Portable CE-system with contactless conductivity detection in aninjection molded polymer chip for on-site food analysis

Holger Beckera**, Holger Mühlbergerb, Werner Hoffmannb, Thomas Clemensc,Richard Klemma, Claudia Gärtnera

amicrofluidic ChipShop GmbH, Carl-Zeiss-Promenade 10, D-07743 Jena, GermanybForschungszentrum Karlsruhe, Institute for Microstructure Technology, D-76021 Karlsruhe,

GermanycCLEMENS GmbH, D-97297 Waldbüttelbrunn, Germany

ABSTRACT

We present a compact portable chip-based capillary electrophoresis system that employs capacitively coupled contactless conductivity detection (C4D) operating at 4 MHz as an alternative detection method compared to the commonly used optical detection employing laser-induced fluorescence. The disposable chip for this system is fabricated out of PMMA using injection molding; the electrodes are screen-printed or thin-film electrodes. The system allows the measurement of small ions like Li, Na, K typically present in foodstuff like milk and mineral water as well as acids in wine.

Keywords: capillary electrophoresis, polymer chip, contactless conductivity detection, C4D

1. INTRODUCTION

While chip-based capillary electrophoresis has established itself as one of the fastest and potentially convenient methods for the analysis of larger molecules in many biotechnological and clinical applications [1], the large and economically attractive field of analysis of foodstuff has not been addressed with the same intensity. This is the more surprising as increased regulatory requirements, the commercial structure of the food industry and the inherent perishable nature of the ingredients make this field an ideal candidate for a chip-based analysis system that can be operated on-site at the food production site. The advantages of the miniaturization technologies in the analytical sciences can be used to the fullest extend, namely by generating a small (portable) and fast analysis system. While the detection method in many of the lab-based capillary-electrophoresis (CE) system is based on optical methods like laser-induced fluorescence [2,3] which allows for a very high detection sensitivity up to the detection of single molecules, the instrumental components necessary for such a detection scheme are normally bulky, expensive and due to the mechanical sensitivity of optical components, not well suited for portable systems. Furthermore, they often require recalibration or adjustment after moving the instrument which would be counterproductive for an on-site analysis tool. Finally, an optical label or dye is necessary for most analytical tasks, requiring a sample preparation step prior to analysis. As an alternative detection method, capacitively coupled contactless conductivity detection (C4D) was proposed, first for conventional, capillary-based systems [4,5], but quickly adopted to chip-based devices [6,7]. This detection method proves to be well suited for the intended use in the analysis of foodstuff, where small inorganic ions are present in comparatively high concentrationswhich would be difficult to detect with optical methods. It allows for a comparatively simple detection scheme, consisting of a pair of electrodes integrated on the outside of the chip plus high-frequency (in our case up to 4 MHz) electronics in the instrument unit that fulfills the requirements for low-cost fabrication of the chips themselves as well as portability of the complete instrument. Furthermore, no labeling of sample molecules is necessary. We have realized such a set-up with a fully integrated chip-based capillary electrophoresis unit, where all the electronics including high-voltage power supplies and the chip itself fit into a metal box with dimensions 19 cm × 12 cm × 8 cm (length, width, height). Readout, power supply and control is realized using a conventional laptop computer.

* hb@microfluidic-chipshop.com phone +49 3641 643121; fax +49 3641 347090; www.microfluidic-chipshop.com

2. PRINCIPLE AND THEORY OF CAPACITIVELY COUPLED CONTACTLESS CONDUCTIVITY DETECTION (C4D)

Capacitively coupled contactless conductivity detection (C4D) was introduced as a further development to overcome several shortcomings of conventional electrochemical detection methods, namely:

a) The exact positioning of the electrodes with respect to the microchannel, which influences the measurement signal, proved to be difficult

b) Due to the contact between electrodes and sample, electrode contamination or fouling can occur which leads to a drift in the signal over time

c) The interaction of the applied separation voltage along the microchannel with the detection voltage can distort the signal or even lead to a crossing of the electrolytic threshold, resulting in bubble formation at the electrode

d) Within or across microchannels, the thick-film electrodes will perturb the flow and are therefore not feasible.

The principal arrangement of the electrodes and the microchannel for the C4D detection scheme can be seen in Fig. 1. An electrode pair is placed on the outside of the chip, separated from the electrolyte by the channel wall. The distance between the electrodes and the cross-section of the microchannel determine the volume of the detection cell. A high frequency signal is applied to one electrode which couples this signal capacitively into the electrolyte (actuator or transmitter electrode). After passing through the liquid inside the detection cell volume, the signal is capacitively coupled into the second electrode, the pick-up or receiver electrode. Figure 2 shows the equivalent circuit representation of this set-up.

Fig. 1. Top and side view of the electrode arrangement with respect to the electrolyte-carrying microchannel and the cover lid.

Fig. 2. Equivalent circuit representation of the electrode configuration shown in Fig. 1. Clid denotes the capacitance between electrode and electrolyte solution through the cover lid, CH the capacitance of the Helmholtz double-layer at the channel wall, Cel and Rel the resistance and capacitance of the electrolyte solution and CS the parasitic stray capacitance (adopted from [8]).

In order to optimize the detection signal which is the absolute value of the complex impedance |Z|, the following relationships have to be taken into account:

,22CXRZ (1)

with R being the real resistance and CX the imaginary capacitive part. R can be expressed as

ElectrodesMicrochannel

Top view

Side view

Cover lid

CS

Clid CH CelCH Clid

Rel

,1AlR

(2)

with being the conductivity of the analyte, l the length of the measurement cell and A its cross-section. As the normal buffer solution already has a finite conductivity, all measurements will be relative conductivity changes with respect to the background electrolyte (BGE). It can be seen from this equation that a narrow microchannel geometry is favorable for this kind of measurement. The complex impedance CX of a capacitor can be expressed as

,2 0 Cr

C AjfdX

(3)

with d as the distance between the two capacitor plates, AC their surface area, f the frequency and r the relative permittivity of the chip material.

It follows from these equations that in order to maximize the capacitive coupling signal, the thickness of the channel wall should be as small as possible, the operating frequency of the system and the relative permittivity of the chip material as high as possible. These constraints can be utilized advantageously in a polymer chip where the electrodes are located on a thin cover foil which closes the microchannel, as described in the following section.

3. INSTRUMENT AND CHIPS

3.1 Instrument

In order to obtain a portable instrument, care was taken to use available miniaturized electronic components which would fit into a metal box with dimensions 19 cm × 12 cm × 8 cm (length, width, height). The principle setup of the instrument can be seen in Fig. 3. The instrument consists of two parts which are hinged together. In the bottom part, the chip can be mounted on a chip holder which contains the contacts for the on-chip C4D electrodes. All high-frequency components are housed in a specially shielded box close to the chip holder to minimize the risk of spurious signals. The operating frequency of the system is 4 MHz which is significantly higher than in the systems published in the literature [9]. The detection signals are sampled at a rate of 10 Hz with a USB-adapter plugged into a laptop computer. Care was taken to select a ADC with a low noise level in order to minimize read-out errors due to the electronics.

The upper part of the systems contains the high-voltage power supply necessary to drive the sample injection and separation. The power supply consists of two miniature regulated unipolar power supply modules (EMCO HighVoltage Corporation, Sutter Creek, USA) which are coupled together to deliver a bipolar driving voltage, allowing the separation of both anions and cations. The maximum current of the power supply is limited to 250 µA which proved to be sufficient even for highly conducting electrolytic solutions; maximum voltage output is 4 kV. The voltage is regulated using a 0-5V control voltage from a digital/analog converter controlled via an USB port.

After a chip is placed on the chip holder, the two parts are closed together and locked using a turning knob (left side of Fig. 3). In doing so, the locking mechanism lifts the chip up and the high-voltage electrodes for sample injection and separation are dipped into the sample and buffer reservoirs, enabling the measurement to start. For safety reasons, an interlock mechanism via a microswitch allows to switch on the high-voltage power source only if the instrument is closed and locked. The complete instrument housing consists out of a 3 mm thick aluminum to ensure mechanical strength for field use. This casing also acts as an additional electromagnetic shield to reduce undesired measurement artifacts.

Readout and control of the instrument is carried out through a specific user-interface software.

Fig. 3. Photograph of the instrument in open position. For operation, both parts are closed together and locked with the locking knob. This automatically lifts the chip holder with the chip and brings the injection and separation electrodes into contact with the liquid in the fluidic reservoirs

3.2 CE-Chips

To generate acceptance in the market for analytical applications in the regular production of foodstuff, the devices used should sell at a reasonable price. In order to allow for such a low-cost disposable chip, the chips are manufactured using injection molding [10]. As chip material, polymethyl-methacrylate (PMMA) is used. The replication of the chips was carried out from a precision machined stainless steel master. The fluidic reservoirs at the channel endings with a fluid volume of 70 µl are already integrated into the molding tool to avoid additional assembly steps [11] as well as ensuring a hermetic seal between the microchannel and the reservoirs. For the C4D detection method, the electrode configuration is of critical importance. Two technologies for the generation of the electrodes on the 60 µm thin cover lid foil (also made from PMMA) have been tested: A low-cost solution is realized using screen-printing, either with a silver or a carbon filled printing paste. Alternatively, a thin-film deposition of metal by sputtering or evaporation through a shadow mask has been carried out. Figure 4 shows a schematic drawing of the chip including al relevant dimensions, Fig. 5 an actual photograph of the chips, the top chip with carbon electrodes, the lower one with silver electrodes. The separation channel has the dimensions 50 µm width and 50 µm in height, with a separation length of 75 mm. The electrodes have a design width of 200 µm with an electrode gap for measuring of 250 µm. A microscopy image of screen-printed electrodes can be seen in Fig. 6a, an equivalent image of the thin-film electrodes is shown in Fig. 6b. It is important to notice that despite the purely manual optical alignment of the chip and the cover lid containing the electrodes with alignment marks, the alignment accuracy is of the order of 10 µm. Furthermore, despite of the comparatively large thickness of the screen-printed electrodes (4-5 µm), the cover lid could be sealed completely without voids on top of the channel.

Upper part Lower part

HV electronicsInjectionand separationelectrodes USB port

Shielded HFelectronics

Locking knob

Detection electrode contacts

Chip holder

Fig. 4. Schematic drawing of the CE-chip. Fig. 5. Photograph of the actual chip. Top chip with carbon electrodes, bottom chip with silver electrodes.

Fig. 6. a) Screen-printed silver electrode on top of the microchannel. Thickness of the electrode is 4-5 µm, its resistance 10 . b) Thin-film gold electrode. Thickness of the electrode is 200 nm, its resistance approx. 20 .

4. EXPERIMENTAL RESULTS

As the targeted applications for the system are the analysis of a variety of foods, investigations into the ionic concentrations of mineral water, wine and milk were carried out.

43

43,5

44

44,5

45

45,5

46

20 30 40 50 60 70 80Time [s]

Vol

tage

[mV

]

Sulfate 100µM

Chloride 100µM

Nitrate 100µM

54,9

55,1

55,3

55,5

55,7

20 30 40 50 60 70

Time [s]

Vol

tage

[mV]

Fig. 7a. Separation of a 100 µM anionic standard in a MES/His buffer

Fig. 7b. Separation of the same anions in a 1:10 diluted mineral water sample. The peaks are in consecutive order: Cl-, NO3

-, SO42-. Nominal

concentration of sulfate according to the label is 2 µM.

In a first experiment, the usability of the system for the detection of both anions and cations due to the bipolar nature of the power supplies was tested. Figure 7a shows the separation of an anions standard, containing sulfate (SO4

2-), chloride (Cl-) and nitrate (NO3

-) ions each at 100 µM concentration. The electrolytic buffer solution was a MES/His (2-Morpho-linoethanesulfonic acid) / L-Histidine buffer (from Fluka, Buchs, Switzerland) with a concentration of 50 mM/25 mM;separation conditions were injection voltage 500 V for 3 s, then separation at 4 kV for 80 s. Figure 7b shows the equivalent measurement of a 1:10 diluted sample of mineral water with identical separation conditions.

41,5

42

42,5

43

43,5

44

10 20 30 40 50

Time [s]Vo

ltage

[mV]

Fig 8a. Measurement of cationic species. Upper curve: Ca2+ ions with 4.4 mM concentration in 1:10 diluted milk. Lower curve LiNaK standard with 125 µM concentration

Fig 8b. Measurement of cationic species in 1:10 diluted mineral water. The peaks are in consecutive order: Ca2+ with a concentration of 73µM, Na+ at 26 µM and Mg2+ at 16 µM concentration

The measurements of cationic species are shown in Fig. 8a and b. The upper curve in Fig. 8a shows calcium ions with a concentration of 4.4 mM in 1:10 diluted milk. It is remarable that the fats and proteins in the milk do not perturb the ionic signal. The lower graph in Fig. 8a shows a LiNaK standard at 125 µM concentration. The buffer system used was again the MES/His buffer with concentrations of 30 mM/10mM; separation conditions were injection voltage 500 V for 6 s, then separation at 4 kV for 120 s. Figure 8b shows the measurement of 1:10 diluted mineral water. The ionic concentrations were calculated from the accompanying analysis certificate. The constituent species are Ca2+ with a concentration of 73 µM, Na+ at 26 µM and Mg2+ at 16 µM concentration.

108

108,5

109

109,5

110

110,5

20 40 60 80 100

Tim e [s]

Volta

ge [m

V]

Fig. 9. Separation of a wine standard. The peaks 1-5 denote various acids contained in the wine (see text)

21,822

22,222,422,622,8

2323,223,423,623,8

24

10 15 20 25 30

Time [s]

Volta

ge [m

V]

Na+Ca2+K+

Li+

1 2 3 4 5

Figure 9 shows the separation of a wine standard. The peaks 1-5 represent the following species: peak 1 sulfate, peak 2 tartaric acid, peak 3 citric acid, peak 4 succinic acid and peak 5 acetic acid. The buffer system used was again the MES/His buffer with concentrations of 10mM/10mM; separation conditions were injection voltage 300 V for 8 s, then separation at 4 kV for 120 s.

5. CONCLUSIONS

We have presented a complete chip-based capillary electrophoresis system based on the principle of capacitively coupled contactless conductivity detection (C4D). Its main features are the compact and portable instrument containing all the high-voltage and high frequency electronics and the disposable injection molded PMMA chip containing the detection electrodes. Several applications from the food industry could be carried out, demonstrating the speed and satisfying separation efficiency of the system. In future work, the various applications will be characterized further, includingdetermining the detection limits for the given electrode configurations. Other electrode geometries will be tested as well as the application of the system for the detection of biomolecules like DNA.

REFERENCES

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