Terpyridine Complexes into a Helical Poly ... - ACS Publications

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Intercalation of Alkynylplatinum(II) Terpyridine Complexes into a Helical Poly(phenylene ethynylene) Sulfonate: Application to Protein Sensing Shanshan Wang,† Charles J. Zeman, IV,† Junlin Jiang,† Zhenxing Pan,† and Kirk S. Schanze*,†,‡ †

Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, United States Department of Chemistry, University of Texas at San Antonio, One UTSA Way, San Antonio, Texas 78249, United States

‡

S Supporting Information *

ABSTRACT: The interactions of two anionic poly(phenylene ethynylene) sulfonate-conjugated polyelectrolytes (mPPESO3− and pPPESO3−) with two alkynylplatinum(II) terpyridine complexes (Pt2+ and Pt3+) were studied. The Pt(II) complexes interact with helical mPPESO3− by intercalation within the polymer helix to form a “guest−host” ensemble. Titration of Pt(II) complexes into an aqueous solution of mPPESO3− gives rise to efficient quenching of the polymer’s fluorescence; meanwhile, triplet metal− metal-to-ligand charge transfer (3MMLCT) state emission from the intercalated Pt(II) complexes appears when the ensembles are excited into the polymer’s absorption band. The 3MMLCT state emission implies that the Pt(II) complexes aggregate or dimerize on the mPPESO3− scaffold. The responses of the mPPESO3− and Pt(II) complex ensembles to various proteins were examined by monitoring the mPPESO3− fluorescence change. Negatively charged proteins recover the mPPESO3− fluorescence more than the positively charged proteins under physiological pH, indicating that electrostatics play an important role in the protein−ensemble interaction. KEYWORDS: intercalation, conjugated polyelectrolyte, platinum terpyridyl complex, fluorescence quenching, protein sensing

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tions.13,14 The ion-screening effects can be significant for nonhelical-structured CPEs, leading to reduced electrostatic interaction between the CPEs and the ionic analytes.15 Proteins are essential biomolecules in living organisms: many proteins are enzymes that catalyze important biochemical reactions, and proteins also perform structural and mechanical functions in maintaining cell shape. 16 Detection and quantification of proteins are critical in the study of metabolism, clinical analysis, and dietary and pharmaceutical processes. Among many protein quantification methods, spectrophotometric assays based on UV absorbance or colorimetric and fluorescence methods give high throughput and are more cost-effective and convenient.17 Many CPE-based spectrophotometric assays for proteins have been developed, taking advantage of the good water solubility and unique optical properties of CPEs.7,18−21

INTRODUCTION Conjugated polyelectrolytes (CPEs), with aromatic conjugated backbones and ionic side groups, have been studied extensively as fluorescence-based sensory materials because of their good water solubility and the amplified quenching effect.1 CPEs with various π-conjugated backbones, such as poly(phenylene ethynylene) (PPE), polyfluorene (PF), polythiophene (PT), and poly(p-phenylene vinylene) (PPV), have been used to sense ions, enzymes, and biological analytes such as proteins and nucleic acids.2−7 Among the sensing mechanisms for biological species, nonspecific interactions play an important role because the hydrophobic backbones of CPEs tend to interact with the hydrophobic domains of biological analytes, while the charged ionic side groups can interact with ionic moieties through electrostatic interactions.8,9 There is a special interest in studying meta-linked CPEs, which adopt a helical conformation in water.10 Helical CPEs mimic double-stranded DNAs and have been demonstrated to exhibit specific interactions with DNA-binding molecules through intercalation within the CPE helix.10−12 In addition, helical CPEs can prevent ion-screening effects in the presence of the salts found in aqueous buffers that are required to stabilize biological species and mimic physiological condi© XXXX American Chemical Society

Special Issue: Hupp 60th Birthday Forum Received: February 1, 2017 Accepted: March 24, 2017

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DOI: 10.1021/acsami.7b01587 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Structures of mPPESO3−, pPPESO3−, Pt2+, and Pt3+.

Figure 2. (a) Normalized UV−vis absorbance (solid lines) and fluorescence (dashed lines) spectra of mPPESO3− (black) and pPPESO3− (red). (b) Normalized UV−vis absorption spectra of Pt2+ (blue) and Pt3+ (green). All of the spectra were measured in 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 aqueous buffer solutions.

fluorescence in the visible range concomitant with protein addition.

Recently, Yam and co-workers performed a thorough study concerning the interaction between alkynylplatinum(II) terpyridine complexes and a linear para-linked CPE with sulfonate side groups (pPPESO3−); they proposed the concept of the “two-component ensemble” of pPPESO3− and Pt(II) complexes.22 Employing this two-component ensemble, they developed a label-free protein sensor for selective detection of human serum albumin (HSA).22 In this sensor, the luminescence intensity of pPPESO3− and the Pt(II) complex change with varying concentrations of HSA. However, this luminescence sensor depends on the emission of the Pt(II) complex in the near-IR region, which has a very low quantum yield,23,24 and thus, a sensitive near-IR emission detector is required. Moreover, the sensing is carried out in citrate buffer at pH 3.0, which is considerably below the physiological pH; under this condition proteins tend to denature.25−28 To enhance the application of CPEs in chemical and biological sensing, it is important to understand the interactions between CPEs and other species, including small organic molecules, ions, and biological macromolecules. In addition, studying how the CPE structure affects the optical properties and fluorescence quenching efficiency will give more insight into the structure−property relationships of CPEs, guiding the design of CPE-based sensors. In this work, we investigated the interactions of two different anionic CPEs (para-linked pPPESO3− and meta-linked mPPESO3−) with two differently charged cationic alkynylplatinum(II) complexes (Pt2+ and Pt3+, with two and three positive charges, respectively) by studying their UV−vis absorption and emission spectral changes. We found that the meta-linked mPPESO3−, which adopts a helical conformation in aqueous solution, affords 10 times stronger binding to the Pt(II) complexes than the para-linked pPPESO3−. Additionally, we explored the effects of proteins on the mPPESO3− fluorescence in the presence of the Pt(II) complexes. A protein sensor that operates at physiological pH was developed, taking advantage of changes in the mPPESO3−

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EXPERIMENTAL SECTION

Materials. Glucose oxidase from Aspergillus niger (GOx), phospholipase D from Arachis hypogaea (peanut), type II (PLD2), bovine serum albumin (BSA), human serum albumin (HSA), hexokinase from Saccharomyces cerevisiae, type III (HX3), dipeptidyl peptidase from porcine kidney (PEP), myoglobin from equine skeletal muscle (MB), horseradish peroxidase, type I (HRP), avidin, and protease from Bacillus licheniformis, type VIII (PRT) were purchased from Sigma-Aldrich. The synthesis and characterization of pPPESO3− and mPPESO3− have been previously reported.10,29 Details concerning the synthesis and characterization of Pt2+ and Pt3+ are provided in the Supporting Information. All of the solutions were prepared using water that was purified by a Millipore purification system (Simplicity ultrapure water system from EMD Millipore). Buffer solutions were prepared using reagent-grade materials from Fisher Scientific or SigmaAldrich. All polymer concentrations are provided in terms of polymer repeat unit (PRU). Instrumental Methods. UV−vis absorption spectra were recorded on a Shimadzu UV-1800 dual-beam spectrophotometer. Corrected steady-state emission spectra were measured on a Photon Technology International (PTI) spectrophotometer. Quartz cuvettes with 1 cm path length were used for all of the measurements.

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RESULTS AND DISCUSSION Optical Characterization of Conjugated Polyelectrolytes and Pt(II) Complexes. The structures of the linear paralinked polymer pPPESO3−, the helical meta-linked polymer mPPESO3−, and the two alkynylplatinum(II) terpyridine complexes Pt2+ and Pt3+ are shown in Figure 1. The optical properties of pPPESO3−, mPPESO3−, Pt2+, and Pt3+ were examined in aqueous pH 7.5 buffer containing 30 mM TrisHCl and 30 mM NaCl (Figure 2). pPPESO3− contains a linear para-linked PPE backbone and has two sulfonate side groups on each polymer repeat unit. It has a major absorption band with a maximum at λ ≈ 444 nm and a broad fluorescence B


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Figure 3. (a) Normalized UV−vis absorbance and (b) fluorescence spectra of pPPESO3− upon addition of Pt2+. (c) UV−vis absorbance and (d) fluorescence spectra of mPPESO3− upon addition of Pt2+. Titrations were done in 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 aqueous buffer. The concentrations of pPPESO3− and mPPESO3− were 45 μM; the excitation wavelengths for (b) and (d) were 430 and 322 nm, respectively.

spectrum with a maximum at λ ≈ 544 nm, similar to the values reported in the literature.29,30 According to the previous studies, pPPESO3− has a red-shifted absorption spectrum and a red-shifted, broad, and structureless fluorescence spectrum with lower quantum efficiency in water than in methanol, which is attributed to polymer aggregation in water.29,30 In contrast to pPPESO3−, mPPESO3− is meta-linked and has only one sulfonate side group per polymer repeat unit. mPPESO3− absorbs in the near-UV with a maximum at λ ≈ 322 nm, and it has a broad, structureless fluorescence band with a maximum at λ ≈ 455 nm. Because of the meta links on every other phenylene unit, the polymer self-assembles in aqueous solution into a helical conformation.10,31 The unstructured fluorescence spectrum of mPPESO3− is attributed as arising from π−π interactions between the phenylene ethynylene units within the helical structure.10 In the helical conformation of mPPESO3−, the PPE backbone adopts a coiled helical structure, with the sulfonate side groups being solvated on the periphery of the helix. The peripheral orientation of the charged groups enhances interchain charge repulsion, which minimizes the formation of interchain aggregates. Consistent with this notion, a previous study showed that mPPESO3− has a much lower hydrodynamic radius in aqueous solution than pPPESO3−, indicating that mPPESO3− does not undergo interchain polymer aggregation.32 The absorption spectra of Pt2+ and Pt3+ exhibit multiple bands for λ > 300 nm: Pt2+ has two absorption bands at 325 and 415 nm, and Pt3+ has absorption bands at 333 and 392 nm. According to the literature, the shorter-wavelength absorption bands are assigned as intraligand π → π* transitions of the terpyridine (tpy) and alkynyl ligands, and the longer-wavelength absorption bands are assigned to dπ(Pt) → π*(tpy) metal-to-ligand change transfer (MLCT) and alkynyl-toterpyridine ligand-to-ligand charge transfer (LLCT). Fluorescence Quenching Studies. The linear and helical CPEs are composed of hydrophobic conjugated backbones and negatively charged hydrophilic side chains. The Pt(II) complexes have aromatic platinum−terpyridine units and

alkynyl ligands with one or two positively charged tetralkylammonium substituents. It is predicted that the CPEs and Pt(II) complexes may interact nonspecifically through both electrostatic and hydrophobic interactions. Additionally, the helical mPPESO3− may allow intercalation of the Pt(II) complexes into the helix to form a “host−guest complex”, considering that the structure of mPPESO3− resembles the DNA double-helix structure10−12 and Pt(II) terpyridine complexes feature DNA-intercalator characteristics.33−37 The interactions between the CPEs and Pt(II) complexes were studied in experiments where the Pt(II) complexes were added to buffer solutions of the CPEs. Figure 3 shows the UV−vis absorption and emission spectral changes of the linear and helical CPEs upon addition of Pt2+. The absorption spectrum of pPPESO3− exhibits a hypochromic effect: upon addition of Pt2+, the absorbance maximum decreases (Figure 3a). In addition, there is a clear red shift of the polymer’s absorption maximum, which is attributed to polymer aggregation resulting from the charge neutralization when Pt2+ interacts with the polymer.22 The absorbance tail from 500−600 nm also increases in intensity, which is due to the absorbance of Pt2+ aggregates formed on the platform of pPPESO3− as reported previously.22,38 In addition to these changes in the absorption spectrum, the fluorescence of pPPESO3− is efficiently quenched by addition of Pt2+ (Figure 3b). The Stern−Volmer (SV) equation was employed to analyze the fluorescence quenching efficiency; the SV plots are shown in Figure 4, and the calculated Stern−Volmer constants (KSV) are listed in Table 1. In 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 buffer, KSV for the fluorescence quenching of pPPESO3− by Pt2+ is 4.4 × 105 M−1. As mentioned above, pPPESO3− can interact with Pt2+ complexes via hydrophobic, π−π, and electrostatic interactions. When these interactions are analyzed, the salts present in the buffer have to be considered with respect to their effect on the aggregation state and the effective charge of pPPESO3−. In aqueous solution, pPPESO3− is aggregated, and the high salt concentration in the buffer enhances the aggregation,39 which C


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previously been shown to enhance the KSV for fluorescence quenching. In view of these factors, we believe that the more efficient fluorescence quenching of mPPESO3− by Pt2+ is related to the helical conformation of this polymer. Pt(II) terpyridine compounds are known DNA intercalators, where the terpyridine moieties intercalate between the base pairs of DNA.42 We speculate that there is a strong intercalation interaction between Pt2+ and mPPESO3− that occurs via insertion of the terpyridine moiety of Pt2+ into the helical turns of the polymer. This intercalation is the result of the π−π and hydrophobic interactions between the conjugated terpyridine units and the π-stacked polymer backbone. Additionally, when Pt2+ intercalates into the polymer helix, the positively charged tetramethylammonium units are oriented outside the helix toward the negatively charged sulfonate groups, thus enhancing the Coulombic interaction between Pt2+ and mPPESO3−. To investigate the effect of the charge of the Pt(II) complex on the CPE fluorescence quenching, similar quenching experiments were carried out using Pt3+. In contrast to Pt2+, Pt3+ has two tetraalkylammonium groups, and thus, the complex features an additional positive charge. Because of the extra charge, Pt3+ will have a stronger Coulombic interaction with the anionic CPEs; Pt3+ is expected to bind more strongly to pPPESO3− and mPPESO3− and cause more efficient fluorescence quenching. The absorbance and fluorescence changes of pPPESO3− and mPPESO3− during the titration with Pt3+ are shown in Figure S1, and generally speaking the findings are quite similar to those seen for addition of Pt2+ to the two CPEs. The KSV value for quenching of the pPPESO3− fluorescence by Pt3+ is only slightly higher than that for Pt2+ (Table 1). This is explained by the high ionic strength of the buffer, which screens the electrostatic interaction between the Pt(II) complexes and the CPE, minimizing the effect of the higher charge on Pt3+ on the quenching efficiency. When Pt3+ is added to mPPESO3−, the Stern−Volmer plot of Pt3+ is very similar to that of Pt2+ (Figure 4), with a slightly lower KSV (Table 1). This is contrary to the expectation that the additional charge on Pt3+ should lead to a stronger binding and thus more efficient fluorescence quenching. The result suggests that the salts in the buffer effectively screen out the electrostatic interaction and that the intercalation is the primary driving force for the Pt(II) complexes to associate with mPPESO3−. It should be noted that all of the SV plots deviate from linearity with upward curvature (superlinear), indicating that the fluorescence quenching becomes more effective at higher quencher concentrations. At low quencher concentrations (0.5 μM), the upward curvature of the SV plot suggests a change of the fluorophore−quencher binding constant and/or the operation of additional quenching pathways. Alkynylplatinum(II) terpyridine complexes can aggregate, templated by a polyelectrolyte.43 In the present system, it is speculated that Pt2+ and Pt3+ self-assemble on mPPESO3− through Pt···Pt and π−π interactions. This assembly may give rise to an effectively larger association constant to the polymer (e.g., a cooperative binding effect). Furthermore, upon the association with the Pt(II) complexes, mPPESO3− becomes less hydrophilic, which might stimulate greater hydrophobic and intercalation interactions with the

Figure 4. Stern−Volmer plots for quenching of pPPESO3− and mPPESO3− fluorescence by Pt2+ and Pt3+ in 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 buffer. The concentrations of pPPESO3− and mPPESO3− were 45 μM. Symbols: (black ■) pPPESO3−/Pt2+; (blue ▲) pPPESO 3−/Pt3+; (magenta ▼ ) mPPESO3−/Pt3+; (red ●) mPPESO3−/Pt2+. The inset shows expansions of the plots for low Pt2+ and Pt3+ concentrations (0−1.0 μM).

Table 1. Stern−Volmer Fluorescence Quenching Constants (KSV) KSV (M−1)a Pt2+ −

pPPESO3 mPPESO3−

Pt3+

4.4 × 10 4.5 × 106 5

5.5 × 105 3.1 × 106

a

KSV values were calculated within the quencher concentration range from 0 to 1.0 μM.

gives rise to enhanced fluorescence quenching by the oppositely charged quencher ions.31,40 On the other hand, the salt in the buffer screens the electrostatic interaction between the polymer and the quencher,41 which decreases the quenching efficiency. Given the comparatively large value of KSV (4.4 × 105 M−1) observed for Pt2+ quenching of pPPESO3− despite the high salt concentration, we posit that hydrophobic and π−π interactions may play significant roles in the fluorescence quenching. Like that of pPPESO3−, the absorbance maximum of mPPESO3− also decreases concomitant with addition of Pt2+ (Figure 3c); however, unlike the case of pPPESO3−, there is not a red shift in the polymer absorption band, which indicates that no significant mPPESO3− interchain aggregation occurs during the titration. In addition, a clear increase in the absorbance around 400−500 nm matches well with the absorbance of the Pt2+ added to the polymer solution. The Pt2+ quenches the fluorescence of mPPESO3− very efficiently (Figure 3d), with KSV = 4.5 × 106 M−1, which is 10 times larger than the KSV for pPPESO3−. The much higher quenching efficiency for mPPESO3− signals that there are different interactions between mPPESO3− and Pt2+ compared with pPPESO3− and Pt2+. Considering the electrostatic interactions, mPPESO3− has one less negative charge than pPPESO3− on each polymer repeat unit; therefore, the effective electrostatic attraction between mPPESO3− and Pt2+ should be less than that between pPPESO3− and Pt2+. In addition, mPPESO3− is not believed to engage in significant interchain aggregation, which has D


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Figure 5. (a) Photoluminescence spectrum of Pt2+ in the titration of mPPESO3− in 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 buffer. (b) Photoluminescence intensities of Pt2+ (at 822 nm) and Pt3+ (at 780 nm) vs various concentrations of Pt(II) complexes. The concentration of mPPESO3− was 45 μM, and the excitation wavelength was 322 nm.

Figure 6. Computational results on the interaction between Pt2+ and mPPESO3− in aqueous buffer. (a, b) Models of mPPESO3− with (a) intercalated Pt2+ and (b) surrounding but nonintercalated Pt2+. The Pt2+ is shown in green as a stick-and-ball model. The mPPESO3− is shown in gray (polymer backbone) and yellow and red (sulfonate groups) shown as a space-filling model. (c) Calculated potential energies of intercalated and nonintercalated models.

concentrations where charge parity between the Pt(II) complexes and the polymer are achieved. Since the near-IR emission arises from the 3MMLCT state of the aggregated (or dimeric) Pt(II) complexes, when the emission intensity no longer increases, it indicates that no more Pt(II) can bind to the mPPESO3− scaffold where the aggregates (or dimers) form. It is reasonable that the charge balance between the polymer and the Pt(II) complexes determines the largest number of Pt(II) complexes that can bind to mPPESO3−. To further support the model that the helical mPPESO3− acts as a scaffold to induce aggregation of the Pt(II) complexes, converse addition of mPPESO3− into Pt2+ and Pt3+ solutions was performed (Figures S3 and S4, respectively). When small aliquots of mPPESO3− were added to the Pt(II) complex solutions, continuous increases in the polymer absorbance around 330 nm were observed because of the high molar absorptivity of the polymer (Figures S3a and S4a). The Pt(II) complex solutions exhibited no emission in the absence of mPPESO3− (Figures S3b and S4d); however, when mPPESO3− was added, the 3MMLCT state emissions of Pt2+ and Pt3+ appeared in the near-IR. This confirmed that in the absence of mPPESO3−, the Pt(II) complexes remain in a nonemissive

Pt(II) complexes. Yam and co-workers proposed an additional Förster resonance energy transfer (FRET) quenching mechanism in the presence of higher Pt(II) concentrations,44,45 which may also contribute to the improved quenching efficiency. Platinum(II) MMLCT Emission in the Near-Infrared Region and Structural Simulations. In addition to their efficient quenching of the fluorescence of mPPESO3−, when Pt2+ and Pt3+ are added to the CPE solutions a new emission band appears in the near-IR region with an intensity that increases with Pt(II) concentration. As shown in Figure 5a, upon addition of Pt2+, an emission band with a maximum at 822 nm increases; on the basis of previous studies, this new emission is attributed to the triplet metal−metal-to-ligand charge transfer (3MMLCT) state of aggregated Pt2+.22,38,43 (We note that the 3MMLCT emission may also emanate from a dimeric state, e.g., (Pt2+)2).46,47 Similarly, when Pt3+ is added to a solution of mPPESO3−, an emission band at around 780 nm, corresponding to the 3MMLCT state of Pt3+, increases with Pt3+ concentration (Figure S2). Interestingly, as shown in Figure 5b, the near-IR emission intensities of Pt2+ and Pt3+ cease to increase at ∼22 and 13 μM, respectively. The concentrations of Pt2+ and Pt3+ at the leveling point match the E


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Figure 7. (a) Fluorescence spectra of a mixture of 45 μM mPPESO3− and 7 μM Pt2+ upon addition of BSA in 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 buffer. (b) Relative fluorescence intensity change at 455 nm as a function of BSA concentration (the intensity was normalized to 1 in the absence of BSA).

“monomeric” state;43 however, in the presence of mPPESO3−, the complexes aggregate (or dimerize) on the mPPESO3− scaffold and become more emissive. Molecular dynamics simulations were carried out in order to better understand the structural basis for the intercalation and aggregation or dimerization of Pt2+ on the mPPESO3− scaffold. The simulations were carried out using the Materials Studio package with the methodology described in the Supporting Information. The simulations were initialized with either (1) all of the Pt2+ complexes inserted into the helix (intercalated) or (2) positioning the Pt2+ complexes outside of the helix (nonintercalated) prior to the start of the calculation. The two systems were constructed with equal pressure, volume, temperature, and number of atoms, so the microscopic potential energy (Umic) from the simulation result reflects the free energy of the system. The results from 250 ps of dynamics calculations on these simulation cells are shown in Figure 6a,b, respectively. Interestingly, as shown in Figure 6c, ΔUmic for the intercalated conformation (−2250 kcal/mol) is especially low compared with that of the nonintercalated conformation (718 kcal/mol). This indicates that intercalation stabilizes the system of mPPESO3− and Pt2+, suggesting that the intercalated form of the complexes is more stable. Close inspection of the simulation model shows that the intercalated Pt2+ is oriented such that the trpy ligand is directed inward toward the core of the helix, while the cationic ammonium units protrude outside the helix where they are able to ion-pair with the anionic sulfonate groups. A key element that is not addressed by the simulations is the structure of the Pt2+ aggregate or dimer that is responsible for the MMLCT emission in the near-IR. Thus, we can only speculate as to the structural features of the CPE−metal complex that facilitate the Pt−Pt interaction that is responsible for the near-IR emission. Given the fact that the simulations suggest a classical intercalated structure for the Pt2+ in the mPPESO3− helix, we suspect that it is most likely that the Pt− Pt interaction arises from intercalated metal complex dimers. Effect of Proteins on the Fluorescence of the mPPESO3−/Pt(II) Assemblies. By intercalating the terpyridine moiety into the mPPESO3−, the Pt(II) complexes aggregate or dimerize on the helical scaffold. This “host−guest ensemble” between mPPESO3− and the complexes is the consequence of π−π interactions combined with hydrophobic and electrostatic interactions. It is interesting to explore how the ensemble responds to proteins, since proteins are charged macromolecules with various extents of hydrophobicity. Toward

this goal, the effect of bovine serum albumin (BSA) on the emission spectrum of the mPPESO3−/Pt2+ ensemble was examined. These studies were inspired by the previous work of Yam and co-workers, who showed that addition of human serum albumin (HSA) to the pPPESO3−/Pt2+ ensemble leads to a decrease in the near-IR MMLCT emission characteristic of the Pt2+ aggregate.22 In the present study, the mPPESO3−/Pt2+ ensemble was preformed by mixing 45 μM mPPESO3− and 7 μM Pt2+ in aqueous buffer; then aliquots of BSA were added to the ensemble solution, and the fluorescence of mPPESO3− was monitored. As shown in Figure 7a, addition of BSA to the ensemble solution induced an increase in the mPPESO3− fluorescence band centered at 455 nm until the concentration of BSA reached approximately 5 μM. Figure 7b plots the intensity of the mPPESO3− fluorescence at 455 nm versus [BSA]; it can be seen that the intensity increases linearly at low BSA concentrations, gradually levels out, and stops increasing after 5 μM. A detection limit of 6 nM for BSA was calculated from the initial linear segment of the plot on the basis of the standard deviation of the response and the slope of the plot. The inset of Figure 7b shows the calibration plot derived from the first six data points, which was used for the detection limit calculation. The recovery of the mPPESO3− fluorescence indicates that BSA disrupts the structure of the mPPESO3−/Pt2+ ensemble. The isoelectric point (pI) of BSA is 4.8, and thus, in the buffer solutions that were used BSA has a net negative charge. Thus, the BSA has an electrostatic attraction with the positively charged Pt2+ and a concomitant electrostatic repulsion with the mPPESO3− in the ensemble. Overall, the electrostatic interactions between the BSA and the ensemble tend to disrupt the binding of Pt2+ to mPPESO3−, consequently leading to recovery of the mPPESO3− fluorescence that is quenched by the intercalated Pt 2+. In addition to the electrostatic interactions, BSA exhibits surfactant characteristics and is capable of enhancing the fluorescence of various CPEs.7 The hydrophobic domains on the protein surface may also interact with both the mPPESO3− and Pt2+, disrupting the ensemble structure. Nine other proteins with different pI values and molecular weights were also studied with respect to their ability to recover the mPPESO3− fluorescence in the mPPESO3−/Pt2+ ensemble, including glucose oxidase (GOx, pI 4.2, 160 kDa), phospholipase D (PLD2, pI 4.65, 200 kDa), human serum albumin (HSA, pI 4.7, 66.5 kDa), hexokinase (HK3, pI 5.25 for P-I and 4 for P-II, 54 kDa), peptidase (PEP, pI 5.2, 280 kDa), F


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increasing when the BSA concentration reaches 7 μM. The effect of different proteins on the mPPESO3− fluorescence recovery is shown in Figure S6. Similarly, proteins with negative net charges at pH 7.5 lead to more mPPESO3− fluorescence recovery than proteins with positive net charges, except for MB and HRP as explained above. Overall, the mPPESO3−/Pt(II) ensemble can discriminate the proteins with negative net charge from those with positive net charge in a pH 7.5 buffer with high ionic strength that mimics physiological saline. The nonspecific electrostatic interactions among the proteins, mPPESO3−, and the Pt(II) complexes are the main cause for the discrimination. For CPEbased protein sensor development, nonspecific interactions, including electrostatic and hydrophobic interactions, are important factors to consider.5,9 However, instead of completely avoiding them, this work shows that the nonspecific electrostatic interactions can give rise to a beneficial sensory response, providing the ability to discriminate among proteins to some extent. For detection of a specific protein, a more complicated CPE system needs to be constructed with a recognition unit for the specific targeting of analyte proteins. In Yam and co-workers’ earlier work, the pPPESO3−/Pt2+ ensemble was used for protein detection in a pH 3 buffer solution; they explained that at higher pH (≥5), the proteins associate with Pt2+, resulting in less significant changes in the visible and near-IR luminescence.22 In comparison, in the present work with the mPPESO3−/Pt2+ ensemble, the protein sensing is done in a pH 7.5 buffer solution and employs the visible fluorescence of the CPE. The detection limit of this sensing system for BSA (6 nM) is comparable to their earlier work (1.25 nM by both visible fluorescence and near-IR luminescence on HSA). The improved sensitivity in pH 7.5 buffer solution is attributed to the stronger binding affinity between mPPESO3− and Pt2+ and the consequently more efficient fluorescence quenching of mPPESO3− than pPPESO3−. In addition, the present sensing system requires one to monitor only the change in the visible fluorescence, which is advantageous over having to utilize the near-IR emission, which is weak and requires more sophisticated instrumentation for detection. For example, the present sensory system could be implemented using a standard fluorescence plate reader that is commonly used for bioassays.

Figure 8. Relative fluorescence intensity changes of 45 μM mPPESO3− and 7 μM Pt2+ mixtures at 455 nm upon addition of different proteins at 1 μM. The proteins were glucose oxidase (GOx), phospholipase D (PLD2), bovine serum albumin (BSA), human serum albumin (HSA), hexokinase (HX3), peptidase (PEP), myoglobin (MB), horseradish peroxidase (HRP), avidin, and protease (PRT). Proteins with pI values smaller than 7.5 are labeled red, those with pI values higher than 7.5 are labeled blue, and that with an unknown pI value is labeled green. The solutions were prepared in 30 mM Tris-HCl, 30 mM NaCl, pH 7.5 buffer.

myoglobin (MB, pI 6.8, 7.2, 17 kDa), horseradish peroxidase (HRP, pI 3−9, 44 kDa), avidin (pI 10, 66 kDa), and protease (PRT, pI 9.4, 27 kDa). Figure 8 shows the mPPESO3− fluorescence intensities at 455 nm after addition of each protein (1 μM) to solutions of the polymer (45 μM) and Pt2+ (7 μM). Among the 10 proteins studied, six (GOx, PLD2, BSA, HSA, HK3, and PEP) recovered the mPPESO3− fluorescence more than 15-fold compared with the control (no protein added). These six proteins have pI values less than 7.5 and thus have negative net charge in the pH 7.5 buffer. Their greater capability to recover the mPPESO3− fluorescence is explained by their electrostatic interactions with the mPPESO3−/Pt2+ ensemble as discussed above for BSA. The other four proteins (MB, HRP, avidin, and PRT), recover the mPPESO 3 − fluorescence less than 7-fold compared with the control. MB has a net charge close to zero at pH 7.5, which leads to weak electrostatic interactions with the mPPESO3−/Pt2+ ensemble and less recovery of the mPPESO3− fluorescence. HRP has a pI covering a large range. Thus, the net charge density of this protein is unclear at pH 7.5, and the low recovery of the mPPESO3− fluorescence is probably caused by a weak electrostatic interaction with the ensemble. Avidin and PRT have positive net charges at pH 7.5 and are electrostatically attracted to mPPESO3− but not to Pt2+. Because the net charge of the mPPESO3−/Pt2+ ensemble is still negative, avidin and PRT probably bind to the ensemble; however, the electrostatic repulsion between the protein and Pt2+ may not be enough to disrupt the intercalation effectively. As a result, the mPPESO3− fluorescence is still largely quenched by Pt2+, and less mPPESO3− fluorescence recovery is observed. The mPPESO3−/Pt3+ complex showed similar responses to BSA and the other proteins. The recovery of the mPPESO3− fluorescence by BSA is shown in Figure S5. The mPPESO3− fluorescence increases linearly at low BSA concentrations; the incremental increase becomes less when the BSA concentration is greater than 1 μM, and gradually the fluorescence stops

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CONCLUSION The interactions between a pair of Pt(II) complexes and the linear and helical conjugated polyelectrolytes pPPESO3− and mPPESO3 were studied. The Pt(II) complexes interact with linear pPPESO3− through electrostatic, hydrophobic, and π−π interactions. With helical mPPESO3−, the Pt(II) complexes intercalate into the polymer helix via π−π interactions between the terpyridine moiety and the π-stacked aromatic backbone of the CPE. The intercalation of the Pt(II) complex into the helical polymer gives rise to a larger association constant by a factor of 10 compared with the linear pPPESO3− that was studied in an earlier report.22 Electrostatic interactions, however, determine the maximum amount of Pt(II) that binds to mPPESO3−. Upon binding, the Pt(II) complexes quench the mPPESO3− fluorescence efficiently; meanwhile, emission appears from the 3MMLCT state, indicating the formation of metal complex aggregates or dimers. The effect of various proteins on the fluorescence of the mPPESO3−/Pt(II) ensembles was studied in a buffer similar to physiological conditions. The ensemble can provide discrimination between G


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proteins with low and high pI values using the visible fluorescence of mPPESO3−, which is easier to detect than the near-IR emission used in the earlier report by Yam and coworkers.22

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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.7b01587. Spectroscopic data for the interaction of Pt3+ with the CPEs, synthetic schemes, procedures, NMR analysis, and a description of the computational methodology (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kirk S. Schanze: 0000-0003-3342-4080 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (Grant CHE-1504727). REFERENCES

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