A SERS-Based Sandwich Assay for Ultrasensitive and Selective

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A SERS-based sandwich assay for ultrasensitive and selective detection of Alzheimer`s tau protein Adem Zengin, Ugur Tamer, and Tuncer Çaykara Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm400968x • Publication Date (Web): 26 Jul 2013 Downloaded from http://pubs.acs.org on July 31, 2013

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A SERS-based sandwich assay for ultrasensitive and selective detection of Alzheimer`s tau protein

Adem Zengin,† Ugur Tamer‡ and Tuncer Caykara†,* †

Gazi University, Faculty of Science, Department of Chemistry, 06500 Besevler, Ankara, Turkey

‡

Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry, 06330 Etiler,

Ankara, Turkey

ABSTRACT: In this study, a simple and highly selective homogeneous sandwich assay was developed for fast and ultrasensitive detection of the tau protein using a combination of monoclonal anti-tau functionalized hybrid magnetic nanoparticles and polyclonal anti-tau immobilized gold nanoparticles as the recognition and surface-enhanced Raman scattering (SERS) component, respectively. The magnetic silica particles were first coated with poly(2hydroxyethyl

methacrylate)

via

surface-mediated

RAFT

polymerization

and

then

biofunctionalized with monoclonal anti-tau, which are both specific for tau and can be collected via a simple magnet. After separating tau from the sample matrix, they were sandwiched with the SERS substrate composed of polyclonal anti-tau and 5,5-dithiobis(2-dinitrobenzoic acid) on gold nanoparticles. The correlation between the tau concentration and SERS signal was found to be linear within the range of 25 fM to 500 nM. The limit of detection for the sandwich assay is less than 25 fM. Moreover, the sandwich assay was also evaluated for investigating the tau specificity on bovine serum albumin and immunoglobulin G.

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INTRODUCTION Alzheimer`s disease is a brain disorder disease that destroys brain cells, causing troubles with long term memory loss, irritability, agglomeration, and mood swings.1-3 Despite the big trouble, there is no definitive diagnosis of Alzheimer`s disease, other than postmortem determination of senile plaques and neurofibrillary tangles in the brain tissue. Neurofibrillary tangles are insoluble twisted fibers formed due to hyperphosphorylated tau agglomeration in brain cells. Tau is a protein with a molecular weight of about 65 kDa. It plays a very important role in the structure of the neurone. In the brains of Alzheimer`s disease individuals, tau is phosphorylated at more than 20 residues, many of which are serine/threonine-proline sites. In healthy individuals, 8-10 of these residues are heterogeneously phosphorylated, and lose the capacity to bind to microtubules. Instead, these phosphorylated tau proteins bind to each other inside nerve cells, tying themselves in ``knots`` known as neurofibrillary tangles.4-9 Level of tau is increased in cerebral spinal fluid of Alzheimer`s disease individuals compared to age-matched controls,10-12 probably due to neuronal and axonal degeneration or accumulation of neurofibrillary tangles.13 Therefore, the reliable, sensitive, and rapid detection of tau is of great importance. To date, the detection method of tau is based on measuring specific optical absorption such as that implemented in the enzymelinked immunosorbent assay (ELISA) method,10,13 localized surface plasmon resonance (LSPR) techniques,14-16 or chromatography.17 These well-known methods, however, do not provide a ready pathway for facile direct detection of the tau molecules within one measuring cycle. These approaches often require a complicated sample preparation or multiple intermediate steps, which are costly and time-consuming. Among numerous protein detection methods, surface-enhanced Raman scattering (SERS) has become an area of intense research as a highly sensitive probe for trace level detection of biomolecules since the demonstration of single molecule detection.18-19 The highly sensitive 2


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vibrational spectroscopic technique of SERS can also potentially provide a method for analysis of proteins such as tau, brovine serum albumin, immunoglobulin G etc., down to single molecules, which was impossible due to the complexity of such biomolecules.20,21 The design of the substrate on which the SERS phenomenon becomes significant is the most critical aspect of a sensitive protein probe.22 Most popular designs involve various engineered substrates such as roughened core-shell-structured nanoparticle films,23,24 metalic and bimetalic nanostructures,25,26 and nanorod substrates.27 In this report, we present a novel homogeneous detection method utilizing monoclonal anti-tau functionalized hybrid magnetic nanoparticle (MNP) probes and polyclonal anti-tau immobilized gold nanoparticles as SERS tags in solution. After the tau targets were separated from the matrix, a sandwich assay procedure was applied using the SERS tags composed of polyclonal anti-tau and 5,5-dithiobis(2-dinitrobenzoic acid) (DTNB) on gold nanoparticles. Furthermore, this method was also tested for the tau specificity on bovine serum albumin (BSA) and immunoglobulin G (IgG). The SERS-based sandwich assay suggested here provides a simple method that is much faster (less than one minute of total measurement time) than current optical methods.

EXPERIMENTAL SECTION Materials. Tetraethyl orthosilicate (TEOS, 98%), 3-methacryloxpropyltrimethoxysilane (MPS, 98%), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC), disuccinimidyl carbonate (DSC), N,N-dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), 5,5dithiobis(2-dinitrobenzoic acid) (DTNB), polyclonal anti-tau, monoclonal anti-tau, tau, brovine serum albumin (BSA) and immunoglobulin G (IgG) were obtained from Aldrich Chemical Co. FluoroTag™ FITC Conjugation Kit

was also purchased from Sigma and Fluorescein

isothiocyanate tau, (FITC-tau) were prepared according to supplier. Azobis(isobutyronitrile) 3


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(AIBN, Acros 98%) was recrystallized from methanol twice and stored at –20 oC. 2-hydroxyethyl methacrylate (HEMA, Aldrich 97%) was passed through a column of activated basic alumina prior to use. N,N-dimethylformamide (DMF, anhydrous, Aldrich 99.8%) was distilled over CuSO4. Tetrahydrofuran (THF, Aldrich) was purified by distillation from calcium hydride followed by distillation from sodium/benzophenone ketyl. Dichloromethane (DCM, Aldrich) was distilled under vacuum after drying with CuSO4. 2-Cyano-2-propyl benzodithioate (chain transfer agent, CPBT) was synthesized as reported earlier.28 Preparation of MPS-modified MNPs. Oleic acid-stabilized maghemite (γ-Fe2O3) nanoparticles and silica-coated MNPs were prepared according to the literature procedures.29,30 The silicacoated MNPs (100 mg) were dispersed in dry toluene (20 mL) and sonicated for 15 min. After the addition of MPS (1.2 mL) and hydroquinone (catalytic amount) (to prevent self polymerization of MPS), the mixture was stirred at 90°C for 40 h, and then the solution was cooled and exposed to air. The MPS-modified MNPs were collected with a magnet, and washed with toluene, toluene/methanol, methanol, and dried under vacuum at room temperature for 24 h. Preparation of CPBT-modified MNPs. The MPS-modified MNPs (100 mg) were dispersed in dry DMF (20 mL). The mixture was sonicated for 10 min, and then CPBT (0.50 mmol) and AIBN (2.5 mmol) were added. After degassing with nitrogen for 30 min under sonication, the flask was flame-sealed. The mixture was stirred at 60 °C for 8h and then at 90 °C for 16h. The solution was cooled and exposed to air, washed with DMF and DCM for several times, and dried under vacuum at room temperature for 12 h. Surface-mediated RAFT polymerization of HEMA from CPBT-modified MNPs. A mixture of HEMA (0.57 g), CPBT-modified MNPs (100 mg), AIBN (0.14 mg), free CPBT (1.95 mg) and dry DMF (5 mL) was added in a dry ampoule and treated in ultrasonic for 10 min. The mixture was purged with nitrogen for 15 min to eliminate the dissolved oxygen, and then the ampoule 4


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was flame-sealed. The polymerization proceeded in an oil bath held by a thermostat at the desired temperature (60 °C). After the desired polymerization time (6 h, 16 h, 32 h, 48 h, and 72h, respectively), the ampoule was rapid cooled by immersing it into liquid nitrogen, and then, the reaction mixture was diluted by THF and centrifuged at 6000 rpm for 10 min to separate the ``free`` poly(HEMA) and poly(HEMA) grafted hybrid MNPs. This cycle of centrifugation and redispersion in THF was repeated four times to make sure that the free polymers were removed completely. The grafted poly(HEMA) were dried in vacuum oven at room temperature. Cleavage of the poly(HEMA) grafted chains from the hybrid MNPs. Cleavage of the poly(HEMA) grafted chains from the hybrid MNPs was carried out according to the literature procedure.31 In a polyethylene tube, the hybrid MNPs (40 mg) was dissolved in 5 mL THF. Into the solution, aqueous HF (48%, 0.3 mL) was added, and reaction mixture was stirred at room temperature for 12 h. The polymer was precipitated by the polymer solution into 10-fold excess of hexane in a polyethylene beaker. The precipitate was collected by filtration and dried in a vacuum oven at 60 oC overnight. The recovered poly(HEMA) was then subjected to gel permeation chromatography (GPC) analysis. Removal of dithiobenzoate end groups from the poly(HEMA) grafted hybrid MNPs. Removal of dithiobenzoate end groups from the grafted polymer chains was carried out according to the literature procedure.32 Briefly, the poly(HEMA) grafted hybrid MNPs (25 mg) were mixed with a solution of AIBN (38 mg) in DMF (10 mL). The mixture was purged with nitrogen for 15 min, and ampoule was septum-sealed. The reaction was carried out at 60 °C for 8 h. The hybrid MNPs were collected with a magnet and washed with DMF for several times, and then dried in vacuum oven at room temperature. Preparation of monoclonal anti-tau functionalized hybrid MNPs as capture probe. The hybrid MNPs (10 mg) were mixed with a solution of DSC (21.5 mg) and DMAP (12.2 mg) in 5


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DMF. The reaction was carried out at room temperature under nitrogen atmosphere for 16 h. The activated hybrid MNPs were collected with a magnet and washed with DMF and DCM for several times, and then dried in vacuum oven at room temperature. To covalent immobilization of monoclonal anti-tau, the activated hybrid MNPs ( 4 mg) dispersed in phosphate-buffered saline (PBS, pH 7.4, 10 mL) were mixed with a solution of monoclonal anti-tau 20 µg/mL in PBS. The mixture was shaken at room temperature for 2 h. The monoclonal anti-tau functionalized hybrid MNPs were collected with a magnet and washed with the PBS solution containing 0.1 M NaCl and 1% BSA for several times. After washing procedure, the nanoparticles were redispersed in PBS solution and stored in a refrigerator prior to use. Preparation of polyclonal anti-tau immobilized gold nanoparticles as SERS tag. Gold nanoparticles with a diameter of 15 ± 8 nm (accounted on 160 nanoparticles) were synthesized by citrate reduction method.33 After purification, the gold nanoparticles were modified with a selfassembled monolayer (SAM) of DTNB (10 mM) in absolute ethanol for overnight. The carboxylic acid groups of DTNB were activated by using a solution of EDC (0.2 M) and NHS (0.05 M). The activated nanoparticles were incubated with polyclonal anti-tau (PBS buffer, 20 µg/mL) for 2 h at room temperature under shaking. The nanoparticles were washed with PBS solution containing 0.1 M NaCl and 1% BSA for several times. After washing procedure, the polyclonal anti-tau immobilized gold nanoparticles were dispersed in PBS solution and stored in a refrigerator prior to use. Preparation of sandwiched assay. The tau solutions with the concentrations in a range from 25 fM to 500 nM were obtained by a serial dilution with PBS. The monoclonal anti-tau functionalized hybrid MNPs (~2 mg) were added into each tau solution and incubated at room temperature for 30 min under shaking. After isolating with a magnet and washing with PBS

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solution containing 0.1 M NaCl and 1% BSA for several times, the tau conjugated hybrid MNPs were further incubated with the suspension of polyclonal anti-tau immobilized gold nanoparticles (500 µL) for 30 min at room temperature under shaking. The resultant sandwich complex was collected with a magnet and washed with PBS solution containing 0.1 M NaCl and 1% BSA for several times, and stored at 4 oC before SERS measurements. After detection of tau in different concentrations, the cross-specificity of the sandwiched assay was also tested by using a solution with equal amounts (500 nM) of tau, BSA and IgG under same conditions. Characterization methods. The transmission electron microscopy (TEM) observations were conducted on FEI Tecnai G2 Spirit electron microscope at an acceleration voltage of 120 kV. The sample for TEM observations was prepared by placing a 10 µL nanoparticle solution on copper grids successively coated with thin films of carbon. No staining was applied. The grazing angleFourier transform infrared (GA-FTIR) spectra were performed using a Thermo Nicolet 6700 spectrometer coupled with a Mercury Cadmium Telluride detector and a smart SAGA grazing angle (80°) attachment. The spectra were taken at a resolution 4 cm-1 after 128 scan were accumulated to achieve an acceptable signal/noise ratio. The x-ray photoelectron (XPS) spectra were recorded on a SPECS XPS spectrometer equipped with a Mg Kα X-ray source. After peak fitting of the C 1s spectra, all the spectra were calibrated in reference to the aliphatic C 1s component at a binding energy of 285.0 eV. The molecular weights and molecular weight distributions were determined by a GPC equipped with a Waters 1515 pump and a Waters 2414 differential refractive index detector (set 30 oC). It uses a series of three linear Styragel columns HT2, HT4, and HT5 at an oven temperature of 45 oC. The eluent was THF at a flow rate of 1.0 mL/min. A series of low-polydispersity polystyrene standards were employed for the GPC

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calibration. The thermogravimetric analysis (TGA) was performed in nitrogen atmosphere at a heating rate of 10 oC/min from room temperature to 600 oC using a Perkin-Elmer Diamond TG/DTA. The magnetic measurements were carried out at room temperature by using a Physical Properties Measurement System-PPMS from Quantum Design. UV-vis spectra were recorded on a Shimadzu UV-vis spectrometer UV-1601 with Shimadzu/UV Probe software. A DeltaNu Examiner Raman microscope (DeltaNu Inc., Laramie, WY) with a 785 nm laser source, a motorized microscope stage sample holder, and a CCD detector was used to detect tau. During the measurements, a 20X objective was used and the laser spot diameter was 30 µm. Samples were measured with 140 mW laser power, for 10 s acquisition time. Baseline correction was performed for all measurements.

RESULTS AND DISCUSSION Scheme 1 shows the procedure for creating magnetic particles coated with poly(HEMA) brushes that bind monoclonal anti-tau molecules. The first step is the formation of a silica coating on the maghemite nanoparticles to prevent oxidation and dissolution of the γ-Fe2O3. The high density of hydroxyl groups on the silica also allows further modification of the nanoparticles through silanization. To coat γ-Fe2O3 nanoparticles with silica, we employed a microemulsion method described by Yi et al.30 For this purpose, the solution of γ-Fe2O3 nanoparticles in toluene was introduced to a solution of a surfactant in cyclohexane and vortexed to minimize particle aggregation during deposition of silica through hydrolysis of TEOS and condensation on the γFe2O3 seeds. The addition of NH4OH formed a reverse microemulsion whereas subsequent addition of TEOS started the growth of the silica shell. The silica-coated MNPs were aged for 48 h and purified by several cycles of centrifugation and redispersion in ethanol and toluene. The

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MNPs have an average diameter of around 14 ± 5 nm (accounted on 140 nanoparticles), Figure 1a). After the silanization, the average diameter of silica-coated MNPs increased to about 64 ± 4 nm (accounted on 170 nanoparticles, Figure 1b). The TEM images showed unaggregated particles with a dark γ-Fe2O3 core, which may consistent of one γ-Fe2O3 nanoparticle (~14 nm), outside a silica shell (~50 nm). Thus, the thickness of the silica shell is about 25 nm. The silica-coated MNPs were transformed into double-bond-bearing spheres that are suitable for immobilization of RAFT agents by condensation of MPS onto the surface of the particle. The formation of the silica layer on the γ-Fe2O3 was confirmed by GA-FTIR and XPS analysis. In the GA-FTIR spectra (Figure 2a), the band at 1090 cm-1 is assigned to the Si-O stretching vibration. In the XPS spectra (Figure 3a), the peaks at 154 eV and 103 eV are assigned as Si 2p and Si 2s, respectively. The absence of Fe peaks at 724, 712, 92 eV is also indicated the formation of a thick silica layer. The C 1s peak at 285.0 eV is probably due to residual ethoxy groups and/or contamination of the nanoparticles during XPS operation. The GA-FTIR spectrum of MPSmodified MNPs is given in Figure 2b. The distinct absorption band at 1628 cm-1 is associated with the vinyl group of MPS. The C=C bonds on the surface of silica serve as RAFT agent bonding sites during the subsequent grafting process. The presence of MPS was also confirmed by the increase in the intensity of the C 1 s peak at 285.1 eV in the XPS survey-scan spectrum (Figure 3b). Coupling of CPBT was achieved via a one-step process in bearing a terminal double bond region of the MPS-modified MNPs from reactions with a mixture of AIBN and CPBT. GA-FTIR analysis was conducted to verify the attachment of CPBT to the MPS-modified MNPs. Compared with the spectrum of the MPS-modified MNPs (Figure 2b), the characteristic C=S bands of CPBT moieties at around 1234 cm-1 and 1171 cm-1 overlap with the strong absorption of SiO2

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(Figure 2a) and the absence of C=C band at 1628 cm-1 confirm the coupling of CPBT on the MNPs. Further evidence of coupling reaction of MPS by CPBT on the surface of MNPs was provided with XPS analysis (Figure 3c). The peaks at about 102, 154, 285 and 532 eV, which are ascribed to Si 2p, Si 2s, C 1s and O 1s, respectively. Apart from these peaks, there appear two new signals at about 162.8 and 226 eV assignable to S 2p and S 2s, respectively, in the survey scan spectrum of the particles after coupling of CPBT. Both GA-FTIR and XPS results indicated that the RAFT agent CPBT was successfully introduced on the surface of MNPs. The CPBT-modified MNPs were then used as a RAFT agent for the surface-mediated RAFT polymerization of HEMA, where the polymer chains were grafted directly from the nanoparticle surface to give magnetic and reactive core-shell hybrid nanoparticles (Scheme 1). The surfacemediated RAFT polymerization was conducted in DMF at 60 oC using AIBN as the initiator ([M] =2.2 M, [AIBN] = 0.88 mM). The ln([M]o/[M]) vs time plots (Figure 4a) was reported. After a typical induction period,34 the first order kinetic plot is linear in all cases indicating that the concentration of active species is constant, which is essential in controlled radical polymerization. Figure 4b shows the evolution of the number-average molecular weight ( M n ) and polydispersity index (PDI) of the grafted poly(HEMA) as a function of monomer conversion. The experimental

M n , GPC (measured by GPC) and theoretical ( M n , theo ) molecular weights were found to be proportional to the monomer conversion. The M n , GPC values are slightly higher than the M n , theo ones and the PDI values are less than 1.32, indicating a well-controlled polymerization consistent with the known traits of living radical polymerization. Moreover, all of the synthesized

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polymers showed rather symmetrical and narrow GPC traces (Figure 4c). Thus, the M n , GPC of

the grafted polymer (corresponding to the thickness of the polymeric shell) can be controlled with polymerization time. The GA-FTIR spectrum of hybrid MNPs is shown in Figure 2d. The absorption band at 1714 cm1

is assigned to the C=O stretching vibrations. The spectrum also shows a strong absorption band

at around 3500-3200 cm-1 and 2900 cm-1, associated with the stretching vibrations of –OH and – CH2 groups of the poly(HEMA) brushes. The surface-mediated RAFT polymerization of HEMA on the silica shell has caused a significant decrease in the intensity of the Si signals in the XPS survey-scan spectrum of the hybrid MNPs (Figure 3d). The C 1s core-level spectrum (Figure 3e) can be curve-fitted into three peak components with binding energies at about 284.9, 286.7, and 288.8 eV, attributed to the C-C/C-H, C-O/C-S, and O-C=O species of the poly(HEMA) brushes grafted on the nanoparticle surface. After growth of poly(HEMA) from silica-coated MNPs, TEM image such as that in Figure 1c shows the formation of a 5.5 nm thick polymer layer on the ~64 nm diameter SiO2- γ-Fe2O3 particles, although the polymeric shell is not discernible because of the lack of contrast with the background.35 Notably, the hybrid MNPs were well dispersed and no agglomeration had occurred. The TGA curves of weight loss versus temperature (Figure 5) provide an estimate of quantity of polymer grown from MNPs. The silica-coated MNPs composed of both thermally stable compounds that remain in the residue (SiO2 and γ-Fe2O3) degradable polymer brushes and organic compounds (MPS and CPBT) that contribute to weight loss. Even with just silica-coated MNPs, however, a small amount of water and residual ethoxy groups from the TEOS give rise to a 1.2% total weight loss (Figure 5a). After MPS and CPBT modifications, TGA curves show the

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total weight loss of 4.9% (Figure 5b) and 6.6% (Figure 5c), respectively. After growth of poly(HEMA) from the particle, TGA curve shows a total weight loss of 19% (Figure 5d), which corresponds to a poly(HEMA) thickness of about ~4.0 nm. This thickness value agrees reasonably well with the TEM thickness of ~5.5 nm. The hybrid MNPs must be highly magnetic for collection with a magnet after tau binding. Figure 6 shows magnetization curves obtained with a vibrating sample magnetometer for MNPs before and after different modification steps. As expected, the saturation magnetism per gram of these particles decreased after silica coating (from 63.7 to 31.2 emu/g) and then decreased further to 14.5 emu/g after growth polymer brushes (72 h, ~72 % monomer conversion) because of the decrease in γ-Fe2O3 content of the nanoparticles. However, this level of saturation magnetization is deemed sufficient for biological applications.36,37 After surface-mediated RAFT polymerization, the ditihobenzoate end groups were removed via AIBN due to their toxicity and potential aminolysis reaction. Figure 7 shows the UV-vis spectra of the hybrid MNPs after treated with AIBN. The absorption at 315 nm was ascribed to the thiocarbonyl group (Figure 7a, b). After treated with AIBN (Figure 7c), the disappearance of absorption peak at 315 nm is the exact proof that the thiocarbonyl group has removed from the polymer chain ends successfully. Bioconjugation of the hybrid MNPs includes activation of the pedant –OH groups of poly(HEMA) with DSC and DMAP, reaction with monoclonal anti-tau and subsequent bioconjugation with FITC-tau (Scheme 2). The fluorescent microscope image in Figure 8a clearly shows that the fluorescein is present only on FITC-tau-conjugated hybrid MNPs, as confirmed by comparison to the dark image of the monoclonal anti-tau functionalized hybrid MNPs in Figure 8b. Visible changes in the fluorescent images qualitatively demonstrate the success of the bioconjugation procedure. However, the presence of FITC-tau could be due to non-specific 12


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binding. In order to separate these contributions we carried out the control experiments where FITC-tau is exposed to clean hybrid MNPs (without anti-tau functionalization) to determine the extent of non-specific adsorption versus selective bonding. As seen Figure 8c, while there were some fluorescence signals due to the non-specific binding of FITC-tau to the hybrid MNPs, the amount could be close to the blank signal observed by SERS (Figure 10a). To prepare the homogeneous sandwich assay, the gold nanoparticles were first modified with a layer of Raman reporter DTNB and then a layer of polyclonal anti-tau. Polyclonal anti-tau can help amplify signal from target tau with low level, as the target tau molecule binds more than one polyclonal anti-tau molecule on the multiple eptitopes. This not only helps to the formation of homogeneous sandwich assay and but also increases sensitivity of SERS platform. Here, the target tau molecules were captured through the monoclonal anti-tau functionalized hybrid MNPs. After washing away the unbound molecules, the captured tau molecules were labeled with polyclonal anti-tau immobilized gold nanoparticles (Scheme 2). The polyclonal anti-tau immobilized gold nanoparticles were used as the SERS tag to generate a strong Raman scattering.38 After the sandwich assay procedure, the monoclonal anti-tau functionalized hybrid MNPs and the polyclonal anti-tau immobilized gold nanoparticles were bound each other via tau molecules, forming aggregate structures as shown in TEM image (Figure 9). The SERS measurements were performed by using aggregated nanoparticles. The monoclonal anti-tau functionalized hybrid MNPs are not SERS active and therefore do not form hot spots with the polyclonal anti-tau immobilized gold nanoparticles. However, there will likely be plasmonic coupling between adjacent the polyclonal anti-tau immobilized gold nanoparticles. When the captured tau molecules were labeled with polyclonal anti-tau immobilized gold nanoparticles, a sharp peak at 1332 cm-1 and peaks at 1053 and 1553 cm-1 appeared because of the DTNB Raman signal.38 Therefore, the resultant SERS spectrum involves only DTNB labeled SERS probe. In 13


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this case, the binding of tau molecules to the antibodies causes a plasmon and the peak at 1332 cm-1 of DTNB increases depending on the concentration of tau. To evaluate the sensitivity of sandwich assay, the relationship between SERS intensity and tau concentration was examined. Figure 10a shows the dependence of SERS intensity on the tau concentration. The blank signal is result from the non-specific adsorption (Figure 8c and Figure 10a). The SERS intensity of the peak at 1332 cm-1 is proportional to the tau concentration. To prevent the fluctuations in the SERS response, SERS experiments were repeated four times and the average signal intensity was plotted to versus tau concentrations between 25 fM and 500 nM. When the concentration was 25 fM, the characteristic peak at 1332 cm-1 became difficult to distinguish from that in blank spectrum, indicating the detection limit. Figure 10b shows the dose-response calibration curve constructed by averaging four reading at the point of each sample in the range of 500 nM to 25 fM. SERS intensity of the designated peak was proportional to the tau concentration within the whole range. The linear regression equation was y = 686.8x + 395.6 with a correlation coefficient (R2) of 0.994. The detection limit was lower than 25 fM. Meanwhile, the monoclonal anti-tau functionalized hybrid MNPs was also exposed to a solution with equal amounts (500 nM) of tau, BSA and IgG to determine its selectivity for the tau-protein over the other molecules. As can be seen in Figure 10c, the SERS intensity obtained from the response of the complex had no apparent difference, indicating that BSA and IgG had almost negligible on the tau detection. This test showed that our sandwich assay was specific for tau detection.

CONCLUSIONS In conclusion, we have developed a SERS-based sandwich assay for fast and sensitive detection of tau by using of monoclonal anti-tau functionalized hybrid MNPs as recognition agent. The 14


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sandwich assay was completed by binding captured molecules with the SERS substrate composed of polyclonal anti-tau and DTNB on gold nanoparticles. This design possesses advantages over current methods such as ELISA, LSPR, and chromatography which contain label-free and rapid detection using a simple and cost-effective substrate fabrication technique. This approach might provide accurate, sensitive, and more selective detection of tau than current protein detection methods with the lowest limit of detection for tau solution of below 25 fM, which is comparable to the sensitivity of conventional optical biosensing methods. An additional clear advantage of this highly sensitive SERS method on an engineered substrate is the speed of detection and simple substrate preparation. Moreover, this method does not require several binding and purification steps an in ELISA which can take hours to complete. Simple preparation of sandwich assay allows for rapid SERS detection in less than several minutes. This method is also cost-effective in that fluorescently labeled proteins are not required for detection.

AUTHOR INFORMATION *E-mail: [email protected]. Telephone: +90 312 202 11 24. Fax: +90 312 212 22 79

ACKNOWLEGMENTS This study was supported by Gazi University Scientific Research Projects Unit, Project no : 05/2011-13. The authors thank to Prof. Z. Suludere for TEM measurements.

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Caption of the figures Scheme 1. Schematic representation of the synthesis of hybrid nanoparticles. Scheme 2. Representative presentation of the preparation of sandwich assay. Figure 1. TEM images of (a) Oleic acid-stabilized MNPs and (b) silica-coated MNPs and (c) hybrid MNPs (Polymerization time is 72 h). Figure 2. GA-FTIR spectra of (a) silica-coated MNPs, (b) MPS-modified MNPs, (c) CPBTmodified MNPs and (d) hybrid MNPs (Polymerization time is 72 h). Figure 3. Survey-scan XPS spectra of (a) silica-coated MNPs, (b) MPS-modified MNPs, (c) CPBT-modified MNPs, survey scan and C1s core-level spectra of (d, e) hybrid MNPs (Polymerization time is 72 h). Figure 4. (a) Kinetic plot, (b) evolution of molecular weights (•) and polydispersities, PDI (ο) with monomer conversions obtained during surface-mediated RAFT polymerization of HEMA from CPBT-modified MNPs and (c) evolution of GPC traces with polymerization time during the surface-mediated RAFT polymerization of HEMA from CPBT-modified MNPs. The poly(HEMA) chains were detached from the hybrid nanoparticles. Figure 5. TGA curves of (a) silica-coated MNPs, (b) MPS-modified MNPs, (c) CPBT-modified MNPs and (d) hybrid MNPs (Polymerization time is 72 h). TGA was performed in nitrogen atmosphere at a heating rate of 10 oC/min. Figure 6. (left) Magnetic hysteresis curves of γ-Fe2O3 (●), silica-coated MNPs (∆), and hybrid MNPs (Polymerization time is 72 h) (□). (right) image of the magnetically collected particles. Figure 7. UV-vis spectra of (a) RAFT agent CPBT, (b) hybrid MNPs, and (c) hybrid MNPs after treated with AIBN to remove the ditihobenzoate groups

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Figure 8. Fluorescence images of (a) FITC-tau-conjugated hybrid MNPs, (b) (a) monoclonal anti-tau functionalized hybrid MNPs and (c) FITC-tau-adsorbed hybrid MNPs (without anti-tau functionalization) Figure 9. TEM image of the sandwich complex Figure 10. (a) SERS spectra of the sandwich complex at different tau concentrations. (b) Doseresponse curve of the above tau assay. (y = 686.8 x + 395.6, R2 = 0.994, n = 4, the four different concentrations are replaced by integers 1-10 in this equation, each error bar indicates the standard deviation of four different readings) and (c) SERS spectra of monoclonal anti-tau functionalized hybrid MNPs exposed to BSA (500 nM) IgG (500 nM), tau (500 nM), and a solution with equal amounts (500 nM) of BSA, IgG and tau

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Scheme 1

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Scheme 2

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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