DISPOSABLE 1H NMR DETECTORS FOR FOOD QUALITY MONITORING: APPLICATION TO BUTTER AND CITRUS JUICE

DISCRIMINATION V. Badilita1*, S.S. Adhikari1,2, N. MacKinnon2, U. Wallrabe1, and J.G. Korvink2

1Lab. for Microactuators, Dept. of Microsystems Engineering – IMTEK, Univ. of Freiburg, GERMANY 2Lab. for Simulation, Dept. of Microsystems Engineering – IMTEK, Univ. of Freiburg, GERMANY

ABSTRACT

We present an inexpensive and disposable single-chip probe for food quality monitoring via 1H nuclear magnetic resonance (NMR) spectroscopy. Successful demonstration is shown for two samples of only 100 nl: (i) a soft emulsion sample – butter, showing the complete 1H-NMR spectrum and (ii) a liquid sample – freshly squeezed lemon juice, and its comparison with freshly squeezed orange juice, to emphasize the signature of citric acid expected for the lemon juice. The miniaturized high-sensitivity, high-resolution sensor enables excellent detection quality and spectral narrowing, which renders it suitable for use with expensive (deuterated) or rare (metabolomics) samples. KEYWORDS: NMR spectroscopy, food quality, microcoils.

INTRODUCTION

Nuclear magnetic resonance spectroscopy (NMR) and imaging (MRI) are two investigation methods well-established in a wide range of fields from fundamental research to applications in engineering or medicine [1]. The main advantage resides in the NMR principle itself which extracts highly specific molecular information without perturbing the molecular processes. Therefore, NMR/MRI is appreciated as a non-invasive, non-destructive technique.

The NMR/MRI potential in the field of food monitoring is huge because NMR spectra have the capability to deliver a unique molecular fingerprint of foodstuffs. At the same time, the analysis procedures have been greatly simplified so that push-button testing is possible delivering a relatively large number of parameters with very basic sample preparation.

The present work goes in the same direction of making NMR more available for daily life applications, the main focus here being food quality monitoring with proof of concept demonstrations. We report herewith significant advancements in the design, fabrication and testing results for our previously reported [2] miniaturized insert for Magic Angle Coil Spinning (MACS) NMR spectroscopy.

STATE OF THE ART

One strategy to tackle the sensitivity issue in NMR/MRI experiments, which is even more severe in the case of mass- and volume-limited samples, is to optimize the RF receiver coils. The sensitivity is significantly improved by miniaturizing the MR detector and bringing it at the chip level. This NMR-on-chip trend became very popular in the past decade with the development of a number of different microscale NMR concepts: stripline probes for NMR [3], microslot waveguides for ultra-small samples [4], or various solenoidal microcoil solutions [5, 6]. Microfluidic integration was the next natural step towards a more complete NMR-on-chip system [7, 8].

Another equally important figure of merit in NMR spectroscopy is the spectral resolution which is hindered by the nuclear spin interactions that broaden the NMR spectral lines, thus reducing the specificity. One way to fight the spectral broadening is represented by the Magic Angle Spinning method [9] when the sample is spun at rotation frequencies of tens of kHz about an axis which makes a very specific angle with the direction of the external magnetic field B0. The value of this angle is 57.4° and is called the “magic” angle. More recently, simultaneous spinning of both the sample and the detector coil has been introduced [10], the method being coined as Magic Angle Coil Spinning (MACS) and offering the advantage of improved filling factor of the detector coil, therefore improved sensitivity, along with the high resolution spectroscopy brought along by spinning at high rates. In the MACS NMR experiment, the

978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001 1710 18th International Conference on MiniaturizedSystems for Chemistry and Life Sciences

October 26-30, 2014, San Antonio, Texas, USA

signal is wirelessly coupled between the pick-up coil and the static coil on the NMR spectrometer via inductive coupling.

FABRICATION OF THE MACS INSERTS

One of challenges brought along by the miniaturization of NMR detectors is the fact that in a relatively small volume, many different materials are stacked, each material having different physical properties. In the NMR context, the crucial property in the magnetic susceptibility. When the static magnetic field B0 of the NMR spectrometer is crossing the interfaces between various materials, the difference in magnetic susceptibility introduces discontinuities in the highly homogenous (up to parts per billion) B0 field, which subsequently broadens the final NMR spectrum of the sample. Moreover, in the case of MACS NMR configuration where the sample and detector are spun about an axis, the entire ensemble has to have circular symmetry in order to minimize the local magnetic field inhomogeneities.

Figure 1: a) NMR probe before and after coil winding with Cu microwire. The sample tube of 500 µm diameter and 800 µm height is photolithographically formed from SU-8. b) 2 mm3 NMR probe after SU-8 encapsulation. c) diced NMR probes.

In the case of the MACS detector, the most severe susceptibility jumps happen at the interface with air and, as previously demonstrated [2], because of the gold wire used for microcoils. Here we have replaced gold with copper wire, which has both a better conductivity and a favorable magnetic susceptibility for aqueous samples. Furthermore, an additional improvement in design with respect to the previous version of the MACS detectors, we encapsulated the entire device in SU-8, thus pushing the air interface further away from the sample – Figure 1. At the same time, SU-8 encapsulation provides much easier handling of the final detector chip for sample loading and measurements. The circular symmetry of the entire structure has been improved by replacing the rectangular on-chip capacitor in the previous version with a circular design.

The on-chip, microfabricated MACS NMR detector consists of a capacitor patterned by UV lithography, electroplated up to 12 µm, and integrated with a wirebonded microcoil. The capacitor and the coil are calibrated to form an LC-microresonator tuned at the Larmor frequency of the 1H-proton in 11.74 T (500MHz). This geometry enables wireless inductive coupling of the NMR signal to the detector coil of the magic angle spinning (MAS) NMR probehead, whilst rapidly spinning the sample-containing probe for high-resolution MAS-NMR.

100 1000Frequency [MHz]

2

2.2

2.4

2.6

2.8

Cap

acita

nce

[pF]

13 fingers circular capacitor

Figure 2: Frequency dependence of the capacitance of a 13 finger circular capacitor.

The capacitance at 500 MHz for 13-finger capacitor is 2.23 pF (Figure 2) with a remarkable fabrication precision of only ±0.03 pF, as measured for 40 structures. The high aspect-ratio sample holders were defined by SU-8 photolithography [2], followed by high-speed copper coil winding – Figure 1. The microcoils typically have 10.5 windings and 58.9 nH of inductance. The SU-8 encapsulation of the entire device and subsequent dicing (Fig.1), results in a robust and easy to handle SU-8 insert, which is easily filled with sample and disposed of after performing the measurement, without additional cleaning steps.

a) b) c)

1711

EXPERIMENTAL As an application for food monitoring, we present in Figure 3a a spectrum of freshly squeezed lemon

juice taken under magic angle spinning (MAS) at 2.5 kHz in a 11.74 T Bruker NMR spectrometer and the corresponding peak assignment. Successful discrimination between lemon juice and freshly squeezed orange juice is confirmed by comparing the absence of the citric acid signature for orange juice. Figure 3b presents a room temperature butter spectrum, displaying a strong peak of saturated fat, along with water and unsaturated fat peaks.

1234567

1H chemical shift [ppm]1234567

1H chemical shift [ppm]

butter500 MHz 1H NMR spectrum

*

butter500 MHz 1H NMR spectrum

*

Figure 3: a) 1H 500 MHz lemon juice spectrum obtained by magic angle spinning at 2.5 kHz. Comparison with orange juice spectrum shows that the citric acid signature is missing in the orange juice spectrum. Signal assignment: suc – sucrose, fru – fructose, glc – glucose, DMP – dimethylproline; b) 1H 500 MHz butter spectrum identifying the saturated fat (~ 1.8 ppm), water (4.7 ppm), triacylglycerides (~ 4.9 ppm), and unsaturated fat (~ 5.4 ppm). Asterisks mark side bands due to spinning at 2.5 kHz. CONCLUSION

This work shows the potential of the microfabricated disposable NMR detectors as a means to protect against damage and contamination the expensive commercial MAS rotors, at the same time avoiding contamination between successive measurements. These detectors can be routinely used for food quality monitoring, for example, to reveal adulteration of citrus fruit juices thorough fraudulent addition of water, sugar, or pulp wash. ACKNOWLEDGEMENTS

S. Adhikari, N. MacKinnon, and J.G.Korvink acknowedge the generous support of the ERC project NMCEL 290586. V. Badilita kindly acknowledges financial support through the DFG project BA 4275/2-1. REFERENCES [1] V. Badilita et al., Soft Matter, 8, 10583-10587, 2012. [2] V. Badilita, et al., PLoS ONE, 7 (8), e42848, 2012. [3] PJM van Bentum et al., J Magn Reson, 189, 104–113, 2007. [4] Y. Maguire et al., Proc Natl Acad Sci USA, 104: 9198–9203, 2007. [5] V. Badilita et al., Lab Chip, 10, 1387–1390, 2010. [6] DL Olson et al., Science, 270, 1967–1970, 1995. [7] J Bart et al., J. Magn. Reson., 201, 175–185, 2009. [8] M. Utz, R. Monazami, J Magn Reson, 198: 132–136, 2009. [9] ER Andrew et al., Nature, 182, 1659–1659, 1958. [10] D. Sakellariou et al., Nature, 447, 694–697, 2007. CONTACT * V. Badilita: phone: +49-761-203-7435; vlad.badilita@imtek.de

a) b)

1712