Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase

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Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots Based on Inner Filter Effect Guoliang Li, Huili Fu, Xuejie Chen, Peiwei Gong, Guang Chen, Lian Xia, Hua Wang, Jin-Mao You, and Yongning Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04193 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on January 29, 2016

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Analytical Chemistry

Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots Based on Inner Filter Effect

Guoliang Li *1,2, Huili Fu1, Xuejie Chen1, Peiwei Gong1, Guang Chen1, Lian Xia1, Hua Wang1, Jinmao You*1,3 and Yongning Wu *2

1

Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal

University, Qufu 273165, People's Republic of China 2

Key Laboratories of Chemical Safety and Health, China National Center for Food

Safety Risk Assessment, Beijing 100050, People's Republic of China 3

Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology,

Chinese Academy of Sciences, Xining 810001, People's Republic of China * To whom correspondence should be addressed

AUTHOR INFORMATION Tel.: 86-537-4456305; E-mail: [email protected] (G.L. Li); [email protected] (J.M. You) ; [email protected] (Y.N. Wu)

1

ACS Paragon Plus Environment


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Abstract A simple and sensitive fluorescent assay for detecting alkaline phosphatase (ALP) based on inner filter effect (IFE) has been proven, which is conceptually different from the previously reported ALP fluorescent assays. In this sensing platform, N-doped carbon dots (CDs) with high quantum yield of 49 % were prepared by one-pot synthesis and was directly used as fluorophore in IFE. P-nitrophenylphosphate (PNPP) was employed to act as ALP substrate, and its enzyme catalytic product (p-nitrophenol (PNP)) was capable of functioning as a powerful absorber in IFE to influence the excitation of fluorophore (CDs). When in the presence of ALP, PNPP was transformed into PNP and induced the absorption band transition from 310 to 405 nm, which resulted in the complementary overlap between the absorption of PNP and the excitation of CDs. Due to the competitive absorption, the excitation of CDs was significantly weakened, resulting in the quenching of CDs. The present IFE-based sensing strategy showed a good linear relationship from 0.01 to 25 U/L (R2 =0.996) and provided an exciting detection limit of 0.001 U/L (signal-to-noise ratio of 3). The proposed sensing approach was successfully applied to ALP sensing in serum samples, ALP inhibitor investigation and phosphatase cell imaging. The presented IFE-based CDs fluorescence sensing strategy gives a new insight on development of the facile and sensitive optical probe for enzyme activity assay because the surface modification or the linking between the receptor and the fluorophore is no longer required. Keywords：Carbon dots, Alkaline phosphatase, Inner filter effect, ALP inhibitor

Introduction Alkaline phosphatase (ALP) is an important biomarker for diagnostics, and the abnormal level of ALP in serum is closely related to various diseases including breast and prostatic cancer, bone disease, liver dysfunction and diabetes1-3. Therefore, the sensitive detection of ALP is of significant importance. Recently, organic molecules and nanomaterial-based signal amplification sensors have gained increased importance in achieving high sensitivity and selectivity of ALP detection with added benefits for rapid analysis and easy miniaturization4-9. Although each of these methods possesses 2


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their own features and gives the different insights for ALP sensing, these approaches usually suffer from some disadvantages including with poor photostability and solubility in water, high cost, requiring a complicated synthesis process, surface modification or using a colorimetric method 4-8. Furthermore, many reported methods are not sensitive enough to accurately screen ALP levels in human serums (40−190 U/L for adults), which greatly limits their practical application in medical diagnosis4,10,11. Thus, it is still challenging to develop ALP diagnostic assays with high sensitivity, selectivity, simplicity, less sample consumption and cost-effectiveness. Fluorescent carbon dots (CDs) have attracted rapidly growing attention in recent years due to their unique optical properties, excellent water dispersibility, chemical and photo stability, the low cost of fabrication and good biocompatibility 12-17, which have garnered much interest as potential competitors to conventional semiconductor quantum dots18. In the last few years, extensive research efforts have been devoted to engineering CDs to improve their quantum yields. Unfortunately, the majority of the CDs synthesized so far have quantum yields of below 10 % (Table S1). Meanwhile, many attempts have been made toward increase emission wavelength of CDs. But most of them emitted blue light (emission under 450 nm), limiting their application in cell imaging. In this study, we reported a simple approach for the preparation of cost effective and highly photoluminescent water-soluble CDs from organic compounds (catechol and ethylenediamine), by one-pot synthesis. It is worthy to note that the new CDs emitted strong green fluorescence (emission at 510 nm upon the excitation of 405 nm) and gave a higher quantum yield of 49 %. Moreover, the application of CDs in sensor development has also become prosperous

18,19

. But most of the reported CDs

were employed for metal ion or small molecule sensing 18,19. The further broadening of CDs application in biosensing is interesting and challenging 18. Inner filter effect is due to the absorption of the excitation or emission light by absorbers in the detection system when the absorption spectra of the absorbers overlaps with the fluorescence excitation or emission spectra of fluorophores. IFE has emerged as an efficient and useful strategy for the design and development of novel sensors by converting the analytical absorption signals into fluorescence signals20-22, which has 3



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been proven to commendably enhance the sensitivity and selectivity compared to other fluorescence quenching mechanisms (e.g. fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET)) 19-22. Meanwhile, IFE based sensor does not require the surface modification of CDs or establishing of any covalent linking between a receptor and a fluorophore, providing considerable flexibility and simplicity in probe fabrication. In this study, a new type fluorescent assay based on IFE via the new CDs was developed for simple, sensitive and selective detection of ALP, which was realized by the following steps. First, the green CDs without any modification were directly employed as the fluorophore with the task of fluorescent signal readout. Second, p-nitrophenylphosphate (PNPP) as a common substrate in colorimetric determination of ALP was capable of functioning as the ALP substrate in this sensing system. The maximum absorption wavelength of its ALP reaction products (p-nitrophenol, PNP) can well overlap with excitation light of the prepared CDs, and in the presence of ALP, the excitation of CDs was significantly weakened by the competitive absorption, resulting in the efficient quenching of CDs. The proposed sensing strategy provided the exciting detection limit of 0.001 U/L (signal-to-noise ratio of 3), which was 1−2 orders of magnitude lower than most of the recently reported sensing platforms 4, 11,13

23

. The

ultrasensitivity of our method may be ascribed to the high quantum yield of CDs and high molar absorptivity of PNP. When applied to serum sample analysis, it exhibited good applicability. This new method required as little as 10 µL of serum sample, which is particularly useful for samples storing and multiple serum information obtaining from the same sample during clinical studies. We further investigated the possibility of applying the established fluorescence approach for ALP inhibitor efficiency evaluation and phosphatase cell imaging. To the best of our knowledge, it is the first trail of employing IFE based fluorescent sensing strategy via fluorescent CDs for ALP sensing. The developed method shows many merits including rapidity, simplicity, lost cost, high sensitivity and excellent selectivity, which has a broad prospect in clinical diagnosis applications. Meanwhile, this study also provides a new insight on the application of CDs to develop the facile and sensitive biosensor. 4


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EXPERIMENTAL SECTION Instrumentation The fluorescence and the absorption spectra were recorded with a Hitachi F-7000 fluorescence spectrophotometer and a Hitachi UV-2910 spectrophotometer, respectively. Fourier transform infrared spectrometry (FT-IR) was conducted on a Nicolet Nexus 670 spectrometer using KBr pellets. The morphologies and sizes of N-doped CDs were characterized by transmission electron microscopy (TEM, Hitachi-600, Hitachi, Japan). X-ray photoelectron spectroscopy (XPS) analyses were carried out using a PHI 5702 spectrometer. Chemicals ALP (alkaline phosphatase), dehydrogenase(GDH), galactosidase (Gal), glucose oxidase (GOX), thrombin and amino acids (e.g. Pro, Tyr, Nleu, Phe, Asp, Trp, His, Ser, Val, Cys, Thr, Ileu, Arg, Glu, Lys, Gly, Ala, Orn, Met and Asn) were purchased from Sigma-Aldrich (Sigma-Aldrich Company, USA). Ethylenediamine, catechol, PNPP and sodium orthovanadate (Na3VO4) were of analytical reagent grade (Shanghai Chemical Reagents Corp., Shanghai, China). High purity water purified with a Milli-Q water purification system (Millipore, Molsheim, France) was used throughout the experiment. Other chemicals were analytical grade from Jining Chemical Reagent (Jining, Shandong Province, China). Preparation of N-doped CDs In brief, 300 µL of ethanediamine and 25 mL of 0.1 M catechol were mixed by ultrasonic and then transferred into a 50 mL teflon-lined autoclave and heated at 180 °C for 12 h. The resultant dark brown mixture was diluted by 20 mL water, and centrifuged to remove the large dots at 12000 rpm for 10 min, and then the supernatant was collected and dialyzed against ultrapure water through a dialysis membrane for 48 h. The as-prepared N-doped CDs were stored at 4 °C for use.

IFE-based Fluorescent Detection of ALP The IFE based fluorescent ALP activity assay was performed under the following procedures. A total of 10 µL ALP with activities from 0 to 100 U/L (or 10 µL of serum 5



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sample) was added into the reaction system (Tris-HCl, pH=9.0), which consisted of 1 mM PNPP and 0.1 µM MgSO4. The reaction solution was incubated at 37 °C for 30 min, after that the mixture was transferred to 1mL CDs solution and then subjected to fluorescence spectral measurements at the excitation wavelength of 405 nm. Cell imaging and cytotoxicity study Mouse macrophage cells (RAW264.7) were provided from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells were grown in high glucose Dulbecco's Modified Eagle Medium (DMEM, 4.5 g of glucose/L) supplemented with 10 % Fetal Bovine Serum (FBS). Fluorescent CDs (0.5 mg/mL) was added into the cells, and the cells were incubated for 20 min at 37 °C. The N-doped CDs loaded cells were washed to remove the excess CDs, and then PNPP (1 mM) was introduced into cells with an incubation time of 1 h and 2 h. Prior to imaging, removed the cell medium, and then washed cells with DMEM for three times. Fluorescent images of cells were acquired on an Olympus FluoView FV1000 laser-scanning microscope with an objective lens (×40). The emission intensities collected in optical windows were collected at 450 - 550 nm upon the excitation at 405 nm for CDs. RAW264.7 were seeded at 5 × 104 cells per well in 24-well plates and incubated for 24 h before treatment, followed by exposure to different concentrations of CDs for an additional 24 h. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)3,5-diphenytetrazolium bromide (MTT) colorimetric procedure. Each experiment was performed at least three times. The cytotoxic effect of the CDs was assessed through the ratio of the absorbance of the CDs treated sample versus the control sample. ALP Inhibitor Investigation In order to investigate the application of the developed fluorescent assay for ALP inhibitor evaluation, 50 µL of Na3VO4 aqueous solution with different concentrations 6


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(from 1 µM to 1 mM ) was added into the ALP reaction system (containing 1U/L ALP, 1 mM PNPP and 0.1 µM MgSO4) at 37 °C water bath for 30 min. The inhibitor treated ALP reaction solution was mixed with 1 mL CDs and then were recorded at the excitation wavelength of 405 nm by fluorescent spectra. RESULTS AND DISCUSSION Characterization of N-doped CDs The as-prepared N-doped CDs were characterized by TEM, XPS and FT-IR spectroscopy, respectively. The morphology of the N-doped CDs is showed in Figure 1A. TEM observation clearly confirms the successfully obtaining C-dots, which display a narrow diameter distribution (ca. 5–7 nm) and excellent monodispersity in aqueous solution (Figure 1A). XPS analysis suggests that the as-prepared CDs are composed of carbon, oxygen and nitrogen (Figure S1, Supporting Information). Combing the analytical results of XPS and FT-IR (Supporting Information), we deduced that the synthesized CDs are N-doped CDs, and have the predominant functional groups, including the hydroxyl, carbonyl group and amine group (Figure S1, Supporting Information). The detail characterization for CDs is presented in Supporting Information. The UV−vis spectra and fluorescence spectra of the N-doped CDs were investigated. The inset in Scheme 1A displays the photograph of N-doped CDs, which was obtained under UV light (365nm). As shown in Scheme 1A, strong green photoluminescence in water can be observed for the N-doped CDs. The UV−vis spectra depicts a maximum absorption peak at 405 nm is observed, and under this excitation wavelength, there is a strong emission peak at 510 nm (Figure 1B). The quantum yield (QY) of the new CDs was calculated using quinine sulfate as reference and the detail presents in Supporting Information. The QY was calculated to be 49 %. The high quantum yield may attribute to the new carbon source and the nitrogen-doping, which was higher than the recently reported CDs (Table S1). Principle of ALP Activity Assay Based on IFE We have demonstrated an IFE based fluorescent assay for facile and ultrasensitive sensing of ALP. The new CDs without any modification were directly employed as 7



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fluorophore with the task of fluorescent signal readout. PNPP was chosen as ALP substrate, and ALP hydrolysis product (PNP) was introduced as absorber. PNPP did not have interference on the excitation of CDs, after catalyzed by ALP, p-nitrophenol (PNP) was released, which induced the great absorbance around 405 nm and resulted in the quenching of CDs due to the IFE effect. PNPP is chosen as the ALP substrate, due to the following advantages: (a) PNPP as a common ALP substrate is cheap, easy to get, and with the excellent enzymatic utilization; (b) PNPP with a maximum UV absorbance at 310 nm has no effect on the excitation of CDs, while its ALP reaction product (PNP) shifts to 405 nm, which has a good overlap with the excitation spectrum of fluorescent CDs; (c) PNPP and its product PNP have good solubility in water. In order to prove this design, we incubated PNPP with different concentrations of ALP, and then the reaction solution was added to 1mL CDs solution and monitored the fluorescence changes of CDs. As shown in Figure S2, the fluorescence emission intensity has no change when without adding ALP, and a gradual decrease of the fluorescence intensity was observed with the increment of the concentration of ALP. The fluorescence intensity of CDs under an excitation wavelength of 405 nm was significantly quenched about 92 % upon an addition of 1 U/mL ALP (Figure S2), indicating the robust IFE on CDs fluorescence intensity and promising the sensitivity of the design. The UV absorption of the ALP reaction systems with different ALP concentrations was also recorded, and after reaction, the solution color obviously changed from colorless to yellow, and the absorptions at 310 and 405 nm were, respectively, decreased and increased gradually (Figure 2), which demonstrated the design of IFE based fluorescence assay. The corresponding color change of PNPP driven by ALP has been employed for the colorimetric sensing of ALP, but this colorimetric technique generally displays a lower sensitivity than that of the fluorescence sensing methods 24. By employing the means of the IFE effect of PNP on CDs, the highly selectivity and sensitivity for ALP detection can be achieved readily. In addition, by characterization of N-doped CDs, the N-doped CD was proven to possess amine groups in its surface. It is possible that between phenolic hydroxyl group of PNP and CDs can form complex formation by electrostatic attractive interaction, and then 8


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induced the fluorescence quenching of CDs. But we found that when the mixed solution was dialyzed, the CDs showed the recovered fluorescence. Meanwhile, in the ALP reaction system, Tris-HCl buffer was used, which introduced abundant compounds with amine groups into the sensing system, thus this deduction was not exist, further ensuring that the fluorescence quenching between PNP and CDs was caused by IFE not by electrostatic attractive interaction for complex formation or energy transfer. Analytical Performance of IFE based Fluorescent Assay for ALP Sensing The fluorescence intensity of CDs changes were recorded after adding different concentrations of ALP from 0.01 to 100 U/L. As shown in Figure 3, the fluorescence intensity of CDs decreased gradually with the increasing ALP concentrations, while the negative control without ALP could not cause changes. A good linear relationship between the relative fluorescence intensity (F0 - F1) and ALP concentration was obtained in the range of 0.01 to 25 U/L (Figure 3 inset, Y= 280.27 X + 112.03, R2 = 0.996). The developed sensing platform provided an ultralow detection limit of 0.001 U/L (signal-to-noise ratio of 3), and this sensitivity is enough for ALP activity assay in biological samples (the normal range of serum ALP in adults is about 40−190 U/L). To evaluate the analytical reliability and application potential, the proposed sensing method was employed to detect ALP in human serum samples. The serum samples were collected from six adult volunteers by Qufu People's Hospital. These samples were respectively submitted to be analyzed by the proposed sensing platform and the clinic method (Supporting Information). In clinical measurement, ALP serum activity was analyzed by molecular absorbance spectrophotometry, using an enzymatic method25. ALP serum activity was measured with a commercial reagent kit (Aeroset AlkPo ® E-3230) at 37 ºC and 405 nm by an automated biochemistry analyzer (Abbott Aeroset ®) 25. The analytical result is presented in Figure 4. The data analyzed by the two methods were almost consistent, and the results of our method and the clinic method were in the range of 43 to 107 U/L and 35-100 U/L, respectively. The recovery of added known amounts of ALP to serum samples was more than 97 %, suggesting the good accuracy. It is to be noted that the new sensing strategy yielded the satisfactory 9



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accuracy with the consumption of 10 µL serum samples, this low sample consumption was of vital importance in clinic studies. Furthermore, we compared our sensing strategy with the most recently reported approaches. Our method showed many advantages including ultralow sensitivity, simple operation process, easy to implement, and less sample consumption. For example, the ultralow detection limit of 0.001 U/mL was achieved, which was significant lower than the reported methods (e.g. 60 U/L4, 0.01 U/mL13, 1.4 U/L11 and 1.1 U/L23). Except for detection sensitivity, the developed method for clinic diagnose should be capable of with the simple operation process and easy to implement. In this study, the proposed sensing strategy avoided the complex surface modifications or construction of sensing probes in organic molecules probes or nanomaterial-based signal amplification sensors 4,11,13,23. The as-prepared CDs were used as the fluorogen, and the ALP reaction solution mixing with CDs were directly analyzed by fluorescence spectrophotometer. Thus, our IFE method was more sensitive and simpler process for ALP sensing. Selectivity of IFE Based Sensing Platform To explore the specificity of the proposed approach, the system was treated with several enzymes in serum including dehydrogenase(GDH), galactosidase (Gal), glucose oxidase (GOX), thrombin, other nonspecific proteins and free amino acids such as human serum albumin (HSA), immune globulin G (IgG) and amino acids (e.g. Pro, Tyr, Nleu, Phe, Asp, Trp, His, Ser, Val, Cys, Thr, Ileu, Arg, Glu, Lys, Gly, Ala, Orn, Met and Asn) under the same conditions (Figure S3). Meanwhile, the interference on fluorescence CDs from metal ions as well as others was also investigated including Mg2+, Zn2+, Cu2+, Fe3+,

K+, Na+, Ca2+, ascorbic acid, glutathione (GSH) and

glucose (Figure S3). The interference experiments indicated the above elements have negligible effect on the fluorescent intensity of CDs, indicating the satisfactory selectivity of the developed sensing approach. Application to ALP Inhibitor Investigation The possibility of applying the assay for evaluating the enzyme inhibitor efficiency was investigated. Na3VO4 as a common ALP inhibitor was employed for inhibiting 10


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assays. With the addition of ALP inhibitor to the assay solution, ALP activity can be inhibited, and fluorescence quenching would be hindered accordingly depending on the added amount of the inhibitor. Figure 5 shows that the fluorescence intensity decreased with the addition of increasing concentrations of the inhibitor. The inhibiting ability was expressed by inhibition ratio (I, %), and the regression equation was I (%) = , where FB is the initial fluorescence intensity in absence of ALP, F0 stands for the fluorescence intensity in presence of ALP without inhibitor, and FI is the fluorescence intensity in presence of ALP with the inhibitor. The regression equation was I (%) = 30.49 + 0.062X (µM, R2=0.996). The detection limit was estimated to be 0.1 µM for Na3VO4 at a signal-to-noise ratio of 3, indicating the ultra-sensitivity. These results clearly showed that our method can be used for the screening of trace ALP inhibitors in drug discovery. Application to Phosphatases Sensing in Living Cells CD as an up-and-coming nanomaterial has been widely employed to cell imaging due to their unique optical properties. But most of them are locked at low excitation (around 350 nm) and emission wavelength (under 450 nm), which limits their application in cell imaging. The as-prepared CDs improved the awkward situation in a certain extent because the emission was driven to green light. The murine macrophage cell line, RAW 264.7, was taken as model for cell imaging. Prior to assess the potential of N-doped CDs in cell imaging, the cytotoxicity of N-doped CDs was evaluated by the MTT assay. Figure S4 shows the viability of macrophage cell line (RAW 264.7) after incubation with the N-doped CDs at various concentrations for 24 h. The results clearly showed that the N-doped CDs exhibited the negligible cytotoxicity to living cells under experimental conditions. The good water solubility, low cytotoxicity and high QY of the N-doped CDs promised the cell imaging application. Firstly, RAW 264.7 cells were incubated with N-doped CDs (0.5 mg/mL, prepared by water) at 37 ℃ for 20 min, and their bright field and fluorescence images are given in Figure 6 A and B, which show a clear cellular contour and a good state, and no evident base fluorescence was observed in the corresponding dark field. As shown in Figure 6, the nucleus was not marked and 11



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the strong fluorescence was observed in the cytoplasm, confirming the high cellular uptake of CDs by RAW 264.7 cells. These results indicated that our CDs could easily cross the cell membrane and come into the cytoplasm of living cells within 20 min. Secondly, the N-doped CDs loaded cells were washed to remove the excess CDs, and then incubated with 1 mM PPNP for phosphatase sensing. As expected, PNPP was hydrolyzed to PNP by phosphatase, and due to IFE, the intensity of fluorescence from RAW 264.7 cells decreased gradually with the prolonging of time (Figure 6 B, D, and F), and after 2 h, weak fluorescence was observed (Figure 6F). In order to further prove the design, the proposed method was applied to sensing ALP in the human liver cells (SMMC-7721), and the satisfactory results were obtained in Figure S5. These results clearly demonstrate that the proposed IFE fluorescence strategy can expediently sense phosphatase in living cells. Conclusion We have demonstrated the development of a new IFE-based fluorescent assay for ALP sensing in buffer solution and living cells, in which CDs as an IFE fluorophore and the hydrolysate of ALP as an IFE absorber. The developed method was prove to be facile, sensitive and with less simple consumption, which provides a potential platform for ALP sensing in clinical diagnosis and trace ALP inhibitor screening in drug discovery. The presented IFE-based strategy in this study would open a new avenue for the facile and rapid enzyme activity detection using CDs nanomaterials, due to the avoiding the complex modification of the fluorophore or covalent bond linking between a receptor and a fluorophore. In the future, the potential of the as-prepared CDs would be exploited in constructing other fluorescence detection platforms. Acknowledgements This work was supported by The National Natural Science Foundation of China (No. 31301595, NO. 21275089 and 21475074) and the Natural Science Foundation of Shandong Province, China (ZR2013BQ019). Supporting Information Quantum yield measurement, characterization of N-doped CDs, ALP serum activity analysis by clinic method, comparison of synthetic methods and quantum yield of the recent CDs, Figures for the selectivity of ALP assays, cell viabilities of N-doped 12


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Figures

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Scheme 1 (A) Preparation procedures of N-CDs using ethanediamine and catechol as precursors by one-pot synthesis, (B) Working principle for ALP sensing based on inner filter effect

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Figure 1 (A) TEM image of the as-prepared N-doped CDs, (B) The absorption and fluorescence spectra of N-doped CDs

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Figure 2 Absorbance spectra of enzymatic reaction solution upon addition of various concentrations of alkaline phosphatase from 0.01 U/L to 200 U/L with 1 mM PNPP and 0.1 µM MgSO4 incubated at 37 °C for 30 min. The inset is the photograph of the enzymatic reaction solutions.

Figure 3 (A) Fluorescence responses of N-CDs upon addition of various concentrations of ALP (from top to bottom 0, 0.01, 0.025, 0.05, 0.1, 1.25, 2.5, 5, 10, 15, 25, 50, 80 and 100 U/L) with 1mM 17



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PNPP and 0.1 µM MgSO4 incubated at 37 °C for 30 min, (B) Plots of F0-F1 versus ALP concentration from 0 to 100 U/L (inset: its corresponding linear relationship from 0.01 to 25U/L ). F0 and F1 are the fluorescence intensities of CDs in the absence and presence of ALP, respectively. Fluorescence spectrum was measured with the excitation at 405 nm.

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Figure 4 Detection of ALP in six adult volunteer serums by our method and the clinic method. 10 µL of serum sample was added into the reaction system (pH=9.0, Tris-HCl buffer), consisting of 1 mM PNPP and 0.1 µM MgSO4, and then incubated at 37 °C for 30 min. The reaction solution was mixed with 1mL CDs. Fluorescence spectrum was measured with the excitation at 405 nm.

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Figure 5 Fluorescence intensity of N-doped CDs in the presence of various concentrations of Na3VO4 (from bottom to top 0, 40, 80, 100, 200, 400, 600, 800 and 1000 µM ) (A) and their calibration plots (B). The inhibitors were incubated with 1 U/L enzymes, 1mM PNPP and 0.1 µM MgSO4. Fluorescence spectrum was measured with the excitation at 405 nm.

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Figure 6 Confocal fluorescence microscopy of CDs (0.5 mg/mL) applied to RAW 264.7 cells. Bright field and fluorescence images of RAW 264.7 cells without PNPP (A and B), bright field and fluorescence images of RAW 264.7 cells in presence of 1 mM PNPP with an incubation time of 1 h and 2 h (C-D and E-F). The emission intensities collected in optical windows were collected at 450 - 550 nm upon the excitation at 405 nm for CDs.

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TOC graphic

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Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase

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