INTRODUCTION

Urea, which is an end product of protein metabolism and the main nitrogen component of urine, is an important biomarker monitored in blood and urine samples to diagnose renal and liver diseases. Urea concentrations above the normal level can be an indication of renal failure, urinary tract obstruction, and gastrointestinal bleeding.^1,2 Conversely, low urea concentrations may be observed in hepatic failure, nephritic syndrome, and cachexia. Therefore, it is essential to develop techniques for the determination of urea in blood. Conventional spectroscopic methods have been used for many years in clinical laboratories for the determination of urea in blood samples.^3,4,5 However, these methods are time consuming due to sample pretreatment and an unsuitable real-time determination of urea. Urease-based biosensors are alternative methods for the determination of urea levels.⁶ To this end, a number of methods have been developed and reported such as potentiometry^7,8,9, voltammetry^10,11, conductometry^12,13,14, ion selective electrode¹⁵, and spectrometry.^16,17

Quantum dots (QDs) are superior to organic dyes with their size-tunable photonic properties, quantum yield, and stability against photobleaching.^18,19,20,21 The luminescence properties of QDs are highly sensitive to changes on their surface. The majority of QD photoluminescent probes are based on the increasing or quenching of the photoluminescence signal, which is caused by chemical or physical interaction. Thus the selective determination of an analyte can be achieved via interaction with functionalized QD or non-functionalized QD.^22,23,24,25

QDs have been widely used as biosensors in biotechnology.^24,25,26,27 Recently, a few applications of QDs for the determination of urea appeared in the literature.^6,17,24 All of these studies are based on the pH change upon the reaction of urea and urease. Although most previous studies have been focused on QDs as a fluorescence sensor, their long lifetime allows the use of the phosphorescence mode, which has more advantages than fluorescence; for example, the spectral interferences from biologic matrices can be easily prevented in the phosphorescence mode.^6,28,29

This study describes a simple and reliable analytical method for the determination of urea in biologic samples using 3-mercaptopropionic acid (MPA)-capped cadmium telluride (CdTe) QDs. The urea concentration was determined in serum samples by monitoring the increase in phosphorescence signal in the presence of urease.

EXPERIMENTAL

Materials

All the measurements were performed using analytical grade chemicals. Deionized water was used throughout the study. CdCl₂, H₂TeO₆, NaBH₄, NaOH, HCl, and urease were obtained from Merck, and the MPA was obtained from Fluka. Trisodium citrate was obtained from Riedel de Haen and ethanol was obtained from Sigma-Aldrich. Dilute solutions of the QD, urease, and urea were prepared daily.

Apparatus

A Varian, Cary Eclipse Luminescence spectrometer equipped with a xenon lamp was used for photoluminescence measurements. All instrumental parameters were controlled using the instrument software. The automatic filter selection mode for both excitation and emission monochromator was used to avoid scattered light. Excitation and emission spectral band passes were 20 nm for both monochromator. The detector voltage was set to 800 V. An Orion 720. A model pH/Ionmeter was used for pH adjustments. Deionized water with 18.2 MΩ.cm was obtained from Millipore Simplicity water purification system. A Nüve NF200 centrifuge and Nüve MK418 magnetic stirrer were used during the synthesis of the QDs. Unicam Mattson 1000 Fourier transform infrared (FTIR) spectrometer was used to obtain IR spectra of the modified QDs to confirm surface modification. The ultraviolet (UV) spectrum of QDs was obtained using a Shimadzu UV-visible (VIS) spectrometer. Transmission electron microscopy (TEM) measurements were performed on a JEOL 2100 HRTEM instrument (JEOL Ltd., Tokyo, Japan). TEM samples were prepared by pipetting 10 µL of QD solution onto copper grids, which were allowed to stand for 10 min.

Synthesis of water-soluble MPA- CdTe QDs

MPA-CdTe QDs were synthesized using a modified method of Yuan et al.³⁰ In the method, 25 mL 0.64 mM CdCl₂ solution and 0.10 g of trisodium citrate was transferred into a single-necked flask. Then, 100 µL, 11.5 M MPA and 0.01 mmol Te (IV) were added respectively, with continuous stirring. The color of the solution becomes bright yellow with the addition of 50 mg of NaBH₄, and it is heated to 90°C for 1 hour with continuous stirring. After cooling, QDs were precipitated with ethanol, centrifuged and dried in vacuum. Each batch resulted in 80-100 mg of dry QD powder, and in order to have a constant QD concentration, a 75-mg portion of the QD powder was re-dissolved in water and diluted to 25 mL volume. At this stage, the pH of the QD solution was adjusted to 11.4 and heated to 96-100°C for a different period of time. An increase in particle size as well as fluorescence emission at longer wavelength was observed.

Procedure for urea determination

Ten milliliters of 3.0 mg/mL MPA-CdTe and 10 mL of 5 units/mL urease solutions were placed into a beaker and the pH was adjusted to 2.5 with 0.01 M HCl. The solution was transferred to a 25-mL volumetric flask and diluted to volume with deionized water. A series of standard solutions was prepared by transferring 1.0 mL of the mixture solution into a test tube and then various volumes of urea standard solution or 0.10 mL of serum samples were added. The volume was completed to 5.0 mL with deionized water. The solutions were mixed and allowed to stand for 10 min at laboratory temperatures. Measurements were performed using the phosphorescence mode with a 0.1 ms delay time and 3 ms gate time. Excitation wavelength was 300 nm and spectral band passes were 20 nm for both excitation and emission monochromators.

Samples

Human serum samples were collected from healthy volunteers. The samples were diluted 50 times with deionized water adjusted to pH 2 with 0.01 M HCl before the measurement procedure. The same samples were analyzed for urea using standard methods used in clinical laboratory in order to test the accuracy of the proposed method.

RESULTS AND DISCUSSION

Characterization of MPA-CdTe QDs

The QDs were characterized using fluorescence, UV-VIS, infrared spectroscopy, and TEM images. After the synthesis procedure (without thermal pretreatment), the QDs had fluorescence emission maximum at 505-510 nm with a full width at half maximum about 35 nm, and almost no phosphorescence signal, as shown in Figure 1. On the other hand, when this QD was heated to 90-100°C (pH 11.4) for different periods of time, an increase in particle size as well as fluorescence and phosphorescence emission intensity was observed. Therefore, the heating period of three hours, which provided intense phosphorescence signal at longer wavelength, was selected to avoid fluorescence background emission from the biologic sample. The phosphorescence spectra of MPA-capped CdTe heated at different periods of time are shown in Figure 1.

The diameter of CdTe QD heated for 180 min (Figure 1) was calculated using the equation given below.²⁷

D = (9.8127 × 10^-7) λ³ − (1.7147 × 10^-3) λ² + (1.0064) λ - 194.84

D is the diameter of the nanocrystals (nm); λ is the wavelength corresponding to absorbance maximum determined as 560 nm from the UV-VIS spectrum, as shown Figure 2b. Calculations showed that the diameter of the MPA-CdTe QDs was 3.34 nm. A TEM image of MPA-capped CdTe QDs is shown in Figure 2b.

FTIR spectroscopy was used to confirm the modification of CdTe QDs with MPA molecules. The spectra of free MPA and MPA-CdTe are given in Figure 3a, 3b. The two bands at 2666 and 2570 cm^-1, which is attributed to hydrogen bonding between acid and thiol groups, disappeared in the IR spectra of MPA capped-CdTe because the MPA is attached to QD through S-atoms. The small peaks that appeared at 2927, 2945, and 2854 cm^-1 were attributed to the asymmetric and symmetric C-H stretching of methylene groups. The appearance of an intense peak at 1570 cm^-1 can be attributed to asymmetric stretching of carboxylic acid.

The effect of pH and concentration of urease on phosphorescence intensity of MPA-capped CdTe QDs

pH is one of the important parameters that affects the photoluminescence intensity of the QDs. Therefore, the effect of the solution pH on the signal intensity of QD was studied using 0.5 mL of 0.04 M Britton–Robinson buffer between pH 2.5-8.0. It was observed that the phosphorescence signal increased linearly as the pH increased from 2.5 to 5.0, and decreased between pH 5.0-8.0. Therefore, a pH between 2.5-5.0 was selected for the determination of urea in the presence of QD and urease. Interestingly, the influence of pH on the fluorescence signal was different than the signal measured in the phosphorescence mode. The effect of pH on the photoluminescence intensity of MPA-CdTe QDs is shown Figure 4.

The effect of urease concentration was studied between 1-7.5 units/mL in the presence of 0.24 mg/L MPA-CdTe and 0.07 mM urea. The maximum signal enhancement was observed when the urease concentration was 5 units/mL, which was then used throughout the experiments.

Determination of urea

Determination of urea is based on the production of ammonia in the presence of urease.

CO(NH₂)₂ + 3H₂O ^urease 2 NH₄⁺ + OH^- + HCO₃^-

The pH of the medium is increased depending on the degradation of urea by urease. Consequently, the phosphorescence signal increased throughout the pH 2.5-5.0 range with increasing urea concentration. In the optimum conditions (0.24 mg/mL CdTe-MPA, 5 units/mL urease and pH 2.5), the calibration was constructed by plotting I-I₀ versus urea concentration (I₀: phosphorescence signal of CdTe-MPA I: phosphorescence signal of CdTe – MPA + 0.0016 - 0.16 mM urea). A linearity in phosphorescence signal was observed between 0.016 -0.16 mM urea concentrations. The phosphorescence signal with increasing urea concentration is shown Figure 5a. Although the fluorescence signal increased with urea concentration, the dynamic range was relatively narrow as shown in Figure 5b. The calibration curve based on phosphorescence signal is given in Figure 6.

The lifetime software of the instrument was used to obtain a decay curve for the phosphorescence emission and the data were used to construct a log intensity versus time graph. The lifetime of the QD was calculated using the -1/slope of this linear line and found as 21.5 µs. (Figure 7).

The proposed method was compared with the methods in the literature. The limit of detection for the proposed method was lower or comparable with the methods such as fluorescence, amperometry, and potentiometry (Table 1). The analytical performance data of the method used for the determination of urea are given in Table 2. The proposed method is relatively simple and free from the interference from the biologic matrix because the phosphorescence signal was used.

Determination of urea in human serum

Serum samples obtained from university laboratory were analyzed for urea using the proposed method. In order to test the accuracy of the proposed method, the same samples were analyzed in a private clinical laboratory; the results of which are shown in Table 3. The precision in terms of percent relative standard deviation, for three parallel determinations, was less than 6.7% and the urea concentrations were consistent with those reported.

CONCLUSION

It was shown that water-soluble and phosphorescent MPA-CdTe QDs can be used for the determination of urea in human blood serum samples. The proposed method is based on enzymatic degradation of urea by urease. In addition, the use of phosphorescence prevents interference such as scatter and autofluorescence from the sample matrix. Compared with conventional room temperature phosphorimetric methods, phosphorescent QDs provide a simpler methodology because no additional chemicals such as heavy atoms and oxygen removal processes are necessary. The results show that the proposed method is accurate, selective, rapid, and simple for urea determination in serum samples and can be applied to other biologic samples.

Conflict of Interest: No conflict of interest was declared by the authors.