New Off-On Sensor for Captopril Sensing Based on Photoluminescent MoO x Quantum Dots.

Molybdenum oxide nanomaterials have recently attracted widespread attention for their unique optical properties and catalytic performance. However, until now, there is little literature on the application of photoluminescent molybdenum oxide nanomaterials in biological and pharmaceutical sensing. Herein, photoluminescent molybdenum oxide quantum dots (MoO x QDs) were synthesized via a facile method, and then, the synthesized MoO x QDs were further applied as a new type of photoluminescent probe to design a new off-on sensor for captopril (Cap) determination on the basis of the fact that the quenched photoluminescence of MoO x QDs by Cu2+ was restored with Cap through specific interaction between the thiol group of Cap and Cu2+. Under optimal conditions, the restored photoluminescence intensity showed a good linear relationship with the content of Cap, ranging from 1.0 to 150.0 μM, with a limit of detection of 0.51 μM (3σ/k). Additionally, the content of Cap in pharmaceutical samples was successfully detected with the newly developed off-on sensor, and the recoveries were 99.4-101.7%, which suggest that the present off-on sensor has a high accuracy.

Introduction

Transition metal oxides, including molybdenum oxide,[1,2] vanadium oxide,[3] titanium dioxide,[4] germanium dioxide,[5] tungsten oxide,[6] nickel oxide,[7] and zinc oxide,[8] have stimulated great interest due to their optical, electrical, and semiconducting properties and catalytic performance. In particular, molybdenum oxide has drawn tremendous attention for its excellent properties, and numerous methods, including the hydrothermal/solvothermal method,[9] physical vapor deposition method,[10] chemical spray pyrolysis,[11] mechanical grinding and sonication,[12] thermal oxidation,[2] thermal evaporation and decomposition, and electrospinning technologies,[13,14] have been developed for the synthesis of molybdenum oxide nanomaterials with a variety of morphologies because the physical and chemical properties of molybdenum oxide nanomaterials are closely related to their morphologies. Until now, molybdenum oxide nanomaterials have been used in many applications, including gas sensors,[1,2] catalysis,[15] solar cells,[16] photochromism,[17] lithium-ion batteries,[18] thin-film capacitors,[18] field-effect transistors,[19,20] antiseptics, and anticancer treatments.[14,21] However, only a few reports have mentioned the application of molybdenum oxide nanomaterials as a photoluminescent probe in biological and pharmaceutical fields, that is, there is still plenty of room for methodological innovation.

Herein, photoluminescent molybdenum oxide quantum dots (MoO QDs) were prepared by a one-pot method at room temperature (Figure a) and a new photoluminescent sensor for captopril (Cap) detection was constructed using MoO QDs as an effective probe. As an angiotensin-converting enzyme (inhibitor), Cap, 1-[(2s)-3-mercapto-2-methylpropionyl]-l-proline, plays a crucial biological role in the treatment of hypertension, coronary heart disease, congestive heart failure, and some types of diseases associated with diabetes.[22−24] Thus far, several methods have been proposed for Cap determination, including voltammetry,[25] chemiluminescence,[26] flow injection spectrophotometry,[27] surface-enhanced Raman spectroscopy,[28] and mass spectrometry.[29] However, certain drawbacks limited the application of the above methods. For example, electrochemical methods are sensitive enough, but the preparation of electrodes is relatively tremendous while chemiluminescence methods are simple and rapid, but they possess the limitation of low detection sensitivity. Raman spectroscopy and mass spectrometry are sensitive enough; however, the need for expensive instruments or complicated procedures limits their application in routine analysis. Therefore, it is necessary to establish a facile and sensitive method for Cap determination in biological fluids and pharmaceutical samples. The sensing platform for Cap developed in this manuscript is shown in Figure b. The system is a combination of MoO QDs and Cu2+, in which Cu2+ can generate a nonluminous complex with the MoO QDs, resulting in the quenching of the photoluminescence of the MoO QDs (switched off) through static quenching processes. However, the formed MoO QDs–Cu2+ complex might be dissociated after the introduction of Cap because Cu2+ displays a higher affinity toward the −SH from Cap. As a consequence, the fluorescence of the MoO QDs is restored (switched on), providing a facile switch-on assay for Cap detection. On the basis of the proposed switch-on assay, the Cap content in the pharmaceutical samples was successfully determined with high sensitivity and good repeatability.

Figure 1

(a) Preparation of MoO QDs and (b) construction of a Cap-sensing platform using MoO QDs and Cu2+.

Results and Discussion

Characterization of the Obtained MoO QDs

The MoO QDs were synthesized at room temperature using commercial MoS2 powder and H2O2 as the precursor and oxidant (Figure a), respectively,[30,31] and the as-prepared MoO QDs had an average diameter of around 2.0 nm (Figure a) and a height of about 1.5 nm (Figure b). From the X-ray photoelectron spectroscopy (XPS) spectra (Figures c,d and S1), it can be clearly seen that Mo4+ and S2– were both oxidized by H2O2 to higher valence states, Mo5+ (230.6 and 232.8 eV) and Mo6+ (232.1 and 235.3 eV) and SO42– (168.5 and 169.7 eV), respectively.[32−34] The average oxidation state, calculated from the XPS peak area proportion of Mo5+ and Mo6+ in the MoO QDs of Mo, was 5.91, suggesting the presence of oxygen vacancies in the obtained MoO QDs, which is in agreement with previous reports.[30−32,34−36] On the basis of these results, it can be concluded that bulk MoS2 powder was first spontaneously exfoliated by H2O2 during the reaction process (Figure a), in which S atoms gradually exited the lattice of MoS2 while O atoms from H2O2 immediately refilled the lattice vacancies because the bonding affinity of Mo—O is stronger than that of Mo—S.[36] Finally, photoluminescent MoO QDs with a maximum absorbance at 317 nm (Figure ) were synthesized, and the strong absorption between 200–400 nm was the reason for the charge transfer of the Mo—O band in the MoO66– octahedron.[37] Moreover, the MoO QDs also showed excitation-dependent emission, and the FL emission peak of MoO QDs progressively shifted to a longer wavelength when the excitation increased from 325 to 475 nm, which might be due to particles of different sizes and the distribution of emissive trap sites on the surface of the MoO QDs. The strong excitation-dependent fluorescence characteristics of the MoO QDs were also consistent with those previously reported in the literature.[35,38]

Figure 2

(a) Transmission electron microscope (TEM) and (b) atomic force microscopy (AFM) images of the obtained MoO QDs. The high-resolution XPS spectra of Mo3d (c) and S2p (d) of MoO QDs.

Figure 3

Optical spectra of MoO QDs. The black line represents the absorption spectrum of MoO QDs and the other lines are photoluminescence spectra of MoO QDs excited at different wavelengths. The excitation wavelengths are 325, 350, 375, 400, 425, 450, and 475 nm, whereas the maximum emission wavelengths are 470, 500, 510, 517, 525, 534, and 539 nm, respectively. Inset: Photographs obtained under visible (left) and 365 nm UV light (right).

Quenching the Fluorescence of MoO QDs by Cu2+

As a common quencher, Cu2+ can quench the fluorescence of organic fluorescent dyes,[39] fluorescent proteins,[40] and carbon dots[41] through electronic transfer or other processes. On the basis of this, the photoluminescence of MoO QDs incubated with Cu2+ was first measured to confirm whether or not the photoluminescence of MoO QDs could be quenched by Cu2+. As shown in Figure a, MoO QDs have a strong photoluminescence emission peak at 527 nm when excited at 405 nm, whereas the photoluminescence was gradually quenched as the concentration of Cu2+ increased, and the maximal quenching efficiency reached nearly 90% when Cu2+ was 75 μM. To elucidate the quenching mechanism, the fluorescence lifetimes of MoO QDs and MoO QDs–Cu2+ were measured (Figure b and Table S1), and the results show that the average fluorescence lifetimes of MoO QDs and MoO QDs–Cu2+ are 4.83 and 5.97 ns, respectively, which indicate that the static quenching mechanism accounts for the decrease in photoluminescence and a nonluminous complex is formed between MoO QDs and Cu2+. The formation of MoO QDs–Cu2+ complexes was also confirmed by ultrafiltration experiments. For ultrafiltration experiments, MoO QDs–Cu2+ solutions were filtered through a 1 kDa MWCO ultrafiltration membrane, through which only free Cu2+ ions in the solution could pass, whereas those adhering to MoO QDs could not. The residues were then subjected to XPS measurements, and the Cu2p peak was clearly observed in the survey spectrum (Figure S4), suggesting the formation of MoO QDs–Cu2+ complexes.

Figure 4

(a) Photoluminescence of the MoO QDs quenched by Cu2+. Cu2+ concentrations from top to bottom are 0, 1.0, 2.5, 5.0, 10.0, 20.0, 40.0, 60.0, 80.0 μM, respectively. Inset is the plot of photoluminescence intensities vs Cu2+ concentration. (b) Fluorescence lifetimes of MoO QDs, MoO QDs–Cu2+ complexes, and MoO QDs–Cu2+–Cap. The data were obtained from three parallel samples.

Construction of an Off–On Sensor for Cap Determination

In view of the effective reaction between Cu2+ and the active thiol groups (−SH) in cysteine,[41] the MoO QDs–Cu2+ system was utilized to detect the thiol-containing Cap tablets. Figure shows the photoluminescence recoveries of the MoO QDs–Cu2+ system with increasing contents of Cap, and it can be clearly seen that the photoluminescence was gradually restored as more and more Cap was added. The reason for the photoluminescence recovery might be as follows: a Cu2+—S bond can be formed between the thiol group of Cap and Cu2+, resulting in the removal of Cu2+ from the surface of MoO QDs via competitive adsorption interactions (Figure b),[41] which enhanced the photoluminescence. The desorption of Cu from MoO QDs–Cu2+ complexes was also confirmed by XPS, and the results showed that the Cu2p peak of the residues disappeared after the MoO QDs–Cu2+–Cap solutions were passed through a 1 kDa MWCD ultrafiltration membrane (Figure S5).

Figure 5

(a) Photoluminescence spectra of MoO QDs–Cu2+ with the addition of Cap. Cap concentrations from the bottom to top are 0.0, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 40.0, 50.0, 75.0, 100.0, and 150.0 μM, respectively. Inset is the plot of photoluminescence changes at 527.0 nm vs Cap concentration. MoO QDs: 0.1 mg mL–1; Cu2+: 75μM; Tris–HCl buffer (pH 7.5): 50 mM; excitation: 405 nm. The data were obtained from three parallel samples. (b) Photoluminescence responses of the off–on sensor for Cap and interferents. MoO QDs: 0.1 mg mL–1; Cu2+: 75 μM; Tris–HCl buffer (pH 7.5): 50 mM; excitation: 405 nm; emission: 527 nm.

The photoluminescence recovery was affected by pH and time, and the photoluminescence intensity reached its maximum value at pH 7.5 within 5 min (Figures S2 and S3). Under optimal conditions, the photoluminescence follows a linear relationship with the Cap content, ranging from 1.0 to 150.0 μM, with a limit of detection of 0.51 μM (3σ/k), which is comparable to that of other methods, as shown in Table S2.

Detection of Cap in Pharmaceutical Samples Based on the New Off–On Sensor

To further evaluate the selectivity of the new off–on sensor for Cap, the photoluminescence responses to other common ions and excipients in antihypertensive pills were investigated. As shown in Figure b, the photoluminescence of other detected substances showed no significant increase even when their concentrations were 10-fold higher than those of Cap, illustrating the high selectivity of the developed off–on sensor, which might be employable for Cap determination in real samples. Therefore, the Cap contents in real pharmaceutical samples were detected to further illustrate the feasibility. To avoid any interference from the sample matrix, a standard addition method was used, spiking each pharmaceutical sample with a known concentration of Cap. The results clearly showed that the sample matrix has no obvious interference, and the actual Cap concentration of the pharmaceutical sample could be obtained from the linear trend produced by the standard addition method (Figure S6). Meanwhile, Cap contents in pharmaceutical samples were successfully detected (Table ), and the standard deviations and recovery (99.4–101.7%) demonstrated that the proposed method for Cap detection has a high accuracy and good repeatability, which can fulfill the needs of real applications.

Table 1

Determination of Cap in Pharmaceutical Samples

sample nos.	measured (±SD, μM)a	value added (μM)a	value found (±SD, μM)a	recovery (%)
1	19.2 ± 1.05	25.0	44.09 ± 0.86	99.4
2	19.19 ± 1.04	50.0	69.52 ± 1.05	101.7
3	19.17 ± 1.29	50.0	69.28 ± 1.08	100.6

The data were obtained from three parallel samples.

Conclusions

In summary, photoluminescent MoO QDs were synthesized at room temperature and a new “off–on” photoluminescent sensor was designed for Cap detection in pharmaceutical samples based on MoO QDs–Cu2+. The quenching and recovery mechanisms were carefully explored. On the one hand, the photoluminescence of MoO QDs was quenched by Cu2+ by a static quenching process, which was attributed to the formation of nonluminous MoO QDs–Cu2+complexes. On the other hand, Cu2+ might be dissociated from MoO QDs due to the stronger binding affinity of Cu2+ to the thiol group of Cap, resulting in restoration of the photoluminescence of the MoO QDs. Moreover, the content of Cap in the pharmaceutical samples was successfully detected with the newly developed off–on sensor; the standard deviations and recoveries (99.4–101.7%) demonstrated high accuracy and good repeatability of the off–on sensor, which fulfill the requirements for real-time applications.

Experimental Section

Reagents

MoS2 powder was commercially obtained from Sigma-Aldrich. NaOH, 30% H2O2, HCl, CuCl2, NaCl, Fe2(SO4)3, NaNO3, CaCl2, Mg(NO3)2, KCl, and Zn(SO4)2 were provided by Shanghai Maikun Chemical Reagents Co., Ltd (Shanghai, China). Tris (C4H11O3) was purchased from Solarbio Company. Cap was supplied from Wanjia (China). Starch, lactose, glucose, sucrose, and dextrin were purchased from Guangzhou Yuwei Chemical Reagents Co., Ltd (Guangzhou, China). All chemicals and solvents were of analytical grade and were used without further purification. Deionized water was used in all experiments.

Apparatus

A JEM-2010 TEM (JEOL Ltd, Japan) with a 200 kV accelerating voltage and an AFM in the ScanAsyst mode were used to obtain the size and height of the MoO QDs, whereas XPS (Thermo) was used to characterized the elemental composition and bonding configuration. The fluorescence lifetime was measured by an FL-TCSPC fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France). The absorption was measured using a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan), whereas a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) or a USB-4000FL spectrophotometer (Ocean Optical) was utilized to record the fluorescence spectra.

Preparation of MoO QDs

MoO QDs were prepared using a one-pot method at room temperature according to our previous studies,[30,31] in which MoS2 and 30% hydrogen peroxide were selected as the precursor and oxidant, respectively. Briefly, 10.0 mg of the MoS2 powder was incubated with 10 mL of the mixing solution (H2O–30% H2O2 = 3:2) for 30 min at room temperature. Thereafter, the pH of the mixture was adjusted to 7.0 using sodium hydroxide (NaOH), with slow stirring for 20 min. Finally, MoO QDs were obtained by centrifuging the resulting mixture at 8000g for 10 min.

Fluorescence Sensing of Cu2+ and Cap

For the detection of Cu2+, 20 μL of MoO QD solution (1 mg/mL), 20 μL of Tris–HCl buffer solution (50 mM, pH 7.5), Cu2+, and H2O were added to a final volume of 200 μL. The final concentration of Cu2+ ranged from 0 to 80 μM. The resulting solutions were excited with a 405 nm laser, and their fluorescence spectra were recorded with a USB-4000FL spectrophotometer.

For the detection of Cap, 20 μL of MoO QDs (1 mg/mL); 20 μL of Tris–HCl buffer solution (50 mM pH 7.5); 15 μL of Cu2+ (1 mM), with a final concentration of 75 μM; different concentrations of Cap solution; and deionized water were added to a final volume of 200 μL. The final concentration of Cap ranged from 0 to 150 μM. After incubation for 5 min, the fluorescence spectra were recorded using a USB-4000FL spectrophotometer equipped with a 405 nm laser light source.

Pharmaceutical Sample Preparation

The three pharmaceutical samples were obtained from Henan Yu pharmaceuticals, Ltd, Shanghai pharmaceutical Co., Ltd, and Hangzhou Minsheng pharmaceutical Co., Ltd, respectively. Four tablets of each sample were ground to a homogenized powder. A portion of the powder containing 12.5 mg of Cap was accurately weighted and dissolved in 10 mL of H2O. After ultrasonication for 20 min, the supernatant was obtained by centrifuging at 500g for 5 min. The obtained sample solutions were diluted 300 fold before use. The standard Cap solution was added into the diluted sample solutions for recovery tests to evaluate the accuracy of the present off–on sensor.