Determination of Lead and Cadmium in Non-mineralized Raw Milk Samples Employing Extraction with Magnetic Restricted Access Carbon Nanotubes Followed by FAAS Analysis

Lead and cadmium were determined in non-mineralized raw milk samples employing dispersive solid-phase extraction with magnetic restricted access carbon nanotubes (M-RACNTs), followed by flame atomic absorption spectrometry analysis. M-RACNTs were obtained by modifying carbon nanotubes (CNTs) with magnetite nanoparticles and then covering with a chemically crosslinked bovine serum albumin (BSA) external layer. The M-RACNT particles were very efficient to capture the metals and exclude proteins from the milk, being easily separated from the sample using a neodymium magnet. The extraction parameters were appraised by multivariate optimization (factorial design and Doehlert matrix). The method was validated and proved to be robust for small variations, simple, fast, sensitive, and selective. The determination coefficient was 0.998 for both Pb2+ and Cd2+ ions. The limit of quantification was 10 µg L−1, for both ions. The developed method was able to determine the metals in concentrations below the maximum residue limits (20 and 50 µg L−1 for Pb2+ and Cd+, respectively, in Brazil), demonstrating that M-RACNTs are promising materials for metal extraction in protein-rich samples, without the need for mineralization procedures.


Introduction
Food safety has become a relevant topic in recent years due to the growing search for a better quality of life and the consumer's right to purchase safe health products. The potential toxicity of foods containing metal concentrations above natural levels is a major concern. In addition, inappropriate technological practices and the indiscriminate use of chemicals in agriculture and animal production have led to the appearance of toxic chemical elements in food (Gonçalves et al. 2008). As an example, the presence of lead and cadmium in foods is worrying due to their relevant toxic effects. Lead can cause disorders of the central and peripheral nervous system, gastrointestinal problems, arterial hypertension, anemia, and renal failure, whereas cadmium can cause neurological and renal alterations, and cancer, among others (Balali-Mood et al. 2021).
Milk is a widely consumed food, and its contamination with lead and cadmium has been reported in the literature (Feizi et al. 2020;Boudebbouz et al. 2022). Thus, simple, precise, and accurate analytical methods are required to quantify both metals in food. Sample preparation is the crucial step in this case, considering the very complex composition of milk (proteins, fats, sugars, etc.). Mineralization protocols using acids and oxidants have been preferred, given that the organic matrix is destroyed and the metals remain in conditions that can be analyzed using atomic absorption spectrometry (AAS) (Barbosa et al. 2016). On the other hand, the drastic temperatures and 1 3 pressures required, together with the acidic and oxidant media, make the process dangerous and time-consuming.
Recently, the use of smart sorbents to extract metals from complex samples has increased, mainly due to the simplicity and security of this procedure. Carbon nanotubes (CNTs) can be an important sorbent in this case, due to their high adsorptive capacity (Herbst et al. 2004; Barbosa et al. 2015). However, the use of commercial CNTs is limited in the preparation of biological samples, given that proteins can bind to the surface of the CNTs, obstructing the binding groups. Thus, in 2014, our group encapsulated commercial CNTs in a crosslinked bovine serum albumin (BSA) external layer, resulting in restricted access carbon nanotubes (RACNTs) (Barbosa et al. 2015(Barbosa et al. , 2016. This material was able to capture metals and exclude proteins from human blood samples in an online solid phase extraction (SPE) procedure coupled to flame atomic absorption spectrometry (FAAS). The exclusion mechanism was based on the electrostatic repulsion of proteins from the sample and from the BSA layer at pH levels higher than the isoelectric point of both (Barbosa et al. 2015(Barbosa et al. , 2016. Magnetic sorbents have also excelled in recent years due to the ability to manipulate them in dispersive solidphase extraction (d-SPE) (Kandasamy et al. 2018). Nanoparticles of iron oxide and cobalt oxide (with an emphasis on magnetite-Fe 3 O 4 ) have been synthesized and incorporated in different conventional sorbents by non-covalent or covalent interactions, conferring magnetic susceptibility to the sorbent (Chen et al. 2009). In general, all conventional sorbents can be modified with magnetite, and these sorbents have several advantages in d-SPE, such as a high efficiency of extraction due to the direct mixture of sorbent and sample, easy separation of sorbent from the sample using an external magnet, and the absence of clogging problems frequently observed with cartridges and packed columns in SPE, among others (Nakhjavan et al. 2021).
Based on the relevant ability of RACNTs to exclude proteins, as well as the advantages of working with magnetic particles in d-SPE, in 2020, our research group incorporated magnetite nanoparticles into RACNTs, resulting in magnetic restricted access carbon nanotubes (M-RACNTs) (Frugeri et al. 2020). This new material was successful used to extract organophosphates from milk samples. Here, we evaluate the performance of the M-RACNTs in the extraction of lead and cadmium from milk samples, followed by direct FAAS analysis. The M-RACNTs were able the extract both elements in a d-SPE, with sufficient extraction efficiency to analyze them at concentrations lower than the maximum residue limits (MRLs). Additionally, proteins from the sample were quantitatively excluded, avoiding the need for a mineralization step.

Reagents and Solutions
All reagents were of analytical grade. Aqueous solutions were prepared with 18.2 ΩW.cm Milli-Q water (Milli-pore®, Bedford, USA). Pb 2+ and Cd 2+ working solutions were prepared from 1000 mg L −1 standard solutions, both from Sigma-Aldrich® (Missouri, USA). Multi-walled CNTs (Sigma-Aldrich), with 50-90 nm (i.d.) × 10 µm (95% purity) were used in the extraction experiments. Ammonium hydroxide (Pro Analysis®, São Paulo, Brazil), glutaraldehyde, BSA, iron(III) chloride hexahydrate, iron(II) sulfate heptahydrate (all from Sigma-Aldrich®), sodium borohydride, and nitric acid (both from Merck®, Darmstadt, Germany) were used in the synthesis of the materials. Laboratory glassware was kept for 24 h in a 10% (v/v) nitric acid bath, washed with deionized water, and dried in an oven before use. To prepare de Bradford reagent, 50 mg of Coomassie brilliant blue (Sigma-Aldrich®), 25 mL of 95% ethanol (Merck®), and 50 mL of 85% phosphoric acid (Sigma-Aldrich®) were added in flask, and the volume was completed with water for 500 mL. Solution was sonicated for 5 h and filtrated before the use.

Synthesis of the M-RACNT
Magnetite particles were synthesized by co-precipitation, according to Mendes et al. (Mendes et al. 2020). First, 50 mL water were added to a glass flask and heated at 50 °C. Then, 80 mL of 0.1875 mol L −1 iron(III) chloride hexahydrate and 0.125 mol L −1 iron(II) sulfate heptahydrate aqueous solution were added to the flask, under agitation and with nitrogen bubbling. Next, 250 mg CNTs were added, and the system was maintained at 50 °C for 20 min under a nitrogen atmosphere. Finally, 2.5 mL of 30% (v:v) NH 4 OH were added dropwise, and maintained under agitation for 30 min in a nitrogen atmosphere. The obtained M-CNTs were separated with a neodymium magnet, washed with water until neutral pH, and dried at 60 °C for 12 h. The synthesis scheme is shown in Fig. 1.
The M-CNTs were covered with BSA according to the procedure of Moraes et al. (De Oliveira Isac Moraes et al. 2013). First, 20 mL 1% (w/v) BSA solution (prepared in phosphate buffer, 50 mmol L −1 , pH 6.0) was percolated at a flow rate of 1 mL min −1 through a cartridge containing 500 mg M-CNTs (see Fig. 1). Then, 5 mL of a 25% (w/v) glutaraldehyde aqueous solution were percolated through the cartridge at a flow rate of 1 mL min −1 , and the system was maintained in standby for 5 h. The particles obtained were separated and dispersed in 10 mL of 1% (w/v) sodium borohydride aqueous solution, under agitation for 15 min. The material obtained was separated with the use of a neodymium magnet, washed several times with water, and dried at 60 °C for 12 h (see Fig. 1) (Mendes et al. 2020).

Characterization of the Materials
Transmission electron microscopy (TEM) images were taken using a FEI TECNAI G2 S-TWIN microscope equipped with a filament of LaB6.
A diffractometer Rigaku (Tokyo, Japan) was employed in x-ray diffraction (XRD) analyses, model Ultima IV, with CuK, α = 1.54051 Ᾰ. The current and voltage were 30 mA and 40 kV, respectively. The 2θ scan interval was from 10 to 80º, with an angular step of 4º min −1 .

Protein Exclusion
The protein exclusion test was performed using a BSA solution (100 mg L −1 ) prepared in phosphate buffer (10 mmol L −1 , pH 4.1) and a spectrophotometer KASUAKI (Model IL-593-BI) operating at 595 nm. Initially, 200 µL of the BSA solution was placed in test tubes with 2.8 mL of Bradford reagent. After 10 min, the absorbance was considered to be 100% BSA. In the next step, 1 mL of the same BSA solution was added to test tubes containing 5 mg of M-RACNT or M-CNT. The tubes were shaken for 30 min. After this time, the supernatant was separated using a neodymium magnet, and 200 µL of the supernatant was treated with Bradford reagent as described above. The absorbance of the extracted BSA solution divided by the absorbance of the non-extracted BSA solution corresponded to the percentage of protein excluded by the M-RACNT or M-CNT. All experiments were performed in triplicate.

Kinetic and Isotherm Adsorption
The adsorption studies were carried out individually for Cd 2+ and Pb 2+ , and the experiments were performed in triplicate. For the kinetics assays, 1 mL of 10 mg L −1 Cd 2+ solution or 1 mL of 5 mg L −1 Pb 2+ solution (prepared in ultrapure water with pH adjusted to 4.5 for Cd 2+ , or 4.0 for Pb 2+ ) was placed in test tubes containing 5 mg M-RACNT. The tubes were shaken for 0,083, 0.5, 2.0, 3.0, 5.0, 10.0, 15.0, and 20.0 min for Cd 2+ and 0,083, 0.16, 0.25, 0.5, 1.0, 2.0, 3.0, 5.0, 8.0, and 12.0 min for Pb 2+ . After each time, the supernatants were immediately separated from the material using a neodymium magnet. The concentrations of the analytes were determined using FAAS, model Shimadzu AA-7000 (Shimadzu®, Tokyo, Japan), equipped with hollow-cathode lamps for both elements and a deuterium lamp for background correction. Analytical signals were obtained as absorbance at 217.0 and 228.8 nm for Pb 2+ and Cd 2+ , respectively. Acetylene and air at 0.8 and 3.6 L min −1 , respectively, were used in the flame composition. The equilibrium adsorption capacity (qe, in mg g −1 ) was calculated according to Eq. 1. Co and Ce (both in mg L −1 ) are the initial and equilibrium concentrations, respectively; m (g) is the sorbent mass; and V (L) is the solution volume.
Additionally, the data were treated according to different kinetic models (pseudo-first-order, pseudo-second-order, fractional-order, and Elovich kinetics models). Both the linear determination coefficients (R 2 ) and the error functions (F error ) (Eq. 2) were used to determine the best adjustment for each analyte. n and p are the number of experiments and parameters of the fitted model, respectively; q i,exp is each value of qe experimentally measured; and q i,theoretical is each value of qe obtained by the fitted model.
For the isotherm absorption studies, 1 mL of Cd 2+ solution (prepared in ultrapure water with pH adjusted to 4.5) at concentrations of 50,60,80,100,120,140,160,180,200, 250, 300, 400, 500, and 600 mg L −1 was added into 14 test tubes containing 5 mg M-RACNT. The tubes were shaken for 5.0 min at 25 °C, and the supernatants were separated using a neodymium magnet. The concentration of Cd 2+ was determined using FAAS (using the same conditions as described before). The same procedure was carried out for the Pb 2+ absorption studies, but using concentrations of 10,15,20,25,30,35,40,45,50,60,70, and 80 mg L −1 (prepared in ultrapure water with pH adjusted to 4.0). The qe values were calculated according to Eq. 1, and the data were fitted to different models (Langmuir, Freundlich, Sips, Khan, Toth, and Redlich-Peterson). All data were treated using OriginLab® software (Northampton, USA).

Multivariate Optimization of the Extraction Procedure
A factorial design 2 5-1 (16 experiments) was carried out with the extraction time (ET), desorption time (DT), eluent concentration (EC), eluent volume (EV), and sorbent mass (SM). Experiments were carried out with a milk sample fortified with 500 µg L −1 of Cd 2+ and Pb 2+ . The significant variables were selected through the Pareto diagram. Response surfaces obtained by the Doehlert matrix were built for Cd 2+ and Pb 2+ , and the maximum values for each variable were selected as working conditions. Data were treated with the software STATISTICA 6.0 (StatSoft®, Tulsa, USA), and the statistical significance was established with a confidence level of 95%.

Optimized Analytical Protocol
Before the d-SPE procedure, the pH of the samples was adjusted to 4.0 or 4.5 for Pb 2+ and Cd 2+ , respectively. The sample volume increased in 1% with the pH adjustment. The samples were vortexed for 10 min and centrifuged for 10 min at 5478 g. Next, 45 mL of the supernatant was transferred to a Falcon tube containing 30 mg of M-RACNTs. The tubes were agitated at ambient temperature for 28 min. A neodymium magnet was used to separate the material from the supernatant, which was discarded. Then, 0.4 mL HNO 3 aqueous solution (1.26 or 1.00 mol L −1 for Pb 2+ and Cd 2+ , respectively) was added to the M-RACNTs, and the flask was vortexed for 5 min. The supernatant was separated for Pb 2+ and Cd 2+ determinations by FAAS (using the same conditions as described in the "Multivariate Optimization of the Extraction Procedure" section).

Figures of Merit
The linearity, limits of detection (LOD) and quantification (LOQ), intra-and inter-day precisions and accuracies were apprised according to FDA and 2002/657/EC guidelines (96/23/EC Commission 2002;FDA 2013). Analytical curves were built by fortifying a pool (n = 3) of commercial raw milk samples with Pb 2+ or Cd 2+ , both from 10 to 150 µg L −1 . Samples were extracted with the M-RACNTs, and the eluates were analyzed by FAAS. The LODs was defined as 3 times the ratio between the standard deviation of the linear coefficient of the analytical curve. The LOQ was defined as the lower concentration that can be quantified with precision and accuracy. Precisions (as relative standard deviation (RSD), Eq. 3) and accuracies (as relative error (RE), Eq. 4) were calculated for 10, 50, and 150 µg L −1 for Pb 2+ , and for 10, 100, and 150 µg L −1 for Cd 2+ . Intra-day tests were carried out on the same day (n = 3), and inter-day tests were performed on 3 consecutive days.
where SD is the standard deviation of the analytical responses, A is the average of the analytical responses, and AC and RC are the analyzed and real concentrations.
Youden's test was performed to evaluate if slight variations in the method significantly affect its responses. Seven parameters were studied: ET, DT, SM, sample volume (SV), EV, EC, and vortex speed (VS) (see Table S1). The method was considered robust when the analytical responses obtained with the optimized parameters were not significantly different from the changed parameters (p ≥ 0.05). The analyses were performed in triplicate with a milk sample fortified with 150 µg L −1 of Pb 2+ and Cd 2+ . The sample pHs were 4.0 and 4.5 for Pb 2+ and Cd 2+ determinations, respectively.

Characterization of the Materials
The morphological structure of Fe 3 O 4 , M-CNTs, and M-RAC-NTs were investigated by TEM. Figure 2 suggests a spherical form of Fe 3 O 4 nanoparticles, as well as their presence in the M-CNT and M-RACNT materials. Also, the M-RACNT images (Fig. 2C) show a more uniform distribution of nanoparticles around the CNTs, possibly due to the coating with BSA, which favors homogenization of the material.  Fig. 3A. The mass losses occurred mainly from 220 to 450 °C and from 600 to 800 °C, and stability was achieved at 830 °C for Fe 3 O 4 , M-CNTs, and M-RACNTs. The mass of iron nanoparticles was preserved at the appraised temperature range due to its inorganic composition. The CNTs lost more than 90% in mass from 690 to 800 °C due to the carbon matrix degradation. On the other hand, M-CNTs lost only 45% in mass from 500 to 1000 °C, attesting that a part of this material is composed of iron nanoparticles. BSA lost 10% in mass from 30 to 490 °C, probably due to its denaturation and degradation, and also lost about 66% of its mass from 250 to 1000 °C due to the carbon matrix degradation. Finally, M-RACNTs lost 6% in mass from 30 to 490 °C, confirming the presence of BSA. Additionally, M-RACNTs lost only 46% in mass from 490 to 750 °C, attesting the presence of thermally stable Fe 3 O 4 particles.
The diffractogram of the Fe 3 O 4 particles showed an XRD pattern (Fig. 3B) referring to the single cubic spinel-type crystalline phase with peaks at 30, 35, 43, 53, 57, and 63 Ө (Nalbandian et al. 2008). The spinel-like cubic crystal structure is characteristic of iron oxide materials. Fe 3 O 4 is an example of this structure, with the oxygen atoms in a face-centered cubic arrangement and the Fe 2+ and Fe 3+ ions occupying the sites between the oxygen ions (Korolev et al. 2008). Fe 3 O 4 was adequately incorporated into the M-CNTs and M-RACNTs due to the similarity of the diffractograms of these particles. Peaks 2Ө = 25° and 43° are characteristic of the graphene present in nanotube diffraction patterns, and the intensity and width of these peaks reflect the orientation of the CNTs in terms of the incident X-ray beam, the

Protein Exclusion
The M-CNTs excluded 5.00 ± 0.54% of the total amount of BSA in the solution. In contrast, the M-RACNTs were able to exclude 25.10 ± 1.99% of the protein. The significantly higher exclusion by the M-RACNTs indicate their characteristic of restricted access material. According to Barbosa et al., the exclusion mechanisms are based on the electrostatic repulsion between the proteins present in the matrix and those in the material's external layer when the pH medium is different from the isoelectric point of the BSA (Barbosa et al. 2015). Additionally, a physical mechanism contributes to the macromolecules' exclusion, preventing their access to the material (de Faria et al. 2017).

Kinetic and Isotherm Adsorption
The equilibrium between the metal ion and the M-RACNTs was achieved at 3.0 min for Cd 2+ and at 5.0 min for Pb 2+ . The data were adjusted to the pseudo-first-order, pseudosecond-order, fractional-order, and Elovich kinetics models (Table 1, Fig. 4A and B). The best adjustment for Cd 2+ was obtained with fractional-order (R 2 0.94041 and F error 4.12619), which considers possible changes in the function of the analytes' initial concentration and extraction time (Ohs et al. 2018). The best adjustment for Pb 2+ was obtained with the Elovich model (R 2 0.97388 and F error 4.15 × 10 −13 ); this model assumes that the material surface is energetically heterogeneous (Salam et al. 2012) and that chemisorption is the main adsorption mechanism (Wang and Guo 2020). However, both metals have a suitable adjustment with fractional-order and the Elovich model.
According to the isotherm adsorption studies ( Fig. 4C and D), the maximum adsorption capacities were 100.7 mg g −1 and 3.9 mg g −1 for Cd 2+ and Pb 2+ , respectively. The data obtained from Cd 2+ extraction tends to follow the isotherm of type III, indicating that every receptor site is occupied by an infinite number of metal ions with the same energy (Khalfaoui et al. 2003). The Pb 2+ isotherm was closer to type IV, indicating surfaces with mesopores (Kajama 2015).
Langmuir, Freundlich, Sips, Khan, Toth, and Redlich-Peterson models were used to fit the data ( Table 2). The Table 1 Results for the kinetic adjusted models to M-RACNTs M-RACNTs, magnetic restricted access carbon nanotubes; R 2 , determination coefficient; F error , error function; qe, the amount of analyte adsorbed in equilibrium per gram of material; t, time; K 1 , pseudo-first-order constant; K 2 , pseudo-second-order constant; K AV , Avrami constant; n AV , Avrami exponent; k, Elovich constant; n, initial adsorption rate Cd 2+ isotherm was only adjusted by Freundlich and Redlich-Peterson isotherms; both presented adjusted R 2 above 0.95 and similar F error values. In turn, the Pb 2+ isotherm was best adjusted with the Khan isotherm (adjusted R 2 0.90775, F error 1.06506). These results indicate a heterogeneous surface with the possibility of multilayer adsorption of the metal ions (Al-Ghouti and Da'ana 2020). CNTs can extract Cd 2+ and Pb 2+ by Van der Waals forces and electrostatic interactions (Salam et al. 2012). The positive metal ions can also be retained in the BSA external layer by electrostatic interactions (which are minimized due to the proximity of the used solution pH and the protein isoelectric point). In addition, the metals possibly bind covalently with the albumin, helping to explain the adjustment with the Elovich kinetic model.

Multivariate Optimization
Initially, the influence of sample pH on extraction efficiency for Pb 2+ and Cd 2+ was investigated (Fig. FS1). The highest analytical signals were obtained at pH 4.0 and 4.5 for Pb 2+ and Cd 2+ , respectively. Additionally, in both pHs, the casein was precipitated (casein isoelectric point is 4.8, (Tuinier and de Kruif 2002)]), promoting an initial cleanup of the sample. Thus, these pH values were selected as the working conditions. The factorial design 2 5-1 , with 16 experiments, 32 in duplicate, for Pb 2+ and Cd 2+ , is presented in Table S2, together with the analytical signals obtained. Additionally, the analysis of variance of the design experiments are shown in Table S3. As can be seen in the Pareto graphics ( Fig. 5A and B), EC, EV, SM, and ET were the variables with significant effects for Pb 2+ , and EV, SM, and ET were the variables with significant effects for Cd 2+ . The SM was fixed at 30 mg for both analytes, and higher masses were not appraised in order to avoid overuse of the M-RACNTs. The EV was fixed at 0.4 mL for Pb 2+ , due to its positive effect in analytical response. The EC and ET were optimized by a Doehlert matrix for Pb 2+ and the EV and ET were optimized for Cd 2+ . Table S4 presents the apprised levels and the analytical responses, and Fig. 5C and D show the response surfaces. The maximum levels were ET = 28 min for both Pb 2+ and Cd 2+ , EC = 1.26 mol L −1 for Pb 2+ , and EV = 0.4 mL for Cd 2+ .

Validation and Application
Tables 3 and 4 show the figures of merit for the determination of Pb 2+ and Cd 2+ in milk samples. In both cases, good linearities were obtained (R 2 > 0.99). The MRLs for Pb 2+ and Cd 2+ (20 and 50 μg L −1 , respectively (Brazilian Health Regulatory Agency)) were within the analytical ranges for both elements (10-150 μg L −1 ), attesting the applicability of the proposed methods. The LOQs (10 µg L −1 in both cases) were at least half of the MRL. Intra-and inter-day precisions (appraised as RSD) were lower than 17.0 and 19.2 for Pb 2+ and Cd 2+ , respectively. Intra-and inter-day accuracy (appraised as RE) were within − 19.9% and 12.5% for Pb 2+ and − 17.9% and 18% for Cd 2+ . These results are in accordance with the used validation guidelines (96/23/EC Commission 2002;FDA 2013). The method proved to be robust to seven studied parameters (Table S1, Fig. FS2). Table 5 shows a comparison of the proposed method with other literature methods used to analyze Pb 2+ and Cd 2+ in milk samples. It can be seen that most of the published works that determined metal ions in milk used the mineralization method. The proposed method avoids the use of high temperatures, pressures, and amounts of acids and oxidants. The M-RACNTs were very efficient for Pb 2+ and Cd 2+ extraction at room temperature, using only 0.4 mL of HNO 3 for the elution step. In addition, the developed methodology presented similar or lower LOQs to other methods using FAAS (Borahan et al. 2019;Capcarova et al. 2019;Hasan et al. 2022;Boudebbouz et al. 2022), sensors (Palisoc et al. 2019), or more sensitive techniques, such as ICP-MS (Sujka et al. 2019), for metal determination. Finally, d-SPE with RACNTs can be considered simpler and less time-consuming than methods employing many cycles of d-SPE (using a narrow-bore tube) followed by a dispersive liquid-liquid microextraction (Parvizzad et al. 2022) and digestion methods. M-RACNTs, magnetic restricted access carbon nanotubes; R 2 , determination coefficient; F error , error function; qe, the amount of analyte adsorbed in equilibrium per gram of material; q s , theoretical capacity of saturation; K L , Langmuir constant; K F , Freundlich constant; n F , Freundlich exponent; K S , Sips constant; n S Sips exponent; a K and b K , Khan constants; K R and a R , Redlich-Peterson constants; K T and a T , Toth constants; t, Toth exponent percolated through them and were also able to retain the metals from the protein matrix with high efficiency. The sample manipulation in the proposed method was very easy, and milk mineralization processes were not required. Validation was completed, demonstrating excellent results for linearity, detectability, precision, and accuracy. It is  Data Availability Data will be made available if requested.

Declarations
Ethics Approval and Consent to Participate This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of Interest
The authors declare no competing interests.