Optical Determination of Tryptophan Using Persistent Luminescence Nanoparticles (PLNPs)

Abstract Tryptophan is an essential amino acid that is an important part of the human diet. In this work, label-free photoluminescence sensing based upon persistent luminescent nanoparticles (PLNPs) Zn0.8Ga2O4: Cr3+ is reported to determine tryptophan. Following 254 nm excitation, the PLNPs released luminescence after storing the absorbed light. Compared to the standard fluorescence, the persistent afterglow of the PLNPs was used for detection and imaging without external illumination, thereby eliminating autofluorescence and scattered light generated by biological substrates. For tryptophan concentrations from 4 to 240 µM, a linear relationship was described by F/F0 = 0.9416 − 0.0024 [Trp] with a correlation coefficient of 0.9911 and a detection limit of 0.19 µM. The recoveries of analyzed samples were between 97.9 and 101.9% providing good accuracy and simple methodology.


Introduction
Tryptophan (Trp) is an aromatic essential amino acid (Sathya et al. 2023), It is also a precursor for many biologically active substances (Zhao et al. 2015).Tryptophan not only participates in cell regulation and metabolism (Savitz 2020), but also produces serotonin, regulates the synthesis of proteins, and modulates immune and digestive functions (Malviya et al. 2019;Ma et al. 2022).This compound plays an integral role in biological processes such as the development of plants, the growth of animals, and the synthesis of proteins (Diem, Bergmann, and Herderich 2000;Mackay et al. 2006;Bista et al. 2009).
In addition, tryptophan influences growth in infants and nitrogen balance in adults (Remko et al. 2011).However, it also causes adverse effects such as pain in the limbs, mouth ulcers, small abdominal pain, shortness of breath, which are all caused by excessive concentrations of Trp (Xu et al. 2016;Zhang, Jamal, et al. 2020;Prabakaran et al. 2021).Mood abnormalities such as anxiety, irritability, insomnia, and depression are caused by its deficiency (Capuron et al. 2011).Due to its importance and toxic effects, the accurate determination of tryptophan is of increasing importance.
Various methods have been developed to determine tryptophan.High performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and capillary electrophoresis have been extensively studied (Forteschi et al. 2015;Goswami, Thakker, and Dhandhukia 2015;Alizadeh and Amjadi 2017).However, these techniques are high-cost, time-consuming, and require tedious sample preparation procedures and sophisticated equipment with specialized personnel (Zhang, Jamal, et al. 2020).Electrochemical methods such as voltammetry and potentiometry methods have also been reported.For example, cyclic voltammetry was used to determine tryptophan (Lima et al. 2022).While these electrochemical methods have advantages, the long-term stability of the electrodes is unsatisfactory and hence practical applications are limited.
Fluorescence offers high sensitivity, ease of use, low cost, and design flexibility (Liu et al. 2012;Sun et al. 2021).However, a continuous and constant external light excitation is required in most methods.As a result, interferences from autofluorescence and scattered light in biological matrices is difficult to eliminate and the signal-to-noise ratio is poor (Liang et al. 2019).Therefore, a highly sensitive, autofluorescence-free, and selective approach is required for the determination of tryptophan in real samples.
Persistent luminescent nanoparticles (PLNPs) are a unique category of optical nanomaterials that are able to store excitation energy and maintain persistent luminescence after excitation (Sun, Wang, and Yan 2018;Lin et al. 2020).In comparison to standard fluorescence techniques, autofluorescence and background noise interference are eliminated by PLNP-based approaches (Maldiney et al. 2014).Various PLNP-based probes have been developed for biosensing (Zhao et al. 2019), as well as bioimaging and therapeutic applications (Wang et al. 2017).Zhang et al. (2018) constructed a PLNP-based label-free sensor for the sensitive and selective determination of organic pollutants.Li et al. (2014) developed a novel nanoprobe using MnO 2 -modified PLNPs based upon the site-specific reaction of manganese dioxide and GSH (glutathione) for the determination and imaging of glutathione in living cells and in vivo.Wang et al. (2021) designed a PLNP-based functionalized adaptive sensor for the selective, sensitive, and autofluorescence-free determination of kanamycin in food.The sensor was functionalized by the aptamer of kanamycin as a recognition unit as well as the separation element.The aptamer complementary DNA (cDNA) was subsequently grafted onto the surface of the PLNPs to give functionalized nanoparticles that were hybridized to prepare a PL turn-on adaptor sensor.However, the determination of amino acids by PLNPs sensors has not yet been reported.
In this work, a persistent luminescent nanomaterial (Zn 0.8 Ga 2 O 4 :Cr 3þ ) was synthesized using a hydrothermal method for the sensitive and selective determination of tryptophan (Li et al. 2015).The analyte absorption overlapped with the photoluminescence excitation spectrum of PLNPs.Therefore, the luminescence of PLNPs was quenched by tryptophan allowing its determination.This approach was employed to determine tryptophan in milk and human serum.

Reagents and instrumentation
Gallium nitrate and the amino acids were acquired from Shanghai Aladdin.Chromium nitrate, zinc acetate, and inorganic salts were purchased from Macklin Reagent Factory (Shanghai, China).All reagents were of analytical grade and were not purified before use.Ultra-pure water was used in all experiments.

Preparation of PLNPs
Cr 3þ -doped Zn 0.8 Ga 2 O 4 nanoparticles were prepared by a direct hydrothermal method (Ai et al. 2018).Zinc acetate (0.8 mL, 1 mol/L), gallium nitrate (1 mL, 2 mol/L), and chromium nitrate (8 mL, 0.5 mol/L) were mixed with vigorous stirring and ultrapure water was added to a total volume of 15 mL.Ammonium hydroxide was added with stirring and the pH was adjusted to 9.5.The liquid rapidly changed from colorless to a milky white suspension with stirring for 30 min (8000 rpm).The suspension was transferred to a Teflon-lined counterpressure kettle (50 mL) and hydrothermally treated at 220 � C for 10 h.
After cooling to room temperature, the white precipitate was separated by centrifugation and washed with 0.01 mol/L hydrochloric acid to remove zinc oxide impurities.Next, the PLNPs nanocrystals were mixed with excess isopropanol and washed by centrifugation.Lastly, the PLNPs nanocrystals were dispersed into ultrapure water and purified by centrifugation.The white precipitate was dried in a vacuum oven at 60 � C for 12 h and milled in agate mortar.The concentration of stored PLNPs was approximately 1 mg/mL.

Optimization of preparation conditions
To optimize the conditions for the preparation of PLNPs, the Zn/Ga ratio, the quantity of Cr 3þ doping, and the pH were varied.The photoluminescence spectra of the solid powders and the afterglow decay curves were measured with excitation at 254 nm.

Handling of actual samples
The human serum was obtained from healthy male volunteers at the University Hospital.All human serum experiments were approved by the Ethics Committee of Shanxi Normal University in accordance with the requirements of the Chinese National Statement on Ethical Conduct in Human Research.The serum samples were diluted 100-fold to remove interferences.
The milk samples obtained from a local market were preprocessed as previously reported (Sa-nguanprang, Phuruangrat, and Bunkoed 2022).10.0 mL of milk were centrifuged at 5000 rpm for 20 min to eliminate fat and 10.0 mL of acetonitrile were introduced and centrifuged at 5000 rpm for 10 min to remove proteins.The supernatant was collected and dried under vacuum at 50 � C and the residue dissolved in 10.0 mL of ultrapure water.The samples were spiked with tryptophan to characterize the recovery.The experiments were performed in triplicate and reported as the average.

Preparation and characterization of PLNPs
The Zn 0.8 Ga 2 O 4 : Cr 3þ PLNPs were prepared by a hydrothermal method and the conditions optimized (Figure S1).The results show that the photoluminescence excitation and emission spectra were the highest and the afterglow decay optimum for Zn/Ga ¼ 0.8/2, doping with 0.004 mmol Cr 3þ , and a pH of 9.5.The PLNPs nanoparticles were confirmed to have good crystallinity under hydrothermal conditions as indicated by narrow and sharp diffraction peaks (Figure 1a), demonstrating the purity and crystallinity of PLNPs (JCPDS No. 86-0415) (Zhou et al. 2017).The surface was demonstrated to include Zn, Ga, Cr, and O by X-ray photoelectron spectroscopy (XPS) (Figure 1b).The PLNPs were shown to have a narrow size distribution, uniform dispersion and homogeneous diameter by TEM (Figure 1c).The infrared spectrum of the PLNPs is shown in Figure 1d.
The emission spectra of PLNPs at different excitation wavelengths show the best response was obtained from 240 to 260 nm (Figure 2a).The excitation spectrum of PLNPs was centered at 254 nm and the emission peak at 694 nm.Luminescence images of PLNPs powders in the absence and presence of excitation are shown in the insets of Figure 2b.The photoluminescence of PLNPs lasted for more than 600 s following excitation for 5 min, demonstrating sustained luminescence (Figure 2c).The photoluminescence excitation and emission spectra with the afterglow decay curve are shown in Figure 3a, c.The excitation and emission spectra with the afterglow decay curve of PLNPs after 20 days are displayed in Figure 3b, d.The optical properties of PLNPs were basically unchanged after 20 days, which demonstrated suitable stability.

Design and principle of label-free photoluminescent sensor
Label-free photoluminescent sensors based on PLNPs were developed for the determination of tryptophan (Scheme 1).To minimize autofluorescence encountered in conventional fluorescence, PLNPs were used as a light source for tryptophan without external excitation.The absorption spectrum of tryptophan overlapped considerably with the photoluminescence excitation spectrum of the PLNP dispersions (Figure 4).Therefore, the luminescence of PLNPs was quenched by tryptophan.

Optimization of the conditions
The reaction time and pH were optimized to maximize the sensitivity.The influence of pH upon the luminescence quenching of PLNPs-Trp was investigated using Britton-Robison buffer at pH values between 3.0 and 12.0 (Figure S2a) and Tris-HCl buffer at pH values from 3.0 to 10.0 (Figure S2b).
The optimum response was obtained at pH 6.0 which was used in subsequent conditions with the Tris-HCl buffer.The influence of time on the PLNPs-Trp system at pH 6.0 demonstrated no significant change in quenching with the reaction time (Figure S2c).Hence, 5 min was selected to be optimum.

Detection of Trp by PLNPs
Under the optimum conditions, various concentrations of tryptophan were sequentially added to the PLNPs to characterize the sensitivity.The emission intensity decreased with the tryptophan concentration from 4 to 240 mM (Figure 5).The linear relationship was described by F/F 0 ¼ 0.9416 − 0.0024 [Trp] with a correlation coefficient of 0.9911.The limit of detection determined by 3r/j, where j is the slope of the calibration relationship and r the standard deviation of 11 blanks, was 0.19 mM.

Selectivity of the PLNPs for tryptophan
To characterize the selectivity and interference resistance, blanks were analyzed to exclude luminescence quenching caused by environmental interferences and the substances that may be present in milk and serum.These include 240 mM tryptophan and 240 mM L-isoleucine, L-arginine, L-aspartic acid, L-leucine, L-alanine, DL-valine, D-cysteine, L-phenylalanine, L-tyrosine, Na þ , Mn 2þ , NH 4 þ , and Ba 2þ (Figure 6a).Following the addition of inorganic ions and amino acids, the luminescence intensity of PLNPs did not significantly change.The determination of tryptophan by PLNPs was unaffected by the presence of several interferences (Figure 6b).The results demonstrate that the persistent luminescence nanoparticles had stable optical properties, excellent selectivity, and suitable anti-interference properties.

Determination of tryptophan in serum and milk
To verify the applicability of the developed label-free luminescence sensor, tryptophan was determined in spiked human serum and milk using the method of standard  addition as described in the experimental section.The results in Table 1 show recoveries from 97.9 to 101.9% and relative standard deviations between 1.17 and 3.51%, illustrating good accuracy and precision.Hence, the persistent luminescent nanoplatform was suitable for the determination of tryptophan in biological samples.
The method in this article was compared with the literature for tryptophan.Table S1 shows the developed protocol offers a wide linear range and a low detection limit.In addition, this scheme is simple, environmentally friendly, and provides a promising strategy for tryptophan determination in biological samples.

Conclusions
A label-free photoluminescence sensing platform based on persistent luminescence is reported for the sensitive and selective determination of tryptophan.The analyte was determined in milk and serum by the developed approach without interference from autofluorescence.This work provided a promising optical strategy for the determination of amino acids in biological samples without background interferences and scattered light generated by in situ excitation.

Acknowledgement
The authors are grateful to the Analysis and Testing Center of Shanxi Normal University.

Figure 2 .
Figure 2. (a) Emission spectra of 1 mg/mL PLNPs as a function of excitation wavelength.(b) Excitation and emission spectra of 20 mg PLNP powder.Inset: luminescence images of the PLNPs in the absence and presence of ultraviolet excitation.(c) Afterglow decay curve of 20 mg PLNPs at 694 nm after 5 min of excitation at 254 nm.

Figure 3 .
Figure 3. (a) Excitation and emission spectra of 20 mg PLNPs.Inset: luminescence photograph of the PLNPs.(b) Afterglow decay curves of 20 mg PLNPs.(c) Excitation and emission spectra of 20 mg PLNPs after 20 days.Inset: luminescence photograph of 20 mg PLNPs after 20 days.(d) Afterglow decay curves of PLNPs after 20 days.Excitation was performed at 254 nm and emission at 694 nm.

Figure 5 .
Figure 5. Variation of emission of PLNPs with the tryptophan concentration.

Table 1 .
Recovery of tryptophan in human serum and milk.