Direct Determination of Redox Statuses in Biological Thiols and

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Functional Nanostructured Materials (including low-D carbon)

Direct Determination of Redox Statuses in Biological Thiols and Disulfides with Noncovalent Interactions of Poly(ionic liquid)s Wanlin Zhang, Yao Li, Yun Liang, Xianpeng Yin, Chengcheng Liu, Shiqiang Wang, Li Tian, Hao Dong, and Guangtao Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09413 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019

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ACS Applied Materials & Interfaces

Direct Determination of Redox Statuses in Biological Thiols and Disulfides with Noncovalent Interactions of Poly(ionic liquid)s Wanlin Zhang,†,‡ Yao Li,§ Yun Liang,† Xianpeng Yin,‡ Chengcheng Liu,† Shiqiang Wang,† Li Tian,† Hao Dong,† Guangtao Li*,† †Department of Chemistry, Key Lab of Organic Optoelectronics and Molecular Engineering, the Ministry of Education, Tsinghua University, Beijing 100084, P. R. China ‡Aerospace Research Institute of Special Material and Processing Technology, Beijing 100074, P. R. China §Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China KEYWORDS: biothiol, redox status, sensor array, poly(ionic liquid), photonic crystal

ABSTRACT: The three most important redox couples, including cysteine (Cys)/cystine (Cyss), homocysteine (Hcys)/homocystine (Hcyss) and reduced glutathione (GSH)/glutathione disulfide (GSSG), are closely associated with human aging and many diseases. Thus, it is highly important to determine their redox statuses at the following two levels: ⅰ) the redox identity in different thiols/disulfides and ⅱ) the redox ratio in a mixture of a specific couple. Herein, by using one

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single AIE-doped (AIE, aggregation-induced emission) photonic-structured poly(ionic liquid) sphere as a virtual sensor array, we realize a direct determination of the redox status without a reducing pre-treatment of disulfides, which will greatly promote the development of highthroughput and simple procedures. The pattern-recognition method uses the multiple noncovalent interactions of imidazolium-based poly(ionic liquid)s (PILs) with these redox species to produce differential responses in both the photonic crystal and fluorescence dual channels. On the one hand, a single sphere enables the direct and simultaneous discrimination of the redox identities of Cys, Cyss, Hcys, Hcyss, GSH and GSSG under the interference of other five commonly occurring thiols. On the other hand, this sphere also allows for not only a direct quantification of the GSH/GSSG ratios without previously determining the individual concentrations of GSH and GSSG, but also the accurate prediction of the ratios in unknown redox samples. To further demonstrate applications of this method, redox mixtures in a biological sample are differentiated. Additionally, quantum calculations further support our assignments for interactions between the imidazolium-based PILs and these redox species.

1. INTRODUCTION Owing to the generation of excessive reactive oxygen species in metabolic disorder processes, the manifested oxidative stress profoundly alters higher-order structures of proteins, which is considered to be closely associated with human aging and many diseases.1 Given that intracellular biothiols play a crucial role in combating oxidative stress and maintaining redox homeostasis by regulating the redox status between the reduced thiols and their oxidized disulfides, the determination of the redox status in thiols and disulfides has attracted extensive interest in recent years.2-8 Particular attention has been paid to three important redox couples, including cysteine


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(Cys)/cystine (Cyss), homocysteine (Hcys)/homocystine (Hcyss) and reduced glutathione (GSH)/glutathione disulfide (GSSG).9-11 The analysis of redox status in biothiols should be performed at two levels: ⅰ) determining the redox identity in different thiols and disulfides;12,13 ⅱ) determining the redox ratio in a mixture of a specific couple.14,15 For the former, since the abnormal alteration of the above six biothiols and disulfides are involved in different pathological processes and illnesses,16 the development of optical probes that can discriminate among these redox species is highly valuable for a better understanding of their respective molecular mechanisms and physiological functions.17 Based on the two unique properties of the S-H group including the strong nucleophilicity and the high binding affinity towards metal ions,18 a variety of optical probes have been designed for these three biothiols,19-26 but unfortunately only very few multi-functional probes can simultaneously distinguish Cys/Hcys/GSH because of their similarities in structure and reactivity.27-32 Meanwhile, detection of their disulfides (containing the S-S group) usually needs prior reduction to the thiol state,10,14,15 further rendering it an unmet challenge for the direct, simultaneous, and discriminative detection of these six biothiols and disulfides, particularly using only a single optical sensor. Besides the above first level, directly and quantitatively dissecting the extent of the reduction and oxidation in a mixture of thiol and its corresponding disulfide is another ongoing challenge. Despite the importance in determining the absolute concentration of GSH or GSSG, many studies have indicated that the assessment of the GSH/GSSG ratio is more meaningful as this ratio is a useful indicator of oxidative stress and health risk.33 Nearly all of the current methods for such ratios are indirect and require a determination of individual concentrations of GSH and GSSG, making them impractical for a high-throughput routine clinical assay. Methodologies that employ a direct detection procedure and minimal sample treatment are more useful.


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Limited by the aforementioned sensing principles of thiols and disulfides, the direct determination of the redox status in these three important biothiol couples has not yet been realized. Toward this end, the substantial development of new sensing materials and recognition mechanisms for this emerging frontier are very rewarding. Recently, several groups reported that imidazolium cations offer sites for formation of hydrogen bonds with various biomolecules including cysteine.34,35 Following this lead, we assumed that imidazolium-based ionic liquids (ILs) might be useful sensing materials for direct discriminations of redox status since ILs possess more abundant noncovalent interactions like hydrogen bonding, electrostatic forces, van der Waals forces, hydrophobic interactions, and π-π interactions.36-39 These interactions promote binding with not only thiols but also disulfides. Thanks to a cross-reactive characteristic,40 these IL based sensing materials are very suitable for an array based sensing protocol, which depends on collected fingerprint information and resultant response patterns are similar to those generated by our tongues and noses for taste and smell.41-48 Therefore, in this article, we used a single poly(ionic liquid) sphere with an inherent photonic structure49-51 and doped aggregation-induced emission (AIE) dye52,53 as a virtual sensor array. This array enables a direct, simultaneous, and discriminative detection of three important redox couples under interference from five commonly occurring thiols (structures see Chart 1). Additionally, this method also enables direct determination of GSH/GSSG ratios and estimation of these ratios in unknown redox samples. Finally, quantum calculations reveal an interaction foundation for a discriminative mechanism of the redox status in these species. Chart 1. Chemical structures of three important redox couples of biothiols/disulfides and five commonly occurring thiols.


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2. EXPERIMENTAL SECTION 2.1 Materials Biothiols and disulfides, including cysteine (Cys), cystine (Cyss), homocysteine (Hcys), homocystine (Hcyss), reduced glutathione (GSH), glutathione disulfide (GSSG), N-acetylcysteine (NAC), penicillamine (PA), 3-mercaptopropionic acid (MPA), 2-thioglycolic acid (TGA) and 2mercaptoethylamine (MEA), were obtained from Beijing Inno-Chem Science and Technology (Beijing, China) and used without further purification. The N2-saturated deionized water, 2-[4-(2hydroxyethyl)-1-piperazinyl]ethanesulfonic acid buffer (HEPES, pH = 7.2) and fetal bovine serum (FBS) were obtained from Alfa Aesar (Ward Hill, USA) and used for the preparation of target biothiol and disulfide solutions. The imidazolium-based ionic liquid monomer (1-propyl-3vinylimidazolium bromide), crosslinker (1,6-di(3-vinylimidazolium) hexane bromide), and AIE luminogen (AIEgen) used in this work were synthesized as described in the Supporting Information Scheme S1. The monodisperse silica particles with seven different diameters (140 nm,


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170 nm, 190 nm, 215 nm, 240 nm, 273 nm and 305 nm) were synthesized by a modified Stöber method.54 2.2 Characterization The sizes of silica nanoparticles and macropore structures were investigated with a scanning electron microscope (SEM) (JEOL, JSM-6460LV). The fluorescence intensities of AIE luminogen in good and poor solvents were measured using a fluorescence spectrometer (Perkin-Elmer, LS55). Reflection images of the template silica colloidal crystal spheres and AIE-doped photonicstructured poly(ionic liquid) spheres were captured by an optical microscope (OLYMPUS, 51M) equipped with a CCD camera (OLYMPUS, UTV0.5XC-3). Fluorescence images of the AIE-doped photonic-structured poly(ionic liquid) spheres were obtained using an inverted fluorescence microscope (OLYMPUS, IX71; excitation filter 330-385 nm; long-pass emission > 420 nm) equipped with a CCD camera (OLYMPUS, DP73). The reflection spectra and emission spectra of the poly(ionic liquid) spheres were recorded using an optical microscope coupled with a fiber optic detector (Ocean Optics, USB2000+). 2.3 Preparation of Poly(ionic liquid) Spheres The AIE-doped photonic-structured poly(ionic liquid) spheres were prepared using a general template replication method.55 First, template silica colloidal crystal spheres were prepared by a sophisticated droplet-based microfluidic technique. Then, the template silica colloidal crystal spheres were immersed in a polymerizable ionic liquid monomer solution which was composed of 0.30 g IL monomer, 0.018 g crosslinker, 100 μL AIEgen solution (3 mM) and 10 μL photoinitiator. Next, the above spheres were exposed to UV light for the polymerization of the monomer solution infused into the interparticle voids of the (opal) templates. Finally, after the removal of silica


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templates with hydrofluoric acid (2%), the resultant spheres with highly ordered 3D inverse opal structures and doped AIE luminogens were successfully harvested. 2.4 Collection of Response Signals Poly(ionic liquid) spheres were put into test tubes and incubated with N2-saturated target biothiol and disulfide solutions on an orbital shaker for 6 hours. Each sensing response to a target analyte was tested with seven individual spheres to establish reproducibility. The reflection images, fluorescence images, reflection spectra and fluorescence spectra of the spheres upon binding analytes were recorded on a microscope equipped with a fiber optic spectrometer in different detection modes. Bragg diffraction peak shifts (∆λ) were obtained from position after binding (λ1) subtracting position before binding (λ0): ∆λ = λ1 - λ0. Fluorescence enhancements for AIE luminogen ∆F/F0 at 515 nm (or at 554 nm) were calculated by obtaining fluorescence intensity before (F0) and after (F1) binding with saccharides at 515 nm (or at 554 nm): ∆F/F0 = F1/F0 - 1. 2.5 Data Analysis, Classification and Prediction A database of response signals was statistically analyzed using principal component analysis (PCA) and hierarchical cluster analysis (HCA) with unsupervised methods to classify each analyte. A cross-validation (leave-one-out) routine based on linear discriminant analysis (LDA) was used to evaluate predictability of the sensor array by leaving one observation out of a set and simultaneously utilizing the other data as a training set to generate a linear discriminant function. The classifications of every data point into different groups could be found from a jackknifed classification matrix. Additionally, prediction of unknown samples was done by a similar procedure to one mentioned above. PCA, HCA, and LDA were performed using software XLSTAT 12.0.


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2.6 Quantum Chemical Calculations. All structural optimizations were carried out with a Gaussian 09 package at the B3LYP/6311+g(d,p) theoretical level. Resulting geometries were confirmed by frequency to ensure no imaginary frequencies. Interaction energies were calculated at the same theoretical level with basis set superposition error (BSSE) correction. To further reveal the chemical origin of these interactions, reduced density gradient (RDG) analyses were performed with Multiwfn software. 3. RESULTS AND DISCUSSION 3.1 Preparation of Poly(ionic liquid) Spheres The AIE-doped photonic-structured PIL spheres were prepared by a general template replication method, and the IL monomer, crosslinker, and AIEgen used here could be seen in Figure 1a. First, using differently sized silica nanoparticles, various silica colloidal crystal spheres as sacrificial templates were readily generated via the droplet-based microfluidic technique (Figure S1). Then, a monomer solution was infused and polymerized in the voids of the templates. Finally, removal of the silica templates yielded uniform AIE-doped photonic-structured PIL spheres (Figure 1b and 1e) in large quantities (the insert image in Figure 1b). The top-view and cross-section SEM images of the obtained PIL spheres (Figure 1c and 1d) show periodic and interconnected macropore structure. This structure not only gives rise to vivid structural colors of an inverse opal because of the periodic pores, but also contributes to detection sensitivity due to high surface area and interconnected channels available for interacting with analytes. The solution of the synthesized dye in good solvents such as CH2Cl2 is almost non-emissive, while upon addition of poor solvents such as hexane into CH2Cl2, the fluorescence is turned on and the brightness of blue-light emission dramatically increases after aggregation (Figure 1f). Thus, the fluorescent dye with typical AIE


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characteristic makes these spheres fluorescent. This combination of photonic crystal (PhC) optical properties and fluorescence (FL) affords the dual channels to transduce the binding behaviors of PIL receptors with analytes into multiplexed optical outputs (Figure 1g). Besides, it was found that these silica colloidal crystal sphere templates and the molar ratios of IL crosslinker played significant roles in the stop-bands of the final poly(ionic liquid) photonic spheres (Figure S2 and S3). This indicates that there are great flexibility and extendibility in constructing various spherebased sensing elements.

Figure 1. (a) Schematic representation of the template replication method and structures of the used ionic liquid monomer, crosslinker and AIEgen. (b) Reflection image, (c) top-view SEM image, (d) cross-section SEM image and (e) fluorescence image of the AIE-doped photonic-


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structured poly(ionic liquid) spheres. Insert in (b) shows large quantities of PIL spheres and exposure time for (e) is 100 ms. (f) Fluorescence spectra verifying that the doped dye is AIE luminogen. Inserts are the fluorescence photographs of solution of dye in CH2Cl2 (left) and in 99% hexane/CH2Cl2 mixture (right) under illumination of a handheld UV lamp. (g) The left and right plots are reflection and fluorescence spectra of the dual-channel spheres. 3.2 Direct and Simultaneous Discrimination of Redox Identity The structural similarity among these biothiols, particularly for Cys and Hcys which differ by a single methylene unit, poses considerable difficulties to design highly specific probes for simultaneously distinguishing one thiol species from another. Unlike a lock and key based sensing strategy, differential sensing or sensor array technologies using cross-responsive sensing elements make it relatively easier to tackle such an intractable problem.41-48 Recently, several groups constructed sensor arrays for the discrimination of biothiols mainly on the basis of interactions between thiols and metal ions/gold nanoparticles.56-64 However, among them, only a few studies can enable a simultaneous discrimination of Cys, Hcys, and GSH.60,63,64 More seriously, an added oxidized form of these biothiols would unquestionably impede sensor arrays for full and direct discrimination of redox identity just considering the low affinity of S-S group towards metals and the similarity in disulfides such as Cyss and Hcyss. Thus, currently no work attempts to address such a significant but neglected issue. To this end, our first goal is to use a single of the above fabricated AIE-doped photonicstructured PIL sphere as a virtual sensor array for direct, simultaneous, and discriminative detection of three important redox couples in biothiols under interference from five commonly occurring thiols (Chart 1). The noncovalent interactions of these PILs with thiols and disulfides are diverse since their interactions are affected by not only the sulfhydryl and disulfide groups but


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also the amino group and carboxyl group. The inherent photonic crystal structure and the doped AIE luminogen in a single sphere provides dual-channel transducers to report binding, as shown in Figure 2. After interaction with these redox species, shrinking or swelling of the PIL photonic hydrogel spheres to different extents leads to the change of distance between two neighboring pores, and according to Bragg’s law, this results in diverse wavelength shifts of Bragg diffraction,65-68 whose changes can be detected by the naked eye (Figure 2a, c and e).

Figure 2. The optical responses of the poly(ionic liquid) spheres to 11 biothiols and disulfides at 100 μM. (a) Reflection images, (c) reflection spectra, (e) histogram of reflection peak shifts; (b)


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fluorescence images (false color, exposure time 30 ms), (d) emission spectra (integration time 200 ms), (f) histogram of the folds of fluorescence enhancements. Scale bars in (a) and (b) are 500 μm. Simultaneously, the alteration of chemical environments around the doped AIE luminogens was responsible for fluorescence enhancements in a typical light-on mode (Figure 2b, d and f). Interestingly, the responses of three biothiols Cys, Hcys, and GSH at both the PhC channel (Bragg peak shifts 39 nm, 135 nm and 24 nm, respectively) and the FL channel (fluorescence enhancements at 515 nm 3.99, 1.57 and 0.40, respectively) were very different. Additionally, the reduced thiol and its oxidized disulfide in a redox couple showed pronounced differences in dual signal channels, indicating that it is promising for direct discrimination of the redox identity without a prior reduction treatment of the disulfides. The recorded complex fingerprint information was analyzed by a statistical multivariate analysis method, principal component analysis (PCA), to generate characteristic patterns for convenient visualization. Intuitively, the PCA plot in Figure 3a depicts full discrimination of all of the 11 redox species and one buffer control. A jackknifed classification matrix with a leave-one-out validation routine demonstrates 100% accuracy (Table S1). The space distances among the clusters of the three biothiols in this plot are very large, showing a very high discriminative resolution for Cys, Hcys, and GSH. The reduced and oxidized forms in an arbitrary redox couple (Cys/Cyss, Hcys/Hcyss or GSH/GSSG) also display a prominent space resolution. Two pharmaceutically important thiol compounds (N-acetylcysteine, NAC; penicillamine, PA) which derive from Cys and three frequently used thiols (3-mercaptopropionic acid, MPA; 2-thioglycolic acid, TGA; 2mercaptoethylamine, MEA) do not produce any interference, exhibiting the charming of leveraging an overall structural features other than only the sulfhydryl group. Figure S4 shows how the transduction signals from two different principles are complementary and make the


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sensing information more efficient. For example, if only using an FL channel, the 11 redox species could not be completely distinguished from each other (Figure S5) and only a 75% correct identification was obtained (Table S2). The contribution of the PhC and FL channels to the principal components (PC1 and PC2) were estimated from the factor loadings (Table S3), indicating the necessity of dual-channel signals for the full discrimination with a miniaturized array. In addition, we further exploited hierarchical cluster analysis (HCA), another unsupervised clustering technique, to evaluate the obtained fingerprint database in a model-free fashion. Analytes of the similar response signals tend to be clustered closely with each other but apart from less similar ones. The resultant HCA dendrogram in Figure 3b also shows obvious discrimination of the target redox species and interfering thiols. Taken together, to our knowledge, this is the first case by using only one single sensing element to realize direct, simultaneous, and discriminative determination of the redox identity in the three most important biothiol/disulfide couples.

Figure 3. (a) PCA plot and (b) HCA dendrogram for the discrimination of 11 biothiols and disulfides at 100 μM by a single poly(ionic liquid) sphere. 3.3 Assay of Cys and Hcys at Different Concentrations


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As shown in Figure S6 and S7, the optical responses of these redox species are concentrationdependent. A crucial issue to be addressed is whether different analytes with varying concentrations will confuse the discrimination of the redox identity. As a representative demonstration, two subtly different biothiols Cys and Hcys at different concentrations were tested. The PCA plot in Figure 4 shows a clear separation for the clusters of Cys and Hcys at all of the tested concentrations ranging from 5 to 500 μM. As the concentrations increase, the space distances between Cys and Hcys become larger, indicating that the higher concentration makes discrimination easier. Remarkably, the distributions of each individual series of clusters for Cys and Hcys are scattered along the increasing concentrations in two different directions. This means that cases for Cys and Hcys at other concentrations would not cause any influence on their identities. Therefore, it is evidential that concentration variations would not make discrimination of the redox identity confusable.

Figure 4. PCA plot for the semiquantitative assay of Cys and Hcys at five different concentrations by the poly(ionic liquid) sphere. 3.4 Quantification and Prediction of Redox Ratio


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A typical process for assessing the GSH/GSSG ratio involves quantifying free GSH concentration, blocking free thiol, reducing disulfide, quantifying GSSG by the newly liberated GSH, and calculating the GSH/GSSG ratio. These methods usually suffer from several major drawbacks associated with complex sample processing, time-consuming implementation, and lowthroughput analysis. Thus, our second goal is to avoid these complicated procedures by directly quantifying the GSH/GSSG ratio, which has not been achieved previously. The multiple noncovalent interactions between PILs and GSH or GSSG make it possible for a direct determination of the redox ratio in a GSH/GSSG mixture. A semiquantitative assay for the known redox mixtures of GSH and GSSG at 11 different molar ratios (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 4:6, 3:7, 2:8, 1:9, 10:0) was performed and optical responses are presented in Figure S8. The PIL spheres exhibit that the Bragg peak shifts increase from 22 nm to 108 nm and the fluorescence enhancements at 515 nm change from 0.40 to 1.51, as the GSH/GSSG ratios increase from 0 to 1. The PCA plot in Figure 5a shows that clusters of all of these 11 different redox ratios, varying from 0 to 1, were successfully discriminated by a single PIL sphere.

Figure 5. (a) PCA plot for the semiquantitative assay of the GSH/GSSG mixtures with different redox ratios by the poly(ionic liquid) spheres. (b) The linear regression of the Euclidean distances


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versus the different GSH/(GSH+GSSG) molar ratios in the range from 0 to 1 for the quantitative analysis. The total concentration of GSH and GSSG is 100 μM. Importantly, a smooth and progressive trend in these clusters suggests a high possibility for a successful quantification of the GSH/GSSG redox ratio. Toward this end, the dependence of optical responses (illustrated by the Euclidean distances, EDs) on different redox ratios was evaluated using a linear regression analysis. Figure 5b displays a linear relationship between the ED values and the GSH/(GSH+GSSG) molar ratios in the range from 0 to 1. The obtained linear regression is warranted to realize the precise prediction of the redox ratios in unknown samples. Table S4 shows that six GSH/GSSG mixture samples with unknown redox ratios were correctly quantified with predicted errors less than 2%. Overall, we developed a direct method with a capability for high-throughput quantification and precise prediction of GSH/GSSG redox ratios, demonstrating great potential value in monitoring the oxidative stress. 3.5 Applications in Real-World Samples Encouraged by the above results, we further studied the performance of our PIL spheres with real-world samples. A typical real biological sample owns two possible features including the existence of mixtures (not merely the pure analyte) and the involvement of a complex background (a high likelihood of interference). For this goal, a series of thiol and disulfide mixtures in a fetal bovine serum background (10% FBS, v/v) as target analytes was investigated using our virtual sensor array. Figure S9 provides resultant response information. As shown in Figure 6, three biothiols and one disulfide (Cys, Hcyss, GSH and GSSG), together with all six of their binary mixtures, one ternary mixture and one quaternary mixture, are discriminated with 100% correct classification (Table S5). Additionally, it is generally accepted that an array based strategy can


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predict unknown samples if they have been adopted as training data sets.41 Herein, 12 blind samples from the 12 training analytes in Figure 6 were correctly classified with 100% predicted accuracy (Table S6).

Figure 6. PCA plot for the discrimination of 1 control analyte, 4 unitary analytes, and 8 mixture analytes in a 10% fetal bovine serum background by poly(ionic liquid) spheres. The concentration for all of the biothiols and disulfide was 100 μM. (A–E) represent Cys, Hcys, GSH and GSSG, respectively. Apart from the above practical application, we also found another interesting example. The high-throughput screening of a resting cell is of great importance in cancer research. In a resting cell, the molar ratio GSH/GSSG exceeds 100:1, which is considered an indicator of the quiescent state of a cell. However, the concentration of GSSG is often present several orders of magnitude lower than that of GSH, which causes trouble for this sensing purpose. As shown in Figure S10, a naked-eye approach was developed for the potential selection of a resting cell since the reflection images of PIL spheres are red, orange, and green upon an addition of a pure GSH sample, a GSH/GSSG mixture sample (redox ration 100:1),

and a pure GSSG sample in the FBS

background, respectively. Therefore, the visual color changes of the spheres from orange to red


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(corresponding Bragg peak wavelengths from 619 to 654 nm) might provide useful information for the screening of a resting cell in the future. 3.6 Theoretical Study of Discriminative Foundation To discover the nature of distinct responses of imidazolium-based poly(ionic liquid)s to various biothiols and disulfides, we used quantum calculations to study the underlying interactions, which lay a discriminative cornerstone for a direct determination of redox statuses. For a simplification of calculations, an ion pair, the basic repeating unit extracted from PILs, was utilized to interact with two redox couples including Cys/Cyss and Hcys/Hcyss. Four structures with their lowest energies optimized by density functional theory (DFT) are shown in Figure 7a-d. Their bond lengths indicate that the relatively acidic C2 hydrogens of the imidazolium rings form hydrogen bonds with these redox species just as reported.34,35 It can be seen that the interaction energies of the ILs with Cys and Hcys are different, which are responsible for the discrimination of the two homologues. Meanwhile, Cyss, the disulfide form of Cys, can directly interact with the ILs and exhibits a pronounced difference in the interaction energies of about 28 kJ/mol. For Hcys and Hcyss, the energy difference becomes larger and is about 35 kJ/mol. These results provide some preliminary understanding of why our strategy is suitable for a direct determination of redox statuses. A calculated interaction energy provides an overall assessment of a molecule’s contribution to an interaction strength. However, weak interactions besides hydrogen bonds aren’t shown, and the detailed role of every individual group of the molecule cannot be obtained. In this vein, a reduced density gradient (RDG) analysis was carried out to visually depict a distribution profile of various weak interactions in the real molecular space.69 The RDG isosurfaces in Figure 7e-h describe the weak interactions and their relative strengths between the IL moiety and the redox species. The


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green or earth green colors indicates the occurrence of weak interactions such as weak hydrogen bonding, van der Waals forces, and hydrophobic interactions. The red regions signify the repulsive interactions from steric hindrance of imidazolium rings and analyte skeletons. It can be conveniently found that different biothiols and disulfides with specific molecular structures lead to correspondingly distinct interaction patterns, which is responsible for a diversification of interaction energies and further results in an excellent discrimination of target analytes.

Figure 7. Optimized structures with the lowest interaction energies for the ion pair and two redox couples: (a) Cys; (b) Hcys; (c) Cyss; (d) Hcyss. Dashed lines denote hydrogen bonds, and the corresponding bond lengths are labeled. C atoms (gray), N atoms (blue), H atoms (white), O atoms (red), and S atoms (yellow) are shown in different colors for clarity. Corresponding reduced density gradient (RDG) isosurface (s = 0.5) for the ion pair and two redox couples: (e) Cys; (f) Hcys; (g) Cyss; (h) Hcyss. Green regions represent weak interactions while red regions represent repulsive interactions. 4. Conclusions


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Given the pathological importance of both the reduced biothiols (Cys, Hcys and GSH) and their oxidized disulfides (Cyss, Hcyss and GSSG), it is highly valuable to determine their redox statuses at the following two levels including the redox identity and the redox ratio. However, the involvement of disulfides in these sensing tasks is often accompanied by a reducing pre-treatment and other complex procedures, rendering impractical for a high-throughput routine clinical assay.70 It is found that the multiple noncovalent interactions of the imidazolium-based poly(ionic liquid)s with these redox species opened a brand-new avenue for the direct determination of redox status, which greatly simplified the sensing implementation. The developed single AIE-doped photonicstructured poly(ionic liquid) sphere could produce differential responses in both the photonic crystal and fluorescence channels. We first realized the direct, simultaneous and discriminative detection of the redox identities in the six biothiols and disulfides without a reducing pre-treatment. Very interestingly, the single PIL sphere also allowed for not only the direct quantification of the GSH/GSSG ratio without the necessity of knowing the individual concentrations of GSH and GSSG, but also the accurate prediction of the ratios in unknown redox samples with predicted errors less than 2%. Additionally, we further demonstrated the significant value of our PIL spheres in practical applications by a successful differential sensing of complex redox mixtures in a biological context. We believe that this method will promote a development of a high-throughput assay for redox status in biothiols and disulfides.

ASSOCIATED CONTENT Supporting Information. Synthesis and characterization of AIE luminogen, preparation and images of poly(ionic liquid) spheres using different opal templates and crosslinker ratios, various


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response histogram profiles, jackknifed classification matrix, illustration of the necessity of complementary signals, concentration dependent responses, prediction for the redox ratios and blind samples. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author [email protected]. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support from the NSFC (No. 21773135, 21473098, 21121004 and 21421064), MOST (2017YFA0204501, 2013CB834502) and the Deutsche Forschungsgemeinschaft DFG (TRR61). REFERENCES (1) Hwang, C.; Sinskey, A. J.; Lodish, H. F. Oxidized Redox State of Glutathione in the Endoplasmic Reticulum. Science 1992, 257, 1496−1502. (2) Yin, C.-X.; Xiong, K.-M.; Huo, F.-J.; Salamanca, J. C.; Strongin, R. M. Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys, Hcy, and GSH. Angew. Chem. Int. Ed. 2017, 56, 13188−13198. (3) Jiao, X.; Li, Y.; Niu, J.; Xie, X.; Wang, X.; Tang, B. Small-Molecule Fluorescent Probes for Imaging and Detection of Reactive Oxygen, Nitrogen, and Sulfur Species in Biological Systems. Anal. Chem. 2018, 90, 533−555. (4) Niu, L.-Y.; Chen, Y.-Z.; Zheng, H.-R.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. Design Strategies of Fluorescent Probes for Selective Detection among Biothiols. Chem. Soc. Rev. 2015, 44, 6143−6160.


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Table of Contents Graphic


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Direct Determination of Redox Statuses in Biological Thiols and

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