Discrimination of Metalloproteins by a Mini Sensor Array Based on

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Discrimination of Metalloproteins by a Mini Sensor Array Based on Bispyrene Fluorophore/Surfactant Aggregate Ensembles Yuan Cao,† Lijun Zhang,† Xinyan Huang,‡ Yunhong Xin,‡ and Liping Ding*,† †

Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, and ‡School of Physics and Information Technology, Shaanxi Normal University, Xi’an 710062, P. R. China S Supporting Information *

ABSTRACT: Fluorescent sensor arrays with pattern recognition ability have been widely used to detect and identify multiple chemically similar analytes. In the present work, two particular bispyrene fluorophores containing hydrophilic oligo(oxyethylene) spacer, 6 and 4, were synthesized, but one is with and the other is without cholesterol unit. Their ensembles with cationic surfactant (CTAB) assemblies realize multiple fluorescence responses to different metalloproteins, including hemoglobin, myoglobin, ferritin, cytochrome c, and alcohol dehydrogenase. The combination of fluorescence variation at monomer and excimer emission of the two binary sensor ensembles enables the mini sensor array to provide a specific fingerprint pattern to each metalloprotein. Linear discriminant analysis shows that the two-ensemble-sensor-based array could well discriminate the five tested metalloproteins. The present work realizes using a mini sensor array to accomplish discrimination of complex analytes like proteins. They also display a very high sensitivity to the tested metalloproteins with detection limits in the range of picomolar concentration. KEYWORDS: supramolecular assemblies, bispyrene, surfactant, aggregate, metalloprotein

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INTRODUCTION Development of novel fluorescent sensors for sensing and recognizing proteins and reporting their presence is important for applications in proteome research, clinical diagnostics, and biomedical research, etc. Almost half of the known proteins and enzymes must associate with a particular metal to function.1 For example, hemoglobin (Hb) and myoglobin (Mb) are ironcontaining proteins and perform important roles in oxygen binding and transport.2 Ferritin (Ferr) is a very important iron storage protein and plays key roles in a variety of biological functions such as aerobic metabolism and homeostasis of ferrous ion level.3 Another iron-containing metalloprotein, cytochrome c (Cyt-c), is a common guest in biological protein recognition processes and works not as an enzyme but as an electron carrier in biological respiration.4 In humans and many animals, as a zinc-containing metalloprotein, alcohol dehydrogenase (ADH) serves to break down alcohols that otherwise are toxic, and they also participate in generation of useful aldehyde, ketone, or alcohol groups during biosynthesis of various metabolites.5 © XXXX American Chemical Society

In addition to genetic and other biological methods, many chemical efforts have been devoted toward sensing and discriminating of biological proteins. Even a relatively small protein, its structure is too complicated to be easily recognized by common synthetic receptors.4 Thus, the development of designed receptors brings enormous challenges in chemical, biological, and medical research. To overcome this challenge, fluorescent sensor arrays have been widely used to detect and identify multiple chemically similar analytes by simulating mammalian nose or tongue.6,7 Sensor arrays are usually composed of a series of nonspecific or semiselective sensing elements that can generate a combined recognition pattern to each analyte.8 This array-sensing method not only recognizes different analytes9 or identifies different analytes in mixed samples10 but also realizes quantitative analysis.11 As a consequence, it has attracted intensive attention and has been Received: October 5, 2016 Accepted: December 2, 2016 Published: December 2, 2016 A

DOI: 10.1021/acsami.6b12646 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Synthesis of Bispyrene-Based Fluorophores 4 and 6

Thus, in the present work, we devoted to developing a mini fluorescent sensor array containing only two sensor elements to realize discrimination of different metalloproteins. Two specially designed bispyrene fluorophores 4 and 6 were synthesized and formed ensembles with cationic surfactant (CTAB) assemblies to act as sensor element. The structure of 4 and 6 is illustrated in Scheme 1. The two fluorescent binary sensor systems, namely, 6/CTAB and 4/CTAB, display both monomer and excimer emission in aqueous buffer solutions. Moreover, the combination of the fluorescent responses at monomer and excimer wavelengths of both binary sensors can generate distinct recognition patterns to the five tested metalloproteins. Linear discriminant analysis (LDA) found that the two-element mini sensor array could well discriminate the five metalloproteins.

applied for sensing and recognizing proteins over the past decade. For example, various fluorescent materials such as small molecules,12,13 conjugated polymers,14,15 quantum dots,16 and nanoparticles17−19 have been used to construct multiple element-based sensor arrays for discriminating proteins. For these methods, a number of different sensor elements were used, and some reports even employed different fluorophorebased sensor elements, which usually leads to complex data collecting process and tedious synthesis efforts. Thus, simple methods for constructing fluorescent sensor arrays or developing small-number sensor arrays are highly demanded. Surfactants are well-known amphiphilic molecules and can form various supramolecular assemblies (e.g., micelles and vesicles) in aqueous solutions.20,21 These assemblies are heterogeneous and possess a hydrophobic core that could encapsulate hydrophobic fluorophores. Surfactant assemblies have shown advantages in term of improving photophysical properties of the encapsulated fluorophores, such as enhancing their fluorescence stability and fluorescence quantum yield in aqueous media and efficiently modulating their fluorescence emission.22,23 Moreover, they can provide noncovalent interaction with various analytes. Thus, the strategy of using surfactant assemblies encapsulating fluorophores provides advantages in term of simplicity, modulating ability, and functioning in aqueous solutions. Up to now, this method has been explored in constructing fluorescent sensor arrays for detecting various targets such as metal ions,9 explosives,24 and even proteins.12,25−28 However, low-element-number arrays based on this methodology are still less developed for discriminating proteins or metalloproteins. Our group has focused on using surfactant assemblies to construct fluorescent sensors22,23,29 and found that the employment of a particular fluorophore with multiple emission bands could empower the supramolecular binary sensor system with discriminative ability.9,30 Pyrene is well-known in exhibiting monomer and excimer emission according to its different aggregate states. We used anionic surfactant SDS assemblies encapsulating three structurally similar bispyrene fluorophores and prepared a three-element sensor array for discriminating lanthanide ions.9 We employed cationic surfactant DTAB assemblies to encapsulate a bispyrene fluorophore and realized discrimination among three different types of proteins.31 However, it is hard to identify different proteins belonging to the same type.

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EXPERIMENTAL SECTION

Chemicals and Instruments. 1,2-Bis(2-aminoethoxy)ethane (EOA, 98%) and cyanuric chloride (CC, 99%) were purchased from Sigma-Aldrich Co. and used without further purification. Pyrene (Alfa, 98%) was used after recrystallization from ethyl alcohol. N,NDiisopropylethylamine was purchased from J&K Scientific Ltd. Analytically pure THF and CHCl3 were dried with anhydrous CaCl2 overnight before use. Aqueous solutions were prepared from Milli-Q water (18.2 MΩ cm at 25 °C). All other reagents are at least analytically pure. 1 H and 13C NMR spectra were measured on Bruker AV 400 MHz NMR spectrometer. The high-resolution mass spectra (MS) were acquired in ESI positive mode using Bruker maxis UHR-TOF mass spectrometer. The FTIR spectra were measured on a Fourier transform infrared spectrometer (Vertex 70v, Bruker, Germany). UV−vis absorption spectra were recorded on a spectrophotometer (U3900, Hitachi Instrument). Steady-state fluorescence measurements were collected using a single photon counting fluorescence spectrometer (FS5, Edinburgh Instruments, UK) with xenon light (150 W) as the excitation source at room temperature (rt). All samples were excited at 353 nm. Time-resolved fluorescence measurements were collected with the time-correlated single photon counting fluorescence spectrometer (FLS920, Edinburgh Instruments, UK). All of the solutions were excited by a laser light (343 nm), and the emission wavelength was fixed at 498 nm. The particle size distribution and dynamic light scattering were measured on a Malvern Zetasizer Nano-ZS90. Surface tension was measured using a Dataphysics DCAT 21 tensiometer equipped with a Wilhelmy plate at 25 °C. Linear discrimination analysis (LDA) of the fluorescence variation was performed using a commercial available software MATLAB2010b with B


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Figure 1. UV−vis absorption and steady-state fluorescence emission spectra of 4 and 6 (1.0 μM) in (a) THF and (b) water (λex = 351 nm). an in-house written program. The detailed written program is provided in the Supporting Information. Synthesis of Bispyrene Fluorophores (4 and 6). The synthesis process of the two bispyrene fluorophores is depicted in Scheme 1. Cyanuric chloride was used to supply three reactive sites, the two of which were connected to two pyrene moieties via a hydrophilic oligo(oxyethylene) spacer to provide fluorophore 4. A cholesterol unit was further used to modify the third reactive site via a diamine spacer to get the other fluorophore 6. Compounds 3 and 5 were synthesized by adopting literatures methods.32,33 The synthesis procedure of compound 4 is as follows: A solution of compound 3 (1.15 g, 2.79 mmol) in THF (50 mL) was added dropwise to a solution of cyanuric chloride (0.25 g, 1.33 mmol) and N,N-diisopropylethylamine (974 μL, 1.25 mmol) in THF (20 mL) at rt under stirring and N2 atmosphere over a period of 4 h, after which the reaction system was stirred at room temperature for extra 48 h. Then the mixture was evaporated under reduced pressure. The resulting yellow-green oil was purified by column chromatography on silica gel column with PE:EA (v:v, 5:1) as eluent. Compound 4 was obtained as a yellow solid after freeze-drying (773 mg, yield 62%). 1H NMR (400 MHz, CDCl3): δ 8.97 (dd, J = 11.6, 8.0 Hz, 2H), 8.68 (t, J = 8.9 Hz, 2H), 8.41−7.90 (m, 14H), 7.14 (t, J = 5.8 Hz, 1H), 6.85 (s, 1H), 6.31−5.82 (m, 2H), 3.70−2.77 (m, 24H). 13C NMR (101 MHz, CDCl3, ppm): δ 169.96, 165.52, 141.63, 134.45, 131.91, 131.73, 131.11, 129.14, 127.90, 127.76, 127.69, 127.33, 125.99, 125.53, 122.45, 121.11, 72.34, 71.55, 69.85, 43.52, 39.75. FTIR (KBr plate, cm−1): 3277 (−NH), 3044 (Ar−H), 2922 (−CH2), 1588 (Ar CC), 1325 (−C−N), 1159 (OSO), 1095 (−C−O−C). MS (ESI) [M + H]+: calcd for C47H46ClN7O8S2, 936.2611; found, 936.2490. The synthesis procedure of compound 6 is as follows: compound 4 (0.42 g, 0.44 mmol) and compound 5 (0.29 g, 0.62 mmol) were added into THF (50 mL) under stirring and N2 atmosphere and then refluxed over a period of 72 h. After this, the mixture was cooled to room temperature and then evaporated under reduced pressure. The resulting yellow-green oil was purified by column chromatography on silica gel column with CH2Cl2:CH3OH (v:v, 35:1) as eluent. Compound 6 was obtained as a yellow solid after freeze-drying (443 mg, yield 73%). 1H NMR (400 MHz, DMSO, ppm): δ 8.98 (d, J = 9.4 Hz, 2H), 8.59 (d, J = 8.1 Hz, 2H), 8.52−8.11 (m, 14H), 7.02 (s, 1H), 6.48−6.19 (m, 3H), 5.20 (s, 1H), 4.27 (s, 1H), 3.13 (dd, J = 71.6, 30.0 Hz, 25H), 2.36−0.56 (m, 48H). 13C NMR (101 MHz, DMSO): δ 165.96, 156.19, 139.94, 134.28, 133.30, 130.93, 130.23, 130.13, 129.74, 127.57, 127.46, 127.41, 127.28, 127.11, 126.91, 124.73, 124.50, 123.99, 123.62, 122.11, 73.33, 69.78, 69.55, 69.38, 56.25, 55.81, 49.63, 42.65, 42.04, 39.40, 38.67, 36.85, 36.27, 35.98, 35.54, 31.56, 31.36, 28.24, 28.08, 27.79, 26.74, 25.17, 24.09, 23.64, 23.05, 22.78, 22.46, 21.47, 20.78, 19.23, 18.85, 14.35, 11.89. FTIR (KBr plate, cm−1): 3382 (−NH), 3046 (Ar−H), 2939 (−CH2), 1703 (Ar CC), 1325 (−C− N), 1328 (OSO), 1032 (−C−O−C). MS (ESI) [M + H]+: calcd for C77H97N9O10S2, 1372.6846; found, 1372.6873. Preparation of the Mini Sensor Array. The mini sensor array is composed of two sensor elements, each of which is prepared from a binary system based on the bispyrene fluorophore/CTAB ensemble. The surfactant solution with 0.06 mM CTAB was first prepared in

HEPES buffer solution (10 mM, pH 7.4). Then, the sensor element solutions were prepared by adding the stock solution of 4 or 6 (400 μL, 2.5 × 10−4 M) in CH3CN into the prepared CTAB solution (100 mL) followed by 30 min sonication, where the final bispyrene concentration was fixed at 1 μM. The two solutions were used for protein sensing experiments and discrimination analysis.

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RESULTS AND DISCUSSION UV−Vis Absorption and Fluorescence Emission of 4 and 6 in Neat Solvents. The UV−vis absorption and steadystate fluorescence emission spectra of 4 and 6 were measured in a good solvent (THF) and a poor solvent (water) to evaluate their basic photophysical properties. As seen in Figure 1a, UV− vis absorption spectra of the two fluorophores in THF almost overlap and contain the typical pyrene absorption peaks at 279 and 351 nm. The molar absorption coefficients of both 4 and 6 in THF were determined to be (6.2 ± 0.1) × 104 and (5.8 ± 0.1) × 104 L mol−1 cm−1, respectively, with R2 values of 0.998 (Figure S1, Supporting Information). The emission spectra of the two fluorophores (1.0 μM) in THF both show strong monomer peaks at shorter wavelengths and weak broad excimer bands at longer wavelengths. Moreover, their fluorescence quantum yields (Φ) in THF were determined to be 0.431 and 0.442 for 4 and 6, respectively. These results show that the basic photophysical properties of two fluorophores, whether has cholesterol attachment or not, are similar in molecularly dissolved state. However, when measured in water, both fluorophores show quite strong excimer emission, suggesting strong aggregation occurs in water (Figure 1b). Moreover, 6 shows almost no monomer emission. Both the absorption and emission intensities of 6 are weaker than those of 4, which could be due to the aggregation-induced quenching effect. These results suggest that the presence of cholesterol unit facilitates aggregation of bispyrene fluorophores in poor solvent. CTAB Effect on UV−Vis Absorption and Fluorescence Spectra of Bispyrene Probes. It is generally known that the photophysical properties of small molecular probes are often modulated by surfactant assembles due to the change of microenvironment inside the milieu as compared to the bulk.34 The UV−vis absorption and fluorescence emission spectra of both bispyrene fluorophores (1.0 μM) in a series of CTAB aqueous buffer solutions (10 mM HEPES, pH 7.4) were measured to investigate surfactant effect on their photophysical properties. The concentration range of CTAB solutions was chosen from 0.04 to 0.08 mM since the critical micellar concentration (CMC) of CTAB was determined to be ca. 0.06 mM by measuring the surface tension of CTAB solutions in the presence of bispyrene fluorophores (Figure S2). The temperC


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Figure 2. UV−vis absorption (a) and fluorescence emission spectra (b) of 6 (1.0 μM) in a series of different concentrations of CTAB buffer solutions (10 mM HEPES, pH 7.4).

Figure 3. Fluorescence spectra of 6/CTAB (1.0 μM/0.06 mM) in aqueous buffer solution (10 mM HEPES, pH 7.4) upon addition of different metalloproteins: (a) Hb, (b) Mb, (c) Ferr, (d) Cyt-c, and (e) ADH (λex = 353 nm).

D


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ACS Applied Materials & Interfaces ature of the testing solutions was controlled at 25 °C by using a thermostatic water bath system. The resulting absorption and fluorescence spectra of 6 in a series of CTAB solutions are shown in Figure 2. When CCTAB is 0.04 and 0.05 mM, the absorption of 6 is broad with a certain degree of turbidity, and the fluorescence spectra exhibit strong excimer but very weak monomer emission (see Figure 2a). These results suggest that probe 6 still strongly aggregates in low CCTAB solutions. When CCTAB is at or higher than 0.06 mM (the CMC), the absorption peaks show fine structures at 379, 353, 281, and 271 nm, and the turbidity disappears. Such results indicate that the aggregation extent of 6 becomes weaker in CTAB micellar solutions. At the same time, the large increase of monomer emission and decrease of excimer emission as seen in Figure 2b approves the enlarged distance among pyrene moieties in high CCTAB solution. The UV−vis absorption and fluorescence emission spectra of 4 (1.0 μM) in different concentration of CTAB buffer solutions were also measured, and the results are shown in Figure S3. Clearly, the cationic surfactant shows a similar modulation effect on the fluorescence emission of 4, where the monomer emission is enhanced along increasing CTAB concentration. Thus, the two bispyrene fluorophores can show both monomer and excimer emission with an appropriate CTAB assembly, providing a possibility for multiple wavelength cross-reactive responses. A parallel comparison of the monomer to excimer ratio, IM/IE, between 6 and 4 in the same CTAB solution reveals two facts: one, the two bispyrene fluorophores exhibit different emission profiles in the same CTAB assemblies as evidenced by the different IM/IE value; two, a higher IM/IE is observed for 6 in high concentrated CTAB solutions (Figure S4). This suggests that the presence of cholesterol unit indeed has an effect on the fluorescence emission of bispyrene fluorophores in the aggregate state and helps to separate the two pyrene moieties in a confined environment. The different fluorescence emission profile further ensures the possible crossreactive responses of the two sensor elements based on bispyrene/CTAB ensembles. Sensing Behaviors of Bispyrene/CTAB Ensemble to Metalloproteins. The sensing behaviors of 6/CTAB and 4/ CTAB to five metalloproteins were first examined, where 0.06 mM CTAB was selected because both probes can emit both monomer and excimer in the solution. The tested metalloproteins have diverse isoelectric points and molecular weights, which include hemoglobin (Hb, pI 6.8, Mw 64.5 kDa), myoglobin (Mb, pI 7.2, Mw 17.0 kDa), ferritin (Ferr, pI 4.4, Mw 440.0 kDa), cytochrome c (Cyt-c, pI 10.7, Mw 12.3 kDa), and alcohol dehydrogenase (ADH, pI 5.4, Mw 141.0 kDa). These proteins are all Fe-containing proteins except that ADH contains Zn. The fluorescence emission spectra of 6/ CTAB and 4/CTAB systems upon titration of the five metalloproteins are shown in Figure 3 and Figure S5, respectively. As to 6/CTAB, the ensemble sensor exhibits two main different response modes according to which metal element is contained in the proteins. The sensor presents turn-off responses to the Fe-containing metalloproteins, where the fluorescence emission was gradually quenched along increasing metalloprotein concentration. This is observed for Hb, Mb, Ferr, and Cyt-c, as displayed in parts a, b, c, and d of Figure 3, respectively. Another mode is ratiometric response and observed for titration of Zn-containing ADH. As seen in Figure 3e, along increasing ADH concentration, monomer

emission was enhanced and accompanied by a decreased excimer emission via an isoemissive point at 430 nm. Similarly, the 4/CTAB ensemble also shows two different response modes to the tested metalloproteins: turn-off responses to Hb, Mb, Ferr, and Cyt-c and ratiometric responses to ADH (Figure S5). Moreover, even for the same response mode, the two fluorescent ensembles display quite different quenching extent to different proteins. As illustrated in Figure S6, the turn-off response of the two fluorescent ensembles varies from protein to protein either monitored at 380 or 498 nm. Take 6/CTAB as an example; the turn-off response at excimer follows an order of Hb > Mb > Ferr > Cyt-c > ADH (Figure S6a). As to 4/ CTAB, the turn-off response at excimer follows an order of Hb > Ferr > Mb > ADH > Cyt-c (Figure S6b). Such results suggest that each fluorescent ensemble show quite different responses to different metalloproteins. Additionally, the two fluorescent ensembles also show some difference in the fluorescence responses to the same metalloprotein. As demonstrated in Figure S7a, when monitored at 380 nm, 6/CTAB shows larger turn-off response to Hb or Mb than 4/CTAB. Similarly, when monitored at 498 nm, the fluorescent ensemble based on 6/CTAB also displays higher turn-off responses to Hb or Mb than 4/CTAB (Figure S7b). More interestingly, for the same fluorescent ensemble, the fluorescence response to a metalloprotein is also different from monomer to excimer. As seen from Figure S6, both fluorescent ensembles exhibit higher response extent at excimer than at monomer to a protein. Thus, the two fluorescent ensembles exhibit not only the typical cross-reactive responses to metalloproteins but also multiple-wavelength cross-reactive responses, which may then empower the two-ensemble mini sensor array of pattern recognition ability. Pattern Recognition of Metalloproteins Using the Two-Element Sensor Array. According to the above fluorescence measurements, the two ensemble sensors may generate a distinct recognition pattern for each metalloprotein by collecting their fluorescence variation at both monomer and excimer. Then, the fluorescence variations, (I0/I) − 1, of the two sensor elements at both monomer (380 nm) and excimer emission (498 nm) to five metalloproteins at 10 nM were collected. As a consequence, four fluorescence signals of the two-element sensor array are combined for generating a recognition pattern for each metalloprotein. The results are displayed in Figure 4. The error bars represent the calculated standard deviation for three individual replicate measurements. It can be seen from the bar charts that the sensor array can generate a distinct recognition pattern to each metalloprotein, suggesting that the present mini sensor array could provide cross-reactive responses to metalloproteins and may discriminate among different metalloproteins. Discrimination Ability of the Sensor Array to Metalloproteins. With the purpose of further exploring the discrimination ability of the two-element sensor array, linear discriminant analysis (LDA), a pattern recognition algorithm, was used to differentiate the fluorescence variation of 6/CTAB and 4/CTAB with target metalloproteins. LDA is a classical statistical approach for supervised dimensionality reduction. Using the defined group classes, it aims to maximize the ratio of the between-class distance to the within-class distance, thus maximizing the class discrimination.6,35 Relative distances between the scores in the canonical space can be correlated with similarities or difference in the responses generated by the E


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four fluorescence responses of the sensor array (2 wavelengths × 2 sensors) for a single analyte. The LDA canonical score plots shown in Figure 5 contain the first two factors. These two factors are necessary to describe at least 90% of the total information (variance) contained in the data set. Clearly, all the five metalloproteins are well separated from each other, demonstrating that they, even at very low concentration, can be effectively discriminated by the mini sensor array. We also examined the recognition ability of the sensor array to unknown samples. For doing so, we used the fluorescence variation data of the sensor array to the five metalloproteins at six different concentrations (10, 20, 30, 50, 70, and 100 μM) as the training set. Thus, a matrix of 2 sensors × 5 metalloproteins × 6 concentrations was built and used to train a linear classifier (LDA). Then, we measured again the fluorescence variation to the five metalloproteins at the six concentrations and used the resulting data of the new matrix as the test set, and the five metalloproteins were treated as unknown samples and labeled as U1, U2, ..., and U5 by a different researcher. The test set data were classified by the linear classifier. As shown in Figure 5d, the five unknown metalloproteins could be well and correctly grouped with the same type of metalloprotein. Using similar method, we further examined the identification ability of the mini array to recognize unknown samples at the same concentration. The fluorescence variation of the mini array to each metalloprotein at a given concentration was measured for five repeated times. As shown in Figure S8, the five different metalloproteins could be correctly recognized by the mini sensor array either at 10, 50, or 100 nM. Such results suggest that the present mini sensor array has powerful recognition ability in identifying multiple different metalloproteins.

Figure 4. Recognition patterns for metalloproteins (10 nM) by collecting fluorescence variation data at two selected wavelengths (380 and 498 nm) of the two sensor elements: 4/CTAB and 6/CTAB.

samples.35 LDA was chosen over the more common principal component analysis (PCA) as it produces greater differentiation and less overlap between groups.36 For each metalloprotein, we tested the protein-induced fluorescence intensity variation against the two sensor ensembles for five times, generating a matrix of 2 sensors × 5 metalloproteins × 5 replicates. LDA analysis reduces dimensions of the matrix of different metalloproteins into canonical factors, and the two factors can be visualized in a two-dimensional (2D) plot. The LDA plots for the two-element sensor array to the five metalloproteins at three different concentrations, namely, 10, 50, and 100 nM, are shown in parts a, b, and c of Figure 5, respectively. Each point in the LDA plot represents the entire

Figure 5. LDA canonical score plots for the response of the mini sensor array to metalloproteins at (a) 10 nM, (b) 50 nM, (c) 100 nM, and (d) to unknown metalloproteins (U1 = Mb; U2 = ADH; U3 = Cyt-c; U4 = Ferr, U5 = Hb). F


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Figure 6. Plots of fluorescence intensity ratio, I0/I, of the two binary ensembles as a function of the concentration of metalloproteins: (a) 6/CTAB; (b) 4/CTAB.

Figure 7. Fluorescence quenching plots of intensity ratio (I0/I) and lifetime ratio (τ0/τ) of fluorescent ensembles to the concentration of myoglobin: (a) 4/CTAB (1.0 μM/0.06 mM); (b) 6/CTAB (1.0 μM/0.06 mM) (10 mM HEPES, pH 7.4). Insets: time-resolved fluorescence decays of fluorescent ensembles in aqueous buffer solution upon titration of myoglobin.

determined to be (7.16 ± 0.48) × 107, (2.57 ± 0.07) × 107, and (3.25 ± 0.16) × 107, respectively. These KSV values are 1−2 magnitudes higher than many previously reported smallmolecule-based sensors.14,40 Meanwhile, the detection limits of 6/CTAB to Hb, Mb, and Ferr are determined to be as low as 11 pM (710 pg/mL), 179 pM (3.0 ng/mL), and 204 pM (89.8 ng/mL), respectively, and that of 4/CTAB to Hb, Mb, and Ferr is determined to be 25 pM (1.6 ng/mL), 317 pM (5.4 ng/mL), and 208 pM (91.5 ng/mL), respectively. The detailed determination process is provided in the Supporting Information. As far as we know, this is the lowest detection limit that has been reported for detecting hemoglobin and myoglobin using a small molecular fluorescent sensor.14,40 Exploration of Sensing Mechanisms. Several experiments were conducted to explore the mechanisms of crossreactive responses of the present sensor array to metalloproteins. The remarkable quenching responses of 6 and 4 to iron-containing metalloproteins are possibly due to the fact that the porphyrin functionality in these proteins could quench the excited state of pyrene by energy or electron transfer. Such phenomena have been extensively reported by others.14,15,26,41 We first carried out time-resolved measurements to explore the quenching mechanism of the two sensor ensembles. The timeresolved fluorescence decays of the two sensor ensembles were measured in the presence of various metalloproteins. Figure 7 illustrates the fluorescence quenching plots of intensity ratio (I0/I) and lifetime ratio (τ0/τ) of 4/CTAB and 6/CTAB to the concentration of myoglobin. The insets are time-resolved

However, a shortcoming of this method is it has to perform with analytes at a known concentration. This is a common issue encountered in developing discriminative sensors or sensor arrays.37,38 Further efforts should be put to developing discriminative sensors or arrays with semiquantitative or quantitative analysis ability.39 Sensitivity of Fluorescent Ensembles to Metalloproteins. The present fluorescent ensembles not only exhibit very strong discrimination ability but also show very high sensitivity. The sensitivity of 6/CTAB and 4/CTAB to the metalloproteins was examined by investigating their turn-off responses. The fluorescence intensity of the fluorescent ensembles was regarded as I0, and the one in the presence of metalloproteins was regarded as I. Figure 6 illustrates the plots of I0/I as a function of metalloprotein concentration ranging from 0 to 40 nM. The error bars represent the calculated standard deviation for three individual replicate measurements. Clearly, the plots are linearly increasing along with metalloprotein concentration. The linear relationship between I0/I and metalloproteins follows the Stern−Volmer equation: I0/I = 1 + KSV[metalloproteins]

(1)

The Stern−Volmer constant KSV represents the sensitivity of a sensor and can be extracted from the plots of I0/I versus [metalloproteins]. The KSV values of 6/CTAB ensemble to Hb, Mb, and Ferr are determined to be (1.08 ± 0.05) × 108, (4.41 ± 0.21) × 107, and (3.67 ± 0.31) × 107, respectively. The KSV values of 4/CTAB ensemble to Hb, Mb, and Ferr are G


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ACS Applied Materials & Interfaces fluorescence decays of the two sensor ensembles upon titration of myoglobin. Clearly, the two sensor ensembles display quite different lifetime variation behaviors compared to the intensity variation. As seen from Figure 7a, the τ0/τ value of 4/CTAB slightly changes along increasing the quencher concentration and stays close to 1, and the decay curves barely change (the inset of Figure 7a). Such result suggests that the nature of myoglobin quenching the 4/CTAB sensor ensemble is static rather than dynamic. This means that metalloprotein− fluorophore complex forms, and myoglobin is possibly binding with the fluorescent ensemble. Similar results were also observed for the titration of Hb, Cyt-c, and ADH. As seen in Figure S9a,c,d, the increasing of these three metalloproteins slightly change the lifetime of 4 in the ensemble. As to the 6/CTAB ensemble, the τ0/τ value changes from 1.00 to 1.62 along increasing myoglobin concentration (see Figure 7b), indicating the lifetime of 6 became shorter when myoglobin was added. Similar phenomena were also observed for Hb, Ferr, and Cyt-c, where the lifetime decreases along increasing the concentration of these three metalloproteins (see Figure S10a−c). These results mean that there is a certain degree of dynamic quenching to the 6/CTAB system. This difference in quenching mechanism may be due to the different structure between 4 and 6, which renders them have different interaction with the same metalloprotein.42 This could be one reason for the different responses of the two sensor elements to the same metalloprotein. In addition, different quenching mechanisms also occur to the same sensor ensemble to different metalloproteins. As to 4/ CTAB, an exception was found for Ferr, which causes some certain of dynamic quenching (Figure S9b). Whereas for 6/ CTAB, the exception was observed for ADH, which exhibits static quenching (Figure S10d). Such results indicate that the fluorescent ensemble has different interactions with different metalloproteins, which also contributes to the cross-reactive responses. As it is well-known, different metalloproteins have diverse isoelectric points. The pI values of the tested five metalloproteins are 6.8 (Hb), 7.2 (Mb), 4.4 (Ferr), 10.7 (Cyt-c), and 5.4 (ADH). As a consequence, they carry different electronic charge and may have different electrostatic interactions with the cationic CTAB assemblies. Thus, dynamic light scattering (DLS) was measured to estimate the size variation of fluorescent ensemble in the presence of various metalloproteins. Figure 8 illustrates the size variation of 6/CTAB upon addition of metalloproteins as an example. Prior to the addition of proteins, the main size of the sensor ensemble is ca. 160 nm. Upon addition of metalloproteins, apparent diameter changes are observed for Ferr and ADH, whereas slight size variation is found for the other three proteins. Clearly, the size change is highly related to the pI values of the added metalloproteins. The low pI values endow Ferr (pI 4.4) and ADH (pI 5.4) with more negative charges at neutral pH and have stronger electrostatic interaction with CTAB assemblies. On the other hand, the higher pI values make Hb (pI 6.8), Mb (pI 7.2), and Cyt-c (pI 10.7) carry less negative charges or even positive charges at pH 7.4, which make them have weaker electrostatic attraction or even electrostatic repulsion with CTAB assemblies. Clearly, the difference in electrostatic interaction between metalloproteins and fluorescent ensemble induce different aggregation variation of surfactant assemblies, which may further modulate the fluorescence emission of the

Figure 8. Size distribution of 6/CTAB (1.0 μM/0.06 mM) before and after addition of various metalloproteins (100 nM).

encapsulated fluorophores.31,43 This could be another reason to account for the cross-reactive responses to metalloproteins. A schematic cartoon describing the cross-reactive sensing process of the mini sensor array is presented in Scheme 2. The two binary sensors based on bispyrene/CTAB assemblies exhibit multiple emission bands including monomer and excimer emission. They have different binding interactions with different proteins, which may induce different aggregation variation of the surfactant assemblies and further leads to different fluorescence variation. The different quenching mechanisms of a protein to the two ensemble sensors also contribute to the cross-reactive responses. Thus, the different fluorescence variation at monomer and excimer of the two sensor elements can generate a distinct recognition pattern to each metalloprotein and realize the discrimination of these proteins.

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CONCLUSION A two-element mini sensor array was constructed for discriminating metalloproteins using the strategy of surfactant assemblies encapsulating two bispyrene fluorophores. UV−vis absorption and fluorescence emission measurements reveal that the cationic surfactant CTAB assemblies could well modulate the photophysical properties of the two bispyrene fluorophores. Sensing behavior studies found that the bispyrene/surfactant aggregates based binary ensembles exhibit not only the typical cross-reactive responses to metalloproteins but also multiplewavelength cross-reactive responses. The combination of fluorescence variation of the two fluorescent ensembles at both monomer and excimer emission could generate a foursignal recognition pattern for each metalloprotein. LDA results show that all the tested metalloproteins are well separated from one another. Moreover, the unknown metalloprotein samples could also be correctly recognized by the mini sensor array. Time-resolved fluorescence decays and DLS measurements suggest that the different quenching mechanism and different electrostatic interaction between metalloproteins and CTAB assemblies may account for the cross-reactive responses of the two sensor elements to metalloproteins. Compared with traditional protein discriminating sensor arrays, three obvious advantages of this mini sensor array make it particularly attractive: (1) the two sensor elements using the same fluorophore, which renders it fast and convenient data collection process; (2) the use of surfactant assemblies provides easy modulation effect, which facilitates the acquisition of crossH


Research Article


Scheme 2. Schematic Representation of Cross-Reactive Sensing Metalloproteins by the Mini Sensor Array Based on Two Bispyrene/CTAB Ensembles

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reactive responses; (3) the use of fluorophores with multiple emission bands endows multiple signals and reduces the number of sensor elements needed. Moreover, the high sensitivity and fast response time make the mini sensor array more attractive in the potential applications in discriminating metalloproteins. A shortcoming of this method is it has to perform with analytes at a known concentration. Further efforts will be put in developing discriminative sensors or arrays with semiquantitative or quantitative analysis ability.

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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/acsami.6b12646. Fluorescence quantum yield measurements, determination of the detection limits, and LDA analysis process, along with results for UV−vis absorption of compounds 4 and 6, CMC determination, CTAB concentration effect on UV−vis absorption and fluorescence emission of 4, fluorescence responses of 4/CTAB to various metalloproteins, comparison of quenching efficiencies of different metalloproteins to 4/CTAB and 6/CTAB, comparison of quenching efficiencies of same metalloprotein to 4/CTAB and 6/CTAB, LDA canonical score plots for identifying unknown samples, and fluorescence quenching plots of intensity ratio and lifetime ratio of 4/CTAB and 6/CTAB to different metalloproteins (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel 86-29-8153-0789 (L.D.). ORCID

Liping Ding: 0000-0002-1010-6066 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from National Natural Science Foundation of China (21573140), the Program of Introducing Talents of Discipline to Universities (B14041), Program for Changjiang Scholars and Innovative Research Team in Universities (IRT_14R33), and Research Funds of Shaanxi Normal University (X2014YB06). I


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Discrimination of Metalloproteins by a Mini Sensor Array Based on

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