Turn-off fluorescence of amino-functionalized carbon quantum dots as effective fluorescent probes for determination of isotretinoin

Accepted Manuscript Title: Turn-off fluorescence of amino-functionalized carbon quantum dots as effective fluorescent probes for determination of isotretinoin Authors: Tayyebeh Madrakian, Somayeh Maleki, Sahar Gilak, Abbas Afkhami PII: DOI: Reference:

S0925-4005(17)30488-4 http://dx.doi.org/doi:10.1016/j.snb.2017.03.071 SNB 21983

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

4-2-2017 12-3-2017 14-3-2017

Please cite this article as: Tayyebeh Madrakian, Somayeh Maleki, Sahar Gilak, Abbas Afkhami, Turn-off fluorescence of amino-functionalized carbon quantum dots as effective fluorescent probes for determination of isotretinoin, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.03.071 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 proof before it is published in its final 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.

Turn-off fluorescence of amino-functionalized carbon quantum dots as effective fluorescent probes for determination of isotretinoin

Tayyebeh Madrakian*, Somayeh Maleki, Sahar Gilak, Abbas Afkhami

Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran

*Corresponding author, Tel. / Fax: +988138272404 E-mail: [email protected], [email protected]

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Graphical Abstract

Highlights     

Amino-functionalized carbon quantum dots were used as effective fluorescence probes. The fluorescence of N-CQDs was quenched in the presence of isotretinoin. The quenching of N-CQDs induced by isotretinoin is static quenching. Isotretinoin was measured at 0.03 μmol L− 1 with good sensitivity and selectivity. The probe has been successfully applied in serum and pharmaceutical samples.

Abstract: Amino-functionalized carbon quantum dots (N-CQDs) as effective fluorescent probes were synthesized by a facile one-pot hydrothermal method via gluconic acid and Nmethylethylenediamine as carbon and nitrogen sources respectively. Then the N-CQDs were applied to determine isotretinoin (INN). The synthesized N-CQDs were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FT-IR), UV-vis absorption and photoluminescence spectroscopy. As the emission of N-CQDs is efficiently quenched by INN, the as-prepared N-

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CQDs are employed as a highly sensitive and selective probe for INN detection. Under the optimal conditions, linear response was observed in the range of 0.08–70.0 μmol L−1 for INN determination. The calculated detection limit was 0.03 μmol L−1. The established method showed a good selectivity for INN among several kinds of ions and biomolecules. The results of this study showed that the as-synthesized N-CQDs could be successfully applied to determine INN in blood serum and pharmaceutical samples.

Keywords: Isotretinoin; Fluorescence probe; Amino-functionalized carbon quantum dots; Hydrothermal method; Gluconic acid; N-methylethylenediamine.

Introduction: Isotretinoin (INN) chemically 3, 7-dimethyl-9 (2, 6, 6trimethylcyclohex-1-enyl) nona-2, 4, 6, 8 tetraenoic acid (Scheme 1), is a retinoid classified as vitamin A. It is used in the treatment of skin disease including acne vulgaris as a topical keratolytic agent. The mechanism of action is believed to inhibit the secretion of sebum and alter the lipid composition of the skin surface. Its effect on regulating cell differentiation led to use of it to treat cystic and nodular acne and also to inhibit neoplastic cells proliferation during past decades [1]. Several techniques have been applied to determine INN such as high-performance liquid chromatography with ultra-violet detection [13], gas chromatography (GC) [4] and others high-performance liquid chromatography with mass spectrometric detection [5-7]. Most of these methods depend on lengthy sample preparation schemes including extraction using an organic solvent, evaporation of solvent and reconstitution prior to HPLC analysis or need column switching techniques (online solid-phase extraction) or require expensive instruments such as LC–MS–MS [3].

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Carbon quantum dots (CQDs) are a group of nanoparticles which consist of carbon with a size below 10 nm. Since the discovery of fluorescent fragments of a batch of single-wall nanotubes in 2004, numerous of papers have investigated the synthetic methods, properties, and application of CDs [8]. Unlike the conventional semiconductor quantum dots (QDs) containing toxic heavy metal elements and chalcogens, CDs are mainly composed of non-toxic C, O, and N elements, so are superior in the aspects of good water solubility, high quantum yield (Φs), outstanding photoluminescence (PL) properties, large stokes shifts, robust chemical inertness, low cytotoxicity , ease of functionalization, and excellent biocompatibility [9]. These are acceptable reasons for wide use of CQDs in the fields of biological labeling, bioimaging, and drug delivery recently. Doping is a common approach to tune the PL properties of photoluminescent materials. Various doping methods with dozens of elements such as P, S, and N have been reported to tune the features of CQDs. N-doping is the most studied a way to increase the emission of the CQDs by inducing an upward shift in the Fermi level and electrons in the conduction band. It was demonstrated that only the nitrogen bonding to carbon can really enhance the PL emission of CQDs with quantum yields of more than 20% [10-12]. The excitation-dependent emissions are general features of CQDs. Several possibilities are proposed including an optical selection of nanoparticles with different size (quantum effect); different distributions of emissive trap sites on each CQD; free zigzag sites with a carbene-like triplet ground state; radiative recombination of excitons [13, 14]. In this paper, a simple N-CQDs -based “turn-off” fluorescence method is proposed to determine INN. The N-CQDs were synthesized by a facile one-pot hydrothermal method with a quantum yield (QY) 35.2% using gluconic acid and N-methylethylenediamine as carbon and nitrogen sources, respectively. The morphology, size, and structure of the synthesized N-CQDs were

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characterized using different techniques. The effective parameters were evaluated and INN was determined under the optimal conditions. Scheme 1 2. Experimental 2.1. Chemicals and reagents All reagents and materials were obtained from Merck Company (Darmstadt, Germany) or Aldrich Company (St. Louis, MO, USA) and used without further purification. Gluconic acid and N-methylethylenediamin were purchased from Merck Company (Darmstadt, Germany) or Aldrich Company (St. Louis, MO, USA). INN stock solution (1.0×10-3 mol L−1) was prepared in double distilled water (DDW) and stored at 4◦C. Phosphate buffer solution (PBS, 0.1mol L−1) of pH 7.0 was used for pH adjustments. For recovery tests, INN tablets (20.0 mg, Roche Company) were purchased from a local pharmacy, and fresh human serum samples were provided from Bessat Hospital Lab. (Hamedan, Iran). DDW was used throughout the work. 2.2. Apparatus A Perkin Elmer (LS50B) luminescence spectrometer was used for INN spectrofluorometric concentration determination. The absorption spectrum was recorded using a single beam UV-miniWPA spectrophotometer. A transmission electron microscope (TEM, Philips-CM10-300 kV) was performed to characterize the morphology of the N-CQDs. The crystal structure of the synthesized N-CQDs was determined using an X-ray diffractometer (XRD, 38066 Riva, d/G. via M. Misone, 11/D (TN) Italy). Size distribution of the N-CQDs in water was measured by using dynamic light scattering (DLS, Nano ZS (red badge) ZEN 3600). The mid-infrared spectra of the synthesized NCQDs in the 400–4000 cm-1 region were recorded by an FT-IR spectrometer (Perkin Elmer model Spectrum GX) using KBr pellets. A Metrohm model 713 pH meters was used for pH measurements. Also, a 40 kHz universal ultrasonic cleaner water bath (RoHS, Korea) was used. 5

2.3. Synthesis of amino-functionalized carbon quantum dots The N-CQDs were prepared by a hydrothermal method [15]. Gluconic acid (1.0 g) and Nmethylethylenediamin (2 mL) were dissolved in DDW water (10 mL). Then the solution was transferred to a Teflon-lined autoclave (100 mL) and heated at 200 ◦C for 5 h. Carbonization of the reactants was detected through colour changing of the solution to dark brown. The reaction mixture was cooled down to room temperature. Next, the aqueous solution was centrifuged at 15000 rpm for 15 min to dislodge the non- fluorescent deposit and got the upper N-CQDs aqueous solution for use. As prepared dark brown solution was further purified through a 0.2 µm membrane filter. Finally, the N-CQDs were diluted using DDW until appropriate signal was obtained 2.4. Procedure for spectrofluorometric detection of INN To study the quenching effect of INN on the fluorescence intensity of N-CQDs, 50 µg mL1

of N-CQDs solution, 1.5 mL of 0.1 mol L−1 PBS (pH 7.0) and different amounts of INN were

successively added into a 5 mL volumetric flask, diluted to the mark with DDW and sonicated for 2 min until the solution was fully mixed. The fluorescence spectra were recorded from 300 nm to 700 nm. The fluorescence intensity of the solution at the maximum emission wavelength at 450 nm (λex= 350 nm) was used for quantitative analysis. 2.5. Pharmaceutical sample solution preparation For pharmaceutical analysis, seven INN capsules were opened carefully using a sharp blade and the content of these capsules was extracted. Then calculated amount (equal to weight of one capsule) of homogenized liquid was imported into a 25 mL volumetric flask and was dissolved in 5 ml of methanol. Afterward, it was sonicated to dissolve it completely and diluted up to the volume with DDW. The content of the flask was sonicated for 15 min and centrifuged for 10 min at 4000 rpm. Then, the obtained mixture was filtered and sufficient volume from this solution was

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used [1, 16]. Finally, the ΔF was measured at the maximum emission wavelength (450 nm) in a 1.0 cm fluorescence quartz cell using the procedure described above. 2.6. Serum sample preparation For serum sample preparation various amounts of INN were spiked to the 1.0 mL of serum sample and then acetonitrile (0.8 mL) was added to remove serum protein. Afterward, the mixture was centrifuged for 15 min at 3500 rpm and the supernatant was taken carefully and transferred into 5.0 mL volumetric flask [17, 18]. Finally, the ΔF was measured using the mentioned procedure. 3. Results and discussion 3.1. Characterization of amino-functionalized carbon quantum dots The N-CQDs were characterized by UV-Vis spectrophotometry, fluorescence spectra, Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and Xray diffraction (XRD). The absorption spectra of N-CQDs were investigated in the range of 200 to 600 nm. The UV–vis absorption spectrum of N-CQDs aqueous solution in Fig. 1A(a) revealed the absorption band was at 340 nm. The emission spectrum (λex=350) of N-CQDs is shown in Fig. 1A(b). The maximum emission wavelength was observed in 450 nm. Excitation-dependent PL behavior was observed, which is common in fluorescent carbon materials. Whether this occurs because of optical selection of differently sized nanoparticles (quantum effect) and/or different emissive traps on the C-dot surface or another mechanism altogether is currently unresolved (Fig. 1B) [10]. The N-CQDs were also characterized by FT-IR spectroscopy (Fig. 2A). The characteristic peaks between 3200 and 3500 cm-1 are assigned to the stretching vibrations of N-H and O-H. The peaks at 2957 cm-1 and 2830 cm-1 are ascribed to the stretching vibrations of C–H. The typical peaks at 1578 cm-1 and 1633 cm-1 are associated with the bending vibrations of CO-

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NH group. The peak at 1471 cm-1 is related to the stretching band of C–N and the peaks at 1384 cm-1 and 1036 cm-1 are ascribed to the vibration of C–O [15, 19]. TEM image of the N-CQDs in Fig. 2B indicates that the particles are most rounds without apparent aggregation. We also studied the DLS for estimation of diameter of N-CQDs (inset in Fig. 2B). It was found that the size distribution range of N-CQDs is mainly between 1 nm to 6 nm with the average diameter about 4 nm. The XRD pattern of the N-CQDs (Fig. 2C) also displayed a broad peak centered at 20° (0.82 nm), which is also attributed to highly disordered carbon atoms [20, 21]. The above evidence indicative that the N-CQDs were successfully synthesized. Furthermore, the QY of N-CQDs was measured 35.2% using the method described in Ref.[22, 23]. Fig. 1 Fig. 2 3.2. Optimization of synthesis of amino-functionalized carbon quantum dots The synthetic method could be used to prepare different types of N-CQDs by tuning the precursor's conditions as depicted in Table 1. In our preliminary experiments, the OH, COOH (from gluconic acid) and NH2 (from N-methylethylenediamin) moieties were found crucial for the formation of N-CQDs. First, if the precursor contains only OH and COOH groups, the QY of as prepared CDs is only 10.3%. However, the QY of the CQDs was enhanced when NH2 groups were added. The highest QY is 35.2% for N-CQDs prepared with (1.0 g) gluconic acid and (2 mL) Nmethylethylenediamin. It is necessary to mention that time and temperature synthesis was investigated. Temperature synthesis of 200 ◦C was found to be the optimal condition for further carbonization and thus further fluorescence intensity. Reaction time is considered as one of the important parameters for N-CQDs synthesis. With increasing duration of synthesis, the polymerlike CDs are converted into carbogenic CDs. Formation of polymer-like CDs and the PL arising

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from the surface/molecule state (perhaps owing to amide-containing fluorophores) take place in a modest reaction time. Whereas in a short or long reaction time, owing to further carbonization, partial carbogenic CDs is formed and the PL is derived from the synergistic effect of the carbogenic core and the surface/molecule state. The carbogenic core plays a greater role in CDs no matter the synthesis time decreases or increases [9, 20]. The optimal synthesis time was found to be 5 h. Table 1 3.3. Fluorescence quenching by INN Interestingly, it found that INN could obviously quench the fluorescence of the N-CQDs. It means that the N-CQDs could be potentially used as an INN sensor. Figure 3 shows the fluorescence emission spectra of the N-CQDs (a) and the N-CQDs -INN (b). The fluorescence quenching mechanism can be usually classified as either static quenching, caused by ground-state complex formation between fluorophores and quenchers or dynamic quenching, caused by collisional encounters, respectively. The static quenching and the dynamic quenching can be distinguished by their dependence on excited-state lifetime and temperature. Higher temperature may result in decreasing the stability of the complex and thus smaller values of the static quenching constant. Dynamic quenching constant is expected to increase with increasing temperature because the higher temperature is likely to result in a larger diffusion coefficient and promote the process of electron transfer. To elucidate the mechanism of fluorescence quenching, the quenching experiments were performed at three different temperatures 283, 303 and 323 K and the fluorescence quenching constant (KSV) was calculated using the well-known Stern–Volmer equation (Eq. 1) [24]: F0 F

= 1 + K SV [Q] = 1 + K q τ0 [Q]

Eq. 1

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Where F0 and F are the steady-state fluorescence intensities in the absence and the presence of quencher, respectively, and [Q] is the initial concentration of quencher. kq is the quenching rate constant. τ0 is the fluorescence lifetime of the unquenched fluorophore. KSV is the Stern–Volmer quenching constant, which can be determined by linear regression of Stern–Volmer equation. The Stern–Volmer equations of the fluorescence of N-CQDs quenched by INN at different temperatures were shown in Table 2. As shown in Table 2, there were good linear relationships between F0/F and the [Q]. The KSV values for the interactions of N-CQDs with INN were decreased with increasing temperature, indicating that the fluorescence quenching was arisen from INN binding and the probable quenching process was static quenching mechanism rather than a dynamic quenching mechanism. In addition, the kq values were greater than the maximum diffusion-collision quenching rate constant (2.0 × 1010 L/mol s) of a variety of quenchers, further indicating that the quenching of N-CQDs induced by the INN is not caused by dynamic collision and mechanism of fluorescence quenching is static [25, 26]. Fig. 3 Table 2

3.4. Optimization of the variables 3.4.1. Effect of pH The results showed that pH value is effective on the fluorescence intensity of the N-CQDs solution in absence and presence of INN (Fig. 4A). As displayed in Fig. 4A(a), the emission of NCQDs in absence INN is highly stable in a pH range from 3.0 to 8.0, whereas the intensity of the N-CQDs had a tendency to decrease with the intensity of the sensing system tending to increase at higher pH. The reason may be attributed to the presence of the carboxyl groups on the surface of

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the CDs that could be dissociated in the basic solutions, which implied that overly basic environment may induce the changes of functional groups and then the electronic transition of some defects would be disrupted or even prohibited [23, 27]. Also, the effect of pH on the fluorescence intensity of the N-CQDs solution in presence of INN was investigated. As shown in Fig. 4A(b), INN wasn’t stable in lower pH of 7.0, so it wasn't possible to investigate its behavior in this range of pH. At higher pH of 7.0, the fluorescence intensity of the N-CQDs solution in presence of INN decreased. The maximum of ΔF (difference in fluorescence intensity of N-CQDs solution in both absence and presence of INN) was observed at pH 7.0 (Fig. 4B). Therefore the pH 7.0, which is close to biological pH value, was chosen as an optimum solution pH for further experiments. Then in pH 7.0 effects of some buffer systems (such as Britton–Robinson (B–R), Tris–HCl and phosphate salt) on the ΔF were tested. Based on the results, Phosphate buffer solution (PBS, 0.03 mol L−1) was the most efficient among mentioned buffer systems. Fig. 4

3.4.2. Effect of the N-CQDs concentration The effect of concentration of the N-CQDs (from 5.0 to 100.0 μg mL-1) on the fluorescence quenching of N-CQDs in the absence and presence of INN was studied. As shown in Fig. 5, the ΔF was increased dramatically with the increase in the N-CQDs concentration to the maximum concentration of 50.0 μg mL-1 and then decreased at higher concentrations. This reduction in fluorescence intensity may be related to self-quenching that take place in high concentration due to the aggregation quenching effect [8, 28]. Therefore, the N-CQDs concentration of 50.0 μg mL1

was chosen in the further experiments.

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Fig. 5 3.4.3. Effect of sonication time For the study the sonication time influences on the fluorescence intensity of N-CQDs in the absence and presence of INN, fluorescence spectrum was recorded at different time intervals of 0 to 10 min after the addition of reagents (Fig. 6). The ΔF was increased dramatically with the increase of sonication time to 2 min and then decreased at higher sonication time. Therefore, fluorescence spectrum was recorded at 2 min after the addition of reagents. Fig. 6

3.5. Calibration curve and detection limit of the method As mentioned above, under the optimal conditions, the emission spectra of N-CQDs with different amounts of INN were recorded. As illustrated in Fig. 7A, the addition of a series of increasing concentrations of INN led to the gradual quenching of the fluorescence intensity of the N-CQDs system. As shown in Fig. 7B, there was a good linear relationship (Eq. 2) between the ΔF and INN concentration in the range of 0.08 to 70.0 μmol L-1.

𝛥𝐹 = 2.891 + 2.4804[𝐼𝑁𝑁], μmol L−1

(Eq. 2 )

The corresponding regression coefficient (R2) is 0.9934. The limit of detection, defined as LOD=3Sb/m, where Sb, m and LOD are the standard deviation of the blank, the slope of the calibration graph and the limit of detection, respectively, was found to be 0.03 µmol L−1 using the criterion of three times the standard deviation of the blank signal. The relative standard deviation (RSD%) in five successive assays for 30 µmol L−1 of INN was 1.7%.

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Fig. 7 3.6. Effect of interferences To evaluation the selectivity of the mentioned detection system, the fluorescence emission responses of N-CQDs system in absence and presence of INN (20.0 µmol L−1) were investigated using several kinds of ions and biomolecules. As the results that are given in Table 3 showed, the common ions such as Na+, K+, Cl−, Br-, SO42-, NO3- and CO32- did not show interference with INN detection. As for the common interference in biological samples for the determination of INN, different concentration of ascorbic acid, glucose, sucrose, fructose, glycine, urea, starch, uric acid, citric acid, metal ions as Mg2+ and Ca2+ had no effective interference in INN detection. Fe2+ and Zn2+ have negligible interference when their concentration is no more than 30-fold of INN. Also the effects of interference tyrosine, cysteine and acetaminophen have been investigated and it found that they do not have any interference with the detection system to the ratio of 20 times. The major interference was observed for Fe3+ in the ratio 1:1. The interfering effect of Fe3+ can be removed by addition phenanthroline solution (10-fold of Fe3+) to the reaction mixture. Thus, the mentioned detection system has excellent selectivity towards the determination of INN in pharmaceutical and biological samples. Table 3

3.7. Detection of INN in pharmaceutical preparation and human serum samples For evaluation the feasibility of the mentioned method for detection in real samples, the developed fluorescence probe was applied to the determination of INN in pharmaceutical preparation and human serum samples. The results are presented in Table 4. The mean results of the three determinations for the pharmaceutical preparation sample of INN were close to the values

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declared on the labels. To check accuracy of the proposed method in pharmaceutical preparation sample and its applicability to the determination of INN in the human serum, the recovery investigation was carried out. The obtained recovery results in Table 4 indicate that the proposed method could be successfully applied to the determination of INN in both of the pharmaceutical preparations and human serum samples. The performance of the mentioned method has been also compared with other analytical systems for determination of INN. The results are shown in Table 5. It was proved that the proposed method is rapid, simple and low cost in compare to some other methods. Also, it has the detection limit of good and wide linear range than other methods. Table 4 Table 5 4. Conclusions In this study, the N-CQDs were synthesized for selective detection of INN. The fluorescence of the N-CQDs was quenched by INN. There is a good linear relationship between the ΔF and the concentration of INN in optimum conditions. The Stern–Volmer quenching constant values for the interactions of N-CQDs with INN were decreased with increasing temperature, indicating that the fluorescence quenching was arisen from INN binding and the probable quenching process was static quenching mechanism. The fluorescence probe showed a low detection limit, wide linear range and high selectivity in detecting INN in compare to some other methods. Furthermore, the proposed fluorescence probes can act as bio-probes for INN detection in pharmaceutical preparations and human serum samples.

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Acknowledgement Biographies Tayyebeh Madrakian received her Ph.D. in Analytical Chemistry in 2000 at Razi University, Kermanshah, Iran. She is currently a Professor of Chemistry at Bu-Ali Sina University, Hamedan, Iran. Her current fields of interest are the development of the methods for analysis of trace amounts of organic and inorganic compounds and her research interests include biomedical signal processing and feature extraction. Abbas Afkhami received his Ph.D. in Analytical Chemistry in 1991 at Shiraz University, Shiraz, Iran. He is currently a Professor of Chemistry at Bu-Ali Sina University, Hamedan, Iran. His research interest comprises the development of new optical chemical and electrochemical sensors for anions, cations and neutral chemical species. Somayeh Maleki is currently a Ph.D. student at Bu-Ali Sina University, Hamedan, Iran. Sahar Gilak was a Bachelor student at Bu-Ali Sina University, Hamedan, Iran.

The authors acknowledge the Bu-Ali Sina University Research Council and Center of Excellence in Development of Environmentally Friendly Methods for Chemical Synthesis (CEDEFMCS) for providing support to this work.

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Biographies Tayyebeh Madrakian received her Ph.D. in Analytical Chemistry in 2000 at Razi University, Kermanshah, Iran. She is currently a Professor of Chemistry at Bu-Ali Sina University, Hamedan, Iran. Her current fields of interest are the development of the methods for analysis of trace amounts of organic and inorganic compounds and her research interests include biomedical signal processing and feature extraction. Abbas Afkhami received his Ph.D. in Analytical Chemistry in 1991 at Shiraz University, Shiraz, Iran. He is currently a Professor of Chemistry at Bu-Ali Sina University, Hamedan, Iran. His research interest comprises the development of new optical chemical and electrochemical sensors for anions, cations and neutral chemical species. Somayeh Maleki is currently a Ph.D. student at Bu-Ali Sina University, Hamedan, Iran. Sahar Gilak was a Bachelor student at Bu-Ali Sina University, Hamedan, Iran.

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Figure Captions Fig. 1 (A) UV-Visible absorption (a) and emission (b) spectra of the N-CQDs. (B) Emission spectra of the N-CQDs with excitation at different wavelengths. Fig. 2 (A) FT-IR spectra of the N-CQDs. (B) TEM image of the N-CQDs. Inset image shows size distribution histogram of the N-CQDs measured by DLS. (C) XRD pattern of the N-CQDs. Fig. 3 Fluorescence emission spectra of the N-CQDs (a) and the N-CQDs mixed with INN (b). Conditions: The concentration of N-CQDs and INN was 50.0 μg mL-1and 70.0 μmol L-1, respectively, in PBS of pH 7.0 at sonication time 2 min. Fig. 4 (A) Effect of pH on the fluorescence intensity of N-CQDs in the absence (a) and presence (b) of INN. (B) Effect of pH on the ΔF. Conditions: The concentration of N-CQDs and INN was 60.0 μg mL-1 and 30.0 μmol L-1, respectively, at sonication time 5 min. Fig. 5 Effect of concentration of theN-CQDs on the ΔF. Conditions: The concentration of INN was 30.0 μmol L-1, in PBS of pH 7.0 at sonication time 5 min. Fig. 6 Effect of sonication time on the ΔF. Conditions: The concentration of INN was 30.0 μmol L-1, in PBS of pH 7.0 and concentration of N-CQDs was 50.0 μg mL-1. Fig. 7 (A) The fluorescence intensity change of the N-CQDs with different concentrations of INN. The curves a–k represents the concentrations of INN of 0.0, 0.08, 1.0, 5.0, 10, 25, 50, 70, 100, 130 and 170 µmol L−1, respectively. (B) A linear relationship between the ΔF value and the concentration of INN in the solution.

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Fig. 1

22

Fig. 2

23

Fig. 3

24

Fig. 4

25

Fig. 5

26

Fig. 6

27

Fig. 7

28

Scheme 1 Chemical structure of INN.

Scheme 1

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Table 1. Other reaction conditions for N-CQDs (In (Teflon)-lined autoclave/200 ºC for 5 h).

Serial

Carbon source

Nitrogen source

(g)

(mL)

Water

QY standard by

(mL)

quinine sulfate (%)

1

1.0

-

10.0

10.3

2

1.0

1.0

10.0

27.6

3

2.0

1.0

10 .0

16.4

4

1.0

2 .0

10 .0

35.2

5

0.5

3.0

10.0

21.5

30

Table 2. The quenching constants for the interaction of the N-CQDs with INN. T (K)

Stern–Volmer equation

Kq (L mol-1 s-1(

283

F0

= 12932[Q] + 1.056

1.29×1012

1.29×104

0.996

303

F0

= 9220.6[Q] + 1.007

9.22×1011

9.22×103

0.999

323

F0

= 6462.1[Q] + 0.977

6.50×1011

6.46×103

0.997

F

F

F

31

Ksv(L mol-1)

R2

Table 3. Interference of some foreign substances for 20.0 µmol L−1 INN.

Interferents

Tolerance level

Na+, K+, Cl−, CO32- , Br-, SO42-, NO3-

200

Glucose, Sucrose, Fructose, Urea

150

Glycine, Uric acid

100

Ascorbic acid, Citric acid, Starch, Mg2+, Ca2+

50

Fe2+, Zn2+

30

Tyrosine, Cysteine, Acetaminophen, Tryptophan, Fe3+*

20

32

*

After removal as described in the text.

Table 4. Determination results of INN in real samples (n=3).

Sample

Serum

Isotretinoin tablet

Added (µmol L−1)

Found (µmol L−1)

Recovery %

RSD %

-

-

-

-

10.0

9.8 ± 0.2

98.0

2.0

50.0

50.7± 0.6

101.4

1.2

-

19.3*± 0.5

96.5

2.6

(20.0 mg ( * in mg

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Table 5. Comparison of the proposed system with previous measurement methods for the determination of INN.

Method

Detection limit (µmol L−1)

Linear range (µmol L−1) Ref.

RP-HPLC

0.14

0.37-1997.0

[2]

HPLC–UV

0.02

0.07-2

[3]

HPLC

0.1

0.1-2.0

[5]

HPLC–ESI-MS

0.02

0.03-5.0

[7]

N -CQDs

0.03

0.08- 70.0

This work

RP-HPLC: Reversed- phase high-performance liquid chromatography HPLC–ESI-MS: High-performance liquid chromatography–electrospray ionization mass spectrometry

34