A Boronate Affinity-Assisted SERS Tag Equipped with a Sandwich

May 5, 2016 - Phenylboronic acid-functionalized, Ag shell-coated, magnetic, monodisperse polymethacrylate microspheres equipped with a glycoprotein-se...
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A Boronate Affinity-Assisted SERS Tag Equipped with a Sandwich System for Detection of Glycated Hemoglobin in the Hemolysate of Human Erythrocytes Duygu Deniz Usta,† Kouroush Salimi,‡ Asli Pinar,▽,∥ Il̇ knur Coban,∥ Turgay Tekinay,†,⊥ and Ali Tuncel*,‡,§ †

Department of Medical Biology and Genetics, Gazi University, 06500, Ankara, Turkey Chemical Engineering Department, ▽Department of Medical Biochemistry, Faculty of Medicine, and §Division of Nanotechnology and Nanomedicine, Hacettepe University, 06800, Ankara, Turkey ∥ Hacettepe University Hospitals Central Laboratory, 06800, Ankara, Turkey ⊥ Life Sciences Application and Research Center, Gazi University, 06830, Ankara, Turkey ‡

S Supporting Information *

ABSTRACT: Phenylboronic acid-functionalized, Ag shellcoated, magnetic, monodisperse polymethacrylate microspheres equipped with a glycoprotein-sensitive sandwich system were proposed as a surface-enhanced Raman scattering (SERS) substrate for quantitative determination of glycated hemoglobin (HbA1c). The magnetization of the SERS tag and the formation of the Ag shell on the magnetic support were achieved using the bifunctional reactivity of newly synthesized polymethacrylate microspheres. The hemolysate of human red blood cells containing both HbA1c and nonglycated hemoglobin was used for determination of HbA1c. The working principle of the proposed SERS tag is based on the immobilization of HbA1c by cyclic boronate ester formation between glycosyl residues of HbA1c and boronic acid groups of magnetic polymethacrylate microspheres and the binding of p-aminothiophenol (PATP)-functionalized Ag nanoparticles (Ag NPs) carrying another boronic acid ligand via cyclic boronate ester formation via unused glycosyl groups of bound HbA1c. Then, in situ formation of a Raman reporter, 4,4′-dimercaptoazobenzene from PATP under 785 nm laser irradiation allowed for the quantification of HbA1c bound onto the magnetic SERS tag, which was proportional to the HbA1c concentration in the hemolysate of human erythrocytes. The sandwich system provided a significant enhancement in the SERS signal intensity due to the plasmon coupling between Ag NPs and Ag shell-coated magnetic microspheres, and low HbA1c concentrations down to 50 ng/mL could be detected. The calibration curve obtained with a high correlation coefficient between the SERS signal intensity and HbA1c level showed the usability of the SERS protocol for the determination of the HbA1c level in any person. KEYWORDS: SERS, boronate affinity chromatography, glycoprotein, sandwich assay, HbA1c, core−shell microspheres

1. INTRODUCTION Surface-enhanced Raman scattering (SERS) is a highly sensitive spectroscopic method in which the Raman signal of molecules is enhanced due to the surface plasmon resonance of the nanostructured surface.1,2 Various SERS tags in the form of nanoparticles, microspheres, and capillary monoliths have been designed for the sensitive detection of various biomolecules (i.e., DNA, proteins, sialic acid, and creatinine) and different types of cancer cells.3−6 Recently, significant attention has been given to the detection of glucose in biological specimens via SERS. Tandem assays of protein and glucose with functionalized core/shell particles based on magnetic separation and SERS were developed.7 Au@SiO2 core/shell nanoparticles were used as substrate for highly sensitive SERS-based determination of glucose and uric acid.8 The gold nanostar@silica core−shell © XXXX American Chemical Society

nanoparticles conjugated with glucose oxidase were also proposed as a SERS biosensor for label-free detection of glucose.9 The boronate affinity interaction was first utilized as an efficient tool for the design of SERS tags for the detection of small saccharide molecules. Highly sensitive SERS detection of glucose was performed by following the alkyne band originating from alkyne-functionalized phenylboronic acid as a SERS probe.10 The 4-mercaptophenylboronic acid (MPBA)-functionalized gold surface of a quasi three-dimensional plasmonic nanostructure array was used as a SERS platform for fast Received: January 5, 2016 Accepted: April 28, 2016

A

DOI: 10.1021/acsami.6b00138 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces detection of fructose in complex media.11 Self-assembled MPBA on aligned Ag nanorods was also used for glucose detection in the physiological range by SERS.12 Another substrate containing two self-assembled monolayers was also tried as a SERS probe by means of the boronic acid and the alkyl spacer groups that serve as molecular recognition and penetration agents, respectively, for the detection of glucose.13 In most boronate affinity-based SERS platforms, MPBA was the common boronate affinity ligand for capturing target biomolecules via reversible boronate ester formation.14,15 The surface-enhanced biosensing of glycan expression in different cell lines by SERS on the plasmonic platforms functionalized with MPBA as the Raman reporter was demonstrated.15 A facile and sensitive sandwich assay using SERS was developed for sensitive detection of saccharides (i.e., glucose, galactose, and fructose) in an aqueous media.16 A highly sensitive and facile “turn-off” SERS sensor based on the etching effect on silver nanoparticles marked with Raman tags of 4-mercaptopyridine was designed for the determination of glucose.17 The low-level detection of glycosyl-functionalized biomolecules was also performed with the SERS platform without using the boronate affinity interaction.18−21 Plasmonic free-standing films made of biocompatible chitosan nanofibers and gold nanoparticles were engineered by a simple protocol and used as a new SERS platform for the detection of glucose.19 SERS imaging of cancer cells and tissues via sialic acid-imprinted nanotags was performed.20 A novel SERS method for sensitive quantitative analysis of haptoglobin (Hp), an acute phase plasma glycoprotein as a prognostic ovarian cancer biomarker, was developed.21 Glycated hemoglobin A1c (HbA1c), which is formed by nonenzymatic glycosylation of hemoglobin, has become a vital clinical indicator for the diagnosis of diabetes.22,23 To date, various analytical techniques have been developed for analyzing HbA1c and glycemic control.24−29 Ishikawa and co-workers recently reported an alternative method for the detection of HbA1c by surface-enhanced resonance Raman spectroscopy (SERRS).30 Here, we demonstrate, for the first time to the best of our knowledge, the application of a diol-sensitive sandwich system via SERS for the sensitive and selective detection of HbA1c. For distinguishing Hb (nonglycated protein) and HbA1c (glycated protein), using boronate affinity chromatography as a tool makes possible the remarkably selective detection of HbA1c. For this purpose, HbA1c was selectively isolated from a solution of hemolysate of human erythrocytes and bound to the phenylboronic acid ligand (MPBA) on Ag shell-coated magnetic polymethacrylate microspheres (MPBAAg@MagPMMS). Then, a sandwich system was constructed by the binding of silver nanoparticles (Ag NPs) carrying both paminothiophenol (PATP) and 4-mercaptobenzoic acid (PMBA) onto the HbA1c present on MPBA-Ag@MagPMMS. The photocoupling of PATP to 4,4′-dimercaptoazo-benzene (DMAB) in situ upon illumination of the laser during the SERS measurement allowed for the determination of bound HbA1c with specific/sensitive Raman peaks using the hemolysate of human erythrocytes as a multicomponent sample mixture. Hence, a SERS-based assay using magnetic microspheres and a SERS nanotag for the detection of HbA1c was constructed first.

WI, USA) and used in the synthesis of monodisperse, porous poly(3chloro-2-hydroxypropyl methacrylate-co-ethylene dimethacrylate) (poly(HPMA-Cl-co-EDMA)) microspheres without further purification. Ethylbenzene (EB), tetrahydrofuran (THF), and absolute ethanol (EtOH) were of HPLC grade and supplied from Aldrich. The stabilizer polyvinylpyrolidone (PVP K-30, Mw: 40,000 Da) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Sodium lauryl sulfate (SLS) and poly(vinyl alcohol) (PVA, 87−89% hydrolyzed, molecular weight: 85,000−146,000) were also obtained from Sigma. 2,2′-Azobis(isobutyronitrile) (AIBN) was supplied from Merck A.G. (Darmstadt, Germany) and recrystallized from methanol before use. The oil soluble initiator benzoyl peroxide (BPO) was supplied from Merck and dried in vacuo at 30 °C. Distilled−deionized (DDI) water (18.2 MΩ cm) from Merck-Millipore Direct-Q3 (Germany) was used in all experiments. FeCl3·6H2O, FeCl2·4H2O, and ethylenediamine (EDA) were obtained from Aldrich. Chloroauric acid (HAuCl4), trisodium citrate (TSC), silver nitrate, 4-mercaptophenylboronic acid (MPBA), 3-aminopropyltriethoxysilane (APTES), and triethylamine (TEA) were supplied from Aldrich. 4-Aminothiophenol (PATP), hemoglobin (Hb), and human blood hemolysate (BCR-405) were obtained from Sigma. 2.2. Synthesis of Ag Shell-Coated Magnetic, Monodisperse Polymethacrylate (Ag@MagPMMS) Microspheres. The synthesis of Ag@MagPMMS is provided in the Supporting Information. 2.3. Characterization of Polymethacrylate Microspheres. The average size, size distribution, and surface morphology of magnetic microspheres were investigated by a scanning electron microscope (FEI, Quanta 200 FEG, USA). The specific surface area of magnetic polymethacrylate microspheres was determined by a surface area and pore size analyzer (Quantachrome, Nova 2200E, UK) using a nitrogen adsorption−desorption method. X-ray diffraction spectra of the magnetic polymethacrylate microspheres, Au nanoparticle-attached magnetic polymethacrylate microspheres, and Ag shell-coated magnetic polymethacrylate microspheres were obtained using a Rigaku X-ray diffractometer (Ultima IV, Japan). The magnetization curves of magnetic polymethacrylate microspheres (MagPMMS), Au nanoparticle-coated magnetic polymethacrylate microspheres (Au NP@ MagPMMS), and Ag@MagPMMS were obtained by a vibrating sample magnetometer (Cryogenic Limited, PPM System, UK). 2.4. MPBA Attachment onto the Ag Shell-Coated Magnetic Polymethacrylate Microspheres. For the attachment of MPBA onto MagPMMS, the ethanolic solutions of MPBA prepared with different MPBA concentrations between 10−6 and 10−2 M were used. Typically, Ag@MagPMMS (60 mg) was added into the ethanolic solution of MPBA (20 mL, 0.1 M). The resulting dispersion was vortexed for 1 min and mechanically stirred for 2 h at room temperature. The microspheres were rinsed with ethanol twice, collected by a magnet, and redispersed in ethanol. MPBA-Ag@ MagPMMS was added to HEPES buffer (5 mL, 50 mM). The dispersion was sonicated for 30 s and shaken for 30 min at room temperature. The microspheres were separated from the buffer by a magnet and washed thoroughly with HEPES buffer three times to remove ethanol. The final precipitate was redispersed in HEPES buffer at pH 8.5 (50 mM, 5 mL). 2.5. Preparation of PATP- and PMBA-Attached Ag NPs. The sandwich system was designed according to the method reported by Bi and co-workers for the determination of glucose with some modifications.16 AgNO3 (1 mM) was dissolved in DDI water (200 mL) by heating to a boil. Then, 4 mL of a 1% w/w aqueous sodium citrate solution was added to the AgNO3 solution. Boiling of the solution was continued for 1 h. The dispersion containing Ag NPs with an average diameter of 60 nm was stirred and cooled to room temperature. The vulcanization of Ag NPs was performed by adding Na2S (2 mM) to 10 mL of the Ag NP dispersion by stirring for 30 min and allowing the solution to age without agitation for 1 h. Then, the as-prepared solution was centrifuged at 10,000 rpm for 10 min and washed with DDI water. Finally, PATP (1 mM) and MPBA (0.5 mM) were bound onto the Ag NPs within an ethanolic medium (10 mL) by stirring at 300 rpm for 10 min, followed by aging for 12 h without stirring in dark. The as-prepared colloidal solution was centrifuged at

2. EXPERIMENTAL SECTION 2.1. Materials. Glycidyl methacrylate (GMA), 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), and ethylene dimethacrylate (EDMA) were supplied from Aldrich Chemical Co. (Milwaukee, B

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ACS Applied Materials & Interfaces 10,000 rpm for 10 min, and the isolated Ag NPs were washed with DDI water three times. 2.6. Attachment of HbA1c onto MPBA-Ag@MagPMMS. For preparation of the HbA1c sample solutions at different concentrations for the SERS measurements, the lyophilized form of commercially obtained hemolysate of human erythrocytes was reconstituted according to the following procedure: The ampule content was kept at room temperature for 1 h, and 1.0 mL of DDI water was slowly added to the lyophilized mixture containing 0.23 mmol/L total Hb (Fe4), 6.29% w/w of HbA1c as the percentage of total Hb, and NaEDTA as the anticoagulant in the reconstituted dispersion.31 The resulting dispersion was then gently mixed by rotation. According to the material information form provided by the supplier, the percentage of the HbA1c peak of all hemoglobin peaks was determined by high resolution HPLC at 15 °C and quantified by absorption at 410 nm.31 Venous blood was also taken and placed in EDTA-containing tubes from three volunteers. The erythrocyte suspension from the wholeblood of each volunteer, prepared according to the literature, was lyophilized.32 The hemolysate was obtained by adding an appropriate volume of DDI water to the lyophilized suspension. Total Hb content of each hemolysate was measured by a Unicel DxH 800 Coulter Cellular Analysis System (Beckman Coulter Canada LP, Canada). HbA1c analysis was performed using Immuchrom HbA1c reagents (ImmuChrom, GmbH, Heppenheim, Germany) in an HPLC system (Shimadzu, Japan). The samples with different HbA1c concentrations used for the SERS measurements were prepared by diluting each hemolysate with pH 8.5 HEPES buffer at different volume ratios. Hence, “the hemolysates of human erythrocytes” obtained from four different sources, with total Hb and HbA1c concentrations predetermined by clinical assays, were used for the preparation of SERS calibration curves for HbA1c determination. Typically, MPBA-Ag@MagPMMS (10 mg) was precipitated from the HEPES buffer by means of a magnet. The sample solution (5 mL, 50 mM HEPES buffer, pH 8.5) containing Hb and HbA1c at a certain concentration was added to precipitated MPBA-Ag@MagPMMS (10 mg). The new dispersion was mixed and shaken for 1 h at room temperature. The sample solutions containing HbA1c with concentrations ranging from 0.1 to 50 μg/mL were used for the adsorption of the respective diol carrying agent onto MPBA-Ag@MagPMMS. Hence, HbA1c in the sample was selectively captured by MPBAAg@MagPMMS via the formation of a cyclic boronate ester between MPBA and diol groups of glycosyl moieties on HbA1c. HbA1c-bound MPBA-Ag@MagPMMS was extensively washed with HEPES buffer (50 mM, pH 8.5) using an external magnet and finally dispersed in HEPES buffer (50 mM, pH 8.5) by vortexing. 2.7. Construction of Diol-Sensitive Sandwich System. The HbA1c-bound SERS tag was incubated with the colloidal solution of PATP:MPBA (2:1 mol/mol)-attached Ag NPs (PATP/MPBA@Ag NPs) in HEPES buffer (0.1 mg in 5 mL, 50 mM, pH 8.5) for 2 h. Then, a certain fraction of PATP/MPBA@Ag NPs were bound onto the HbA1c present on MPBA-Ag@MagPMMS by the formation of cyclic boronate ester between the glycosyl residues on bound HbA1c and MPBA ligand on Ag NPs. The unbound PATP/MPBA@Ag NPs were removed with the supernatant obtained by the precipitation of HbA1c@MPBA-Ag@MagPMMS carrying bound fraction of PATP/ MPBA@Ag NPs (i.e., the sandwich system, PATP/MPBA@AgNPs@ HbA1c@MPBA-Ag@MagPMMS) by an external magnet. The sandwich system was washed three times with fresh HEPES buffer (50 mM, pH 8.5) to remove any physically adsorbed constituents. The washed microspheres were redispersed within the fresh HEPES buffer (5 mL, 50 mM, pH 8.5) for SERS measurements. 2.8. SERS Measurements. An aliquot (300 μL) of the sandwich system-bound and HbA1c-attached SERS tag dispersion was deposited on a glass substrate, and a cast film of the SERS tag was obtained by evaporation of the liquid at room temperature. SERS measurements were made using the cast film of the SERS tag. All SERS measurements were performed using the WITEC Alpha 300S Confocal Raman spectrometer module. A NIR laser with 785 nm wavelength was used for excitation. Laser power was measured using an optical power and energy meter console (Thorlabs, PM100D,

USA). For single Raman measurements, 50× objective, 20 s integration time, and 0.67 mW excitation power were used. 2.9. Effect of Plasmon Coupling on SERS. To test the effect of plasmon coupling on the SERS behavior of the sandwich system constructed (i.e., the sandwich obtained using MPBA-Ag@ MagPMMS), a reference sandwich system without an Ag shell on the MagPMMS was prepared. In the synthesis of the reference sandwich system, 3-aminophenylboronic acid (APBA) was selected as the appropriate phenylboronic acid (PBA) ligand because the covalent attachment of APBA onto the hydroxyl-carrying magnetic microspheres was easier via its amino group.33 For this purpose, MagPMMS with hydroxyl functionality (0.4 g) was reacted with 3-glycidoxypropyltrimethoxysilane (GPTMS, 2 mL) within toluene (20 mL) under reflux for 6 h. GPTMS-attached MagPMMS was washed with toluene, ethanol, and water by magnetic separation and then reacted with APBA (0.5 g) at 50 °C for 4 h in an aqueous Na2CO3 solution at pH 9.0. APBA-attached magnetic polymethacrylate microspheres (APBA-MagPMMS) were then used as the base component of the reference sandwich system (i.e., PATP/MPBA-AgNPs@HbA1c@ APBA-MagPMMS). PBA content on the outher surface of APBAMagPMMS (mmol PBA/m2) was determined by FTIR-ATR (FTIRSMART-IR, Thermo-Scientific, USA) using the CC stretching peak belonging to the aromatic ring of APBA at 1590 cm−1. On the other hand, PBA content on the outer surface of MPBA-Ag@MagPMMS was determined using the S atom content of Ag@MagPMMS by X-ray photoelectron spectroscopy (XPS, SPECS EA 300 equipped with a monochromatic Al Kα X-ray source, SPECS GmbH, Germany) because FTIR-ATR was not suitable for this support due to the Ag shell. For evaluating the effect of plasmon coupling on the SERS behavior of the sandwich system constructed, the SERS response of the reference sandwich was investigated with different HbA1c concentrations using APBA-MagPMMS as the base material. For this purpose, the human erythrocyte suspensions diluted with HEPES buffer (50 mM, pH 8.5) containing HbA1c at different concentrations (i.e., 1.0−2.5 μg/mL, 1 mL) were interacted with APBA-MagPMMS (10 mg) by following the protocol used for the interaction of HbA1c with MPBA-Ag@MagPMMS. The reference sandwich without the Ag shell coating on MagPMMS was obtained by using the same procedure followed for the Ag shell-carrying MagPMMS (i.e., section 2.7). SERS spectra of the reference sandwich obtained with different HbA1c concentrations were recorded using laser excitation at 785 nm and compared with the sandwich system described as PATP/MPBAAgNPs@HbA1c@MPBA-Ag@MagPMMS in section 2.7.

3. RESULTS AND DISCUSSION 3.1. Characterization of SERS Tag. A boronate affinityassisted SERS substrate with magnetic form was designed. The magnetic form was preferred for easier isolation of the SERS active tag following binding of the glycated protein. Monodisperse, macroporous poly(HPMA-Cl-co-EDMA) microspheres 5.8 μm in size were selected as the starting material for the synthesis of the magnetic SERS tag. Various SERS tags have been synthesized using monodisperse polystyrene microspheres as the starting material in the past few years.34,35 The inertial character of polystyrene-based materials involves more tedious and time-consuming derivatization protocols in the synthesis of SERS tags. In our study, a new type of monodisperse, porous microsphere with bifunctional character [i.e., poly(HPMA-Cl-co-EDMA) microspheres] was first tried as the starting material. The derivatization routes used for the synthesis of the SERS tag are schematically described in Figures S1−S4. In the first stage, monodisperse, porous poly(HPMACl-co-EDMA) microspheres were obtained by a seeded polymerization technique (Figure S1).36 The magnetization was performed via their chloropropyl functionality, whereas their hydroxyl functionality was used for the formation of a silver shell on the magnetic polymer microspheres. For C

DOI: 10.1021/acsami.6b00138 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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broken bead (Figure 2A). As mentioned before, the silver shell around the polymer core was formed by the seed-mediated

magnetization, poly(HPMA-Cl-co-EDMA) microspheres were reacted with EDA to obtain covalently bound primary amine groups (Figure S2). The magnetization of primary aminefunctionalized microspheres was performed according to the literature.37,38 Iron ions (Fe2+ and Fe3+) were impregnated onto the microspheres using primary amine groups, and they were subsequently precipitated with ammonium hydroxide to form magnetite (Fe3O4) nanoparticles within the polymer microspheres (Figure S2).37,38 Following the synthesis of magnetic microspheres, additional primary amine functionality was generated by the covalent attachment of APTES via the hydroxyl group of magnetic microspheres (Figure S3). In the next stage, Au nanoparticles obtained by citrate reduction were attached to the magnetic microspheres via their primary amine functionality generated by APTES attachment onto the microspheres (Figure S4). In the last stage, the Ag shell was obtained on the surface of magnetic microspheres via “seedmediated growth” according to the literature (Figure S4).35 SEM photographs of plain poly(HPMA-Cl-co-EDMA) microspheres and magnetic polymethacrylate microspheres obtained from the poly(HPMA-Cl-co-EDMA) microspheres are given in Figure 1. As seen in Figure 1A, nearly

Figure 2. SEM photographs showing (A) Au nanoparticles within macropores of aminated magnetic polymethacrylate microspheres (photograph taken from a broken microsphere), (B) the size distribution, and (C) the surface morphology of Ag@MagPMMS. Magnification: (A) 200,000×, (B) 4,000×, and (C) 35,000×.

growth of Au nanoparticles attached to the magnetic polymer microspheres. SEM photographs of Ag@MagPMMS are given in Figure 2B and C. As seen in Table 1, slightly higher average size with respect to the previous stage was obtained from the SEM photograph of Ag@MagPMMS (Figure 2B). A marked decrease in SSA was observed by the formation of the Ag shell on the magnetic polymethacrylate microspheres (Table 1). This decrease was explained by the accumulation of Ag NPs within the pores of magnetic microspheres. The comparison of Figure 1B and 2C showed that an apparent change in the surface morphology of magnetic microspheres occurred upon the formation of the Ag shell around the magnetic microspheres. X-ray diffraction spectra of MagPMMS, Au NP@MagPMMS, and Ag@MagPMMS microspheres are given in Figure 3. The peaks belonging to Fe3O4, Au, and Ag were clearly observed in the XRD spectra of the respective microspheres. The magnetization curves of different microspheres are given in Figure 4. As seen here, all microsphere types exhibited superparamagnetic behavior. The highest saturation magnetization was obtained with the polymer microspheres. The saturation magnetization decreased by the deposition of Au and Ag onto the polymer microspheres. Ag@MagPMMS gave lower saturation magnetization due to the mass increase that occurred upon the deposition of Ag onto the polymer microspheres. The aggregation behavior of Ag@MagPMMS in aqueous media was investigated by comparing with some commonly used nanoparticles. In these experiments, Ag NPs (60 nm) and magnetic Fe3O4 nanoparticles (MNPs, 21 nm) were included for comparison. The aggregation response was determined by the absorbance measurement at 500 nm in a UV−visible spectrophotometer for an aqueous particle dispersion at pH 7.4 and containing NaCl at a certain concentration.37,38 The experimental details are given in the Supporting Information. In these runs, the ratio of the absorbance of the aqueous particle dispersion containing NaCl (pH 7.4) to the absorbance of the particle dispersion obtained in DDI water (i.e., the medium

Figure 1. SEM photographs of (A) poly(HPMA-Cl-co-EDMA) microspheres and (B) MagPMMS. Magnification: 18,000×. Insets for both particles show the size distribution. Inset magnification: 4,000×.

monodisperse polymer microspheres were obtained in the porous form. No significant changes were observed in the surface morphology by the magnetization of polymer microspheres (Figure 1B). The properties of bare and magnetic microspheres are given in Table 1. A slight increase was observed in the mean size of Table 1. Size Properties and Specific Surface Area (SSA) of Polymethacrylate Microspheres at Different Stages of the Synthesis microsphere type

size (μm)

CV (%)

SSA (m2/g)

poly(HPMA-Cl-co-EDMA) microspheres MagPMMS Ag@MagPMMS

5.8 6.0 6.2

5.1 4.1 4.2

45 42 10

the polymer microspheres after magnetization. The slight decrease in specific surface area should probably be related to the accumulation of magnetic Fe3O4 nanoparticles within the pores of the polymer microspheres. The attachment of Au NPs onto MagPMMS was confirmed by scanning electron microscopy. Gold nanoparticles attached on the surface of the macropores of MagPMMS were observed in the form of white dots in the SEM photograph taken from a D

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concentrations was also checked by optical microscopy. As seen in the optical micrographs in the right panel of Figure S5, Ag@ MagPMMS were observed in the form of invidiual microspheres in the aqueous buffer media prepared with different NaCl concentrations up to 500 mM. This property can be considered as an advantage, making easier the use of the proposed SERS tag in different biological fluids. Higher particle size (5.8 μm) and the surface chemistry are likely the factors that make Ag@MagPMMS more resistant to aggregation. 3.2. SERS Study. Recently, various SERS platforms were successfully designed for sensitive and reliable detection of different biomolecules and cancer cells using PATP as the SERS probe.41−49 The characteristic SERS signals are generated by photocoupling of PATP to 4,4′-dimercaptoazobenzene (DMAB) in situ upon illumination of a laser during the SERS application.17 The SERS response obtained by using PATP as the Raman probe and its in situ photocoupling behavior were extensively investigated.50−52 The sandwich system used for the detection of HbA1c in the hemolysate of human erythrocytes also containing Hb by SERS is shown in Figure 5. The selected boronate affinity ligand MPBA was attached to the Ag shell on the magnetic polymethacrylate microspheres via the interaction between silver and thiol groups. Phenylboronic acid is found in the anionic tetrahedral form at pH 8.5, and the selective binding of diol-carrying agent (HbA1c in our case) should occur via the formation of cyclic boronate ester between diol-carrying agent and anionic tetrahedral form (Figure 5B, HbA1c@MPBA-Ag@ MagPMMS).53 Then, Ag NPs carrying both MPBA and PATP (PATP/MPBA-Ag NPs) were interacted with the HbA1c bound to MPBA-Ag@MagPMMS, and a certain fraction of PATP/MPBA-Ag NPs were bound to the HbA1c present on MPBA-Ag@MagPMMS by the formation of cyclic boronate ester between the glycosyl residues on bound HbA1c and the MPBA ligand on Ag NPs. Hence, the sandwich system PATP/ MPBA-AgNPs@HbA1c@MPBA-Ag@MagPMMS was obtained (Figure 5C). The photocoupling of PATP to 4,4′dimercaptoazobenzene (DMAB) in situ upon illumination of a 785 nm laser during the SERS measurements allowed for the determination of bound HbA1c on the magnetic microspheres with the Raman peaks originated from DMAB formed in situ on the bound Ag NPs (Figure 5D). Then, the extent of DMAB formed on the bound fraction of Ag NPs proportional to the HbA1c bound to MPBA-Ag@MagPMMS controls the SERS signal intensity. Ag@MagPMMS was first interacted with the MPBA solution prepared in HEPES buffer (50 mM, pH 8.5). For removing physically adsorbed MPBA, the microspheres were extensively washed with HEPES buffer using a magnet and redispersed in the same buffer. An aliquot from the dispersion was placed on a glass slide, and the SERS spectrum was recorded using the film formed by the dried microspheres after the evaporation of water at room temperature. A typical SERS spectrum of MPBAAg@MagPMMS is given in Figure S6. As seen here, SERS peaks were observed at 698, 1000, 1024, 1070, 1184, 1285, 1490, and 1583 cm−1. Very similar SERS spectra with different MPBA-functionalized SERS tags were also obtained by different groups.11−13 A Raman band appearing at 698 cm−1 was assigned to the bending of C−C ring and C−S stretching modes. The peaks at 1000 and 1024 cm−1 were due to the bending of C−C in the phenyl ring and bending of C−H, respectively.54,55 The peaks at 1070 and 1184 cm−1 showed the B−OH and B−C stretching modes of the phenylboronic acid

Figure 3. X-ray diffraction spectra of (A) poly(HPMA-Cl-co-EDMA) microspheres, (B) magnetic polymethacrylate microspheres, (C) Au nanoparticle-decorated magnetic polymethacrylate microspheres, and (D) Ag shell-coated magnetic polymethacrylate microspheres.

Figure 4. Magnetization curves of (A) MagPMMS, (B) Au NP@ MagPMMS, and (C) Ag@MagPMMS.

containing no salt) was utilized as the indicator for particle aggregation.39,40 The variation of the absorbance ratio with the salt concentration is shown in Figure S5 for the aqueous dispersions of Ag@MagPMMS, Ag NPs, and MNPs. In Figure S5, the zero point on the x-axis corresponds to the particle dispersion obtained in DDI water, whereas the other points were obtained with different NaCl concentrations within 50 mM of phosphate buffer at pH 7.4. As seen in Figure S5, apparent decreases in the absorbance ratio were observed for both Ag NPs and MNPs due to the aggregation observed by starting from very low NaCl concentrations (i.e., 20 mM). However, no significant decrease was observed in the absorbance of the Ag@MagPMMS dispersion up to a salt concentration of 500 mM. This behavior showed that Ag@ MagPMMS was much more resistant against aggregation with respect to the nanoparticle types commonly used in the fabrication of SERS tags. The aggregation in the aqueous dispersions of Ag@MagPMMS containing NaCl at different E

DOI: 10.1021/acsami.6b00138 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 5. (A) Attachment of MPBA to Ag@MagPMMS; (B) binding of glycated (HbA1c) and nonglycated (Hb) proteins to MPBA-Ag@ MagPMMS via cyclic boronate ester formation by the interaction of phenylboronic acid ligand with the diol moiety; (C) interaction of the sandwich system by the formation of cyclic boronate ester between the glycosyl residues on bound HbA1c and the MPBA ligand on Ag NPs; and (D) formation of the photocoupling product (DMAB) under laser irradiation and detection of HbA1c.

group, respectively.12 The peak at 1583 cm−1 was due to the CC stretching mode of the phenyl group.13 The Raman spectrum in Figure S5 clearly indicated that MPBA was attached to the Ag shell on the magnetic polymethacrylate microspheres. On the other hand, the signals obtained at different spots taken from different magnetic microspheres gave similar peak intensities (data not shown). This finding was evaluated as evidence for the homogeneous distribution of MPBA molecules on the uniform Ag shell obtained on the magnetic microspheres. The hemolysate of human red blood cells containing both HbA1c and Hb was used as the sample for the determination of HbA1c with a boronate affinity-based SERS substrate equipped with a sandwich system. The working principle of the system is based on the immobilization of HbA1c by the cyclic boronate ester formation between glycosyl residues of HbA1c and boronic acid groups of Ag shell-coated magnetic polymethacrylate microspheres and the binding of Raman reporter (PATP)-functionalized Ag NPs also carrying boronic acid ligand via cyclic boronate ester formation via remaining (unused) glycosyl groups of bound HbA1c. Then, in situ formation of DMAB from the selected reporter, PATP, under 785 nm laser irradiation allowed the determination of PATP on the bound Ag NPs. Principally, the extent of PATP present on the magnetic microspheres should be proportional to the extent of HbA1c bound to the magnetic SERS tag, which should be also proportional to the HbA1c concentration in the hemolysate. For investigating the usability of the sandwich system for determination of the HbA1c concentration, the hemolysates of human red blood cells obtained from whole-blood samples

taken from three volunteers and a commercially supplied hemolysate of human red blood cells were used. Total Hb and HbA1c levels of hemolysates obtained from the three volunteers were determined by clinical assays and are given in Table 2. Total Hb and HbA1c levels of commercially Table 2. Total Hb and HbA1c Concentrations of Hemolysates of Human Red Blood Cells Obtained from Different Sources As Determined by Clinical Assays source

hemolysate code

Hb (g/dL)

HbA1c (% mol)

HbA1ca (μg/mL)

commercial volunteer 1 volunteer 2 volunteer 3

A0 A1 A2 A3

1.5 5.3 7.3 5.1

6.3 4.9 7.1 9.9

950 2160 5180 5050

a The HbA1c concentration (μg/mL) was calculated from total Hb and HbA1c values determined by clinical assays.

supplied human erythrocyte suspension are also included in Table 2 as the values provided by the supplier.31 HbA1c concentration in terms of μg of HbA1c/mL of hemolysate was also calculated for each sample using the values obtained from clinical assays and are given in Table 2. The hemolysates obtained from four different sources were serially diluted using HEPES buffer (50 mM, pH 8.5) with different volume ratios for varying the HbA1c concentration in the diluted hemolysates. The SERS spectra of diluted hemolysates obtained from each donor were then recorded. Typical SERS spectra obtained by the dilution of commercially supplied hemolysate of human red blood cells F

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investigated by using a reference sandwich system prepared with the magnetic microspheres not containing a Ag shell (i.e., MagPMMS). The representative SERS spectra for the sandwich system constructed (i.e., PATP/MPBA-AgNPs@HbA1c@ MPBA-Ag@MagPMMS) and the reference sandwich system not containing a Ag shell on the magnetic microspheres (PATP/MPBA-AgNPs@HbA1c@APBA-MagPMMS) obtained with a HbA1c concentration of 2.5 μg/mL are given in Figure 7A. The surface PBA contents of APBA-MagPMMS and MPBA-Ag@MagPMMS were determined to be 0.76 and 0.84 mmol/m2, respectively. In other words, the surface PBA contents of MPBA-Ag@MagPMMS and APBA-MagPMMS were very similar. Hence, the amount of HbA1c and the number of MPBA/PATP-Ag NPs captured on the outer surfaces of these supports via cyclic boronate ester formation should be very close during the formation of the corresponding sandwiches. This case normally involves the generation of SERS peaks belonging to the in situ Raman reporter with similar intensities using the sandwich systems obtained with both supports. However, the peak areas at 1140 and 1436 cm−1 in the SERS spectrum obtained with the sandwich containing a Ag shell carrying MagPMMS (i.e., MPBA/PATP-Ag NPs@ HbA1c@MPBA-Ag@MagPMMS) were more than 2 times higher with respect to the corresponding peaks of the sandwich obtained without using the Ag shell (i.e., MPBA/PATP-Ag NPs@HbA1c@APBA-MagPMMS) (Figure 7B). Hence, the plasmon coupling between Ag NPs and Ag@MagPMMS should be the reason for extra enhancement to the SERS of the selected Raman reporter.48−51 Thus, using a sandwich system containing both Ag NPs and Ag@MagPMMS instead of a SERS platform containing Ag NPs and MagPMMS allowed for the generation of reasonably stronger Raman peaks with the selected SERS probe. For evaluating the SERS behavior of the sandwich system with the hemolysates obtained from different sources, the area of the peak from DMAB at 1436 cm−1 in the SERS spectra of hemolysates obtained by the serial dilution of each original hemolysate with HEPES buffer was plotted against the HbA1c concentration (Figure 8). A similar plot was also sketched for the peak at 1140 cm−1 and is given in Figure S7. The peak areas at 1436 and 1140 cm−1 were reported as the mean of four measurements with satisfactorily small standard deviation values. For all hemolysate sources, the peak area at the

with HEPES buffer are exemplified in Figure 6. As seen here, the Raman bands at 698, 1000, and 1024 cm−1 were assigned to

Figure 6. Typical SERS spectra obtained by the dilution of hemolysate of human red blood cells containing HbA1c at different concentrations using the PATP-MPBA@AgNPs@HbA1c@MPBA-Ag@MagPMMS sandwich system with commercially obtained hemolysate of human red blood cells.

the bending of C−C ring/C−S stretching, C−C bending in the phenyl ring, and C−H bending in the phenylboronic acid group, respectively.54,55 The B−OH stretching of the same group should also contribute to the peak at 1070 cm−1.12 The SERS tag capturing HbA1c and PATP/MPBA-functionalized Ag NPs displayed clear SERS peaks at 1436, 1390, and 1140 cm−1. These peaks resulted from in situ-generated DMAB from the photocoupling transformation of PATP on the Ag NPs under 785 nm laser irradiation.19,56 Raman shifts at 1436 and 1390 cm−1 are related to the NN stretching vibrations of DMAB.56 In situ-formed DMAB should also contribute to the peak at 1070 cm−1.19,50,56 On the other hand, the peak at 1574 cm−1 is related to the contributions from both MPBA and DMAB/PATP.1 As seen in Figure 6, the intensities of peaks at 1140 and 1436 cm−1 originating from the in situ-formed Raman reporter during the SERS measurement, DMAB, showed a clear change proportional to the HbA1c concentration in the sample mixture. The effect of plasmon coupling between Ag NPs and Ag@ MagPMMS on the SERS behavior of the sandwich system was

Figure 7. (A) Representative SERS spectra for the sandwich system constructed (PATP/MPBA-AgNPs@HbA1c@MPBA-Ag@MagPMMS) and the reference sandwich system not containing a Ag shell on magnetic microspheres (PATP/MPBA-AgNPs@HbA1c@APBA-MagPMMS) with a HbA1c concentration of 2.5 μg/mL for both sandwiches. (B) Bar diagram showing the effect of plasmon coupling between PATP-carrying Ag NPs and phenylboronic acid-functionalized Ag@MagPMMS for HbA1c concentrations of 1.0, 1.5, and 2.5 μg/mL in the hemolysate of human erythrocytes. G

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Figure 8, the peak area at 1436 cm−1 could be well-correlated with the HbA1c concentration for all hemolysates obtained from different sources with the square of the coefficient of variation (R2) values close to 0.99 within the HbA1c concentration range of 0.05−100 μg/mL. This range was reasonably broader with respect to the typical clinical range of HbA1c concentration. On the basis of the SERS spectra obtained, the lowest detection limit of HbA1c concentration with the proposed protocol could be stated to be 0.05 μg/mL. The strong SERS response obtained by the plasmon coupling between the constituents of the constructed sandwich system (i.e., Ag NPs and Ag@MagPMMS) allowed for the determination of low HbA1c concentrations down to 50 ng/ mL. The variation of the peak area with the HbA1c concentration expressed on a linear scale is also included in the Supporting Information to provide information regarding the saturation point of the SERS signal obtained by the sandwich system (Figure S8). As seen in Figure S8, the increase in the peak area after the HbA1c concentration reached 40 μg/mL was very small for both peaks. On the other hand, the linear tendency for the variation of the peak area with the HbA1c concentration sketched on a logarithmic scale was observed up to 100 μg/mL (Figure 8). Hence, one can conclude that the sensitivity of the determination decreases for HbA1c concentrations higher than 40 μg/mL. For generating a SERS calibration curve that can be used for estimation of the HbA1c level in a real hemolysate, the hemolysates obtained from four different sources (i.e., the commercial hemolysate and hemolysates obtained from three volunteers) were diluted with HEPES buffer by fixing the dilution ratio to a constant value for all of the hemolysates. Representative SERS spectra of four hemolysates diluted with a constant volume ratio of 1:100 μL:μL are given in Figure S9. Variations of the peak areas at 1436 cm−1 with the HbA1c concentration in the diluted hemolysate predetermined by the clinical assay are presented in Figure 9 for the hemolysates prepared with different dilution ratios ranging from 1:100 to 1:1000 μL:μL. In this figure, each hemolysate (i.e., each donor) is shown by a circle with a separate color. As seen here, a separate calibration curve was obtained for each dilution ratio. In other words, a linear correlation between the peak area at 1436 cm−1 and HbA1c concentration in the diluted hemolysate

Figure 8. Variation of the peak area at 1436 cm−1 with the HbA1c concentration in the hemolysates of human red blood cells either commercially obtained or prepared with the whole-blood samples taken from three volunteers diluted with HEPES buffer at pH 8.5.

Raman shift of 1436 cm−1 showed a linear increase with the increasing HbA1c concentration expressed in logarithmic scale. The results in Figure 8 and Table 2 showed that a diluted hemolysate prepared from an original hemolysate with higher HbA1c concentration provided a stronger SERS signal at a constant HbA1c concentration. Although the behavior in Figure 8 could not be directly used for the determination of the HbA1c concentration in a real sample, it was useful to see the SERS response of an original hemolysate with the respect to the others. Note that a similar behavior was also observed for the other characteristic Raman shift of DMAB at 1140 cm−1 (Figure S7). When total Hb and HbA1c levels of the commercial erythrocyte sample were considered,31 the HbA1c concentration of 6.29% mole total Hb found in the original hemolysate with a concentration of 0.23 mmol/L (i.e., 1.5 g/dL) corresponds to 467 μg/mL. The typical widest range that can be encountered in the clinical measurement of HbA1c is 2− 15% mole total Hb.57,58 Hence, the lowest and highest HbA1c concentrations in a diluted hemolysate should be 0.3 and 2.4 μg/mL, respectively, when a dilution ratio for obtaining a HbA1c concentration of 1 μg/mL is desired in a diluted hemolysate prepared from the selected original hemolysate. In

Figure 9. Calibration curves for HbA1c determination in real samples prepared with different dilution ratios using the hemolysates of human erythrocytes from a commercial product and three volunteers. The R2 values are given on the plot. H

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ACS Applied Materials & Interfaces was established for each dilution ratio with an R2 value higher than 0.98 by using original hemolysates with different HbA1c contents. Good consistency was also observed between the SERS spectra used for Figure 8 (sketched using a variable dilution ratio) and Figure 9 (sketched using a constant dilution ratio). When a given hemolysate of human red blood cells obtained from any donor with an unknown HbA1c level is diluted with HEPES buffer, using a certain dilution ratio, the HbA1c concentration in the diluted hemolysate can be determined by using both the calibration curve belonging to the selected dilution ratio in Figure 9 and the SERS peak area at 1436 cm−1 obtained with the diluted hemolysate. Then, the HbA1c level of the original, unknown hemolysate can be calculated by using the HbA1c concentration of the diluted sample and the dilution ratio selected. The straight line obtained with the dilution ratio of 1:100 μL:μL seemed to be more appropriate for the determination of the HbA1c concentration in a broader range for an unknown sample. The straight lines with higher slopes obtained by using the dilution ratios lower than 1:100 (i.e., 1:250, 1:500, and 1:1000) can be also used for the determination of the HbA1c concentration in narrower ranges with respect to 1:100. The approach presented here can be considered as an appropriate route for the derivation of a calibration curve with the real samples by considering the potential interferences for the determination of HbA1c level in the hemolysate of human erythrocytes. In conclusion, the behavior of Figure 9 clearly indicated that the boronate affinity SERS tag equipped with a sandwich system could be appropriately used for the determination of HbA1c in a real sample of hemolysate of human erythrocytes. On the other hand, the presence of nonglycated Hb in the hemolysate of the human erythrocyte suspension may be considered as a potential disturbance interfering with the proposed SERS measurement protocol based on the boronate affinity interaction between the target molecule (HbA1c) and the phenylboronic acid ligand on the SERS tag. Nonglycated Hb could potentially be nonspecifically adsorbed onto the SERS tag via interactions and then not be able to be removed by the washing protocol using HEPES buffer at pH 8.5. This hypothesis was tested by taking a SERS spectrum of the aqueous solution of nonglycated Hb on the SERS tag developed by following the protocol used for the determination of HbA1c in the hemolysate. In this measurement, total Hb concentration in the aqueous medium was set to 6.4 g/dL as a satisfactorily high and representative value for a typical hemolysate of human erythrocytes (Table 2). A comparison of the SERS spectrum of an aqueous solution containing only nonglycated Hb and the SERS spectrum of the diluted hemolysate containing both nonglycated Hb (6.4 g/dL) and HbA1c (10 μg/mL) is given in Figure 10. As seen here, no peak was detected at both 1140 and 1436 cm−1 (i.e., the wavelengths used for the determination HbA1c concentration in the hemolysate). Hence, this comparison indicated that the interaction of the developed SERS tag with a medium containing nonglycated Hb did not result in an interference in the proposed measurement protocol. Nonglycated Hb might be removed by the washing protocol applied or might not provide any SERS band originating from itself. The stability of the SERS spectra obtained by using PATP as a SERS probe for the detection of HbA1c was also examined. SERS spectra obtained at different times using a HbA1c concentration of 1 μg/mL are given in Figure S10. Here, the

Figure 10. Raman spectra acquired from nonglycosylated hemoglobin in water and HbA1c in the hemolysate of human erythrocytes for a commercially obtained hemolysate of human red blood cells.

time was defined after spreading an aliquot of the dispersion (∼50 μL) containing the HbA1c-bound sandwich system onto the glass slide. This behavior showed that a reproducible and reliable SERS response could be obtained under the selected conditions by in situ photocoupling of PATP to DMAB upon illumination of 785 nm laser.

4. CONCLUSIONS A magnetic tag with a sandwich system was synthesized for the detection of HbA1c by boronate affinity-assisted SERS. MPBAAg@MagPMMS was used for the selective isolation of HbA1c from the hemolysate of human erythrocytes containing nonglycated Hb. HbA1c bound onto the magnetic microspheres could be effectively quantified by the SERS tag equipped with a sandwich system constructed based on the boronate affinity binding of Ag NPs carrying an in situ formed Raman reporter. The main advantages of the magnetic SERS tag designed in this study are as follows: (i) Monodisperse porous polymethacrylate microspheres used as starting material for the magnetic SERS tag were obtained with reproducible and well-controlled size and porous properties by the seeded polymerization protocol. (ii) The chemistry of the starting material (i.e., the bifunctional character of polymethacrylate microspheres) was suitable for the synthesis of a SERS tag in magnetic form. The magnetization was performed using the chloropropyl group, whereas the coating of the Ag shell was achieved via hydroxyl functionality. (iii) The SERS protocol is based on the boronate affinity capture of the target biomolecule. The magnetic polymethacrylate microspheres can be isolated from the aqueous medium within 5−10 s by using an external magnet. In other words, the isolation of the SERS tag was quickly performed during the successive washings applied for the removal of undesired constituents following the interaction with HbA1c. (iv) Ag shell-coated magnetic polymethacrylate microspheres used as support for the proposed SERS tag can be easily dispersed in different media with a broad polarity, pH, and ionic strength range. Hence, resistance to aggregation, particularly with respect to the larger particle size, is an advantage that allows the use of this SERS tag for glycoprotein detection in various biological fluids with different ionic strengths. I

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(5) Gong, T.; Cui, Y.; Goh, D.; Voon, K. K.; Shum, P. P.; Humbert, G.; Auguste, J. L.; Dinh, X. Q.; Yong, K. T.; Olivo, M. Highly Sensitive SERS Detection and Quantification of Sialic Acid on Single Cell using Photonic-Crystal Fiber with Gold Nanoparticles. Biosens. Bioelectron. 2015, 64, 227−233. (6) Alula, M. T.; Yang, J. Photochemical Decoration of Magnetic Composites with Silver Nanostructures for Determination of Creatinine in Urine by Surface-Enhanced Raman Spectroscopy. Talanta 2014, 130, 55−62. (7) Kong, X.; Yu, Q.; Lv, Z.; Du, X. Tandem Assays of Protein and Glucose with Functionalized Core/Shell Particles Based on Magnetic Separation and Surface-Enhanced Raman Scattering. Small 2013, 9, 3259−3264. (8) Quyen, T. T. B.; Su, W. N.; Chen, K. J.; Pan, C. J.; Rick, J.; Chang, C. C.; Hwang, B. J. Au@SiO2 Core/Shell Nanoparticle Assemblage used for Highly Sensitive SERS-based Determination of Glucose and Uric Acid. J. Raman Spectrosc. 2013, 44, 1671−1677. (9) Al-Ogaidi, I.; Gou, H.; Kareem, A.; Al-kazaz, A.; Aguilar, Z. P.; Melconian, A. K.; Zheng, P.; Wu, N. A gold@silica Core−Shell Nanoparticle-based Surface-Enhanced Raman Scattering Biosensor for Label-free Glucose Detection. Anal. Chim. Acta 2014, 811, 76−80. (10) Kong, K. V.; Jun, C.; Ho, H.; Gong, T.; Lau, W. K. O.; Olivo, M. Sensitive SERS Glucose Sensing in Biological Media using Alkyne Functionalized Boronic Acid on Planar Substrates. Biosens. Bioelectron. 2014, 56, 186−191. (11) Sun, F.; Bai, T.; Zhang, L.; Ella-Menye, J. R.; Liu, S.; Nowinski, A. K.; Jiang, S.; Yu, Q. Sensitive and Fast Detection of Fructose in Complex Media via Symmetry Breaking and Signal Amplification Using Surface-Enhanced Raman Spectroscopy. Anal. Chem. 2014, 86, 2387−2394. (12) Sun, X.; Stagon, S.; Huang, H.; Chen, J.; Lei, Y. Functionalized Aligned Silver Nanorod Arrays for Glucose Sensing through Surface Enhanced Raman Scattering. RSC Adv. 2014, 4, 23382−23388. (13) Torul, H.; Ç iftçi, H.; Dudak, F. C.; Adıguzel, Y.; Kulah, H.; Boyacı, I. H.; Tamer, U. Glucose Determination based on a Two Component Self-Assembled Monolayer Functionalized Surface Enhanced Raman Spectroscopy (SERS) Probe. Anal. Methods 2014, 6, 5097−5104. (14) Li, S.; Zhou, Q.; Chu, W.; Zhao, W.; Zheng, J. Surface-Enhanced Raman Scattering Behaviour of 4-Mercaptophenyl Boronic Acid on Assembled Silver Nanoparticles. Phys. Chem. Chem. Phys. 2015, 17, 17638−17645. (15) Tabatabaei, M.; Wallace, G. Q.; Caetano, F. A.; Gillies, E. R.; Ferguson, S. S.; Lagugné-Labarthet, F. Controlled Positioning of Analytes and Cells on a Plasmonic Platform for Glycan Sensing using Surface Enhanced Raman Spectroscopy. Chem. Sci. 2016, 7, 575−582. (16) Bi, X.; Du, X.; Jiang, J.; Huang, X. Facile and Sensitive Glucose Sandwich Assay using In Situ-Generated Raman Reporters. Anal. Chem. 2015, 87, 2016−2021. (17) Qi, G.; Jia, K.; Fu, C.; Xu, S.; Xu, W. A Highly Sensitive SERS Sensor for Quantitative Analysis of Glucose based on the Chemical Etching of Silver Nanoparticles. J. Opt. 2015, 17, 114020−114027. (18) Smith, S. R.; Seenath, R.; Kulak, M. R.; Lipkowski, J. Characterization of a Self-Assembled Monolayer of 1-Thio-β-dGlucose with Electrochemical Surface Enhanced Raman Spectroscopy Using a Nanoparticle Modified Gold Electrode. Langmuir 2015, 31, 10076−10086. (19) Severyukhina, A. N.; Parakhonskiy, B. V.; Prikhozhdenko, E. S.; Gorin, D. A.; Sukhorukov, G. B.; Möhwald, H.; Yashchenok, A. M. Nanoplasmonic Chitosan Nanofibers as Effective SERS Substrate for Detection of Small Molecules. ACS Appl. Mater. Interfaces 2015, 7, 15466−15473. (20) Yin, D.; Wang, S.; He, Y.; Liu, J.; Zhou, M.; Ouyang, J.; Liu, Z. Surface-Enhanced Raman Scattering Imaging of Cancer Cells and Tissues via Sialic Acid-Imprinted Nanotags. Chem. Commun. 2015, 51, 17696−17699. (21) Perumal, J.; Balasundaram, G.; Mahyuddin, A. P.; Choolani, M.; Olivo, M. SERS-based Quantitative Detection of Ovarian Cancer Prognostic Factor Haptoglobin. Int. J. Nanomed. 2015, 10, 1831−1840.

(v) The use of a sandwich system containing both Ag NPs and Ag@MagPMMS for the detection of HbA1c resulted in an enhanced SERS signal intensity due to plasmon coupling between Ag NPs and Ag@MagPMMS. (vi) The proposed SERS tag allowed for the determination of the HbA1c concentration over a broad range (i.e., 0.05−100 μg/mL). The entire detection range of the proposed protocol was also reasonably wider with respect to the clinical range (i.e., 0.32−2.4 μg/mL). This property allows the use of this protocol for sensitive and reliable determination of HbA1c and probably other glycated proteins in different biological fluids. Very low HbA1c concentrations down to 50 ng/mL could be detected by sufficiently high SERS signal intensities. The broad detection range originating from the SERS signal enhancement by the plasmon coupling between Ag NPs and Ag@MagPMMS is one of the most important superior properties of the proposed SERS tag.

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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.6b00138. Experimental procedure for the synthesis of magnetic SERS tag; aggregation response of Ag shell-coated, magnetic, polymethacrylate microspheres; SERS spectrum of MPBA-functionalized, Ag-attached, magnetic, polymethacrylate microspheres; variation of peak area with the HbA1c concentration for the SERS peaks at 1140 and 1436 cm−1; SERS spectra of hemolysates of human erythrocyte suspensions diluted with a volume ratio of 1:100 μL:μL; and SERS spectra obtained by in situ photocoupling of PATP to DMAB upon illumination of a 785 nm laser for different amounts of time (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +90-312-299 21 24. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Specials thanks are extended to the Turkish Academy of Sciences (TÜ BA) for their support to A.T. as a full member. The authors also thank “The Scientific and Technological ̇ AK) for supporting D. D.U. Council of Turkey” (TÜ BIT ̇ AK, BIDEB, through the M.S. scholarship program (TÜ BIT 2210-A). The authors sincerely thank Prof. Feza Korkusuz from Hacettepe University, Faculty of Medicine for his valuable suggestions on the clinical assays.

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REFERENCES

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DOI: 10.1021/acsami.6b00138 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX