AIE nanoassemblies for discrimination of glycosaminoglycans and

Jun 27, 2019 - AIE nanoassemblies for discrimination of glycosaminoglycans and heparin quality control. Zhiyu Yang. Zhiyu Yang. More by Zhiyu Yang...
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AIE Nanoassemblies for Discrimination of Glycosaminoglycans and Heparin Quality Control Zhiyu Yang,† Xia Fan,‡ Wenjing Cheng,† Yubin Ding,*,†,§ and Weihua Zhang*,† †

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Jiangsu Key Laboratory of Pesticide Science, Department of Chemistry, College of Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China ‡ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China § State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, People’s Republic of China S Supporting Information *

ABSTRACT: The discrimination of glycosaminoglycans (GAGs) is a challenging task but of great importance to ensure their safe use in clinics. Herein, four supramolecular AIE nanoassemblies denoted as PDDA-TPE100, PDDA-TPE75, PDDA-TPE50, and PDDATPE25 were synthesized by loading different amounts of the negatively charged AIEgen TPE onto the surface of the positively charged polymer PDDA. These AIE nanoassemblies were utilized for the construction of a fluorescent sensor array, which was able to discriminate various GAGs on the basis of a compaction to displacement reaction mechanism. LDA and HCA results revealed that GAGs including Hep, Chs, HA, CTS, DS, and OSCS can be discriminated with 100% accuracy. The four-sensor array was then simplified to a three-sensor array using PDDA-TPE100, PDDATPE75, and PDDA-TPE50 as the sensors, which was determined to be still powerful in discrimination of various GAGs, accurate identification of unknown GAG samples, sensitive detection of trace OSCS contaminant in Hep, and identification of Hep and other biologically abundant anions. Moreover, the three-sensor array was even successfully applied for differentiating GAGs in serum media.

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INTRODUCTION Glycosaminoglycans (GAGs) are a family of unbranched linear polysaccharides involved in many biological functions.1,2 Some of the GAGs display numerous therapeutic activities and are used as drugs for treatment of various kinds of diseases.3,4 Particularly, heparin (Hep), with the highest negative charge density of all GAGs, is widely used in clinics, especially as an anticoagulant in surgery and kidney dialysis machines.5,6 Along with the clinical importance of Hep, there is a great deal of concern about the quality control of this unique biomolecule.7 These concerns come from the fact that Hep shares a similar main repeating disaccharide unit with other GAGs (Scheme S1); thus, it is technically challenging to discriminate between Hep and other GAGs.8,9 Moreover, it is vital to detect other potential GAGs contaminants in Hep before it is used in clinics.10,11 Although NMR and MS spectrometry and HPLC-based analytical methods are powerful for the characterization of GAGs and even detection of other GAG contaminants in Hep, these methods require expensive equipment and experienced data analysis skills.12,13 In recent years, the design and synthesis of novel small organic molecule based optical sensors has become a hot research topic, as they show the merits of fast response, high sensitivity, ease of operation, and even the ability of naked eye detection of various analytes.14−19 © XXXX American Chemical Society

However, the design of a specific receptor for Hep is very challenging, due to the fact that the structure of Hep has high degrees of heterogeneity, making the electrostatic and hydrogen-bonding interactions between the sensor and Hep too complicated to be fully understood.20−25 The selectivity of fluorescent sensors for Hep relies to a large extent on the highest negative charge density property of Hep. From this point of view, the capability of traditional fluorescent sensors based on the “lock-and-key” recognition mode for GAG sensing is very limited. As an alternative approach, cross-reactive sensor arrays which mimic the human gustatory and olfactory systems show the capability of discriminating similar analytes by recognizing their unique patterns.26,27 In this approach, there is no need to design specific receptors for target analytes, whereas the combined response of several arrayed sensors will generate different patterns for the identification of each analyte.28−31 Considering that the structures of GAGs are complicated and have high degrees of heterogeneity, pattern-based recognition could be considered as an ideal approach to discriminate different GAGs and identify their purity.32 However, literature Received: May 31, 2019 Accepted: June 27, 2019 Published: June 27, 2019 A

DOI: 10.1021/acs.analchem.9b02516 Anal. Chem. XXXX, XXX, XXX−XXX

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weight of its repeating unit (161.7 g/mol). UV−visible absorption spectra were recorded on a Shimadzu UV-2600 spectrophotometer. Fluorescence titration experiments were carried out using a Horiba Fluoromax-4 fluorescence spectrophotometer with a quartz cuvette (path length 1 cm). The slit width for fluorescence emission spectral measurements was set at 3 nm. The particle size was measured by dynamic light scattering (DLS) using a Marvin ZS90 Zetasizer. TEM images were recorded on a Hitachi HT7700 transmission electron microscope. The fluorescence emission intensity of a 96-well plate at 472 nm was recorded by a SpectraMax M5 microplate reader (λex = 340 nm). The compound TPE was synthesized according to the literature.43 Fluorescence Spectrum Measurements. All fluorescence emission spectrum measurements were carried out at room temperature. A stock solution of TPE (1 mM) was prepared with DMF, while stock solutions of PDDA (10 mM), Hep (3.2 mg/mL), Chs (0.64 mg/mL), HA (0.64 mg/mL), CTS (0.64 mg/mL), DS (0.64 mg/mL), and OSCS (0.64 mg/ mL) were prepared using 10 mM PBS (pH 7.4). The stock solutions were diluted by 10 mM PBS before fluorescence measurements. Discrimination Experiments. The four AIE nanoassemblies were synthesized by mixing PDDA (100 μM) and TPE (20 μM for PDDA-TPE100, 15 μM for PDDA-TPE75, 10 μM for PDDA-TPE50, and 5 μM for PDDA-TPE25) in PBS. Each nanoassembly occupied a single column successively in the 96well plate. For a 5 row × 4 column region in the above 96-well plate, each GAG was added at a set concentration to finally generate a 6 GAG × 5 replicates × 4 sensors training data matrix. The fluorescence emission intensity of the plate was then read by a microplate reader for further data analysis.

reports about sensor arrays for GAG pattern recognition are rare.33−37 The development of reasonable strategies for the design of sensor arrays for discriminating different GAGs and Hep quality control is still highly needed. Aggregation-induced emission (AIE) is a photophysical phenomenon observed for a unique family of propeller-shaped molecules, such as tetraphenylethene (TPE), which is nonemissive when it is dispersed in solution but emits bright fluorescence in the aggregated form.38 AIE molecules are considered to be ideal for designing fluorescent sensory materials and imaging agents.39−41 On the basis of our previous work about an AIE−peptide probe for Hep sensing,42 herein we propose a supramolecular approach to construct AIE fluorescent sensor arrays for the reliable discrimination of GAGs and heparin quality control. When a simple AIE molecule, such as TPE, was mixed with a commercially available positively charged polymer, such as poly(diallyldimethylammonium chloride) (PDDA), four supramolecular AIE nanoassemblies denoted as PDDA-TPE100, PDDA-TPE75, PDDA-TPE50, and PDDA-TPE25 were synthesized (Scheme 1). These AIE nanoassemblies can be utilized to Scheme 1. Compaction−Displacement Approach for Designing AIE Nanoassemblies for GAG Recognition: (a) Direct Displacement Process for High Loading; (b) Compaction to Displacement Process for Low Loading

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RESULTS AND DISCUSSION Considering that an aromatic carboxyl group has a pKa value of about 4.2, we envisioned that the compound TPE is negatively charged in neutral and alkaline solutions due to the dissociation of its −COOH group to generate −COO−. It will then be able to bind with positively charged molecules to form a supramolecular complex. We chose PDDA as the supramolecular host for TPE because PDDA is a large, positively charged polymer that can accommodate many TPE molecules, which will enable aggregation of TPE and generate strong AIE fluorescence. The obtained fluorescent nanoassemblies would be able to interact with specific analytes and act as sensory materials. When investigating the AIE properties of TPE, we discovered that its fluorescence stayed weak during changes in different fractions of PBS in DMF/PBS mixed solvent (Figure S1), indicating that it is indeed soluable in weakly basic PBS (pH 7.4) at the set concentration.44 Upon addition of PDDA to the PBS solution of TPE, significant fluorescence turn-on was observed with a maximum emission peak developed at ∼466 nm (Figure 1a). This phenomenon indicated the formation of a PDDA-TPE nanoassembly by aggregation of the TPE molecules on the surface of PDDA, resulting in “turn-on” of the AIE fluorescence of TPE. The reaction between PDDA and TPE was determined to be fast and can be accomplished within less than 1 min (Figure S2). The calibration curve suggested that about 100 μM of PDDA was needed to accommodate 20 μM of TPE and saturate the fluorescence changes (Figure 1b). On the basis of this result, four AIE nanoassemblies, denoted as PDDA-TPE100, PDDA-

construct fluorescent sensor arrays for effective discrimination of different GAGs in both PBS and diluted serum with 100% accuracy via a compaction to displacement mechanism. Moreover, the AIE nanoassembly based sensor array was also able to identify unknown GAGs accurately and detect the OSCS contaminant in Hep with a low detection limit of 1 wt %.

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EXPERIMENTAL SECTION Materials and Instruments. All reagents were of analytical grade and were used as received. Heparin (Hep) sodium salt from hog intestine was purchased from TCI (Shanghai) Development Co., Ltd. Chondroitin sulfate sodium salt (Chs) from bovine trachea and hyaluronic acid (HA) sodium salt from Streptococcus equi were purchased from Sigma-Aldrich. Chitosan (CTS, MW ≈ 30000) and dextran sulfate sodium salt (DS, MW ≈ 500000) were purchased from Shanghai Macklin Biochemical Co., Ltd. Fetal bovine serum (FBS) was supplied by Zhejiang Tianhang Biotechnology Co., Ltd. Poly(diallyldimethylammonium chloride) (PDDA) was purchased from Shanghai Titan Scientific Co., Ltd. The concentration of PDDA was determined by the molecular B

DOI: 10.1021/acs.analchem.9b02516 Anal. Chem. XXXX, XXX, XXX−XXX

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TPE75, PDDA-TPE50, and PDDA-TPE25, were obtained by changing the loading levels (100%, 75%, 50%, and 25%) of TPE on the PDDA host, respectively. We envisioned that differences in the loading level of TPE will have a significant influence on the sensing behavior of the AIE nanoassembly. Dynamic light scattering (DLS) characterization of these four AIE nanoassemblies indicated that their particle sizes increased almost linearly with higher loadings of TPE (Figure S3a−e). Transmission electron microscopy indicated that these nanoassemblies are particles with spherical morphology (Figure S3f). Keeping in mind that Hep is a highly negatively charged biomolecule and bears many hydrogen-bonding sites, we then checked whether Hep was able to displace TPE from the AIE nanoassemblies. When PDDA-TPE100 was titrated with Hep in PBS, the fluorescence emission intensity decreased immediately upon the addition of Hep and was almost fully quenched when 51.2 μg/mL of Hep was added (Figure 2a and Figure S4a). This fluorescence quenching behavior could be ascribed to the displacement of aggregated TPE molecules on PDDATPE100 with Hep (Scheme 1a). Interestingly, different fluorescence response behaviors were observed when other AIE nanoassemblies such as PDDA-TPE75, PDDA-TPE50, and PDDA-TPE25 were titrated with Hep (Figure S4b−d). For those AIE nanoassemblies, the addition of Hep initially enhanced the fluorescence intensity to a maximum value and then fluorescence quenching was observed when more Hep was added (Figure 2b−d). More importantly, it can be noticed that, with a lower loading of TPE, more Hep was needed to saturate the fluorescence enhancement process. This phenom-

Figure 1. (a) Fluorescence titration profile of TPE (20 μM) with increasing amounts of PDDA (0−140 μM) in 10 mM pH 7.4 PBS buffer solution. λex = 340 nm. Inset: photograph showing the fluorescence “turn on” behavior of TPE after addition of PDDA under a 365 nm UV lamp. (b) Corresponding calibration curve of PDDA.

Figure 2. Calibration curves of the AIE nanoassemblies with (a) 100%, (b) 75%, (c) 50%, and (d) 25% loading levels of TPE upon titration with Hep in 10 mM pH 7.4 PBS buffer. C

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Figure 3. Discrimination of GAGs (at 51.2 μg/mL) with the AIE sensor array: (a) fluorescence response patterns (fringerprints) of various GAGs, where each error bar shows the standard deviation of five replicates; (b) heat map derived from the fluorescence response patterns of tested GAGs; (c) HCA dendrogram of the AIE sensor array responses to GAGs; (d) LDA canonical score plots for the first two factors of response patterns of tested GAGs.

strongest fluorescence quenching response toward OSCS, followed by Hep, DS, Chs, HA, and CTS, while PDDA-TPE75 showed the strongest fluorescence quenching response toward DS, followed by OSCS, Hep, Chs, HA, and CTS and PDDATPE50 showed the strongest fluorescence quenching response toward OSCS, followed by Hep, DS, Chs, CTS, and HA. In addition, the combined fluorescence signals obtained from the four AIE nanoassemblies were dependent on each GAG. Groups of relatively low, medium, and high emission intensity signals were observed for Hep, Chs, and HA respectively. For OSCS, it generated the lowest group of emission intensities. For CTS, its reaction with PDDA-TPE100 showed the strongest fluorescence intensity among all GAGs. However, the emission intensity of CTS upon reaction with PDDATPE50 was lower than that of HA. It was also interesting to find that the emission intensity of DS upon reaction with PDDA-TPE50 was stronger than that with PDDA-TPE75, which is totally different from all other GAGs. These results indicated that the four AIE nanoassemblies of our array were cross-reactive toward the tested GAGs. The distinctive response patterns of each GAG generated by the AIE sensor array can be easily recognized in the heat map (Figure 3b). Hierarchical clustering analysis (HCA) was then applied to understand the origins of the response patterns. The dendrogram in Figure 3c shows that each kind of GAG occupied a separated cluster according to their Euclidean distance, suggesting the discriminatory power of the sensor array. It is evident that the tested GAGs were divided into mainly two groups in the HCA dendrogram. Analysis of the relationship between the dendrogram and the chemical structures of the GAGs can give some information on the

enon could be ascribed to the reason that the compaction state of the TPE molecules on the PDDA surface is dependent on their loading levels (Scheme 1b). At lower loading levels, the AIE molecule TPE was less compacted and the rotation of its phenyl groups was not fully inhibited, resulting in weak AIE fluorescence. However, the addition of a suitable amount of Hep led to tight compaction of the TPE molecule on PDDA, generating stronger AIE fluorescence, and further addition of Hep replaced TPE and quenched the AIE fluorescence. Since the AIE nanoassemblies with different TPE loading levels show different compaction to replacement response behaviors toward Hep, they could be considered for the construction of a fluorescent sensor array to discriminate GAGs. Therefore, six important GAGs, Hep, chondroitin sulfate (Chs), hyaluronic acid (HA), chitosan (CTS), dextran sulfate (DS), and oversulfated chondroitin sulfate (OSCS), were chosen for a test of the sensor array’s discrimination ability. Of these GAGs, Hep, Chs, HA, CTS, and DS were selected for investigation because they all play important roles in clinical usage and OSCS was selected because it is a potential artificial GAG contaminant in Hep.45 According to the titration study, which indicated that 51.2 μg/mL of Hep was needed to displace all TPE from the PDDA-TPE100 nanoassembly, GAGs at this concentration were initially submitted against our AIE sensor array for discrimination. By monitoring of the fluorescence emission intensity at 472 nm, distinctive fluorescence response patterns of above GAGs were generated by the AIE sensor array (Figure 3a). It can be seen that the fluorescence response signals toward different GAGs were dependent on each AIE nanoassembly. For example, PDDA-TPE100 showed the D

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clinically used Hep may lead to severe adverse reactions, including angiodema, hypotension, and even death.49,50 To investigate the ability of our three-sensor array’s ability to detect trace OSCS in Hep, various amounts of OSCS were spiked into Hep (weight %: 0, 1, 3, 5, 10, 20, 30, 40, 50) and then submitted to the three-sensor array for discrimination. When the total concentration of Hep and OSCS was set at 51.2 μg/mL, the obtained LDA plot showed that the cluster of pure Hep was clearly separated from the OSCS-contaminated samples, including that with only 1 wt % of OSCS contamination (Figure 4a). Moreover, an HCA dendrogram

discrimination mechanism (Scheme S1). The blue trees consist of three GAGs, including Hep, DS, and OSCS, which share a similar character of a relatively high degree of sulfonation. For the other three GAGs including Chs, HA, and CTS under the red HCA tree, they also share a common character of being rich in hydroxyl groups. This result indicated that the substituent types of the GAG may work as the principal driving force for discrimination. We then applied a statistical method, linear discriminant analysis (LDA), to test whether our AIE sensor array was able to differentiate these GAGs. Each GAG was parallel tested five times against the four AIE nanoassemblies to give a 6 GAGs × 4 sensors × 5 replicates training data matrix. After data processing, the 30 training GAG samples were separated into 6 groups with 100% accuracy. Four canonical factors were generated (97.06%, 1.76%, 1.02%, and 0.16%) that represent linear combinations of the data matrix. Since the cumulative percentage of the first two factors reached a value of 98.82%, a 2D canonical score plot was drawn with the first two canonical factors (Figure 3d). It can be seen from the 2D plot that the six GAGs were clearly clustered into six different groups and all groups were well separated from each other without any connection and overlapping, suggesting that our AIE sensor array was powerful for discriminating these GAGs. Moreover, GAGs at other concentrations (12.8, 25.6, 38.4, 64, 76.8, and 89.6 μg/mL) were also submitted to our sensor array to further evaluate the practicability of the sensor array. The result confirmed that the AIE sensor array was able to discriminate GAGs at various concentrations (Figure S5). To make a sensor array more practical, it is meaningful to reduce the numbers of the sensors in the array. Considering this, we noticed that the fluorescence intensity of PDDATPE25 is always low upon addition of various GAGs. The result suggested that this sensor exhibited a low contribution to the discrimination process. Thus, a simplified three-sensor array was constructed with PDDA-TPE100, PDDA-TPE75, and PDDA-TPE50. We were delighted to find that the simplified three-sensor array was also very powerful in discriminating GAGs at various concentrations (Figure S6). Different GAGs were successfully identified in separated clusters with 100% accuracy without obvious overlapping. Encouraged by this result, we used the three-sensor array constructed by PDDATPE100, PDDA-TPE75, and PDDA-TPE50 for further investigations. The simplified three-sensor array was then tested on its ability to discriminate unknown GAG samples. A total of 25 randomly prepared GAG samples was submitted to the threesensor array to give a data matrix (25 samples × 3 sensors × 1 replicate), which was then processed by LDA as the test data for discrimination. It was determined that 23 unknown samples were correctly identified and the accuracy rate was calculated to be 92% (Table S1). This suggested that the three-sensor array is reliable for identifying unknown GAG samples. Due to the fact that Hep is not artificially synthesized but is of animal origin, the purity of Hep is of great concern.46 After confirming the three-sensor array’s ability to discriminate various GAGs and identify unknown GAG samples, we further investigated whether the three-sensor array was able to detect trace GAG contaminants in Hep. Of all the potential contaminants, OSCS is the most special one.47 In comparison to other natural occurring contaminants such as Chs and HA, this artificially synthesized highly sulfated GAG is more harmful.48 It was reported that the presence of OSCS in

Figure 4. (a) LDA canonical score plots for the first two factors of response patterns of Hep and OSCS-contaminated Hep at a total concentration of 51.2 μg/mL. (b) HCA dendrogram of the threesensor array responses to Hep and OSCS-contaminated Hep at 51.2 μg/mL.

showed that Hep and OSCS-contaminated Hep was divided into two clusters according to their Euclidean distance (Figure 4b). In addition, Hep samples contaminated with 10% OSCS were also divided into two HCA trees. A similar result was obtained when the total concentration of Hep and OSCS was submitted at 38.4 μg/mL (Figure S7). These results indicated that the three-sensor array was powerful in the discrimination of pure and trace OSCS contaminated Hep samples, making the clinical use of Hep more safe. To ensure the practicability of the sensor array in biological systems, we then tested the ability of the three-sensor array to differentiate Hep from biologically abundant anions such as phosphate and pyrophosphate, with Na4P2O7, Na3PO4, and ATP as the examples. It was found that the cluster of Hep is far away from the clusters of Na4P2O7, Na3PO4, and ATP, and those biological anions were even successfully discriminated by the sensor array (Figure 5a). The discrimination of Hep from phosphates and pyrophosphate anions can be achieved with E

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PDDA-TPE100, PDDA-TPE75, PDDA-TPE50, and PDDATPE25 were utilized for the construction of an AIE sensor array which was able to discriminate various GAGs, including Hep, Chs, HA, CTS, DS, and OSCS, with 100% accuracy. More importantly, the four-sensor array can be simplified to a threesensor array using PDDA-TPE100, PDDA-TPE75, and PDDATPE50 as the sensors, which was still powerful in discriminating various GAGs, identifying unknown GAG samples, and detecting trace OSCS contamination in Hep with high sensitivity. The three-sensor array was even successfully applied for differentiating GAGs in serum media. In general, this work proposed a strategy for the construction of supramolecular nanoassembly based fluorescent sensor arrays for the effective and reliable discrimination of GAGs. We believe these results are meaningful for further development of practical sensor arrays for quality control of GAG drugs in clinics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b02516. Chemical structures of the GAGs, AIE properties of TPE, reaction speed between TPE and PDDA, DLS and TEM characterization data of the AIE nanoassemblies, fluorescence titration spectra, LDA analysis, and identification of unknown GAGs (PDF)

Figure 5. (a) LDA canonical score plots for the first two factors of response patterns of Hep and biologically abundant anions (at 51.2 μg/mL). (b) LDA canonical score plots for the first two factors of response patterns of Hep, Chs, HA, CTS, and DS (at 51.2 μg/mL) in PBS containing 2% serum.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for Y.D.: [email protected]. *E-mail for W.Z.: [email protected].

the three-sensor array at various concentrations with 100% accuracy (Figure S8). These results indicated that the threesensor array is promising for biological applications. Moreover, considering that GAGs such as Hep, Chs, HA, CTS, and DS exhibit numerous therapeutic activities and are widely used in clinics, it is important to identify these five GAGs in blood samples to avoid possible medical accidents. Therefore, Hep, Chs, HA, CTS, and DS were dissolved in diluted serum (2%) solution to mimic clinical samples and submitted to the threesensor array for discrimination. As shown in Figure 5b, it was determined that Hep, Chs, HA, CTS, and DS were clearly separated into five independent clusters in 2% serum with 100% accuracy. These results indicated that the AIE nanoassembly based sensor array could be considered for practical application in clinics.

ORCID

Yubin Ding: 0000-0002-2208-6509 Weihua Zhang: 0000-0003-1994-5306 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the Fundamental Research Funds for the Central Universities (KYTZ201604, KYZ201750), National Key R&D Program of China (2017YFD0200506), and the State Key Laboratory of Fine Chemicals (KF1810). We also thank Dr. Jing Wang at the Department of Mathematics at Nanjing Agricultural University for insightful discussions about the data analysis.

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CONCLUSIONS In conclusion, four AIE supramolecular nanoassemblies denoted as PDDA-TPE100, PDDA-TPE75, PDDA-TPE50, and PDDA-TPE25 were synthesized using the positively charged polymer PDDA as the host and the negatively charged AIEgen TPE as the guest by adjusting the loading levels of TPE. It was discovered that these four nanoassemblies showed different compaction to displacement fluorescence response behaviors toward Hep. Under low loading levels, the presence of Hep initially led to tight compaction of TPE and enhanced the AIE fluorescence, while a further increase in the amount of Hep displaced TPE and quenched the AIE fluorescence. On the basis of this reaction mechanism,

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REFERENCES

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DOI: 10.1021/acs.analchem.9b02516 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.9b02516 Anal. Chem. XXXX, XXX, XXX−XXX