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A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide
Accepted Manuscript Title: A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide Authors: Pedro J. Rivero, Elia Iba˜nez, Javier Goicoechea, Aitor Urrutia, Ignacio R. Matias, Francisco J. Arregui PII: DOI: Reference:
S0925-4005(17)30933-4 http://dx.doi.org/doi:10.1016/j.snb.2017.05.110 SNB 22394
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-9-2016 17-5-2017 18-5-2017
Please cite this article as: Pedro J.Rivero, Elia Iba˜nez, Javier Goicoechea, Aitor Urrutia, Ignacio R.Matias, Francisco J.Arregui, A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.05.110 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide
Pedro J. Rivero*1, Elia Ibañez2, Javier Goicoechea2, Aitor Urrutia2, Ignacio R. Matias3 and Francisco J. Arregui2.
1
Materials Engineering Laboratory, Department of Mechanical, Energetic and Materials
Engineering, Public University of Navarre. Campus Arrosadía S/N, 31006, Pamplona, Spain. 2
Nanostructured Optical Devices Laboratory, Department of Electrical and Electronic
Engineering, Public University of Navarre, Campus Arrosadía S/N, 31006, Pamplona, Spain. 3
Institute of Smart Cities, Public University of Navarre, Campus Arrosadía S/N, 31006, Pamplona, Spain.
*email: [email protected]
RESEARCH HIGHLIGHTS Synthesis of silver and gold nanoparticles by using a chemical reduction method. The changes of the absorbance strength maxima of both LSPR absorption bands are measured. A remarkable difference in sensitivity of both LSPR absorption bands are clearly observed. A self-referenced colorimetric sensor for the quantitative determination of hydrogen peroxide. A novel perspective for the chemical detection of other reactive oxygen species
Abstract In this work, a self-referenced colorimetric sensor for the quantitative determination of hydrogen peroxide is presented. This optical sensor is based on the presence of the Localized Surface Plasmon Resonances of silver and gold nanoparticles which are capped with the same encapsulating agent of poly(diallylammonium chloride) (PDDA). These metallic nanoparticles are synthesized by a chemical reduction method of their corresponding inorganic precursors and characterized by UV-Vis spectroscopy and transmission electron microscopy, respectively. A remarkable difference in sensitivity related to both LSPR absorption bands is observed as a function of variable molar concentration of the target molecule. The LSPR band of the silver nanoparticles is gradually decreased whereas the LSPR of the gold nanoparticles is practically unaltered when the hydrogen peroxide molar concentration is increased as a result of its better chemical stability. This stable absorbance LSPR band of the AuNPs is used as an optical reference and the molar concentration of the target molecule is obtained by measuring the changes of the absorbance strength maxima of both LSPR absorption bands. As a result, a very good sensitivity with a high robustness and a linear response over a wide concentration range from 1.25 µM to 1250 µM is obtained. This self-referenced method opens up a new perspective for the chemical detection of other reactive oxygen species or well for the determination of other type of analytes in the future.
Keywords Localized Surface Plasmon Resonance (LSPR), optical colorimetric sensor, silver and gold nanoparticles, self-referenced sensor, hydrogen peroxide detection.
1. Introduction In the last decades, the rapid proliferation of the nanotechnology has led to the fabrication and use of an increasing number of nanomaterials and nanoparticles in fields as diverse as biology, catalysis, chemistry, construction, electronics, medicine, textile or optics, among others [1-8]. One of the most explored characteristics of the noble metal nanoparticles (essentially silver, gold or copper) is a phenomenon known as Localized Surface Plasmon Resonance (LSPR), the frequency at which conduction electrons collectively oscillate in response to the alternating electric field of an incident electromagnetic radiation. As a result, an intense absorption band in the visible region with a specific coloration of the resultant nanoparticles is obtained [9, 10]. A wide variety of chemical routes for the synthesis of metal nanoparticles with different shapes can be found in bibliography [11-14], although the most common approach involves the chemical reduction of metal salts in the presence of an adequate encapsulating agent [15]. The encapsulating agent plays an important role for the synthesis of nanoparticles with a specific morphology because it limits the growth of the particles, directs their shape and provides colloidal stability [16]. The wavelength location of this LSPR absorption band shows a great dependence over several factors such as the resultant shape and size of the nanoparticles, the refractive index of their surrounding medium or the environmental in which the metal nanoparticles are dispersed [17-19]. In addition, other key factor is the average distance between neighboring metal nanoparticles because when the average particle distance is shortened to a distance below the diameter of the particles, an aggregation of the nanoparticles is performed. This aggregation leads to a total color change as a consequence of the LSPR coupling between nanoparticles and as a result, a red-shift of the LSPR absorption band is obtained [20]. This sensing mechanism based on the aggregation of silver or gold nanoparticles is the basis for the
design of a wide number of colorimetric biosensors because it is studied the change in coloration generated by the plasmon coupling between nanoparticles upon aggregation [2125]. However, other colorimetric sensing mechanisms have used the excellent optical properties of the noble metal nanoparticles as a colorful reporter by measuring the changes in the absorbance strength for the quantitative determination of a wide variety of analytes for biological or medical purposes [26-30]. One of the analytes that are causing a great interest in the medical community is the hydrogen peroxide because it is well-known Reactive Oxygen Specie (ROS) [31-35]. An excessive accumulation of this analyte in the body can trigger some diseases because the hydrogen peroxide is the responsible of causing tissue damage and DNA fragmentation. In addition, it is the product of reactions catalyzed by a large number of oxidase enzymes, and therefore its monitorization can lead to different bio-sensing applications. Therefore, the analytical determination of H2O2 also has an important significance in other totally different fields such as industry, food or environmental analysis. According to this multidisciplinary presence, the design and development of new methodologies for the quantitative determination of H2O2 are required. Most of the analytical methods for its quantification are based on enzymes, using different techniques such as chemiluminiscence, spectrofluometry and electrochemistry [36-38]. However, these techniques show several drawbacks such as the use of high sophisticated instrumentation, costly performance, need of a total immobilization of the enzymes in order to obtain a good sensitivity, long assay time, low selectivity or a poor reproducibility. From all these backgrounds, the use of other alternative method is required for the determination of this target molecule. Among all them, LSPR optical sensors based on the incorporation of metal nanoparticles are an ideal candidate due to their high sensitivity, great selectivity, simplicity and cost effective detection. Previous works have reported the quantification of hydrogen peroxide by using LSPR optical colorimetric sensors [26, 32, 39-42]
based on the optical changes of a unique LSPR absorption band related to only silver nanoparticles. In this work, it is reported for the first time a dual reference LSPR optical colorimetric sensor based on the combined colloidal dispersions of gold and silver nanoparticles with the same encapsulating agent for the quantitative determination of hydrogen peroxide. The metallic nanoparticles have been synthesized by a chemical reduction step from their corresponding inorganic salts. From the experimental results, it is concluded that a remarkable difference in sensitivity related to both LSPR absorption bands is observed as a function of variable molar concentration of the target molecule, making possible the design of a robust self-referenced LSPR optical detection method. As a promising result, a very good sensitivity and a linear response over a wide concentration range from 1.25 µM to 1250 µM of target molecule is presented. In addition, the experimental results show a lower quantification limit than previously reported enzymebased biosensors [43-48]. The hydrogen peroxide concentration range of the biosensor presented in this work is suitable for some biomedical applications such as oxidative stress estimation, measuring the hydrogen peroxide in human urine [31], as a simplified test for biomonitoring the glucose in blood or human serum [26, 33, 35, 40], or even for heavy metal ion detection such as mercury due to the catalytic reactivity of AgNPs [34]. Finally, the resultant dual LSPR optical sensor opens up a new perspective for the design and development of biosensors for monitoring other reactive oxygen species or well other analytes in the future.
2. Experimental section 2.1. Reagents and materials Poly(diallyldimethylammoniumchloride) (PDDA) (Mw. 15000), silver nitrate (>99% titration) solution in water 0.1 N,
gold (III) chloride trihydrate (HAuCl4.3H2O), borane
dimethylamine complex (DMAB) and hydrogen peroxide (H2O2) were purchased from Sigma
Aldrich, and used without further purification. The ultrapure H2O was purified to above 18.2 MΩ using a Mili-Q water system (Millipore).
2.2. Synthesis of the silver and gold nanoparticles A wet method based on the chemical reduction of metal salts has been used for the synthesis of capped AgNPs and AuNPs, respectively. As a source of silver and gold ions were the silver nitrate (AgNO3) and Gold (III) chloride trihydrate (HAuCl4. 3H2O), respectively. A reducing agent such as DMAB was the candidate for the reduction of the silver and gold ions to the corresponding nanoparticles. In addition, a polyelectrolyte such as PDDA has been used as a capping agent of the nanoparticles. An important aspect of this chemical method is the high stability of the synthesized nanoparticles (AgNPs, AuNPs) because no changes of the resultant coloration were observed after 6 months of storage without showing any sign of aggregation for both types of nanoparticles at room condition. For the synthesis of PDDA capped AgNPs, 0.100 mL of 0.1 N of AgNO3 was mixed with 15 mL of 25 mM of PDDA and stirred for 1 hour. After that, the addition of 0.500 mL of 0.1 M DMAB to the solution was performed with a constant stirring of 1 hour. A color change from transparent to yellow is indicative that capped-silver nanoparticles (PDDA-AgNPs) were successfully synthesized. For the synthesis of PDDA capped AuNPs, 0.100 mL of 0.1 N of HAuCl4.3H2O was mixed with 15 mL of 25 mM of PDDA and stirred for 1 hour. Then the addition of 0.025 mL of 0.1 M DMAB to the solution was performed with a constant stirring of 1 hour. The color of the solution was changed from pale yellow to red-wine, showing the formation of the capped-gold nanoparticles (PDDA-AuNPs). Finally, once both types of nanoparticles have been successfully synthesized, an equal volume ratio of 1:1 was taken in a glass vial and kept under continuous stirring at room temperature. After a prescribed reaction time of 30 minutes, a light orange coloration has
been observed, indicating that the combination of PDDA-AgNPs and PDDA-AuNPs (referred as PDDA-(Ag+Au)NPs) was obtained in a same solution.
2.3. Characterization of the metallic nanoparticles UV-visible (UV-Vis) spectroscopy has been used to characterize the optical properties of the PDDA-(Ag+Au)NPs. All absorbance spectra were taken from 350 to 900 nm on the UV-Vis spectrophotometer at room temperature. A Jasco V-630 spectrophotometer was used to perform all the measurements. In all cases of study, ultrapure water has been used as an optical reference. A transmission electron microscopy (TEM) was used to characterize the morphology and distribution of the nanoparticles. Samples for TEM were prepared by drop-casting the solution onto a collodion-coated copper grid. And then, the samples were dried up at room conditions. In addition, TEM observations were carried out with a Carl Zeiss Libra 120 using four different samples (PDDA-AgNPs, PDDA-AuNPs, PDDA-(Ag+Au)NPs and PDDA-(Ag+Au)NPs after addition of hydrogen peroxide). All the measurements have been performed at room condition. In order to evaluate the cross-sensitivity of the LSPR of the metallic nanoparticles with sample refractive index variations, different solutions of glycerin in water have been prepared with concentrations that provided refractive indices of 1.333, 1.340, 1.347, 1.362 and 1.3777, respectively. UV-Vis spectra have been performed by mixing 750 µL of metallic nanoparticles (PDDA-AgNPs and PDDA-AuNPs separately) with 750 µL of glycerin aqueous solution with a specific value of the refractive index. All the measurements were made at room conditions. The refractive index of the samples has been characterized using a Mettler-Toledo Refracto 30GS refractometer.
2.4. Detection of hydrogen peroxide concentration The analysis procedure was realized as follows. Firstly, 1.950 µL of PDDA-(Ag+Au)NPs was mixed with 50 µL of hydrogen peroxide water solution at different molar concentration. In this protocol the overall sample volume was kept constant (2 mL) and the only variable was the
amount of H2O2 added in order to avoid optical variations due to the dilution. The time lapse between the addition of the analyte and the optical characterization of the sample was established to 30 minutes for all samples as a convention. Finally, under these experimental conditions and after the addition of analyte, the optical changes of the PDDA-(Ag+Au)NPs have been investigated by UV-Vis measurements in order to implement as a potential colorimetric sensing application. The experiments have been performed for triplicate for the detection of the hydrogen peroxide molar concentration and the error bars indicate the standard deviation for the three measurements. In Figure 1, it is shown a schematic representation of the experimental process and the resultant setup used for the detection of peroxide concentration using PDDA coated silver and gold nanoparticles (PDDA-(Ag+Au)NPs).
Figure 1: Schematic representation of the procedure for obtaining PDDA-(Ag+Au)NPs and experimental setup used for hydrogen peroxide detection as a function of optical colorimetric changes.
3. Results and discussion 3.1 Synthesis of the metal nanoparticles First of all, the metal nanoparticles (AgNPs, AuNPs) have been successfully synthesized at room ambient using a chemical reduction method as a function of an adequate protective and reducing agent. This aspect is corroborated by UV-Vis spectroscopy in Figure 2 where the existence of a plasmon resonance band, known as Localized Surface Plasmon Resonance (LSPR) at a well-defined wavelength position, is clearly observed. This phenomenon occurs when the conduction electrons in metal nanostructures collectively oscillate, as a result of their interaction with an incident electromagnetic radiation. In this figure, the wavelength location related to the LSPR of the capped PDDA-AgNPs with a yellow color is observed at 402 nm, whereas the LSPR of the capped PDDA-AuNPs with a red-wine color is observed at 520 nm. This difference in the wavelength position of both LSPR absorption bands by using the same capping agent (PDDA) is of vital importance for a further combination in a specific molar ratio with the aim to obtain two well-defined LSPR bands. As it was commented in the Experimental Section, after a prescribed reaction time of both types of nanoparticles, a light orange coloration related to PDDA-(Ag+Au)NPs is observed and the corresponding UV-Vis spectrum clearly shows both LSPR peaks at 400 nm and 520 nm, respectively.
Figure 2: UV-Vis spectra of the synthesized PDDA-AgNPs (yellow coloration), PDDA-AuNPs (red-wine coloration) and PDDA-(Ag+Au)NPs (light orange coloration) with the specific location of the LSPR absorption bands.
3.2. Quantitative determination of hydrogen peroxide concentration Once it has been obtained two well-separated LSPR peaks in a spectral range of 120 nm for a same sample solution (PDDA-(Ag+Au)NPs), variable molar concentrations of hydrogen peroxide were tested in order to find out the sensing ability of both LSPR absorption bands. In Figure 3, it can be appreciated the difference behavior in sensitivity of both LSPR absorption bands to the hydrogen peroxide concentrations from 1.25 µM to 1250 µM. When the molar concentration of H2O2 is increased, a gradual reduction of the absorbance strength of the LSPR band corresponding to the AgNPs is clearly observed, whereas the absorbance strength of the LSPR band related to the AuNPs is almost unaltered. In addition, an important aspect observed in this work is that a significant amount of bubbles are generated when PDDA-(Ag+Au)NPs are mixed with the hydrogen peroxide. This phenomenon of bubbles formation has been
corroborated in previous works with only silver nanoparticles [39-42] where a violent reaction is generated by the catalytic reaction between silver and hydrogen peroxide. In this work, the polyelectrolyte of PDDA which is acting as a protective agent of the nanoparticles is used as a good candidate for catalyzing the hydrogen peroxide decomposition.
Figure 3: UV-Vis spectra changes of the PDDA-(Ag+Au)NPs in the presence of different molar concentrations of H2O2 from 1.25 µM to 1250 µM.
In order to corroborate this great difference in sensitivities of both types of metallic nanoparticles to the hydrogen peroxide, the optical dependence with H2O2 concentration of PDDA-AgNPs was studied separately from the PDDA-AuNPs. This separate analysis is helpful to determine the exact nature of the optical changes (chemical decomposition, aggregation, etc.) induced by H2O2 in the final combined biosensor. In Figure 4a, it is shown the UV-Vis spectra changes of the PDDA-AgNPs as a function of variable H2O2 molar concentration from 1.25 µM to 1250 µM. In this figure, it can be clearly observed that an increase of the molar concentration induces a gradual decrease of the absorbance strength related to the LSPR absorption band, as it has been previously observed in Figure 3. In addition, no red-shift
related to LSPR band at 400 nm was observed what it means that no changes in the aggregation state of the AgNPs are obtained. This gradual decrease of the absorbance value related to the LSPR band has been observed in previous works by using other different encapsulating agents [41, 42]. The reason for changing the intensity of the LSPR absorption band is associated to a catalytic reaction between the AgNPs and hydrogen peroxide through the PDDA layer which is acting as a protective layer. As a result, a gradual decomposition of the silver nanoparticles is obtained with a successive oxidative process. Due to this, the LSPR absorption band is drastically decreased for higher H2O2 molar concentrations (from 1.7 a.u. for 1.25 µM to 0.4 a.u. for 1250 µM) which is associated to the change from metal (Ag) to oxide nature (Ag2O), inducing a loss in the plasmonic modes without any coupling of light which is directly associated to the decrease of the intensity of the LSPR absorption band at 400 nm. Furthermore, it is important to remark that this H2O2-induced LSPR band annihilation is only observed for PDDA-AgNPs, while LSPR associated to PDDA-AuNPs showed a high stability. In Figure 4b, it is shown UV-Vis spectra of the PDDA-AuNPs solutions when are exposed to a H2O2 molar concentration as high as 1250 µM. The UV-Vis spectra corroborate that PDDAAuNPs are almost unaltered after the addition of the analyte with a remarkable stability in the location of the LSPR band. The experimental results clearly indicate that PDDA is acting as a robust protective agent for both types of metallic nanoparticles (AgNPs and AuNPs) without any change in their corresponding aggregation state. Figure 5 shows a picture of the samples for PDDA-AgNPs, PDDA-AuNPs and combined PDDA-(Ag+Au)NPs as a function of variable molar concentration of the analyte. As it can be deduced of this figure, a colorimetric change from light yellow (control) to a total transparent color (1250 µM) is observed for PDDA-AgNPs sample. However, no color changes of the PDDA-AuNPs samples were obtained as a function of the molar concentrations of the target molecule. As a consequence, it is important to remark the better chemical stability of the AuNPs because no variation of this LSPR band at 520 nm has been
observed. Finally, this difference in sensitivity as well as resultant coloration for both type of metal nanoparticles is also observed for the PDDA-(Ag+Au)NPs samples, where initially a light orange coloration is presented (control), but a totally change in the resultant coloration towards light reddish color is appreciated for the highest H2O2 concentration (1250 µM), indicating the presence of AuNPs and oxidized AgNPs.
Figure 4: (a) UV-Vis spectra changes of the PDDA-AgNPs (control sample) in the presence of variable molar concentrations of H2O2 from 1.25 µM to 1250 µM; (b) UV-Vis spectra changes of the PDDA-AuNPs (control, black line) and after the addition of 1250 µM of hydrogen peroxide (red line).
Figure 5: Naked eye picture of the evolution of colorimetric changes of PDDA-AgNPs, PDDA-AuNPs and PDDA-(Ag+Au)NPs in the presence hydrogen peroxide molar concentrations from 1.25 µM to 1250 µM.
An important aspect to take in consideration is that LSPR phenomenon strongly depends on the refractive index of the surrounding medium, although other factors such as shape, size or interparticle distance also play a key role in the location of the LSPR absorption bands [9-
13]. Due to this, in order to discard if there is any influence of the refractive index in the resultant absorbance of the LSPR, the refractive index of all sample solution dispersions after the addition of hydrogen peroxide were measured. It is important to remark that the refractive index variation was negligible for the whole H2O2 range, remaining constant at 1.333. Consequently the real effect that is affecting to the optical properties of the sensing solution is only the oxidation of the AgNPs. In order to characterize the dependence of the LSPR absorption bands with refractive index changes, a very high variation of the refractive index of the nanoparticle dispersions have been performed by mixing different amounts of glycerin solutions. The experiment results were carried out separately for PDDA-AgNPs and for PDDA-AuNPs dispersions in order to see if there was any difference in sensitivity. In Figure 6, it is shown the UV-Vis spectra changes related to PDDA-AuNPs (Fig. 6a) as well as PDDA-AgNPs (Fig. 6b) for variable refractive index values from 1.333 to 1.377. There is a slight variation of the intensity of the LSPR bands, showing a growing pattern for AuNPs, whereas a decreasing pattern is observed for AgNPs. In addition, both nanoparticle dispersions showed a slight red-shift of their LSPR absorption bands (5nm for PDDA-AgNPs, and 3 nm for PDDA-AuNPs), which it is coherent with the results previously reported [49-52]. It has to be pointed out that the refractive index variations were very high, and even in these extreme conditions the LPSR absorption bands remained very stable compared with the changes observed when H2O2 is added to the samples.
Figure 6: UV-Vis spectra changes of the PDDA-AuNPs (a) and PDDA-AuNPs (b) in the presence of different glycerin solutions with variable refractive index values of 1.333, 1.340, 1.347, 1.362 and 1.377, respectively.
To sum up of the experimental results previously obtained, the oxidation related to AgNPs is the responsible of this great decrease in the absorbance of the LSPR absorption band at 400 nm for the PDDA-AgNPs as well as for PDDA-(Ag+Au)NPs samples when H2O2 molar concentration is gradually increased. However, no significant changes of the LSPR absorption band associated to the PDDA-AuNPs samples was obtained as a function of the molar concentrations of the target molecule. In addition, an important aspect to take in consideration is that LSPR related to AuNPs is used as an optical reference although this reference can´t be used to protect the measurements against other undesired effects that can interfere with the estimation of the concentration of H2O2. For example, the intensity-based nature of the absorbance measurements makes them very sensitive to slight dilutions of the sample. In this sense, the optical reference that provides the gold nanoparticles within the sample itself is very valuable, since any undesired effect that may interact with the measurement (dilution, temperature, etc.) could also affect to the AuNPs, showing our approach an enhanced robustness These changes in the dispersions were also studied using TEM. In Figure 7, it is shown the micrographs of the different nanoparticle dispersions. In Figure 7d, it can be seen how the silver nanoparticles change their aspect significantly associated to their chemical decomposition. These results are consistent with other recently published works [40, 41].
Figure 7: TEM micrographs of different colloidal dispersions for PDDA-AgNPs (a); PDDA-AuNPs (b); combined PDDA-(Ag+Au)NPs (c) and combined PDDA-(Ag+Au)NPs after exposure to hydrogen peroxide molar concentration of 500 μM (d). All the samples show the same scale bar of 100 nm.
This great difference in sensitivities can be used for the design of a dual reference colorimetric sensor due to this great difference in sensibilities of both LSPR absorption bands. More specifically, the LSPR band of the PDDA-AuNPs can be used as a wavelength fixed reference, while the LSPR band of the PDDA-AgNPs can be used to estimate the molar concentration of the target molecule due to its variation in absorbance. In addition, in order to evaluate the validity and stability of this dual reference probe colorimetric detection of hydrogen peroxide, a standard calibration curve has been performed as a function of the absorbance ratio of the LSPR bands at 520 nm to 400 nm (Abs520/Abs400) by varying different molar concentrations of H2O2 as it can be appreciated in Figure 8.
Figure 8: Linear fitting of absorbance ratio (Abs520/Abs400) changes for PDDA-(Ag+Au)NPs sample in the presence of variable molar concentrations of H2O2 from 1.25 µM to 1250 µM. Error bars indicate the standard deviation for three measurements.
As a summary of the experimental observations obtained by the previous Figures 2 and 3, upon incremental additions of H2O2 to the solution mixture of PDDA-(Ag+Au)NPs, the LSPR absorption peak at 520 nm was almost unaltered whereas the LSPR absorption peak at 400 nm was decreased. When an absorbance ratio (Abs520/Abs400) is plotted with respect to H2O2 concentration, a good sensitivity and linear relationship has been obtained in the range of 6.25-1250 µM with a favourable correlation of estimation (r2) of 0.9985. In addition, the value of limit of detection (LOD) was calculated based on the minimum quantity of H2O2 that could give a change in the absorbance ratio (taken to be 3 times the standard deviation at the minimum H2O2 concentration) and was found to be 1.2 µM. It is important to remark that this detection limit is comparable to the detection limit of 1µM reported in previous works [40, 41]. This sensor offers an enhanced robustness in the measurements thanks to the intrinsic optical reference provided by the PDDA-AuNPs which makes possible to estimate very
precisely the degradation of the PDDA-AgNPs regardless of other potential issues such as the dilution of the sample. The experimental results reported here indicate that this is the first time that a colorimetric self-referenced sensor based on the simultaneous incorporation of silver and gold nanoparticles in a same sample solution (PDDA-(Ag+Au)NPs) is presented in the bibliography for hydrogen peroxide detection. Finally, this self-referenced LSPR-optical sensor platform can be used for potential biosensing applications in the detection of other oxygen reactive species or well of other analytes in the future.
4. Conclusions In summary, we provide the first report for the design of a self-referenced colorimetric sensor for the quantitative determination of a target molecule such as hydrogen peroxide. In this study, it is observed a great difference in sensitivity related to the LSPR absorption bands of a sample solution composed of both PDDA-coated silver and gold nanoparticles (PDDA(Ag+Au)NPs). It is possible to estimate the molar concentration of the target molecule by measuring the absorbance strength of both LSPR absorption bands. A gradual decrease of the LSPR band (PDDA-AgNPs) is associated to the catalytic decomposition of the hydrogen peroxide which induces the aggregation and oxidation of the AgNPs. However, the LSPR band (PDDA-AuNPs) is practically unaltered thanks to the better chemical stability of the AuNPs. This aspect is the key for the design of this self-referenced LSPR optical detection method because the LSPR (PDDA-AuNPs) can be used as a wavelength fixed reference, while LSPR (PDDAAgNPs) can be used to determine the concentration of the hydrogen peroxide by establishing an absorbance ratio of the maxima absorbance of both LSPR bands. On the basis of this mechanism, a good sensitivity and a linear response over a wide concentration range from 1.25 to 1250 µM of target molecule is presented. In addition, it is important to remark that this colorimetric probe based on metal nanoparticles presents the advantages of being simple,
cost effective and a single step method without using sophisticated analytical instruments. Furthermore the referenced measurement of the AuNP/AgNP absorption ratio provides a high robustness against issues such as the dilution of the samples. Taking into account these advantages, this self-referenced LSPR optical colorimetric sensor for hydrogen peroxide detection opens up new perspectives for biosensing applications.
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Author’s biography
Pedro J. Rivero obtained his MS degree in Chemistry and Biochemistry in 2006 and 2007 respectively from the Catholic University of Navarra, Pamplona, Spain. He graduated in Materials Engineering Postgraduate Master from the Public University of Navarra (UPNA) and Science and Polymer Technology Postgraduate Master from the National Distance Education University (UNED) in 2010 and 2011, respectively. In 2011, he obtained a predoctoral scholarship and he received the PhD degree in 2014, working in the Department of Electrical and Electronic Engineering at UPNA. He has worked as a visiting researcher in the School of Physical Sciences at the Dublin City University (DCU), and in the Faculty of Engineering at the University of Lisbon. In 2016, he is working as Assistant Professor in the Department of Mechanical, Energetic and Materials Engineering at UPNA. . He has authored 18 scientific international publications and 3 book chapters (international edition).His main areas of interest are the research and development of nanostructured functional thin films, synthesis of metallic nanoparticles, optical fiber devices and their engineering applications. Elia Ibañez has received her MS degree Industrial Tecnnologies engineering from the Public University of Navarra (UPNA), Pamplona, Spain. Her main research lines are the synthesis of nanoparticles, fabrication of thin films and their engineering applications. Javier Goicoechea received his MS degree in electrical engineering and the PhD degree in 2003 and 2008 respectively from the Public University of Navarra (UPNA), Pamplona, Spain. In 2004 he obtained a predoctoral scholarship from the Spanish Ministry of Culture and Science. In 2010 he became lecturer at the Public University of Navarra. His research interest includes new thin films and nanomaterials for optical fiber sensor applications. He has authored more than 26 scientific international publications and 4 book chapters (international edition). He is Associate Editor of the journal “Optical Engineering” (SPIE).
Aitor Urrutia received his MS degree in telecom engineering, and the MSc degree in communications engineering in 2009 and 2011, respectively from Public University of Navarra (UPNA), Spain. In 2012, he obtained a predoctoral scholarship and he received the PhD degree in 2015, working in the Department of Electrical and Electronic Engineering at UPNA. He has worked as a visiting researcher in the School of Physical Sciences at the Dublin City University, and in the Faculty of Engineering at the University of Nottingham. He is author or coauthor of 13 international publications in journals and 18 national/international conference papers. His main areas of interests are the research and development of nanostructured functional coatings, optical fiber devices, sensors, and other engineering applications. Ignacio R. Matias received the M.S. degree in Electrical and Electronic Engineering and his Ph.D. degree in Optical Fiber Sensors from the Polytechnic University of Madrid (UPM), Madrid, Spain, in 1992 and 1996, respectively. He became a Lecturer at the Public University of Navarra (Pamplona, Spain) in 1996, where presently he is a Permanent Professor. He has coauthored more than 300 chapter books, journal and conference papers related to optical fiber sensors and passive optical devices and systems. He is a Senior Editor of IEEE Sensors Journal. His main interests are optical fiber sensors and nanostructured materials, although he frequently participates in several publications on domotics. Francisco J. Arregui is a Full Professor at the Public University of Navarra, Pamplona, Spain. He was part of the team that fabricated the first optical fiber sensor by means of the layer-bylayer self-assembly method at Virginia Tech, Blacksburg, VA, USA, in 1998. He is the author of more than 350 scientific journal and conference publications (cited around 3200 times, h-index of 32), most of them related to optical fiber sensors based on nanostructured coatings. He has been advisor of more than 70 Master Thesis and 7 PhDs. Francisco J. Arregui has been Founding Editor-in-Chief of “Journal of Sensors” as well as an Associate Editor of the Journals “IEEE Sensors Journal”, “Journal of Sensors” and “International Journal on Smart Sensing and Intelligent Systems”. Dr. Arregui regularly cooperates as evaluator for Science Agencies of
different countries as well as participates in the Technical Program Committee of sensorrelated conferences (IEEE Sensors, Eurosensors, ICST, SPIE). He is also editor of the books “Sensors based on nanostructured materials” and “Optochemical Nanosensors” and cofounder of several technology companies, among them, Nadetech Innovations S.L. and EverSens S.L.
S0925-4005(17)30933-4 http://dx.doi.org/doi:10.1016/j.snb.2017.05.110 SNB 22394
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Sensors and Actuators B
Received date: Revised date: Accepted date:
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Please cite this article as: Pedro J.Rivero, Elia Iba˜nez, Javier Goicoechea, Aitor Urrutia, Ignacio R.Matias, Francisco J.Arregui, A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.05.110 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A self-referenced optical colorimetric sensor based on silver and gold nanoparticles for quantitative determination of hydrogen peroxide
Pedro J. Rivero*1, Elia Ibañez2, Javier Goicoechea2, Aitor Urrutia2, Ignacio R. Matias3 and Francisco J. Arregui2.
1
Materials Engineering Laboratory, Department of Mechanical, Energetic and Materials
Engineering, Public University of Navarre. Campus Arrosadía S/N, 31006, Pamplona, Spain. 2
Nanostructured Optical Devices Laboratory, Department of Electrical and Electronic
Engineering, Public University of Navarre, Campus Arrosadía S/N, 31006, Pamplona, Spain. 3
Institute of Smart Cities, Public University of Navarre, Campus Arrosadía S/N, 31006, Pamplona, Spain.
*email: [email protected]
RESEARCH HIGHLIGHTS Synthesis of silver and gold nanoparticles by using a chemical reduction method. The changes of the absorbance strength maxima of both LSPR absorption bands are measured. A remarkable difference in sensitivity of both LSPR absorption bands are clearly observed. A self-referenced colorimetric sensor for the quantitative determination of hydrogen peroxide. A novel perspective for the chemical detection of other reactive oxygen species
Abstract In this work, a self-referenced colorimetric sensor for the quantitative determination of hydrogen peroxide is presented. This optical sensor is based on the presence of the Localized Surface Plasmon Resonances of silver and gold nanoparticles which are capped with the same encapsulating agent of poly(diallylammonium chloride) (PDDA). These metallic nanoparticles are synthesized by a chemical reduction method of their corresponding inorganic precursors and characterized by UV-Vis spectroscopy and transmission electron microscopy, respectively. A remarkable difference in sensitivity related to both LSPR absorption bands is observed as a function of variable molar concentration of the target molecule. The LSPR band of the silver nanoparticles is gradually decreased whereas the LSPR of the gold nanoparticles is practically unaltered when the hydrogen peroxide molar concentration is increased as a result of its better chemical stability. This stable absorbance LSPR band of the AuNPs is used as an optical reference and the molar concentration of the target molecule is obtained by measuring the changes of the absorbance strength maxima of both LSPR absorption bands. As a result, a very good sensitivity with a high robustness and a linear response over a wide concentration range from 1.25 µM to 1250 µM is obtained. This self-referenced method opens up a new perspective for the chemical detection of other reactive oxygen species or well for the determination of other type of analytes in the future.
Keywords Localized Surface Plasmon Resonance (LSPR), optical colorimetric sensor, silver and gold nanoparticles, self-referenced sensor, hydrogen peroxide detection.
1. Introduction In the last decades, the rapid proliferation of the nanotechnology has led to the fabrication and use of an increasing number of nanomaterials and nanoparticles in fields as diverse as biology, catalysis, chemistry, construction, electronics, medicine, textile or optics, among others [1-8]. One of the most explored characteristics of the noble metal nanoparticles (essentially silver, gold or copper) is a phenomenon known as Localized Surface Plasmon Resonance (LSPR), the frequency at which conduction electrons collectively oscillate in response to the alternating electric field of an incident electromagnetic radiation. As a result, an intense absorption band in the visible region with a specific coloration of the resultant nanoparticles is obtained [9, 10]. A wide variety of chemical routes for the synthesis of metal nanoparticles with different shapes can be found in bibliography [11-14], although the most common approach involves the chemical reduction of metal salts in the presence of an adequate encapsulating agent [15]. The encapsulating agent plays an important role for the synthesis of nanoparticles with a specific morphology because it limits the growth of the particles, directs their shape and provides colloidal stability [16]. The wavelength location of this LSPR absorption band shows a great dependence over several factors such as the resultant shape and size of the nanoparticles, the refractive index of their surrounding medium or the environmental in which the metal nanoparticles are dispersed [17-19]. In addition, other key factor is the average distance between neighboring metal nanoparticles because when the average particle distance is shortened to a distance below the diameter of the particles, an aggregation of the nanoparticles is performed. This aggregation leads to a total color change as a consequence of the LSPR coupling between nanoparticles and as a result, a red-shift of the LSPR absorption band is obtained [20]. This sensing mechanism based on the aggregation of silver or gold nanoparticles is the basis for the
design of a wide number of colorimetric biosensors because it is studied the change in coloration generated by the plasmon coupling between nanoparticles upon aggregation [2125]. However, other colorimetric sensing mechanisms have used the excellent optical properties of the noble metal nanoparticles as a colorful reporter by measuring the changes in the absorbance strength for the quantitative determination of a wide variety of analytes for biological or medical purposes [26-30]. One of the analytes that are causing a great interest in the medical community is the hydrogen peroxide because it is well-known Reactive Oxygen Specie (ROS) [31-35]. An excessive accumulation of this analyte in the body can trigger some diseases because the hydrogen peroxide is the responsible of causing tissue damage and DNA fragmentation. In addition, it is the product of reactions catalyzed by a large number of oxidase enzymes, and therefore its monitorization can lead to different bio-sensing applications. Therefore, the analytical determination of H2O2 also has an important significance in other totally different fields such as industry, food or environmental analysis. According to this multidisciplinary presence, the design and development of new methodologies for the quantitative determination of H2O2 are required. Most of the analytical methods for its quantification are based on enzymes, using different techniques such as chemiluminiscence, spectrofluometry and electrochemistry [36-38]. However, these techniques show several drawbacks such as the use of high sophisticated instrumentation, costly performance, need of a total immobilization of the enzymes in order to obtain a good sensitivity, long assay time, low selectivity or a poor reproducibility. From all these backgrounds, the use of other alternative method is required for the determination of this target molecule. Among all them, LSPR optical sensors based on the incorporation of metal nanoparticles are an ideal candidate due to their high sensitivity, great selectivity, simplicity and cost effective detection. Previous works have reported the quantification of hydrogen peroxide by using LSPR optical colorimetric sensors [26, 32, 39-42]
based on the optical changes of a unique LSPR absorption band related to only silver nanoparticles. In this work, it is reported for the first time a dual reference LSPR optical colorimetric sensor based on the combined colloidal dispersions of gold and silver nanoparticles with the same encapsulating agent for the quantitative determination of hydrogen peroxide. The metallic nanoparticles have been synthesized by a chemical reduction step from their corresponding inorganic salts. From the experimental results, it is concluded that a remarkable difference in sensitivity related to both LSPR absorption bands is observed as a function of variable molar concentration of the target molecule, making possible the design of a robust self-referenced LSPR optical detection method. As a promising result, a very good sensitivity and a linear response over a wide concentration range from 1.25 µM to 1250 µM of target molecule is presented. In addition, the experimental results show a lower quantification limit than previously reported enzymebased biosensors [43-48]. The hydrogen peroxide concentration range of the biosensor presented in this work is suitable for some biomedical applications such as oxidative stress estimation, measuring the hydrogen peroxide in human urine [31], as a simplified test for biomonitoring the glucose in blood or human serum [26, 33, 35, 40], or even for heavy metal ion detection such as mercury due to the catalytic reactivity of AgNPs [34]. Finally, the resultant dual LSPR optical sensor opens up a new perspective for the design and development of biosensors for monitoring other reactive oxygen species or well other analytes in the future.
2. Experimental section 2.1. Reagents and materials Poly(diallyldimethylammoniumchloride) (PDDA) (Mw. 15000), silver nitrate (>99% titration) solution in water 0.1 N,
gold (III) chloride trihydrate (HAuCl4.3H2O), borane
dimethylamine complex (DMAB) and hydrogen peroxide (H2O2) were purchased from Sigma
Aldrich, and used without further purification. The ultrapure H2O was purified to above 18.2 MΩ using a Mili-Q water system (Millipore).
2.2. Synthesis of the silver and gold nanoparticles A wet method based on the chemical reduction of metal salts has been used for the synthesis of capped AgNPs and AuNPs, respectively. As a source of silver and gold ions were the silver nitrate (AgNO3) and Gold (III) chloride trihydrate (HAuCl4. 3H2O), respectively. A reducing agent such as DMAB was the candidate for the reduction of the silver and gold ions to the corresponding nanoparticles. In addition, a polyelectrolyte such as PDDA has been used as a capping agent of the nanoparticles. An important aspect of this chemical method is the high stability of the synthesized nanoparticles (AgNPs, AuNPs) because no changes of the resultant coloration were observed after 6 months of storage without showing any sign of aggregation for both types of nanoparticles at room condition. For the synthesis of PDDA capped AgNPs, 0.100 mL of 0.1 N of AgNO3 was mixed with 15 mL of 25 mM of PDDA and stirred for 1 hour. After that, the addition of 0.500 mL of 0.1 M DMAB to the solution was performed with a constant stirring of 1 hour. A color change from transparent to yellow is indicative that capped-silver nanoparticles (PDDA-AgNPs) were successfully synthesized. For the synthesis of PDDA capped AuNPs, 0.100 mL of 0.1 N of HAuCl4.3H2O was mixed with 15 mL of 25 mM of PDDA and stirred for 1 hour. Then the addition of 0.025 mL of 0.1 M DMAB to the solution was performed with a constant stirring of 1 hour. The color of the solution was changed from pale yellow to red-wine, showing the formation of the capped-gold nanoparticles (PDDA-AuNPs). Finally, once both types of nanoparticles have been successfully synthesized, an equal volume ratio of 1:1 was taken in a glass vial and kept under continuous stirring at room temperature. After a prescribed reaction time of 30 minutes, a light orange coloration has
been observed, indicating that the combination of PDDA-AgNPs and PDDA-AuNPs (referred as PDDA-(Ag+Au)NPs) was obtained in a same solution.
2.3. Characterization of the metallic nanoparticles UV-visible (UV-Vis) spectroscopy has been used to characterize the optical properties of the PDDA-(Ag+Au)NPs. All absorbance spectra were taken from 350 to 900 nm on the UV-Vis spectrophotometer at room temperature. A Jasco V-630 spectrophotometer was used to perform all the measurements. In all cases of study, ultrapure water has been used as an optical reference. A transmission electron microscopy (TEM) was used to characterize the morphology and distribution of the nanoparticles. Samples for TEM were prepared by drop-casting the solution onto a collodion-coated copper grid. And then, the samples were dried up at room conditions. In addition, TEM observations were carried out with a Carl Zeiss Libra 120 using four different samples (PDDA-AgNPs, PDDA-AuNPs, PDDA-(Ag+Au)NPs and PDDA-(Ag+Au)NPs after addition of hydrogen peroxide). All the measurements have been performed at room condition. In order to evaluate the cross-sensitivity of the LSPR of the metallic nanoparticles with sample refractive index variations, different solutions of glycerin in water have been prepared with concentrations that provided refractive indices of 1.333, 1.340, 1.347, 1.362 and 1.3777, respectively. UV-Vis spectra have been performed by mixing 750 µL of metallic nanoparticles (PDDA-AgNPs and PDDA-AuNPs separately) with 750 µL of glycerin aqueous solution with a specific value of the refractive index. All the measurements were made at room conditions. The refractive index of the samples has been characterized using a Mettler-Toledo Refracto 30GS refractometer.
2.4. Detection of hydrogen peroxide concentration The analysis procedure was realized as follows. Firstly, 1.950 µL of PDDA-(Ag+Au)NPs was mixed with 50 µL of hydrogen peroxide water solution at different molar concentration. In this protocol the overall sample volume was kept constant (2 mL) and the only variable was the
amount of H2O2 added in order to avoid optical variations due to the dilution. The time lapse between the addition of the analyte and the optical characterization of the sample was established to 30 minutes for all samples as a convention. Finally, under these experimental conditions and after the addition of analyte, the optical changes of the PDDA-(Ag+Au)NPs have been investigated by UV-Vis measurements in order to implement as a potential colorimetric sensing application. The experiments have been performed for triplicate for the detection of the hydrogen peroxide molar concentration and the error bars indicate the standard deviation for the three measurements. In Figure 1, it is shown a schematic representation of the experimental process and the resultant setup used for the detection of peroxide concentration using PDDA coated silver and gold nanoparticles (PDDA-(Ag+Au)NPs).
Figure 1: Schematic representation of the procedure for obtaining PDDA-(Ag+Au)NPs and experimental setup used for hydrogen peroxide detection as a function of optical colorimetric changes.
3. Results and discussion 3.1 Synthesis of the metal nanoparticles First of all, the metal nanoparticles (AgNPs, AuNPs) have been successfully synthesized at room ambient using a chemical reduction method as a function of an adequate protective and reducing agent. This aspect is corroborated by UV-Vis spectroscopy in Figure 2 where the existence of a plasmon resonance band, known as Localized Surface Plasmon Resonance (LSPR) at a well-defined wavelength position, is clearly observed. This phenomenon occurs when the conduction electrons in metal nanostructures collectively oscillate, as a result of their interaction with an incident electromagnetic radiation. In this figure, the wavelength location related to the LSPR of the capped PDDA-AgNPs with a yellow color is observed at 402 nm, whereas the LSPR of the capped PDDA-AuNPs with a red-wine color is observed at 520 nm. This difference in the wavelength position of both LSPR absorption bands by using the same capping agent (PDDA) is of vital importance for a further combination in a specific molar ratio with the aim to obtain two well-defined LSPR bands. As it was commented in the Experimental Section, after a prescribed reaction time of both types of nanoparticles, a light orange coloration related to PDDA-(Ag+Au)NPs is observed and the corresponding UV-Vis spectrum clearly shows both LSPR peaks at 400 nm and 520 nm, respectively.
Figure 2: UV-Vis spectra of the synthesized PDDA-AgNPs (yellow coloration), PDDA-AuNPs (red-wine coloration) and PDDA-(Ag+Au)NPs (light orange coloration) with the specific location of the LSPR absorption bands.
3.2. Quantitative determination of hydrogen peroxide concentration Once it has been obtained two well-separated LSPR peaks in a spectral range of 120 nm for a same sample solution (PDDA-(Ag+Au)NPs), variable molar concentrations of hydrogen peroxide were tested in order to find out the sensing ability of both LSPR absorption bands. In Figure 3, it can be appreciated the difference behavior in sensitivity of both LSPR absorption bands to the hydrogen peroxide concentrations from 1.25 µM to 1250 µM. When the molar concentration of H2O2 is increased, a gradual reduction of the absorbance strength of the LSPR band corresponding to the AgNPs is clearly observed, whereas the absorbance strength of the LSPR band related to the AuNPs is almost unaltered. In addition, an important aspect observed in this work is that a significant amount of bubbles are generated when PDDA-(Ag+Au)NPs are mixed with the hydrogen peroxide. This phenomenon of bubbles formation has been
corroborated in previous works with only silver nanoparticles [39-42] where a violent reaction is generated by the catalytic reaction between silver and hydrogen peroxide. In this work, the polyelectrolyte of PDDA which is acting as a protective agent of the nanoparticles is used as a good candidate for catalyzing the hydrogen peroxide decomposition.
Figure 3: UV-Vis spectra changes of the PDDA-(Ag+Au)NPs in the presence of different molar concentrations of H2O2 from 1.25 µM to 1250 µM.
In order to corroborate this great difference in sensitivities of both types of metallic nanoparticles to the hydrogen peroxide, the optical dependence with H2O2 concentration of PDDA-AgNPs was studied separately from the PDDA-AuNPs. This separate analysis is helpful to determine the exact nature of the optical changes (chemical decomposition, aggregation, etc.) induced by H2O2 in the final combined biosensor. In Figure 4a, it is shown the UV-Vis spectra changes of the PDDA-AgNPs as a function of variable H2O2 molar concentration from 1.25 µM to 1250 µM. In this figure, it can be clearly observed that an increase of the molar concentration induces a gradual decrease of the absorbance strength related to the LSPR absorption band, as it has been previously observed in Figure 3. In addition, no red-shift
related to LSPR band at 400 nm was observed what it means that no changes in the aggregation state of the AgNPs are obtained. This gradual decrease of the absorbance value related to the LSPR band has been observed in previous works by using other different encapsulating agents [41, 42]. The reason for changing the intensity of the LSPR absorption band is associated to a catalytic reaction between the AgNPs and hydrogen peroxide through the PDDA layer which is acting as a protective layer. As a result, a gradual decomposition of the silver nanoparticles is obtained with a successive oxidative process. Due to this, the LSPR absorption band is drastically decreased for higher H2O2 molar concentrations (from 1.7 a.u. for 1.25 µM to 0.4 a.u. for 1250 µM) which is associated to the change from metal (Ag) to oxide nature (Ag2O), inducing a loss in the plasmonic modes without any coupling of light which is directly associated to the decrease of the intensity of the LSPR absorption band at 400 nm. Furthermore, it is important to remark that this H2O2-induced LSPR band annihilation is only observed for PDDA-AgNPs, while LSPR associated to PDDA-AuNPs showed a high stability. In Figure 4b, it is shown UV-Vis spectra of the PDDA-AuNPs solutions when are exposed to a H2O2 molar concentration as high as 1250 µM. The UV-Vis spectra corroborate that PDDAAuNPs are almost unaltered after the addition of the analyte with a remarkable stability in the location of the LSPR band. The experimental results clearly indicate that PDDA is acting as a robust protective agent for both types of metallic nanoparticles (AgNPs and AuNPs) without any change in their corresponding aggregation state. Figure 5 shows a picture of the samples for PDDA-AgNPs, PDDA-AuNPs and combined PDDA-(Ag+Au)NPs as a function of variable molar concentration of the analyte. As it can be deduced of this figure, a colorimetric change from light yellow (control) to a total transparent color (1250 µM) is observed for PDDA-AgNPs sample. However, no color changes of the PDDA-AuNPs samples were obtained as a function of the molar concentrations of the target molecule. As a consequence, it is important to remark the better chemical stability of the AuNPs because no variation of this LSPR band at 520 nm has been
observed. Finally, this difference in sensitivity as well as resultant coloration for both type of metal nanoparticles is also observed for the PDDA-(Ag+Au)NPs samples, where initially a light orange coloration is presented (control), but a totally change in the resultant coloration towards light reddish color is appreciated for the highest H2O2 concentration (1250 µM), indicating the presence of AuNPs and oxidized AgNPs.
Figure 4: (a) UV-Vis spectra changes of the PDDA-AgNPs (control sample) in the presence of variable molar concentrations of H2O2 from 1.25 µM to 1250 µM; (b) UV-Vis spectra changes of the PDDA-AuNPs (control, black line) and after the addition of 1250 µM of hydrogen peroxide (red line).
Figure 5: Naked eye picture of the evolution of colorimetric changes of PDDA-AgNPs, PDDA-AuNPs and PDDA-(Ag+Au)NPs in the presence hydrogen peroxide molar concentrations from 1.25 µM to 1250 µM.
An important aspect to take in consideration is that LSPR phenomenon strongly depends on the refractive index of the surrounding medium, although other factors such as shape, size or interparticle distance also play a key role in the location of the LSPR absorption bands [9-
13]. Due to this, in order to discard if there is any influence of the refractive index in the resultant absorbance of the LSPR, the refractive index of all sample solution dispersions after the addition of hydrogen peroxide were measured. It is important to remark that the refractive index variation was negligible for the whole H2O2 range, remaining constant at 1.333. Consequently the real effect that is affecting to the optical properties of the sensing solution is only the oxidation of the AgNPs. In order to characterize the dependence of the LSPR absorption bands with refractive index changes, a very high variation of the refractive index of the nanoparticle dispersions have been performed by mixing different amounts of glycerin solutions. The experiment results were carried out separately for PDDA-AgNPs and for PDDA-AuNPs dispersions in order to see if there was any difference in sensitivity. In Figure 6, it is shown the UV-Vis spectra changes related to PDDA-AuNPs (Fig. 6a) as well as PDDA-AgNPs (Fig. 6b) for variable refractive index values from 1.333 to 1.377. There is a slight variation of the intensity of the LSPR bands, showing a growing pattern for AuNPs, whereas a decreasing pattern is observed for AgNPs. In addition, both nanoparticle dispersions showed a slight red-shift of their LSPR absorption bands (5nm for PDDA-AgNPs, and 3 nm for PDDA-AuNPs), which it is coherent with the results previously reported [49-52]. It has to be pointed out that the refractive index variations were very high, and even in these extreme conditions the LPSR absorption bands remained very stable compared with the changes observed when H2O2 is added to the samples.
Figure 6: UV-Vis spectra changes of the PDDA-AuNPs (a) and PDDA-AuNPs (b) in the presence of different glycerin solutions with variable refractive index values of 1.333, 1.340, 1.347, 1.362 and 1.377, respectively.
To sum up of the experimental results previously obtained, the oxidation related to AgNPs is the responsible of this great decrease in the absorbance of the LSPR absorption band at 400 nm for the PDDA-AgNPs as well as for PDDA-(Ag+Au)NPs samples when H2O2 molar concentration is gradually increased. However, no significant changes of the LSPR absorption band associated to the PDDA-AuNPs samples was obtained as a function of the molar concentrations of the target molecule. In addition, an important aspect to take in consideration is that LSPR related to AuNPs is used as an optical reference although this reference can´t be used to protect the measurements against other undesired effects that can interfere with the estimation of the concentration of H2O2. For example, the intensity-based nature of the absorbance measurements makes them very sensitive to slight dilutions of the sample. In this sense, the optical reference that provides the gold nanoparticles within the sample itself is very valuable, since any undesired effect that may interact with the measurement (dilution, temperature, etc.) could also affect to the AuNPs, showing our approach an enhanced robustness These changes in the dispersions were also studied using TEM. In Figure 7, it is shown the micrographs of the different nanoparticle dispersions. In Figure 7d, it can be seen how the silver nanoparticles change their aspect significantly associated to their chemical decomposition. These results are consistent with other recently published works [40, 41].
Figure 7: TEM micrographs of different colloidal dispersions for PDDA-AgNPs (a); PDDA-AuNPs (b); combined PDDA-(Ag+Au)NPs (c) and combined PDDA-(Ag+Au)NPs after exposure to hydrogen peroxide molar concentration of 500 μM (d). All the samples show the same scale bar of 100 nm.
This great difference in sensitivities can be used for the design of a dual reference colorimetric sensor due to this great difference in sensibilities of both LSPR absorption bands. More specifically, the LSPR band of the PDDA-AuNPs can be used as a wavelength fixed reference, while the LSPR band of the PDDA-AgNPs can be used to estimate the molar concentration of the target molecule due to its variation in absorbance. In addition, in order to evaluate the validity and stability of this dual reference probe colorimetric detection of hydrogen peroxide, a standard calibration curve has been performed as a function of the absorbance ratio of the LSPR bands at 520 nm to 400 nm (Abs520/Abs400) by varying different molar concentrations of H2O2 as it can be appreciated in Figure 8.
Figure 8: Linear fitting of absorbance ratio (Abs520/Abs400) changes for PDDA-(Ag+Au)NPs sample in the presence of variable molar concentrations of H2O2 from 1.25 µM to 1250 µM. Error bars indicate the standard deviation for three measurements.
As a summary of the experimental observations obtained by the previous Figures 2 and 3, upon incremental additions of H2O2 to the solution mixture of PDDA-(Ag+Au)NPs, the LSPR absorption peak at 520 nm was almost unaltered whereas the LSPR absorption peak at 400 nm was decreased. When an absorbance ratio (Abs520/Abs400) is plotted with respect to H2O2 concentration, a good sensitivity and linear relationship has been obtained in the range of 6.25-1250 µM with a favourable correlation of estimation (r2) of 0.9985. In addition, the value of limit of detection (LOD) was calculated based on the minimum quantity of H2O2 that could give a change in the absorbance ratio (taken to be 3 times the standard deviation at the minimum H2O2 concentration) and was found to be 1.2 µM. It is important to remark that this detection limit is comparable to the detection limit of 1µM reported in previous works [40, 41]. This sensor offers an enhanced robustness in the measurements thanks to the intrinsic optical reference provided by the PDDA-AuNPs which makes possible to estimate very
precisely the degradation of the PDDA-AgNPs regardless of other potential issues such as the dilution of the sample. The experimental results reported here indicate that this is the first time that a colorimetric self-referenced sensor based on the simultaneous incorporation of silver and gold nanoparticles in a same sample solution (PDDA-(Ag+Au)NPs) is presented in the bibliography for hydrogen peroxide detection. Finally, this self-referenced LSPR-optical sensor platform can be used for potential biosensing applications in the detection of other oxygen reactive species or well of other analytes in the future.
4. Conclusions In summary, we provide the first report for the design of a self-referenced colorimetric sensor for the quantitative determination of a target molecule such as hydrogen peroxide. In this study, it is observed a great difference in sensitivity related to the LSPR absorption bands of a sample solution composed of both PDDA-coated silver and gold nanoparticles (PDDA(Ag+Au)NPs). It is possible to estimate the molar concentration of the target molecule by measuring the absorbance strength of both LSPR absorption bands. A gradual decrease of the LSPR band (PDDA-AgNPs) is associated to the catalytic decomposition of the hydrogen peroxide which induces the aggregation and oxidation of the AgNPs. However, the LSPR band (PDDA-AuNPs) is practically unaltered thanks to the better chemical stability of the AuNPs. This aspect is the key for the design of this self-referenced LSPR optical detection method because the LSPR (PDDA-AuNPs) can be used as a wavelength fixed reference, while LSPR (PDDAAgNPs) can be used to determine the concentration of the hydrogen peroxide by establishing an absorbance ratio of the maxima absorbance of both LSPR bands. On the basis of this mechanism, a good sensitivity and a linear response over a wide concentration range from 1.25 to 1250 µM of target molecule is presented. In addition, it is important to remark that this colorimetric probe based on metal nanoparticles presents the advantages of being simple,
cost effective and a single step method without using sophisticated analytical instruments. Furthermore the referenced measurement of the AuNP/AgNP absorption ratio provides a high robustness against issues such as the dilution of the samples. Taking into account these advantages, this self-referenced LSPR optical colorimetric sensor for hydrogen peroxide detection opens up new perspectives for biosensing applications.
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Author’s biography
Pedro J. Rivero obtained his MS degree in Chemistry and Biochemistry in 2006 and 2007 respectively from the Catholic University of Navarra, Pamplona, Spain. He graduated in Materials Engineering Postgraduate Master from the Public University of Navarra (UPNA) and Science and Polymer Technology Postgraduate Master from the National Distance Education University (UNED) in 2010 and 2011, respectively. In 2011, he obtained a predoctoral scholarship and he received the PhD degree in 2014, working in the Department of Electrical and Electronic Engineering at UPNA. He has worked as a visiting researcher in the School of Physical Sciences at the Dublin City University (DCU), and in the Faculty of Engineering at the University of Lisbon. In 2016, he is working as Assistant Professor in the Department of Mechanical, Energetic and Materials Engineering at UPNA. . He has authored 18 scientific international publications and 3 book chapters (international edition).His main areas of interest are the research and development of nanostructured functional thin films, synthesis of metallic nanoparticles, optical fiber devices and their engineering applications. Elia Ibañez has received her MS degree Industrial Tecnnologies engineering from the Public University of Navarra (UPNA), Pamplona, Spain. Her main research lines are the synthesis of nanoparticles, fabrication of thin films and their engineering applications. Javier Goicoechea received his MS degree in electrical engineering and the PhD degree in 2003 and 2008 respectively from the Public University of Navarra (UPNA), Pamplona, Spain. In 2004 he obtained a predoctoral scholarship from the Spanish Ministry of Culture and Science. In 2010 he became lecturer at the Public University of Navarra. His research interest includes new thin films and nanomaterials for optical fiber sensor applications. He has authored more than 26 scientific international publications and 4 book chapters (international edition). He is Associate Editor of the journal “Optical Engineering” (SPIE).
Aitor Urrutia received his MS degree in telecom engineering, and the MSc degree in communications engineering in 2009 and 2011, respectively from Public University of Navarra (UPNA), Spain. In 2012, he obtained a predoctoral scholarship and he received the PhD degree in 2015, working in the Department of Electrical and Electronic Engineering at UPNA. He has worked as a visiting researcher in the School of Physical Sciences at the Dublin City University, and in the Faculty of Engineering at the University of Nottingham. He is author or coauthor of 13 international publications in journals and 18 national/international conference papers. His main areas of interests are the research and development of nanostructured functional coatings, optical fiber devices, sensors, and other engineering applications. Ignacio R. Matias received the M.S. degree in Electrical and Electronic Engineering and his Ph.D. degree in Optical Fiber Sensors from the Polytechnic University of Madrid (UPM), Madrid, Spain, in 1992 and 1996, respectively. He became a Lecturer at the Public University of Navarra (Pamplona, Spain) in 1996, where presently he is a Permanent Professor. He has coauthored more than 300 chapter books, journal and conference papers related to optical fiber sensors and passive optical devices and systems. He is a Senior Editor of IEEE Sensors Journal. His main interests are optical fiber sensors and nanostructured materials, although he frequently participates in several publications on domotics. Francisco J. Arregui is a Full Professor at the Public University of Navarra, Pamplona, Spain. He was part of the team that fabricated the first optical fiber sensor by means of the layer-bylayer self-assembly method at Virginia Tech, Blacksburg, VA, USA, in 1998. He is the author of more than 350 scientific journal and conference publications (cited around 3200 times, h-index of 32), most of them related to optical fiber sensors based on nanostructured coatings. He has been advisor of more than 70 Master Thesis and 7 PhDs. Francisco J. Arregui has been Founding Editor-in-Chief of “Journal of Sensors” as well as an Associate Editor of the Journals “IEEE Sensors Journal”, “Journal of Sensors” and “International Journal on Smart Sensing and Intelligent Systems”. Dr. Arregui regularly cooperates as evaluator for Science Agencies of
different countries as well as participates in the Technical Program Committee of sensorrelated conferences (IEEE Sensors, Eurosensors, ICST, SPIE). He is also editor of the books “Sensors based on nanostructured materials” and “Optochemical Nanosensors” and cofounder of several technology companies, among them, Nadetech Innovations S.L. and EverSens S.L.