Designed Amphiphilic Polystyrene as Surfactant for Oligo(p

Article Cite This: ACS Appl. Polym. Mater. 2019, 1, 1230−1239

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Designed Amphiphilic Polystyrene as Surfactant for Oligo(p‑phenylenevinylene)-Incorporated PS Nanobeads and Visual Detection of Bilirubin in Human Blood Serum Sarabjot Kaur Makkad†,‡ and S. K. Asha*,†,‡ †

Polymer Science and Engineering Division, CSIR-National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008, India Academy of Scientific and Innovative Research, New Delhi 110025, India

‡

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S Supporting Information *

ABSTRACT: Amphiphilic polystyrene having pendant glucuronic acid (PS-DGlu) was designed and systematically synthesized to be used as a stabilizing agent in styrene miniemulsion polymerization, while covalently incorporating oligo(p-phenylenevinylene) (OPV)-based fluorophore to prepare PSG-OPV-n. This OPV fluorophore was incorporated with an aim to work as a signal transducer, while glucuronic acid on the surface of PS nanobeads would act as the water solubilizing moiety for PS to enable it to function as surfactant and also serve as an interaction site for free bilirubin to facilitate noncovalent interaction via hydrogen bonding. Efficient energy transfer from OPV to bilirubin was observed, owing to the appreciable spectral overlap between emission of OPV and absorption of bilirubin. Visual color change from blue to bluish green was observed under an UV lamp after the addition of bilirubin into the polymer. Selectivity of the sensor was checked among the pool of other interferences, such as glucose, sucrose, metal ions, cholesterol, and biliverdin. The limit of detection was found to be as low as 20 nM, which is far less than the clinical range for causing jaundice (50 μmol/L). Moreover, the developed sensor showed its effectiveness toward real time monitoring of free bilirubin in human serum. KEYWORDS: amphiphilic homopolymer, functionalized polystyrene nanobeads, oligo(p-phenylenevinylene), sensing, bilirubin

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INTRODUCTION Amphiphilic polymers find application in a wide range of areas from materials to biology, such as drug delivery, catalysis, electronics, biosensing, etc.1−3 In this aspect, amphiphilic block copolymers for PS-b-poly(vinyl alcohol), PS-b-poly(acrylic acid), etc., where one block is hydrophilic while the other is hydrophobic, have been extensively investigated. These amphiphilic block copolymers can exhibit a broad range of self-assembled morphologies, which is driven by the mutual immiscibility of the constituent blocks with each other or in solvent. However, synthesis of such an amphiphilic copolymer is rather challenging mainly because, even if one block is synthesized in a controlled manner, the generation of a second block would be constrained due to incompatible solubility of these amphiphiles in common organic solvents.4 Instead, the synthesis of amphiphilic homopolymers are more facile, demanding, and controlled where an 1:1 ratio of hydrophilic and hydrophobic unit could be exactly maintained along the long polymer chain.5 Introducing amphiphilicity at such a small length scale would enable each monomer unit of polymer to behave as a single surfactant moiety; thereby enhancing its surfactant property. However, unlike amphiphilic copolymers, the self-assembly of such a homopolymer is usually driven by intramolecular phase separation.6 The group © 2019 American Chemical Society

of Thayumanavan has reported an amphiphilic polystyrene (PS) homopolymer, which could be used as a reaction medium for photochemical reactions in water7 with superior selectivity compared to block copolymer micelles or small molecule surfactants.6 The same group also demonstrated that the selfassembly in such amphiliphilic homopolymer could exhibit nanocontainer properties for separation as well as drug delivery application.8 Along this line, we have designed and developed a protected glucuronic acid-substituted styrene monomer, which, upon polymerization followed by hydrolysis, afforded the water-soluble PS bearing glucuronic acid as a pendant unit. This water-soluble PS was then utilized as a surfactant for the miniemulsion polymerization of styrene covalently incorporating oligo(p-phenylenevinylene) (OPV) to generate glucuronic acid-functionalized fluorescent PS nanoparticles (NPs). To the best of our knowledge, this is the first report of an amphiphilic homopolymer being used as a surfactant for stabilizing styrene miniemulsion polymerization to generate glucuronic acidfunctionalized PS NPs. Polymeric surfactants impart interesting properties to the final latex as compared to lower molecular Received: March 12, 2019 Accepted: April 22, 2019 Published: April 22, 2019 1230

DOI: 10.1021/acsapm.9b00222 ACS Appl. Polym. Mater. 2019, 1, 1230−1239

Article

ACS Applied Polymer Materials Table 1. Sample Designation, DLC, Size, and Its PDI, Solid Content, and Zeta Potential (ζ) samples

PS-DGlu (mg)

dye in feed (mg)

dye incorporated (mg)

DLC (%)

size (nm)

PDI

solid content (%)

ζ-potential (mV)

PS-OPV-1 PS-OPV-2 PS-OPV-3 PS-OPV-4 PS-OPV-5

10 20 30 40 50

30 30 30 30 30

11.4 7.8 8.2 6.7 8.8

0.11 0.08 0.08 0.07 0.09

163 222 224 328 192

0.06 0.05 0.05 0.07 0.06

6 10 6 8 20

−35 −52 −36 −48 −55

emphasized on the use of amphiphilic PS as a surfactant for stabilizing styrene miniemulsion polymerization to generate glucuronic acid-functionalized PS NPs, which were further used for the successful detection of bilirubin in human blood serum among a pool of other competitive interference, such as proteins, metal ions, cholesterol, sugars, biliverdin, etc. It is anticipated that functionalization of the polymer beads with receptors specific for different analytes would open up new avenues of exploration of fluorescence-based sensing for other biologically relevant analytes too. Also, this would be the first report of novel amphiphilic PS surfactant and quantification of free bilirubin in human blood serum using materials which are easy to synthesize and scale up and using a method which is more sensitive and fast compared to the current clinically practiced method.

weight conventional surfactant, such as low critical micellar concentration (CMC), lower foaming, and additionally modifying the surface of nanobeads with specific functional polymers.9 Glucuronic acid was chosen as the pendant with an aim to serve two main functions. It imparts water solubility to the PS polymer, thereby enabling it to act as a surfactant in miniemulsion polymerization. Second, it can provide interaction sites for bilirubin to enhance sensor−analyte synergy via hydrogen bonding. OPV was chosen as a sensing material because of its nice spectral overlap with bilirubin, thus favoring the energy transfer process. Bilirubin is produced as a breakdown byproduct of red blood cells (RBCs), which gets metabolized in liver and finally excreted from the body in the form of bile.10 However, any disruption to this normal metabolic pathway due to any reason, including viral or bacterial infection causing excess breakdown of RBCs, leads to excess production of free bilirubin in the body.11,12 Free bilirubin is extremely fatal, and its excess accumulation above the normal level, i.e., 50 μmol/L, in human serum is directly related to liver malfunctioning.13,14 Thus, it is extremely crucial to accurately determine the concentration of free bilirubin in human serum in order to diagnose liver disorders and jaundice.14,15 Researchers across the globe have been looking at alternative approaches for a quick and accurate bilirubin assay. There exist only a handful of literature reports on the fluorometric detection of free bilirubin in human serum. Some of the pioneer work in this area has been addressed by Santhosh et al., Ellairaja et al., and Du et al.16−18 For example, Santhosh et al. reported protein-labeled fluorescent biomolecules for monitoring of free bilirubin in serum.16 Ellairaja et al. reported picomolar detection of free bilirubin in human biofluids using an imine-based fluorescent small molecule.17 Very recently, a metal−organic framework (MOF)-based highly efficient bilirubin sensor was reported by Du et al. with a faster response time, lower detection limit (picomolar), and wide range of analyte concentration.18 However, most of these sensors are accompanied by attenuation in their fluorescence intensity, i.e., emission quenching upon adding analyte. In this context, our previously reported polyfluorenebased biosensor could achieve selective sensing of bilirubin with color change from blue to light green.19,20 However, for practical applications, a conjugated polymer-based biosensor is not so feasible due to the difficulties in the reproducible synthesis and scale up. On the other hand, commercial polymers, such as PS, are relatively low cost and are produced and consumed in large quantities. Compared to a conjugated polymer-based substrate that is more expensive and synthetically more challenging, a design based on commercially available and easily scalable PS is more attractive as a sensor substrate for quick and accurate estimation of free bilirubin. These polymeric nanobeads could be developed using watersoluble polymeric surfactant.21−24 Thus, the present work is

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EXPERIMENTAL SECTION

Materials. 4-Bromostyrene, D-glucuronic acid, azidotrimethylsilane (TMS-N3), tin(IV) chloride, bis(triphenylphosphine) palladium(II) dichloride [Pd(PPh3)2Cl2], copper(I) iodide (CuI), 4-ethynyl trimethylsilane, tetrabutylammoniumfluoride (TBAF, 1 M in THF), azobis(isobutyronitrile) (AIBN), styrene, hexadecane (HD), and potassium persulfate (KPS) were purchased from Aldrich and used without further purifications. Human blood serum was obtained from Chelaram Diabetes Institute, Pune, and stored at −80 °C. Other reagents and solvents, such as tetrahydrofuran (THF), dichloromethane (DCM), triethylamine (Et3N), acetic anhydride, iodine, copper sulfate (CuSO4), sodium ascorbate, sodium thiosulfate pentahydrate, sodium bicarbonate, etc., were purchased locally. All the solvents were dried using standard procedures. Polymerizable fluorophore (OPV) was synthesized according to a previously reported procedure.25 Measurements. The instrument techniques used for characterization of monomers and polymers, such as dynamic light scattering (DLS) (for size and zeta potential), Fourier-transform infrared spectroscopy (FTIR), sonicator, NMR, GC−MS analysis, field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), absorption, and fluorescence spectrophotometry, are the same as those described in our previous report.25 For a solid sample, powders were mixed with KBr to make pellets, while, for a liquid sample, their dilute solution was directly drop casted and infrared spectra collected in the attenuated reflectance (ATR) mode in the range of 4000−600 cm−1. Molecular weight was determined using a PL-220 GPC instrument in choloroform, and the flow rate was maintained as 1 mL/min. For DLS, the average of three consecutive readings of freshly prepared samples was taken in order to minimize error. For FESEM and TEM, diluted samples (2 μL/mL) were taken and drop casted onto a silicon wafer and carbon-coated copper grid, respectively, and solvent was dried at room temperature. Synthesis of Pendant D-Glucuronic Acid-Functionalized PS (PSDGlu, 7). A styrene monomer with a pendant-protected glucuronic acid (5) was synthesized by an azide alkyne click reaction, followed by its polymerization and deprotection to obtain PS-DGlu; the details of the synthesis are provided in the Supporting Information (SI). Preparation of PSG-OPV-n Using PS-DGlu (7) as Surfactant. The PS-DGlu polymer was used as a surfactant in the miniemulsion polymerization process for synthesizing novel D-glucuronic acidfunctionalized fluorescent PS nanobeads. The organic phase consisted 1231


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ACS Applied Polymer Materials

Scheme 1. Schematics of Synthesis of Glucuronic Acid-Functionalized Styrene Monomer and Its Further Polymerization via Miniemulsion Route To Obtain Glucuronic Acid-Functionalized PS Nanobeads

Figure 1. Labeled 1H NMR spectrum of PS functionalized with protected D-glucuronic acid-(PS-PGlu) 6 recorded in CDCl3. Sensing of Free Bilirubin in Human Serum. A fixed amount of 100 μL of human blood serum was added to the varying concentration of bilirubin in the range of 1 × 10−6 to 3 × 10−5 M. These solutions were then added to a fixed concentration of polymer (0.1 mg/3 mL) in ches buffer at pH = 10 to make the final volume of 3 mL. The changes in the emission and absorption intensity of the polymer after the addition of different concentrations of bilirubin were recorded at 18 °C with a slit width of 1 nm. One separate experiment was conducted using the same volume (3 mL) of polymer and serum without adding bilirubin at pH = 10.

of styrene (1 g), HD (48 mg), and polymerizable OPV dye (30 mg), while the aqueous phase consisted of water (4 g), initiator (ACVA) (16 mg), and a varying amount of PS-DGlu polymer (values are mentioned in Table 1). The organic phase was then added dropwise to the aqueous phase and kept for pre-emulsification at room temperature for another 1 h, followed by sonication under an icecooled condition for 20 min. The miniemulsion was then allowed to polymerize at 70 °C for 20 h with a stirring speed of 750 rpm. After quenching the polymerization with two drops of 1 wt % hydroquinone, the obtained latex was purified by dialysis using a 6 kDa MW cutoff membrane for 3 days. Calculation of Dye Loading Content (DLC). Three milligrams of dried polymer was taken in 3 mL of THF, and its absorbance was recorded using absorption spectroscopy. This absorbance was used to calculate the dye loading from the molar absorptivity of OPV in THF (40 360 L mol−1cm−1) at its absorption maxima. Sensing of Free Bilirubin in Aqueous Medium. The stock solution of polymer was prepared at a concentration of 0.1 mg/3 mL in ches buffer at pH 10. Varying concentrations of bilirubin solution, ranging from 1 × 10−6 to 5 × 10−5 M in ches buffer at pH = 10, were prepared and kept in the dark. All the solutions were kept in the dark at 18 °C. The changes in emission and absorption intensity of the polymer after the addition of different concentrations of bilirubin were recorded at 18 °C with a slit width of 1 nm.

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RESULTS AND DISCUSSION Synthesis and Structural Characterization. A styrenebased monomer with a pendant-protected glucuronic acid (5) was synthesized by an azide alkyne click reaction (Scheme 1). 4-Ethynyl styrene (3) was synthesized from 4-bromostyrene (1) via Sonogashira coupling, followed by its deprotection,26 while 2,3,4 trio-acetyl 1-azido 1-deoxy D-glucuronic acid methyl ester (4) was synthesized using a three step procedure,27 as shown in Scheme S1. These molecules were structurally characterized by proton NMR spectroscopy along with mass spectrometry and FTIR (details provided in Figure S1−S10). Appearance of a new peak for triazole H in proton NMR at 1232


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Figure 2. Comparison between protected (PS-PGlu) vs deprotected (PS-DGlu) polymers using (A) FTIR and (B) contact angle measurement. (C) Calculation of HLB value of deprotected polymer (PS-DGlu) for investigating its surfactant property, where MW is the repeating mass of the respective monomer unit. (D) Determination of CMC of the PS-DGlu polymer by DLS.

8.06 ppm (Figure S11) confirmed the “click” reaction, which was further supported from its HRMS spectra (Figure S12). This monomer was polymerized via free radical polymerization using AIBN as an initiator to give the protected Dglucuronic acid PS polymer, PS-PGlu (6) (Scheme 1). The structural characterization was performed with 1H NMR spectroscopy, and Figure 1 gives the labeled spectra of PSPGlu recorded in CDCl3. Figure S13 shows the 1H−1H COSY spectra of 6, where a peak along the diagonal corresponded to the signals in 1H NMR. It depicted the coupling among its protons, for instance, cross peaks between protons of j and e, g and a, and b. The molecular weight, determined by gel permeation chromatography (GPC), showed an Mn value of 16 800 with a polydispersity index (PDI) value of around 1.8 (Figure S14). FTIR spectra of 6 showed an intense peak at 1762 cm−1, corresponding to the CO stretching frequency from acetate groups (Figure 2A). PS-PGlu (6) was then deprotected by treatment with sodium methoxide in methanol to give a watersoluble polymer consisting of PS bearing D-glucuronic acid (PS-DGlu, 7) (Scheme 1). This PS-DGlu polymer (7) was found to be soluble in water and MeOH, in contrast to its protected polymer (PS-PGlu, 6), indicating the deprotection of acetate and methyl ester groups to free-COOH and −OH groups (1H NMR spectrum of PS-DGlu in water is provided in Figure S15). The deprotection of these functionalities was further confirmed from its FTIR spectrum28 and contact angle measurement. The FTIR spectrum (Figure 2A) showed an appearance of a broad peak at 3573−2595 cm−1 for the combined O−H and C−H stretching frequencies, while a peak at 1762 cm−1 for CO stretching of the acetyl groups was shifted to 1727 cm−1, accounting for the CO stretching of the deprotected carboxylic acid group. Furthermore, contact angle was drastically changed from 121° for PS-PGlu to 26.4°

for the PS-DGlu polymer because of the transition from a hydrophobic to hydrophilic environment (Figure 2B). The surfactant property of PS-DGlu was explored for stabilizing the styrene miniemulsion polymerization incorporating the OPV-based fluorophore to prepare the D-glucuronic acid-functionalized fluorescent PS nanobeads (PSG-OPV-n). To evaluate the surfactant property of PS-DGlu, its hydrophilic to lipophilic balance (HLB value) was evaluated using Griffin’s method29,30 according to eq 1 HLB = (WH/WH + WL) × 20

(1)

where WH and WL are the weight fraction of the hydrophilic and lipophilic blocks present in the polymer, respectively.31,32 Using this formula, the HLB value for the PS-DGlu polymer was calculated to be 14.06 (Figure 2C). On this note, a typical surfactant having an HLB value in the range of 8−18 has potential to stabilize oil in water emulsion.33 This prompted the appropriateness of the polymer (PS-DGlu, 7) to act as a surfactant to stabilize the styrene miniemulsion polymerization in water. To validate its surfactant nature, its CMC was calculated using DLS, where scattering intensity was plotted against the concentration of PS-DGlu in water9 (Figure 2D). With the formation of micelle in the solution, a sudden change in the scattering intensity is highly expected, thus the minimum concentration of polymer, at which the scattered intensity sharply increased, was used to evaluate CMC. The point of the intersection of two linear regression lines was considered as the CMC of the polymer, and it was found to be 0.2 mg/mL. This low value of the CMC further justified its ability to act as a surfactant and thereby stabilize the miniemulsion. To test the actual fate of 7 as a surfactant, it was introduced to stabilize the styrene miniemulsion having a polymerizable OPV to yield the D-glucuronic acid-functionalized OPV1233


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Scheme 2. Synthesis of Glucuronic Acid-Functionalized PS Nanobeads Incorporating OPV (PSG-OPV-n) via Miniemulsion Polymerization

one (PSG-OPV-5) with the highest value of zeta potential, high dye incorporation, solid content, and molecular weight was finally chosen for bilirubin detection studies. OPV moieties from the polymer were the active fluorophore units and, therefore, would serve as the sensor to signal the presence of analyte through a change in emission property. The glucuronic acid moieties on the nanobeads surface were introduced to serve two purposes. First, it would act as a water solubilizing moiety for the PS polymer to enable it to act as a surfactant. Second, it would serve as an interaction site to bilirubin via noncovalent interaction, such as hydrogen bonding, and thereby facilitate efficient energy transfer from the fluorophore (OPV) to bilirubin, resulting in quick and efficient sensing. Photophysical Studies. Although PS alone has some weak blue emission due to π−π stacking,35 this emission is negligible, and thus a suitable fluorophore needs to be incorporated to impart emission from the PS backbone; thus, OPV-based polymerizable fluorophore was incorporated into the PS backbone to impart blue emission from the resulting nanobeads. Selection of OPV as the suitable fluorophore was done by studying the possibility of its spectral overlap with that of bilirubin. The OPV moiety has a characteristic emission in the range of 400−550 nm (λem = 446 nm) and thus showed a fair spectral overlap with the absorption spectra of bilirubin, which lie in the range of 350−500 nm (λabs = 455 nm). Figure S21 shows spectral overlap between the normalized emission of OPV and normalized absorption of bilirubin. Therefore, efficient energy transfer from OPV to bilirubin was highly expected. All of the glucuronic acid-functionalized PS nanobeads (PSG-OPV-n) contained OPV and were blue emitting, as confirmed by their CIE coordinate diagram (Figure S22), but PSG-OPV-5 was selected for further sensing studies as per the criteria explained above. When the emission and excitation spectra of PSG-OPV-5 were recorded in the ches buffer, the corresponding maxima were observed at 390 and 445 nm, respectively. Both these spectra showed characteristic OPV peak emissions from 400 to 550 nm and excitation from 300 to 435 nm. Also, the emission spectra recorded in human serum at pH 10 showed only a slight difference in the emission intensity as compared to that in ches buffer at the same pH (Figure S23A,B). Additionally, in accordance with our previous observations,25 the emission of OPV from PS nanobeads remained unaffected by the change in pH and temperature over a wide range. Sensing Study of Free Bilirubin in Ches Buffer. Bilirubin consists of an open chain structure of four pyrrole molecules having two pendant carboxylic acids, which are known to possess several intramolecular hydrogen bonds and

incorporated PS nanobeads in water. Different amounts of PSDGlu, with a fixed amount of styrene and OPV, were used to prepare a series of functionalized PS nanobeads (PSG-OPV-1 to PSG-OPV-5) using 4-4′ azobis(4-cyanovaleric acid) (ACVA) and HD as the initiator and hydrophobe, respectively (Scheme 2 and Table 1). The size and stability of the obtained PS nanobeads were analyzed by DLS measurement in water (Figure S16). The size of all the PS nanobeads (PSG-OPV-1 to PSG-OPV-5) was found well within the nanometer range, varying from 163 to 328 nm, and the PDI values remained below 0.1, showing monodisperse particles. The spherical morphology and size uniformity of the nanobeads were further confirmed by microscopic analysis using FESEM and TEM. Figure S17 shows the FESEM images of some of the representative polymers, which pointed out that increasing the amount of macrosurfactant (PS-DGlu) led to more uniformity in the size of nanobeads. Figure S18 showed the TEM images of PSOPV-5 as a representative sample. A clear core−shell structure was discernible with a PS core diameter of 85 nm and an overall size of 133 nm, including a hydrophilic shell. The stability as well as the negative surface charge of nanobeads in water were assured by their zeta potential values, which varied from −35 to −55 mV (Table 1). The highly negative zeta potential values of all nanobeads were attributed to the presence of −OH and −COOH groups from the macrosurfactant and a few −COOH from ACVA. Surface functionalization with glucuronic acid was further confirmed using FTIR spectra, which showed broad peaks at 3406 and 2506 cm−1, corresponding to −OH and −COOH groups, while a peak around 1718 cm−1 belonged to the CO stretching of free acid (Figure S19). The percent solid content, which is the measure of percent dry weight of polymer in 1 mL latex, was found to be in the range of 6−20% (Table 1). The polymer was found to be soluble in chloroform (CHCl3), in which their molecular weight was determined. They were found to have an appreciable high molecular weight, with MW ranging from 37 500 to 272 000 Da, and a PDI value within 2.5 (Table S1). The amount of dye in feed was much less when compared to the amount of monomer, and thus, a signal corresponding to fluorophore could not be traced via NMR spectroscopy. However, its presence could easily be seen in absorption spectroscopy, from which DLC could be estimated using its molar absorptivity in THF (40 360 L mol−1cm−1), as described in our previous article.34 Their DLC values are given in Table 1 and varied within the range of 0.07−0.11% (Figure S20). Although all the polymer nanobeads contained OPV dye and were surface-functionalized with D-glucuronic acid, all could be utilized for bilirubin sensing, but among them, the 1234


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Figure 3. (A) Emission and (B) excitation spectra of PSG-OPV-5 recorded after the addition of varying amounts of bilirubin (1 × 10−6 to 5 × 10−5 M). (C) normalized emission spectra of PSG-OPV-5 recorded after the addition of varying amounts of bilirubin (1 × 10−6 to 5 × 10−5 M) at 446 nm. (D) Bar graph comparing the quenching intensity vs bilirubin concentration. Each measurement was done twice, and their average was plotted along with their respective standard deviation.

OPV-5. Figure 4A shows the blue emission from the polymer; bilirubin is nonemissive due to its poor quantum yield in any solvent, including water (Figure 4B). The partially quenched blue emission of OPV, along with the enhanced green emission of bilirubin, shows up as a distinct bluish green emission in Figure 4C. The gradual shift of the emission maxima was further confirmed by their CIE coordinate diagram, which showed a distinct shift of the x and y-coordinates from 0.15 and 0.12 (blue) for PSG-OPV-5 without bilirubin to 0.19 and 0.42 (bluish green) after the addition of 5 × 10−5 M of bilirubin to polymer (Figure 4D). Mechanism of Sensing. In order to examine the possibility for existence of different sensing mechanisms for the quenching of OPV emission on interaction with bilirubin, quenching of OPV emission was further analyzed using the Stern−Volmer equation. Figure 4E shows the relation between emission intensity of OPV with respect to concentration of bilirubin. The inset shows the linear range of Io/I vs bilirubin concentration from 1 × 10−7 to 1 × 10−5 M. The linearity of the plot was further checked by linear fitting of data, which gave an R2 value of 0.976, showing the perfect linear fit (inset Figure 4E). Additionally, from the slope of the linear fit, the value of the Stern−Volmer constant (Ksv) was found to be 262 008 M−1, which caused the high selectivity of the PSGOPV-5 sensor toward bilirubin. To the best of our knowledge, this is one of the highest quenching constant values among other reported bilirubin sensors in literature to date. The S−V plot clearly indicated the possibility of involvement of more than one sensing mechanism, which may include formation of

thus are reported to have low water solubility at pH 7.4 (cellular pH). However, it is established that pH 10 is sufficient to break these intramolecular hydrogen bonds and to impart water solubility to it.20,36 Therefore, studies conducted for sensing of bilirubin in the literature are usually reported at pH = 10. To check the effectiveness of PSG-OPV-5 for detection of free bilirubin at pH = 10, preliminary studies were carried out using a stock solution of polymer and bilirubin in ches buffer at this pH. The details of the polymer and bilirubin sample preparations are given in the Experimental Section (all the experiments were conducted in the dark due to bilirubin being light sensitive). For carrying out the sensing studies in ches buffer, a fixed concentration of PSG-OPV-5 (0.03 mg/mL) was added to the varying concentrations of bilirubin (ranging from 1 × 10−6 to 3 × 10−5 M in ches buffer at pH = 10) under dark, and their emission, excitation, and absorption spectra were recorded immediately. From the emission spectra (Figure 3A), it could be seen, that with the increase in bilirubin concentration, OPV emission at λmax = 446 nm gradually decreased. The same was further supported by decreasing intensity in excitation spectra with an increase in the concentration of bilirubin (Figure 3B). One could easily observe the enhancement in green emission after the addition of bilirubin on normalizing the spectra at 446 nm (Figure 3C). An instant quenching of ≈36% was observed at a concentration of 3 × 10−6 M, which finally reached to >50% quenching at 5 × 10−6 M and almost 97% quenching at 3 × 10−5 M (Figure 3D). Figure 4A,B,C shows the visual color change from blue to bluish green observed under a hand-held UV lamp upon addition of bilirubin to PS1235


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Figure 4. (A) Photograph showing (A) PSG-OPV-5 (blue emission), (B) bilirubin (no emission), and (C) PSG-OPV-5 after bilirubin addition (bluish green emission) in the ches buffer at pH 10. (D) CIE coordinate diagram of PSG-OPV-5 without and with varying bilirubin concentrations. (E) Plot of changes in emission intensity of PSG-OPV-5 vs concentration of bilirubin and their linear range using Stern−Volmer equation. (F) Schematics depicting the mechanism of bilirubin sensing.

the ground state complex between OPV and bilirubin, inner filter effect (IFE), energy transfer (ET), etc. The absorption spectra of the polymer recorded on various analyte concentrations did not show any shift or appearance of a new peak in the presence of bilirubin (Figure S24), and thus, they excluded any possibility of the formation of the ground state complex between the two. However, a small spectral overlap between the excitation spectra of OPV and absorption spectra of bilirubin, as shown in Figure S25, also indicated the possibility of IFE between the two, thereby reducing the emission intensity of OPV due to competitive absorption at higher bilirubin concentrations. The core−shell structure, as evidenced by the TEM image (Figure S18), supported the presence of a hydrophilic hairy shell on the particle surface. This hydrophilic shell (radius ∼24 nm) formed by the glucuronic acid around the tight PS hydrophobic core (diameter ∼85 nm) could be expected to direct the watersolubilized bilirubin in close contact (∼10 nm) with the PS, where it could interact with the OPV molecules near the surface via long-range energy transfer. This core−shell structure design also had the added advantage of allowing only the surface-bound OPV to participate in energy transfer to bilirubin, retaining partial emission from OPV (away from the surface) to augment the weak green emission of bilirubin. The disperse nature of the sensor thus provides a built-in mechanism for fine-tuning the emission color in contrast to a completely soluble system, where full access of the analyte to the sensor could result in complete quenching of the latter’s emission. Thus, it can be inferred, that the bluish green emission obtained after the addition of bilirubin in Figure 4C is the combined effect of energy transfer, inner filter effect, and

some unquenched emission from OPV. Figure 4F depicts the mechanism of bilirubin sensing. As bilirubin is a biomolecule, the selectivity of PSG-OPV-5 was checked from among the library of other biomolecules, such as NaCl, KCl, glucose, sucrose, cholesterol, etc., which might interfere with the emission of the polymer. In order to check the same, to a fixed concentration of polymer (0.1 mg/3 mL) was added the fixed concentration of each of the analytes (3 × 10−5 M), and their emission spectra were recorded (Figure S26A); an average of two such measurements is plotted in Figure S26B. From the figure, it could be inferred, that no appreciable quenching was observed upon the addition of analytes at a concentration of 3 × 10−5 M, other than bilirubin, which exhibited almost 97% quenching at a similar concentration. This, together with a high quenching constant value, signifies the high selectivity of polymer toward bilirubin. The OPV emission quenching was also used to determine the limit of detection (LOD) for bilirubin. For this, different concentrations of bilirubin (from 10 to 120 nM) were added to fixed concentration (0.1 mg/3 mL) of PSG-OPV-5, and respective emission quenching of OPV was recorded. Considering that a signal-to-noise ratio (S/N) of 3 is generally accepted for estimation of LOD,37,38 20 nM was estimated as the LOD for bilirubin, which indicated high sensitivity. A comparison of the bilirubin sensors reported in literature is tabulated in Table S2, which highlights the merits of our PSbased sensor in terms of type of response (distinct color tuning versus quenching), response time, and lower LOD. Sensing Studies of Free Bilirubin in Human Serum. As shown in the previous section, the sensor could be successfully applied in water for bilirubin detection with an LOD as low as 1236


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Figure 5. (A) Excitation, (B) emission spectra, and (inset) normalized emission spectra of PSG-OPV-5 recorded after the addition of varying concentrations of bilirubin (1 × 10−6 to 3 × 10−5 M) in human blood serum.

best of our knowledge, this is the first report on glucuronic acid functionalized PS nanobeads (PSG-OPV-n) mentioned in the literature so far. The glucuronic acid would impart water solubility to the PS-DGlu polymer to enable it to act as a surfactant in miniemulsion polymerization, which is also reported for the first time in literature. Additionally, the glucuronic acid on the PS nanobeads served as an interaction site for the selective detection of bilirubin via noncovalent interaction. Due to its excellent water dispersibility and good spectral overlap between emission of OPV and absorption of bilirubin; detection of free bilirubin was first targeted in water. Among a series of PSG-OPV-n, PSG-OPV-5 was chosen because of its higher zeta potential, solid content, molecular weight, and high OPV incorporation. Instant visual detection of bilirubin under UV lamp could be possible, where the blue emission of the polymer turned bluish green instantly after bilirubin addition. This could be explained on the basis of the combined effect of energy transfer, inner filter effect, and some unquenched OPV, which was further supported by its emission, excitation, and absorption spectra as well as CIE coordinate diagram. The interference from other biomolecules, such as glucose, sucrose, metal ions, cholesterol, etc. was checked. The limit of detection was found to be as low as 20 nM, which is much lower than the clinically applicable range of 50 μmol/L. Finally, the polymer nanobeads were checked for real time monitoring of bilirubin in human blood serum, where PSG-OPV-5 was found to be selective against its structural homologue, i.e., biliverdin. Thus, a highly selective and sensitive visual sensor for bilirubin in human blood serum could be developed.

20 nM. To verify the sensor for real time biosensing application, the sensor was further applied for bilirubin detection in human blood serum, which contained dissolved proteins, metal ions, triglycerides, glucose, hormones, and water (in short, blood plasma without any clotting factor). In order to explore further, 100 μL of human blood serum was added to each vial containing an 1 × 10−6 to 3 × 10−5 M bilirubin concentration. This mixture was then added to a fixed polymer concentration (0.1 mg/3 mL) in ches buffer at pH = 10 under dark, followed by recording its emission, excitation, and absorption spectra almost immediately. Excitation and emission spectra showed a marked decrease in OPV emission, with the increasing concentration of bilirubin similar to studies done in water (Figure 5A,B). Instant quenching of ≈24% was observed with an 1 × 10−6 M bilirubin concentration, which enhanced to more than 55% for 6 × 10−6 M, and appreciable quenching was observed with a concentration of 3 × 10−5 M. Normalizing emission spectra at 446 nm clearly showed enhancement in the green emission region after the addition of bilirubin (inset Figure 5B). Strong absorption below 300 nm in the absorption spectra of samples in human serum (Figure S27) indicated the presence of other analytes, such as proteins, cholesterol, etc., which tend to absorb in this region. Finally, the interference was checked from structure homologue of bilirubin, i.e., biliverdin. Biliverdin closely resembles bilirubin in structure, differing by the presence of one additional double bond at the C10 position making the dipyrromethene units in conjugation. Figure S28 clearly signifies the drastic drop in emission of OPV upon addition of bilirubin, while a less appreciable change was observed in the case of biliverdin. This proved a good selectivity of the sensor toward bilirubin over other structural homologues, even in human blood serum.

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ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsapm.9b00222.

CONCLUSION A tailor-made water-soluble glucuronic acid bearing PS polymer (PS-DGlu) was synthesized, which not only stabilized the PS nanobeads in the size range of 160−328 nm but also surface-functionalized the nanobeads with glucuronic acid together with successful covalent incorporation of OPV as fluorophore in styrene miniemulsion polymerization. To the

The structural characterization of the monomers using NMR, FTIR, and HRMS; absorption spectra; FESEM and TEM images; GPC, CIE coordinate diagram, and DLS of polymers (PSG-OPV-n); emission and excitation spectra of PSG-OPV-5 with and without human blood serum; absorption spectra after bilirubin addition; 1237


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nation of Bilirubin in Human Blood Serum. Sens. Actuators, B 2013, 185, 337−344. (16) Santhosh, M.; Chinnadayyala, S. R.; Kakoti, A.; Goswami, P. Selective and Sensitive Detection of Free Bilirubin in Blood Serum using Human Serum Albumin Stabilized Gold Nanoclusters as Fluorometric and Colorimetric Probe. Biosens. Bioelectron. 2014, 59, 370−376. (17) Ellairaja, S.; Shenbagavalli, K.; Ponmariappan, S.; Vasantha, V. S. A Green and Facile Approach for Synthesizing Imine to Develop Optical Biosensor for Wide Range Detection of Bilirubin in Human Biofluids. Biosens. Bioelectron. 2017, 91, 82−88. (18) Du, Y.; Li, X.; Lv, X.; Jia, Q. Highly Sensitive and Selective Sensing of Free Bilirubin Using Metal-Organic Frameworks-Based Energy Transfer Process. ACS Appl. Mater. Interfaces 2017, 9, 30925− 30932. (19) Senthilkumar, T.; Asha, S. K. Self-Assembly in Tailor-Made Polyfluorenes: Synergistic Effect of Porous Spherical Morphology and FRET for Visual Sensing of Bilirubin. Macromolecules 2013, 46, 2159−2171. (20) Senthilkumar, T.; Asha, S. K. Selective and Sensitive Sensing of Free Bilirubin in Human Serum Using Water-Soluble Polyfluorene as Fluorescent Probe. Macromolecules 2015, 48, 3449−3461. (21) Stoffelbach, F.; Belardi, B.; Santos, J. M. R. C. A.; Tessier, L.; Matyjaszewski, K.; Charleux, B. Use of an Amphiphilic Block Copolymer as a Stabilizer and a Macroinitiator in Miniemulsion Polymerization under AGETATRP Conditions. Macromolecules 2007, 40, 8813−8816. (22) Save, M.; Manguian, M.; Chassenieux, C.; Charleux, B. Synthesis by RAFT of Amphiphilic Block and Comblike Cationic Copolymers and Their Use in Emulsion Polymerization for the Electrosteric Stabilization of Latexes. Macromolecules 2005, 38, 280− 289. (23) Riess, G.; Labbe, C. Block Copolymers in Emulsion and Dispersion Polymerization. Macromol. Rapid Commun. 2004, 25, 401−435. (24) Liu, S.; Armes, S. P. Polymeric Surfactants for the New Millennium: a pH-responsive, Zwitterionic, Schizophrenic Diblock Copolymer. Angew. Chem., Int. Ed. 2002, 41, 1413−1416. (25) Makkad, S. K.; Asha, S. K. π-Conjugated Chromophore Incorporated Polystyrene Nanobeads as Single Optical Agent for Three-Channel Fluorescent Probe in Bioimaging Application. ACS Biomater. Sci. Eng. 2017, 3, 1788−1798. (26) Tosin, M.; Murphy, P. V. Synthesis of α-Glucuronic Acid and Amide Derivatives in the Presence of a Participating 2-Acyl Protecting Group. Org. Lett. 2002, 4, 3675−3678. (27) Malkoch, M.; Thibault, R. J.; Drockenmuller, E.; Messerschmidt, M.; Voit, B.; Russell, T. P.; Hawker, C. J. Orthogonal Approaches to the Simultaneous and Cascade Functionalization of Macromolecules Using Click Chemistry. J. Am. Chem. Soc. 2005, 127, 14942−14949. (28) Ramanathan, T.; Fisher, F. T.; Ruoff, R. S.; Brinson, L. C. Amino-Functionalized Carbon Nanotubes for Binding to Polymers and Biological Systems. Chem. Mater. 2005, 17, 1290−1295. (29) Griffin, W. C. Calculations of HLB values of Non-ionic Surfactants. J. Soc. Cosmet. Chem. 1954, 5, 249−259. (30) Zhang, J.; Dubay, M. R.; Houtman, C. J.; Severtson, S. J. Sulfonated Amphiphilic Block Copolymers: Synthesis, Self-Assembly in Water and Application as Stabilizer in Emulsion Polymerization. Macromolecules 2009, 42, 5080−5090. (31) Tan, B.; Grijpma, D. W.; Nabuurs, T.; Feijen, J. Crosslinkable Surfactants Based on Linoleic acid-functionalized Block Copolymers of Ethylene Oxide and ε-Caprolactone for the Preparation of Stable PMMA Latices. Polymer 2005, 46, 1347−1357. (32) Garnier, S.; Laschewsky, A. New Amphiphilic Diblock Copolymers: Surfactant Properties and Solubilization in Their Micelles. Langmuir 2006, 22, 4044−4053. (33) Tadros, T. F. In Applied Surfactants: Principles and Applications; Wiley-VCH Verlag: Weinheim, Germany, 2005.

spectral overlap between bilirubin absorption and PSGOPV-5 excitation; and a table summarizing the molecular weight of polymers (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Fax: 0091-20-25902615. ORCID

S. K. Asha: 0000-0002-3999-4810 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been financially supported by the Science & Engineering Research Board funded project SB/S1/OC-66/ 2013. Sarabjot Kaur thanks CSIR for the Shyama Prasad Mukherjee (SPM) fellowship. We acknowledge Dr. Mahesh Kulkarni and his students Rajeshwari Rathore, Yugendra Patil, and Arwind Chaurasiya for providing and helping us with human blood serum.

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REFERENCES

(1) Kale, T. S.; Klaikherd, A.; Popere, B.; Thayumanavan, S. Supramolecular Assemblies of Amphiphilic Homopolymers. Langmuir 2009, 25, 9660−9670. (2) Savariar, E. N.; Aathimanikandan, S. V.; Thayumanavan, S. Supramolecular Assemblies from Amphiphilic Homopolymers: Testing the Scope. J. Am. Chem. Soc. 2006, 128, 16224−16230. (3) Zhu, Y.; Liu, L.; Du, J. Probing into Homopolymer SelfAssembly: How Does Hydrogen Bonding Influence Morphology? Macromolecules 2013, 46, 194−203. (4) Zhang, J.; Dubay, M. R.; Houtman, C. J.; Severtson, S. J. Sulfonated Amphiphilic Block Copolymers: Synthesis, Self-Assembly in Water, and Application as Stabilizer in Emulsion Polymerization. Macromolecules 2009, 42, 5080−5090. (5) Zhang, J.; Liu, K.; Müllen, K.; Yin, M. Self-Assemblies of Amphiphilic Homopolymers: Synthesis, Morphology Studies and Biomedical Applications. Chem. Commun. 2015, 51, 11541−11555. (6) Basu, S.; Vutukuri, D. R.; Shyamroy, S.; Sandanaraj, B. S.; Thayumanavan, S. Invertible Amphiphilic Homopolymers. J. Am. Chem. Soc. 2004, 126, 9890−9891. (7) Arumugam, S.; Vutukuri, D. R.; Thayumanavan, S.; Ramamurthy, V. Amphiphilic Homopolymer as a Reaction Medium in Water: Product Selectivity within Polymeric Nanopockets. J. Am. Chem. Soc. 2005, 127, 13200−13206. (8) Basu, S.; Vutukuri, D.; Thayumanavan, S. Homopolymer Micelles in Heterogeneous Solvent Mixtures. J. Am. Chem. Soc. 2005, 127, 16794−16795. (9) Munoz-Bonilla, A.; van Herk, A. M.; Heuts, J. P. A. Preparation of Hairy Particles and Antifouling Films Using Brush-Type Amphiphilic Block Copolymer Surfactants in Emulsion Polymerization. Macromolecules 2010, 43, 2721−2731. (10) Bonnett, R.; Davies, J. E.; Hursthouse, M. B. Structure of Bilirubin. Nature 1976, 262, 326−328. (11) Fevery, J. Bilirubin in Clinical Practice: a Review. Liver Int. 2008, 28, 592−605. (12) Koolman, J.; Roehm, K. H. Color Atlas of Biochemistry, 2nd ed.; Thieme: Stuttgart, Germany, and New York, 2005. (13) Silbernagl, S.; Despopoulos, A. Color Atlas of Physiology, 6th ed.; Thieme: Stuttgart, 2009; p 252. (14) Boyer, T. D.; Manns, P. P.; Sanyal, A. J. Hepatology: A Textbook of Liver Disease, 6th ed.; Saunders: London, 2011; p 1079. (15) Feng, Q.; Du, Y.; Zhang, C.; Zheng, Z.; Hu, F.; Wang, Z.; Wang, C. Synthesis of the Multi-walled Carbon Nanotubes-COOH/ graphene/gold Nanoparticles Nanocomposite for Simple Determi1238


Article

ACS Applied Polymer Materials (34) Makkad, S. K.; Asha, S. K. Surface Functionalized Fluorescent PS Nanobead Based Dual-Distinct Solid State Sensor for Detection of Volatile Organic Compounds. Anal. Chem. 2018, 90, 7434−7441. (35) Kuo, A. Fluorescence Resulting from π-stacking in Polystyrene Solutions. CheM 2011, 1, 80−86. (36) Brodersen, R. Bilirubin: Solubility and Interaction with Albumin and Phospholipid. J. Biol. Chem. 1979, 254, 2364−2369. (37) Shrivastava, A.; Gupta, V. B. Methods for the Determination of Limit of Detection and Limit of Quantitation of the Analytical Methods. Chronicles of Young Scientists 2011, 2, 21−25. (38) Liu, S. G.; Luo, D.; Li, N.; Zhang, W.; Lei, J. L.; Li, N. B.; Luo, H. Q. Water Soluble Nonconjugated Polymer Nanoparticles with Strong Fluorescence Emission for Selective and Sensitive Detection of Nitro-Explosive Picric Acid in Aqueous medium. ACS Appl. Mater. Interfaces 2016, 8, 21700−21709.

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