Multi-branched gold-mesoporous silica nanoparticles coated with molecularly imprinted polymer for label-free antibiotic SERS analysis

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1 SUPPORTING INFORMATION Multi-branched gold-mesoporous silica nanoparticles coated with molecularly imprinted polymer for label-free antibiotic SERS analysis Sergio Carrasco, a Elena Benito-Peña, a Fernando Navarro-Villoslada, a Judith Langer, b Marta N. Sanz-Ortiz, b Javier Reguera, b Luis M. Liz-Marzán, b * and María C. Moreno-Bondi a * a Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, Ciudad Universitaria s/n, Madrid (Spain) b BioNanoPlamonics Laboratory, CIC biomagune, Paseo de Miramón 182, Donostia- San Sebastián (Spain). Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Paseo Miramón 182, Donostia-San Sebastián (Spain). Ikerbasque, Basque Foundation for Science, Bilbao (Spain) * Luis M. Liz Marzán, Ikerbasque Professor, llizmarzan@cicbiomagune.es. María C. Moreno Bondi, Professor of Analytical Chemistry, mcmbondi@ucm.es. Phone: Fax: S1

2 Experimental Materials and regents Sodium citrate ( 99%), tetrachloroauric(iii) acid trihydrate (HAuCl 4 3H 2 O, 99.9%), hexadecyltrimethylammonium bromide (CTAB, 99%), L-ascorbic acid (AA, 99%), silver nitrate (AgNO 3, 99%), tetraethyl orthosilicate (TEOS, 99%), phenylmagnesium bromide (PhMgBr, 1M in anhydrous THF), carbon disulphide (CS 2, anhydrous, 99%), Boc-Lphenylalanine (Boc-Phe-OH, 99%), N-(2-hydroxyethyl)piperazine-N -(2-ethanesulfonic acid) buffer solution (HEPES, 1M), ampicillin (AMPI, anhydrous, > 96%), ethanol (EtOH, synthesis grade, 96%), hydrochloric acid (HCl, 37%, w/w), ammonium hydroxide solution (28-30% NH 3, w/w), acetonitrile (MeCN, 99.9%), tetrahydrofuran (THF, anhydrous, 99.9%), triethylamine (Et 3 N, 99%), toluene ( 99.9%) and acetic acid (AcOH, 99.7%) were purchased from Sigma-Aldrich and used as received. Methacrylic acid (MAA, 99% with 250 ppm monomethyl ether hydroquinone as inhibitor), 2-hydroxyethyl methacrylate (HEMA, 97% with 250 ppm monomethyl ether hydroquinone as inhibitor) and ethylene glycol dimethacrylate (EDMA, 98% with ppm monomethyl ether hydroquinone as inhibitor) used as monomers were also purchased from Sigma and purified by passing them through a packed column with an ion exchange resin (inhibitor removers, Aldrich) prior to their use. Enrofloxacin (ENRO, > 98%) was from TCI Europe N.V., penicillin G streptomycin (PENGS, U ml -1 ) from Invitrogen and 4- (chloromethyl)phenyltrichlorosilane (4-CPC, 97%) from Alfa Aesar. Radical initiator, 2,2 - azobis(2,4-dimethylvaleronitrile) (ABDV, 98%) was purchased from Wako, and it was recrystallized from cold absolute ethanol and stored at -20 ºC. Distilled deionized water was obtained with a Milli-Q water purification system Millipore (resistivity 17.8 MΩ cm at 25 ºC). The ph of all buffered solutions was adjusted with a GLP 22 ph meter from Crison (Barcelona, Spain) and nanoparticle suspensions were centrifuged, depending on the S2

3 suspension volume, in the following centrifuge models: Universal 320R (V > 2 ml) or Mikro 220R (for small plastic tubes, V 2 ml) from Hettich Lab Technology (Germany). Characterization Optical extinction spectra, were acquired using an Agilent 8453 UV-Vis-NIR photodiode. SERS spectra were recorded using a Renishaw Invia Raman microscope equipped with two Peltier-cooled charge-coupled devices (CCD) detectors, a Leica microscope with two gratings with 1200 and 1800 lines/mm and band-pass filter optics. Two laser sources, of 633 and 785 nm, operating at different potencies and CCD acquiring images at different times were used to optimize the operation conditions. Measurements were finally performed using 785 nm laser working at 1% of its total potency (2 mw) and with 10 s as acquisition time. Automatic baseline (background) corrections were applied for all acquired Raman spectra with software OriginPro 8 SR2 v (OriginLab Corporation, MA, USA), by using 50 baseline anchor points connected by interpolation and a smoothing according the adjacentaveraging method with a weighted average based on 10 points of window without boundary restrictions. Transmission electron microscopy (TEM) images were collected using a JEOL JEM- 1400PLUS instrument operating at 120 kv, with a LaB 6 electron source and a GATAN US1000 CCD camera (2k 2k). Images were acquired and stored with software Gatan DigitalMicrograph version (32-bits, Gatan Microscopy Suite, Gatan Inc.). Nanoparticle sizes and shell thicknesses were measured (n = 100) with software ImageJ 1.48v (32-bits, Wayne Rasband, National Institutes of Health, USA). Synthesis of gold nanoparticles Au@citrate. Citrate-capped gold nanospheres were synthesized according the citrate reduction method described by Turkevich et al. 1 Briefly, 25 ml of 1% (w/w) citrate solution was added to 500 ml of boiling 0.5 mm HAuCl 4 solution under vigorous stirring. After 15 S3

4 min of boiling, the deep-red dispersion of ca. 14 nm diameter gold nanospheres was cooled at room temperature and kept at 4 ºC for a long-term storage. Au@CTAB. 250 ml of as-synthesized Au@citrate solution was slowly added to 250 ml of 0.1 M CTAB solution under stirring at 30 ºC. After 10 min, Au@CTAB nanoparticles were centrifuged at 9000 rpm for 4 h, washed with H 2 O (3, 7500 rpm, 4 h), and resuspended in 35.7 ml of H 2 O to show a Au 0 concentration of 3.5 mm. This value was calculated from the extinction spectra of the colloid suspension using the absorbance value at 400 nm. 2,3 Synthesis of silica coatings Au@mSiO 2. Protection of gold nanospheres with radial mesoporous silica shells has been described elsewhere. 4 The same procedure was followed with minor modifications. Briefly, in 1 L round bottom flask 150 ml of EtOH were mixed with 340 ml of 6 mm CTAB solution under mechanical stirring at 30 ºC. Then, 0.2 ml of NH 3 and 25 ml of a 3.5 mm Au@CTAB solution were sequentially added. After 10 min, 354 µl of TEOS was added dropwise, setting the temperature to 60 ºC and keeping the reaction vessel under mechanical stirring for 72 h. Au@mSiO 2 nanoparticles were centrifuged at 7500 rpm for 1 h, washed with EtOH (1 ), 1 M HCl in EtOH to remove CTAB from the silica pores (2 ) and, again, with EtOH to remove the excess of HCl (3 ). The sample was treated at 300 ºC for 6 h to increase the stability of the silica shell in water and re-suspended in 12 ml of EtOH, to give a final Au 0 concentration of 7.3 mm. As the concentrations of Au 0 in this and following experiments cannot be derived from the extinction spectra of the nanocomposites (due to the presence of organic solvents and silica and/or polymer layers that modify the refractive index of the composite materials), the concentration values given are those calculated considering no nanoparticles losses during the washing and centrifugation steps. Further silanization and functionalization with RAFT agent were performed at fixed concentration levels of [Au 0 ] = 0.5 mm in EtOH and [Au 0 ] = 0.3 mm in THF, respectively; functionalized gold-silica S4

5 nanoparticles bearing the RAFT agent were concentrated at [Au 0 ] = 3.2 mm in MeCN; polymerizations were performed at [Au 0 ] = 0.75 mm in MeCN, and the final gold-silicapolymer composites were stored at [Au 0 ] = 1.5 mm for morphological and analytical characterization. Synthesis of RAFT agent Synthesis of dithiobenzoate magnesium bromide, DMB ml of anhydrous THF was placed inside a two-neck round-bottom flask under a continuous Ar stream for 20 min. 30 ml of 1 M PhMgBr in THF was added and then 5 ml of CS 2 dropwise. The reaction proceeded at 50 ºC for 1 h with magnetic stirring. The crude mixture was immediately used without further purification. Pre-polymerization mixtures Molecules used as templates, ENRO (483 mg, 1.34 mmol), for molecularly imprinted polymer (MIP) precursor, or Boc-Phe-OH (357 mg, 1.34 mmol), for control imprinted polymer (CIP) precursor, were dissolved in 20 ml MeCN with the addition of MAA (460 µl, 5.63 mmol). Mixtures were stored, covered from light, at 4 ºC for 2 h to favour the formation of the complex template-functional monomer. Prior to their use, diluent monomer HEMA (673 µl, 5.63 mmol), cross-linker EDMA (2.58 ml, mmol) and radical initiator ABDV (117 mg, 0.47 mmol) were dissolved in the previous solutions. Pre-polymerization mixtures were gently purged with Ar for 10 min. Fabrication, functionalization and polymerization of multi-branched gold nanospheres As shown in Figure 1 (main text), two different strategies have been followed in order to prepare multi-branched gold-polymer nanocomposites, bau@msio using Au@mSiO 2 nanospheres with mesoporous silica shells as seeds, depending on the order of branching, functionalization and polymerization processes. S5

6 1) Branching-functionalization-polymerization approach. Different concentrations of 2 nanocomposites were assayed in order to create the gold branches in a one-step procedure. First, different seed solutions were created by mixing 17, 34, 51, 68 and 136 µl of Au@mSiO 2 dispersion with 483, 466, 449, 432 and 364 µl of H 2 O, respectively (giving a Au 0 concentration of 0.25, 0.5, 0.75, 1 and 2 mm). A similar procedure has been described before. 4 Briefly, 10 ml of H 2 O was placed in a glass vial. Under vigorous magnetic stirring, 50 µl of 50 mm HAuCl 4 solution, 10 µl of 1 M HCl, 500 µl of different Au@mSiO 2 seed concentrations, as previously described, 100 µl of 3 mm AgNO 3 and 50 µl of 0.1 M AA were added consecutively. After 5 min, reaction has finished, yielding five different bau@msio 2 dispersions, depending on the initial concentration of the seed. On the other hand, the growing of the gold tips was also evaluated through a step-bystep procedure. For that purpose, 3 ml of Au@mSiO 2 seeds was centrifuged at 7500 rpm for 30 min and re-suspended in 45 ml of H 2 O. Under vigorous stirring, 50 µl of 1 M HCl, 250 µl of 50 mm HAuCl 4 solution, 500 µl of 3 mm AgNO 3 and 250 µl of 0.1 M AA were added consecutively. After 5 min, 250 µl of the reaction mixture was extracted for spectroscopic characterization. The addition cycle was repeated for ten times. b1au@msio 2 nanoparticles were centrifuged at 3000 rpm for 10 min, washed with H 2 O (2 ), EtOH (2 ) and re-suspended in 40 ml of EtOH. These branched nanocomposites were functionalized with the RAFT agent according the following procedure: first, the silica shells were functionalized by adding 1.8 ml of silane 4-CPC and 1.3 ml of Et 3 N. Under vigorous agitation, the reaction takes place inside plastic tubes for 2 h. Silanized b1au@msio CPC nanoparticles were centrifuged at 3000 rpm for 10 min, washed with EtOH (3 ) and resuspended in the same volume amount of anhydrous THF as the original seed used for silanization, i.e., 40 ml. In the next step, 25 ml of DMB crude mixture were added and the reaction was left to proceed inside plastic tubes, covered with aluminum foils, for 2 h, under continuous stirring. b1au@msio nanoparticles were centrifuged at 3000 rpm for 10 S6

7 min, and the pellet was washed with THF (3 ), toluene (3 ), EtOH (2 ), MeCN (3 ) and finally re-suspended in 7 ml of MeCN. For the polymerization step, 3 ml of b1au@msio suspension were mixed with 10 ml of MIP/CIP precursor solutions and polymerized inside a 25 ml round-bottom flask under magnetic stirring at 60 ºC for 1 h, followed by a curing step at 70 ºC for 10 min. After centrifugation (3000 rpm, 10 min), b1au@msio nanocomposites were washed with EtOH:AcOH (2%, v/v) (5 ), EtOH (3 ) and MeCN (3 ). The particles were finally re-suspended in 6.4 ml of MeCN. 2) Functionalization-polymerization-branching approach. Initially, 3 ml of Au@mSiO 2 seeds was mixed with 37 ml of EtOH and functionalized and polymerized, following the same conditions as described before for the branched nanostructures, except for the branching steps, yielding Au@mSiO nanoparticles. The polymer nanocomposites were centrifuged at 3000 rpm for 10 min, washed with EtOH : AcOH (2%, v/v) (5 ) and EtOH (4 ). The particles were finally re-suspended in 40 ml of EtOH ([Au 0 ] = 0.56 mm). Gold tips were grown following the same step-by-step procedure as described above. In this case, 6.8 ml of the previous colloid was centrifuged (3000 rpm, 10 min), washed with H 2 O (2 ), and resuspended in 7.7 ml of H 2 O. Ten consecutive additions, consisting of 8.3 µl of 1 M HCl, 42 µl of 50 mm HAuCl 4 solution, 83 µl of 3 mm AgNO 3 and 42 µl of 0.1 M AA, were carried out under vigorous magnetic stirring, in order to obtain b2au@msio nanoparticles. 200 µl aliquots were extracted, after each addition, to evaluate the reaction progress. Nanocomposites were centrifuged at 3000 rpm for 10 min, washed with H 2 O (2 ), MeCN (2 ) and re-suspended in 2.6 ml of MeCN. For the preparation of b2au@mip/cip nanoparticles, an additional etching step was included between polymerization and branching steps. For that purpose, 33.2 ml of Au@mSiO was centrifuged (3000 rpm, 10 min) and re-suspended in 40 ml of a mixture H 2 O:HF (10%, v/v) under stirring, inside a 50 ml plastic tube. After 1 h, Au@MIP/CIP particles were centrifuged at 4500 rpm for 20 min, S7

8 and washed with H 2 O (5 ). In the last step, the pellet was re-suspended in 37.5 ml of H 2 O, and the branching occurred during ten consecutive additions, under vigorous magnetic stirring, consisting of 42 µl of 1 M HCl, 208 µl of 50 mm HAuCl 4 solution, 417 µl of 3 mm AgNO 3 and 208 µl of 0.1 MAA. 180 µl aliquots were extracted, after each addition, to evaluate the reaction progress. Nanoparticles b2au@mip/cip were centrifuged at 3000 rpm for 10 min, washed with H 2 O (2 ), MeCN (2 ) and re-suspended in 12.5 ml of MeCN. SERS activity Raman spectra of pure (powder) antibiotic and a solution 0.1 mm ENRO in MeCN, in the presence of 0.5 mm gold nanostars, prepared as described elsewhere, 6 were recorded in order to determine the SERS active vibrational modes of the antibiotic. The b1au@msio b2au@msio and b2au@mip nanocomposites were incubated, at different nanoparticle and ENRO concentrations levels, according to Table S1, in order to confirm their SERS-activity. Table S1. Concentration and amount of reagents for the incubation of the nanocomposites. Experiment [Au 0 ] [ENRO] Nanocomposite ENRO MeCN HEPES (25 mm, (nm) (1) (nm) (µl) (µl) (µl) ph 7.5) (µl) (2) (3) (4) (2) (3) (4) 200 (1) All stock dispersions of nanocomposites used for this study were previously prepared to show a final Au 0 concentration of 1.5 mm in MeCN, in terms of initial gold seed. The concentration of ENRO dilutions used was: (2) 1.2 µm, (3) 120 nm and (4) 12 nm in MeCN. Each incubation was performed by triplicate. The selected amounts of gold composites and ENRO (see Table S1) were mixed to final volume of 400 µl, with MeCN:HEPES (25 mm, ph 7.5) 50:50 (v/v), following a procedure described previously. 7 After incubation (10 min or 1 h), nanocomposites were centrifuged at 4500 rpm for 5 min, washed with 0.4 ml of MeCN:HEPES (25 mm, ph 7.5) (50:50, v/v) and 0.4 ml of MeCN (2 ) and re-suspended in 0.2 ml of MeCN. A 5-µL drop was deposited S8

9 onto the surface of a quartz slide and, after total solvent evaporation, it was measured in the Raman confocal microscope, by triplicate (see Characterization section for measurement conditions). Analytical characterization of the SERS nanosensor For calibration purposes, the b1au@msio and b1au@msio nanocomposites were incubated (10 min) with ENRO at the concentrations collected in Table S2. Table S2. Incubation conditions (10 min) for the b1au@msio nanocomposites to evaluate the analytical characteristics of the SERS nanosensor. Calibration level [Au 0 ] (nm) (1) [ENRO] (nm) Nanocomposite (µl) ENRO (µl) MeCN (µl) HEPES (25 mm, ph 7.5) (µl) (2) (3) (4) (5) (6) (7) (8) (1) All stock dispersions of nanocomposites used for this study were previously prepared to show a final Au 0 concentration of 1.5 mm in MeCN, in terms of initial gold seed. The concentration of ENRO dilutions used was: (2) 15.2 nm, (3) 30.5 nm, (4) 76.2 nm, (5) nm, (6) nm, (7) nm and (8) 1525 nm in MeCN. Each incubation was performed by triplicate. S9

10 Cross-reactivity To demonstrate their selectivity, and nanocomposites were incubated with different antibiotics considered as potential interferents (IM) for ENRO quantification, such as AMPI and PENGS, as well as with the template molecule used for the fabrication of the CIP, Boc-Phe-OH. Incubation (see Table S3) and measurements in Raman confocal microscope were performed as described in the previous sections. Table S3. Incubation conditions (10 min) for the nanocomposites to evaluate the cross-reactivity of the SERS nanosensor. [Au 0 ] (nm) (1) [IM] (nm) Nanocomposite (µl) IM stock (µl) (2) MeCN (µl) HEPES (25 mm, ph 7.5) (µl) AMPI PENGS Boc-Phe-OH (1) All stock dispersions of nanocomposites used for this study were previously prepared to show a final Au 0 concentration of 1.5 mm in MeCN, in terms of initial gold seed. (2) The concentration of interferent dilutions used was 5.7 µm in MeCN. S10

11 Results and discussion TEM micrographs Figure S1. TEM micrographs of ( ݎ మ = 25 ± 4 nm): a) as-synthesized, and after functionalization with 4-CMPTCS silane, without further modifications (b), or after a thermal annealing consisting of 300 ºC for 6 h (c). Black arrow in (b) indicates a silica aggregation coming from partially destroyed shells. Mesoporosity after functionalization without thermal processing was totally lost. Scale bar: 100 nm. Figure S2. TEM micrographs of b1au@msio2 nanoparticles obtained through a direct branching of a) 0.75 mm Au@mSiO2 ([Au3+]/[Au0] = 6.6), b) 1 mm Au@mSiO2 ([Au3+]/[Au0] = 5.0), c) 2 mm Au@mSiO2 ([Au3+]/[Au0] = 2.5). Length of gold branches decreased as seed concentration increased. Scale bar: 50 nm. Figure S3. TEM micrographs of a) and b) Au@mSiO2@MIP, c) and d) Au@MIP, after silica etching of the previous nanoparticles. No mesoporous channels were observed after silica removal, resulting in gold cores embedded in a polymeric matrix. Scale bars: 100 nm (a, c) and 50 nm (b, d). S11

12 Figure S4. TEM micrographs of a) and b) c) and d) obtained through a direct branching using 0.5 mm seeds and [Au3+]/[Au0] = 6.2 and 5.8, respectively. Black arrows in (d) indicate small gold colloids (< 2 nm) integrated by atoms reduced far from the gold core. Scale bars: 100 nm (a, c) and 50 nm (b, d). Figure S5. TEM micrographs of b2au@mip nanoparticles obtained through a step-by-step branching of initial 0.5 mm Au@MIP used as seed and increasing ratios of [Au3+]/[Au0] = a) 0.6, b) 1.1, c) 1.7, d) 2.3, e) 2.9, f) 3.4, g) 4.0, h) 4.6, i) 5.2, j) 5.8. Scale bar: 50 nm. SERS activity Figure S6. Raman spectra of ENRO, pure powder (red spectra) and 0.1 mm antibiotic in the presence of 0.5 mm suspension of gold nanostars (blue spectra) using a) 633 nm and b) 785 nm lasers as excitation sources. No Raman signals were recorded for 0.1 mm antibiotic in the absence of gold nanostars. Measurement conditions: 10 s acquisition time and lasers operating at 1% of their total potency (2 mw). S12

13 Selectivity of the SERS nanosensor Figure S7. Calibration plot, in logarithm x-axis, of ( ) and b1au@msio ( ) incubated with different ENRO concentrations for 10 min and their respective linear fittings (n = 3). Cross-reactivity of the SERS nanosensor Figure S8. Chemical structures of the analytes used in the cross-reactivity study: a) enrofloxacin (ENRO), b) Boc-L-phenylalanine (Boc-Phe-OH), c) ampicillin (AMPI), d) penicillin G streptomycin (PENGS). All molecules show a carboxylic acid-carboxylate group (in red, bold), not involved in the recognition event, with a symmetric O-C-O stretching Raman mode around 1400 cm -1, used for quantification. References (1) Enustun, B. V.; Turkevich, J. Coagulation of Colloidal Gold. J. Am. Chem. Soc. 1963, 85, (2) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Determination of Size and Concentration of Gold Nanoparticles from UV Vis Spectra. Anal. Chem. 2007, 79, (3) Hendel, T.; Wuithschick, M.; Kettemann, F.; Birnbaum, A.; Rademann, K.; Polte, J. In Situ Determination of Colloidal Gold Concentrations with UV Vis Spectroscopy: Limitations and Perspectives. Anal. Chem. 2014, 86, (4) Sanz-Ortiz, M. N.; Sentosun, K.; Bals, S.; Liz-Marzán, L. M. Templated Growth of Surface Enhanced Raman Scattering-Active Branched Gold Nanoparticles within Radial Mesoporous Silica Shells. ACS Nano 2015, 9, (5) Roy, D.; Guthrie, J. T.; Perrier, S. Graft Polymerization: Grafting Poly(styrene) from Cellulose via Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerization. Macromolecules 2005, 38, (6) Senthil Kumar, P.; Pastoriza-Santos, I.; Rodriguez-Gonzalez, B.; Javier Garcia de Abajo, F.; Liz-Marzan, L. M. High-yield synthesis and optical response of gold nanostars. Nanotechnology 2008, 19, (7) Carrasco, S.; Benito-Pena, E.; Walt, D. R.; Moreno-Bondi, M. C. Fiber-optic array using molecularly imprinted microspheres for antibiotic analysis. Chem. Sci. 2015, 6, S13