Keywords

3.1 Introduction

Metal nanoparticles (NPs) find application in a wide array of fields including catalysis [1] chemical and bio sensors [2, 3], antibacterial substances [4] and drug delivery systems [5]. Noble metal nanoparticles , in particular, such as those of Au and Ag show intense Localized Surface Plasmon Resonance (LSPR) absorption in the visible region [6] and have potential applications as optical biosensors [7, 8] or catalytic systems [9, 10] for initiating a variety of organic reactions. The high surface area provided by these nanoparticles make them extremely efficient as catalytic materials to carry out reactions that are otherwise difficult to initiate [11–16]

The use of metal nanoparticles as analytical and bioanalytical sensors has been receiving significant attention because of their unusual optical, electronic, and chemical properties [17–19]. A large number of methods have been developed for the fabrication of metal nanoparticles. However, the major disadvantages in most of the methods include use of toxic reducing chemicals, poor nanoparticle size distribution, and inhomogeneous dispersion of the nanoparticles in the polymer host [20]. Ionizing radiation (Gamma radiation, Electron Beam, etc.) induced radiolytic route is an efficient alternative technique for fast and one step synthesis of uniformly dispersed metal nanoparticles [21–24]. The use of ionizing radiation for the synthesis of noble metal nanoparticles is promising, as reactive water radiolytic species (eaq −, H⋅) with sufficiently high reduction potential, are generated in-situ on irradiation of aqueous precursor solutions. The easy handling of radiation parameters, namely, absorbed dose and dose rate, offers better control over the size and the size distribution of the metal nanoparticles. Moreover, radiation technology offers the added advantage of being a clean, ambient and room temperature process devoid of use of any external chemical reducing agents. The reduced metal atoms gradually coalesce to form metal nanoparticles whose size is regulated by suitable capping/stabilizing agents, such as PVP. The Localized Surface Plasmon Resonance (LSPR) band of these metal nanoparticles can be utilized as a highly sensitive tool for estimation of various analytes, thereby providing an appropriate, simplified, cheap and rapid alternative to more sophisticated detection techniques. The LSPR wavelength is extremely sensitive to the local environment around the nanoparticles, which facilitates their use as sensing devices [25].

The choice of capping/stabilizing agent during synthesis of metal nanoparticles is also crucial since it decides the overall stability and morphology of the particles generated, which in turn decides the long term utility of the system as a facile catalytic or a sensor system. Therefore, it is imperative to use capping agents that provide optimum stability to the nanoparticle systems and make their use economically viable. For instance, the use of biocompatible polymers like Poly (N-vinyl-2-pyrrolidone) (PVP) as the capping agent [26, 27] further enhances the viability of radiation synthesized metal nanoparticles to detect biologically relevant molecules without disturbing the natural environment of the biological samples in which the estimation is usually done.

In this chapter, we highlight some of the recent applications of radiolytically synthesized noble metal nanoparticles (Au and Ag) as LSPR based optical sensors for the estimation of trace levels of various important analytes.

3.2 Synthesis of Ag/Au Nanoparticles

60Co ‐ gamma and Electron beam irradiation are the two primary radiation sources conventionally used for radiolytic synthesis of noble metal nanoparticles. Prior to irradiation of the samples, radiation doses of the sources are determined by suitable dosimetry techniques, such as Fricke dosimetry [28] for 60Co gamma sources and radiochromic film dosimeter for electron beam sources. When an aqueous solution containing Ag+ or Au3+ precursor ion solution of known concentration, a stabilizer (PVP, Methacrylic acid etc.) and 2-propyl alcohol is purged with N2 and subjected to radiation, radiolysis of water takes place. As a result, reactive transient species, namely, eaq −, H•, •OH, etc., are generated (Eq. 3.1). 2-propyl alcohol present in the reaction medium reacts with H• and •OH to give 2-propyl radical, a mild reducing agent (Eq. 3.2). Simultaneously, the oxidizing radical •OH gets eliminated in the process. The reducing species present in the system viz. eaq − and 2-propyl radical reduce the metal ion to metal in zero valent state.

(3.1)
$$ {}^{\bullet}\mathrm{O}\mathrm{H}/{\mathrm{H}}^{\bullet }+{({\mathrm{C}\mathrm{H}}_3)}_2\mathrm{C}\mathrm{H}-\mathrm{O}\mathrm{H}\to {({\mathrm{C}\mathrm{H}}_3)}_2{\mathrm{C}}^{\bullet }-\mathrm{O}\mathrm{H}+{\mathrm{H}}_2\mathrm{O}/{\mathrm{H}}_2 $$
(3.2)
$$ {\mathrm{M}}^{\mathrm{n}+}+{\mathrm{n}\mathrm{e}}_{\mathrm{aq}}^{-}\to {\mathrm{n}\mathrm{M}}^0 $$
(3.3)
$$ {\mathrm{M}}^{\mathrm{n}+}+n{({\mathrm{C}\mathrm{H}}_3)}_2{\mathrm{C}}^{\bullet }-\mathrm{O}\mathrm{H}\to {\mathrm{n}\mathrm{M}}^0+n{({\mathrm{C}\mathrm{H}}_3)}_2\mathrm{C}=\mathrm{O}+n{\mathrm{H}}^{+} $$
(3.4)

Subsequently, metal atoms in the zero valent state undergo coalescence to form metal colloids whose size and shape are controlled by the concentration and nature of capping/stabilizing agent used. The functional groups present in the capping agent , such as >C = O and >N- groups (in case of PVP), facilitate anchorage and subsequent stabilization of metal nanoparticles. The progress of the reduction reaction can be monitored spectrophotometrically since Ag/Au nanoparticles absorb strongly in the UV-visible region (LSPR band).

The extent of reduction of metal ions to metal atoms depends on the amount of absorbed radiation dose. The minimum dose required to achieve near complete reduction of a given concentration of precursor ions is termed as the saturation dose . Therefore, irradiations are generally carried out so as to impart saturation dose to the precursor ions in order to ensure near complete reduction of metal ions to metal atoms in the zero valent state.

3.3 Characterization of Metal Nanoparticles

The characterization of metal nanoparticles is important to determine the extent of reduction, the size, shape and morphology of the particles formed. The most commonly used techniques for nanoparticle characteriazation are UV-visible spectrophotometry, Transmission Electron Microscopy (TEM ), X-ray Diffraction (XRD) Analysis , Atomic Force Microscopy (AFM ) and Light Scattering based techniques, such as Particle Size Analyzer (PSA ).

Noble metal nanoparticles, such as those of gold and silver, show characteristic intense absorption in the UV-visible region, which is attributed to the Localized Surface Plasmon Resonance (LSPR) based absorption . The intensity, wavelength of absorption maxima and the width of the LSPR band rely on several factors, such as the precursor ion concentration, morphology of the particles formed, as well as on the local environment of the nanoparticles (nature of solvent, capping agents, etc.). These parameters, therefore, provide us with a fair idea about the overall nature of the nanoparticles formed. For instance, spherical Ag nanoparticles in the size range of 8–10 nm show characteristic LSPR band at around 400 nm. The yield of Ag nanoparticles, manifested by the intensity of the LSPR band of Ag nanoparticles, increases with increase in absorbed radiation dose till all precursor Ag+ ions are exhausted. However, an increase in particle size (when the interparticle spacings become less than the nanoparticle diameter) conventionally leads to a shift in the LSPR band toward higher wavelength (red shift), whereas a decrease in particle size can lead to a blue shift in the peak position. The FWHM of the LSPR band can also be used to predict the distribution in particle size, with a broad size distribution typically resulting in a correspondingly broad LSPR band. Anisotropic nanoparticles, if formed, can also result in the appearance of more than one band depending on the aspect ratio of the particles formed (transverse and longitudinal bands), instead of a single band as is the case with spherical particles.

Although UV-visible spectroscopy provides us with a rough idea about the nature of particles formed, the most reliable technique for accurately determining the size and shape of metal nanoparticles is Transmission Electron Microscopy (TEM) . TEM analysis method can be used to capture images of particles with diameters in the nanometer regime. The particle size distribution and the shape of the particles formed can also be determined accurately using this technique. Besides TEM analysis, Atomic Force Microscopy ( AFM), Scanning Electron Microscopy (SEM) and Light Scattering techniques, such as Particle Size Analyzer are also used to gather information about nanoparticle morphology. X-ray Diffraction (XRD ) analysis can also provide valuable information about the composition of nanoparticles, their purity, size, shape, distribution, orientation, etc. XRD, in fact, offers unparalleled accuracy in the measurement of atomic spacing and is the technique of choice for determining strain states in thin films. The intensities measured with XRD can provide quantitative and accurate information on the atomic arrangements at interfaces. Zeta Potential analysis is another technique that is often used to determine the stability of nano colloids. The magnitude of the zeta potential gives an indication of the stability of the nanoparticle suspension system. If all the particles in suspension have a large negative or positive zeta potential then they will tend to repel each other and there will be less tendency for the particles to come together to form agglomerates.

All these techniques have been widely used, either individually or in combination, to characterize metal nanoparticle systems. These techniques have been immensely useful in providing new dimensions to the field of nanomaterials and have opened up new areas of research, which in turn, have yielded remarkable outputs.

3.4 Applications of Radiation Synthesized Noble Metal Nanoparticles: LSPR Based Sensor Applications

The LSPR band of noble metal nanoparticles is a highly sensitive and potent tool that can be employed for estimation of a vast array of biologically and environmentally relevant analytes. Radiolytically synthesized Au and Ag nanoparticles in particular offer an attractive option as LSPR based optical sensors for estimation of a number of important analytes. The highly intense, unique and tunable LSPR bands of Au and Ag NPs can be efficiently exploited owing to the distinct changes they exhibit in response to variations in their chemical environment. For instance, DNA-modified gold nanoparticles [29–31] and thiol-functionalized gold nanoparticles have been recently used for colorimetric estimation of Hg2+ ions [32–34]. Gold NPs functionalized with an antibody anti-CA15-3-HRP were also implemented in a traditional ELISA immunoassay to detect a breast cancer biomarker present in blood [35]. Pregnancy test kits , one of the most widely used devices in the world today, also make use of gold nanoparticles and their colorimetric properties. Carbohydrate stabilized nanoparticles have been successfully used to detect carbohydrate binding proteins, such as lectin concanavalin A [36]. Aggregation based chemical sensing has also been used to monitor a large number of enzymatic reactions. The list of such applications is endless as large scale, cutting edge research in the field of nanoscience is opening up new dimensions in the field with each passing day. Newer, more sophisticated materials and nano based techniques are being designed which have immensely contributed in areas previously unheard of. All these possibilities make nanomaterials one of the brightest prospects for the future. Some of the recent work done in the field of radiolytically synthesized metal nanoparticles based sensors is discussed in this chapter.

A sensor is a device that uses a particular reaction for detecting target analytes in the presence of interfering substances. Schematic of a typical sensor device with different essential components is shown in Fig. 3.1 . In case of metal nanoparticles based sensors, the nanoparticle system acts as the recognition element that selectively interacts with the target analyte under study. The change in the LSPR band characteristics of metal nanoparticles induced by nanoparticle-analyte interaction serves as a measurable output, which can be correlated to the analyte concentration. The detector most commonly used to detect such outputs is a simple UV-visible spectrophotometer. Based on the spectrophotometer output, linear relations can be obtained between analyte concentrations and LSPR band intensities, which can subsequently be used to determine unknown concentrations of the given analyte.

Fig. 3.1
figure 1

Schematic representation of the principle of operation of a sensor

Full size image

3.4.1 PVP Stabilized-Au NPs for H2O2 Estimation

Hydrogen peroxide is widely used as an oxidant, a disinfectant and a bleaching agent in various industries, such as textile, paper and pulp, pharmaceutical industries [37]. Its presence in the environment is detrimental to human health since it causes irritation to eye, skin and mucous membrane. Hydrogen peroxide is also produced in stoichiometric amounts during the oxidation of biological analytes (e.g., glucose) by dissolved oxygen in the presence of corresponding oxidase. Hence micro and trace level determination of hydrogen peroxide is considerably important in clinical chemistry, analytical biochemistry and environmental science. Existing methods for the determination of hydrogen peroxide include titrimetry [38], spectrophotometry [39], kinetic flow-injection method [40], fluorescence [41], enzymatic method [42], chromatographic techniques [43] and electrochemical methods [44]. However, most of these conventional techniques are invariably complicated, time-consuming, expensive as well as inconvenient for point-of-use applications. Recently developed methods for determination of hydrogen peroxide include the use of Au NPs and a peroxidase-catalyzed reaction [45]. Radiolytically synthesized Au NPs have been proved to be highly efficient for such detection applications since they are green, clean systems devoid of any external chemical reagents (which are generally present in chemically synthesized nanoparticle systems) and therefore, provide minimum scope of interference during detection or estimation [8].

We have recently developed a detection method for H2O2 estimation, which is based on radiolytically synthesized PVP stabilized-Au NPs [8]. Substrate orthophenylenediammine (o-PDA) undergoes catalytic oxidation by H2O2 in presence of enzyme Horse radish peroxidise (HRP). The oxidation product of o-PDA has a weak absorption peak at 427 nm. Addition of PVP stabilized-Au NPs of pre determined concentration has been found to enhance the absorption intensity of the oxidation product of o-PDA. This may be due to the interaction of Au NPs with the functional groups of 2, 3- diaminophenazine, which is the final oxidation product of o-PDA [46, 47] (Fig. 3.2). With varying concentration of H2O2 there is a systematic increase in the intensity of the absorption peak of o-PDA (λmax = 427 nm) in presence of PVP stabilized Au NPs, as shown in Fig. 3.3. The response is linear in the range of 2.5 × 10−6 mol dm−3 to 2.0 × 10−4 mol dm−3 H2O2 concentration (Fig. 3.3 inset). Lower concentrations of H2O2 can also be determined by simply reducing the concentration of the substrate o-PDA while keeping all other reagents and experimental procedures intact. The overall response of the system is linear in the H2O2 concentration range of 1.0 × 10−7 mol. dm−3 to 2.0× 10−4 mol dm−3, with a minimum detection limit of 1.0 × 10−7 mol. dm−3

Fig. 3.2
figure 2

Enzymatic oxidation of o-PDA

Full size image
Fig. 3.3
figure 3

UV–vis spectra o f reaction medium containing o-PDA, HRP, H2O2 and Au NPs in citrate buffer with increasing H2O2 concentrations Inset: Linear plot of OD410 nm vs H2O2 concentration (Revised from Ref. [8])

Full size image

3.4.2 PVP Stabilized-Au NPs for Hg2+ Estimation

Au NPs have also been found to be effective in the detection of Hg2+ in aqueous solutions. Mercury is one of the most widespread pollutants found in nature, existing in a variety of different forms such as metallic, ionic and as part of organic and inorganic salts and complexes [29]. The highly toxic nature of mercury necessitates its quantification, particularly in aquatic ecosystems, up to extremely low concentration levels [48]. Colorimetric methods of Hg2+ detection using metal nanoparticles, such as those of gold and silver, have emerged as an attractive technique owing to their simplicity, accuracy and robustness. Techniques based on Au-Hg amalgam formation induced aggregation of Au NPs, catalytic reduction of Hg2+ by Ag NPs [30] have been reported.

Recently, radiolytically synthesized PVP stabilized spherical Au nanoparticles have been employed by us for the detection and estimation of trace levels of Hg2+ in aqueous solutions [49]. These sensors work on the principle of preferential interaction of Hg2+ with PVP, which gets manifested as a change in the LSPR band characteristics of Au NPs with varying concentration of Hg2+ ions (Fig. 3.4). The newly designed sensor system was found to have reasonably good specificity and showed linear response within a concentration range of 0–100 nM Hg2+ ion concentration (Fig. 3.4 inset). The simplicity and acuracy of this technique makes it a potential candidate for real life applications.

Fig. 3.4
figure 4

UV–vis spectra of PVP-Au-NPs solution at varying concentrations of Hg2+ ion. Inset: Linear plot of OD vs Hg2+ ion concentration (Revised from Ref. [49])

Full size image

3.4.3 PVP Stabilized-Ag NPs for Uric Acid Estimation

Uric acid represents the major catabolite of purine breakdown in humans . The normal concentration of uric acid in blood samples is reported to be in the range 150–420 μM [50]. High levels of uric acid in the blood (hyperuricemia or Lesch-Nyhan syndrome) are linked with gout and other conditions including increased alcohol consumption, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease [51, 52]. On the other hand, abnormally low uric acid levels are symptoms of diseases, such as multiple sclerosis. Hence estimation of uric acid in blood can be used as a diagnostic tool for monitoring a large number of diseases. Furthermore, uric acid is an antioxidant in human adult plasma and is involved in various pathological changes [53]. In view of this, numerous techniques have been developed over the years for detection and estimation of uric acid levels.

Recently, a gamma radiolytically synthesized PVP stabilized-Ag NPs based optical sensor has been fabricated for use as a uric acid biosensor [7]. Uric acid undergoes enzymatic degradation in presence of enzyme Uricase under optimum assay conditions of 37 °C and pH 7.4 (Fig. 3.5). Hydrogen peroxide is generated as one of the reaction products, which is known to be a strong oxidizing agent. This, in turn, causes oxidation/degradation of silver nanoparticles, resulting in a decrease in intensity of the LSPR band. The schematic of working principle of PVP stabilized-Ag NPS based uric acid biosensor is presented in Fig. 3.5.

Fig. 3.5
figure 5

Enzymatic d egradation of uric acid in presence of Uricase and schematic of working principle of PVP stabilized-Ag NPS based uric acid biosensor

Full size image

For the evaluation of PVP stabilized-Ag NPs solution as a uric acid biosensor, a predetermined volume of Uricase stock solution is added to different concentrations of uric acid in a phosphate buffer medium at room temperature. Addition of Ag NPs solution to these reaction mixtures results in a variation in the LSPR band of the silver nanoparticles with increasing concentration of uric acid, as evident from the UV-visible spectra recorded in the wavelength range 250–650 nm (Fig. 3.6) . The LSPR band intensity decreases gradually with increase in uric acid concentration due to the degradation of Ag NPs. The decrease is accompanied by a slight red shift in the absorption maxima of the band, which is attributed to the partial oxidation of nano-Ag [54–57]. The shift in λmax might also be due to the slight aggregation caused by the destruction of the PVP shell stabilizing the nanoparticles followed by decrease in the distance between the nanoparticles [58].

Fig. 3.6
figure 6

UV–vis spectra of PVP stabilized-Ag NPs solution (PVP, Mol wt. = 40kD) with increasing concentration of uric acid. Inset: Linear plot of change in OD of Ag NPs vs uric acid concentration (Revised from Ref. [7])

Full size image

The change in particle size is evident from the TEM images of PVP stabilized-Ag NPs recorded before and after addition of uric acid (Fig. 3.7a and b). While the control (Ag-NPs) exhibited an average particle size of 8–10 nm, those in presence of uric acid were found to have size in the range of 10–20 nm. These observations were also substantiated using AFM analysis.

Fig. 3.7
figure 7

TEM micrograph and particle size distribution of PVP stabilized-Ag NPs prepared using PVP of molecular weight 40 kD (a) before addition of uric acid (b) after addition of uric acid

Full size image

The decrease in the magnitude of zeta potential indicates the tendency of the particles to form agglomerates [59]. In addition, zeta potential also indicates the presence of an oxidized surface layer. Zeta potential measurements can, therefore, be used in this case to further confirm the partial oxidation of the silver nanoparticles by in-situ generated hydrogen peroxide. With increase in uric acid concentration, the zeta potential values become less negative (Fig. 3.8), although the stabilities of the nanoparticle suspensions are not disturbed significantly. This is probably due to partial neutralization of the negative charge by Ag+ ions generated via partial oxidation of nano Ag. It has been well established that electrostatic stabilization of nanoparticles would typically require a zeta potential above 30 mV or below −30 mV [60]. Therefore, in the present study, the values of zeta potentials clearly suggest that the stability of the PVP stabilized-Ag NPs suspension is predominantly based on steric stabilization by the PVP polymer. In contrast to electrostatic stabilization, steric stabilization with nonionic polymers is independent of pH and electrolyte concentration. Accordingly, steric stabilization is useful for prevention of agglomeration of nanoparticles in physiological media .

Fig. 3.8
figure 8

Zeta potential of Ag NPs as a function of uric acid concentration (Revised from Ref. [7])

Full size image

3.4.3.1 Estimation of Uric Acid in Bovine and Human Serum Samples

The concentration of uric acid in serum samples is normally found in the micromolar range. Therefore, the proposed method was effectively applied for determination of uric acid concentration in serum samples. To minimize interference from proteins present in the serum sample, samples were initially deproteinized and then subjected to the estimation protocol. The concentrations of uric acid in both bovine and human serum samples were estimated using the calibration plot generated using standard uric acid solutions of known concentrations.

3.4.4 PMA Stabilized-Ag NPs for Dopamine Estimation

Dopamine (DA) belongs to the class of catecholamines , which are compounds containing a dihydroxyphenyl group and an amine group [61]. These biomolecules act as neurotransmitters and aid in the functioning of the brain and nerve signal transductions. Dopamine deficiency can lead to diseases, such as parkinsons, whereas mental disorders, such as schizophrenia and bipolar disorder have been attributed to excess dopamine in the system. In recent years, many methods have been developed for the estimation of dopamine in pharmaceutical preparations and biological samples. Many of these techniques for neurotransmitter detection, however, require expensive and sophisticated instrumentation or complicated sample preparation and time consuming processes. Thus, the development of a new, sensitive, fast and practical method for neurotransmitter detection still remains a great challenge.

Recently, we have developed a sensor for Dopamine, which was based on radiolytically synthesized polymethacrylate (PMA) stabilized-Ag NPs system . Dopamine, or 3,4 dihydroxy phenethylamine is a two electron donor and behaves as a mild reductant. The functional groups can interact with polymethacrylate stabilized-Ag NPs (Fig. 3.9) when present in solution, resulting in the alteration of the LSPR band characteristics of the nanoparticle system [13]. In fact, in this case, a pronounced colour change is observed, thereby making the interaction very conspicuous. This phenomenon can be quantified by recording the absorption spectra of PMA stabilized-Ag NPs in presence of varying concentrations of DA. The observed increase in LSPR band intensity with increase in DA concentration can be attributed to the reduction of residual Ag+ ions present in the system, since the dose delivered in this case was less than the saturation dose required for complete reduction of Ag+ ions. These unreduced Ag+ ions are adsorbed onto the surface of the reduced Ag nanoclusters, resulting in an increase in their reduction potentials. These surface adsorbed Ag+ ions are subsequently easily reduced by DA molecules, which act as mild reducing agents in presence of NaOH by losing an H+ ion. Higher DA concentrations will lead to increased concentrations of freshly reduced Ag+ ions, which are either deposited on the radiolytically formed Ag clusters or can form new Ag nanoclusters on their own. Thus, there is a possibility of increase in the Ag NP size as well as in the size distribution, which is manifested by the increase in intensity and broadening of the LSPR band. There is no shift in the peak position, because for spherical nanoparticles, a small change in particle size does not affect the plasmon band position.

Fig. 3.9
figure 9

Schematic of Ag NPs stabilization by polymethacrylate chains

Full size image

3.4.4.1 Estimation of Dopamine in Presence of Ascorbic Acid

Conventionally, DA was estimated by electrochemical analysis. A common problem associated with electrochemical analyses at unmodified electrodes is the lack of selectivity due to the presence of interfering compounds. For instance, ascorbic acid (AA) oxidizes at nearly the same potential as DA. Also AA coexists with DA in the extracellular fluids of mammalian brain. Therefore, it is imperative to study the interference of AA in the estimation of DA.

The interference of AA in DA estimation was studied by adding 1.0 × 10−4 mol.dm−3 AA to PMA stabilized-Ag NPs in presence of different concentrations of DA. The intensity of the LSPR band of PMA stabilized-Ag NPs at ~415 nm gradually increases with increasing DA concentration even in the presence of AA. It was observed that with optimization of experimental parameters, the detection system could be made free of interference from AA concentrations as high as 1.0 × 10−4 mol dm−3 within the DA estimation range of 5.27 × 10−7 to 1.05 × 10−5 mol.dm−3.

3.5 Conclusion

In the recent past, there has been rapid growth in the field of nanoscience and technology. The applications of nanomaterials have permeated into areas previously undreamt and unheard of. Countless sensors and analytical tools have been designed and developed which have immensely benefited mankind and shall continue to do so in the near future. It can only be envisaged that their popularity will grow manifold in the coming years with each new breakthrough and innovation. The future of mankind, we can sanguinely conclude, lies in switching over from the macro to the nano.