Neutral Dye-Doped Silica Nanoparticles for Electrogenerated

Subscriber access provided by WEBSTER UNIV

C: Physical Processes in Nanomaterials and Nanostructures

Neutral Dye Doped Silica Nanoparticles for Electrogenerated Chemiluminescence Signal Amplification Sagar Kesarkar, Stefano Valente, Alessandra Zanut, Francesco Palomba, Andrea Fiorani, Massimo Marcaccio, Enrico Rampazzo, Giovanni Valenti, Francesco Paolucci, and Luca Prodi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b11049 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Neutral Dye Doped Silica Nanoparticles for Electrogenerated

Chemiluminescence

Signal

Amplification Sagar Kesarkar, Stefano Valente, Alessandra Zanut, Francesco Palomba, Andrea Fiorani,§ Massimo Marcaccio, Enrico Rampazzo, Giovanni Valenti, Francesco Paolucci,* Luca Prodi* Dipartimento di Chimica “Giacomo Ciamician”, Università di Bologna, Via Selmi 2, 40126 Bologna, Italy. §

Present Address: Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama

223−8522, Japan. Corresponding Author * Francesco Paolucci ([email protected]) * Luca Prodi ([email protected])

ACS Paragon Plus Environment

1

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 23

ABSTRACT Immunoassay is probably the primary in vitro technique employed in medical diagnostic. Its different setups include often the well-known and sensitive analytical method based on electrochemiluminescence (ECL), and approaches relying on nanotechnology have the potentiality to further enhance ECL signal response and detection capability. With this intent, we studied the ECL behavior of dye doped silica-PEG core-shell nanoparticles (DDSNPs) doped with neutral Ru(II) complexes in a very large doping range. We found that nanoparticles maintain comparable morphology and dimensions as the amount of Ru(II) neutral complexes which are covalently linked to their silica matrix increases, meanwhile ECL shows an enhanced response. The 9-fold ECL signal amplification obtained when compared to [Ru(bpy)3]2+ doped silica nanoparticles, is due to the synergy between the behavior of neutral Ru(II) complex and the DDSNP architecture: these results pave the way for the development of more efficient probes in ECL

technology..


2

Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction. Electrogenerated chemiluminescence (ECL) is a technique where light is emitted from excited species generated at the surface of an electrode through an electrochemical stimulus.1-4 Since the absence of excitation light source strongly increases the signal-to-noise ratio, ECL possesses a very low detection limit for label detection (up to sub-picomolar concentration) and it has more than six orders of magnitude of dynamic range.5-8 Due to its remarkable advantages, the past decade has witnessed a huge growth in the ECL research field.9-14 ECL applied to immunoassays is nowadays a leading technique in ultrasensitive bioanalysis, with huge potentialities in diagnostic nanomedicine.4, 11, 15-17 The most extensively studied ECL system is tris(2,2’-bipyridyl)ruthenium(II), [Ru(bpy)3]2+, as luminophore and tri-n-propylamine (TPrA) as co-reactant, with leading companies, such as Roche Diagnostics and MesoScale, having libraries of more than 150 bio- and immuno-assays.16 Although [Ru(bpy)3]2+ derivatives in the presence of various coreactant provide efficient ECL signal in water, in recent years, new systems based on other Ru(II) or Ir(III) complexes, or on tailored nanomaterials have been developed to push forward the instrumental detection capacity.10, 14, 18–20

In particular, dye doped silica nanoparticles (DDSNPs) present significant advantages over

other nanomaterials, such as their accessible synthesis methodology, high surface-to-volume ratio, excellent hydrophilicity, and their biocompatibility.22 The nanoparticle shell provides protection from luminescence quenching caused by oxygen and water,23 increasing the photostability of luminophores entrapped within each nanoparticle, compared to bare luminophores, and provides signal enhancement due to the large number of dyes per nanoparticle.24


3


Page 4 of 23

Since the first application reported by Santra et al.,25 [Ru(bpy)3]2+-doped silica nanoparticles have attracted increasing attention for ECL bioanalysis with several analytical applications.26 However, dye leakage prevented a clear interpretation of the ECL experiments and compromised their effective use. This issue was later addressed

21, 27

by covalent bonding of ruthenium dyes within

silica matrix, that inhibits leakage and provides doping reliability. Recently, we showed that the efficiency of ECL generation from dye doped silica NPs is not only governed by the dye/NP ratio. In fact, we demonstrated that the ECL brightness strongly depends on the surface charge properties of the nanomaterial.28 Based on these findings, here we report an attractive strategy for the ECL signal enhancement obtained by controlling the surface charge of the nanomaterial. Choosing a neutral Ru(II) heteroleptic complex as doping agent, in fact, the final charge of the nanoparticles does not depend on the doping level with a consequent 9-fold increase in the ECL efficiency. As synthetic procedure for prepare the nanoparticles, we chose direct micelles assisted methods in order to obtain i) very reproducible morphological properties, ii) large doping regime, iii) high colloidal stability in water and iv) to prevent the dye leakage.

Experimental Section Materials. All reagents and solvents were used as received without further purification. Dimethylformamide (DMF, ≥ 99.8%), Ethylene glycol (≥ 99%), Dimethyl Sulfoxide (DMSO, ≥ 99.5%), Dichloromethane (DCM, ≥ 99.8%), Acetonitrile (CH3CN, 99%), Diethyl Ether (Et2O, ≥ 99.7%), Tetraethyl orthosilicate (TEOS, 99.99%), Chlorotrimethylsilane (TMSCl, ≥ 98%), 3-


4

Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Triethoxysilyl)propyl isocyanate (TEPI, 95%), Acetic acid (AcOH, ≥ 99.7%), Hydrochloric acid (HCl, 37%), Triethyl-amine (TEA, ≥ 99.5%), N,N-Diisopropylethylamine (DIPEA 99.5%), 2(dibutylamino)ethanol (DBAE, 99%), Acetone, Hexane, Ethanol (EtOH) and Ethylene glycol (99.8%) Pluronic F127, were purchased from Sigma-Aldrich. RuCl3 •3H2O, Bathophenanthrolinedisulfonic acid disodium salt trihydrate (Na2BPS•3H2O, 98%), 2,2′-bipyridine (bpy, ≥ 99%), Lithium chloride (LiCl, ≥ 99 %), Sodium chloride (NaCl, ≥ 99.5%), Sodium sulfate (Na2SO4 ≥ 99.0%), Potassium chloride (KCl ≥ 99.0%), sodium phosphate monobasic dihydrate (NaH2PO4•2H2O ≥ 99.0%), sodium phosphate dibasic (Na2HPO4 ≥ 99.5%), were purchased from Fluka. Synthesis of Ru(bpy)(L)Cl2·2H2O Under inert nitrogen atmosphere a mixture of RuCl3·3H2O (162.52 mg, 0.622 mmol, 1 eq.), ligand L, 4-(4’-methyl-[2,2’-bipyridin]-4-yl)butan-1-amine, (150 mg, 0.622 mmol, 1 eq.), 2,2’-bipyridine (bpy) (97.07 mg, 0.622 mmol, 1 eq.) and LiCl (158.13 mg, 3.73 mmol, 6 eq.) were dissolved into DMF (∼ 3.0 mL) under magnetic stirring and then refluxed at 155 °C overnight (see Scheme S1 Step 1). The reaction was then cooled at room temperature and 4 mL of acetone was added to it. The flask was kept in the fridge to promote the precipitation. The black-brown precipitate obtained was then separated by vacuum filtration and washed with three aliquots of 5 mL of acetone, then three of 5 mL of water and finally with three aliquots of 5 mL of hexane. At the end, the clean precipitate was dried under high vacuum. The final yield calculated was 144 mg, (38.2%). Synthesis of Ru(bpy)(L)BPS Under inert nitrogen atmosphere the complex Ru(bpy)(L)Cl2•2 H2O (100 mg, 0.165 mmol, 1 eq.) and the ligand BPS bathophenanthroline disulfonic acid disodium salt trihydrate (97.44 mg, 0.165 mmol, 1 eq.) were dissolved in 3 ml of a degassed


5


Page 6 of 23

mixture of water and ethylene glycol (1:1) under magnetic stirring (see Scheme S1 Step 2). The mixture was then refluxed at 120°C overnight. The reaction was then cooled at room temperature and 4 mL of acetone was added to the solution to promote the precipitation. The brown precipitate obtained was then separated by vacuum filtration and washed with three aliquots of 5 mL of acetone and finally with three aliquots of 5 mL of water. At the end, the clean precipitate was dried under high vacuum. The final yield calculated was 120 mg, (70.5%). Synthesis of Ru(bpy)(L)BPS-TES. The complex Ru(bpy)(L)BPS (x mol%, 1 eq.) was inserted in a 4 mL glass scintillation vial and dissolved in DMF (800 μL) under magnetic stirring for 30 minutes (see Scheme S2). The amount of Ru(bpy)(L)BPS required to reach the desired doping level, was calculated based on the moles of TEOS reported in the Table S1. Triethylamine (TEA) (1.1 eq.) and 3-(Triethoxysilyl)propyl isocyanate (TEPI) (1.2 eq.) were then added into the vial and the stirring was kept overnight at 30 °C. The reaction mixture was then injected directly during the nanoparticles synthesis. Preparation of covalently doped (bpy)(L)BPS-TES core-shell silica-PEG nanoparticles. [Ru@NPs]. Pluronic F127 (100 mg) and NaCl (67 mg) were inserted in a 4 mL glass scintillation vial and dissolved in DCM (1.5 mL) under magnetic stirring for around 30 minutes (see Scheme S3). The solvent was then removed with rotavapor at room temperature and the sample dried under high vacuum. The white solid was then redispersed in 1M acetic acid solution (AcOH) (800 μL) by stirring it at 30 °C and after complete solubilization, the solution of complex Ru(bpy(L)BPSTES was injected directly in the same glass vial. The mixture was stirred for another 3 hours at 30 °C. Tetraethylorthosilicate (TEOS) (175 μL, 0.784 mmol) was then added to the resulting aqueous homogeneous solution and the day after, trimethylsilylchloride (TMSCl) (10 μL) was finally added and the mixture was kept overnight at 30 °C, always under magnetic stirring. The dialysis


6

Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


purification steps were carried out versus water under gentle stirring with regenerated cellulose dialysis tubing (Sigma, mol wt. cutoff > 12 kDa, avg. diameter 33 mm). The whole amount of Ru@NPs solution (1.6 mL) was inserted in the tube and after the dialysis diluted to a total volume of 20 mL with water. The final concentration of the Ru@NPs solution was measured considering the volume after dialysis. The solution was first centrifugated for 10 minutes at 8000 rpm, and then the supernatant solution was filtered first with a 0.45 μm RC, then with 0.20 μm RC filters. The exact amount of reagents used for each sample preparation is shown in Table S1 below Photochemical measurements. Quartz cuvettes were used for both absorbance and emission measurements (optical path length of 1 cm). UV−Vis absorption spectra were recorded at 25 °C by means of PerkinElmer Lambda 45 spectrophotometer. The fluorescence spectra were recorded with a PerkinElmer Lambda LS 55 fluorimeter and with a modular UV−Vis−NIR spectrofluorimeter Edinburgh Instruments FLS920 equipped with a photomultiplier Hamamatsu R928P. The latter instrument connected to a PCS900 PC card was used for the time correlated single photon counting (TCSPC) experiments (excitation laser λ = 410 nm). Corrected fluorescence emission and excitation spectra (450 W Xe lamp) were obtained with the same instrument equipped with both a Hamamatsu R928P photomultiplier tube (for the 500−850 nm spectral range). Nanoparticle solutions were diluted with milli-Q water. Luminescence quantum yields (uncertainty ±15%) were recorded on air-equilibrated solutions using Ru(bpy)32+ as reference dye. (QY = 0.018 in aerated acetonitrile solution). When necessary, deoxygenated samples were prepared by flowing N2 through the solutions housed in a customized airtight quartz cuvette equipped with a closure cap. Trasmission Electron Microscopy (TEM) images and Dynamic Light Scattering (DLS). TEM images of DDSNPs were obtained with a Philips CM 100 microscope, operating at 80 kV,


7


Page 8 of 23

and using 3.05 mm copper grids (Formvar support film, 400 mesh). Two aliquots of 2 μL of nanoparticles solution first diluted with milli-Q water (1:50) were dropped on the grid, waiting for the solvent to evaporate before the second deposition. The grid was then dried under vacuum. The TEM images showing the denser silica cores were analysed with ImageJ software, considering around a few hundred nanoparticles. The obtained histogram was fitted according to Gaussian distribution, obtaining the average diameter for the silica nanoparticles core completed with calculated standard deviation and PDI. Hydrodynamic diameter (dH) distributions of nanoparticles were obtained in water at 25 °C with a Malvern Nano ZS DLS instrument equipped with a 633 nm laser diode. Samples were first treated with 0.45 μm and 0.20 μm RC filters and then housed in disposable polystyrene cuvettes of 1 cm optical path length. The width of DLS hydrodynamic diameter distribution is indicated by PDI (Polydispersion Index). In the case of monomodal distribution (Gaussian), calculated by means of cumulated analysis, PDI = (σ/Zavg)2, where σ is the width of the distribution and Zavg is the average diameter of the particles population, respectively. ζ-Potential experiments. ζ-Potential values of nanoparticles were determined using a Malvern Nano ZS instrument. Samples were housed in disposable polycarbonate folded capillary cell (DTS1070, 750 μL, 4 mm optical path length). Electrophoretic determination of ζ-Potential was made under Smoluchowski approximation in aqueous media at moderate electrolyte concentration. Measurements conditions: ζ-Potential ± SD (n = 4), [NPs] = 2 μM, [PB] = 1 mM, [KCl] = 1 mM, pH = 7.4, T = 25 °C, samples filtered with a 0.20 μm RC syringe filter before analysis. Electrochemical and ECL measurements. ECL and electrochemical measurements were carried out with an AUTOLAB electrochemical station (Ecochemie, Mod. PGSTAT 30). Nanoparticles suspension was diluted with a phosphate buffer (PB, pH = 7.4). For ECL generation,


8

Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30 mM 2-(dibutylamino)ethanol (DBAE) was added as oxidative coreactant. ECL was obtained in single oxidative steps (sweep steps) by generating the oxidized forms of the amine according to known heterogeneous ECL mechanism. The working electrode consisted of a platinum sideoriented 2 mm diameter disk sealed in glass, while the counter electrode was a platinum spiral and the reference electrode was an Ag/AgCl (3M) electrode. The ECL signal generated by performing the potential step program was measure with a photomultiplier tube (PMT, Hamamatsu 4220p) placed, at a constant distance, in front of the working electrode and inside a homemade dark box. A voltage in the range 750 V was supplied to the PMT. The light/current/voltage curves were recorded by collecting the pre-amplified PMT output signal (by an ultralow noise Acton research model 181) with the second input channel of the ADC module of the AUTOLAB instrument.

Results and Discussion. We synthesized a new family of DDSNPs samples using Ru(BPS)(L)(bpy)-TES (BPS: bathophenanthroline disulfonate, TES: triethoxysilane) derivative that was covalently linked to the NPs silica core (Figure 1). We used photophysical, electrochemical and ECL characterization to evaluate the influence of different dye doping levels on ECL efficiency in the presence of 2(dibutylamino)ethanol (DBAE) as oxidative-reduction coreactant. We also considered the influence of NPs ζ-potential on ECL emission properties.29


9


Page 10 of 23

Figure 1. Overall structures and molecular components of the DDSNPs of this work. (A) Surfactant Pluronic F127 and neutral Ru(II) complex molecular structure, (B) Sketch of DDSNP, (C) TEM micrograph of DDSNP and diameter distribution. The neutral complex Ru(BPS)(L)(bpy), was synthesized using a two-step process (see Scheme S1 in the Supporting Information) and its triethoxysilane derivative was synthesized as shown in Scheme S2. The ligand (L), 4-(4’-methyl-2,2’-bipyridin-4-yl)-butan-1-amine, was synthesized according to previously reported procedures.30 The DDSNPs were obtained with a direct micelle assisted method31, 28 with slight modifications in the synthetic procedure (see Scheme S3). We prepared a set of six DDSNPs samples named Ru@NP3, Ru@NP8, Ru@NP11, Ru@NP12, Ru@NP19, Ru@NP21, where the values from 3 to 21 correspond to the average number of dyes within each NP. These samples were synthesized by increasing the amount of Ru(BPS)(L)(bpy)TES introduced during the synthetic process from 0.05% to 2.4% (mol Ru(BPS)(L)(bpy)/mol TEOS x100, see Table S1). The dye inclusion for every sample of Ru@NPs was estimated by UV-Visible spectra, by measuring the concentration of Ru(bpy)(L)BPS complex presents in the dispersion, then dividing it by the concentration of NPs, determined as previously reported.32 The absorption and emission


10

Page 11 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spectra reported in Figure 2 show that the shape and the position of the peaks of the Ru(bpy)(L)BPS@NPs samples are very similar to each other, as well as for Ru(bpy)(L)BPS in DMSO solution (orange line). This is attributed to similar environments and ground state conditions that do not modify the absorption properties, revealing an average from 3 to 21 ruthenium complexes per NP, with the maximum at 2.4% doping level.

Figure 2. Absorption and emission spectra in water at different doping levels of Ru@NP3, black; Ru@NP8, red; Ru@NP11, green; Ru@NP12, blue; Ru@NP19, cyan; Ru@NP21, magenta. Comparison with reference dyes, Ru(bpy)(L)BPS in DMSO (absorption spectra, orange line) and [Ru(bpy)3]2+ in ACN (emission spectra, violet line). The emission spectra for the Ru@NPs were recorded in aerated water (Figure 2) as well as in deaerated (Figure S2) conditions, in order to consider the protection effect of the silica matrix toward the dyes inside the nanoparticle. The trends of the quantum yield and of average excited state lifetimes (Table S2, Figure S3) in aerated and deaerated water showed noticeable influence of dye inclusion in the silica matrix while negligible changes were observed as function of the doping level (Figure 3).


11


Page 12 of 23

Figure 3. Average excited state lifetimes (blue, aerated; green, deaerated) and luminescence quantum yields (black, aerated; red, deaerated) values for Ru@NPs samples in water. (λex = 410 nm). The morphological properties of the DDSNPs were studied by TEM and DLS techniques (reported in Table S3), revealed a silica core diameter (dcore) of ∼10 nm and a hydrodynamic diameter (dH)

owing to PEG shell of ∼25 nm, respectively (see also Figures S4). Despite the different doping

degrees, the Ru@NPs samples showed very close core diameters allowing the storage of many ruthenium dyes. Our previous study evidenced how the increase in the doping level of positively charged [Ru(bpy)3]2+ triethoxysilane derivative in DDSNPs was able to mitigate the silica negative charge and finally bearing slightly positive ζ-potential values at high doping regimes (Figure S5).28 During the ECL process, this impaired the activation of emission events, since the approach of the positively charged and active form of DBAE coreactant toward the NP surface was unfavorable. For this reason, we choose a neutral complex to minimize this effect. As a consequence, the ζpotential of the DDSNPs doped with the neutral complex Ru(bpy)(L)BPS, was only slightly influenced, even at very high doping levels (Figure 4 and Table S3).


12

Page 13 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. ζ-potential of Ru@NPs vs doping percentage. Conditions: [Ru@NPs] = 2μM, [KCl] = 1mM; [phosphate buffer] = 1mM, pH = 7.0, T = 25°C (all samples were filtered with a 0.2 μm RC syringe filter). Since we obtained Ru@NPs where the ζ-potential is almost independent from the doping level, we investigated ECL properties of this set of six DDSNPs samples. ECL experiments were performed in phosphate buffer (PB, pH = 7.4) using DBAE (30 mM) as a sacrificial coreactant and the ECL intensities were compared with ECL behavior of the [Ru(bpy)3]2+ doped NPs. The current-potential-ECL intensity profiles are shown in Figure S6. The CV curves are dominated by the coreactant oxidation with a peak at 0.8 V. The potential-ECL profile are consistent with “oxidative-reduction” coreactant mechanism (see Figure 5a). As expected, the ECL from Ru@NP21 shows a single maximum intensity associated with the coreactant oxidation process (Scheme 1, eq.1-4 and 7) and a weak ECL signal in the region of the direct dye oxidation (Scheme 1, eq.1, 2, 5-7). 33, 34, 35 Scheme 1. Mechanism for the “Oxidative-Reduction” Coreactant ECL generation. P1 is the product of the homogeneous DBAE● oxidation. DBAE  DBAE●+ + e−

(eq.1)


13


DBAE●+  DBAE● + H+

(eq.2)

DBAE● + Ru@NP  P1 + Ru−@NP

(eq.3)

DBAE●+ + Ru−@NP  P1 + Ru@NP*

(eq.4)

Ru@NP  Ru+@NP + e−

(eq.5)

DBAE● + Ru+@NP  P1 + Ru@NP*

(eq.6)

Ru@NP*  Ru@NP + hν

(eq.7)

Page 14 of 23

It is quite interesting to note that for the NPs doped with neutral complexes, the ECL mechanism remains unaffected even at increased doping level. In fact, Ru@NP21, shows the same ECLpotential profile compared with the Ru@NP11, having a lower doping level with both the ECL mechanism active (see comparison of black and red signal in Figure 5a).

Figure 5. Normalized ECL intensity vs potential of DDSNPs doped with (a) neutral complex Ru@NP11 (black), Ru@NP21 (red) and (b) with charged [Ru(bpy)3]2+ RuBPY@NP4 (black),


14

Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RuBPY@NP24 (red). Blue dashed line is guide to the eye for two mechanisms. Set up conditions: DBAE = 30 mM, PB = 100 mM (pH = 7.4), scan rate = 0.1 V·s−1; PMT bias = 750 V; voltage scan between 0 V and +2.0 V vs. SCE reference electrode. On the other hand, the switching in the mechanism was observed at higher doping level in the case of the charged [Ru(bpy)3]2+ derivate (RuBPY@NPs Figure 5b). In the RuBPY@NPs case, the pathway involving the direct oxidation of the dye (Scheme 1, eq.1, 2, 5-7) is the dominant even at high doping level (RuBPY@NP24) and this feature is strictly connected with the change in the ζpotential among the two different families of NPs. As matter of fact, if we compare the overall ECL intensity, at the same doping level, of Ru@NP with the ECL intensity recorded for NPs doped with charged [Ru(bpy)3]2+ complexes (RuBPY@NP-24 in Figure 6 with 24 [Ru(bpy)3]2+ complexes), DDSNPs doped with neutral complexes showed a remarkable increase in the ECL signal (∼9-fold) (Figure 6). This overall increase in the ECL intensity in the case of nanoparticles doped with neutral complex (Ru@NP) is connected with the nanoparticles surface charge and the accessibility of DBAE radical cations.


15


Page 16 of 23

Figure 6. Integrated ECL emission of Ru@NPs showing increase in the output signal vs number of dyes per NP (red) and comparison with charged [Ru(bpy)3]2+ complexes doped NPs, RuBPY@NPs24 (black). Instrumental conditions: DBAE = 30 mM, PB = 100 mM (pH = 7.4), scan rate = 0.1 V·s-1; PMT bias = 750 V; voltage scan between 0 V and +2.0 V vs. SCE reference electrode. Conclusions. In conclusion, doping silica core PEGylated NPs with neutral ruthenium complexes, instead of charged ones, results in an order-of-magnitude increase of the ECL emission intensity. Such a remarkable result was obtained through the engineering of the DDSNPs surface charges so as to favour the interaction of the positively charged coreactant intermediates with the embedded luminophores. Although the direct analytical application of this family of dye doped nanoparticles is still under investigation in our laboratories, our work highlights important strategic choices in the DDSNP approach to obtain ultra-bright labels for highly sensitive bioanalysis.

AUTHOR INFORMATION Sagar Kesarkar: 0000-0001-7731-0267 Stefano Valente: 0000-0002-1432-8985 Alessandra Zanut: 0000-0001-8917-9825 Francesco Palomba: 0000-0002-2249-2961 Andrea Fiorani: 0000-0001-8413-6439 Massimo Marcaccio: 0000-0001-9032-0742


16

Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Enrico Rampazzo: 0000-0003-2290-859X Giovanni Valenti: 0000-0002-6223-2072 Francesco Paolucci: 0000-0003-4614-8740 Luca Prodi: 0000-0002-1630-8291

Notes The authors declare no competing financial interests. Supporting Information Synthesis details, Photophysical characterization, TEM and DLS analysis and ECL-CV propriety of Ru@NP3-21. ζ-Potential of RuBPY@NPs vs. doping percentage [Figure S1-S6]. The Supporting Information is available free of charge on the ACS Publications website.

ACKNOWLEDGMENT We thank the University of Bologna, Italian Ministero dell’Istruzione, Universita e Ricerca (MIUR-project PRIN 2010) and FARB, Fondazione Cassa di Risparmio in Bologna.

REFERENCES (1) Bard, J. A. (Ed.) Electrogenerated Chemiluminescence. Marcel Dekker, New York, 2004.


17


Page 18 of 23

(2) Richter, M. M. Electrochemiluminescence (ECL). Chem. Rev. 2004, 104, 3003- 3036. (3) Valenti, G.; Fiorani, A.; Li, H.; Sojic, N.; Paolucci, F. Essential role of electrode materials in electrochemiluminescence applications. ChemElectroChem 2016, 3, 1990- 1997. (4) Sojic, N.; Bouf, L.; Kuhn, A. Luminescence in electrochemistry applications in analytical chemistry, physics and biology; 2017. (5) Miao, W. Electrogenerated chemiluminescence and its biorelated applications. Chem. Rev. 2008, 2506- 2553. (6) Forster, J. R.; Bertoncello, P.; Keyes E. T. Electrogenerated chemiluminescence. Annu. Rev. Anal. Chem. 2009, 2, 359- 385. (7) Liu, Z.; Qi, W.; Xu, G. Recent Advances in Electrochemiluminescence. Chem. Soc. Rev. 2015, 44, 3117- 3142. (8) Hesari, M.; Ding, Z. Review- Electrogenerated chemiluminescence: Light years ahead. J. Electrochem. Soc. 2016, 163, H3116- H3131. (9) Zhuo, Y.; Wang, H. J.; Lei, Y. M.; Zhang, P.; Liu, J. L.; Chai, Y. Q.; Yuan, R. Electrochemiluminescence biosensing based on different modes of switching signals. Analyst 2018, 143, 3230- 3248. (10) Bertoncello, P.; Stewart, A. J.; Dennany, L. Analytical applications of nanomaterials in electrogenerated chemiluminescence. Anal. Bioanal. Chem. 2014, 406, 5573- 5587.


18

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(11) Ge, L.; Yu, J.; Ge, S.; Yan, M. Lab-on-Paper-based devices using chemiluminescence and electrogenerated chemiluminescence detection. Analytical and Bioanalytical Chemistry 2014, 406, 5613- 5630. (12) Li, L.; Chen, Y.; Zhu, J. J. Recent advances in electrochemiluminescence analysis. Anal. Chem. 2017, 89, 358- 371. (13) Bertoncello, P.; Ugo, P. Recent advances in electrochemiluminescence with quantum dots and arrays of nanoelectrodes. ChemElectroChem 2017, 4, 1663- 1676. (14) Sun, J.; Sun, H.; Liang, Z. Nanomaterials in electrochemiluminescence sensors. ChemElectroChem 2017, 4, 1651- 1662. (15) Valenti, G.; Rampazzo, E.; Kesarkar, S.; Genovese, D.; Fiorani, A.; Zanut, A.; Palomba, F.; Marcaccio, M.; Paolucci, F.; Prodi, L. Electrogenerated chemiluminescence from metal complexes-based nanoparticles for highly sensitive sensors applications. Coord. Chem. Rev. 2018, 367, 65- 81. (16) Parveen, S.; Aslam, M. S.; Hu, L.; Xu, G. Electrogenerated chemiluminescence, protocols and applications; SpringerBriefs in Molecular Science; Springer Berlin Heidelberg: Berlin, Heidelberg, 2013. (17) Juzgado, A.; Soldà, A.; Ostric, A.; Criado, A.; Valenti, G.; Rapino, S.; Conti, G.; Fracasso, G.; Paolucci, F.; Prato, M. Highly sensitive electrochemiluminescence detection of a prostate cancer biomarker. J. Mater. Chem. B 2017, 5, 6681- 6687. (18) Kerr, E.; Doeven, E. H.; Barbante, G. J.; Connell, T. U.; Donnelly, P. S.; Wilson, D. J. D.; Ashton, T. D.; Pfeffer, F. M.; Francis, P. S. Blue Electrogenerated chemiluminescence from


19


Page 20 of 23

water-soluble iridium complexes containing sulfonated phenylpyridine or tetraethylene glycol derivatized triazolylpyridine ligands. Chem. Eur. J. 2015, 21, 14987- 14995. (19) Zhao, Y.; Yang, X.; Han, D.; Qi, H.; Gao, Q.; Zhang, C. Cyclometalated iridium-complexbased label-free supersandwich electrogenerated chemiluminescence biosensor for the detection of micro-RNA. ChemElectroChem 2017, 4, 1775- 1782. (20) Kerr, E.; Doeven, E. H.; Barbante, G. J.; Hogan, C. F.; Hayne, D. J.; Donnelly, P. S.; Francis, P. S. New perspectives on the annihilation electrogenerated chemiluminescence of mixed metal complexes in solution. Chem. Sci. 2016, 7, 5271- 5279. (21) Valenti G.; Rampazzo E.; Bonacchi S.; Khajvand T.; Juris R.; Montalti M.; Marcaccio M.; Paolucci F.; Prodi L. A versatile strategy for tuning the color of electrochemiluminescence using silica nanoparticles. Chem. Commun. 2012, 48, 4187- 4189. (22) Zhai,

Q.;

Li, J.; Wang, E.

Recent

advances

based

on

nanomaterials

as

electrochemiluminescence probes for the fabrication of sensors. ChemElectroChem 2017, 4, 1639- 1650. (23) Zanarini, S.; Rampazzo, E.; Della Ciana, L.; Marcaccio, M.; Marzocchi, E.; Montalti, M.; Paolucci, F.; Prodi, L. Ru(bpy)3 Covalently doped silica nanoparticles as multicenter tunable structures for electrochemiluminescence amplification. J. Am. Chem. Soc. 2009, 131, 22602267. (24) Bagwe, R. P.; Yang, C.; Hilliard, L. R.; Tan, W. Optimization of Dye-doped silica nanoparticles prepared using a reverse microemulsion method. Langmuir 2004, 20, 83368342.


20

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(25) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal. Chem. 2001, 73, 4988- 4993. (26) Chen, M. M.; Wang, Y.; Cheng, S. B.; Wen, W.; Zhang, X.; Wang, S.; Huang, W. H. Construction of highly efficient resonance energy transfer platform inside a nanosphere for ultrasensitive electrochemiluminescence detection. Anal. Chem. 2018, 90, 5075- 5081. (27) Li, M.; Chen, Z.; Yam, V. W.; Zu, Y. Multifunctional ruthenium(II) polypyridine complexbased core–shell magnetic silica nanocomposites: magnetism, luminescence, and electrochemiluminescence. ACS Nano 2008, 2, 905- 912. (28) Valenti, G.; Rampazzo, E.; Bonacchi, S.; Petrizza, L.; Marcaccio, M.; Montalti, M.; Prodi, L.; Paolucci, F. Variable doping induces mechanism swapping in electrogenerated chemiluminescence of Ru(bpy)32+ core–shell silica nanoparticles. J. Am. Chem. Soc. 2016, 138, 15935- 15942. (29) Liu, X.; Shi, L.; Niu, W.; Li, H.; Xu, G. Environmentally friendly and highly sensitive ruthenium(II)

tris(2,2'-bipyridyl)

electrochemiluminescent

system

using

2-

(dibutylamino)ethanol as co-reactant. Angew. Chem. Int. Ed. 2007, 46, 421- 424. (30) Della Ciana, L.; Zanarini, S.; Perciaccante, R.; Marzocchi, E.; Valenti, G. Neutral and dianionic Ru(II) bathophenanthrolinedisulfonate complexes: A route to enhance electrochemiluminescence performance in aqueous media. J. Phys. Chem. C 2010, 114, 36533658.


21


Page 22 of 23

(31) Huo, Q.; Liu, J.; Wang, L.; Jiang, Y.; Lambert, T. N.; Fang, E. A new class of silica crosslinked micellar core-shell nanoparticles. J. Am. Chem. Soc. 2006, 128, 6447- 6453. (32) Kesarkar, S.; Rampazzo, E.; Valenti, G.; Marcaccio, M.; Bossi, A.; Prodi, L.; Paolucci, F. Iridium(III)‐doped

core-shell

silica

nanoparticles:

Near-IR

electrogenerated

chemiluminescence in water. ChemElectroChem 2017, 4, 1690- 1696. (33) Miao, W.; Choi, J. P.; Bard, A. J. Electrogenerated chemiluminescence 69: The Tris(2,2‘bipyridine)ruthenium(II), (Ru(bpy)32+)/Tri-n-propylamine (TPrA) system revisited- A new route involving TPrA•+ cation radicals. J. Am. Chem. Soc. 2002, 124, 14478-14485. (34) Imai, K.; Valenti, G.; Villani, E.; Rapino, S.; Rampazzo, E.; Marcaccio, M.; Prodi, L.; Paolucci, F. Numerical simulation of doped silica nanoparticle electrochemiluminescence. J. Phys. Chem. C 2015, 119, 26111- 26118. (35) Daviddi, E.; Oleinick, A.; Svir, I.; Valenti, G.; Paolucci, F.; Amatore, C. Theory and simulation for optimising electrogenerated chemiluminescence from Tris(2,2’-bipyridine)ruthenium(II)-doped silica nanoparticles as tripropylamine. ChemElectroChem 2017, 4, 17191730.


22

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC GRAPHICS


23

Neutral Dye-Doped Silica Nanoparticles for Electrogenerated

Recommend Documents