Materials Science and Engineering C 53 (2015) 331–335

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Self-assembled micro-structured sensors for food safety in paper based food packaging M. Hakovirta ⁎, B. Aksoy, J. Hakovirta Alabama Center for Paper and Bioresource Engineering, Auburn University, AL 36849, United States

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Article history: Received 3 March 2014 Received in revised form 10 January 2015 Accepted 21 April 2015 Available online 22 April 2015 Keywords: Packaging Food safety Chemical sensor Diatoms Paper Ammonia

a b s t r a c t Natural self-assembled microstructured particles (diatomaceous earth) were used to develop a gas sensor paper with detection mechanism based on visible and distinct color changes of the sensor paper when exposed to volatile basic nitrogen compounds. The coating formulation for paper was prepared by applying diatomites, polyvinyl alcohol (PVOH), and pH sensitive dyes on acidic paper substrate. The surface coating was designed to allow a maximum gas flow through the diatomite sensors. The produced sensor paper was tested for sensitivity using different ammonia concentrations and we observed a sensitivity lower limit at 63 ppm. As a comparison, the results show comparable sensitivity levels to carbon nanotube based sensor technologies reported in literature. © 2015 Published by Elsevier B.V.

1. Introduction Food safety is one of the major health issues facing the global economy and is gaining an increasing amount of awareness in the biosensor and food packaging area. It is reported that each year a total of 48 million Americans become sick from contaminated food. Also, an estimated 128,000 of these cases do require hospitalization and 3000 cases result in death [1]. The economic effect of foodborne illnesses and food safety in the US is an estimated burden of $77.7 billion each year [2]. An efficient detection of microbial contamination, chemicals and toxins in food is the solution to the prevention and recognition of problems related to health and safety [3]. Although, science and technology has recently advanced very sophisticated technologies to detect foodborne threats, including the latest development in nanotechnology, scalable low cost and rapid detection methodologies are still lacking [4,5]. The traditional techniques such as culture and colony counting methods, immunology-based and polymerase chain reaction (PCR) based methods unfortunately take up to several hours or even a few days in order to reach a result [6–8]. Spectrometric methods have also been developed [9]. These methods are rapid, however, require expensive equipment and trained personnel for evaluation [10–12]. Clearly this is inadequate for certain applications, and recently many

⁎ Corresponding author at: Stora Enso AB, P.O.Box 70395, WTC, SE-107 24 Stockholm, Sweden. E-mail address: [email protected] (M. Hakovirta).

http://dx.doi.org/10.1016/j.msec.2015.04.020 0928-4931/© 2015 Published by Elsevier B.V.

researchers are focusing towards the progress of rapid, less costly and more qualitative first response methods [13–15]. Novel bio-molecular techniques for food pathogen detection exist and are currently being developed to improve the characteristics of food biosensors. Key attributes include sensitivity, selectivity, and detection speed [16–20]. The sensor solution has to also be robust and reliable, effective and suitable for in-situ analysis [21–23]. New technologies show high potential but further research and development is needed in order to have low cost, accurate and rapid detection methods that can be integrated to the food supply chain and thus to improve the overall food safety of the global system [24–28]. Food packaging is an essential medium for preserving food quality, minimizing food wastage and reducing preservatives used in food. The packaging also provides the containment protection against physical or chemical damage and gives critical information to the end users the consumers and distributers on the content and its use. Integration of sensor technology to food packaging will provide the food safety related element that can dramatically increase the total safety of the food supply chain. The objective of this research was to study the use of diatomaceous earth as a micro-structured media for food safety sensors. The naturally occurring diatomaceous earth consists of fossilized remains of diatoms that are types of algae with a hard-shell structure. Traditionally diatoms have been used in paper based materials, as for example filler in paper making, filtration media, mild abrasive, absorbent material, and a thermal insulator. Perhaps its most famous application is for the stabilization material for dynamite [29]. The functionality of gas sensor paper is based on pH sensitive dye that indicates spoilage of meat or fish

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Fig. 1. SEM image of sensor paper surface structure with 500× magnification and coat weight of 6 g/cm2. A cellulose fiber can be seen in the middle of the image.

products by color change. The reaction depends on the chromogenic material. For the detection of ammonia, produced during spoilage, pH indicators such as bromophenol blue or bromocresol purple can be used [30,31]. The ammonia gas acts as a Lewis-base and induces the color change due to hydrogen release. Other gasochromic materials are complexes and their color chance is induced through changes in the ligand field [32]. By adding the diatomaceous earth material as a medium to the sensor paper, we developed a low cost, highly selective and sensitive detection method that can easily be integrated in the paper production process. 2. Experimental section 2.1. Materials Diatomite, supplied by World Minerals (Diafil295), had an average surface area of 30.1 m2/g. The diatoms used for this research composed of 86–93% SiO2. Polyvinyl alcohol (PVOH) was supplied by Sekisui Specialty Chemicals (Selvol 523) and bromocresol purple, a pH sensitive dye from Alfa Aesar, was used to create the coating for the sensor paper. Acidic paper was used as a substrate for the sensor paper prototype development. 2.2. Coating formulation

Fig. 3. SEM image of the sensor paper surface using SEM and with 4000× magnification and coat weight of 6 g/cm2. The barrel like structure and micron sized open pores are clearly visible.

dry PVOH powder to cold water under agitation and heating the mixture to 85 °C. A de-foamer was added before the PVOH addition. The solution was held at this temperature for 35–40 min to assure complete dissolution and hydration of PVOH. The PVOH solution was cooled to 25 °C before addition to the slurred diatomite particles at a pigment to binder ratio of 5:1 (pigment: 100 parts and binder: 20 parts) under slow agitation. Finally, pH sensitive dye is added into the coating formulation between 0.05 parts and 1 part. The coatings were mixed in a laboratory mixer for 20–30 min under low shear to ensure that no air is entrapped into the coating and cause foaming. Application of the coatings on acidic paper was made with standard industry technique of drawdown rods that give users the ability to fine-tune coating thickness quickly and easily. The coat weights were varied by rod number and the coating solids content. The coat weights applied were between 6 and 20 g/m2. 2.3. Surface characterization and sensor testing The prepared samples were analysed using optical microscopy. Scanning electron microscopy (SEM, Zeiss DSM 940) equipped with an energy dispersive spectroscopy (EDS) detector was used to observe the surface morphologies of the coating, its thickness and diatom structures. A profilometer (Bruker/Veeco Dektak 150) was also used

PVOH was selected as a binder in the coating formulation because of its high binding power and high water absorption capacity. The solution of PVOH was prepared at 20% solids by adding the required amount of

Fig. 2. SEM image of sensor paper surface structure with 1000× magnification and coat weight of 6 g/cm2.

Fig. 4. Averaged particle surface size distribution of diatomite coated paper with coat weights of 6 g/cm2.

M. Hakovirta et al. / Materials Science and Engineering C 53 (2015) 331–335

Fig. 5. Sensor paper surface using SEM and 10,000× magnification with coat weight of 20 g/cm2.

to test the roughness of the sensor paper surface, FTIR (Thermo iN10 FTIR Spectrometer) was used to make chemical analysis of the paper surface chemistry and porosity tester (Bendtsen ME-113) was used to evaluate the surface porosity and thus the effective gas sensing area described by the fraction of void space in the material surface. Paper based sensors were tested for sensitivity using ammonium hydroxide solution since ammonia is one of the total basic nitrogen (TBN) components emitted during spoilage of meat products. A 5 mmol ammonium hydroxide solution was prepared for sensitivity tests. The solution was placed in ice bath to limit the formation and evaporation of dissolved ammonia gas. Amounts between 1 μL to 20 μL were withdrawn from the ammonium hydroxide solution and placed in 2 mL vials. Immediately after solution placement, top of the vials were covered with the paper gas sensor and the bottom of the vials were heated to 70 °C by inserting the vials in hot water bath for 1 min. The purpose of the heating was to increase the ammonia gas concentration and to accelerate the evaporation rate of the gas from the ammonium hydroxide solution. Vials were occasionally carefully stirred and they were kept in the hot water bath while its top was in contact with the gas sensor for several minutes or until a distinct color change in the sensor was observed. 3. Results and discussion 3.1. Structure and coating The surface morphology of the sensor paper can be seen in Figs. 1–3. A good dimensional comparison between the diatoms and the cellulose fiber is shown in the middle of the image of Fig. 1. The size distribution

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Fig. 7. Sensor paper surface cross-section using SEM and 2000× magnification and coat weight of 6 g/cm2. The cross section shows detailed morphology of the PVOH diatomite matrix coating.

of the diatoms was investigated by visual analysis and was averaged from 10 randomly selected areas (100 μm × 100 μm) with coat weight of 6 g/cm2. The average size distribution can be seen in Fig. 4. Roughness value (Ra) was measured by first subtracting a linear regression from scanned profiles to level the profiles. Three different samples had Ra values ranging from 5.7 μm to 6.9 μm, average being 6.2 μm. The cross-section SEM images were used to evaluate the thickness of the coating and the diatom particle distribution in the z-direction. We used targeted coating weights of 6 g/m2 and 20 g/m2 and this corresponds to 100 μm and 235 μm, respectively. Higher magnification with the SEM was used to see the diatom pores that were visible mainly in micro-scale and to investigate if the diatom pores were filled with the PVOH coating (Figs. 5–7). The SEM images show that the coating formulation is homogenously spread and that the majority of the micro-pores are open. This is essential to maximize the large surface area of the diatoms and their favorable structure for gas permeation. Using the SEM and its energy dispersive spectroscopy technique, we scanned random areas of the sensor paper ranging from 2.5 × 10−11 m2 to 1.0 × 10−8 m2. As the electrons penetrate approximately constant mass, the spatial resolution is a function of density. In the case of silicates like diatoms (average density is 3 g/cm−3), the typical spatial resolution is about 2 μm. However, with larger grain size on the surface a few micrometer resolution is more likely [33,34]. Calibration was done using Cu standard and carbon coating was used to avoid any charge effect. Carbon peak in the spectra was omitted from the spectral analysis. The result can be seen in Table 1. FTIR measurements from the sensor paper surface were performed by collecting spectra from three regions on the coated and un-coated side of the sensor paper as the background. The spectra showed unique carbonyl and aromatic moieties detected on the sensor coated side of our sample (Fig. 8). From comparing the spectra to the literature, we can conclude that the detected moieties can be linked to PVOH, the main component of the coating formulation [35]. Aromatic (benzene) moieties are specific to the dyes that are used in the coating formulation.

Table 1 SEM-EDS measurements of the sensor paper with coat weight of 6 g/cm2.

Fig. 6. Sensor paper surface cross-section using SEM and 500× times magnification and coat weight of 6 g/cm2. The cross section shows the PVOH diatomite matrix coating.

Element

Weight %

Atomic %

OK Mg K Al K Si K Ca K Fe K

58.7 0.4 1.5 38.6 0.3 0.4

71.5 0.3 1.1 26.8 0.1 0.2

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Fig. 8. FTIR spectra from three different regions of a sensor paper surface and coat weight of 6 g/cm2. The graph shows carbonyl and aromatic moieties detected on the coated side of the sample.

3.2. Detection sensitivity Paper based sensors were tested for sensitivity using ammonium hydroxide solution. 5 mM stock solutions were prepared and these solutions were added in 2 mL vials. Sensors were then exposed to

various amounts of diluted ammonium hydroxide solution in the vial and their TBN compound sensitivities were determined by a distinct color change from yellow to blue–purple. The steady-state paper sensor response to the vapor with its known concentration was obtained after adding varying volumes in the vial (Fig. 9.). The vapor concentration of

Fig. 9. Response of paper-based gas sensor with different coating formulations to various amounts of ammonium hydroxide solution; (a), 6 g/m2 coat weight and 0.5 part dye; (b), 20 g/m2 coat weight and 0.5 part dye; (c), 6 g/m2 coat weight and 1 part dye addition; and (d), 20 g/m2 coat weight and 1 part dye.

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ammonia Cg (ppm) inside the vial was calculated using the equation [36,37]: Cg ¼

10C 1 V 1 dRT P 0 V cM ;

in which C1 (wt.%) is concentration in the liquid, V1 (μL) ammonium hydroxide solution volume in the vial, d (g/mL) the density, R the gas constant, T (K) temperature, P0 (atm) the pressure in the vial, Vc (l) the vial volume and M the molecular weight. The color change effect can be seen in Fig. 9. The lowest detectable limit was with 1 μL of ammonium hydroxide, equivalent to 63 ppm NH3. Different coating formulations were also used to test several levels of volatile ammonia. The combinations of coating weight and the dye were 6 g/m2 coat weight and 0.5 part dye, 20 g/m2 coat weight and 0.5 part dye, 6 g/m2 coat weight and 1 part dye and 20 g/m2 coat weight and 1 part dye (Fig. 9). In all measurements the color change was very distinct and uniform in all samples. In case of the diatom containing coating formulation, the color change was also stable and remained in the observed level for about 5 days. We do not expect any interference with other gases in the experiments. 4. Conclusion Our research has proven that we are able to immobilize diatoms in a polyvinyl alcohol matrix. The created diatom–PVOH coating was successfully engineered to expose the diatom morphology with microstructures creating a good platform for paper based gas sensor application in food packaging. This unique formulation shows high sensitivities in the volatile basic nitrogen detection at 63 ppm, representing comparable sensitivity to that reported for nanosensors for nitrogen gas detection [38,39]. The benefits for our application include the beneficial morphology for gas sensor, the adaptability in paper material processes, its low cost (USD 1 b per lb.), its safety of use in food applications and its availability for scaled up low cost industrial processes. Therefore, we will continue to work on using in-vitro food samples for the detection of bacteria based volatile basic nitrogen compound emission as well as looking for other opportunities in immobilization of bioassay enabling antibodies, enzymes and salts to this very attractive nature made nanostructured media. Acknowledgments The authors would like to thank Mr. Mike Buchanan from Georgia Institute of Technology for his support with FTRI analysis. Further we thank Dr. Brian Wright for his efforts in helping with technology transfer issues related to this research. We would also like to thank Auburn University for its continuous support to our research efforts in the field of paper and bioresource engineering. References [1] Centers for Disease Control and Prevention (CDC), http://www.cdc.gov/ foodborneburden/2011-foodborne-estimates.htmlDec. 2010. [2] R. Scharff, Economic burden from health losses due to foodborne illness in the United States, J. Food Prot. 75 (2012) 123–131. [3] Wang R.-F, W.-W. Cao, C.E. Cerniglia, A universal protocol for PCR detection of 13 species of foodborne pathogens in food, J. Appl. Microbiol. 83 (1997) 727–736. [4] M.R. Adams, M.O. Moss, Food Microbiology, 2nd edition The Royal Society of Chemistry, Cambridge U.K., 2000. 479. [5] S. Ampuero, J.O. Bosset, The electronic nose applied to dairy products: a review, Sensors Actuators B Chem. B94 (2003) 1–12. [6] J. Commas-Riu, N. Rius, Flow cytometry applications in the food industry, J. Ind. Microbiol. Biotechnol. 36 (2009) 999–1011. [7] S. Flint, J.-L. Drocourt, K. Walker, B. Stevenson, M. Dwyer, I. Clarke, D. McGill, A rapid, two-hour method for the enumeration of total viable bacteria in samples from commercial milk powder and whey protein concentrate powder manufacturing plants, Int. Dairy J. 16 (2006) 379–384.

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Self-assembled micro-structured sensors for food safety in paper based food packaging.

Natural self-assembled microstructured particles (diatomaceous earth) were used to develop a gas sensor paper with detection mechanism based on visibl...
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