selectivity of the recommended aptasensor for aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) both at a concentration of 50.0 ng mL^− 1, as well as for a mixture of both in the presence of 50.0 ng mL^− 1 of OTA, was investigated. The results are shown in Fig. S3. The data depicted in this figure reveal that there was no significant interference for OTA detection when AFM1 and AFB1 were present either individually or simultaneously. These results indicate that the aptasensor exhibits high selectivity for OTA detection and has potential for practical application. The repeatability of the aptasensor was assessed by incubating OTA (1.0 ng mL^–1) on a BSA/Apt/TB@Zn–MOF/GCE. Then, five repeated differential pulse voltammograms were recorded, and the ΔI values were calculated. The relative standard deviation (RSD) of these measurements was determined to be 4.8%. To examine the reproducibility of the aptasensor, five BSA/Apt/TB@Zn–MOF/GCE were separately prepared incubated with OTA (1.0 ng mL^− 1). The ΔI values were then calculated from the differential pulse voltammograms of the prepared aptasensors. The result showed an RSD of 7.5% (n = 5) for this test. Stability is another crucial factor for a suitable aptasensor. To assess the stability of the proposed aptasensor, it was utilized to detect OTA (1.0 ng mL^− 1) while being stored in a refrigerator for a period of six days. The results showed that the response of the aptasensor reached approximately 96.7% of the initial value after three days and about 94.6% after six days.

These findings indicate that the repeatability, reproducibility, and stability of the aptasensor are acceptable. However, it is important to note that each prepared aptasensor is designed for single use only, making it a disposable aptasensor for OTA measurement, despite the advantages it offers.

Measurement of OTA in coffee powder

The feasibility of using the proposed aptasensor to determine OTA in real samples without complicated pretreatment was investigated by measuring OTA in a sample of coffee powder. To do this, coffee samples contaminated with 0.5, 1.0, and 5.0 ng mL^− 1 OTA were prepared and placed on different BSA/Apt/TB@Zn–MOF/GCE surfaces. These samples were then incubated for 2 hours. The OTA concentration in the spiked samples was determined using the aptasensor, with three repeated measurements. The data in Table 2 show that the OTA concentrations obtained with the aptasensor are approximately the same as the spiked values. The range of recoveries and relative standard derivations were within 96.0%-104.2% and 4.6%-7.1%, respectively. Based on these results, the recommended aptasensor can be considered a good option for accurately measuring unknown OTA concentrations in food.

Table 2

Quantitative measurements and percentage recovery results (n = 3) of OTA determination in a coffee powder sample using the proposed aptasensor
Sample	Added (ng mL^–1)	Found (ng mL^–1)	RSD%	Recovery %
Coffee	-	<Detection limit	-	-
	0.5	0.48	4.6	96.0
1.0	0.98	6.5	98.0
	5.0	5.21	7.1	104.2

In this study, an electrochemical aptasensor for the determination of OTA was developed. The aptasensor was created by encapsulating TB in a Zn-based MOF as a substrate. The large surface area of the Zn-MOF provides a suitable substrate for loading large amounts of aptamers. Additionally, the TB encapsulated in the Zn-MOF acts as an electroactive tag, producing an electrochemical signal. By using differential pulse voltammetry, OTA can be measured in the concentration range of 1.0×10^− 4 to 200.0 ng mL^− 1, with a low detection limit of 2.1×10^− 5 ng mL^− 1. Furthermore, the aptasensor exhibited acceptable selectivity, repeatability, reproducibility, and stability for OTA determination. The results demonstrate that the aptasensor, OTA can effectively measure OTA in coffee powder sample.

Ethical approval
This article does not involve any experiments conducted on human or animal subjects.

Conflict of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

The authors are grateful to Yazd University in Yazd, Iran for their support in conducting this research.

Author Contribution

Elaheh Amini–Nogorani: Writing- Original draft preparation, Investigation, Formal analysis, Software. Hamid R. Zare: Supervision, Conceptualization, Reviewing and Editing, Validation. Fahime Jahangiri–Dehaghani: Writing- Original draft preparation, Formal analysis, Conceptualization, Software. Ali Benvidi: Data curation

Data availability

I do not have any research data outside the submitted manuscript file.

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No competing interests reported.

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Scheme 1 Schematic of the synthesis process of (A) TB@Zn-MOF and (B) fabrication of aptasensor and OTA sensing mechanism
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You are reading this latest preprint version

Quantification of ochratoxin A in a coffee sample utilizing an electrochemical aptasensor fabricated through encapsulation of toluidine blue within a Zn- based metal-organic framework

Status:

Version 1

Abstract

Figures

Introduction

Experimental

Apparatus and methods

Materials

Synthesis of TB@Zn-MOF

Fabrication of the aptasensor

Preparation of solution from coffee powder

Results and discussion

Characterization of TB@Zn-MOF

Sensing mechanism of OTA using the proposed aptasensor

Tracking the fabrication steps of the OTA aptasensor

Optimization of experimental parameters

Analytical performance of the designed aptasensor

Study of the selectivity, repeatability, reproducibility, and stability of the aptasensor

Measurement of OTA in coffee powder

Conclusion

Declarations

Ethical approval
This article does not involve any experiments conducted on human or animal subjects.

Conflict of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

Author Contribution

Data availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Quantification of ochratoxin A in a coffee sample utilizing an electrochemical aptasensor fabricated through encapsulation of toluidine blue within a Zn- based metal-organic framework

Status:

Version 1

Abstract

Figures

Introduction

Experimental

Apparatus and methods

Materials

Synthesis of TB@Zn-MOF

Fabrication of the aptasensor

Preparation of solution from coffee powder

Results and discussion

Characterization of TB@Zn-MOF

Sensing mechanism of OTA using the proposed aptasensor

Tracking the fabrication steps of the OTA aptasensor

Optimization of experimental parameters

Analytical performance of the designed aptasensor

Study of the selectivity, repeatability, reproducibility, and stability of the aptasensor

Measurement of OTA in coffee powder

Conclusion

Declarations

Ethical approval This article does not involve any experiments conducted on human or animal subjects.

Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

Author Contribution

Data availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Ethical approval
This article does not involve any experiments conducted on human or animal subjects.

Conflict of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.