Chiral recognition of tryptophan enantiomers using chitosan-capped silver nanoparticles: Scanometry and spectrophotometry approaches

Author’s Accepted Manuscript Chiral recognition of tryptophan enantiomers using chitosan-capped silver nanoparticles: scanometry and spectrophotometry approaches Marzieh Jafari, Javad Tashkhourian, Ghodratollah Absalan www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(17)31044-5 https://doi.org/10.1016/j.talanta.2017.10.005 TAL18003

To appear in: Talanta Received date: 30 July 2017 Revised date: 3 October 2017 Accepted date: 4 October 2017 Cite this article as: Marzieh Jafari, Javad Tashkhourian and Ghodratollah Absalan, Chiral recognition of tryptophan enantiomers using chitosan-capped silver nanoparticles: scanometry and spectrophotometry approaches, Talanta, https://doi.org/10.1016/j.talanta.2017.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chiral recognition of tryptophan enantiomers using chitosan-capped silver nanoparticles: scanometry and spectrophotometry approaches Marzieh Jafari, Javad Tashkhourian*, Ghodratollah Absalan* Professor Massoumi Laboratory, Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran

[email protected] [email protected] [email protected] [email protected] *Corresponding authors: Tel.: +98 (71) 3613-7137, +98 (71) 3613-7127; Fax: +98 (71) 3646-0788

ABSTRACT A new, fast and inexpensive colorimetric sensor was developed for chiral recognition of tryptophan enantiomers using chitosan-capped silver nanoparticles. The function of the sensor was based on scanometry and spectrophotometry of the colored product of a reaction solution containing a mixture of chitosan-capped silver nanoparticles, phosphate buffer and tryptophan enantiomers. The image of the colored solution was taken using the scanometer and the corresponding color values were obtained using Photoshop software which subsequently were used for optimization of the experimental parameters as the analytical signal. Two types of color values system were investigated: RGB (red, green and blue values) and CMYK (cyan, magenta, yellow and black values). The color values indicated that L-tryptophan had better interaction than D-tryptophan with chitosancapped silver nanoparticles. A linear relationship between the analytical signal and the concentration of L-tryptophan was obtained in the concentration range of 1.3×10−5-

4.6×10−4 mol L−1. Detection limits, were obtained to be 2.1×10−6, 2.4×10−6 and 3.8×10−6 mol L−1 for L-tryptophan based on R (red), G (green) and B (blue) values, respectively. Graphical abstract

Without Trp HO O

O

O

NH2

HO

With LTrp

NH2 O

HO HO

1: CS-

HO

O

OH

H2 N

AgNP

OH

With DTrp

CSAgNP

1 Absorbance

HO

2

1.2

2: CS-AgNP + L3: CS-AgNP + D-

0.8

3

0.4

0 350

450

550

650

750

Wavelength (nm)

Keywords: Chiral recognition; Tryptophan; Scanometry; Spectrophotometry; Chitosan;

Silver nanoparticles

1. Introduction Tryptophan (Trp), 2-amino-3-(1H-indol-3-yl) propanoic acid, plays a significant role in the metabolism pathway (Fig. S1). L-tryptophan (L-Trp) is an “essential” amino acid that must be supplied in human diet. L-Trp acts as a building block in biosynthesis of proteins and serves as a biochemical precursor for many biological active molecules including tryptamine, melatonin, serotonin, auxins and niacin [1]. D-tryptophan (D-Trp) is an

important intermediate in preparation of synthetic peptide antibiotics as well as immunosuppressive agents in the pharmaceutical industry [2]. Generally, the study of enantiomeric recognition of chiral biomolecules can provide important information about the recognition process in biological systems, pharmaceutical sciences, and biochemistry. The selective recognition of an individual enantiomer is difficult due to the similarity of enantiomers in physical and chemical properties and molecular configurations. Thus, it is important to find effective and rapid methods for the chiral recognition of enantiomers [3]. Some analytical methods have been used for chiral recognition of tryptophan enantiomers such as spectrofluorometry [4], chromatography [5, 6], electrochemistry [7, 8] and spectrophotometry [9, 10]. The main principle in chiral recognition is selecting a proper chiral selector which has recognition site for discrimination between enantiomers [2]. In this regard different chiral selectors including amino acids [11-13], cyclodextrins [7, 14], hemoglobin [15, 16], ionic liquids [6], carbohydrates [17] and chitosan [8, 18, 19] have been reported for recognition of enantiomers. Chitosan (CS), a deacetylated derivative of chitin, is an optically active natural polysaccharide. The excellent properties of CS such as hydrophilicity, biocompatibility, biodegradability, antibacterial and non-toxicity make it an ideal candidate as a chiral selector for recognition of enantiomers [8]. The amine groups of glucosamine units in chitosan are important chelating sites for metal ions [20] and can be used for synthesis of some metal nanomaterials. Regarding silver-based nanoparticles, chitosan-capped silver nanoparticles (CS-AgNPs) show as an emerging group of bio-nanostructured materials [21]. The size, surface, and shape dependence of the physical, optical, and electronic properties of nanomaterials make them fascinating components in modern materials research [22]. In this regard, noble metal nanoparticles, especially gold (Au) and silver (Ag) nanoparticles have been intensely used by chemists owing to their unique optical and spectral properties. The extinction coefficients of gold or silver nanoparticles are much higher than that of organic dyes, thus their absorbances are detectable at nanomolar ranges [22].

Due to having a high efficient plasmon excitation [23, 24] and remarkable photophysical and photochemical properties [25], silver nanoparticles have been increasingly explored as colorimetric sensors for chiral recognition. However, the maximum wavelength of the analyte (λmax) must be sharp enough to have accurate and precise measurements. On the other hand, the linear dynamic range of the analyte is restricted by both chemical and instrumental factors, and thus measurements are not applicable on non-transparent samples. To overcome these problems, scanometry is the technique of choice where a flatbed scanner is used to measure the reflection of light from the solution of interest even if it is turbid. In contrast to conventional spectrophotometric methods, the sharpness of the λmax of the analyt is not a serious problem because the measured color intensity is analyzed based on different color values [26, 27]. In this study, a new, simple, fast, inexpensive and selective colorimetric sensor was developed for chiral recognition of tryptophan enantiomers using chitosan-capped silver nanoparticles. After scaning the optical cells containing the sample solution of CSAgNPs and L- or D-tryptophan, the color values of each optical cell was analyzed. To examine the validity of the scanometry, the analysis of solutions was done using spectrophotometric method as well.

2. Experimental 2.1. Reagents Both L- and D-tryptophan were purchased from Merck and their stock solutions (3.0×10−2 mol L−1) were diluted with deionized water to prepare the working solutions. Silver nitrate (AgNO3), sodium borohydride (NaBH4), acetic acid (CH3COOH), sodium citrate, potassium hydrogen phthalate (KHP) and boric acid were purchased from Merck. Chitosan was obtained from Sigma-Aldrich. To prepare phosphate buffer solution (PBS, 0.01 mol L−1) with pH values in the range of 2.5-10.0, the NaOH solution was gradually added to a selected volume of H3PO4 solution until the pH meter showed the desired pH value. All chemicals were of analytical grade and were used without further purification.

2.2. Apparatus The UV/Vis spectra were measured by an Ultrospec 4000 Pharmacia Biotech, UK, at room temperature using 1.0-cm Helma quartz cells. A CanoScan 4200F (Canon) scanner equipped with a cold cathode fluorescent light source (CCFL) and a charge coupled device (CCD), as the detection system, was used. The CCFL was a three-wavelength source (for red, green and blue regions). The resolution of the scanner was regulated at 300 ppi. The cells were built by using a Plexiglas® sheet. Calculation of the R, G, and B color values was performed with an optional histogram palette from Adobe Photoshop element CS3 ver.12.0 (Adobe System Inc., CA, USA). Dimension of working “region of interest” (ROI) was 20×20 pixels. The CMYK values were derived from RGB values using MATLAB Software. An eppendorf micropipette was used for injecting samples into the cells. The pH values of buffer solutions were measured by a Metrohm (780) pH meter. The ultrasound irradiation was carried out by Ultrasonic Processor (Heilscher, UP200H, Germany) equipped with a nitrogen gas (99.9% purity) tank. The fouriertransform infrared (FTIR) spectra of CS-AgNPs in aqueous solutions were recorded by using an FTIR spectrometer (Spectrum RXI, PerkinElmer) equipped with CaF2 circular cell windows. The transmission electron microscopy (TEM) micrographs were obtained by a transmission electron microscope (Zeiss, model EL10C) operated at an accelerating voltage of 80 kV. The X-ray diffraction (XRD) patterns were acquired with a Bruker D8 Advance X-ray diffractometer using the Cu Kα radiation line (40 kV, 300 mA) of 0.154 nm and were interpreted by using the X'Pert High Score software.

2.3. Synthesis of chitosan-capped silver nanoparticles Chitosan-capped silver nanoparticles (Fig. S2) [28] were synthesized based on reduction of AgNO3 by NaBH4 in the presence of chitosan as the stabilizer and protection agent [29]. Briefly, 25.0 mg chitosan was dissolved in 50.0 mL of 4% aqueous acetic acid and was sonicated for 5 min. Then, 20.0 mL of this solution was mixed with 50.0 mL of

aqueous AgNO3 solution (6.0 mmol L−1) and reaction was performed at room temperature under vigorous magnetic stirring for 30 min. Afterwards, an aqueous solution of NaBH4 (1.0 mL, 58.0 mmol L−1) was added to the reaction mixture. After stirring for 2 h, the brownish-yellow dispersion of CS-AgNPs was collected and stored at room temperature for subsequent tests.

2.4. Preparation of cells array By using a laser beam, a series of cylindrical cells (cavities) with inner diameters of 1.5 cm, as demonstrated in Fig. S3, were fabricated in the body of a 0.6-cm thick Plexiglas® sheet [26]. The cells were aligned to 5 columns and 4 rows with a total of 20 cells in the Plexiglas® sheet. The similarity of the Plexiglas® cells was demonstrated in Fig. S4 and Table S1 in ESI.

2.5. Procedure for chiral recognition of tryptophan enantiomers CS-AgNPs (1.0 mL) was added to 500.0 μL PBS (pH=3.5) in a test tube. Then, 20.0 μL of L- and/or D-Trp, with desirable concentration, was injected to this solution (test solution). A blank solution was prepared in the same manner without adding tryptophan enantiomers. After 30 min, the UV-visible spectra of the test solution was recorded. Then, 700.0 μL of the test solution was transferred into the cell of Plexiglas® sheet and the color of the solution was scanned. Using Photoshop software, specific areas of a scanned image were selected for RGB (red, green and blue) analysis and the values were averaged to increase the precision. The effective intensity of any color value was calculated as follows: Ar = −log(Rs / Rb)

(1)

Ag = −log(Gs / Gb)

(2)

Ab = −log(Bs / Bb)

(3)

where Ar, Ag and Ab are the effective intensities for red, green and blue colors, in order. The subscripts “s” and “b” refer to parameters corresponding to sample and blank

solutions, respectively [30]. For assessment of different types of color values, MATLAB software was used to get CMYK values where C stands for cyan, M for magenta, Y for yellow, and K for black. It should be mentioned that CMYK color model is the subtractive of RGB color model [31]. The differences between CMYK values of the blank solution were compared with the CMYK values of solutions containing L- and/or D-tryptophan.

3. Results and Discussion 3.1. Characterization of chitosan-capped silver nanoparticles The synthesized chitosan-capped silver nanoparticles (CS-AgNPs) were characterized using TEM, FTIR, XRD and UV-Vis spectroscopies. The UV-Vis spectrum of the chitosan capped silver nanoparticles is shown in Fig. 1A. The λmax of the silver nanoparticles was observed at 404 nm showing the characteristic surface plasmon resonance (SPR) band of the silver nanoparticles [32]. The TEM image (Fig. 1B) showed that CS-AgNPs were uniformly dispersed in aqueous solution with an average diameter of 15±2 nm. The concentration of chitosancapped silver nanoparticles was calculated using Beer’s law and the extinction coefficient (ε) was measured at 404 nm [32]: ln ε = 1.4418 lnD + 18.955

(4)

where D is the diameter (nm) of CS-AgNPs as obtained from TEM image. The concentration of CS-AgNPs was found to be 6.9 (± 0.9)×10−9 mol L−1. Figure 1C-a demonstrates the XRD pattern of chitosan. The peaks at 2θ values of 11.7° and 20.2° are in agreement with those reported in literature [33, 34]. The broadening of the peaks could be due to the amorphous nature of chitosan structure [35]. The XRD pattern of the synthesized chitosan capped-silver nanoparticles is also shown in Fig. 1C-b. The peak appeared at 2θ value of 11.7° corresponds to chitosan. The prominent peaks at 38.1°, 42.7° and 64.4° (2θ values) corresponding to (111), (200) and (220) sets of lattice planes, respectively, refer to a face centered cubic (fcc) structure for CS-AgNPs [36]. The diffraction peak at 2θ of 29.1° can be attributed to non-reduced

AgNO3 [37]. Figure 1D represents the FTIR spectra of chitosan and chitosan-capped silver nanoparticles. The absorption peaks in FTIR spectrum of chitosan (Fig. 1D-a) have been denoted as O–H stretching at 3435 cm−1, C–H and C–N stretching at 2920 cm−1, N–H bending at 1647 cm−1, N–H angular deformation in –CONH plane at 1350 cm−1 and C– O–C band stretching at 1100 cm−1. The observed shift in the FTIR peaks of CS could be due to interaction of CS with Ag (Fig. 1D-b). It should be noted that the intensity of the hydroxyl peak in CS-AgNPs is lower. The peak intensity in 1387 cm−1 is related to C–N stretching in CS-AgNPs [33, 36]. These results confirmed the binding of Ag with –OH and –NH2 groups of chitosan. 3.2. Chiral recognition of tryptophan enantiomers For studying the chiral recognition of Trp using scanometric and spectrophotometric methods, solutions containing 1.0 mL of CS-AgNPs (6.9×10−9 mol L−1), 0.5 mL PBS (0.01 mol L−1, at pH 3.5) and 20.0 μL of D- and/or L-tryptophan (1.0×10−2 mol L−1) were prepared in the individual test tubes. Blank solutions were prepared similar to sample (reaction) solution without adding tryptophan enantiomers. After 30.0 min, the UV-Vis spectra of solutions were recorded (Fig. 2A). Then, 700 μL of each sample was transferred into the Plexiglas® cells. The cells were scanned afterward and the image of each cell was analyzed, Fig. 2B. According to the results, no color change in CS-AgNPs solution was observed in the presence of D-Trp whereas color changed from yellowish to brown in the presence of L-Trp. The observations were confirmed using the TEM image of the reaction solution (Fig. 2C,D). Thus, CS-AgNPs was used for chiral recognition of D- and L-Trp. As shown in TEM photograph, there was no aggregation in the CS-AgNPs in the presence of D-tryptophan whereas CS-AgNPs aggregated at high concentrations of L-tryptophan. In the presence of higher concentration of tryptophan (4.6×10−4 mol L−1), the hydrogen bond formation is possible between carboxylic groups of tryptophan molecules adsorbed on surface of individual CS-AgNPs resulting in aggregation of these particles [32].

The RGB values of scanned images were obtained using Photoshop software and then, effective intensities (Ar, Ag, Ab) of red, green and blue values of samples and blanks solutions were obtained (Fig 3A,B). The CMYK values and the difference between cyan, magenta, yellow and black values of sample and blank solutions were obtained from the RGB values (Fig. 3C,D) using MATLAB software. The results showed that the highest color value change was achieved for blue (B) and yellow (Y) values. Thus, the changes corresponding to B and Y values were used for performing optimization of the experimental conditions. It should be noted the response of CS-AgNPs to some available chiral amino acids and biomolecules such as cysteine, cysteine, penicillamine, mandelic acid and tyrosine was tested. The results showed that both enantiomers of each compound could induce the CS-AgNPs aggregation and consequently no chiral recognition was possible for these compounds. However, chiral recognition only happened for Trp enantiomers. In the following, effect of some parameters such as pH, incubation time, buffer type and concentration, volume ratio of CS-AgNPs to buffer as well as the reaction solution volume which was injected in the Plexiglas® cell were investigated.

3.3. Effect of pH For studying the effect of pH, the sample solutions were prepared using phosphate buffer solution with different pH values. After 30 min, 700 μL of each sample was transferred into the Plexiglas® cells to be scanned (Fig. S5). As seen, the chiral recognition was possible at acidic pH. In acidic solution, the CS-AgNPs are rich in –OH groups which can significantly interact with carboxylic groups of Trp (pKa1, carboxyl =2.4, pKa2, amine =9.4). However, CS-AgNPs are not stable at alkaline pH as reported [28]. The cell image showed that the best pH for chiral recognition of L- and D-Trp was 3.5. The blue value (B) and its effective intensity for each cell at different pHs are shown in Fig. 4A,B. Also, the yellow values (Y) and difference in the Y values of the sample and the blank solutions (∆(YT-Yb)) were calculated at different pHs and the results are shown in Fig. 4C,D.

To verify the scanometric results, the corresponding spectrophotometric data for each sample were collected at different pH. As shown in Fig. S6, the highest difference in plasmon resonance peak (λmax) was obtained for chiral recognition of tryptophan enantiomers at pH 3.5.

3.4. Effect of incubation time In order to study the reaction (incubation) time on chiral recognition of Trp enantiomers, sample solutions were scanned at different reaction time periods and the image of each cell was analyzed with the Photoshop software (Fig. S7). The results obtained for B and Y values are shown in Fig. 5. As the results indicated the color values levelled off after a reaction time of 30 min. The UV-Vis spectra of solutions at different incubation time periods were recorded and difference in absorbance at λmax are shown in Fig. S8. 3.5. Buffer type and buffer concentration effect Effect of phosphate, acetate, citrate, KHP and universal buffers were investigated in chiral recognition of Trp enantiomers. As shown in Fig. S9, in citrate and KHP buffers with pH=3.5, the color of CS-AgNPs changed from brownish yellow to colorless which could be easily detected by the naked eye. This showed that CS-AgNPs were damaged in this buffers. The results indicated that chiral recognition was not possible in acetate and universal buffers but PBS can be appropriate for resolving the Trp enantiomers. The color values for RGB and CMYK were obtained and the results are shown in Fig. 6. The UV-Vis spectra of CS-AgNPs solutions, with and without Trp enantiomers in different buffers, are shown in Fig. S10. Also, the chiral recognition of Trp enantiomers were investigated in three different concentrations of PBS (Fig. S11). The results showed that a concentration of 1.0×10−2 mol L−1 PBS was suitable to obtain the maximum difference in color values between Trp enantiomers (Fig. S12). The high concentration of PBS inhibited interaction between Trp enantiomers and CS-AgNPs. Thus, 1.0×10−2 mol L−1 PBS was selected as the optimum concentration. It should be mentioned that the presence of buffer is necessary for

performing the analysis as no color change occurred without using buffer after a reaction time of 30 min. The UV-Vis spectra of CS-AgNPs solutions with and without Trp enantiomers, in different buffers, are shown in Fig. S13. 3.6. Volume ratio of CS-AgNPs to buffer The volumet ratio of CS-AgNPs (6.9×10−9 mol L−1) to phosphate buffer (1.0×10−2 mol L−1) solution (CS-AgNPs : PBS) was studied at three different ratios (0.5:1, 0.75:0.75, and 1:0.5). By increasing the ratio of nanoparticls, the number of interaction sites between Trp and silver nanoparticles increases. Thus, the ratio of 1:0.5 was chosen as the optimum ratio. Also, the effect of sample volume injected into a cell was studied. The results are presented in Figs. S14 and S15 in ESI.

3.7. Possible interaction mechanism Chitosan chains have an extended twofold helical conformation stabilized by O-3…O-5 hydrogen bonds (Scheme 1), which is similar to that of DNA [8]. The helix structure of the molecule has a possible role in chiral recognition of enantiomers from the supramolecular point of view. The chiral recognition may be attributed to the selective formation of hydrogen bonds between CS and Trp isomers [8]; the –OH groups of the surface of CS-AgNPs (Fig. S2) can interact with amine and carboxyl functional groups in Trp molecules. At pH=3.5, the surface of CS-AgNPs is protonated and can form H-bond with negatively-charged carboxylic group and amine group of Trp as reported [38]. In terms of spatial direction, it seems that L-Trp has better interaction than D-Trp with CSAgNPs due to its appropriate orientation.

3.8. Analytical figures of merit Different concentrations of Trp enantiomers were injected to CS-AgNPs and solutions were scanned (Fig. S16). Under the optimum experimental conditions, the calibration curves were plotted based on blue and yellow values (Fig. 7). A good linear relationship between the effective intensity of the product and the concentration of L-Trp was

obtained in the concentration range of 1.3×10−5 –4.6×10−4 mol L−1. The mathematical equations of the calibration curves are presented in Table 1. For obtaining the reproducibility of the method, 10 sample solutions containing 20 μL of 1.0×10−2 mol L−1 L-Trp were analyzed and the average of RSD% (relative standard deviation) for R, G and B color values were 1.0, 1.2 and 1.8, respectively. Detection limits, based on three times of the standard deviation of the blank divided by the slope of the linear regression equation, were obtained as 2.1×10−6, 2.4×10−6 and 3.8×10−6 mol L−1 for L-Trp using R, G and B values, respectively. The repeatability (intra-day) and intermediate precision (inter-day) were also obtained by analyzing three different concentrations of L-tryptophan under optimum experimental conditions. The results summarized in Table 2 indicate that the developed scanometric method provide a good precision for determination of L-Trp. The UV-Vis spectra of reaction mixture are shown in Fig. S17. The peak observed at 550 nm is due to increasing the size of nanoparticles and aggregation or complexation of nanoparticles [32]. Because of this, a good linear range was not obtained based on surface plasmon resonance of chitosan-capped silver nanoparticles. The SPR band shifted after adding L-tryptophan and a new peak was observed at a higher wavelength when concentration of L-trp was high. Thus, a proper wavelength for plotting the suitable calibration curve was not achieved. However, the detection limit was obtained to be 1.8×10−6 mol L−1 based on spectrophotometric data.

3.9. Synthetic sample analysis The color values were obtained in the presence of different mole ratios of L-Trp with a total concentration of 9.2×10−5 mol L−1 of tryptophan enantiomers. Figure S18 shows the scanned images of reaction solutions in the presence of tryptophan enantiomers mixture. The B and Y color values are shown in Fig. S19. Also, the reaction solutions were analyzed using spectrophotometry and the corresponding absorption data, at 550 nm, were used for drawing the calibration curve (Fig. S20). The results showed that the enantiomeric composition of tryptophan could be determined from the corresponding

scanometry and spectrophotometric linear calibration curves.

4. Conclusion For chiral recognition of Trp enantiomers, a simple, fast and cost-effective method was developed based on scanometry technique. The RGB and CMYK color models are in agreement and good results in reproducibility and repeatability were obtained. The linear concentration range of 1.3×10−5–4.6×10−4 mol L−1 was obtained for determination of Ltryptophan from scanometry. Detection limits were found as 2.1×10−6, 2.4×10−6 and 3.8×10−6 mol L−1 for L-Trp based on R, G and B values, respectively, and 1.8×10−6 mol L−1 based on spectrophotometry. The RSD% for R, G and B color values were 1.0, 1.2 and 1.8, respectively. The results of both scanometry and spectrophotometry were compared and verified each other. To have a comparison between the developed method and those reported in literature for determination of Trp, the available data have been collected in Table 3. As it is presented, the developed method was found to have a wider linear dynamic range with an acceptable detection limit compared to other methods reported in this table.

Acknowledgments The authors wish to express their gratitude to Shiraz University Research Council for the support of this work.

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nanocomposite

in

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sensing

for

Tryptophan

enantiomers,

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Caption and legends for figures Fig. 1. (A), The UV-Vis spectrum of the chitosan-capped silver nanoparticles dispersion; (B), The TEM photograph of chitosan-capped silver nanoparticles; (C), The XRD patterns, and (D), The FTIR of chitosan (a) and chitosan-capped silver nanoparticles (b). Fig. 2. (A), The UV-Vis spectra of blank (a), L-Trp (b) and D-Trp(c) with concentration of 1.3×10−4 mol L−1. (B), Scanned images of the reaction solutions. The TEM images of CS-AgNPs in the presence of (C), L-Trp; and (D), D-Trp at concentration of 4.6×10−4 mol L−1. Fig. 3. (A) The RGB value analysis. (B) The effective intensity of solutions containing LTrp and D-Trp. (C) The CMYK value analysis. (D) The difference in CMYK value of solutions containing L-Trp and D-Trp from CMYK value of the blank solution Fig. 4. (A), The blue value; and (B), effective intensity of the blue value for L-Trp (♦), DTrp (▲) and blank (●) solutions. (C), The yellow value; and (D), the difference between yellow values of L-Trp (♦) and D-Trp (▲) with blank solution (∆(YT-Yb)) Fig. 5. (A), The blue values; and (B), effective intensity of the blue values of L-Trp (♦), D-Trp (▲) and blank (●) solutions at different interaction times. (C), The yellow values; and (D), the difference between the yellow values of L-Trp (♦) and D-Trp (▲) with blank solution at different interaction times Fig. 6. (A), The blue values; and (B), effective intensities of the blue values for L-Trp (♦), D-Trp (▲) and blank (●) solution at different buffers. (C), The yellow values; and (D), difference between the yellow values of L-Trp (♦) and D-Trp (▲) with blank at different buffers Fig. 7. Calibration curves plot based on (A) RGB color model, and (B) the effective intensities of RGB values. Calibration curves plot based on (C) CMYK color model, and (D) the difference between CMYK of L-Trp solution and that of the blank solution Scheme 1 Extended twofold helical conformation of chitosan

1

0.6

400 0.4

A

C 300

0.2

0 300

400

500

600

700

Wavelength (nm)

800

Count (a.u.)

Absorbance

0.8

200

100

(111 )

a (200)

(220)

b

0 10

B

30

50 2θ (degree)

2920

1647

70

D 1350 1100 3435

1620

1109

3447 1387

Fig. 1

90

B

A

b

1.2

Absorbance

Blank

Blank + L-Trp

a 0.8

c C

0.4

0 350

450

550

650

750

Wavelength (nm)

Fig. 2

20

D

Blank + D-Trp

250

0.45 R G B

0.35

Effective Intensity

RGB Value

200

A

150

100

D-Trp 0.25

0.15

50

0.05

0

-0.05 Blank

D-Trp

L-Trp

R

G

B

0.45

0.8

C

C

D

L-Trp 0.35

∆ (CMYK Value)

M

0.6

CMYK Values

B

L-Trp

Y K

0.4

0.2

D-Trp

0.25

0.15

0.05

-0.05

0 Blank

D-Trp

CT-CB

L-Trp

Fig. 3

21

MT-MB

YT-YB

KT-KB

0.45

160

A

B 0.35 0.25

Ab

Blue Value

120

80

L-Trp D-Trp

0.15 40

Blank L-Trp D-Trp

0.05 -0.05

0

2.5 3.0 3.5 4.0 5.0 6.0 7.0 8.0 9.0 10.0

2.5 3.0 3.5 4.0 5.0 6.0 7.0 8.0 9.0 10.0

pH

pH 0.45

0.8

C

D

0.35

∆ (YT-Yb)

Yellow Value

0.6

0.4

0.2

Blank L-Trp D-Trp

0.25 L-Trp D-Trp

0.15 0.05

-0.05

0

2.5 3.0 3.5 4.0 5.0 6.0 7.0 8.0 9.0 10.0

2.5 3.0 3.5 4.0 5.0 6.0 7.0 8.0 9.0 10.0

pH

pH

Fig. 4

22

160

0.45

0.25

Ab

Blue Value

B

0.35

A

120

80

0.15 40

Blank L-Trp D-Trp

L-Trp D-Trp

0.05 -0.05

0 10

15

25

30

35

10

40

15

30

35

40

0.45

0.8

0.35

C ∆ (YT-Yb)

0.6

Yellow Value

25

Time (min)

Time (min)

0.4

D

0.25

0.15 L-Trp

0.2

Blank L-Trp D-Trp

D-Trp

0.05

-0.05

0 10

15

25

30

35

10

40

Time (min)

15

25

30

Time (min)

Fig. 5

23

35

40

Blank L-Trp D-Trp

0.57

A

120

B

L-Trp D-Trp

0.38

Ab

Blue Value

160

80 0.19 40

-0.01

0 Citrate

Acetate Universal

KHP

Citrate

PBS

Acetate Universal

KHP

PBS

Buffer Type

Buffer Type 0.8

D L-Trp D-Trp

0.4

∆ (YT-YB)

0.6

Yellow Value

C

Blank L-Trp D-Trp

0.4

0.2

0.3

0.2

0.1

0

0.0 Citrate

Acetate Universal

KHP

PBS

Citrate

Acetate Universal

Buffer Type

Buffer Type

Fig. 6

24

KHP

PBS

0.8

250

B

A R G B

150

Effective Intensity

RGB Values

200

100

Ar

0.6

Ag Ab

0.4

0.2

50 0

0 0

10

20

Conc. of L-Trp

30

40

(10−5

L−1)

mol

0

50

10

20

Conc. of L-Trp

1

30

40

(10−5

L−1)

mol

50

0.5 C M Y K

0.8

C

D

CT-Cb MT-Mb

0.4

YT-Yb

∆ (CMYK)

CMYK Values

KT-Kb

0.6

0.4

0.3

0.2

0.1

0.2

0

0 0

10

20

30

40

0

50

Conc. of L-Trp (10−5 mol L−1)

Fig. 7

25

10

20

30

40

Conc. of L-Trp (10−5 mol L−1)

50

OH OH HO

5O

NH2 O

3 HO

OH O H 2N O OH

HO O

NH2 O HO

OH O

H 2N O

OH

HO O

NH2 OH

Scheme 1

26

Table 1 Mathematical equations of calibration curves based on color values for both RGB and CMYK systems Color model

RGB

CMYK

Color

Equation

R2

Color

Equation

R2

Red

y= −1.3573x+202.58

0.9936

Ar

y= 0.0035x+0.0010

0.9841

Green

y= −1.4706x+169.98

0.9978

Ag

y= 0.0048x+0.0323

0.9933

Blue

y= −1.4555x+85.991

0.9719

Ab

y= 0.0128x+0.0990

0.9992

Cyan

0

0

∆(CT−Cb)

0

0

Magenta

y= 0.0023x+0.1565

0.9803

∆(MT−Mb)

y= 0.0023x+0.6380

0.9803

Yellow

y= 0.0061x+0.5646

0.9743

∆(YT−Yb)

y= 0.0061x+0.1386

0.9743

Black

y= 0.0053x+0.2055

0.9936

∆(KT−Kb)

y= 0.0053x+0.0095

0.9936

y: color values x: concentration of L-Trp

Table 2 Repeatability and intermediate precision of L-Trp determination Concentration (mol L−1) 2.0×10−5 1.1×10−4 3.0×10−4

Repeatability (n=3) (Intra-Day) Mean response* ±SD %RSD 83.9 ± 0.8 1.0 70.4± 0.4 0.6 41.9 ± 0.8 1.9

Intermediate precision (n=3) (Inter-Day) Mean response* ±SD %RSD 84.1 ± 1.0 1.2 70.7 ± 0.8 1.1 42.1 ± 0.9 2.1

*Blue value

Table 3 Comparison of the developed method with some method reported for determination of tryptophan in litrature Method Electrochemistry Spectrophotometry Electrochemistry HPLC Electrochemistry Scanometry

Linear range (mol L−1) 5×10−4-2.5×10−3 0.2×10−6-10.0×10−6 5.0×10−5-5.0×10−3 0.5×10−6-4.0×10−6 3.0×10−5-30.0×10−5 1.3×10−5-4.6×10−4

27

Detection limit (mol L−1) 1.7×10−7 1.0×10−7 1.7×10−5 1.0×10−8 2.2×10−5 2.1×10−6

Ref. 39 10 7 40 41 This Work