Magnetic Molecularly Imprinted Chitosan Combined with a Paper-Based Analytical Device for the Smartphone Discrimination of Tryptophan Enantiomers

The separation of enantiomers plays a critical role in pharmaceutical development, ensuring therapeutic efficacy, safety, and patent protection. It enables the production of enantiopure drugs and enhances our understanding of the properties of chiral compounds. In this study, a straightforward and effective chiral detection strategy was developed for distinguishing between tryptophan (TRP) enantiomers. The approach involved the preparation of a magnetic molecularly imprinted chitosan (MMIC) for preparation of the sample, which was combined with a nitrocellulose membrane (a paper-based analytical device, PAD) integrated with D-TRP covalently grafted with polymethacrylic acid (PAD-PMA_D-TRP). Discriminating between the TRP enantiomers was achieved using AuNPs as a colorimetric probe. Indeed, the presence of D-TRP rapidly induced the aggregation of AuNPs due to its strong affinity to PAD-PMA_D-TRP, resulting in a noticeable change in the color of the AuNPs from red to purple. On the other hand, L-TRP did not induce any color changes. The chiral analysis could be easily performed with the naked eye and/or a smartphone. The developed method exhibited a detection limit of 3.3 µM, and it was successfully applied to detect TRP in serum samples, demonstrating good recovery rates. The proposed procedure is characterized by its simplicity, cost-effectiveness, rapidity, and ease of operation.


Introduction
The majority of drugs, pharmaceuticals, and biologically active compounds are mixtures of chiral isomers, which have similar chemical and physical properties. However, in most cases, one isomer exhibits the desired biological and toxicological effects, while the other may be inactive or have different pharmacological effects [1,2]. For instance, the tragic incident in the early 1960s involving thalidomide revealed that only the (R)-enantiomer provided pain relief, while the (S)-enantiomer caused severe deformities in unborn children [3]. Another example is TRP, which has two enantiomers with distinct activities [4,5]. L-TRP is a vital component of proteins and is a precursor to melatonin and serotonin, which aids in sleep and improvements in mental health. On the other hand, D-TRP, a non-protein amino acid, does not participate in the metabolic pathways of living systems but it is commonly used in the synthesis of immunosuppressant and peptide antibiotics [6][7][8]. The importance of chiral molecules in the production of pharmaceutical and biologically active compounds has led to a significant demand for effective separation techniques to purify (resolution = 1 nm). Fourier transform infrared (FT-IR) spectra were collected using aIR Affinity-1S spectrophotometer (Shimadzu, Japan) in the attenuated total reflectance (ATR) mode and across the range of 4000-500 cm −1 . The UV absorption spectra were measured by a double beam UV/vis spectrophotometer (model T80+, PG Instruments, Wibtoft, Leicestershire, UK). The pictures for the discrimination of TRP were taken with an Android smartphone with a 13 MP camera. Nanopure water was prepared by a Wasser Lab Ultramatic Plus (Type I) system (Barbatáin, Navarra, Spain) and used in all experiments.

Synthesis of the Magnetite Nanoparticles
The synthesis of Fe 3 O 4 nanoparticles was carried out using the method described in [32]. In brief, a three-necked flask was prepared under a N 2 atmosphere, and a solution containing 13.56 g of FeCl 3 ·6H 2 O and 4.96 g of FeCl 2 ·4H 2 O dissolved in 250 mL of distilled water was prepared. Subsequently, 20 mL of ammonium hydroxide was introduced into the flask, and the mixture was vigorously stirred for 40 min at 80 • C. The resulting Fe 3 O 4 nanoparticles were then collected using an external magnet and washed with distilled water to eliminate any residual chemicals. The obtained precipitate was finally dried at 50 • C in a vacuum oven.

Synthesis of Molecularly Imprinted Chitosan
The method of preparing MMIC was as follows. Initially, 100.0 mg of chitosan was dissolved in 10.0 mL of a 1.0% (v/v) aqueous acetic acid solution. At the same time, 6.0 mg of the TRP template molecules was added into the chitosan solution, followed by the addition of 100 mg of Fe 3 O 4 and stirred at room temperature for 4 h. Subsequently, 0.5 M sulfuric acid was added to the solution and stirred for 2 h. After that, the TRP template molecules were removed by an ethanol solvent. A magnetic non-imprinted chitosan (MNIC) was prepared following the same procedure, except for the addition of the template.

Synthesis of Gold Nanoparticles
Gold nanoparticles were synthesized according to our previous study [33]. Initially, a cylindrical glass vessel was placed in a water bath containing 1.25 mL of a 1.0 mM HAuCl 4 aqueous solution. To ensure a consistent temperature, the sample vessel remained in the water bath at an ambient temperature throughout the process, as the local heating from sonication affected the solution's temperature. After 4 min of sonication, 250 µL of an aqueous solution of 38.8 mM sodium citrate dihydrate was added to the vessel. This addition caused an immediate change in the solution's color to grey. Continuous sonication led to a subsequent transformation of the solution's color to dark red after 5.5 min, indicating the formation of the colloid AuNPs.

Synthesis of D-TRP-Grafted Polymethacrylic Acid (PMA_D-TRP)
PMA_D-TRP was prepared according to the following procedure: 20 mg of D-TRP was introduced into a beaker containing 50 mL of DMSO, followed by the addition of 700 µL of MAA and 10 mL of EGDMA. Then 10 mg of CuCl 2 was added to link the monomer to D-TRP covalently, and 3.3 mg of AIBN was used to initiate the process of polymerization. The blend was deaerated with nitrogen for 5 min and the reaction was carried out at 60 • C for 15 min using a domestic microwave. The material thus obtained was washed and dried in oven at 60 • C.

Preparation of the Paper-Based Analytical Device (PAD)
For this, 10 mg of the developed material (PMA_D-TRP) was dispersed in 5 mL of nanopure water and filtrated under a vacuum to entrap the PMA_D-TRP in the nitrocellulose membrane's pores (porosity: 0.45 µm). After filtration, the paper was dried in an oven for 15 min at 40 • C. The paper was then cut into small disks for further use and named PAD-PMA_D-TRP.

Adsorption Study
For this study, 10 mg of MMIC/MNIC was introduced into 1 mL of D-TRP with a concentration range of 30-320 µM. The mixtures were shaken at a room temperature for 15 min. After the process of adsorption, the sorbent was separated by centrifugation at 1000 rpm for 2 min. The concentrations of the obtained supernatants were determined by the UV-spectrophotometer at a wavelength of 280 nm. The adsorption capacity (Q e ) was calculated by Equation (1):

Solid-Phase Extraction Combined with Smartphone Detection
Solid-phase extraction is a versatile and effective technique of preparing samples that enables the selective extraction of the target analytes from complex sample matrices. In this work, the proposed MMIC was used as the sorbent in a solid-phase extraction technique for the total isolation of TRP. The task of discriminating between the TRP enantiomers was performed using the developed PAD-PMA_D-TRP. The procedure was as follows. First, 10 mg of MMIC was introduced into an Eppendorf tube containing 1 mL of a TRP solution. The blend was shaken for 30 min at room temperature. After adsorption, the TRP was eluted with 1 mL of methanol and measured by UV spectroscopy at λ = 280 nm to determine the total concentration of L/D-TRP. lulose membrane's pores (porosity: 0.45 µm). After filtration, the paper was dried in an oven for 15 min at 40 °C. The paper was then cut into small disks for further use and named PAD-PMA_D-TRP.

Adsorption Study
For this study, 10 mg of MMIC/MNIC was introduced into 1 mL of D-TRP with a concentration range of 30-320 µM. The mixtures were shaken at a room temperature for 15 min. After the process of adsorption, the sorbent was separated by centrifugation at 1000 rpm for 2 min. The concentrations of the obtained supernatants were determined by the UV-spectrophotometer at a wavelength of 280 nm. The adsorption capacity (Qe) was calculated by Equation (1):

Solid-Phase Extraction Combined with Smartphone Detection
Solid-phase extraction is a versatile and effective technique of preparing samples that enables the selective extraction of the target analytes from complex sample matrices. In this work, the proposed MMIC was used as the sorbent in a solid-phase extraction technique for the total isolation of TRP. The task of discriminating between the TRP enantiomers was performed using the developed PAD-PMA_D-TRP. The procedure was as follows. First, 10 mg of MMIC was introduced into an Eppendorf tube containing 1 mL of a TRP solution. The blend was shaken for 30 min at room temperature. After adsorption, the TRP was eluted with 1 mL of methanol and measured by UV spectroscopy at λ = 280 nm to determine the total concentration of L/D-TRP. Then 10 µL of the supernatant was added to the developed PAD-PMA_D-TRP containing 40 µL of the dispersed AuNPs

Characterization Studies of Fe 3 O 4 and MMIC
The FTIR technique was used to analyze the chemical composition and functional groups of the proposed materials ( Figure 1A TEM was used to characterize the size and morphological structure of the prepared materials, namely Fe3O4 and Fe3O4-chitosan. The developed Fe3O4 particles exhibited a mean diameter of approximately 12.03 ± 3.1 nm (n = 13) (see Figure 1C). However, MMIC ( Figure 1D) showed dense particles due to the presence of the polymer, which modified a large number of the Fe3O4 nanoparticles. These results strongly confirmed the successful modification of the magnetic nanoparticles with chitosan.  TEM was used to characterize the size and morphological structure of the prepared materials, namely Fe 3 O 4 and Fe 3 O 4 -chitosan. The developed Fe 3 O 4 particles exhibited a mean diameter of approximately 12.03 ± 3.1 nm (n = 13) (see Figure 1C). However, MMIC ( Figure 1D) showed dense particles due to the presence of the polymer, which modified a large number of the Fe 3 O 4 nanoparticles. These results strongly confirmed the successful modification of the magnetic nanoparticles with chitosan.

Transmission Electron Microscopy for AuNPs
TEM images were acquired to assess the behavior of AuNPs when exposed to D-TRP or L-TRP. As depicted in Figure 2A,B, the presence of D-TRP led to the aggregation of AuNPs. Conversely, in the presence of L-TRP, the AuNPs remained dispersed and exhibited a monodisperse distribution.

Transmission Electron Microscopy for AuNPs
TEM images were acquired to assess the behavior of AuNPs when exposed to D-TRP or L-TRP. As depicted in Figure 2A,B, the presence of D-TRP led to the aggregation of AuNPs. Conversely, in the presence of L-TRP, the AuNPs remained dispersed and exhibited a monodisperse distribution.

Adsorption Study
The adsorption study played a crucial role in the characterization of MIPs by providing insights into the binding capacity. It contributed to an understanding of the behavior of MIPs and aided in the development of cavities that were efficient and highly specific to the analyte.
The binding isotherms of D-TRP on the MMIC and MNIC at 25 °C are presented in Figure 3. As the concentration of D-TRP increased, both the MMIC and MNIC demonstrated higher binding capacities. However, due to the presence of multiple cavities, the MMIC exhibited a higher adsorption capacity compared with the MNIC. This observation was supported by the higher values of the imprinted factor, which represents the ratio of the adsorption capacity of MMIC to MNIC. The imprinted factor values ranged between 1.5 and 2, providing further evidence for the successful development of D-TRP cavities on the surface of the MMIC.

Adsorption Study
The adsorption study played a crucial role in the characterization of MIPs by providing insights into the binding capacity. It contributed to an understanding of the behavior of MIPs and aided in the development of cavities that were efficient and highly specific to the analyte.
The binding isotherms of D-TRP on the MMIC and MNIC at 25 • C are presented in Figure 3. As the concentration of D-TRP increased, both the MMIC and MNIC demonstrated higher binding capacities. However, due to the presence of multiple cavities, the MMIC exhibited a higher adsorption capacity compared with the MNIC. This observation was supported by the higher values of the imprinted factor, which represents the ratio of the adsorption capacity of MMIC to MNIC. The imprinted factor values ranged between 1.5 and 2, providing further evidence for the successful development of D-TRP cavities on the surface of the MMIC.

Transmission Electron Microscopy for AuNPs
TEM images were acquired to assess the behavior of AuNPs when exposed to D-TRP or L-TRP. As depicted in Figure 2A,B, the presence of D-TRP led to the aggregation of AuNPs. Conversely, in the presence of L-TRP, the AuNPs remained dispersed and exhibited a monodisperse distribution.

Adsorption Study
The adsorption study played a crucial role in the characterization of MIPs by providing insights into the binding capacity. It contributed to an understanding of the behavior of MIPs and aided in the development of cavities that were efficient and highly specific to the analyte.
The binding isotherms of D-TRP on the MMIC and MNIC at 25 °C are presented in Figure 3. As the concentration of D-TRP increased, both the MMIC and MNIC demonstrated higher binding capacities. However, due to the presence of multiple cavities, the MMIC exhibited a higher adsorption capacity compared with the MNIC. This observation was supported by the higher values of the imprinted factor, which represents the ratio of the adsorption capacity of MMIC to MNIC. The imprinted factor values ranged between 1.5 and 2, providing further evidence for the successful development of D-TRP cavities on the surface of the MMIC.

Discriminating between TRP Enantiomers
Discriminating between the two enantiomers of tryptophan is important for several reasons, including protein synthesis, the production of neurotransmitters, drug development, and understanding metabolism and health. In the present work, the discrimination was performed after the extraction of total TRP using the developed MMIC. Indeed, a paper-based analytical device was developed for discriminating between the TRP enantiomers. This device is based on a membrane modified with polymethacrylic acid grafted with D-TRP (PAD-PMA_D-TRP). For visual detection, AuNPs were used as colorimetric probes because of their sensitive aggregation with TRP. Indeed, the AuNPs changed color from red to purple through the aggregation phenomenon stimulated by TRP. The newly developed PAD-PMA_D-TRP exhibited high affinity to D-TRP, providing a rapid aggregation of AuNPs. However, there was no interaction between PAD-PMA_D-TRP and L-TRP, and thus the AuNPs maintained their red coloration (Scheme 2). This approach enabled the convenient discrimination of TRP enantiomers using a smartphone or even with the naked eye.

Discriminating between TRP Enantiomers
Discriminating between the two enantiomers of tryptophan is important for several reasons, including protein synthesis, the production of neurotransmi ers, drug development, and understanding metabolism and health. In the present work, the discrimination was performed after the extraction of total TRP using the developed MMIC. Indeed, a paper-based analytical device was developed for discriminating between the TRP enantiomers. This device is based on a membrane modified with polymethacrylic acid grafted with D-TRP (PAD-PMA_D-TRP). For visual detection, AuNPs were used as colorimetric probes because of their sensitive aggregation with TRP. Indeed, the AuNPs changed color from red to purple through the aggregation phenomenon stimulated by TRP. The newly developed PAD-PMA_D-TRP exhibited high affinity to D-TRP, providing a rapid aggregation of AuNPs. However, there was no interaction between PAD-PMA_D-TRP and L-TRP, and thus the AuNPs maintained their red coloration (Scheme 2). This approach enabled the convenient discrimination of TRP enantiomers using a smartphone or even with the naked eye. Scheme 2. Detailed mechanism of discriminating between TRP enantiomers.

Smartphone Detection
To enable on-site quantitative evaluation of the color change exhibited by AuNPs in the presence of TRP, we combined our PAD-PMA_D-TRP with a smartphone. This allowed us to monitor alterations in the RGB values as the red color of the AuNPs transitioned to a purplish-blue upon the addition of TRP. The standard RGB scale uses wholenumber values ranging from 0 to 255 to represent the intensity of each color channel: red, green, and blue. In this scale, [255, 255, 255] corresponds to pure white, while [0, 0, 0] represents absolute black [35][36][37][38][39]. By utilizing smartphone-based RGB detection, we were able to observe and record the changes in color. The RGB values were measured by Image J software 1.53t, and the difference between the red and blue (R-B) values was plo ed against the concentration of TRP.

Optimization of the Detection Procedure
The volume of AuNPs, the pH conditions, and the time of color developement are the most important parameters to be optimized in order to achieve an effective means of discriminating between TRP enantiomers.
As mentioned above, AuNPs were used as the colorimetric probe for the discrimination of TRP. Thus, the volume of AuNPs is a relevant parameter to be optimized. Figure  4A shows that 40 µL of AuNPs was the most suitable volume to be added to the PAD-PMA_D-TRP for the colorimetric discrimination of L-TRP and D-TRP enantiomers. Scheme 2. Detailed mechanism of discriminating between TRP enantiomers.

Smartphone Detection
To enable on-site quantitative evaluation of the color change exhibited by AuNPs in the presence of TRP, we combined our PAD-PMA_D-TRP with a smartphone. This allowed us to monitor alterations in the RGB values as the red color of the AuNPs transitioned to a purplish-blue upon the addition of TRP. The standard RGB scale uses whole-number values ranging from 0 to 255 to represent the intensity of each color channel: red, green, and blue. In this scale, [255, 255, 255] corresponds to pure white, while [0, 0, 0] represents absolute black [35][36][37][38][39]. By utilizing smartphone-based RGB detection, we were able to observe and record the changes in color. The RGB values were measured by Image J software 1.53t, and the difference between the red and blue (R-B) values was plotted against the concentration of TRP.

Optimization of the Detection Procedure
The volume of AuNPs, the pH conditions, and the time of color developement are the most important parameters to be optimized in order to achieve an effective means of discriminating between TRP enantiomers.
As mentioned above, AuNPs were used as the colorimetric probe for the discrimination of TRP. Thus, the volume of AuNPs is a relevant parameter to be optimized. Figure 4A shows that 40 µL of AuNPs was the most suitable volume to be added to the PAD-PMA_D-TRP for the colorimetric discrimination of L-TRP and D-TRP enantiomers.
pH effect of the BR buffer was studied in the pH range of 4-11.5. A good interaction between D-TRP and AuNPs was achieved at a pH value of 4. However, the L-TRP had no effect on the AuNPs' coloration. Furthermore, the effect of time on the ability to discriminate between the TRP enantiomers was also examined ( Figure 4C). The addition of D-TRP showed a significant decrease in the R-B intensity compared with L-TRP. A significant difference between L-TRP and D-TRP was obtained at 25 min. Therefore, this time was utilized for subsequent experiments.  Figure 5A shows the calibration curves of D-TRP and L-TRP obtained using different concentrations ranging from 10 to 300 µM. As shown in the photographs, the color of the PAD-PMA_D-TRP color changed from pink to purple, and became more intense with an increasing concentration of D-TRP; thus, aggregation of the AuNPs occurred. However, with the addition of L-TRP, the PAD-PMA_D-TRP showed no significant color change, indicating the low interaction between L-TRP and the developed material. To quantitatively evaluate the results, the color density was converted into the R-B color intensity. This relationship can be described by the equation R-B = −0.15X + 57, where X represents the concentration of the analyte. The correlation coefficient for this linear relationship was 0.986, indicating a strong association. The limit of detection (LOD) for D-TRP was determined to be 3.3 µM, whereas the LOD for L-TRP could not be calculated due to the absence of a significant color change. These findings were based on three individual assays (n = 3). The aggregation of AuNPs depends on the pH of the solution ( Figure 4B). Indeed, the pH effect of the BR buffer was studied in the pH range of 4-11.5. A good interaction between D-TRP and AuNPs was achieved at a pH value of 4. However, the L-TRP had no effect on the AuNPs' coloration. Furthermore, the effect of time on the ability to discriminate between the TRP enantiomers was also examined ( Figure 4C). The addition of D-TRP showed a significant decrease in the R-B intensity compared with L-TRP. A significant difference between L-TRP and D-TRP was obtained at 25 min. Therefore, this time was utilized for subsequent experiments. Figure 5A shows the calibration curves of D-TRP and L-TRP obtained using different concentrations ranging from 10 to 300 µM. As shown in the photographs, the color of the PAD-PMA_D-TRP color changed from pink to purple, and became more intense with an increasing concentration of D-TRP; thus, aggregation of the AuNPs occurred. However, with the addition of L-TRP, the PAD-PMA_D-TRP showed no significant color change, indicating the low interaction between L-TRP and the developed material. To quantitatively evaluate the results, the color density was converted into the R-B color intensity. This relationship can be described by the equation R-B = −0.15X + 57, where X represents the concentration of the analyte. The correlation coefficient for this linear relationship was 0.986, indicating a strong association. The limit of detection (LOD) for D-TRP was determined to be 3.3 µM, whereas the LOD for L-TRP could not be calculated due to the absence of a significant color change. These findings were based on three individual assays (n = 3).

Selectivity Study
A selectivity study plays a pivotal role in ensuring accurate and reliable analyses b identifying the target analytes and eliminating interference. The response of the newl developed PAD-PMA_D-TRP was tested with other amino acids, including cysteine, m thionine, histidine, and L-TRP. Figure 5B presents the results obtained under the optim conditions. It is evident that only D-TRP exhibited a lower R-B response compared wit the other amino acids. Notably, L-cysteine was found to cause aggregation of the AuNP However, the use of the MMIC in the sample's preparation effectively removed interfe ences such as L-cysteine thank to its specific cavities, which were complementary only t TRP in terms of the shape, size, and functional groups. By combining the MMIC with th newly developed PAD-PMA_D-TRP, successful discrimination of TRP enantiomers wa achieved.

Enantioselective Measurement of L/D Tryptophan Mixtures
The primary objective was to investigate whether the discriminative sensing capabi ity of AuNPs could be utilized for determining the enantiomeric percentage using th PAD-PMA_D-TRP. Since TRP commonly exists as enantiomeric pairs, it is crucial to asses the influence of one enantiomer on the other. With the proposed approach, we were ab to directly evaluate the performance of the PAD-PMA_D-TRP in determining the percen age and confirming the enantioselective separation and purification of TRP in a racem solution using AuNPs. Indeed, after the preparation of the samples using the develope MMIC, the concentration of L/D-TRP in the resulting supernatant could be determined b measuring the total concentration at 280 nm using UV spectroscopy. Subsequently, th percentage of each enantiomer was determined using the PAD-PMA_D-TRP.
An interesting observation is related to the change in color from red to purple, whic is dependent on the enantiomeric ratio. Figure 6 illustrates that the aggregation of AuNP was selectively induced by D-TRP, leading to the precipitation of D-TRP with AuNPs. A a result, the excess of the other enantiomer remained in the solution, thereby enablin enantioseparation. The graph in Figure 6 demonstrates a linear decrease in the R-B inten

Selectivity Study
A selectivity study plays a pivotal role in ensuring accurate and reliable analyses by identifying the target analytes and eliminating interference. The response of the newly developed PAD-PMA_D-TRP was tested with other amino acids, including cysteine, methionine, histidine, and L-TRP. Figure 5B presents the results obtained under the optimal conditions. It is evident that only D-TRP exhibited a lower R-B response compared with the other amino acids. Notably, L-cysteine was found to cause aggregation of the AuNPs. However, the use of the MMIC in the sample's preparation effectively removed interferences such as L-cysteine thank to its specific cavities, which were complementary only to TRP in terms of the shape, size, and functional groups. By combining the MMIC with the newly developed PAD-PMA_D-TRP, successful discrimination of TRP enantiomers was achieved.

Enantioselective Measurement of L/D Tryptophan Mixtures
The primary objective was to investigate whether the discriminative sensing capability of AuNPs could be utilized for determining the enantiomeric percentage using the PAD-PMA_D-TRP. Since TRP commonly exists as enantiomeric pairs, it is crucial to assess the influence of one enantiomer on the other. With the proposed approach, we were able to directly evaluate the performance of the PAD-PMA_D-TRP in determining the percentage and confirming the enantioselective separation and purification of TRP in a racemic solution using AuNPs. Indeed, after the preparation of the samples using the developed MMIC, the concentration of L/D-TRP in the resulting supernatant could be determined by measuring the total concentration at 280 nm using UV spectroscopy. Subsequently, the percentage of each enantiomer was determined using the PAD-PMA_D-TRP.
An interesting observation is related to the change in color from red to purple, which is dependent on the enantiomeric ratio. Figure 6 illustrates that the aggregation of AuNPs was selectively induced by D-TRP, leading to the precipitation of D-TRP with AuNPs. As a result, the excess of the other enantiomer remained in the solution, thereby enabling enantioseparation. The graph in Figure 6 demonstrates a linear decrease in the R-B intensity with an increasing enantiomeric percentage of D-TRP, ranging from 0% to 100%, indicating the optimized assay's ability to quantify the enantiomeric composition accurately.
Biosensors 2023, 13, x FOR PEER REVIEW 10 of 13 sity with an increasing enantiomeric percentage of D-TRP, ranging from 0% to 100%, indicating the optimized assay's ability to quantify the enantiomeric composition accurately.

Real Sample
With the aim of using the proposed PAD-PMA_D-TRP for a biological sample, it was utilized for the determination of D-TRP in a human serum sample. Samples spiked with 30 and 100 µM of D-TRP were tested. The results are presented in Table 1 along with satisfactory recoveries of 88.6-106.4%. The obtained results confirmed the applicability of the developed device for the detection of D-TRP in real biological samples.

Conclusions
The discrimination of enantiomers is necessary in order to understand their distinct biological activities, ensure the safety and efficacy of chiral drugs, and achieve analytical accuracy. This study presents a straightforward approach for the chirally selective sensing of TRP enantiomers using AuNPs as a colorimetric probe. The combination of MMIC with the developed PAD-PMA_D-TRP enabled the chiral recognition of TRP. The detection of TRP enantiomers could be easily read with the naked eye and/or a smartphone. Remarkably, the proposed sensor demonstrated the capability to detect D-TRP in human serum, suggesting its potential as a valuable platform for analyzing real samples. This paperbased device represents the first example of a simple, cost-effective, and user-friendly platform for enantioselective sensing applications. The success of this application opens up new possibilities for designing innovative enantiosensing strategies in the future.

Real Sample
With the aim of using the proposed PAD-PMA_D-TRP for a biological sample, it was utilized for the determination of D-TRP in a human serum sample. Samples spiked with 30 and 100 µM of D-TRP were tested. The results are presented in Table 1 along with satisfactory recoveries of 88.6-106.4%. The obtained results confirmed the applicability of the developed device for the detection of D-TRP in real biological samples.

Conclusions
The discrimination of enantiomers is necessary in order to understand their distinct biological activities, ensure the safety and efficacy of chiral drugs, and achieve analytical accuracy. This study presents a straightforward approach for the chirally selective sensing of TRP enantiomers using AuNPs as a colorimetric probe. The combination of MMIC with the developed PAD-PMA_D-TRP enabled the chiral recognition of TRP. The detection of TRP enantiomers could be easily read with the naked eye and/or a smartphone. Remarkably, the proposed sensor demonstrated the capability to detect D-TRP in human serum, suggesting its potential as a valuable platform for analyzing real samples. This paper-based device represents the first example of a simple, cost-effective, and user-friendly platform for enantioselective sensing applications. The success of this application opens up new possibilities for designing innovative enantiosensing strategies in the future. Funding: Abdelhafid Karrat gratefully acknowledges financial support from the Erasmus+ KA107 (EU) Program of the University of Cádiz (Spain), through "Servicio Español para la Internacionalización de la Educación" (SEPIE). The Spanish authors thank the Agencia Estatal de Investigación (AEI), the Ministerio de Ciencia e Innovación of Spain, and FEDER funds (EU) for the "Multibioanalysis" research project (Proyecto de Generación del Conocimiento, PID2021-122578NB-I00) financed by MCIN/AEI/10.13039/501100011033/FEDER, EU "ERDF: A way of making Europe". They also thank "Plan Propio 2022-2023" from the University of Cadiz for providing funding through the "Acceso al uso de Servicios Centrales de Investigación. Financiación de Actividades" (Ref: SC2022-001) and the "Proyectos Noveles para Impulsar su Carrera Científica" (Ref: PR2022-025, SENSPOT) programs.

Institutional Review Board Statement:
It should be noted that this study was carried out within the framework of a research project of the 'Plan Estatal 2021', entitled: "Development and validation of a multiparametric device for the continuous analysis of biomedical samples-MULTIBIOANALYSIS". 'Proyecto de Generación del Conocimiento', PID 2021-122578NB-I00 financed by the Agencia Estatal de Investigación (AEI), Ministerio de Ciencia e Innovación of Spain, and FEDER (EU) funds "FEDER A way of making Europe": MCIN/AEI/10.13039/501100011033. This project includes a research work applied to biological simples (human blood, plasma, and serum), such as the ones used in the present work. The study was conducted in accordance with the Declaration of Helsinki, and the aforementioned project and, consequently, the present study, in accordance with the Spanish regulation 'Real Decreto 1090/2015, de 4 de diciembre, por el que se regulan los ensayos clínicos con medicamentos, los Comités de Ética de la Investigación con medicamentos y el Registro Español de Estudios Clínicos', was approved by the Research Ethics Committee of Cadiz (Spain), on 12 May 2023, registry/protocol number 15.23, resolving that the aforementioned project, involving human samples, is viable, that it presents sufficient methodological rigor, that the evaluation of economic costs is correct, and that with respect to the ethical aspects, the project meets the necessary requirements of suitability of the protocol in relation to the objectives of the study.
Informed Consent Statement: Informed consent was obtained from the participant involved in this study to publish this paper.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.