Holographic sensors for the determination of ionic strength

Analytica Chimica Acta 527 (2004) 13–20

Holographic sensors for the determination of ionic strength Alexander J. Marshall1 , Duncan S. Young, Satyamoorthy Kabilan, Abid Hussain, Jeff Blyth, Christopher R. Lowe∗ Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK Received 16 June 2004; received in revised form 11 August 2004; accepted 11 August 2004 Available online 5 October 2004

Abstract Holographic sensors for monitoring ionic strength have been fabricated from charged sulphonate and quaternary ammonium monomers, incorporated into thin, polymeric hydrogel films which were transformed into volume holograms. The diffraction wavelength or reflected colour of the holograms was used to characterise their swelling or de-swelling behaviour as a function of ionic strength in various media. The effects of co-monomer structure, buffer composition, ion composition, pH and temperature were evaluated, whilst the reversibility and reproducibility of the sensor was also assessed. An acrylamide-based hologram containing equal molar amounts of negatively and positively charged monomers was shown to be able to quantify ionic strength independent of the identity of the ionic species present in the test solution. The sensor was fully reversible, free of hysteresis and exhibited little response to pH between 3 and 9 and temperature within the range 20–45 ◦ C. The system was successfully used to quantify the ionic strength of milk solutions, which contain a complex mixture of ions and biological components. © 2004 Elsevier B.V. All rights reserved. Keywords: Hologram; Sensor; Hydrogel; Ionic strength

1. Introduction The measurement of electrolyte content and ionic strength is important in the environmental, agricultural, food, beverage, biotechnology and biomedical industries. For example, in the healthy body, electrolytes are subject to tight regulation by the kidneys due to balancing absorption and excretion processes. Disorders of electrolyte homeostasis may result from metabolic disturbances associated with coronary heart disease, angina pectoris, acute myocardial infarction, diabetes mellitus, dehydration, renal failure and chronic alcohol abuse [1]. Currently, there is an urgent requirement for inexpensive, mass-producible point-of-care diagnostics for the measurement of electrolytes in biological fluids such as ∗

Corresponding author. Tel.: +44 1223 334160; fax: +441223334162. E-mail addresses: [email protected] (A.J. Marshall), [email protected] (C.R. Lowe). 1 Tel.: +44 1233 334152. 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.08.029

blood, urine, saliva, sweat and tears to aid diagnosis and monitor the progress of electrolyte therapies. Furthermore, in the environmental sector, the monitoring of water quality, in particular dissolved salt is a crucial aspect of the maintenance of public health. The monitoring of water quality and salinity is also of major importance in the food and beverage industry as well as in many industrial processes. Whilst it is important to be able to determine the concentration of individual electrolytes, it is often desirable to obtain a general measure of electrolyte content in many cases. In such situations, sensors that can determine ionic strength are of interest [2]. In this context, there is much interest in the use of stimuli-sensitive hydrogels as ionic strength sensors since the hydration of these polymers is known to vary with the ionic strength of the bathing solution [3–5]. Hydrogels can be prepared to undergo large, reversible changes in solventswollen volume in response to specific stimuli including pH [6 and Refs. therein], ions [4,7], temperature [8,9], electric fields [10] solvents [11] and specific chemical [12,13] or

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biological analytes [14]. Such hydrogels are often referred to as stimuli-sensitive or “smart” polymers and a sensor can be constructed by coupling this selective swelling to a suitable transduction mechanism. For example, a polymer bead has been used to control the position of a reflecting surface relative to two parallel fibre optics, one for illumination and the other for collection of the modulated light [15–17]. Variation in the size of the polymer bead, as a function of ionic strength, determined the intensity of the collected signal, although the inherent fragility of this arrangement precluded its use as a practical sensor. More recently, a sensor system based on measuring the change in pressure accompanying polymer-swelling has been described [18]. A number of polymer-swelling based optical sensor principles have been introduced which rely on diffractive or interferometric techniques to monitor salt concentration. The metal-island coated swelling polymer over mirror system (MICSPOMS) is an optical reflection interference system that has been applied to construct chemical sensors and the feasibility of this approach for ionic strength sensing has been demonstrated [19–22]. However, despite the fact that the sensor displays a strong visually perceptible colour change, the spectral peak shape is complex, making measurements difficult, and fabrication requires very precise deposition of the sensing polymer films. An alternative transduction approach comprises a crystalline colloidal array of polymer spheres (∼100 nm diameter) polymerised within a hydrogel that swells or shrinks reversibly in the presence of appropriate analytes [23–25]. The crystalline colloidal array diffracts light at visible wavelengths in a manner governed approximately by Bragg’s law and determined by the lattice spacing: mλ = 2nd sin θ where m is the diffraction order, λ the wavelength of light in vacuo, n the average refractive index of the system, d the spacing of the diffracting plane and θ is the glancing angle between the incident light propagation direction and the diffracting planes. This colloidal array has been made sensitive to pH and ionic strength through partial hydrolyzation of the supporting acrylamide hydrogel [25,26]. Whilst these crystalline colloidal arrays represent an attractive approach for the fabrication of visually readable sensors, we have proposed an alternative and more generic technique that exploits the concept of a simple reflection hologram as the interactive element in a truly mass-producible biochemical sensor [6,11,27–32]. When holographic diffraction gratings are illuminated by white light they act as sensitive wavelength filters. Conventionally, the gratings comprise a gelatin-silver halide photographic emulsion and are fabricated by passing a single collimated laser beam through a holographic plate backed by a mirror. Interference between the incident and reflected beams produces a latent image [33], which after development and fixing steps, manifests as a modulated refractive index in the form of fringes lying in planes parallel to the gelatin surface

and approximately half a wavelength apart within the 10 ␮m thickness of the gelatin film. Under white light illumination, the developed grating acts as a reflector of the light for a specific narrow band of wavelengths and holographically recreates the monochromatic image of the original mirror used in its construction. The constructive interference between partial reflections from each fringe plane gives a characteristic spectral peak with a wavelength governed by the Bragg equation [28]. Any physical, chemical or biological mechanism that changes the spacing of the fringes (d), for example, by causing the supporting polymeric medium to swell, or the average refractive index (n), will generate observable changes in the wavelength (colour) of the reflection hologram [27]. In this report, we describe a further development of the technology in which appropriate hydrogels have been used as the basis for ionic strength sensitive holograms.

2. Experimental 2.1. Materials All chemicals were of analytical grade unless otherwise stated. 1,1 -Diethyl-2,2 -cyanine iodide (photosensitising dye), 2,2 -azobis(2-methylpropionamidine) dihydrochloride (AIBA), 3-(trimethoxysilyl)propyl methacrylate, 2-acrylamido-2-methyl-1-propanesulphonic acid (AMPS), (3-acrylamidopropyl)trimethylammonium chloride (75% (w/v) solution in water) (ATMA), 4-methylaminophenol sulphate (Metol), calcium chloride dihydrate, magnesium chloride (1 M, volumetric standard), methacrylamide, potassium bromide, potassium chloride, sodium carbonate, sodium chloride, disodium hydrogen phosphate, sodium nitrate and silver nitrate (1 M, volumetric standard), and were purchased from Aldrich Chemical Company, The Old Brick Yard, Gillingham, Dorset, U.K. Acrylamide (Electrophoresis Grade), and N,N -methylene-bis-acrylamide (MBA) (Electrophoresis Grade) were purchased from Sigma Chemical Company, Fancy Road, Poole, Dorest, U.K. 2-(N-morpholino) ethanesulphonic acid (hydrate) (MES), and chloroactetate were purchased from Acros Organics, Janssens, Pharmaceuticalaan, 3A, 2440, Geel, Belgium. Sodium thiosulphate (Hypo) and acetic acid (Glacial) were purchased from Fisher Scientific Ltd., Bishop Meadow Road, Loughborough, Leicestershire, LE115RG, U.K. Sodium carbonate was purchased from BDH (Merck) Ltd., Poole Dorset, U.K., whilst Bicine was purchased from Avocado (Research Chemicals Ltd.), Shore Road, Heysham, Lancs., U.K. Whole cows milk was purchased from a local store. 2.2. Equipment and instrumentation Microscope slides (Super Premium, 1–1.2 mm thick, low iron) were purchased from BDH (Merck) Ltd., Poole Dorset, U.K. Aluminized, 100 ␮m polyester film (grade MET401) was purchased from HiFi Industrial Film Ltd.,

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Stevenage, U.K. A UV exposure unit (∼350 nm, model number: 555-279) was purchased from RS Components, Birchington Road, Weldon Industrial Estate, Corby, Northants, NN179RS. A frequency-doubled Nd:YAG laser (350 mJ, 532 nm, Brilliant B, Quantel, France) was used in all hologram recording work whilst holograms were analysed using an LOT-ORIEL MS127i Model 77480 imaging spectrograph in single channel mode with a 256 × 1024 pixel InstaSpec IV CCD detector and processing software. 2.3. Synthesis of sensing polymer films The required volumes of monomer solutions to give a prepolymer solution with the correct molar ratio of the monomers were mixed and diluted with deionised water to form a 45.2% (w/v) stock solution. A 5% (w/v) solution of the free radical initiator 2,2 -azobis(2-methylpropionamidine) dihydrochloride (AIBA) in water was added to a final concentration 5% (v/v) in the solvent and the mixture briefly vortexed. A 100 ␮l aliquot of monomer solution was pipetted onto the polyester side of an aluminised polyester sheet resting on a clean flat surface. A glass microscope slide, presubbed with methacryloxypropyl-triethoxysilane as described previously [11], was then gently lowered, silane treated side down, onto the solution and any trapped air bubbles gently squeezed out. The films were polymerised by UV initiated free radical reaction at 20 ◦ C under UV light for 30 min. Polymerised films were carefully peeled off the aluminised polyester substrate whilst submerged in deionised water. To remove any unpolymerised material and by products of the polymerisation reaction, films were washed several times in deionised water. Prior to hologram construction, the edges of each film were cleaned with a scalpel blade to remove any excess polymer material to give a smooth, flat polymer surface. 2.4. Hologram construction All photosensitization, exposure and development work was carried out under bright red safe lighting. An of aqueous silver nitrate solutions (0.3 M; 400 ␮l) was pipetted as an elongated droplet onto a clean glass sheet and the preformed polymer film lowered onto it, polymer side down. This was left for 2 min to allow the solution to soak into the polymer before excess surface liquid was removed using a soft rubber squeegee. The film was dried briefly under a tepid air current and then submerged polymer side uppermost, in a continuously agitated bath of 4% (w/v) potassium bromide solution (40 ml, in methanol/water 1:1 (v/v)), cyanine dye (1 ml, 0.1% (w/v) 1,1 -diethyl-2,2 -cyanine iodide in methanol) and ascorbic acid (5 mg/l) for 1 min. Films were removed to a continuous stream of water and washed thoroughly before being dried and stored in a light tight box. Photosensitive films were placed polymer side downwards in an exposure bath [6] filled with deionised water. Photosensitive films were exposed to five 10 ns pulses from the

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Nd:YAG laser, washed in deionised water and immersed, polymer side up, in developer solution. Developer solution was a modified formula based on the standard Saxby developer [33] and was made up as two separate solutions, which were mixed immediately before hologram development. Solution A contained 40 g/l ascorbic acid and 6 g/l 4-methylaminophenol in distilled water and solution B contained 100 g/l anhydrous Na2 CO3 and 30 g/l NaOH in distilled water. The holograms were developed by immersion in an agitated solution with a 4:1 (v/v) A:B developer solution. The time spent in the developer was typically around 10 s leading to an optical density of 1–2. In all cases, films were removed to a stream of water before immersion into a bath of stop solution (5% (v/v) acetic acid) for approximately 1 min to arrest development. Excess dye and undeveloped silver bromide were removed by immersion in agitated 20% (w/v) sodium thiosulphate for 4 min, when slides were rinsed under running tap water, and then in methanol for a further 5 min to remove all traces of the cyanine dye. Completed holographic gratings were rinsed in deionised water and air dried before being stored in sealed polythene bags until use. 2.5. Hologram interrogation and testing Holographic devices were interrogated using an in-house built reflection spectrophotometer as described previously [29]. Individual holographic sensors, approximately 8–9 mm wide, were placed, polymer side facing inward, into 4 ml cuvettes into which 1 ml samples of test solution were introduced. These test solutions were kept at a constant temperature of 30 ± 0.1 ◦ C, unless otherwise stated, using a thermostated jacket with a circulating water bath and stirred at a constant rate with a magnetic microflea/stirrer arrangement. Holographic sensors were tested using the following pH buffer systems: chloroacetate (pH 3.0), acetate (pH 4.0 and 5.0), MES (pH 6.0 and 7.0) and Bicine (pH 8.0 and 9.0). For each solution, the buffer components were used at a concentration of 10 mM and the final ionic strength of the solutions was fixed for 200 mM using NaCl. Salt and buffer solutions were prepared in ultra pure deionised water.

3. Results and discussion 3.1. Fabrication of ionic strength-sensitive holograms A series of holograms comprising polymers bearing charged groups were synthesised and tested to select one with the appropriate characteristics for use as an ionic strength sensor. Thin acrylamide-based hydrogel films (∼10 ␮m thick) were fabricated with positively charged monomers (20 mol% (3-acrylamidopropyl)trimethylammonium chloride, ATMA), negatively charged monomers (20 mol% 2acrylamido-2-methyl-1-propanesulphonic acid, AMPS), or both (10 mol% ATMA and 10 mol% AMPS) (Fig. 1). Control films were also produced in the absence of any

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Fig. 1. Ionised monomers used in holographic sensor construction.

ionised monomer and in all cases 5 mol% N,N -methylenebis-acrylamide (MBA) was included as a crosslinker. The copolymer films were synthesized by solution polymerisation in water by UV-initiated free radical polymerisation in a simultaneous copolymerisation and crosslinking reaction. Methacrylamide was added to the prepolymer mixture because crosslinked polyacrylamide alone produced mechanically weak films that were easily damaged by shear and were therefore unsuitable for use as holographic recording materials. Copolymers of acrylamide and methacrylamide at a ratio 2:1 mol% were far more robust and adhered well to the silanized slides, thus, making them practical to work with. For use as holographic recording materials, it was necessary to optimize the prepolymer composition and polymerisation protocol to produce clear, transparent films that showed no evidence of phase separation. Holograms were fabricated in the preformed polymer films by immersing them sequentially in solutions of silver nitrate and potassium bromide containing a photosensitising dye, 1,1 -diethyl-2,2 -cyanine iodide [34]. Following exposure of the film to laser light [30] and a conventional photographic development step, an interference fringe pattern comprising ultra-fine (<20 nm diameter) grains of metallic silver (Ag0 ) spaced λ/2 apart is generated within the thickness of the polymer film. Illumination of the developed grating under white light recreates the monochromatic image of the plane mirror used in its construction with the constructive interference at each fringe plane resulting in a characteristic spectral peak with a wavelength governed by the Bragg equation. Any physical or chemical mechanism that alters the spacing (d) of the fringes, for example, by changing the swelling state of the supporting hydrogel film, or the average refractive index (n), results in observable changes in the diffraction wavelength (colour) of the reflection hologram. 3.2. Hologram testing The holograms displayed no holographic image when dry, presumably because in their collapsed state they replayed in the UV region of the spectrum. When the dry holograms were

immersed in deionised water they absorbed water and a blue holographic image was visible with a replay wavelength of ∼492 nm. All the constructed holograms replayed at a wavelength of about 492 nm, which is consistent with the exposure wavelength of 532 nm. The slight decrease of replay wavelength compared to the construction wavelength is normal for reflection holograms since processing removes material from the hologram interior resulting in a slight shrinkage of the polymer film [33]. All holograms were tested for their sensitivity to NaCl concentrations up to 500 mM at 30 ◦ C. Fig. 2(a) shows the diffraction spectrum of a polyacrylamide-co-AMPS-coATMA hologram as a function of NaCl concentration. As the salt concentration of the bathing medium was increased from 0 to 500 mM, the peak wavelength of the diffraction spectrum red-shifted by 58 nm from its initial value of 492 nm to a final value of 550 nm as the hologram changed colour from blue,

Fig. 2. (a) Spectrograph showing the effect of increasing salt concentration at 30 ◦ C on the diffraction spectra of a polyacrylamide hologram containing 5 mol% MBA as crosslinker, 10 mol% ATMA and 10 mol% AMPS. (b) Peak diffraction wavelength as a function of salt concentration at 30 ◦ C of a polyacrylamide hologram containing 5 mol% MBA, 10 mol% ATMA and 10 mol% AMPS.

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Fig. 3. The effect of increasing salt concentration at 30 ◦ C on the diffraction wavelength of a series of polyacrylamide holograms containing 5 mol% MBA and either no ionised monomer (䊉), 20 mol% AMPS (), 20 mol% ATMA (
) or 10 mol% AMP and 10 mol% ATMA (). The error bars represent the standard deviation of three separate measurements using the same holographic sensor.

through green to yellow. The peak shape of the diffraction spectrum was maintained throughout this process. Fig. 2(b) shows a calibration curve depicting the non-linear relationship between peak diffraction wavelength and salt concentration for the polyacrylamide-co-AMPS-co-ATMA hologram. Fig. 3 shows the response of the polyacrylamide-coAMPS-co-ATMA hologram co-plotted with those for holograms constructed with only one type of ionised monomer and with that obtained for a neutral control. Unlike the polyampholytic polyacrylamide-co-AMPS-co-ATMA hologram, those holograms constructed with only positively or negatively charged monomers blue-shifted as the salt concentration of the bathing medium was increased. This is similar to the responses observed with the MICSPOMS system [20] and the polymerised crystalline colloidal array [25] in response to increasing ionic strength. In contrast, the control hologram constructed with neutral monomers displayed a small red-shift of 8 nm under the same conditions. Swelling of hydrogels is encouraged by the hydrophilic nature of the polymer chains but is limited by the degree of crosslinking of the network [35]. Hydrogels containing fixed charges along the polymeric backbone swell to a greater extent than their uncharged counterparts [3–5] as the presence of fixed charges on the network generates a Donnan potential causing counterions to migrate into the gel to maintain electroneutrality. The concentration of mobile ions inside the gel is therefore greater than in solution giving rise to an osmotic pressure that causes the gel to imbibe more water [37]. Combined with charge repulsion between fixed charges in the network and an increase in the hydrophilicity of the polymer backbone, this osmotic pressure effect leads to a higher degree of equilibrium swelling [36]. As the ionic strength of the bathing medium increases, the swelling decreases due

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to the fact that the concentration of ions within the gel phase and the bulk solution begin to equalize and hence the osmotic swelling pressure decreases. These changes in volume can be followed by monitoring the diffraction wavelength of the incorporated Bragg grating. As the volume of the polyanionic (20 mol% AMPS) and polycationic (20 mol% ATMA) holograms decreases, the spacing of the associated Bragg planes is concomitantly decreased, resulting in a shorter wavelength being reflected by the holographic mirror. On the other hand, the polyampholytic hologram (10 mol% AMPS and 10 mol% ATMA) was observed to swell as the NaCl concentration was increased and displayed a so-called “antipolyelectrolyte effect” [36–39]. The positive and negative charges in the hydrogel attract one another to form an ionically crosslinked network. These electrostatic crosslinks restrict the swelling of the hydrogel as the length of the polymer chain between crosslinks is decreased, leading to a greater loss of entropy for a given expansion [37]. Externally added salts interrupt these ionic interactions and decrease the physical crosslinking in the network, thereby causing the hydrogel layer to swell and concomitantly redshifting the diffraction wavelength. The polyampholytic hologram was chosen for further study as an ionic strength sensor by virtue of the fact that its response mechanism resulted in a red-shift across the visible spectrum. Hence, a visually perceptible response to salt concentration is obtained offering the potential for use in a semi-quantitative format in the absence of a spectrometer. In contrast, the polycationic and polyanionic holograms blueshift in response to increasing salt concentration and this displaces the diffraction spectra further into the blue/violet end of the visible spectrum. Furthermore, it is well known that polyelectrolyte gels swell less in the presence of multivalent counterions at a given ionic strength [40–46]. Qualitatively, such ions satisfy the requirement for a corresponding number of monovalent counterions in the Donnan equilibrium and therefore the mobile counterion concentration, and hence osmotic potential in the gel phase at a given ionic strength, is decreased. On a more quantitative level, however, swelling equilibria in the presence of divalent ions does not fit Donnan equilibrium theory [3,46,47] and more complex swelling processes have been observed that are not yet well understood. The polyampholytic hologram was not susceptible to this complication in the measurement of ionic strength in the presence of multivalent ions, and responded only to the ionic strength of the solution. 3.3. Sensor characterisation and testing in crude biological samples The polyampholytic hologram was tested with a series of clinically and environmentally relevant ions. The effect of increasing ionic strength at 30 ◦ C, obtained by varying the concentration of a number of different salts, on the holographic diffraction wavelength is shown in Fig. 4. Clearly, the response of the sensor is independent of the nature of the

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Fig. 4. The effect of a range of different salts on the diffraction wavelength of a polyacrylamide hologram containing 5 mol% MBA, 10 mol% ATMA and 10 mol% AMPS. The holographic sensor was tested with KCl (䊉), NaCl (), NaNO3 (
), MgCl2 () and CaCl2 ().

ion under investigation and the electrostatic interactions that control the hydration state of the hologram are dependent only on the ionic strength of the bathing medium. The effect of variation in pH in 1 pH step units over the range pH 3.0–9.0 was determined at a fixed ionic strength of 200 mM and at 30 ◦ C. When the polyampholytic hologram was tested using the following pH buffer systems: chloroacetate (pH 3.0), acetate (pH 4.0 and 5.0), MES (pH 6.0 and 7.0) and Bicine (pH 8.0 and 9.0) the diffraction wavelength was observed to vary by ∼1 nm over this pH range. This is consistent with the fact that the ionisation state of the sulphonic acid groups (pKa ∼ 1.9) is not expected to change over the pH range investigated, whilst the network quaternary amine bears a permanent positive charge. In addition, the combined effects of pH and ionic strength were investigated at 30 ◦ C using a series of sodium phosphate buffers between pH 3.0 and 7.4. For each pH value, three buffer solutions were prepared at ionic strength values of 15, 75 and 150 mM. In line with the above result, the pH dependency at a given ionic strength was also less than 1 nm. Furthermore, the resultant diffraction wavelength as a function of ionic strength for each pH buffer system, including those prepared with multivalent phosphate anions, corresponded to the curve depicted in Fig. 4 suggesting that the presence of the various buffer salts used in the pH experiments did not affect the hydration of the hologram beyond the effects of ionic strength. These findings highlight the suitability of these holograms for use as reference sensors, particularly for hydrogel and pH indicator based sensing systems, to allow compensation for the background ionic strength of the monitored media. Finally, to assess the utility of the device to determine ionic strength in crude biological samples, a polyampholytic hologram was tested in whole cows milk warmed to 30 ◦ C. Milks are very complex biological fluids containing thousands of different components. The approximate composi-

Fig. 5. The response of a polyampholytic hologram in the presence of serial milk dilutions at 30 ◦ C co-plotted with the response to various ion solutions (from Fig. 4).

tion of whole cows milk by weight is water, lactose (4.6%), milk fat (3.7%), minerals (0.65%) and protein (3.4%), whilst the ionic strength is approximately 0.08 M [47]. Three fresh whole milk samples were tested. In each case, an unadulterated sample, a sample diluted two-fold and one diluted 10-fold (both with deionised water) were tested (i.e., approximately 80, 40 and 8 mM ionic strength milk samples) and in between readings, the holographic sensor was washed with deionised water. Fig. 5 shows the diffraction wavelength of the hologram in the presence of the serial dilutions of milk, contrasted to the response with various ion solutions. The calibrated holographic response correlated well with the defined values of the ionic strength of the milk solutions demonstrating the utility of these sensors to measure ionic strength in complex biological samples. As the reflected signal was monitored through the rear of the device that is not in contact with the test solution, and because the wavelength rather than the intensity of the reflected light is measured, the analytical signal from the hologram was seemingly unaffected by the light scattering properties of the milk sample. Previously, reflection holograms have been demonstrated to be suitable for use in highly coloured or turbid samples when tested in a series of different alcoholic beverages [11]. 3.4. The effect of temperature Many hydrogels undergo changes in hydration in response to temperature, which affects the ability of the solvent to interact with the polymer [8,9,48]. The diffraction wavelength of the polyampholytic hologram was tested, as a function of temperature, in 5 ◦ C steps from 20 to 45 ◦ C, in deionised water in the absence and presence of 200 mM NaCl. In each case, a 7 nm increase in diffraction wavelength was observed as the temperature was increased from 20 to 45 ◦ C. These findings are similar for polyacrylamide based holographic

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pH sensors containing 4, 6 and 8 mol% acrylic acid, which displayed a negligible response to temperature at any point along the pH titration curve [6]. Similar results demonstrating low temperature dependency were obtained for IPCCA sensors fabricated from carboxylated acrylamide gels [26]. 3.5. Reversibility of the holographic ionic strength sensors A key issue in the application of holographic ionic strength sensors for real-time measurements in biological samples is the swelling and de-swelling kinetics of the hydrogel film supporting the holographic interference fringe structure. Fig. 6 shows the change in diffraction wavelength as a function of time when a polyampholytic hologram was tested with increasing concentrations of salt from 0 to 150 mM ionic strength and back again. This was preformed initially with NaCl solutions and then repeated with MgCl2 . After each change in salt concentration, the diffraction wavelength of the hologram shifted as a new equilibrium swelling position was established, and in each case, a new stable diffraction wavelength was obtained within 30 s. The resultant diffraction wavelength at a given ionic strength was within ±2 nm irrespective of whether the salt concentration was increased or decreased. The response of the sensor was therefore fully the reversible when tested with increasing or decreasing salt concentrations indicating that the complexation between the pendant positive and negative charges within the hydrogel are reformed when the ionic strength in the gel phase is decreased. These observations suggest that the sensor is suitable for continuous real time sensing of dynamic changes in salt concentration. Furthermore, the interference fringe structure of the holograms was not permanently altered during such ex-

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periments since the holograms could be swelled/de-swelled many times and always returned to the same peak shape and reflectivity. Individual holographic sensor elements were stable enough for multiple ionic strength tests, attesting to the reversibility and general robustness of the sensor system, although long-term stability in crude sample needs to be assessed.

4. Conclusions A variety of different hydrogel compositions containing pendant groups bearing fixed charges have been synthesised as thin polymer films (∼10 ␮m thick) and transformed into reflection holograms. The replay wavelength of such sensors was used to characterise the volume changes that occur when the hydrogels are exposed to solutions of different ionic strength. A polyacrylamide-co-AMPS-co-ATMA hologram, fabricated with an equal concentration of both positively and negatively charged monomers, showed rapid and fully reversible responses to changes in ionic strength in the visible wavelength range. Furthermore, the response to ionic strength was found to be near independent of pH and temperature over the ranges pH 3–9 and 25–45 ◦ C, respectively. Unlike hydrogel based sensors fabricated with a charged group of a single polarity [18,20,25,26], the holographic sensors responded solely to ionic strength and did not display complex responses in the presence of multivalent anions or cations. The polyacrylamide-co-AMPS-co-ATMA hologram was able to quantitate ionic strength in whole milk samples, which are opaque and contain a multitude of different ionic components. Because these sensors are planar and operate in a reflection format, they are highly suitable for miniaturisation in an array. Future work will involve utilizing the ionic strength sensors described here as reference sensors to compensate for the effects of background ionic strength on the pH response of pH-sensitive holographic sensors [6] as well as characterisation of their utility in various industrial processes and diagnostic systems.

Acknowledgements The authors thank the Biotechnology and Biological Research Council (BBSRC), for a studentship (A.J.M.) and Blanca Madrigal for useful discussions.

References Fig. 6. The effect of two cycles of step changes in ionic strength within the range 0–150 mM on the replay wavelength response of a polyacrylamide-coATMA (10 mol%)-co-AMPS (10 mol%) hologram at 30 ◦ C. The hologram was initially tested with NaCl solutions and then the procedure was repeated with MgCl2 solutions. The spikes on the figure are artefacts resulting from emptying the cell containing the hologram and refilling it with a new salt solution.

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