Perylene Derivatives As Useful SERRS Reporters, Including

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Perylene Derivatives As Useful SERRS Reporters, Including Multiplexing Analysis Eleonora Tenori,† Andrea Colusso,§ Zois Syrgiannis,*,† Aurelio Bonasera, Silvio Osella,‡ Adrian Ostric,† Roberto Lazzaroni,*,‡ Moreno Meneghetti,*,§ and Maurizio Prato*,† †

Center of Excellence for Nanostructured Materials, Dipartimento di Scienze Chimiche e Farmaceutiche, INSTM unit of Trieste, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy § Nanostructures and Optics Laboratory, Dipartimento di Scienze Chimiche, University of Padova, via Marzolo 1, Padua, Italy ‡ Laboratory for Chemistry of Novel Materials, University of Mons−UMONS, Place du Parc 20, 7000 Mons, Belgium S Supporting Information *

ABSTRACT: Five perylene bisimide (PBI) derivatives were designed and synthesized, on the basis of quantum-chemical calculations. The influence of halogen substituents on the shape and energy of the frontier orbitals and the Raman spectra were calculated, in the prospect use in surface-enhanced resonance Raman scattering (SERRS) studies. The corresponding experiments confirmed a very strong SERRS response in the presence of pristine (i.e., uncoated) gold nanoparticles. These spectra can be used for multiplexing measurements, namely measurements in which, by using a single laser excitation, one can recognize the simultaneous presence of several analytes. KEYWORDS: perylene bisimides, SERRS, Raman spectroscopy, multiplexing analysis

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INTRODUCTION Surface-enhanced Raman scattering (SERS) has generated significant interest as an analytical spectroscopic technique, exploiting molecular specificity and sensitivity at the single molecule level.1 In a SERS experiment, weak Raman signals are enhanced when an analyte of interest is adsorbed on a nanoscale-roughened noble metal surface.2 Current models explaining the experimentally observed signal enhancements are based on two contributions: (i) the electromagnetic (EM) and (ii) the “chemical” enhancement (CE). The EM mechanism is the most important one and relates to the enhancement of local electromagnetic fields due to resonant excitation of plasma oscillations (localized surface plasmons) in the metallic nanostructure.3,4 Strong intensity enhancements also occur for nanosized (10−100 nm) surfaces, as in the case of nanoparticles (NPs).5 The enhancement is particularly strong in the so-called “hot spots”, i.e., nanoscale crevices and interstices in nanostructures6 and in aggregates, suggesting that it is the interaction between excited localized plasmons on different particles that generates large Raman enhancements.7 The SERS effect has been observed for a large variety of molecules adsorbed on the surface of metals, and in particular those for which it is possible to excite the localized plasmons in the UV−vis−NIR spectral range, i.e., Ag, Cu, and Au, which have become, by far, the dominant substrates for SERS. Among these substrates, gold nanoparticles (AuNPs) play an important role because of their localized surface plasmons in the Vis to NIR range, imparting interesting optical properties, in addition to their easy functionalization, chemical stability and biocompatibility.8 © XXXX American Chemical Society

Recently, it has been reported that the Raman enhancement effect can be obtained with gold nanoparticles under in vivo conditions for tumor detection in live animal models, opening the way to advanced biomedical applications.9 One of the most important factors for sensitive and reproducible SERS measurements is the control of the NP aggregation. To achieve such control, different approaches have been investigated. Usually, the addition of an appropriate SERS reporter to a suspension of NPs leads to partial aggregation of the particles with the generation of hot spots. Aggregation can also be obtained via physical processes such as centrifugation or electrodeposition. In these cases, the SERS reporter is deposited after the nanoparticles have aggregated.10 Regarding the reporters, a large variety of organic compounds have been considered in the literature.11 Because of their extended π-conjugated systems, perylene derivatives appear to be particularly promising for electronic or biosensing devices. However, investigations of their interaction with metals mostly deal with photoemission spectroscopy studies of molecular layers on single crystalline metal surfaces.12 In terms of Raman studies, series of perylene bisimides (PBIs) were deposited onto silver, gold, or mixed Ag−Au films,13,14 as well as onto In and Ag layers.15 A significant enhancement of Special Issue: Advances towards Electronic Applications in Organic Materials Received: April 25, 2015 Accepted: July 31, 2015

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DOI: 10.1021/acsami.5b03586 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

washed with Et2O (2 × 30 mL). The resulting solid was dried in vacuum to afford the iodine salt of 1 (0.32 gr, 98%) as a dark red powder. The characterization of the material is in agreement with the already reported synthetic procedures.18 For the exchange of the counterion, the precursor iodide salt was solubilized in a mixture of MeOH/HCl conc. and the solution was stirred for 5 h at 70 °C. The solution was then cooled to r.t., diethyl ether was added and the precipitate was filtered and washed with toluene (2 × 30 mL) and Et2O (2 × 30 mL). The resulting solid was dried in vacuum to afford 1 (2.19 g, 80%) as a dark red powder. 1H (TFAd, 270 MHz), δH (ppm): 8.88 (d, J = 8.10 Hz, 4H), 8.80 (d, J = 7.83 Hz, 4H), 4.83 (bt, 4H), 3.84 (bt, 4H), 3.36 (s, 12H). 13C (TFAd, 67.5 MHz), δC (ppm): 36.4, 55.6, 64.6, 123.5, 126.7, 128.7, 131.6, 135.5, 138.6, 167.8. IR (powder, ν cm−1): 1694, 1657 (>CO), 1593 (−CC−), 1439, 1403, 1343 (>C−N-R). ESI-MS, m/z calcd for C34H34Cl2N4O4: 632.20; found: 281.1[(M2+ − 2Cl−)/2]. UV−vis (TFA 0.01 mM solution) λmax: 468, 499, 536 nm; λem: 552, 591 nm (λexc: 350 nm). General Procedure for the Synthesis of Bay-Substituted PBIs. One hundred milligrams of bisbrominated, tetrachlorinated, or tetrabrominated perylene-3,4,9,10-tetracarboxylic bisanhydride were suspended in dry DMF (5 mL) with 2 equiv of the corresponding amine and 1 mL of acetic acid in a pressure-tight microwave tube. The suspension was sonicated for a a few minutes before heating under microwave irradiation at 60W for 10 min. The maximum temperature was set at 200 °C. After cooling the color turned dark red and the mixture appeared more homogeneous. 50 mL of NaOH 1 M was added to the starting material and stirred for 20 min. The precipitate was filtered, washed abundantly with water until pH neutralization, and dried in vacuum. The synthesis and the characterization of all the bisimides used in this work are reported in the Supporting Information. Au NPs. The gold nanoparticles were obtained through an innovative approach, namely LASiS (Laser Ablation Synthesis in Solvents) using 9 ns pulses at 1064 nm of a Nd:YAG laser (Quantel YG-981 × 10−10).25 The laser beam is focused with a lens (10 cm focal length) on a Au target at the bottom of a cell (3 × 3 × 4 cm3) filled with 15 mL of 1 × 10−5 M NaCl bidistilled water solution. Fluences of 10 J cm−2 are used for obtaining nanoparticles with an average diameter of 20 nm.23,26 LASiS is a “green” technique for the synthesis of stable noble metal nanoparticles in water or in organic solvents, in that it can be carried out without using stabilizing agents. The nanoparticles obtained via LASiS thus have a free surface, allowing an easy functionalization with a large variety of molecules.21 The stability of the colloidal solution derives from the negative surface charges that the particles show after the laser ablation synthesis. For the nanoparticles synthesized in water, ζ-potential measurements show values exceeding −30 mV, which guarantees the stability of nanoparticles in solution.23 Estimates of the average diameter and concentration of the nanoparticles have been obtained following a published methodology based on the fitting of the UV−vis spectra.27 Raman and SERRS Characterization. Raman spectra were recorded with a microRaman inVia (Renishaw) equipped with two laser sources (He−Ne (632.8 nm) and diode laser (785 nm)) and directly interfaced to a Leika DM-LM confocal microscope. A 20× microscope objective was usedfor the measurements, with a spot diameter on the sample of 8−10 μm. The samples for SERRS measurements were produced by mixing 1 mL Au NPs (1 × 10−9 M) and 1 mL PBI (1 × 10−7 M) solutions in water, stirring for 30 min, and depositing 50 μL of the resulting solution on a glass slide, generating a spot of about 1 mm in diameter, while the spot diameter of the laser on the sample is about 8−10 μm. The final amount of 1 for the SERRS experiments is therefore 2.5 × 10−9 moles (in a slightly lower range for all the other compounds).

the Raman signals was observed for all the perylene derivatives, clearly indicating the occurrence of surface-enhanced Raman scattering. Interestingly, depending on the electronic properties of the reporter, the SERS signals can be further enhanced, a phenomenon called the SERRS effect, namely SurfaceEnhanced Resonance Raman Scattering. SERRS occurs when the wavelength of the excitation laser matches an electronic absorption of the molecule. Thus, both enhancement from the plasmon resonance (SERS) and molecular resonance (or preresonance) from the reporter contribute to generate very intense scattering.16 The additional signal enhancement due to such molecular electronic resonance makes SERRS a highly sensitive vibrational spectroscopic technique. Perylene derivatives perfectly fulfill the above-mentioned condition for SERRS, because they have absorption profiles in resonance or preresonance with the exciting laser lines that are commonly used, for example, in biological applications.17,18 Perylene is a good fluorophore and perylene-gold nanoparticle binary systems have shown fluorescence quenching, together with SERS enhancement.19 SER(R)S has a major advantage over fluorescence in that the signals have extremely small line-widths compared to the broad spectra of fluorescence emission. Therefore, it is possible, by using molecules with SER(R)S activity, to carry out multiplexed detection, namely, in one spectrum excited with a single laser line, one can detect SERRS signals from several reporters.20,21 Herein, we report a combined theoretical/spectroscopic study of a series of core-halogenated perylene derivatives. On one hand, quantum-chemical calculations are performed to understand the influence of the substitution on the shape and energy of the frontier orbitals, which are involved in the electronic excitation. On the other hand, the PBI compounds are made to interact with gold nanoparticles and their SERRS response is investigated, with the assistance of theoretical Raman spectra. The potential of these AuNPs/PBI systems in terms of multiplexing analysis is then assessed on the basis of the spectroscopic results.

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EXPERIMENTAL SECTION

Materials. Chemicals and solvents for all the synthetic procedures and characterization analyses were purchased from Sigma-Aldrich and TCI and used as received if not differently specified. Deuterated solvents were purchased from Cambridge Isotope Laboratories. The characterization and the synthesis of the gold nanoparticles were reported previously.21,22 2-Br-tetracarboxylic bisanhydride, 4-Brtetracarboxylic-bisanhydride, and 4-Cl-tetracarboxylic-bisanhydride were synthesized by methods described in the literature.23,24 N,N′-Bis(2-(trimethylammonium)ethylene)perylene-3,4,9,10-tetracarboxylic acid bis-imide bis chloride salt (1) has been synthesized starting from the corresponding iodide salt, which was synthesized through a microwave-assisted procedure. One hundred milligrams of perylene-3,4,9,10-tetracarboxylic bisanhydride were suspended in dry DMF (5 mL) with 2 equiv. of N,N-dimethyl ethylene diamine in a pressure-tight microwave tube. The suspension was sonicated for a few minutes before heating under microwave irradiation at 50W for 10 min. Five cycles. The maximum temperature was set at 200 °C. After cooling, the color of the medium turned dark red and the mixture appeared more homogeneous. Fifty milliliters of 1 M NaOH were added to the starting material and stirred for 20 min. The precipitate was filtered and washed thoroughly with water until pH neutralization and dried in vacuum. For the methylation of the tertiary amines, the precursor diamine (0.25 gr, 0.46 mmol) and MeI (40 mmol) were solubilized in toluene (20 mL) and the mixture was refluxed for 5 h. The solution was cooled to r.t. and the precipitate was filtered and

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RESULTS AND DISCUSSION

As a first step, we converted the hydrophobic PBI derivatives into more water-friendly materials. According to the literature,18 this can be achieved by introducing a short organic pendant group bearing a quaternary ammonium salt at the NB


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halogens (namely F, Cl, and Br) causes a gradual distortion of the aromatic system (Figure 2). As expected, the tilting angle between the two halves of the molecule is largest for the tetrasubstituted compounds (36.0 and 37.0 degrees for 4 and 5, respectively) with respect to the disubstituted molecules (2 vs 5), and it increases with the size of the halogen atom (3 vs 2 and 4 vs 5, see Figure 2, Table S3). This is related to the steric hindrance of the systems with different degrees of substitution; substituents in 1,12 and 6,7 positions are facing each other, and the largest the atomic radius, the highest the distortion of the perylene core required to reduce the steric repulsion. The simulated Raman spectrum of 1 shows six main peaks at 1704, 1562, 1429, 1366, 1271, and 1074 cm−1, related to different vibrational modes (to take into account the dependence of vibrational modes on the functional and basis set, a scaling factor of 0.951 is used) (Table 1).30 The peak at 1704 cm−1 is attributed to the symmetric stretching of the carbonyl groups. The following two peaks are related to the symmetric and antisymmetric stretching modes of the carbon atoms of the aromatic core. The band located at 1366 cm−1 is attributed to the wagging modes of the hydrogen atoms connected to the sp3 carbon atoms at the imide position. The last two peaks, at 1271 and 1074 cm−1, are bending movements of the aromatic hydrogen atoms, like scissoring and rocking vibrational modes. We assume that the frequency of these vibrations would not be affected upon grafting on the gold surface since that process occurs through electrostatic interactions with the positively charged terminal amine of 1, without involving the aromatic core. Subsequently, the possibility of modifying the Raman features through substitution at bay position was investigated. Since it is reported in literature17 that the electronic and optical properties of PBIs can be easily tuned by bay substitution, we selected a family of perylene derivatives where halogen substituents (F, Cl, Br) were introduced in the aromatic core (Scheme 1). The theoretical calculations indicate that (i) the addition of different halogen substituents does not affect the general shape of the frontier orbitals, and (ii) the halogen atoms at the bay position appear to stabilize the HOMO and LUMO levels, because of their electron-accepting character.31,32 Additional differences arise from steric factors, which affect the stabilization of the frontier orbitals. In the case of 4, the HOMO and LUMO levels show a 0.42 eV stabilization in comparison with 1; this effect is the combination of the electron-withdrawing character of the substituents, which tends to stabilize both frontier orbitals, and the geometric distortion, which reduces the π-conjugation in the backbone and increases the HOMO−LUMO gap. Besides those electronic effects, the main aim of the halogen substitution is to introduce controlled geometrical modifications of the central scaffold of the chromophore that will impact on the vibrational modes giving rise to the Raman signals. The Raman spectra simulations for these compounds revealed a similar pattern of peaks to unsubstituted 1, with the vibrational modes shifted toward lower wavenumbers and a new peak related to the presence of the halogen atoms (1532, 1540, 1512, and 1512 cm−1 for 2, 3, 4 and 5, respectively). We focused on that peak because it is related to the stretching mode involving the carbon atoms that bear the halogen substituents. The presence of the substituents gradually redshifts the peak position, with a maximum shift of 40 cm−1 for the tetra-substituted compounds (the frequencies for all the compounds are reported in Tables 1 and Tables S4−S7). This

imide positions. The presence of the ammonium cation provides a useful anchor point onto AuNPs, which possess a negative surface charge.21 In addition, the water solubility is improved, a desired feature for applications in biology. This led us to the synthesis of five perylene derivatives, namely 1-5 (Scheme 1). The representative absorption spectra of those compounds are presented in Figure 1. In all the spectra, welldefined vibronic fine structures appear for the S0 → S1 transition. Scheme 1. Synthetic Procedure for the Compounds Investigated in This Studya

a (a) (i) N,N-Dimethylethylenediamine, DMF, MW; (ii) MeI, reflux, HCl/MeOH; (b) (i) Br2, H2SO4, HNO3; (ii) N,N-dimethylethylenediamine, DMF, acetic acid, MW; (iii) MeI, reflux; (c) (i) chlorosulfonic acid, I2, heating; (ii) N,N-dimethylethylenediamine, DMF, acetic acid, MW; (iii) MeI, reflux; (d) (i) Br2, H2SO4, HNO3; (ii) N,N-dimethylethylenediamine, DMF, acetic acid, MW; (iii) MeI, reflux; (f) KF, 18-crown-6, sulfolane, heating.

Quantum Chemical Calculations. To assess the potential of PBIs as SERRS reporters, we examined their vibrational normal modes with quantum-chemical calculations. The geometry of the PBIs has been optimized and their IR and Raman spectra have been simulated at the HSEh1PBE28/6311+G(d,p) level of density functional theory (DFT).29 Presence of the halogen substituents at the bay positions is likely to affect the geometry of the molecular scaffold. Taking 1 as the benchmark molecule (since the perylene core is fully planar and unsubstituted), the introduction of different C


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Figure 1. Normalized absorption spectra of 1, 2, 3, 4, and 5 in TFA (left) and in aqueous NaCl solution (right) ([PBI] = 1 × 10−5 M).

Figure 2. Schematic representation of the distortion of the perylene core caused by the presence of halogen substituents in the bay-positions.

atoms), the structural distortion of the aromatic core, the loss of conjugation, and the shift in the Raman stretching mode. Surface-Enhanced Resonance Raman Spectroscopy. The calculated Raman spectra of 1 are in good agreement with the experimental spectra, which present typical PBI peaks at 1095, 1304, 1384, 1460, 1583, 1703 cm−1 (Figure 3, Table 1).33,34 SERRS spectra, as recalled above, can be recorded when the molecules are close to the plasmonic nanoparticles and

Table 1. Theoretical and Experimental Raman Signals of 1 before and upon Deposition on AuNPs frequency (cm−1) peak

theoretical

experimental

@AuNPs solution

A B C D E F

1074 1271 1366 1429 1562 1704

1095 1304 1384 1460 1583 1703

1084 1296 1379 1456 1576 1695

trend is fully consistent with the structural distortion due to the presence of the halogen substituents, as described above. The presence of four substituents causes the highest distortion of the perylene core and the conjugation is partly lost. This loss of conjugation weakens the C−C bonds; as a consequence their vibrational mode is shifted to lower wavenumbers. As depicted in Figure 2, in which a highly substituted molecule (5; four Br atoms) and a less substituted one (2; two Br atoms) are compared, the aromatic core of the latter is less distorted (24.4 vs 37.0 degrees) and shows a calculated C−C stretching band that is less shifted down in energy (1532 vs 1512 cm−1). Interestingly, the difference between 5 and 4 (four Cl atoms) is negligible, consistent with the similar distortion angle (36 and 37 degrees, respectively). In contrast, when comparing 2 and 3 (two F atoms), the calculated peaks positions are clearly distinct (1532 vs 1540 cm−1), consistent with the significant structural difference between 2 and 3 (24.4 and 6.6°, respectively). The calculations clearly underline the relationship between the substitution pattern (type and number of halogen

Figure 3. Simulated (left) and experimental (right) Raman spectra of the investigated PBI derivatives (experimental Raman spectra recorded at 633 nm excitation wavelength). D


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(the mixture of the AuNPs with 1 will be referred to as 1@ AuNPs). The resulting experimental Raman spectrum of 1@ AuNPs presents typical PBI peaks at 1084, 1296, 1379, 1456, 1576 and 1695 cm−1 (Figure 5, left). Compared to the spectrum of the pristine molecule, the peaks present a slight red-shift that can be attributed to the intimate interaction of 1 with the gold nanoparticles (Table 1). Compound 1 is found to be a good organic reporter for SERRS analysis since its signals are intense and well-defined for a 1 × 10−7 M concentration of the PBI. It is noteworthy that the Raman signature of 1 can still be easily identified for concentrations as low as 1 × 10−9 M (see Figure S29). The calculated Raman spectra of 2−5 are also in good agreement with the experimental spectra of the perylene derivatives (Figure 3 and in Tables S4−S7). Their presence in AuNP solutions induces the same aggregation of the nanoparticles as in the case of derivative 1, as expected (Figures S15−S18). The SERRS spectra of 2−5, obtained using the same approach used for 1, namely by mixing the AuNP and PBI solutions and depositing 50 μL on a glass substrate, are reported in Figure 5 (right) for 1 × 10−7 M PBI solutions. They show intense peaks as in the case of 1, as expected for SERRS spectra.

especially in hot spots, which are easily generated when the nanoparticles are interacting (i.e., when they are aggregated). In fact, molecules like the PBIs can readily adsorb on uncoated particles, such as those generated by Laser Ablation Synthesis in Solvents (LASiS), creating bridges between nanoparticles and inducing their aggregation. This is what can be observed when the PBI concentration is increased in a AuNP solution. The spectra recorded at different PDI concentrations (Figure 4)

Figure 4. UV−vis spectra of aqueous solutions of AuNPs with increasing concentrations of 1 ([AuNPs] = 1 × 10−9 M, [1] = 1 × 10−8 to 1 × 10−6 M).

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MULTIPLEXING MEASUREMENTS SERRS is an ideal technique for the analysis of multiple analytes. In many cases the nanostructures showing SERRS signals also contain targeting units for recognizing analytes to be detected.22 The presence of a SERRS reporter signals, after the targeting process, allows detecting the presence of the targeted analyte.22 Several analytes can be detected simultaneously by combining nanostructures with different targeting units and different SERRS reporters if their spectra can be distinguished. One also notes that for such multiplexing analysis, only one laser line is ideally needed for excitation. Based on the SERRS spectra obtained for compounds 1−5, and summarized in Tables 1 and 2 (see also Figure 5), we show the possibility of using their spectra for a multiplexing approach. For an easy interpretation of multiplex analysis, it is desirable to obtain Raman peaks, generated from each dye, with similar intensities. In most cases, the dyes reported in the literature, despite they are present at the same concentration, show large variations in the intensities of their signals because of differences in the efficiency of the resonances of the exciting laser line with the different molecules. In our case, compounds 2-5 have similar intensities at the same concentration of

show that increasing the concentration of 1 (from 1 × 10−8 M up to 1 × 10−6 M) induces the appearance and growth of an extinction band in the spectral region of 600−800 nm where localized plasmon resonances of large particles or aggregates can be observed.23,35,36 This band gradually shifts to lower energies when increasing the PBI concentration, which is the signature of the formation of increasingly large aggregates. SERRS spectra can be obtained when the laser excitation for generating the Raman signal is in resonance with the localized surface plasmon resonance and with a molecular excitation. Here we recorded the spectra using the 633 nm excitation of a He−Ne laser because it is important for many applications and in particular for biological applications. The absorption spectra of Figure 4 shows that the aggregates of nanoparticles can enter in resonance with the 633 nm radiation. Furthermore, Figure 1 shows that PBIs absorb light near 600 nm, which constitutes at least a preresonance condition with the 633 nm excitation. Thus, the conditions are met for observing the SERRS effect. For the SERRS spectra, we stirred together a 10−9 M aq. solution of AuNPs and a 1 × 10−7 M aq. solution of 1 for 30 min and deposited 50 μL of the mixture on a glass substrate

Figure 5. SERRS spectra of 1@AuNPs (left) and of 2, 3, 4, 5 @AuNPs (right) (recorded using the 633 nm excitation line). E


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This is shown in Figure 6 with the pink spectrum. For the other combinations, similar spectra are recorded and reported in Figure S26−S28. A multivariate statistical analysis allows in any case a quick identification of the presence of all the SERRS reporters. This is a feature specific to SERRS spectra, which cannot apply for example to the more usual fluorescence spectra. Fluorescence studies need different excitation energies and show bands with much larger bandwidths, which do not allow an easy identification of the spectra if more than one component is present.

Table 2. Experimental Raman Frequencies of 2, 3, 4, and 5@ AuNPs frequency (cm−1) @AuNPs peak

2

3

A B C D E F G H

1249 1293 1317 1358 1455 1548 1587 1696

1254 1293 1354 1456 1548 1591 1702

4

5

1342 1447 1540 1593 1707

1311 1371 1442 1544 1582 1689

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CONCLUSION Five water-soluble PBI derivatives were synthesized and characterized. Despite the changes induced by the bay substitution of the perylene core, the lower energy electronic excitation spectrum does not show large variations. This is of major interest because the resonance or preresonance conditions are not affected by the substitution in the bay position and the SERRS signals can show similar enhancements. The calculated vibrational spectra show features that allow distinguishing the different molecules to a sufficient extent, which therefore opens the possibility of using them in multiplexing experiments. Experimental data confirm the calculations and show that this series of PBIs can be exploited for multiplexing analysis, for instance, for applications in biomedicine.

deposited solutions on the glass slides (Figures S24−S25) because of their very similar electronic absorption spectrum. However, not all five compounds can be used simultaneously, because of the coincidence of some of their vibrational bands. Comparing their Raman spectra, we concluded that for a reasonable multiplexing analysis, it is possible to combine up to three of these derivatives. The possible combinations are represented in Table 3. Table 3. Combinations of the Five PBI Derivatives for Multiplexing Analysis compds 1 2 3 4 5

combination A combination B X X

X

X X

X

X

ASSOCIATED CONTENT

S Supporting Information *

X X

X X

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combination C combination D

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b03586. Characterization data for compounds 1−5, spectrophotometric titrations of the AuNPs with the compounds 1− 5, Raman spectra of 1−5@AuNPs and the tables with the theoretical and experimental frequencies, TEM images of the aggregated AuNPs, detailed information on the theoretical studies (PDF)

X

We performed multiplexing analysis of all A−D combinations, confirming the utility and versatility of this approach. As an example, only the combination of 1, 2, and 4 (combination A in Table 3) is reported. The other combinations (B−D) are reported in the Supporting Information. The SERRS spectra of the single molecules and the multiplexing experiment are reported in Figure 6. All the peaks of the single molecules can be distinguished in the multiplexing spectrum and they can be easily deconvoluted using for example the band at 1249 cm−1 for the spectrum of 2, which when eliminated allows us to easily pinpoint the peaks at 1304 and 1384 cm−1 for 1 and the peak at 1342 cm−1 for 4.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the University of Trieste, Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (INSTM), Ministero dell’Università e della Ricerca (MIUR) (FIRB prot. RBAP11ETKA and Cofin. Prot. 2010N3T9M4). The work in Mons is supported by the Science Policy Office of the Belgian Federal Government (PAI 7/5), the OPTI2MAT excellence program of Région Wallonne, and FNRS-FRFC.

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REFERENCES

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Figure 6. Multiplexing experiment using 1, 2, and 4 (combination A) and excitation wavelength at 633 nm. F


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Perylene Derivatives As Useful SERRS Reporters, Including

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