A SERS-based pH sensor utilizing 3-amino-5-mercapto-1,2,4-triazole functionalized Ag nanoparticles.

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A SERS-based pH sensor utilizing 3-amino5-mercapto-1,2,4-triazole functionalized Ag nanoparticles† * and Jolanta Bukowska Piotr Piotrowski, Beata Wrzosek, Agata Krolikowska ´ We report the first use of 3-amino-5-mercapto-1,2,4-triazole (AMT) to construct a surface-enhanced Raman scattering (SERS) based pH nano- and microsensor, utilizing silver nanoparticles. We optimize the procedure of homogenous attachment of colloidal silver to micrometer-sized silica beads via an aminosilane linker. Such micro-carriers are potential optically trappable SERS microprobes. It is demonstrated that the SERS spectrum of AMT is strongly dependent on the pH of the surroundings, as the transformation between two different adsorption modes, upright (A form) and lying flat (B form) orientation, is provoked by pH variation. The possibility of tuning the nanosensor working range by changing the concentration of AMT in the surrounding solution is demonstrated. A strong correlation between the pH response of the nanosensor and the AMT concentration in solution is found to be

Received 17th June 2013 Accepted 3rd December 2013

controlled by the interactions between the surface and solution molecules. In the absence of the AMT monomer, the performance of both the nano- and microsensor is shifted substantially to the strongly

DOI: 10.1039/c3an01197f www.rsc.org/analyst

acidic pH range, from 1.5 to 2.5 and from 1.0 to 2.0, respectively, which is quite unique even for SERSbased sensors.

Introduction Gold and silver nanoparticles functionalized with various molecules have received special attention in recent years due to their possibility of acting as very sensitive sensors or biosensors in chemical/biochemical analysis. Among various optical nanosensors, these nanoparticles exploiting huge enhancement of Raman scattering (surface-enhanced Raman scattering – SERS), which is a result of surface plasmon resonance generated by the metal nanostructures, exhibit unique sensitivity. Gold and silver nanoparticles of various shapes and sizes such as colloidal particles,1–3 nanoshells,4 nanocubes,5 nanorods6,7 or nanoparticle assemblies8,9 have been used to enhance Raman signals in SERS-based sensors. The pH sensing capability has important implications in biology and medicine. SERS sensors can indicate the pH of the environment either using routinely the intensity change10 or less frequently the shi of the maximum of the Raman band upon variation of pH as the pHsensing index.11 Another interesting approach is pH-dependent aggregation of metal nanoparticles, resulting in visualization of the morphology of the cells with SERS signals and local photothermal cancer therapy.12 An attractive application of SERS nanosensors is probing and imaging of pH in living cells.13 To

Department of Chemistry, University of Warsaw, Pasteur Street 1, 02-093 Warsaw, Poland. E-mail: [email protected]; Fax: +48 22 822 59 96 † Electronic supplementary 10.1039/c3an01197f

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date, two compounds were used exclusively as pH-dependent nanoprobes in living cells: one containing a pH sensitive carboxyl group (4-mercaptobenzoic acid – pMBA)14,15 and the second one containing an amino group (2-aminothiophenol – 2ATP or 4-aminothiophenol – 4-ATP).16 Good SERS-based sensors must exhibit a strong response to pH changes and be capable of measuring pH in their local vicinity continuously over a wide range of pH values. Moreover, in the case of intracellular sensors they must retain their chemical functionality inside living cells. It has been demonstrated that a SERS sensor based on pMBA as a reporter molecule is responsive over a pH range of 6–8 in the case of silver nanoparticle clusters2 or 4–9 in the case of hollow gold nanospheres.17 Measurements over a wider pH range are enabled by using a surface-enhanced hyper-Raman scattering (SEHRS) sensor based on silver nanocolloids.14 Amino-derivatives of thiophenol (2-ATP and 4-ATP) exhibit response to pH over the range of 3–6.16,18 Nanoparticles of metal colloids, which are very efficient SERS supports, do not ensure homogeneous spatial distribution inside a cell, because they undergo aggregation processes. Another obstacle to application of nanometer-sized metallic particles is related to issues such as optical trapping and the possibility of scanning the SERS probes with nanometric accuracy, for example inside the living cells.19,20 These problems may be overcome by using micrometer-sized dielectric beads covered with metal nanoparticles, which have been shown to be effective in detecting emodin inside a cell membrane.19 In this case the gold-coated beads were optically trapped and the SERS

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spectra for the cell membrane in contact with the metallized microprobe were recorded.19 Local pH inside individual live glioma cancer cells was also monitored with the aid of micrometer-sized silica beads covered with silver nanoparticles, coated with pMBA as a Raman reporter.21 In this paper, we report the development of a system for pH sensing, based on silver nanoparticles modied with 3-amino-5mercapto-1,2,4-triazole (AMT) as SERS reporter molecules, which offers some improvement over the state of the art. As shown in our previous paper, AMT adsorbs primarily on Ag and Au surfaces by forming a thiolate bond with the metal.22 However, two molecular forms of AMT on Ag and Au electrodes were identied, dependent both on the concentration of AMT in the solution and on pH values. In the presence of a sufficient amount of the adsorbate molecules in the vicinity of the metal surface, AMT adsorbs through the metal–sulfur bond, in more or less perpendicular orientation to the metal surface (A form). However, in the case of decient concentration of the adsorbate at the surface, the AMT molecules interact with the metal also through the nitrogen atoms of the deprotonated triazole ring, lying almost at on the metal surface (B form). The relative amount of the respective structure depends critically on both the concentration of the adsorbate in the solution and the pH value of the surroundings.

Experimental section Instrumentation Raman measurements were carried out with a LabRAM HR800 (Horiba Jobin Ivon) Raman spectrometer coupled to an Olympus BX61 confocal microscope. The spectrometer was equipped with a charge-coupled device detector cooled by Peltier modulus. The confocal pinhole size was set to 200 mm and the holographic grating with 600 grooves per mm was used. All Raman spectra were excited with 532 nm radiation: the second harmonic of an optically pumped Nd:YAG laser. Backscattered light was collected through a 10 (in SERS measurements for pH nanosensors) or 100 (in SERS measurements for pH microsensors) objective. The maximum light beam power was about 100 mW at the head of the laser, while the lter lowering the laser intensity by 1000 was used during SERS microsensor measurements. The system was calibrated using the 520 cm1 silicon band. Scanning electrode microscopy (SEM) measurements were carried out in order to image the SERS microsensor. SEM images were obtained with a Merlin eld emission scanning electron microscope (Carl Zeiss Germany) with a LaB6 cathode. The images were collected with a secondary electron detector (In-Lens SE). An electron accelerating bias of 3 kV was used. Materials Hydroxylamine hydrochloride (99.9%), (3-aminopropyl)trimethoxysilane (97%), (3-aminopropyl)triethoxysilane ($98%) and 3-amino-1,2,4-triazole-5-thiol (95%) were purchased from Aldrich. Silver nitrate, sodium hydroxide, hydrochloric acid, trisodium phosphate, disodium hydrogen phosphate, sodium

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dihydrogen phosphate and phosphoric acid were supplied by POCH. All inorganic chemicals were of analytical reagent grade. Aqueous solutions (5%) of SiO2 and SiO2–NH2 functionalized microspheres with a diameter of 0.462 mm and 2.12 mm (further denoted as 0.5 and 2 mm for simplicity) were purchased from MicroParticles GmbH. All reagents were used without further purication. Ultra-pure water (18 MU cm1) was used to prepare all solutions. The concentration of the phosphate buffer solutions was 50 mM.

Preparation procedures Silver colloid was prepared according to the procedure by Leopold and Lendl.23 The UV-Vis spectrum of the colloid exhibited maximum at 406 nm. Based on the SEM images, the average nanoparticle diameter was determined to be around 18 nm. Samples to investigate the pH dependence of SERS nanosensors were prepared as follows: the buffer solution and silver colloid were mixed in a 1 : 10 ratio; the respective volumes being 200 ml (buffer) and 2 ml (sol). Subsequently, this solution was mixed with 102 M solution of 3-amino-5-mercapto-1,2,4triazole (AMT) in a 10 : 1 volume ratio. The nal sample was placed on a watch glass and the SERS spectra were collected by focusing an exciting laser beam through a 10 objective. In order to measure the pH dependent SERS response of the nanosensor without excessive AMT molecules, the Ag nanoparticle suspension and AMT solution were mixed in a 10 : 1 ratio, reaching a nal AMT concentration of 103 M in solution. Next, the mixture was centrifuged to eliminate the nonadsorbed AMT molecules. The gray-greenish precipitate was separated from the supernatant and re-dispersed in the volume of water equal to the initial. Samples of such prepared colloid were taken and buffer was added in a next step, while the other conditions (buffer to sol mixing ratio and the method of spectrum collection) were maintained as for the experiment in the presence of AMT in solution. Both 0.5 mm and 2 mm silica beads were utilized to construct pH microsensors. The surface of the beads was functionalized by mixing the commercial suspension of SiO2 spheres (original 5% suspension diluted 40 times) with 0.1% solution of (3-aminopropyl)trimethoxysilane (APTMS) for 10 minutes.19 Centrifugation of unattached silane molecules was performed twice for 20 minutes to prevent later agglomeration of silver particles. Amino-silanized microbeads and the silver colloid were mixed in a 1 : 1000 ratio (9 ml of the microbead suspension and 9 ml of the silver sol) and stirred for 15 minutes. Next, the suspension of the microprobes was centrifuged to remove the unattached Ag particles. To obtain a pH microsensor, AMT molecules were attached to the surface of the silver nanoparticles, which coated SiO2 microbeads. It was achieved by mixing 102 M AMT solution with a suspension of the silica microprobes in a 1 : 10 ratio (20 ml of AMT solution and 200 ml of microprobe suspension). Amino-silanization of the glass surface was performed by immersing a HF-cleaned glass plate in a 0.5% aqueous solution of (3-aminopropyl)triethoxysilane (APTES) under constant stirring for 10 minutes. Unattached silane molecules were removed


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Paper Table 1

Analyst Parameters derived from the fit of the pH response to the Boltzmann function for the studied sensors

AMT concentration in the surrounding solution Microsensor


Nanosensor

A B C S

0.91 103 M

0.91 105 M

0.91 107 M

None

None

0 (arbitrary) 1.776 0.090 9.932 0.226 1.004 0.171

0.109 0.038 1.956 0.190 6.908 0.180 0.699 0.126

0.121 0.900 4.065 0.861

0.463 0.031 2.388 0.072 1.942 0.026 0.187 0.020

0.128 0.048 2.411 0.348 1.600 0.081 0.210 0.052

by rinsing the glass in ultra-pure water. Such functionalized plates acted as a substrate for immobilization of the pH microsensor. 200 ml of the microsensor suspension was dropped on a silanized plate and le to dry out, then rinsed with ultra-pure water to remove non-reporter AMT molecules. The pH-dependent SERS experiment was performed by dropping 200 ml of HCl solution of a respective pH on a plate with immobilized sensors and the measurement was carried out just aerwards. Data treatment The SERS intensities calculated as the band heights of the two overlapping peaks around 1330 and 1360 cm1, tted rst with the mixed Gaussian–Lorentzian curves, were used to plot the pH response curves for the nano- and microsensor. Experimental points constituting the pH response of the nano- and microsensors were next tted with the sigmoid Boltzmann function, given by the following equation: yðxÞ ¼ B þ

0.017 0.078 0.273 0.191

chemical states in each form of AMT result in different molecular interactions between the molecule and the metal substrate. Non-deprotonated molecules (A form) form a monolayer tilted with respect to the surface, stabilized by hydrogen bonding between amino groups (of neighboring AMT molecules and/or between adsorbed AMT and the molecules in the solution) and p–p stacking of the heterocyclic rings. The chemical structure and geometry typical for this form are favored at low pH values and higher surface coverage of AMT molecules, i.e. also by the presence of the excess AMT molecules in solution, which were not attached to the surface. The ring–metal interactions are facilitated in the case of the deprotonated B form, which results in more parallel orientation of the AMT molecules with respect to the metal surface. Preferential adsorption of AMT molecules in the B form on silver was observed for the basic pH and sub-monolayer surface coverage.22

AB Cx S

1þe

A and B parameters denote the limiting values (top and bottom) of the Boltzmann curve: A is the y value for x / +N, while B is for x / N. Parameter C corresponds to the pH value at which the response is halfway between the maximum and minimum values. Parameter S describes the slope of the curve, with a larger value denoting a shallow curve. The tting procedure provided the following values of the Boltzmann function parameters, listed in Table 1.

Results and discussion 1. SERS features of AMT adsorption modes There are two possible binding sites of the AMT to the silver substrate: the mercapto group and the nitrogen atoms (of both the substituent group and the heterocyclic ring). SERS spectra distinctive for two different adsorption modes of AMT on colloidal Ag (reduced with hydroxylamine) are presented in Fig. 1. The top spectrum, further referred to as A form, is attributed to the non-deprotonated AMT molecules (with NH2 groups), while the bottom spectrum corresponds to the deprotonated form (with imine H–N]C moiety), denoted as B form. The above described structures have been conrmed by XPS and SERS measurements in our previous studies.22 Various


Fig. 1 SERS spectra (532 nm excitation) representative for the two various adsorption modes of AMT molecules on a silver surface: A form (top spectrum) and B form (bottom spectrum). Inset: schematic representations of the two binding modes ascribed to A form (nondeprotonated, perpendicular orientation) and B form (deprotonated, lying flat). AMT molecules in the A form are presented as a more probable isomer of 3-mercapto-5-amino-1,2,4-triazole. Spectra were recorded for AMT adsorbed at silver sol (hydroxylamine reduced).

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Schematic representations of the monolayer structure typical for A and B forms are shown in the inset of Fig. 1. The most striking intensity changes observed in the SERS spectra of these two forms involve the bands located around 1330 cm1 and 1360 cm1, which were assigned to the vibrational modes of the triazole ring.24 The bands around 1330 cm1 and 1360 cm1 can be therefore considered the marker bands of the A and B forms, respectively, while the intensity ratio of these two bands can be used to quantify the relative surface concentration of these two forms. As their relative content is affected by the pH of the surroundings, AMT functionalized silver nanoparticles can be employed to construct a SERS-based pH sensor.

2. Response of the SERS-based pH nanosensor The principle of operation of the SERS based pH sensor relies on the enhancement of the Raman signal of reporter molecules linked to the metal nanoparticles. In the case of metal colloid suspensions, the reproducibility of the enhancement is limited because of their tendency to form clusters upon aging. We used the freshly prepared silver nanoparticles fabricated with hydroxylamine as a reducing agent. At rst we studied the SERS response of the pH nanosensor. A series of representative pH dependent SERS spectra is shown in Fig. 2. The pH of the suspension was adjusted by means of phosphate buffers in the range attainable for the conjugated acid–base pairs of this system. What is important, the spectrum of the colloidal Ag in contact with Na3PO4, in the absence of AMT, does not exhibit any spectral features in the investigated spectral range (data not shown). Aer sensor fabrication (involving mixing of colloidal suspension with the buffer and further adsorption of AMT molecules) the nal pH of the sample was veried again and these latter values are given in Fig. 2. For this specic experiment, AMT molecules not attached to the metal were also present in solution. The nal AMT concentration in solution was equal to 0.91 103 M, further denoted as 103 M for simplicity. The spectra in Fig. 2 evidence clearly that there is a pH sensitive response of the SERS signal, typical for AMT molecules. The intensity ratio of the A and B form marker bands present around 1330 cm1 and 1360 cm1 respectively can be correlated with the pH of the environment. As can be seen in Fig. 2, band component characteristic for the A form is dominant for acidic pH values, while the contribution of the B structure increases upon alkalization. As a parameter probing the pH, the intensity ratio of the A and B form characteristic bands was chosen. This approach is quite novel, as it was mostly a normalized intensity of a single band, which has so far been employed to evaluate the SERS response of a pH sensor in the literature.2,16,25 According to our knowledge, only one paper reporting the ratio of the two bands corresponding to n(C^C) stretching vibrations of 4-ethynylpyridine was used to indicate the pH of the solution.26 The use of the ratio of two SERS band intensities, each responding opposite to the varying activity of hydronium ions, increases the sensitivity of the sensor toward pH detection. Molecular vibrations of AMT selected for analysis are situated in a similar

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Fig. 2 Representative series of pH dependent SERS spectra (532 nm excitation) of 103 M AMT in silver sol at a pH value of: (A) 5.3, (B) 6.5, (C) 10.2, (D) 10.4, (E) 10.7, (F) 10.9, (G) 11.0, (H) 11.4 and (I) 11.8. Marker bands of A and B forms, spotted with the lines, are located around 1330 and 1360 cm1, respectively. Spectra were smoothed, scaled and shifted for the clarity of presentation.

spectral range. Therefore to determine the A and B form intensity ratio accurately, the band tting procedure was necessary. The ratio of the heights of the two overlapping peaks around 1330 and 1360 cm1 (taken at real maxima) tted with the mixed Gaussian–Lorentzian curves was calculated to construct a plot of the relative intensity of A to B form of AMT molecules (IA/IB) as a function of pH. The resulting plot for spectra collected in the 103 M AMT solution is presented in Fig. 3, marked by the points with the plus symbols. Experimental points were tted with the sigmoid Boltzmann curve (equation and tting parameters for this and all the systems studied here are given in Table 1 in the Experimental section). The fragment of the curve exhibiting the largest slope corresponds to the biggest signal change of the sensor per pH unit. The sensor will be hence the most sensitive to the pH of the surrounding medium in this range. The nanosensor constructed in this way, working in a 103 M AMT solution, is the most reliable pH indicator in the pH range 8–12. This is unfortunately beyond the intracellular pH range, which for a typical mammalian cell can vary from 4.7 in lysosome to 8.0 in mitochondria. However, optical sensors operating in strongly basic media are still rare,26,27 while pH glass electrodes can suffer from alkaline errors.


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Plots of the intensity ratio of the SERS (532 nm excitation) bands characteristic for A form: 1330 cm1 and B form: 1360 cm1 of AMT molecules in silver sol as a function of varying pH. The presented plots correspond to different pH-dependent SERS responses of the nanosensor for varying concentrations of the AMT monomer in solution (see the legend). The lines connecting the data points are the result of the sigmoid Boltzmann fits (see the Experimental section for the details of the fit for each curve). Fig. 3

The change of pH is not the only factor inuencing the equilibrium between the A and B forms of AMT within the monolayer, as the conversion between chemical states of the molecules is accompanied by the geometry change. The deprotonated B form favors the parallel to the metal surface orientation of the AMT molecules, leading to metal–ring interactions. AMT molecules in the A form adopt an upright orientation, which enables higher packing density of the monolayer. Therefore, there is a coupling between the pH and the concentration effect: transition between A and B forms is dependent on both pH and concentration of the AMT monomer in solution. For that reason, the concentration effect was studied, performing an experiment similar to the one presented above, but involving solutions providing nal concentrations of the free AMT monomer of 0.91 105 M and 0.91 107 M, further simplied as 105 M and 107 M. The responses of such prepared SERS-based pH sensors are plotted in Fig. 3: marked with the dot and asterisk symbols for the experimental data collected for 105 M and 107 M AMT in solution respectively. The acquired parameters of the Boltzmann ts (see the Experimental section) are quite different than these selected for the 103 M concentration, which is a consequence of the altered pH dependence of the SERS signals of the studied systems. These results demonstrate that the response of the nanosensor described herein toward pH changes is inuenced by the concentration of the non-adsorbed AMT molecules in solution. This suggests that there is either a multilayer formation or a different sub-monolayer coverage, both of which may provide various orientations of the AMT molecules in the monolayer at given pH; according to the surface selection rules, the resulting SERS intensity will be also affected. This behavior manifests in the changed working range of the nanosensor: the pH can be determined with the biggest precision in the 5–9 and 2–6 range for the 105 M and 107 M AMT concentration, respectively. At a rst glance these results show the possibility of tuning the This journal is © The Royal Society of Chemistry 2014

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operating pH range of the AMT based SERS nanosensor. However, in the case of application of such a prepared nanosensor to the real biological or chemical system, the amount of the excessive AMT molecules in contact with the monolayer, if any, can be out of experimentalist control. If the changed pH behavior of the nanosensor was signicant only for the submonolayer coverage, it would enable it to operate unambiguously at the concentration ensuring the full AMT monolayer coverage of the silver nanoparticles. Anyhow, our observations demonstrate how essential the monomer concentration can be for the SERS-based nanosensor performance. For any SERS-based nanosensor involving chemical transformation of the molecules, additionally accompanied by the orientation changes of the molecules in the monolayer, it should be veried whether it is exclusively related to the pH value of the surrounding media and whether the pH response remains unchanged upon varied concentration of the SERS reporter monomer. For the system examined herein it can be undoubtedly stated that a higher AMT concentration in solution results in a higher surface coverage of the formed monolayer. Higher surface coverage facilitates in turn adopting the A form, as the high packing density hinders “lying down” of the AMT molecules. Therefore, a higher pH value (more basic) is required to deprotonate AMT molecules and force their parallel orientation (B form). The performance of the nanosensor consistent with this statement is clearly visible for the curves presented in Fig. 3. The gradual transformation of the A form into the B form takes place for higher pH values, as the concentration of the AMT monomer in solution increases. Coefficient C (see the Experimental section for details) of the Boltzmann t of the plots in Fig. 3 can be useful in evaluating the sensor behavior discussed here. This coefficient corresponds to the pH value for which the SERS intensity ratio is halfway between the bottom and the top of the curve and it reaches a value of 4.1 for the lowest AMT concentration, 6.9 for the intermediate concentration and approaching 9.9 for the highest examined concentration. The approach presented here provides mathematical tools for quantitative description of the qualitative changes occurring in the studied system, which will be also helpful in its further application as a SERS microsensor.

3. Fabrication of the SERS-based pH microsensor The long distance goal of this work was fabrication of a pH microsensor. Change of the sensor size scale is expected to provide higher control of the placement of the device in the studied object and hence improve the precision of the local pH measurement. The microsensor consists of four elements: SiO2 microbead (1) coated with the Raman reporter molecules (2), adsorbed on silver nanoparticles (3), which are attached via aminoalkoxysilane linkers (4) to the microsphere. The silica microbead serves as an optically trappable substrate, by being a carrier of the metal nanoparticles, which latter enhance the SERS spectrum of the Raman reporter molecules. Trapping of SERS-active metal nanoclusters is efficient only for the short times, since for the nanometer-sized particles the gradient force in the propagation direction becomes overwhelmed by the

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scattering force exerted by the trapping beam.20 SERS-active probes prepared in a manner described above were demonstrated to promise efficient optical manipulation, precise placement and scanning with nanometric accuracy in living cells.19 However, in this work we tested different colloidal silver nanoparticles, obtained with hydroxylamine as the chemical reducing agent, while Petrov et al.19 studied the citrate reduced Ag nanoparticles, synthesized according to the slightly modied Lee and Meisel procedure.28 Comparison of these two types of silver nanoparticles prepared with different chemical reducing agents, performed by Sanchez-Cortes et al. revealed substantial differences between them in terms of morphology, aggregation, adherence to glass and SERS activity.29 Hydroxylamine reduced Ag nanoparticles were found to be aggregated more easily and to show higher tendency toward immobilization on glass, while forming more homogenous lms than the citrate reduced ones. Regrettably, both types of colloidal Ag nanoparticles were demonstrated to display some impurities aer their immobilization, detectable with SERS. However, the advantage of the hydroxylamine reduced colloid is the absence of interference bands in the important range of the Raman shi and lack of thermal degradation of hydroxylamine and/or its products, observed for citrate. The critical issue for the successful fabrication of the SERS microsensor according to the scheme described in this paragraph is establishing the protocol, which secures dense and homogenous coating of the microbeads with the silver nanoparticles, operating in a reproducible manner. In the case of the reduction of Ag+ with hydroxylamine, the residual oxidation products are molecular nitrogen and nitrogen oxides.29 We decided to use an aminosilane linker to immobilize previously synthesized metal nanoparticles, as the successful attachment of hydroxylamine-reduced gold and silver nanoparticles to glass and silica functionalized with APTMS (3-aminopropyltrimethoxysilane) has already been reported in the literature.19,30,31 We have tested SiO2 microbeads of two distinct sizes: 0.5 and 2 mm diameter and compared the beads functionalized with the APTMS in our laboratory with those terminated commercially with NH2 groups. Metal coating was achieved via simple mixing of the amino groups modied silica beads with the metal colloid, under continuous stirring. At rst we optimized the relative amount of the silica microsphere suspension and colloidal silver nanoparticles. We examined various volumetric ratios of the microbeads to colloidal Ag: 1 : 10, 1 : 100 and 1 : 1000, starting with the 2 mm silica beads, functionalized with APTMS. The concentration of APTMS employed in order to introduce amino groups in the rst step was 0.1% (v/v), to avoid formation of metal nanoparticle multilayer.19 Scanning electron microscopy (SEM) imaging of the surface of such modied SiO2 microspheres was used to verify their coverage with the silver nanoparticles. The most uniform and dense metal coating was observed for the 1 : 1000 (v/v) ratio of the SiO2 microbeads to the colloidal Ag, as can be seen in the SEM image in Fig. 4a. This procedure worked reproducibly and produced comparable packing densities for various spheres, as can be seen in Fig. 4b. This ratio of the reagents was therefore applied also for the attachment of the Ag nanoparticles to the commercially amino-modied silica

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Fig. 4 SEM (scanning electron microscopy) images of the respective 2 mm silica microbeads mixed with colloidal Ag in a 1 : 1000 (v/v) ratio: (a and b) beads modified with amino groups in the laboratory (using APTMS) and (c) commercially functionalized with amino groups; (d) purified (centrifuged) only after the final step of fabrication (involving the use of APTMS), (e and f) purified after each step of the synthesis (with APTMS). Varying contrast between the images is a result of different electrostatic characteristics of various samples.

beads, resulting in packing density comparable to that of the APTMS modied beads. A SEM image showing the typical metal coverage for the 2 mm silica beads functionalized commercially with amino groups is presented in Fig. 4c. For the lower microbead to metal nanoparticle ratio, i.e. 1 : 10 and 1 : 100, the metal surface coverage of the 2 mm silica microspheres was unsatisfactory (see Fig. S1a and b† for the APTMS functionalized microspheres). These results suggest that for some reason for hydroxylamine reduced silver colloid, a large excess amount of the metal nanoparticles over SiO2 is essential for the high frequency of collisions between the metal and silica microbeads, which is reasonable. But surprisingly, for 1 : 10 and 1 : 100 ratios the barrier for attachment of the metal nanoparticles to the surface (SiO2) seems to be higher than particle–particle agglomeration one, as evidenced by the clearly visible metal clusters, separated from the beads (see Fig. S1a and b†). A similar and even more pronounced effect was observed when we attempted to coat 0.5 mm beads with the hydroxylamine reduced colloidal silver. For both the APTMS-modied and commercially amino-terminated silica beads, formation of the silver clusters outside the SiO2 microspheres was favored, as can be seen in


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Fig. S1c and d, respectively.† For the commercially functionalized particles, the beads remained totally not covered with the Ag nanoparticles (see Fig. S1d†), which may suggest a temporal degradation of the surface layer providing NH2 groups. In the case of the microbeads (of both studied sizes) modied with APTMS in the laboratory, some amorphous or crystalline forms surrounding the silica spheres can be clearly seen in the SEM images of the composites (Fig. 4a and b; Fig. S1a–c†). We believe this residual is excessive APTMS, therefore we decided to remove it from the reaction mixture through centrifugation. Our protocol of the microsensor fabrication involving APTMS consists of two steps: modication of the SiO2 surface with the aminosilane and mixing the functionalized silica beads with the hydroxylamine reduced colloidal silver nanoparticles in a volume ratio 1 : 1000. SEM images of the microbeads centrifuged aer the nal step only and aer each step of the synthesis are compared in Fig. 4d and e. This second procedure resulted in a substantial improvement of the microsensor quality. Removal of the excessive reagents aer the second step was insufficient to purify. Therefore, large agglomerates of the silica microbeads, in a form of long chains of the SiO2 microspheres, merged by metal nanoparticles and “glued” together with remaining silane can be still clearly visible in Fig. 4d. This feature would make their application as mobile SERS sensors impossible. Encouragingly, the satisfying purity was accomplished aer centrifugation both aer silanization, as well as aer mixing with colloidal Ag. In this case nicely separated probes composed of single or at most a few silica microspheres can be observed (see Fig. 4e). What is important, the purication method did not affect the metal nanoparticles coating of the microbeads, as evidenced from the SEM image in Fig. 4f, showing no sign of a lower packing density of the Ag nanoparticles compared to the non-centrifuged microsensor (compare with Fig. 4c). Therefore, for the further SERS experiments we used silver coated, APTMS modied, 2 mm size silica microbeads, centrifuged aer each step of fabrication to eliminate excessive silane and metal nanoparticles.

4. pH sensing with Ag modied microbeads, utilizing AMT as a Raman reporter Next, the microprobes constructed according to the procedure described in the previous section were tested as SERS pH microsensors. In order to adsorb SERS reporter molecules, the previously produced suspension of microsensors was mixed with the AMT solution, reaching a nal concentration of 103 M of AMT molecules. To verify the efficiency of the AMT adsorption on microsensors, a SERS mapping experiment was performed for such a prepared suspension, immobilized on glass (using 3aminopropyltriethoxysilane, APTES) and dried. The optical image of the examined area (size: 22 mm 23.5 mm) and the corresponding Raman map plotting the distribution of integral intensity of the B form characteristic band in the range of 1335– 1370 cm1 are shown in Fig. 5a and b, respectively. Under experimental conditions (spectra collected in air) AMT molecules exist exclusively in the B form, as shown by acquired spectra and demonstrated by our previous studies of AMT adsorption.22 For


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Fig. 5 (a) Optical microscopic image (in reflected light) of the examined area of the microsensors modified with Ag nanoparticles and mixed with the 103 M AMT solution, with the visible microbeads (spherical features of 2 mm diameter) deposited onto the glass substrate; (b) corresponding SERS (532 nm excitation) map plotting the distribution (red color) of the integral intensity of the marker band of the B form of AMT molecules (range: 1335–1370 cm1). The step size of the SERS map was 0.5 mm. White arrows mark some Ag nanoparticle aggregates.

this reason only B marker band distribution was analyzed. The results conrm successful AMT adsorption on microsensors. There is a good correlation between the location of the microbeads spotted with an optical microscope and the maxima of the intensity of the AMT characteristic SERS band (intense red color in Fig. 5b). The dimensions of the two additional objects visible in the Raman map in Fig. 5b (marked with white arrows) are certainly too large to be the microsensors studied here. We attributed these features to the presence of the residual, not fully centrifuged silver nanoparticles, which are also capable of AMT chemisorption and Raman scattering enhancement. Having evidence of an efficient amplication of Raman signals by silver modied silica microbeads, we have tested microprobes fabricated in the same manner as SERS-based pH sensors. Prior to these measurements, the microsensors were attached to the glass substrate to eliminate the risk of ushing the SERS microprobes upon contact with the applied buffer solution. To immobilize the pH microsensors, the aminosilanization procedure of the glass was applied again (the details are given in the Experimental section). Next, the SERS spectra of the AMT molecules adsorbed on microsensors exposed to solutions of different pH were collected. Before the contact of a given microsensor with the environment of varying pH, the AMT modied microprobes attached to the glass substrate were rinsed with water to remove loosely adsorbed and/or not bound Raman reporter molecules. In this case the intensity ratio of the SERS bands characteristic for A and B forms of AMT was sensitive to pH only under the strongly acidic conditions (the pH was adjusted using HCl solution). SERS spectra acquired for the pH varying from 1.25 to 2.37 showing the spectral region diagnostic for A and B forms are presented in Fig. 6a. The resulting plot, displaying the change of the IA/IB SERS bands as a function of the pH, is shown in Fig. 6b. It demonstrates that the detection limit of the microsensor reaches strongly acidic values, in the absence of the AMT monomer in the examined solution. The working range of the microsensor is now shied to the low pH values. This means that at zero

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concentration of AMT in solution, protonation of the monolayer and adaptation of upright conguration is possible only under strongly acidic conditions. These observations are consistent with the results for the pH nanosensors, for which the sensitivity range was moved toward lower pH values with the decreasing concentration of AMT monomer in solution. Moreover, the steepness of the sigmoid curve in plot in Fig. 6b is larger than for the nanosensor shown in Fig. 3 (smaller values of S parameter), allowing higher precision of pH measurements in the range from 1.0 to 2.0.

5. Dependence of A/B form equilibrium on the AMT solution concentration and SERS response of the nanosensor in the absence of non-reporter AMT molecules

Fig. 6 (a) SERS spectra (532 nm excitation) obtained for the microsensor utilizing Ag nanoparticles with attached AMT molecules, collected in the pH sensitive range: 1.25–2.37. Depicted positions of marker bands of A and B forms, spotted with the lines, are located around 1330 and 1360 cm1, respectively. Spectra were smoothed, scaled and shifted for the clarity of presentation. (b) SERS response to the varying pH for the same type of microsensor, coated with AMT. The presented plot was constructed taking the ratio of the bands characteristic for the A form (1330 cm1) and B form (1360 cm1) of AMT molecules. The experimental points and the sigmoid Boltzmann fit (see the Experimental section for the details of the fitting parameters) are shown.

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The pH response of the microsensor formed from 103 M AMT in solution is totally different from that observed for the nanosensor (compare relevant plots in Fig. 6b and 3). To explain this performance we attempted to estimate the AMT concentration in solution resulting in the formation of the monolayer on Ag nanoparticles, composed of AMT molecules exclusively in the A form (upright orientation). For this purpose SERS spectra of AMT on colloidal Ag (in suspension) adsorbed from AMT solutions of varying concentrations were collected at natural pH and the results can be seen in Fig. S2.† This experiment showed the evolution from the typical spectrum of the B form, characteristic for concentrations of AMT in solution around 106 M or lower, to the SERS pattern typical exclusively for the A form for concentrations in a range of 103 M and higher. Although for the microsensor the AMT monolayer is formed from a comparable bulk concentration as for the nanosensor, in this case the extremely acidic pH is a prerequisite to force the silver bound molecules to adapt perpendicular orientation. The only difference between these two experiments is the absence of the excessive AMT molecules in solution for the microsensor. These results suggest that the A form is stabilized more by interactions through hydrogen bonds with the molecules in the bulk in the presence of a sufficient amount of AMT in the solution than by interactions of adsorbed AMT molecules with the adjacent molecules at the surface. When the equilibrium between the surface and bulk molecules is disturbed (like in the case of total removal of non-adsorbed AMT molecules), it is probably exclusively the surface coverage which becomes decisive for the pH behavior. To verify this statement, we examined a pH response of the nanosensor in the absence of the AMT molecules in solution as well. The SERS reporter molecules were adsorbed on silver nanoparticles from 103 M AMT in solution and subsequently molecules which had been not rmly bound to the surface were removed from the analyzed sample. Acquired SERS response (shown in Fig. 7) to varying pH demonstrates that the working range of the nanosensor is now shied to strongly acidic pH, similar to the microsensor under equivalent conditions (Fig. 6b). The nanosensor is capable of measuring pH in its local vicinity continuously over the range of 1.5–2.5 pH units, which is pretty close to the results obtained for the microsensor. The difference may come from different amount of unattached AMT molecules feasible to be removed


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Fig. 7 SERS response to the varying pH for the nanosensor utilizing Ag nanoparticles, modified with AMT. Spectra used to draw the plot were collected after the removal of AMT molecules not bound to the metal. The presented plot was constructed using the ratio of the SERS intensities of A and B form marker bands. The experimental points and the sigmoid Boltzmann fit (see the Experimental section for the details of the fitting parameters) are shown.

from these two systems. This experimental nding conrms the hypothesis above that excessive AMT molecules in solution are responsible for the pH and AMT concentration dependent equilibrium observed for the nanosensor in contact with freely diffusing AMT molecules (see Fig. 3). For the nanosensor in the absence of the AMT molecules in solution, in the case of adsorption carried out for AMT concentrations below 103 M we can expect a sub-monolayer surface coverage (according to the results presented in Fig. S2†). Hence, lower packing density can only result in a further down-shi of the pH working range, as it will favor the B structure form of AMT molecules. 6. Prospects for the application of the nano- and microsensor working in strongly acidic regions Sensing as strongly acidic pH as this achieved for the nano- and microsensor constructed here with high precision can be still useful in biology, medicine and industry related elds. Acidophilic bacteria which are known to thrive at highly acidic pH, usually lower than 4,32 can be not considered the objects of intracellular pH sensing in this range. The reason is that these microorganisms held their intracellular pH close to neutral, as they developed mechanisms to pump acid out of the cell to protect DNA.33 Nevertheless, the pH of natural (sulfuric pools) and manmade (coal and metal ore mines) environments of the acidophiles can be examined by means of pH probes depicted in this paper. There are still some cell compartments recognized to work under weak acidic conditions, like endosomes or lysosomes, but they function in a pH range from 4.5 to 6.0.34,35 However, several intracellular enzymes from acidophiles are known to be functional at much lower pH than the cytoplasmic one. Performance of carboxylesterase and three a-glucosidases from Ferroplasma acidiphilum (a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7) was studied in vitro.36 All the studied enzymes were active and stable in the pH


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range 1.7–4.0 and their pH optima were found to be of much lower values than the mean intracellular pH of 5.6. These results may suggest the existence of yet undetected cellular compartmentalization, providing cytoplasmic pH patchiness and low pH environments for the examined enzymes, which drives the need to establish the tools to verify this hypothesis. Another motivation to seek the variety of pH probes capable of sensing acidic intracellular conditions is their potential application in the studies of plant vacuoles, whose typical range of pH is 5.0–5.5,37 but for the citrus fruits it can be as low as 2.0.38 The record for the most acidic vacuole belongs to the brown algae Desmerestia, with a lumenal pH below 1.0, owing to accumulation of H2SO4.39 There are also many reports that relate some cellular dysfunction to abnormal pH values in organelles. It has been demonstrated that in nervous system diseases, such as stroke, Parkinson's or Alzheimer's disease, the common characteristics are decreased pH or acidosis at both tissular and cellular levels.40,41 Particularly the activity of some Alzheimer's disease associated enzymes is expected to be altered under acidic conditions.40 It was also shown that dysregulated pH, such as a reversed gradient, i.e.: higher intracellular pH and a lower extracellular pH, facilitates cancer progression.42 The given examples refer rather to the small deviation of the intracellular pH from the typical close to the neutral value of 7.2. However, the presence of the strongly acidic phagosomes in breast cancer cells was also conrmed by means of a uorescence indicator.43 Recently Tang et al.44 introduced a new uorescent dye capable of monitoring pH in the entire physiological range and visualized the acidic and basic compartments in the native and acidied HeLa cells. All these examples demonstrate that development of intracellular sensors covering a broad range of pH (supported by imaging techniques) could provide an insight into the molecular basis for pH-dependent cell structure and behaviour that are relevant to the cancer cell biology and nervous system disease mechanism. Therefore, development of the new SERS-based pH sensors described here, offering the precise measurement of intracellular pH in a strongly acidic pH range, is of great importance. The extracellular pH of solid tumors was also demonstrated to be highly acidic compared to normal tissue, as a result of a consequence of high glycolysis and poor perfusion.45 Hence, providing potential microprobes capable of pH sensing in this range and offering control of the location can be extremely useful for tumor diagnosis. There is also a high demand for the pH sensors functioning in strongly acidic aqueous solutions (pH below 3), as traditional glass pH electrodes suffer from serious limitations in this range including electrical interference, occurrence of acid error and possible damage. Performance of the alternative uorescence based sensors in extremely acidic solutions is also difficult, because of the problems with chemical instability of the uorophore,46 saturation of the receptors,47,48 and reduced selectivity49 under these conditions. The potential applications of the pH sensors fabricated here for strongly acidic conditions in the area of aqueous solutions can be industrial wastewater evaluation, environmental protection or determination of the acidity of stomach acid in health care.

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Conclusions We have successfully fabricated a SERS-based pH nano- and microsensor, utilizing silver nanoparticles functionalized with AMT. Monitoring a pH sensitive transformation between the two distinct AMT orientations: A form (perpendicular orientation) and B form (parallel orientation) with the SERS technique was employed as a pH indicator. The pH working range of the nanosensor was strongly dependent on the amount of the AMT in solution, being in contact with the Raman reporter modied Ag nanoparticles. Aer the removal of the non-adsorbed AMT molecules, the pH nanosensor enabled highly sensitive detection of pH under strongly acidic conditions: from 1.5 to 2.5 pH units. The obtained SERS response of the microsensor (molecules of the SERS reporter attached to silica microbeads coated with Ag nanoparticles) in the absence of AMT in solution was similar, changing the most for the pH value varying from 1.0 to 2.0. Sensing such strongly acidic pH makes the sensors constructed here suitable for vacuole studies, environmental protection application, or even examination of pathological changes at the cellular level. However, although the nano- and microsensor described here was thoroughly characterized and tested in the laboratory, they have not yet been applied in any real-life system.

Acknowledgements This work was supported by the grant from Polish Ministry of Science and Higher Education “Iuventus Plus” (no. IP2011 027371). The measurements involving SEM were carried out using equipment purchased with the support of the project nancing agreements POIG.02.02.00-14-024/08-00 (CePT project). Special thanks are owed to Dr Marianna Gniadek for performing SEM analysis.

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