An ink-jet printed, surface enhanced Raman scattering paper for food screening

Abstract

A printable, surface enhanced Raman scattering (SERS) active test strip, with in situ growth of gold nanoparticles on completely untreated paper, is demonstrated to screen for bio-toxins below the maximum residue levels in the European Union, in food. The cheap disposable strip produced from as-purchased printing paper can also be used for SERS mapping of fluorescent dyes.

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1 Introduction

Food safety is an obvious global threat following the melamine-adulterated infant formula and pet food reported in China, several incidents involving phthalates in soft drinks, and maleic acid in rice-based food products. Such toxic agents are generally detected in laboratories using mass spectroscopic, liquid chromatographic or bio-assay techniques. The availability of portable Raman spectrometers, together with noble metal nanoparticle (NP) enhanced Raman scattering, opens up the possibility for on-site toxic screening via plasmonics. Surface enhanced Raman scattering (SERS) has developed into a frontline tool for chemical analysis, sensing,¹ and the identification and detection of a wide range of adsorbate molecules down to a single molecule.² In many cases, sensing has been carried out on labeled molecules.³ The signal enhancement involves a 10⁶ times increase in the Raman scattering (RS) cross section, σ, which is believed to be because of a surface plasmon-aided stronger electromagnetic field at the analyte site,⁴ in addition to a possible enhancement by a chemical charge transfer effect.⁴ Together with organic and inorganic molecules, SERS is becoming increasingly important for bio-sensing^5,6 and for the quality control of food and drinks.^7,8 In order to have a high sensitivity of detection via SERS, it is imperative to have a good SERS platform containing the metal (Au, Ag) NPs of a specific density, size, morphology, and of course the nature of the support on which the metal NPs are dispersed is important. Au and Ag NPs can be prepared using many different processes^9–11 in which a gold- or silver-containing salt is reduced by citrates, sugars (glucose, fructose and sucrose),¹⁰ or boranes working at specific temperatures, and at different reaction rates. One common method to synthesize AuNPs is from the reduction of hydrochloroauric acid (HAuCl₄), with sodium citrate (C₆H₅O₇Na₃). In one of the recent developments, lecithin, a component of human sweat, has been used as a reducing agent to grow AuNPs, at room temperature, using HAuCl₄.¹² Physical vapour deposition methods have also been used to grow Au and Ag NPs.^13,14

Solid SERS substrates are very common for the type of analytical application that we are considering. The advantages of using a solid substrate is that both top-down and bottom-up fabrication is possible, of which the former has high precision in controlling the location and dimension of the plasmon-generating structures. This is important because previously reported studies lead one to believe that exceedingly small structures and gaps (<10 nm) are required to generate the hot spots typically associated with high SERS activity.¹⁵ These SERS platforms are developed using nanolithography methods,¹⁶ including electron-beam (e-beam) lithography,¹⁷ a nanoimprint lithography technique,¹⁸ a photolithography technique,^19–22 together with several examples of template-based¹⁸ and sputtering methodologies^13,23 to generate the metallic nano-patterns. Successful fabrication and application of uniform SERS substrates have been demonstrated in commercially available Klarite™ substrates that use inverted gold pyramidal^16,24 structures. But these substrates are difficult to use on-site and outside the laboratory because of practical constraints such as size, flexibility, contamination, and cost. Furthermore, the fabrication of these rigid substrates is energy intensive and leaves behind substantial waste.

On the other hand, because of the problems associated with a rigid SERS substrate, researchers have attempted to make flexible substrates with reasonable success. Flexible SERS substrates can be easily incorporated into spectroscopic assemblies without the requirement of special handling or laboratory environments. Noble metal NPs have been grown on supports including flexible polymers,^25,26 fibers,²⁷ carbon nanotubes,²⁸ and paper.²⁹ The recent development of flexible SERS substrates uses nanoimprinting,³⁰ electrospinning,^31,32 thermal inkjet technology,³³ shadow mask assisted evaporation,³⁴ and an oblique angle deposition method.³⁵ The nanoimprinting and electrospinning approach for polymer and nanofiber substrates are either complicated or room temperature incompatible. But not all of these have been used for SERS application presumably because of the strong background Raman signals from the substrate itself, or other prohibitory factors arising from the NP loading density, or background fluorescence.

In this study we demonstrate in situ growth of AuNPs, on chemically unmodified, as-purchased printing paper, using an inkjet printing process to obtain a sensitive SERS paper for analytical purposes such as sensing and mapping. With this SERS strip, and a portable Raman spectrometer, loaded with a chemical library, it is possible to demonstrate high-sensitivity screening of toxic food outside the laboratory without the need for any expert handling.

2 Preparation and characterization of the SERS strip

2.1 Preparation of the SERS strip

The preparation of AuNPs on paper uses a bio-ink consisting of a combination of an aqueous solution of lecithin (Acros, Belgium), and potassium iodide (KI, 99.5%, Acros, Belgium) mixed in a 1 : 1 by volume ratio. The solution was then injected as an ink into an empty cartridge of an inkjet printer. A computer-generated, user-defined pattern was printed out on plain A4 paper (72 kg m⁻³, Taiwan). No hydrophobization of the A4 paper was performed. The pattern is initially invisible to the naked eye, but when immersed in an aqueous solution of HAuCl₄ (HAuCl₄·3H₂O, 99.99%, ACS reagent, Acros, Belgium) at room temperature, the bio-ink reduces the HAuCl₄ and in situ growth of AuNPs proceeds and visible purple patterns develop on the paper signifying the presence of AuNPs. The developing time can be from 2–24 h, depending on the concentration of the bio-ink and the HAuCl₄. A representative photograph of the SERS strip is shown in Fig. 1 and the procedure to prepare it is shown in Fig. S1 (ESI†). The colour of the AuNP pads, pink-purple, on the SERS strips depends on the AuNP size and density, and those without AuNPs appear white (Fig. 1a). Fig. 1b shows the SERS strip, being developed, compared to the commercial pH paper (green). The printing technique is capable of generating complex patterns, as shown in Fig. 1c, which is the National Yang-Ming University, Taiwan, logo (inset, Fig. 1c). The sharpness of the patterns is governed by the printer head, and the diffusion of the reactants on the wet paper. For smaller and sharper patterns, for applications such as blood typing,³⁶ better printers and low wettability paper may be used. The approximate material cost of preparing one such AuNP pad is about 0.1 USD. To control the AuNP density or size, the reactant concentrations had to be changed. In the present case, the reactant concentration varied from 25–75 mM for lecithin, 2–10 mM for HAuCl₄, but the KI concentration was fixed at 100 mM.

Fig. 1 (a) Optical image of the SERS strip showing purple AuNP patterns on paper. (b) Comparison between commercial pH strips and the new SERS strips. (c) Optical image of complex patterns (Logo of National Yang Ming University) using the same technique for printing the SERS strips. Inset in (c) shows the original logo of National Yang-Ming University.

Although there are few reports on using silver-coated paper,³⁷ the first patterned and printed SERS strips came from Yu and White.³⁸ They used as-grown AgNP solution, whose viscosity is chemically controlled by glycerol, and the ‘ink’ is introduced into the printer cartridges for printing. This method is technically well controlled; however, it is more complicated because of the use of centrifugation, filtration and requires chemical treatment for making the cellulose paper hydrophobic. The ‘ink’ used in these printing processes may clog the printer head, and storage of this ink within the cartridges is challenging. Using a printer with a microelectromechanical system (MEMS)-based actuation and filtration may partly solve the corrosion or clogging issue, but its industrial sustainability is not known. In addition, the chemicals used in the surface modification process may complicate the spectroscopic background. These steps are redundant in the process described here. The nanoparticle density, controlled by repeated printing in the published report,³⁸ is apparently larger than in our process, which we control by the reactant concentration only. The other difference between the published work and the work described in this paper is that we have used the common A4 paper for printing as against the specialized chromatography paper³⁸ used previously. There has been a recent report on disposable screen-printed SERS assay on solid substrates³⁹ following basically similar steps, but different chemicals, as described by Yu et al.³⁸ This report mentioned that a screen-printed paper-based SERS substrate was the weakest. However, none of these substrates were tested for detecting toxins in real food that had a smaller Raman scattering cross section than dyes such as the rhodamine 6G (R6G) used in these previously published papers. The problem of millimetre-scale paper wrinkling (Fig. 1b) observed here because of long periods of immersion is not a serious problem, because the laser beam of the Raman spectroscopic setup is only a few microns across and data collection is not disturbed once focus is achieved.

2.2 Characterization of the AuNPs

The sensing performance will depend on the AuNP characteristics shown in Fig. 2. The SERS signal will increase with the strength of the electric field in the surface plasmons generated in the metal NPs when properly irradiated. The calculations of Garcia-Vidal and Pendry⁴⁰ showed that the metal inter-particle spacing (d) contributes more to the enhancement factor in SERS than the radius (r) of the metal NPs. They predicted that metal NPs touching each other, in a monolayer, to yield d = 2r would produce the highest SERS sensitivity. The morphology of the AuNPs were studied by transmission electron microscopy (TEM; Jeol JEM 2000-EXII) and a field emission scanning electron microscope (SEM; Jeol JSM-6700F) using exfoliated sections of the printed paper. Fig. 2a and b show TEM and SEM images, respectively, of the AuNPs on paper at the edges of the pattern. The AuNP density is not very high, possibly because of reactant diffusion. However, the SEM from the pattern-edge helps in obtaining a size distribution of the AuNPs. Fig. 2c shows a typical SEM image at the centers of the pattern which had higher AuNP density and could explain the Raman results. A series of TEM and SEM images were then used to obtain the AuNP size distribution (Fig. 2d), which peaked at around 30 nm. The images indicate the formation of AuNPs not only on the surface of the paper but also embedded in it. The UV-VIS spectra (Fig. 2e) (JASCO V-670) show the characteristic surface plasmon band of AuNP at 540 nm (red curve) in the purple region. The small red shift of the absorption band from 520 nm indicates some larger AuNPs or more possibly some agglomeration. This result is consistent with the size distribution observed from the SEM/TEM measurements. In the absence of the AuNPs, on the white portion of the plain paper, there are obviously no absorption bands (black curve, Fig. 2e).

Fig. 2 Typical (a) TEM and (b) SEM images of the AuNPs grown in situ on the printed paper at the edge of the pattern. (c) SEM of the AuNPs at the central part of the pattern. (d) Particle size distribution of the AuNPs estimated from the electron microscopy images. (e) UV-VIS spectrum of (top curve, red) the AuNPs printed on paper forming the SERS strips, and (bottom curve, black) the plain paper.

In the current study, the density of the AuNPs is not as high as those proposed by the d = 2r condition⁴⁰ (Fig. 2a–c), nor is their size as small as 10 nm, which is the size required to give an AuNP plasmon absorption peak at 520 nm. Thus, there is scope for further optimization of the AuNP density. The AuNP size distribution, and more importantly its density, will depend upon the reactant concentration. Thus, there is a need to optimize the SERS strip for sensitivity. The optimization steps, via change of reactant concentrations, are shown in Fig. S2 (ESI†) by which we arrived at a concentration of 2 mM for HAuCl₄, and 25 mM for lecithin for the preparation of our SERS strip, as the concentration of KI was fixed at 100 mM. The use of a low concentration for HAuCl₄ is similar to that which is generally used for the preparation of a colloidal solution that looks wine-red in colour. Higher concentrations of HAuCl₄ would result in larger AuNPs and severe agglomeration (inset c, Fig. S2 (ESI†)). A smaller concentration of lecithin is required for two reasons. First, the lecithin solution is sticky and it is easy to clog the printer head. Heavy deposition of lecithin would invariably result in precipitates on the printed patterns and they would appear dark after developing (inset c, Fig. S2 (ESI†)). Thus, the dark colour of the patterns, although it looks more distinct, is actually inferior to the low-contrast pink-purple patterns for SERS functionality.

3 Sensing performance of the SERS strips and its use in food screening

3.1 General calibration of the SERS strip

For a basic understanding of the SERS performance of the strips, 20.0 μL of an aqueous solution of the standard molecule rhodamine 6G (R6G, 99%, Acros, Belgium), at different concentrations, were dispersed on to the SERS strips using a micropipette. The SERS measurements were made using a Jobin Yvon HR 800 commercial Raman spectrometer with 632 nm excitation (HeNe laser, beam diameter ∼2 μm, 10 mW). Experimental conditions used a single collection with an exposure time between 3–30 s. The RS data of the plain paper (Fig. 3a(i)), and the AuNP pad on the paper (Fig. 3a(ii), is shown in Fig. 3b(i) and (ii), respectively. These two spectra constitute our background signal, which is reasonably clean. The fact that the background signal from the paper or the AuNP pads is clean (Fig. 3b(i) and (ii)) is extremely useful otherwise it would interfere with the analyte signal.

Fig. 3 (a) Schematic illustrations of the SERS experiments performed on the AuNP strips with rhodamine 6G as the analyte. (i) Plain paper; (ii) paper with the AuNP pad; R6G dispersed on the strip and measured on the (iii) paper, and (iv) AuNP pad. (b) Corresponding Raman spectra obtained at each stage shown in (a).

The analyte, a solution of 1 μM R6G, is then detected on the strip. The RS and SERS spectra obtained from the white paper with R6G (Fig. 3a(iii)), and from the AuNP pads containing R6G (Fig. 3a(iv)) are shown in curve (iii) and (iv) of Fig. 3b, respectively. Curve (iii) (Fig. 3b) demonstrates weak R6G signals. However, curve (iv) (Fig. 3b) shows the strong fingerprint signals of R6G, at 612, 774, 1363 and 1510 cm⁻¹, because of the presence of AuNPs on the purple pad.

In addition, concentration-dependent SERS (on the AuNP paper) and conventional RS (on the plain paper) measurements have been done to check the sensitivity and are presented in Fig. S3 (ESI†). These demonstrate the SERS activity of the strips. The relative standard deviation (RSD = standard deviation/mean) curve, shown at the bottom of each panel in Fig. S3 (ESI†), is used to estimate the reproducibility of the SERS signals and is calculated by a method reported previously.⁴¹ A flat RSD would imply the best reproducibility. The RSD values for R6G were ∼15%, which needs to be improved. This is where the lithographically prepared substrates with precise control of the nanostructure morphology and density are ahead of the SERS strips. But, as mentioned earlier, with maximization of the AuNP density on the paper, this problem can be resolved.

3.2 Performance of the SERS strip in detecting toxic food contaminants

For real use of these SERS strips as a tool for varied molecular detection in drug administration, forensic science, or food safety protocols, it is imperative to study their sensitivity and reproducibility using relevant molecules and their concentrations. For this purpose our choice was malachite green (MG) (Fig. S4 (ESI†)), a common fungicide used in marine animal culture, and iprodione (Fig. 4), a pesticide commonly used in fruits and vegetables, and to prevent Botrytis bunch rot in grapes, all over the world. Iprodione has low solubility in water, and thus farmers use a modified iprodione (50%) solution to spray their crops. Here, we have used this modified real agricultural product (50% iprodione, Wonderful Agriculture (VN) Co. Ltd., Long An Province, Vietnam) solution for our sensing experiments. A concentration-controlled aqueous solution of the real agricultural product was sprayed on the surface of oranges, and thereafter collected by rubbing the SERS strips on the fruit surface. For MG, the solution was drop-coated. The SERS strips were then dried under a nitrogen flow. The experimental design included repeated measurement of different concentrations of these molecules at five different locations on the optimized AuNP pads, and on the plain paper. The repeated measurements were to obtain the RSD.

Fig. 4 SERS spectra of real agricultural iprodione (50%) measured over a range of concentrations on the AuNPs: (a) 1 M (b) 1 × 10⁻² M (c) 1 × 10⁻³ M (d) 1 × 10⁻⁵ M (e) 1 × 10⁻⁶ M, collected from five randomly selected points on the purple part of the SERS strip (with AuNPs). (f–j) Corresponding Raman scattering (not SERS) spectra of concentration dependent (the same as in a–e) iprodione on the white part of the SERS strips (without AuNPs). The RSD of the five spectra are shown at the bottom of each plot in black. The acquisition time for (a–e) is 6 s, whereas for (f–j) is 30 s. The arrows indicate iprodione peak position.

Fig. 4 and S4 (ESI†) shows the comparison of SERS (panels a–e), measured on the AuNP pads, and the corresponding RS data (panels f–j), measured on plain paper, of real agricultural iprodione, and of MG, at different concentrations. Concentration dependent SERS signals of Iprodione (Fig. 4a–e) and MG (Fig. S4a–e (ESI†)), measured on the AuNP pads, shows increasing intensities with increasing concentration. However, individual SERS intensities are higher than the corresponding RS intensities, as shown in the adjoining panels (Fig. 4 and S4 (ESI†), panels f–j). The diagnostic peak for the iprodione is at 998 cm⁻¹ (Fig. 4). There is evidence of fluorescence from the analytes, especially at 800 cm⁻¹ for iprodione. These signals had a larger line width and were not because of the presence of RS. In these cases, the efficacy of the SERS strip is also demonstrated and the analytes could be detected at low concentrations.

Each country has its own rules for the maximum allowed levels of such toxic food additives measured as ‘maximum residue levels (MRL)’. The MRL values of MG and iprodione for the European Union (EU) is 1 ppb, and 10 mg kg⁻¹, respectively. From the results shown in Fig. 4 and S4 (ESI†), the limits of detection (LoD) for iprodione and MG were 10 μM, and 1 nM, respectively, which were below the MRL of the EU. The LoD was determined experimentally as the concentration at which the analyte signals were detectable with a spectral signal-to-noise ratio (S/N) equal to or more than 3.0. The data shown in Fig. 4e, and S4e,† for iprodione and MG, respectively, are the lowest concentrations measured with a S/N ratio >3, and thus, the LoD in this research. Although we believe that the AuNP density and the sensitivity of the SERS strips could be further increased, the current sensitivity levels, of at least the MRL for these molecules, are practically and commercially acceptable.

Fig. 5 shows the variation of the prominent peaks of the analyte molecules (R6G, MG, and iprodione) as a function of their concentrations when measured on the AuNPs and on plain paper, i.e., for the SERS and RS cases, respectively. To start with, Fig. 5a shows the variation of the 610 cm⁻¹ line of R6G as a function of its concentration (Fig. S3 (ESI†)) and fitted with the Langmuir adsorption equation. Fig. 5b and c show the variation of the 1613 cm⁻¹ line of MG, and the 998 cm⁻¹ line of Iprodione, respectively, as a function of its concentration. The Langmuir equation relates the coverage or adsorption of the molecules on a solid surface to its concentration, with which the SERS and RS intensities vary, at a fixed temperature. The good fit to the data indicates good agreement of the experimental and theoretical predictions of the concentration-dependence studies. When plotted on a log scale, the concentration-dependent SERS signals yield a linear variation (Fig. S5a–c (ESI†)). At low analyte concentrations, expressing sub-monolayer adsorption, the signal increases rapidly with increasing concentration (Fig. 5a–c). However, at higher concentrations, reaching a state of monolayer coverage or more, the signal saturates. The slope of the curves (Fig. S5a–c (ESI†)) indicates sensitivity of the SERS measurements. Fig. 5 includes the data for both SERS, on the AuNP pads, and for the corresponding RS studies carried out on the plain paper. The comparison clearly indicates the efficacy of the AuNPs used in the SERS measurements.

Fig. 5 Variation of the intensity of the signal at (a) 610 cm⁻¹ of R6G, (b) 1613 cm⁻¹ of malachite green, and (c) 998 cm⁻¹ of iprodione, as a function of their respective concentrations plotted on a linear scale. Raman (black curve) (measured without AuNPs) and SERS (red curve) (measured with the AuNPs) signals are compared in each plot to show the efficacy of the AuNPs. Inset in (c) shows the same variation over the whole range of the concentrations studied. The lines joining the data points are a fit according to the Langmuir adsorption equation. The error bars indicate standard deviations of five independent measurements.

3.3 SERS mapping

Lastly, SERS mapping was carried out to demonstrate molecular imaging on the strips. All the molecules that were used in the detection experiments could be used for mapping purposes; however, here we chose two kinds of fluorescent dyes frequently used in cell staining, (i) fluorescein, and (ii) Nile red. This was to demonstrate the versatility of the AuNP strip against a strong fluorescence that normally suppresses the Raman signals. Solutions of these dyes (100 μM) were dispersed on to plain paper (as control) and the AuNP pads. RS was performed to select specific features to scan for. An area of 10 μm × 10 μm was scanned for each sample to map the intensity of the specific Raman features from the chosen dyes, under the same experimental conditions. The sample stage had a X–Y resolution of ∼0.7 μm.

The results of the Raman mapping of fluorescein (∼1540 cm⁻¹ band) on plain paper (Fig. 6a) and the AuNP pads (Fig. 6b) are presented. Similar data for Nile red can be found in Fig. S6 (ESI†). As expected, the AuNP pads show remarkably enhanced and distinct molecular imaging, compared to the plain paper, at the given concentration of the dye dispersion, which is assumed to be identical on the two surfaces. The pixel intensity (Fig. 6) is a function of the number of dye molecules as well as the AuNP aided enhancement at that location. The SERS strips, together with their potential to be used as a molecular mapping surface, add strength to the emerging field of paper-based devices for biological assays.^36,42,43

Fig. 6 SERS mapping for molecular imaging of fluorescein, 100 μM, on (a) plain paper, and (b) AuNP pad.

4 Conclusions

In summary, we have demonstrated the simple fabrication and use of a flexible SERS strip for possible on-site toxicity screening of food and drinks. The SERS strip is made on plain printing paper by ink-jet printing. A combination of lecithin and KI is used to reduce chloroauric acid at room temperature. The gold NPs are formed in situ on the printed patterns. The SERS strips demonstrate reasonably high sensitivity for the detection of common Raman-active molecules, such as R6G, and toxic materials in food, such as malachite green, and iprodione. The method is scalable, with potential for use in other kinds of printing materials. The flexible paper substrate is free of background Raman signals or fluorescence, and we believe its porous nature helps in holding the toxic analytes when rubbed onto the surfaces of fruits/vegetables, a feature that is not readily possible for rigid substrates, for subsequent detection via SERS.

Acknowledgements

The authors would like to acknowledge financial support from National Science Council, Grant nos (NSC-101-2112-M-010-003-MY3, and 101-2120-M-001-010), and Aim for the Top University plan of MoE, Taiwan.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Fabrication and optimization procedures of the SERS strips; SERS and conventional Raman scattering data for malachite green, and rhodamine 6G on the SERS strip and paper; SERS mapping of Nile red on the SERS strip. See DOI: 10.1039/c4ra04821k

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