Developing a miniaturized continuous flow ...

Developing a miniaturized continuous flow electrochemical cell for biosensor applications M. Ilie*a,b , E. Ovreiub, R. Dejanaa, V. Fogliettia, L. Nardıc, A. Mascıc, B. Lanzad, L. Della Setac, M-R. Monterealic, W. Vastarellac, R. Pıllotonc a CNR/IFN, Via Cineto Romano 42, Rome, Italy b Univ. Politehnica Bucuresti, LAPI, Bd Iuliu Maniu 1-3, cam 135, 161071 Bucharest, Romania c ENEA, SP 061,C.R. Casaccia, Via Anguillarese 301, 00060 Santa Maria di Galeria, Rome, Italy d ENEA, C.R. Portici, Via Vecchio Macello 80055, Portici (Napoli), Italy ABSTRACT The development of a miniaturized electrochemical cell for biosensor application regards both the structuring of an array of electrodes in a fluidic chamber and their connections to the control & processing unit The sensitivity of the chronoamperometric measurement performed with the cell is increased by: (a) integrating the reference electrode on the same chip with the counter- and working- electrodes, (b) designing a specific pattern of the gold electrodes and (c) serially distributing them along the pipeline reservoir. Borosilicate glass is used as substrate for the electrodes, allowing, due to its transparency, an accurate and easy pad to pad alignment of the up-side-down chip versus a PCB soldered on a standard DIL 40 socket. This alignment is necessary to accomplish the elastomer-based-solderless electric contact, between chip and PCB. The solderless contact significantly improves both reliability and signal processing accuracy. The reservoir and its cover are micromachined out of silicone rubber respectively photosensitive glass in order to easy disassemble the fluidic chamber without any damage. Both thickness and elasticity of the photosensitive glass rend the device less brittle. A plug-in -plug-flow device with improved characteristics has been obtained with a modular structure that allows further extension of the number of electrodes. Keywords: electrochemical cell, electrodes array, chronoamperometric measurements, fluidic chamber

1. INTRODUCTION The biosensors, as functional analogs of chemoreceptors, are based on the direct spatial coupling of immobilized biologically active molecules with a signal transducer and an electronic amplifier. They use biological systems at different levels of integration to specifically recognize the substance to be determined (the analyte). The first step of this recognition is the specific complex formation by interaction of the immobilized biologically active molecules with the studied substance. The physicochemical changes caused either by the complex formation or by the chemical conversion of the analyte (e.g. owing to enzymes) might be of electrical nature (charge or enzyme activity) and therefore they can be indicated by means of transducers, based on potentiometric or amperometric electrodes, that perform the second step of the recognition: transduction of the physicochemical effect into an electrical signal. In this way species as H+, OH-, CO2, NH3, H2O2 can be identified in a so-called electrochemical biosensor. Further on, the electric signal has to be amplified and processed. The electrochemical indication prevails over all other methods of transduction [1]. Screen-printed electrodes are widely used as transducers in electrochemical biosensors [2]. The miniaturization of the electrodes using techniques borrowed from the semiconductor industry and their integration in a continuous flow micro-cell opens wide and interesting perspectives for the development of biosensors, offering the possibility to increase both the speed and the sensitivity of detection. Such a micro-cell, working in continuous flow, has been previously approached by the authors [3]. Its functional tests have revealed some characteristics that should be improved: (a) electrode behavior (sensitivity), (b) the reliability of both electric and fluidic connections (c) alignment accuracy. The causes of these drawbacks have been analyzed and new technical solutions have been adopted as follows.

2. EXPERIMENTAL RESULTS The electrochemical biosensor micro-cell consists of a set of working electrodes (WE), a counter-electrode (CE), and a reference electrode (RE) placed inside a reservoir provided with inlet and outlet openings for the fluid flow. Advanced Topics in Optoelectronics, Microelectronics, and Nanotechnologies IV, edited by Paul Schiopu, Cornel Panait, George Caruntu, Adrian Manea, Proc. of SPIE Vol. 7297, 729724 · © 2009 SPIE CCC code: 0277-786X/09/$18 · doi: 10.1117/12.823688 Proc. of SPIE Vol. 7297 729724-1

Electric connections allow the transmission of incoming and outgoing electric signals between the micro-electrodes and the external control unit. Fluidic connections allow the solution to flow through the microfluidic chamber placed on the suction side of an external peristaltic pump. The improvements of each constructive part are presented below. 2.1. The constructive parts of the miniaturized cell 2.1.1. The micro-electrodes In order to improve the sensitivity, the configuration of the working electrodes is now changed from a 2-dimensional array to a line. This configuration of the working electrodes allows the analyte to reach the electrodes one after another, in a serial manner, reducing thus the possibility that a down-stream electrode could give any signal before being reached by the analyte. The current measured on one electrode of the previous micro-cell [3] was about 5 nA and quite noisy; the size of an WE was 0.005 mm2 and that of a CE was 0.08 mm2, with a ratio of 1:16 between the surfaces of WE and respectively CE. In order to improve both the sensitivity and the signal-to-noise ratio, the shape of CE and of each WE are newly designed so that the WE/CE surface ratio reaches 1:50 (with a WE area of 0.016 mm2). The reference Ag/AgCl electrode, with an active surface of 0.116 mm2, is integrated on the chip. Chemical vapor deposition (CVD) of Cr and Au as well as optical lithography have been used in order to pattern the electrodes on a borosilicate glass substrate; CVD of Ag followed by lift-off were used to accomplish the RE. The patterns of the electrodes are presented in the fig.1.a,b,c.

a)-Reference electrode (RE)

Fig. 1 Integrated electrodes pattern: b) -Counter electrode (CE )

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c)-Working electrode (WE)

2.1.2. The microfluidic chamber The serial configuration of the electrodes requires a pipeline-shaped reservoir in order to determine an improved plugflow of the analyte liquid carrier, restricting the stream lines to a straight area. We adopted a sandwich solution for the reservoir and its openings. As a reservoir material we choose the silicone rubber, casted, polymerized and mechanically embossed. This strongly reduces the breakage risks related to the connection of the flexible capillaries used as pipe-line (allowing both an elastic connection to the chip and the easy disassembling of the cell without damage). UV-exposure, thermal development and selective wet etching have been performed on a thick (1.5 mm) photosensitive glass plate (FOTURAN®) for obtaining the cover with openings. The selective wet etching of the photosensitive glass led to an improvement of the transversal shape of the inlet and outlet openings and thus of the fluid flow. The reservoir and the cover with openings are presented in the exploded view of fig.2.a. The two parts have been assembled without any gluing over the chip (fig. 2.b.). The cover has been obtained in a thicker (1.5 mm) photosensitive glass sample than the previous one (0,5 mm), rendering the micro chamber more robust. 2.1.3. The fluidic connections Home-made mini-pedestals (fig 2.a) obtained from an ordinary glass micro-pipette by means of small scale glass blowing [4] are glued with araldite over the openings of the cover plate (fig. 2.c) allowing the insertion of the flexible

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capillaries used as pipe-lines to pass on the other side of the PCB . A top view of the assembled device is presented in the fig.2.c.

a Exploded view ; from left to right: glass fluidic connections, cover (of photo sensitive glass-before structuring), silicone reservoir, chip on borosilicate glass.

b The up-side-down glass chip aligned versus the PCB; from up to bottom: glass chip, silicone reservoir, PCB

c The chip is connected under the lower side of the PCB by means of the elastic ‘zebra’ connectors Top view of the device.

Fig.2

2.1.4. Electric connections The electric connections from the electrodes up to the flat cables that lead to the external controller consist mainly of Printed Circuit Boards (PCBs): the device board (DB) and (b) the family board (FB). The chip electrodes are connected to the DB, while the flat cables are connected to the FB. The standard DIL 40 socket and its corresponding ZIF (zero insertion force) socket provide a plug-in connection between the two PCBs. Further on, standard connectors allow the signals to pass towards the multiplexer. On the chip, the small size electrodes (0.116 mm2) are linked to bigger pads (3 x 0.6 mm2) situated outside the reservoir area. A custom designed printed circuit board (DB) is manufactured by us in order to easily connect the pads to the socket. The DB is provided with both pads of the same size as those on the chip as well as small holes for soldering the socket pins. The pad-to-pad connection between the chip and the DB is achieved by means of the so called ‘zebra’ connectors FUJIPOLY® Gold 8000. These connections are based on flexible elastomeric elements consisting of low durometer silicone rubber cores surrounded by flat metallic gold conductors vulcanized in a row parallel to each other. The ‘zebra’ connectors are pressed between the chip and the DB by means of two little screws (fig.2 c) and an additional plate underneath the chip. Thus, a contact resistance of about 200Ω is achieved. The DB is also provided with a slot (fig. 2 b, c) that allows both the fluidic connections to pass through it and the visual inspection of the reservoir channel during the measurement. 2.2. Functional tests The cell is very easy to plug in the in-house-built modular addressing system that applies and receives the signals to and from the cell, by polarizing the selected WE and by reading the current that passes through it. The schematic view of both electric and fluidic systems (block arrows) is presented in the fig. 3. The signal acquired from the WE is processed in the potentiostat unit and then in the computing unit that finally displays the dependency of the current vs time during the sequential injection of several diluted solutions of H2O2 (0.5· 10-3M, 1· 10-3M and 0.1· 10-3M) in a carrier phosphate buffer solution with pH=7.00 and a concentration 50 · 10-3M.

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Long term test of fluidic connections proved 6 week functioning without fail.

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3. CONCLUSIONS ¾

A plug-in plug-flow micro-cell with continuous flow of the analyte has been obtained, with a volume in the range of 0.006 – 0.010 ml and with vertical connections of capillaries (with inner diameters of 0.4 – 0.8 mm), with a specific pattern of the electrodes integrated on borosilicate glass rather than on silicon.

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Rigid glass fluidic connections as well as flexible elastomer-based solderless electric connections between the chip and the fluidic respectively electric system have been developed.

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The reservoir has been manufactured in silicone rubber instead of glass (thus allowing both an elastic connection to the chip and the easy disassembling of the cell without damage).

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The cover has been obtained in a thicker (1.5 mm) photosensitive glass, increasing the robustness of the device.

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The functional tests performed so far have proved not only a higher reliability, but also a lower electric resistance from the cell electrodes to the connector (1kOhm). Work is in progress to completely characterize the quality of the electrodes.

REFERENCES [1] [2]

[3] [4]

F. Scheller and F. Schubert, Biosensors, Techniques and instrumentation in analytical chemistry, volume 11, Elsevier, 1992 W. Vastarella, B. Lanza, A. Masci, R. Pilloton, Screen printed electrodes for biosensor application: reproducibility, sensitivity and stability, Proceedings of the 9th Italian Conference Sensors and Microsystems, Ferrara, Italy, 8-11 February 2004, Editors C. Di Natale, a. o., World Scientific publishing Co. Pte. Ltd. , ISBN 981-256-386-5, pp.1924 J. Maly, M. Ilie, V. Foglietti, E. Cianci, A. Minotti, L. Nardi, A. Masci, W. Vastarella, R. Pilloton, Sensors and Actuators B 111-112 (2005) 317-322 W. Vastarella, M. Ilie, E. Cianci, A. Coppa, V. Foglietti, L. Nardi, A. Masci, R. Pilloton, Improved capillaries connection to a continuous flow glass micromachined micro-cell, sent for publication in the Proceedings of the XI Conference of AISEM, Feb. 8-10, 2006, Lecce, Italy

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