Turn-on Fluorescent

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Diketopyrrolopyrrole based ratiometric/turn-on fluorescent chemosensors for citrate detection in near-infrared region by aggregation-induced-emission mechanism Yandi Hang, Jian Wang, Tao Jiang, Niannian Lu, and Jianli Hua Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03715 • Publication Date (Web): 08 Jan 2016 Downloaded from http://pubs.acs.org on January 11, 2016

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Analytical Chemistry

Diketopyrrolopyrrole Based Ratiometric/Turn-on Fluorescent Chemosensors for Citrate Detection in Near-Infrared Region by Aggregation-Induced-Emission Mechanism Yandi Hang, Jian Wang, Tao Jiang, Niannian Lu, Jianli Hua* Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China. ABSTRACT: This work reports two new diketoprrrolopyrrole based fluorescent chemosensors (DPP-Py1 and DPP-Py2) using symmetrical diamides as recognition group for selective and fast detection of citrate in near-infrared region. To our delight, DPP-Py1 is a ratiometric sensor, while DPP-Py2 is a turn on fluorescent sensor. It is worth noting that the DPPPy1 has higher accuracy, sensitivity with a relatively lower detection limit (1.8 × 10-7M) and better stability in different pH buffers than DPP-Py2. Scanning electron microscopy, dynamic light scattering analyses, 1H NMR titration and 2D-NOESY NMR suggested that the fluorescence increment of the probes DPP-Py1 and DPP-Py2 for citrate could probably be originated from aggregation-induced- emission (AIE) on the basis of the complexation of the pyridinium based symmetrical diamides-DPPs with carboxyl anions of citrate. Our work may provide a simpler and faster mean for qualitative and quantitative analysis of citrate through AIE mechanism.

Citrate is an organic tricarboxylate that plays important roles in the biochemistry of human beings since it is one of the crucial metabolites in Krebs cycle (citric acid cycle) of virtually every aerobic cell.1 Its levels in biological fluids have been used to diagnose some pathological states, for instance, reduction of citrate in urine is associated to kidney dysfunction such as nephrolithiasis and glycogen storage disease.2,3 In addition, citrate levels are markedly decreased in malignant prostate cancer tissue and provide the most consistent characteristic change in the onset and progression of prostate cancer.4 Conventional analytical methods for determination of citrate in biological systems including gas chromatography, high performance liquid chromatography,5 polarography,6 spectrometry7 and capillary electrophoresis,8 which are complicated and time consuming or require expensive equipment. Consequently, to develop fast, simple and selective methods for citrate still attracts increasing attention in research field. Currently, fluorescent sensor as a novel technology has been widely used in testing ions,9-12 small molecules13 and imaging14,15 analyst in vivo due to its excellent sensitivity, visualization, easy operation and lower requirement of equipment. In recent years, some colormetric and fluorescent sensors for detection of citrate are reported,16-18 but many of them based on an Indicator-Displacement Assay (IDA) which generally contains two steps.19,20 Firstly, the fluorescent sensors can form complexes with metals result in changing fluorescent signals. Then, with the addition of citrate into the mixture, citrate can compete with the sensors for metal cations leading to a recovery of fluorescence. However, in this technique, analytes can be

detected only if their binding constant values with metal cations are higher than those between sensors and metal cations. As a result, these sensors recognize analytes indirectly and often show lower selectivity which hamper their application in biosystems. The aggregation-induced-emission (AIE) is a unique photophysical phenomenon observed by Tang and colleagues,21-23 which is completely opposite to conventional aggregation-caused-quenching (ACQ). Recently, a variety of AIE-active compounds are developed for fluorescent sensing24-27 and bioimaging.28-30 It is interesting that some molecules exhibit fluorescence both in solution and solid states due to conjugation-induced rigidity (CIR) in twisting molecules taking advantages of both ACQ and AIE luminogens.42 1,4-Diketo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP) and its derivatives are a class of excellent red fluorescent emission dyes with brilliant light, weather, and heat stability, which have been extensively applied in polymer solar cells,31, 32 OLEDs,33 organic solar cells,34,35 fluorescent probes,36-37 two-photon absorption,38 and dye sensitizing solar cell39-41 applications. In our previous works, DPP derivatives functionalized with electrondonating triphenylamine groups can exhibit red to nearinfrared (NIR) emission and large Stokes shift, which have been used in bioimaging. As far as we know, there are rare reports about sensors based on DPP through AIE mechanism.37 According to our previous work, the DPP derivatives which are modified with triphenylamine analogues have the possibility to exhibit the AIE phenomenon. Herein, we used the DPP functionalized with triphenylamine groups as fluorophores, pyridinium based symmet-

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rical diamides as a recognition group to synthesize two fluorescent sensors DPP-Py1 and DPP-Py2 (Scheme 1) for citrate detection. Under our design, both DPP-Py1 and DPP-Py2 would have AIE properties, but only DPP-Py2 is AIE active which is nearly non-emission in buffers but emits remarkable red fluorescence in NIR region when the citrate is added. However, it is interesting that DPPPy1 emitted strong yellow-green fluorescence in buffer without citrate and emerged a new stronger red emission after the addition of citrate. Meanwhile, the distinct variation of ratio two different luminescence indicates that DPP-Py1 can be used as a ratiometric sensor for detection of citrate. The remarkable changes of fluorescence of probes are ascribed to the formation of aggregation of DPP-Py1 and DPP-Py2 with citrate. The two new probes both exhibit good sensitivity and selectivity for citrate detection with a fast response (only several seconds) and can also be applied to determine citrate in biosystems.

EXPERIMENTAL SECTION General. 1H and 13C NMR spectra were recorded on a Bruker AM 400 MHz spectrometer with tetramethyl silane (TMS) as the internal reference. Absorption spectra were measured with a Varian Cary 500 UV-Vis spectra photometer. Fluorescence spectra were measured with a Horiba Fluoromax-4 Fluorescence spectrometer. Electrospray ionization and time-of-flight analyzer (ESI-TOF) mass spectra were recorded with a Waters Micromass LCT mass spectrometer. Matrix assisted laser desorption ionization and time-of-flight analyzer (MALDI-TOF) were recorded with an Applied Biosystems 4700 Proteomics Analyzer. The SEM micrographs were recorded with a JEOL JSM-6360 scanning electron microscope (SEM). N, N-dimethylformamide (DMF) was refluxed with calcium hydride and distilled before use. Tetrahydrofuran (THF) was pre-dried over 4Å molecular sieves and distilled under an argon atmosphere from sodium benzophenone ketyl immediately before use.

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guests of equal concentrations were prepared in the solvents used in the experiment. Then host and guest solutions were mixed in different proportions maintaining a total volume of 3 mL of the mixture. Then absorbance of the solutions of different compositions were recorded. The concentration of the complex, i.e., [HG], was calculated using the equation [HG] = ΔA/A0 × [H] where ΔA/A0 indicate the absorbance intensities. [H] corresponds to the concentration of pure host. Mole fraction of the host (χH) was plotted against concentration of the complex [HG]. In the plot, the mole fraction of the host at which the concentration of the host–guest complex concentration [HG] is maximum, gives the stoichiometry of the complex. For binding constant values, Binding constant values were determined by absorption methods using eqn. (1). A = (A0 + AKCG)/(1 + KCG)

(1)

Where A represents absorbtance intensity; A0 represents the intensity of pure host; CG are the corresponding concentration of the guest; K is the association constant. The association constants and correlation coefficients (R) were obtained by a non-linear least-square analysis of A vs. CG for eqn. (1). For sensitivity measurements, Stock solutions of DPPPy1 and DPP-Py2 were prepared in DMSO (1 mM). Citrate was dissolved in a 10 mM, pH 7.4 HEPES buffer (containing 10 mM HEPES). Fluorescence and absorption titrations were carried out by sequentially adding 0-80 µL aliquots of citrate solution to a 20 µL stock solution of DPPPy1 and DPP-Py2 followed by addition of 680 mL DMSO and an aqueous HEPES buffer to acquire a solution of 1.0 mL. The UV and FL measurements of the resulting solutions were then performed immediately. For calculation of LOD, Plotting the FL intensity (value of the emission maxima) as a function of citrate concentration for determination of the limit of detection (LOD). LOD were calculated by the equation 3δ/slope (δ is a standard deviation of the measured value of the emission maxima of blank solution for seven times).

3,6-bis(4-bromophenyl)-2,5-dihexyl-2,5-dihydropyrrolo [3,4-c]pyrrole-1,4-dione (compound 1)41 and 4-(bis(4methoxyphenyl)-amino)phenyl boronic acid37 were prepared according to previous literature protocols. All other reagents and proteins were purchased from SigmaAldrich and used as received. The solutions for analytical studies were prepared with deionized water treated with a Milli-Q System (Billerica, MA, USA).

For pH effect measurements, different pH ranging from 3-9 of HEPES buffer were prepared. Equal concentrations of citrate were added to DPP-Py1 or DPP-Py2 solutions with different pH of HEPES buffer.

UV-vis and fluorescence (FL) spectra. Stock solutions of DPP-Py1 and DPP-Py2 were prepared in DMSO (2 mM). Aliquots of the stock solution were transferred to 10 mL volumetric flasks. After addition of appropriate amounts of DMSO, water was added dropwise under vigorous stirring to furnish 4.0 × 10-5 M solutions with different water fractions (0-90 vol%). The UV and FL measurements of the resulting solutions were then performed immediately.

Scanning electron microscope (SEM). The SEM micrographs were recorded with JEOL JSM-6360. The samples used were prepared by dispersion on quartz plate, and then dried at room temperature prior to measurement.

For binding stoichiometries measurements, the stoichiometry was determined by the continuous variation method (Job Plot). In this method, solutions of host and

For selectivity measurements, equal concentrations of other anions were added to DPP-Py1 or DPP-Py2 solutions. The concentrations of the anions were 40 µM for DPP-Py1 and DPP-Py2, respectively.

H NMR titration 1H NMR titration spectra were measured via the addition of various equivalents citrate anion into DPP-Py1 or DPP-Py2 in DMSO-d6, respectively, at room temperature. 1


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Scheme 1. Synthesis of the target DPP-Py1 and DPP-Py2

RESULTS AND DISCUSSION Synthesis. The synthesis of target DPP-Py1 and DPPPy2 is shown in Scheme 1. In the first step, alkyl DPP core was functionalized with triphenylamine derivatives through a palladium-mediated Suzuki cross-coupling reaction to form compounds 2 and 3, which were then treated with bis(pinacolato)diboron to produce DPP intermediates 4 and 5. Palladium-mediated Suzuki crosscoupling reaction between DPP intermediates and 6 gave the 7 and 8, which were finally treated with 2-chloro-Nmethylacetamide to produce the desirable DPP-Py1 and DPP-Py2. All the new compounds were well characterized by 1H NMR, 13C NMR, and HRMS (Supporting Information). Primary optical measurements. With the compounds in hand, their AIE features were firstly measured using UV-vis and fluorescence (FL) spectroscopic techniques. The UV-absorption and FL spectra of DPP-Py1 in DMSO/water mixture solutions with different water fractions (fw, the volume percentage of water in DMSO/water mixtures) are shown in Figure 1. It was different from our initial anticipation that the DPP-Py1 exhibited strong fluorescence at 564 nm in pure DMSO and its absorption maximum (λabs) located at 500 nm (Figure 1a, b). However, when the water was added, the emission at 564 nm decreased, while a new emission around 650 nm emerged and increased dramatically but then decreased when fw > 50% (Figure 1c). This particular property may be caused by its structure. Both triphenylamine and DPP core are

luminophores. The independent triphenylamine has blue emission, while the DPP core which is not modified has orange emission. After the combination between the two fluorophores, their emissions in solutions are bathochromically shifted because of the intramolecular charge transfer effect. As the CIR effects in twisting molecules filling the gap of ACQ and AIE properties,42 the DPP-Py1 also has fluorescence in solid states. However, the solid emission of DPP-Py1 located at 650 nm which red shifts by approximately 90 nm. This phenomenon could be explained that the solid emission of the DPP core plays a major role because its fluorescence intensity is much stronger than the triphenylamine’s. Thereby, the aggregates formed gradually when the water was added slowly leading to the yellow-green emission decreasing and red emission increasing remarkably. In a sharp contrast, DPPPy2 was nearly non-emissive in pure DMSO and its absorption maximum (λabs) located at 505 nm (Figure 1d, e). When fw was 40 %, the obvious fluorescence was around 670 nm (NIR) emerged and saturated at fw = 60 %, indicating that DPP-Py2 is AIE-active. (Figure 1e, f). However, the fluorescence intensity decreased when the water fraction was higher than 60%. The phenomenon can be explained that the DPP-Py2 is an amphiphilic molecular and it may dissolve partly when the fw > 60%. In addition, the emission of DPP-Py1 and DPP-Py2 had little change (Figure 1c, f), when the fw = 30%, so we choose this water fraction as environmental condition for the detection of citrate.



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Figure 1. Normalized UV-absorption spectra of a) DPP-Py1 (4×10 5 M) and d) DPP-Py2 (4×10 5 M), and fluorescence (FL) emission spectra of b) DPP-Py1 and e) DPP-Py2 in DMSO with increasing water fractions (0-90 vol%). Plotting the FL intensity (value of the emission maxima) change of c) DPP-Py1 (emission maxima: 564 nm and 652 nm) and f) DPP-Py2 (emission maxima: 672 nm) in DMSO as a function of increasing water fractions.

Detection of citrate. With their primary AIE properties determined, we further probed the ability of DPP-Py1 and DPP-Py2 for detection of citrate in aqueous solution (DMSO/HEPES, 7/3, v/v, pH = 7.4, 10 mM HEPES). As shown in Figure 2, with the titration of citrate from 0-20 equivalents into DPP-Py1 solutions, a slightly decrease in fluorescence intensity at 564 nm was observed. In contrast, strong turn-on fluorescence intensity changes at 650 nm were found (Figure 2b), which demonstrated that DPP-Py1 was a ratio-metric sensor for citrate. Ratiometric measurements, which involves two signals, changes differentially with analysts concentration and the ratio of the signals are independent of the probe concentration and environment. In our case, we chose the I650/I564 ratio as the signal output when citrate was added. Actually, the I650/I564 ratio increased linearly when citrate concentration was 0 to 40 μM and the limit of detection (LOD) for citrate (Figure 2c) was determined to be 1.8 × 10-7M. Compared to DPP-Py1, DPP-Py2 behaved as a “turn-on” fluorescent sensor for citrate. The probe was nearly nonemissive in the absence of citrate, obviously, ﬂuorescence intensity of DPP-Py2 at 672 nm enhanced gradually by increasing the concentration of citrate and ultimate enhancement factor was about 10-fold (Figure 2e). Meanwhile, the detection produced satisfactory linearity at a low concentration (0-100 µM) and the limit of detection (LOD) for citrate (Figure 2f) was determined to 8.9 × 10-7 M. The detection ranges of the DPP-Py1 and DPP-Py2 were also determined at 0-5 mM and 0-20 mM, respective-

ly (Figure S1). Corresponding absorption titration experiments were also carried out (Figure 2a and 2d). Both absorption bands of compounds decreased gradually with the increasing of citrate, which implied that the DPP-Py1 and DPP-Py2 may form particles through the coordination with citrate. In addition, the absorption of DPP-Py1 not only decreased but also was bathochromically shifted by about 9 nm. For further verify the hypothesis that the enhancement of red fluorescence of DPP-Py1 and DPP-Py2 were attributed to the aggregation of the compounds by coordination of DPP-Py1 with citrate. Scanning electronic microscope (SEM) and dynamic light scattering (DLS) were used to visualize the morphology of the probes in the absence and presence of citrate. There were scarce nanoparticles could be observed without citrate, indicating that DPP-Py1 and DPP-Py2 were nearly dissolved in solutions (Figure 3a, c and Figure S2a, c). In sharp contrast, the size of particles of probes large increased after the addition of citrate, in which nanoparticles with an average diameter of 200 nm were observed by SEM as shown in Figure 3a and 3c. DLS measurements were further carried out to determine the hydro-dynamic particle size of the whole nanoparticle range, indicating that DPP-Py1 and DPPPy2 had a relative narrow size distribution with a mean size of around 248 nm and 219 nm (Figure S2b and S2d) in aqueous solution. The nanoparticle diameter obtained from the SEM were measured under vacuum state, while


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Figure 2. Titration absorption spectra of a) DPP-Py1 (4×10 5 M) and d) DPP-Py2 (4×10 5 M) in HEPES buffer (10 mM, 70% DMSO, pH 7.4) upon addition of 0.0−6.0 equivalents of citrate. Titration fluorescence spectra of b) DPP-Py1 (4×10 5 M) and e) DPP-Py2 in HEPES buffer (10 mM, 70% DMSO, pH 7.4) upon addition of 0.0−20.0 equivalents of citrate. c) Plotting the fluorescence intensity (ratio of I652/I564) as a function of citrate concentration for determination of the limit of detection (LOD) of DPPPy1. f) Plotting the fluorescence intensity (values of emission maxima) as a function of citrate concentration for determination of the limit of detection (LOD) of DPP-Py2. Inset (b): photographs of DPP-Py1 solution before and after addition of citrate a UV lamp with 365 nm incident light. Inset (e): photographs of DPP-Py2 solution before and after addition of citrate under a UV lamp with 365 nm incident light.

the DLS result was the hydrodynamic diameter of the micelle measured in the solution state. Therefore, the particle sizes tested by SEM was smaller than that by DLS. This suggests that the fluorescence increment of DPPPy1 and DPP-Py2 in NIR region could probably be originated from an AIE on the basis of the complexation of DPP-Py1 and DPP-Py2 with citrate (Scheme 2). In addition, the binding stoichiometries of DPP-Py1 and DPPPy2 with citrate were evaluated to be 1 : 1 by the Job method (Figure S3) and analysis of the absorbance data gave the binding constant values 3.11 ×105M-1 and 1.02 ×105M-1, respectively (Figure S4). 1

H NMR titration. The binding of DPP-Py1 and DPPPy2 (12.0 mM) with citrate was further understood through 1H NMR titration experiments. As can be seen in Figure 4, 1H NMR spectrum of DPP-Py1 before addition of citrate clearly showed the chemical shifts of pyridiniummeta-amide Ha proton (δ = 12.08 ppm), pyridinium ring protons Hb (δ = 9.65 ppm) and the amide protons Hc ( δ = 8.79 ppm). The amide Hc and pyridinium protons Hb of DPP-Py1 moved to the upfield obviously, while amide Ha died away with the increasing concentration of citrate.

Figure 3. Scanning electronic microscope imaging of a) DPP-Py1 and c) DPP-Py2 without citrate; b) DPP-Py1 and d) DPP-Py2 in the presence of citrate.

The similar chemical shifts of DPP-Py2 can also be observed in Figure S5. The 2D-NOESY was also used to show interaction between special hydrogens, in which the spec-



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tra indicated that only DPP-Py1 did not have this intermolecular NOEs (Figure S6a). However, there were NOEs between hydrogens (Hd) of methylene adjacent to carbonyl of DPP-Py1 and hydrogens (Hi) of methylene of citrate (Figure S6b), suggesting that Hi was close to the pyridinium based symmetrical diamides. The NOEs of DPP-Py2 before and after addition of citrate were similar to the DPP-Py1’s (Figure S6c and S6d). Therefore, SEM and DLS analyses, 1H NMR titration and 2D-NOESY NMR indicated that the fluorescence increment of the probes DPP-Py1 and DPP-Py2 for citrate could probably be originated from the complexation of the pyridinium based symmetrical diamides-DPPs with carboxyl anions of citrate (Scheme 2).

Figure 4. 1H NMR titration of DPP-Py1 with citrate in DMSO-d6. Ha is the hydrogen of amide which links the pyridine, Hb is the hydrogen of pyridine and Hc is hydrogen of amide which links the terminal methyl group (See Scheme 2)

Scheme 2. The suggested hydrogen binding structure and illustrate of the AIE mechanism

Figure 5. a) The ratio of I650/I564 of DPP-Py1 and b) fluorescence intensity variation of DPP-Py2 with or without citrate in the presence of a variety of carboxylates, Phosphate (such as ATP, ADP, PPi), some amino acids (such as Arginine and GSH) and metal ions (10 equivalents for DPP-Py1 and DPPPy2). Blue bar: Probes without citrate; Red bar: Probes with citrate. For the original FL spectra, see Figure S7.

The selectivity and pH influences of DPP-Py1 and DPP-Py2. In order to extend the probes’ application into biosystems, their selectivity and pH stability were investigated. To examine the selectivity, DPP-Py1 and DPP-Py2 were incubated with several other carboxylates (such as malate, tartrate), Phosphate (such as ATP, ADP, PPi), some amino acids (such as Arginine and GSH) and metal ions. As shown in Figure 5a and 5b, other analytes could not induce any remarkable change of fluorescence. By sharp contrast, the addition of citrate into solutions of DPP-Py1 and DPP-Py2 in the presence of other interferences lead to ev-ident enhancement of fluorescence in NIR region. Hence, DPP-Py1 and DPP-Py2 have good selectivity for citrate among other analytes. To examine the pH effects, several pH values ranging from 3 to 9 HEPES


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buffers were used to instead of initial HEPES buffer (pH = 7.4). We can observe from Figure 6a that the I650/I564 ratios of DPP-Py1 and DPP-Py1 with citrate remained almost unchanged in HEPES buffer with different pH values. Meanwhile, the fluorescence intensity of DPP-Py1 and DPP-Py2 with citrate fluctuated in a small area along with the change of pH values, but the intensity of DPP-Py2 still show an obvious enhancement after addition of citrate (Figure 6b). These results demonstrate that DPP-Py1 and DPP-Py2 are selective sensors for citrate and available for detection of citrate in biosystems.

Figure 6. a) The ratio variation of I650/I564 of DPP-Py1 and b) fluorescence intensity variation of DPP-Py2 in the different pH values of HEPES buffer ranging from 3.0-9.0 before and after addition 10 equivalents of citrate. For the original FL spectra, see Figure S7.

Preliminary analytical application. To further establish the real utility of the proposed probes in biosystems, we applied sensors in quantification of citrate in artificial urine samples. The artificial urine contains fundamental organic and inorganic salts in order to simulate an analogous condition of urine. The DPP-Py1 and DPP-Py2 were spiked different concentrations of citrate dissolved in artificial urine, respectively. The recovered citrate concentrations were determined according to the lowconcentration plotting shown in Figure 2c and 2f. The result shows good recovery values by using DPP-Py1 and DPP-Py2 as probes (average deviation = 2.14 % and 2.38 %, respectively. Figure 7), which confirmed that the DPP-Py1 and DPP-Py2 sensors had potential capacity to quantify citrate in real biological systems. Since concentration of citrate in serum can increase to about 2 mM in critically ill patients,43, 44 we then explored the two probes for detection of citrate in serum samples. Different concentrations of citrate dissolved in pretreated fetal bovine serum (FBS) were prepared. The fluorescence spectra were carried out by sequentially adding different aliquots of citrate solution to DPP-Py1 and DPP-Py2 in HEPES buffer. A ﬁnal concentration of citrate was 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mM, respectively. The results were shown in Figure S8, although the signals were interfered slightly by serum, the sensors could still qualitatively detected citrate in serum samples, which means that DPP-Py1 and DPP-Py2 are potential in detecting citrate qualitatively in serum as its level is relevant for human health.

Figure 7. The spiked and measured concentrations of citrate by a) DPP-Py1 and b) DPP-Py2 in HEPES buffer ((10 mM, 70% DMSO, pH 7.4), respectively. a) Black bar: the spiked concentrations of citrate, from left to right: 10.0, 20.0 and 40 μM; Red bar: the measured concentrations of citrate, from left to right: 10.22, 19.43, and 40.76 μM). The average deviation is 2.14 %. b) Black bar: the spiked concentrations of citrate, from left to right: 20.0, 40.0 and 80 μM; Red bar: the measured concentrations of citrate, from left to right: 19.52, 41.21, and 78.61 μM). The average deviation is 2.38 %. Citrate solution were prepared in urine, both DPP-Py1 and DPP-Py2 were 4.0 × 10-5M.

CONCLUSION In summary, two new DPP-Py1 and DPP-Py2 fluorescent sensors for detection of citrate were developed. Compared with the conventional IDA method, our method is much faster (consume serval seconds) and easier to carry out. It is worth noting that DPP-Py1 as a ratiometric sensor has higher accuracy, sensitivity with a relatively lower detection limit (1.8 × 10-7M) and better stability in different pH buffers than DPP-Py2 which is a fluorescence turn on sensor. The red fluorescence variation of DPP-Py1 and DPP-Py2 could probably be originated from aggregation-induced-emission on the basis of the complexation of the pyridinium based symmetrical diamidesDPPs with carboxyl anions of citrate, which were confirmed by absorbance analysis, 1H NMR titration, 2DNOESY NMR, SEM and DLS images. Also, the utility of DPP-Py1 and DPP-Py2 in quantitation citrate in urine and serum indicates the promise of probes for the detection of citrate in real biosystems. To sum up, our work may provide a simpler and faster mean for qualitative and quantitative analysis of citrate.

ASSOCIATED CONTENT Supporting Information Synthetic procedures of DPP-Py1 and DPP-Py2, supporting Figures S1-S8, and compound characterization data (1H NMR, 13C NMR, and ESI-TOF mass). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]


Analytical Chemistry Notes

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The authors declare no competing financial interest.

ACKNOWLEDGMENT For financial support of this research, we thank the National Basic Research 973 Program (2013CB733700), and NSFC/China (21421004, 21172073, 21372082, 21572062 and 91233207).

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