Gold Superparticles Functionalized with Azobenzene Derivatives

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Gold Superparticles Functionalized with Azobenzene Derivatives: SERS Nanotags with Strong Signals Ying Ma, Kittithat Promthaveepong, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01074 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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ACS Applied Materials & Interfaces

Gold Superparticles Functionalized with Azobenzene Derivatives: SERS Nanotags with Strong Signals Ying Maa*, Kittithat Promthaveeponga, and Nan Lib* a

Department of Biomedical Engineering, National University of Singapore, 4 Engineering Drive

3, Engineering Block 4, Singapore 117583. b

Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang

Technological University, 70 Nanyang Drive, Singapore 637457, Singapore

KEYWORDS: Azobenzene, gold superparticles, SERS nanotag, sensor

1 Environment ACS Paragon Plus


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ABSTRACT

The Surface-enhanced Raman spectroscopy (SERS) nanotag was proposed as a substitute for fluorescent dye for imaging and biosensors several decades ago. However, its weak signal and poor reproducibility has hindered its application. Here, we report a new strategy to form Au superparticles (AuSPs) with high SERS enhancement via one-pot formation and self-assembly of Au nanoparticles (NPs). An azobenzene-carrying Raman reporter was synthesized to exhibit a large Raman cross-section and multiple bands. The self-assembly of the Raman reporter on AuSPs generated SERS nanotags with intense signals. A Raman reporter carrying boronic acid and azobenzene groups displayed six distinctive bands. Its corresponding SERS nanotag demonstrated a high sensing ability towards glycoprotein through aggregation-induced SERS enhancement or as a substitute for labeled antibodies in an immunoassay of the glycoprotein.


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Introduction The development of optical probes for biomedical applications is currently of broad interest in science, engineering, and medicine1. Fluorescence (FL) is currently the gold standard in biological imaging and detection because of highly developed instrumentation and the wide availability of efficient fluorophores such as organic dyes and quantum dots (QDs). However, given the broadness of its emission bands (>50 nm for organic dyes and ∼30 nm for QDs), FL-based technology is difficult to multiplex and often leads to “crosstalk” among various channels. Some FL-based imaging methods, such as the development of fluorescent dyes with tunable emissions stimulated by the different targets, have been developed to avoid this problem

2-5

. However, it is still challenging to

image a sample with more than one FL marker. Moreover, two or more lasers are often required to excite fluorophores emitting in reasonably spaced spectral regions. Finally, FL is also prone to photobleaching and can be obscured by autofluorescence, a common issue in cell and tissue imaging6. Surface-enhancement Raman scattering (SERS) has been used to design nanoprobes known as “SERS nanotags,” which combine metallic plasmonic nanoparticles (NPs) and organic Raman reporter molecules. SERS nanotags produce characteristic Raman signals (fingerprints) and can be used to sense the targeted molecules by laser Raman spectroscopy or SERS microscopy. This is akin to fluorescent dyes and QDs

7-8

.

Compared with fluorescent dyes or QDs, SERS nanotags exhibit three major advantages: 1) High signal intensity. The SERS signal intensity can be much stronger than the FL signal generated from fluorescent dyes provided that the surface enhancement factor is high; 2) Narrow bandwidth (~5 nm vs. 50 nm for fluorescent dyes), allowing their use for 3

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multiplexing applications; 3) Extreme short lifetime. This can prevent photobleaching, energy transfer, or quenching of reporters in the excited state, rendering high photostability to the SERS tags6. Therefore, they were proposed as substitutes for FL probes for biosensors9-10 or biological imaging several decades ago. However, they are not yet routinely used for biomedical applications. The major reason lies in their weak SERS signals due to the limited hotspots and poor reproducibility of most SERS nanotags11. A typical SERS nanotag has two components: metallic NPs possessing a high density of hotspots for SERS enhancement and Raman reporters with unique fingerprints1. Although Raman reporters are also a crucial factor for high-quality SERS nanotags, most researchers focus on the development of substrates with high SERS enhancement rather than the design of new Raman reporters. The reason may lie in the difficulties of designing and synthesizing new Raman reporters. In this study, we developed SERS nanotags with intense signals and multiple bands using the AuSP as good SERS substrates and an azobenzene-carrying molecule as a Raman reporter. AuSPs were synthesized through a simple, one-pot strategy using 3-aminophenyl boronic acid (APBA) as a reducing agent. APBA can reduce HAuCl4 to form small AuNPs with the oxidation product poly (3-aminophenylboronic acid) (PAPBA) attached to their surface as a capping agent. The AuNPs self-assemble into AuSPs through the π-π stacking of PAPBA. An azobenzene-carrying Raman reporter, bis [4,4’-(dithiodiphenylazo)-phenol] (DTDPAP), was synthesized and modified on AuSPs to form SERS nanotags with intense signals and multiple bands. We also developed a sugar-responsive SERS nanotag with

bis

[4,4’-(dithiodiphenylazo)-1-(N,N’-dimethylamino)-3-phenylboronic 4


acid]

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(DTDPA-DMAPBA) as a Raman reporter, which displayed 6 intense bands and good sensing ability towards the glycoprotein horseradish peroxidase (HRP). Furthermore, this SERS nanotag was successfully used as a substitute for a labeled antibody in an immunoassay of glycoproteins with high sensitivity.

Experimental section Materials HAuCl4, 3-aminophenylboronic acid (APBA), 4,4′-dithiodianiline (DTDA), phenol, 3dimethylaminophenylboronic acid (DMAPBA), sodium nitrite (NaNO2), horseradish peroxidase (HRP), erythropoietin (EPO) and anti-erythropoietin receptor antibody (EROR) were purchased from Sigma. All other reagents were analytical grade and used as received. Ultrapure water (resistivity up to 18.2 MΩ cm) was used throughout the experiment. Characterization Transmission electron microscopy (TEM) (JEOL JEM 2100F TEM) was used to study the morphology of particles. The size of the particles and their standard deviation (SD) were calculated by measuring over 200 individual particles. UV−vis absorption spectra were measured by a Varian Cary 60 spectrophotometer. Dynamic light laser scattering (DLS) measurements were performed using a Malvern DLS Zetasizer Nano S instrument. Nuclear magnetic resonance (NMR) spectra were acquired on a 600 MHz NMR spectrometer (Premium Shielded Narrow Bore, Agilent Technologies, CA, USA) at room temperature. SERS spectra were recorded using an XploRA PLUS Raman microscope

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(Horiba/JY, France) using a 785-nm laser excitation source. The incident laser power was maintained at 1 mW, and total accumulation times of 20 s were employed. Synthesis of DTDPAP DTDPAP was synthesized according to the reported azo coupling reaction (Scheme 1a)12. Simply, 4,4′-dithiodianiline (DTDA) (2.3 mmol) was dissolved in 20 ml 0.5 M HCl and cooled in ice water. Subsequently, NaNO2 (5.0 mmol) in 2 ml cooled water was added dropwise. After stirring the mixture for 20 min, 4, 4’-dithiodiphenyldiazonium (DTDPDA) was formed. Afterward, phenol (5.5 mmol) in 10 ml 1 M NaOH was added dropwise into the DTDPDA solution. The solution was continually stirred for 5 min, and its pH was adjusted to 10 with 1 M NaOH. The eventual mixture was continually stirred at 4 °C for 30 min and room temperature for 1 h. The final precipitate was collected by centrifugation at 25,000×g for 5 min and washed with water three times. Yellowish DTDPAP was obtained after drying by lyophilization (yield=95%). 1HNMR (CDCl3): 6.9 (d, 2H, Ph), 7.5 (m, 2H, Ph), 7.7 (2H, Ph) 7.8 (2H, Ph). MS (ESI) m/z: calc’d for C24H18N4O2S2: 458.1; [M+H]+, found: 459.2 (See Figure S1a). Synthesis of Bis [4,4’-(dithiodiphenylazo) -1-(N, N’-dimethylamino)-3-phenylboronic acid] (DTDPA-DMAPBA) DTDPA-DMAPBA was synthesized according to the reporter method with some minor modifications (Scheme 1b)13. DTDPDA (2.3 mmol) was prepared using the method mentioned above. DMAPBA (5.5 mmol) in 10 ml 1 M HCl was subsequently added to the DTDPDA solution dropwise. After adjusting the pH to 10 with 1 M NaOH, the mixture was stirred at 4 °C for 30 min and room temperature for 1 h. The resulting precipitates were collected by centrifugation at 25,000 ×g for 5 min and washed with cold 6


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water three times. Reddish DTDPA-DMAPA was collected after drying by lyophilization. (Yield=96%). The results of proton NMR (1H NMR (CDCl3)): 7.87 (2H, Ph), 7.76 (1H, Ph), 7.71 (2H, Ph), 6.89 (1H, Ph), 6.8 (d, 1H, Ph), 3.06 (s, 6H, N (CH3)2). MS (ESI) m/z: calc’d for C28H30N6B2O4S2:546.1; [M+H]+, found: 547.3 (See Figure S1b). Synthesis of AuSPs AuSPs were synthesized using a wet-chemical method. Simply, 9 µl 10% HAuCl4 (2.4 µmol) was added to 1 ml 11.5 mg/ml sodium dodecyl sulfate (SDS) aqueous solution. Subsequently, different volumes of APBA (10 mg/ml in water) were injected. The mixture was continually stirred for 30 min and centrifuged at 16,000×g for 5 min. The collected pellets were washed three times with water and dissolved in water for further characterization. To study the evolution of AuSPs, the reaction solutions were centrifuged at specific times, and the pellets were collected for TEM measurement. Formation of SERS nanotags SERS nanotags were prepared by dispersing AuSPs into 1 ml water containing 1 mM Raman reporters. After 4 h stirring, AuSPs modified with Raman reporters (SERS nanotags) were collected by centrifugation and washed three times with water. The resulting SERS nanotags were dissolved in water and stored in the refrigerator for further usage. SERS measurement of individual SERS nanotags To measure the SERS spectra, 20 µL of the diluted SERS nanotag solution was deposited on a silicon wafer. After drying in air, a dark-field image was taken, and SERS spectra of the individual SERS nanotags were recorded. To study the response of SERS 7



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nanotags (with DTDPA-DMAPBA as a Raman reporter) to HRP, SERS nanotag solutions were mixed with different concentrations of HRP solutions for 30 min. Their corresponding SERS spectra were measured using a round cuvette with a diameter of 2 cm and depth of 1 cm. For each concentration, 5 samples were measured to calculate the SD. SERS-based immunoassay The immunoassay was performed on a microplate. A total of 100 µl EPO-R solution (10 µg/mL in PBS containing 0.05 M sodium carbonate) was injected into a microplate and incubated at 37 °C for 2 h. After thorough washing with PBS (0.01 M containing 0.1% Tween), 200 µL 0.2% gelatin in PBS was added and incubated for 2 h. The microplate was washed with PBS again, and 100 µl of the EPO solutions with varied concentrations were added and incubated at 37 °C for 1 h. After washing with PBS, 100 µl DTDPA-DMAPBA-modified SERS nanotag solution was injected. After incubation for another hour at 37 °C, the excess SERS nanotag solution was removed, and the microplate was washed with PBS three times. The SERS spectra were measured after the plate was dried in air.

Results and Discussion Synthesis of AuSPs APBA is a derivative of aniline (ANI), and it can reduce HAuCl4 to form AuNPs 14-16. Upon addition of APBA to a solution of HAuCl4 containing SDS, the yellowish solution turned to red and purple in 2 min. After 30 min, the sample was centrifuged. TEM images of the collected pellets show spherical Au superparticles (AuSPs) with a mean size of 8


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115.8±4.5 nm (Figure 1a). AuSPs consist of high-density, small NPs with a d-spacing of 0.24 nm corresponding to the (111) lattice plane of Au crystals (Figure 1b)17. The XPS spectrum of AuSPs reveals the presence of the elements Au, C, and N (Figure 1c). The Au 4f region exhibits a pair of peaks at 83.9 and 87.6 eV assigned to the Au 4f7/2 and Au 4f5/2 peaks, respectively (Figure 1d), suggesting the formation of AuNPs18. The N 1s peak can be fitted into the three peaks corresponding to positively charged nitrogen N+ (N1, 401 eV), amine (–NH–) (N2, 399.2 eV) and imine (=N–) (N3, 398.4 eV)19, indicating the existence of a PAPBA polymer within the AuSPs (Figure 1e). Figure 2a shows the TEM images of samples collected at different reaction times. A large number of small AuNPs formed within the first minute; their sizes varied from units up to ~20 nm (Figure 2a). Aggregated AuNPs were present after 3 min, and uniform AuSPs were present after 5 min. AuSPs with similar size and morphology revealed the completion of their growth in 5 min. The growth mechanism of AuSPs is proposed in Figure 2b: APBA reduces HAuCl4 to form AuNPs, and the corresponding oxidant product PAPBA adsorbs on the AuNP surface as a capping agent. PAPBA subsequently self-assembles AuNPs into AuSPs via π-π stacking. This accords with our previous report that π-π stacking of poly (N-(3-amidino)-aniline) (PNAAN) can enable self-assembly of Au nanoplates into Au nanoflowers20. Reaction conditions dramatically affect AuSP formation. APBA has a lower redox potential at a basic pH, resulting in an increased reduction rate with an increased solution pH value21. Figures S2a-d show TEM images of AuSPs prepared at various pH values. Spherical AuSPs exhibited decreasing sizes of 370.2±22.5, 115.8±4.5, 79.4±3.8, and 55.3±2.8 nm when the reaction was conducted in 1 mM HCl, H2O, 1 mM and 5 mM 9



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NaOH solutions, respectively. Furthermore, the high APBA/HAuCl4 ratio induced fast reduction of HAuCl4, leading to small AuSPs (APBA/HAuCl4 ratios of 5/1, 10/1, 20/1 and 30/1 generated AuSPs with sizes of 480±32, 115.8±4.5, 78.6±3.8, and 57.6±4.5 nm, respectively) (Figure S2e-h). The size of the AuSP is governed by the nuclei number of the AuSPs, which is directly proportional to the formation rate of small AuNPs. For a given amount of precursor, a larger number of nuclei results in smaller final particles22. Therefore, at the fixed HAuCl4 concentrations, the fast reduction of HAuCl4 produced more AuNPs in the early stage of the reaction, resulting in a greater number of AuSPs with a smaller size. SERS enhancement of AuSPs The AuSP surface contains a large number of sharp tips, which are hotspots for SERS enhancement. Moreover, the presence of high-density nanogaps on the AuSP surface can generate high-density hotspots

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. Therefore, such AuSP can be used as a good SERS

substrate for the development of SERS nanotags. AuSPs were well-dispersed on a silicon wafer after self-assembly of DTDA on them to form SERS nanotags (Figure 3a). This was confirmed by the isolated bright dots under dark-field microscopy (Figure 3b). The corresponding SERS spectrum of a single SERS nanotag shows typical SERS bands at 1076 and 1587 cm-1 (Figure 3c), which are assigned to a mixed mode from the C-C stretching, the C-H in-plane bending vibration, and the parallel C-C vibration stretching vibration25-26. After measuring over 100 SERS nanotags, each of them presents reliable SERS signals, with an SD of 26.8% as calculated by the peak intensity at 1076 cm-1. The enhancement factor (EF) was calculated to be 2.1×107

27

. Compared with the reported

single-particle SERS using etched octahedral particles (the EF of ~105)28 or well-defined 10


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Au nanobridge nanogap particles (Au-NNP) (90% of the analyzed particles generated detectable SERS signals)29, the as-synthesized AuSP shows better SERS performance with an easy method for their preparation. Although single DTDA-modified SERS nanotags displayed good SERS signals, their signal/noise ratio is low. To improve the signals of SERS nanotags, a new azobenzenecarrying Raman reporter (DTDPAP) was synthesized, and the synthetic procedure is shown in Scheme 1a. DTDPAP has two key domains: 1) the disulfide bond allows for its self-assembly on AuSPs via the formation of the Au-S bond, and this can significantly improve the stability of SERS nanotags; 2) the azobenzene moiety demonstrates a large Raman cross-section and multiple peaks compared with DTDA 30-31. After self-assembly of DTDPAP on AuSPs to form SERS nanotags (Figure 4a), their SERS spectrum is shown in Figure 4b. Compared with that of the DTDA-carrying SERS nanotag (black curve in Figure 4b), two new bands at 1145 and 1433 cm-1 assigned to the typical “b2” bands of an azobenzene derivative were observed

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. Moreover, their

signal/noise ratio significantly improved. We measured the SERS spectra of ten SERS nanotags and noticed that each of them displayed intense SERS signals (Figure 4c). SD was calculated to be 10.5% according to the peak at 1145 cm-1. This SD value is much lower than that when using DTDA as a Raman reporter (26.8%). Furthermore, four characteristic peaks are present (Figure 4d); thus, multiple bands are provided for. In comparison, DTDA-modified SERS nanotags only contain 2 bands. SERS nanotags carrying multiple bands are important because they can provide multiple identifications and prevent false positives in complex biological targeting. Formation of sugar-responsive SERS nanotags 11



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To demonstrate the practical application of SERS nanotags, a Raman reporter DTDPA-DMAPA containing both azobenzene and boronic acid moieties was synthesized. We employed AuSPs with a size of 55 nm (shown in Figure S2d) as a SERS substrate and DTDPA-DMAPA as a Raman reporter to prepare the SERS nanotags. After self-assembly of DTDPA-DMAPA, the AuSP surface was covered both with boronic acid and azobenzene groups (Figure 5a). The boronic acid group can react with 1,2-diols, such as sugar, glycoprotein or even the glycosylated domains on bacteria or cell surfaces, to form a five-membered ring (Figure 5b)34-40. HRP is a glycoprotein rich in mannose groups, one of the sugars with good reactivity towards boronic acid41. Therefore, the addition of HRP to a SERS nanotag solution can induce their aggregation due to the presence of multiple binding sites of the boronic acid group on an HRP molecule (Figure 5c). Figure 5d presents a broad UV absorption peak between 400-800 nm for SERS nanotags, and the presence of 1 µg/mL HRP induced an apparent decrease of their absorbance, indicating the aggregation of AuSPs. The DLS spectra of AuSPs show a narrow peak at 58 nm, which is consistent with the size measured by TEM (55 nm). The peak became broad, and its position shifted to 328 nm after incubation with HRP, revealing the aggregation of AuSPs. The SERS nanotags with DTDPA-DMAPA as a Raman reporter show 6 typical bands at 1075, 1169, 1333, 1377, 1415 and 1576 cm-1 (Figure 6a). After incubation with different concentrations of HRP solutions, the intensities of all the peaks increased with increasing HRP concentrations (Figure 6b). These enhanced SERS signals were caused by the aggregation-induced generation of SERS hotspots42. Figure 6c demonstrates the good linear relationships between peak intensities and log [HRP concentration] from 12


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1 ng/ml to 1 µg/ml (R2 = 0.990). The limit of detection (LOD) was calculated to be 0.2 ng/mL by the equation of 3σ/slope (Figure 6c). Compared with the reported method using a sandwich-like strategy (linear range of 1 ng/mL to 10 µg/mL)35, this sensor shows comparable performance. Application of SERS nanotags to immunoassay Owing to the high affinity of DTDPA-DMAPA-modified SERS nanotags to glycoproteins, we proposed a simple method to detect glycoproteins via an immunoassay. As shown in Figure 7a, a glycoprotein antibody is adsorbed on a microplate, which captures its corresponding glycoprotein. Subsequently, the boronic acid carrying SERS nanotags is captured by the glycoproteins via interaction of its sugar domain and the boronic acid group. The glycoprotein concentration can thus be measured by the SERS intensity of the attached SERS nanotags. To demonstrate the feasibility of this approach, we performed immunoassay experiments for the profiling of EPO. EPO is a glycoprotein that is responsible for the proliferation of erythrocytes in the human body. As a blood doping agent, it can enhance athletic performance by increasing the number of erythrocytes43. It was listed as a prohibited substance by the International Olympic Committee in 199044. The SERS spectra of the immunoassay in Figure 7b indicates that the EPO concentration-dependent SERS intensity increases. The peak intensity linearly increased with log [EPO] concentrations in the range of 1.45×10-14 to 3.71×10-12 M (R2 = 0.992) with an LOD of 6×10-15 M (Figure 7c), which is much lower than the normal concentrations of EPO in blood (10-12-10-11 M)45 and in urine (10-14-10-13 M) 46 for healthy individuals. Therefore, we believe that this sensitive SERS-based immunoassay has great potential for clinical assays. Due to the generality of the binding ability between boronic 13



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acid and sugar, we envision that the as-synthesized SERS nanotags can be used as a general substitute for labeled antibodies in the profiling of sugar-carrying molecules.

Conclusion In summary, we have developed a new strategy to synthesize SERS nanotags with high intensity and multiple bands. AuSPs, which were prepared through a one-pot formation and self-assembly of small AuNPs ,showed good SERS enhancement. A newly prepared azobenzene-carrying Raman reporter demonstrated strong SERS signals and multiple peaks compared favorably to the commercial Raman reporter. A sugarresponsive SERS nanotag showed six unique bands and good sensing ability towards the glycoprotein HRP. Moreover, these SERS nanotags also demonstrated good performance when applied as a substitute to labeled antibodies in an immunoassay of glycoproteins. We expect that these SERS nanotags have great potential for the monitoring of pathogens or the targeting of cancer cells via recognition of the sugar residuals on their surface.

ASSOCIATED CONTENT Supporting Information. Mass spectra of DTDPAP and DTDPA-DMAPBA, TEM images of AuSPs prepared in different reaction media, and with different ratios of NAAN/HAuCl4. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Ying Ma) 14


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[email protected] (Nan Li)

ACKNOWLEDGMENT This work was supported by research funding from the Singapore Millennium Foundation and the National Medical Research Council for funding support (NMRC/CIRG/1359/2015).

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Scheme 1 Synthetic route of (a) DTDPAP and (b) DTDPA-DMAPBA.

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Figure 1. (a) TEM image of AuSPs, (b) a lattice structure of AuNPs marked in the circled area. (c) XPS survey spectrum of AuSPs. (d, e) Au 4f and N 1s core level spectra of AuSPs. The solid lines are the fits to the data.

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Figure 2. (a) TEM images of products acquired at different reaction times. (b) Schematic illustration of the AuSP formation.

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Figure 3. (a) Schematic illustration of DTDA-modified SERS nanotags. (b) Dark-field image of isolated SERS nanotags on a silicon wafer. (c) SERS spectra of an individual SERS nanotag.

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Figure 4. (a) Schematic illustration of a DTDPAP-modified SERS nanotag. (b) SERS spectra of (black curve) DTDA- and (red curve) DTDPAP- modified SERS nanotags. (c) SERS spectra of ten DTDPAP-modified SERS nanotags and (d) SERS intensity of SERS nanotags at different SERS bands.

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Figure 5. Schematic illustration of (a) DTDPA-DMAPBA modified SERS nanotag, (b) reaction of the boronic acid with sugar and (c) HRP-induced SERS nanotag aggregation. (d) UV absorption spectra of SERS nanotags in the (black curve) absence of and (the red curve) presence of 1 µg/mL HRP. (e) The corresponding DLS spectra of SERS nanotags in d.

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Figure 6. SERS spectra of (a) DTDPA-DMAPBA modified SERS nanotag solution and (b) SERS nanotags in the presence of different concentrations of HRP. (c) The SERS intensity vs. HRP concentration recorded at various SERS bands.



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Figure 7. (a) Schematic demonstration of a SERS-based immunoassay for glycoprotein detection, (b) SERS spectra of immunoassays for different concentrations of EPO. (c) The SERS intensity vs. EPO concentrations recorded at various SERS bands.


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Gold Superparticles Functionalized with Azobenzene Derivatives

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