Glutathione-Capped ZnS Quantum Dots-Urease Conjugate as a Highly Sensitive Urea Probe

Quantum dots (QDs) possess characteristic chemical and optical features. In this light, ZnS QDs capped with glutathione (GSH) were synthesized via an easy aqueous co-precipitation technique. Fabricated QDs were characterized in terms of X-ray diffraction (XRD), high resolution transmission electron microscope (HRTEM), Fourier transform infrared (FTIR) and Zeta potential analyses. Optical properties were examined using photoluminescence (PL) and ultraviolet–visible (UV–visible) spectroscopies. Moreover, GSH-capped ZnS QDs were evaluated as an optical probe for non-enzymatic detection of urea depending on the quenching of PL intensity of ZnS QDs in the presence of urea from concentration range of 0.5–5 mM with a correlation coefficient (R2) of 0.995, sensitivity of 0.0875 mM−1 and LOD of 0.426 mM. Furthermore, GSH-capped ZnS QDs-urease conjugate was utilized as an optical probe for enzymatic detection of urea in the range from 1.0 µM to 5.0 mM. Interestingly, it was observed that urea has a good affinity towards ZnS QDs-urease conjugate with a linear relationship between the change of PL intensity and urea concentration. It was found that R2 is 0.997 with a sensitivity of 0.042 mM−1 for mM concentration (0.5–5 mM) and LOD of 0.401 mM. In case of µM concentration range (1–100 µM), R2 was 0.971 with a sensitivity of 0.0024 µM−1 and LOD of 0.687 µM. These data suggest that enzyme conjugation to capped QDs might improve their sensitivity and applicability.


Introduction
Quantum dots (QDs) are semiconductor nanosized materials in the range from 1 to 10 nm. They exhibit unique electronic, chemical and optical features [1,2]. Owing to the tiny size of these semiconducting QDs, they can produce high photoluminescence quantum yield when compared to their bulk materials [3]. Moreover, they can resist photobleaching for long time, and hence can be included in the design of optical biosensors [4][5][6], photovoltaic [7,8], bioimaging [9] and light emitting diode [10]. The most commonly utilized types of QDs are composed of semiconductors of periodic group II-VI (CdTe, CdSe, CdS, ZnSe, ZnS, PbS, PbSe, PbTe, SnTe) [11]. ZnS is an II-VI compound semiconductor with a wide direct band gap (3.72 eV at room temperature), wide wavelength pass band and high refractive index [12]. Moreover, it is the cutting-edge QDs which has a large surface to volume ratio, high exciton binding energy and Bohr exciton radius, wide bandgap and quantum confinement effect [12]. In addition, ZnS is non-toxic chemically stable and environmentally safe compound [13].
For successful involvement of QDs for sensing purposes in biological system, they should be in an aqueous or colloidal medium. QDs can be prepared in a hydrophobic form followed by a consecutive solubilization step or they can be directly prepared in an aqueous media. Direct aqueous preparation method compared to other preparation methods is easy, reproducible and less toxic. Furthermore, this method usually produces QDs highly water soluble, biologically compatible and stable [14]. In order to maintain the luminescence of QDs for good sensing performance, the semiconductor core material should be protected from oxidation and degradation [14]. The main approach by which QDs can be prepared with the required features is their surface functionalization or capping with some compatible biomolecules [15].
Many biocompatible molecules can be used to functionalize QDs. Among these molecules is reduced glutathione which is composed of three amino acids including; L-glutamate, L-cysteine and glycine [16]. It can serve as a capping agent via its thiol group, besides comprising two carboxylic and one amino group which can be further utilized to couple other sensing biomolecules [14]. GSHcapped QDs were previously utilized to determine heavy metals depending on quenching of the fluorescence of QDs in an amount proportional to the concentration of heavy metals. In a similar manner, glucose concentration was determined depending on quenching in the fluorescence of GSH-capped QDs in presence of H 2 O 2 produced from the catalytic reaction of glucose oxidase [17].
The similar dimensions of QDs with some important biomacromolecules such as proteins and nucleic acids opened the door to conjugate QDs with different biomolecules [18]. Conjugation of QDs with versatile biomolecules including enzymes is an essential step that might enhance utilizing nanomaterials in the pharmaceutical and medical sectors. These recognition molecules usually enhance the sensitivity and selectivity of the conjugated QDs to specially recognize certain molecules [19]. In biosensing, molecular recognition is an essential requirement based on high affinity between complementary components such as antibody/antigen, receptor/hormone or enzyme/substrate. This high recognition property is usually included in the design of multiple biosensors based on the production of signals proportional to the concentration of analytes under investigation [20]. Semiconductor QDs can act as transducer for biosensors to detect urea, creatinine, glucose, cysteine and ascorbic acid [21,22].
Enzyme labeling of QDs surface might alter their surface properties leading to a modulation in their optical properties [23,24]. Enzyme reactions catalyzing proton transfer causes a change in the pH of the proximity area around the enzyme only without affecting the entire solution pH resulting in poor signal transfer. As an alternative, a pH sensitive QDs can be conjugated to an enzyme to maximize the signal via taking the privilege of both characteristic QDs' PL and specific enzymatic reactions [25].
Urea as an analyte to be examined is of clinical importance as an essential parameter in diagnosing renal or hepatic disorders. It is a final metabolic waste, along with uric acid and creatinine produced from the catabolism of nitrogen-containing compounds such as protein in mammals. Moreover, urea level usually gives information about the nutritional status of the body and many pathogens infecting the gastrointestinal or urinary tracts which can produce urea, and hence it could be utilized to detect the presence of some pathogenic bacteria [25]. Various procedures have been reported for the determination of urea in tissue or body fluids including; ion chromatography, electrochemical and enzymatic assays [26]. Urea can also be enzymatically detected based on its hydrolysis in the presence of urease yielding ionic products (NH 4 + and HCO 3 − ) which can be further determined by various techniques such as voltammetry, potentiometry, conductance or spectrophotometry. However, all these methods have limitations because of the buffering effect of the bulk sample [27].
In this study, GSH-capped ZnS QDs are conjugated to urease via carbodiimide coupling reaction which is the most common method involved in covalent conjugation. Carbodiimide molecules first activate the carboxylic groups of GSH on the surface of ZnS QDs to form acyliosurea active intermediate that will be then replaced with the primary amine groups of the enzyme to form an amide bond between ZnS QDs and urease ( Fig. 1) [19]. Afterwards, the physicochemical properties of the prepared conjugate will be examined, then GSH-capped ZnS QDs-urease conjugate will be utilized as an optical probe for enzymatic detection of urea based on the quenching of PL intensity of enzyme-conjugated QDs in a wide range of urea from 1.0 µM to 5 mM.

Synthesis of GSH-capped ZnS QDs
GSH-capped ZnS QDs was prepared by facile co-precipitation method. First, 10 mL of 0.1 M Zn(Ac) 2 .2H 2 O was added to 10 mL of 0.1 M R-glutathione, followed by pH adjustment to 10.5 by 0.1 M NaOH till complete clearance of turbidity from the solution. The solution was stirred for 30 min under nitrogen protection, then10 mL of 0.1 M Na 2 S was immediately injected into the solution and left under stirring for another 20 min. The solution was heated for 2 h at 100 ℃ to form GSH-capped ZnS QDs. Finally, ZnS QDs were finally precipitated by adding ethanol: QDs solution at volume ratio 2:1 and centrifugation at 7000 rpm for 20 min (Focus instrument, Spain), followed by washing twice and drying in a vacuum oven (Binder, Bohemia, USA) at 40 ̊ C O/N to obtain white fine powder.

Preparation of GSH-capped ZnS QDs-urease conjugate
GSH-capped ZnS QDs-urease conjugate was fabricated by carbodiimide coupling reaction via formation of amide bond between activated carboxylic groups of QDs and the primary amines of urease [28]. In brief, GSH-capped ZnS QDs (10.0 mg) were dissolved in 9.0 mL phosphate buffer (pH7.4, 0.01 M). Afterwards, EDC (2.0 mg, 1.0 mM) and NHS (2.17 mg, 1.0 mM) were added one after the other, then they were left for 30 min to allow activation of carboxylic groups located on the surface of the ZnS QDs. After that, 1.0 mg of freshly prepared urease solution (1.0 mg/mL) was added to reach final reaction volume of 10.0 mL, then this reaction mixture was left under mild magnetic stirring for 3.0 h to allow the conjugation reaction to proceed at room temperature. Finally, the obtained solution was centrifuged at 6000 rpm and the obtained pellet was collected and dried in a vacuum oven at 40 °C O/N and stored at 4 °C for further characterization and analyses experiments [29]. The pH of the reaction was selected to be 7.4 as this pH is the optimum one for urease [30]. On the other hand, the conjugate was also prepared by electrostatic interaction by mixing 10.0 mg ZnS QDs with 1.0 mg urease in 10 mL phosphate buffer (pH7.4, 0.01 M), then they were allowed to self-assemble overnight at 5 °C. The obtained complex was also separated by centrifugation as previously mentioned. All the prepared formulations were examined in terms of their PL values and the formula with the highest PL value was selected for the upcoming examinations [25].

Physicochemical characterization of GSH-capped ZnS QDs-urease conjugate
Fourier-transform infrared spectroscopy analysis was conducted for prepared samples in order to study their structure. Each sample was grinded with KBr powder (1:100) then, the spectrum of each sample was detected in the range of 4000-400 cm −1 using Spectrum BX11 Infrared spectrometer (FTIR LX 18-5255 Perkin Elmer). X-ray diffraction (XRD) analysis was performed using powder X-ray diffractometry ((X-ray 7000 Shimadzu, Japan). Scanning of diffraction patterns were conducted from 10 to 80 degree using copper characteristic X-ray wavelength of 1.54 Å with scanning rate of 10 scan /min. Surface charge or Zeta potential of GSH capped-ZnS QDs and GSH capped-ZnS QDs-urease conjugate were determined by NanoZS/ZEN3600 Zetasizer (Malvern Instruments Ltd., UK). Aqueous solutions of the assigned samples were placed in a universal folded capillary cell equipped with platinum electrodes. The Zeta potential values were determined from the mean electrophoretic mobility by Laser Doppler Anemometry (using a Zetasizer Malvern Nano-ZS). Morphological examination was conducted using JEOL JEM 2100F HRTEM microscope at an accelerating voltage of 200 kV. Samples were prepared for visualization by dispersing the dried powder in ethanol and allowing a drop to dry for 30 s on carbon coated fine copper grid (3 mm in diameter) prior to visualization. For studying the spectral absorbance properties of the prepared formulations, 100 µL of each sample was diluted with 3 mL deionized water and then scanned in the range from 200 to 900 nm using UV-visible spectrometer (Thermo scientific evolution 300-USA). The emission spectra of the prepared samples were studied using Perkin Elmer LS 55 fluorescence spectrophotometer. All the measurements were run in triplicates at room temperature. Both excitation and emission slits were fixed at 10 nm.

Detection of urea
For non-enzymatic detection of urea, 10 mM stock solution of urea was prepared by dissolving 60.06 mg in 100 mL deionized water, then 500 µL of GSH-ZnS QDs were added to 1.0 mL of phosphate buffer ( pH 7.1) containing different urea concentrations (0.5-5 mM) and incubated for 10-40 min at 25 °C. To examine the selectivity of the prepared QDs to detect urea, similar experiments were performed using different analytes to act as interfering agents for urea. The interfering analytes were glucose, ascorbic acid, cholic acid and dopamine with a concentration of 2.0 mM. For enzymatic detection of urea, the same procedure was followed, except that GSH-ZnS QDs-urease conjugate was included instead of GSH-ZnS QDs.
For optimizing analytical parameters, the slope of calibration curve was used based on Eq. [1] [31].
where F° and F represent PL intensity of QDs in the absence and presence of the bioanalyte molecule, C is the concentration of the bioanalyte molecule. The slope (sensitivity) and intercept of the calibration curve are represented by a and b, respectively. The quenching efficiency (QE) was calculated using Eq. [2] [32].
The limit of detection (LOD) of the bioanalyte molecule was estimated from the following Eq. [3] [31,33]: where SD and s are standard deviation and slope, respectively.

Structural Properties of QDs
FTIR spectra of pure GSH, GSH-ZnS QDs and GSH-ZnS QDs-urease conjugate exhibit a broad peak at 3000-3500 cm −1 which is assigned to O-H or N-H indicating the presence of amino and hydroxyl groups on the surface of the ZnS QDs. This confirms that -COOH and NH 2 groups are not involved in any chemical bonding with ZnS QDs ( Fig. 2A). The peak at 1550 −1400 cm −1 is attributed to carboxylate group's ν s (-COO − ). The strong peak at 1600-1650 cm −1 corresponds to ν asy N-H bending vibration, whereas the peak at 1380 cm −1 is related to ν C-N groups. The vibration stretching peak at 1230 cm −1 is due to ν s (C-O) while vibration stretching peak at 1000-1100 cm −1 is assigned to (-C-OH) peak. The peak at 630 cm −1 stretching vibration of Zn-S can be explained based on the interaction between ZnS QDs and capping agent via thiol group [33][34][35][36][37]. It is observed that the vibration stretching peak of S-H (2520 cm −1 ) of pure GSH disappears which might confirm successful interaction between thiols group of GSH and the surface of ZnS QDs. For GSH-ZnSurease conjugate, it is noted that the intensity of peak (OH/ NH) at 3500 cm −1 increases with slight shifting for peaks at 1644 cm −1 in comparison to pure urease (1650 cm −1 ) and GSH-ZnS (1612 cm −1 ) and appearance of a new broad peak at 1050 cm −1 is corresponding to C-O. A peak at 1644 cm −1 appeared in the spectrum of free urease and GSH-ZnS QDsurease conjugate is related to the N-H bend of peptide bond. The band at 2900 cm −1 is due to the C-H stretching vibrations of -CH 2 -groups, while the broad peak at about 3345 cm −1 might be attributed to the -OH groups on the surface of QDs or -NH 2 groups of urease molecules. The slight shifting of some peaks and increasing the intensity of other peaks could be related to conjugation between urease and ZnS QDs.

Crystallinity of GSH-ZnS QDs and GSH-ZnS QDs-urease Conjugate
The crystal structure of GSH-capped ZnS QDs and GSHcapped ZnS-urease conjugation (GSH-ZnS-urease) were identified via XRD. As depicted in Fig. 2B and listed in where n is integer number, λ is the wavelength of X-ray radiation, and θ is Bragg's diffraction angle. As listed in Table 1, the d-spacings for both samples calculated at (111) diffraction plane are slightly lower than the standard database [38]. It is observed that the percentages of contraction in the d-spacing are 0.28 and 0.48% for the GSH-ZnS-QDs and GSH-ZnS-urease conjugation samples, respectively. Similarly, the lattice parameter "a" of the face-centered cubic unit cell for both samples is estimated through Eq. [6] [40]: where d hkl is the interlayer spacing (d-spacing) between atoms and hkl are the miller indices of the atomic planes.
(5) n = 2d hkl sin( ) The lattice parameter "a" is lower than the standard database for both samples, as shown in Table 1 [38]. Consequently, the reduction in the lattice parameter "a" results in a compressive microstrain (ε) through the structure. The microstrain is calculated from the differences in the lattice parameters as compared to the standard database [38]. where β hkl ( 2θ ) is full width at half maximum (FWHM), K is shape factor of 0.9, and λ is the wavelength of X-ray. As listed in Table 1, the mean crystallite size of the GSH-ZnS-QDs sample, which corresponds to the most intense peak (111) is 2.6 nm and the average crystallite size is 2.43 ± 0.69 nm. On the other hand, the mean crystallite size of the GSH-capped ZnS-urease conjugate is reduced to 1.33 nm and the average crystallite size is also declined to 1.57 ± 0.35 nm.
Interestingly, D values can be also calculated by applying

Williamson-Hall (W-H) expression [Eq. 8] [42]:
where is the microstrain fluctuations in the crystal. As shown in Fig. 2C, D, the D values were extracted from the

Morphological Analysis
It is observed that the diameter of GSH-ZnS QDs is 5.59 ± 1.56 nm with well-resolved lattice fringes having an interplanar spacings of about 0.27 nm (Fig. 3). SAED pattern is in accordance with the data obtained using XRD analysis indicating the crystalline nature of the synthesized ZnS QDs [44][45][46]. GSH-ZnS QDs-urease conjugate has an

Optical and Surface Charge Properties
GHS-capped ZnS QDs display a predominant peak at 280 nm due to transition of electrons from valance band to conduction band while GSH-capped ZnS QDs-urease conjugate show a characterized peak at 278 nm with higher intensity and slight shift owing to the conjugation between QDs and the enzyme (Fig. 4A). These sharp optical absorption edges and well defined excitonic features indicate that the synthesized ZnS QDs have a relatively narrow size distribution [36,47]. The optical direct band gap (Eg) value of QDs was determined according to Tauc's plot relation (αhν = α o (hν-Eg.) n ), where (hν) and (α o ) are photon energy and a constant, respectively [48]. The values of Eg   [46,47,49].
Zeta potential of GSH-capped ZnS QDs is found to be of about −24 mV, whereas GSH-capped ZnS QDs-urease conjugate's zeta potential is −17.9 mV (Fig. 4C, D). This relative decrease in the value of zeta potential after enzyme conjugation might be due to involvement of the carboxylic groups on the surface of QDs in the formation of amide bond with primary amine groups of urease, confirming successful conjugation [48]. Furthermore, this decline in zeta potential might be also related to the presence of urease enzyme on the surface of the QDs with its isoelectric point of about 5.0-5.2 urase which is nearly acidic and may aid in the decrease in the zeta potential value [50].
To examine the emission properties of the fabricated ZnS QDs, PL spectra of GSH-capped ZnS QDs and GSH-capped ZnS QDs-urease conjugate are conducted at room temperature and illustrated in Fig. 5. PL spectra of GSH-capped ZnS QDs at different excitation wavelengths from 280 to 350 nm are shown in Fig. 5A, in which GSH-capped ZnS QDs display an excitation independent emission at 430 nm.
At an excitation wavelength of 330 nm, the fluorescence intensity is the highest. The independence of the emission of GSH-capped ZnS QDs could be related to the passivating of surface of ZnS QDs by glutathione. In addition, the reduction of the surface defects via non-radiative recombination of electrons and holes could further enhance this mechanism [51,52]. As displayed in Fig. 5B, there is a reduction in the PL spectra of GSH-capped ZnS QDs-urease conjugate in comparison to GSH-capped ZnS QDs due to conjugation of the enzyme on the surface of the QDs.

Detection of Urea
The large surface area of QDs is beneficial for covalent conjugation of some macromolecules for specific biorecognition, such as antibodies, peptides, nucleic acids or small molecules, for different applications as fluorescent biosensors [48,53]. According to the optical properties of GSHcapped ZnS QDs, it can be used as a sensor for urea. The effect of incubation time of urea with GSH-capped ZnS QDs on PL emission is illustrated in Fig. 5C. Results reveal that the reaction between them occurred rapidly within 10 min, in which a notable quenching in the PL intensity is observed. After 20-30 min, the PL spectra are slightly decreased, and hence, the incubation time of 15 min is selected for all the upcoming experiments [48]. As shown in Fig. 5D, the PL intensity of GSH-capped ZnS QDs decreases with increasing the urea concentration. In addition, it is observed that the urea has a good affinity towards ZnS QDs with a linear relationship between the change of PL intensity (quantum efficiency, QE) versus urea concentration. The correlation coefficient (R 2 ) is found to be 0.9956 with a sensitivity of 0.0875 mM −1 and limit of detection of 0.427 mM as estimated from the linear equation of calibration curve (Inset in Fig. 5D). This fluorescence quenching might be due to energy or electron transfer, in which the quenching of a fluorophore may occur from Forster resonance energy transfer (FRET), photoinduced electron transfer (PET), static or dynamic quenching and inner filter effect phenomena. FRET is possible when the UV-visible spectrum of biomolecules overlaps with the emission spectrum of the fluorophore [54]. However, the emission spectrum of GSH-ZnS QDs do not overlap with the absorption spectrum of the biomolecules. This reveals that FRET cannot be suggested fluorescence quenching mechanism. There is no spectral shift observed for GSH-capped ZnS QDs upon urea addition, and hence, the aggregation induced quenching (self-quenching) is also negligible. Consequently, the fluorescence quenching of GSH-capped ZnS QDs could be mainly because of the photoinduced electron transfer between the biomolecule and electron-donating GSH-capped ZnS QDs. Subsequent addition of electron deficient biomolecules to electron-rich GHS-capped ZnS QDs, the net charge separation between GSH-capped ZnS QDs and the biomolecules is expected to reduce. As a result, the fluorescence intensity of GSHcapped ZnS QDs will decrease through excited state electron transfer to the biomolecules [55].
PL spectra of GSH-capped ZnS QDs-urease conjugate are examined with different urea concentrations in two concentration ranges from 1 to 100 µM and from 0.5 to 5 mM at pH 7 and 15 min incubation time. As shown in Fig. 6, PL intensity of GSH-capped ZnS QDs decreases with increasing urea concentration. From results illustrated in Fig. 6A, it is noticed that urea has good affinity towards GSH-ZnS QDs urease conjugate with a linear relationship between the change of PL intensity (QE) and urea in the concentration range from 0.5 to 5 mM. R 2 was 0.9967 with a sensitivity of 0.042 mM −1 and LOD of 0.401 mM for the mM concentration range. For µM concentration range (1-100 µM), R 2 is 0.971 with a sensitivity of 0.0024 µM −1 and LOD of 0.687 µM, which are calculated from the linear equation of the calibration curve (Fig. 6B).
Moreover, when examining the efficiency of the produced probe in different pH values, it is observed that pH 7 produces the best results due to enhanced interaction between urea and GSH-capped ZnS QDs (Fig. 7A) [48]. The pH value is an important parameter which has a significant effect on immobilization of urease and the activity of the produced biosensor. The enzymatic reaction between urea and urease occurs in a two-step mechanism. First, urease reacts with urea to produce ammonia and carbamate, then in the second step, the carbamate hydrolyzes to ammonia and carbonic acid [46,[56][57][58].NH 2 CONH 2 + 3H 2 O → 2NH + 4 + CO 2 + OH − As a result, increasing the concentration of urea causes an increase in pH from 7.64 in case of pure conjugate to 8.03 when incubated with 0.5 mM urea and to 9.61 when incubated with 5 mM urea and these results in a decline in the PL intensity.

Selectivity and Interference of GSH-capped ZnS QDs-urease Conjugate Towards Urea Detection
Along with high sensitivity requirement, high specificity is also crucial especially in real sample detection. To verify the performance of GSH-capped ZnS QDs as a urea biosensor, its selectivity towards urea in the presence of some other biomolecules including; glucose, ascorbic acid, dopamine and cholic acid is investigated. Each bioanalyte is examined at concentration of 2.0 mM, pH 7 and 15 min incubation time. The quenching effect of urea is found to be the most superior (39%) in comparison to glucose, ascorbic acid,  [46,[55][56][57][58][59][60]]. Among all the tested biomolecules, urea reveals the highest quenching efficiency which could be attributed to adsorption of urea on the surface of GSH-capped ZnS QDs (Fig. 7B) [57]. The specific selectivity toward urea for the developed sensor is examined by comparing its response to interfering species commonly found in biological samples [61] such as glucose (2 mM), cholesterol (0.5 mM) and a mixture of them. As shown in Fig. 7C, the PL spectra for these interfering species do not show a considerable response compared to that of urea. As a result, it could be concluded that the prepared optical probe is selective towards urea sensing in the existence of other interfering species. This enhanced selectivity could be attributed to covalent conjugation of urease to GSH-capped ZnS QDs, suggesting the possible feasibility of this biosensor to selectively detect urea in real human plasma samples [62,63].

Application of GSH-capped ZnS QDs-urease Conjugate as Urea Biosensor in Biological Media
To examine the efficiency of the prepared GSH-capped ZnS QDs-urease conjugate to determine urea in biological fluids, Dulbecco's Modified Eagle's Medium is utilized as a model to mimic biological fluids since it includes high concentrations of most cellular components such as glucose, vitamins, amino acids, inorganic salts and other cellular components.
DMEM medium is spiked with known concentrations of urea (0.5-5 mM). For each urea concentration, it is measured with GHS-capped ZnS QDs-urease conjugate. Results displayed in Fig. 7D indicate that the PL intensity is decreased when concentration of urea increased, which is in accordance with the assigned calibration curve of urea in the mM concentration range, indicating that conjugation of urease to the surface of GSH-capped ZnS QDs improves its selectivity towards urea biosensing [27].

Conclusion
In this study, a sensitive probe based on GSH-capped ZnS QDs-urease conjugate was prepared by co-precipitation and conjugation methods for urea detection. FTIR results confirmed the interaction between thiol group (S-H), surface of ZnS QDs and GSH-capped ZnS QDs-urease via covalent bonding. GSH-capped ZnS QDs were utilized as an optical probe for non-enzymatic urea detection based on the quenching of PL intensity of GSH-capped QDs in the presence of urea in the concentration range of 0.-5 to 5 mM with R 2 of 0.9956, sensitivity of 0.0875 mM −1 and LOD of 0.426 mM.
In addition, GSH-capped ZnS QDs-urease conjugate was used as an optical probe for enzymatic detection of urea based on the quenching of PL intensity of enzyme-conjugated QDs in urea concentration range from 1 µM to 5 mM. Interestingly, it was observed that urea has good affinity towards GSH-capped ZnS QDs-urease conjugate with a linear relationship between the change of PL intensity and urea concentration. R 2 was 0.9967 with sensitivity of 0.042 mM −1 for mM concentration (0.5-5 mM) and LOD of 0.401 mM. In case of µM concentration range (1-100 µM), the R 2 was 0.971 with sensitivity of 0.0024 µM −1 and LOD of 0.687 µM.
Authors Contribution All the authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by WM, EF, MS, SE and SAS. The first draft of the manuscript was written by WM, EF and SAS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Funding Declaration
The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

Conflict of Interest
Authors have no relevant financial or non-financial interests to disclose.
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