DEVELOPMENT OF A SILICON MICROPROBE FOR NO DETECTION 1
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Marcelo B.A. Fontes , Jorge Santiago-Avilés , Rogério Furlan ✝University of São Paulo (Brazil) *University of Pennsylvania (USA) ABSTRACT
An array of electrochemical sensors - amperometric detection - was developed using silicon planar technology. Our main purpose is to detect the activity of free radicals such as nitric oxide (NO) at cellular dimensions. In this paper we focused on the process sequence used to produce silicon microprobes, based on plasma etching. Typical dimensions of the structures are: a length of 10 mm, a width of 1 mm, a tip width of 60 µm, and a thickness of 30 µm. Four different probe designs were adopted in order to test mechanical integrity. The defined process using plasma etching revealed to be feasible, although the lateral walls of the obtained probes resulted very rough. Preliminary mechanical tests were performed using probes with a thickness of 300 µm. Probes with wider shapes seem to have a better combination of higher fracture force and possibility to place more electrodes close to the tip.
Keywords: silicon microprobe, silicon micromachining, electrochemical sensor, amperometric detection, NO activity INTRODUCTION Microelectrode arrays based on silicon technology have been used with success on several applications, such as action potentials [1] and single neuron activity [2] detection. Due to the small dimensions involved, microfabrication presents many advantages including: batch fabrication with many electrodes, easy shape definition by computer designed masks, and possibility of signal processing circuits integrated on the same device. In this paper, we describe the development steps of a general purpose silicon based electrochemical sensor, having a needle shape probe. Our primary focus is to detect the activity of free radicals - such as nitric oxide (NO), at cellular dimensions [3,4]. The NO molecule have attracted attention in the recent years because the many interactions in the human body. In the atmosphere it is a noxious chemical, but in the body, in small controlled doses, it is extraordinarily beneficial. It helps to maintain blood pressure by dilating blood vessels, helps to kill foreign invaders in the immune response, is a major biochemical mediator of penile erection and is probably a major biochemical component of long-term memory [5]. NO measurement in biological systems is difficult due to the small amounts present, usually in the nanomolar range, and the rapid interaction with oxygen. The detection of the activity of such molecules in small concentrations is vital in many studies in vitro and in vivo as well as in diagnostic and therapy [6]. There are many different ways and techniques used to detect NO [7]: Electron Paramagnetic Resonance (EPR) detection threshold about 10-9 Mol, Spectrophotometry - 10-9 Mol, Chemiluminescence - 10-11 Mol, Gas Chromatography less sensitive than EPR and chemiluminescence, and Amperometric - 10-9 Mol. The last one, adopted in this work, is easy to be implemented using silicon planar technology.
✝Further authors information:
Laboratório de Sistemas Integráveis (LSI), São Paulo, SP, Brazil, 05508-900, Tel. +55 11 818 5657, Fax. +55 11 818 5665. E-mails :
[email protected],
[email protected],
[email protected].
PROBE DESIGN We have developed an electrochemical cell with a microelectrode array which enables to perform multichannel recording at different positions. This system is composed of: a silver/silver chloride reference electrode, a platinum counter electrode and 10 gold working electrodes, placed on a silicon substrate that has a needle shape. Four different probe designs, Figure 1, were adopted in order to test mechanical integrity. Structures A and B are wider than C and D, what could be detrimental in terms of cellular application, but they allow the interconnection lines and associated electrodes to get close to the tip.
Figure 1- Probes shapes and dimensions (in microns). The front side of the probe, Figure 2, contains gold interconnection lines and bonding pads. The central line defines the reference electrode. Five lines on each side of this electrode contain working electrodes defined on their ends, with areas of 10 µm x 10 µm. The back side is covered with platinum, defining the counter electrode.
Figure 2 - Top part of the probe showing interconnection lines and bonding pads.
FABRICATION The probes were fabricated according to the follow sequence, schematically represented in Figure 3:
Figure 3 - Probe fabrication steps. a)Wet oxidation of 2" silicon wafers (p type, orientation: , resistivity: 1 - 10 Ωcm) to obtain at least 1 µm of SiO2; b)Patterning of the silicon oxide film, using the needle shape mask (mask 1). This step has to be performed at this point of the fabrication sequence in order to maintain the integrity of the polyimide film, which is applied later; c)NiCr e-gun evaporation (adhesive material); d)Gold film e-gun evaporation (200 nm). One could optionally use a gold electrodeposition in order to increase considerably the gold thickness; e)Gold and NiCr electrode lines (20 µm wide) and bonding pads (150 µm x 150 µm) patterning with mask 2; f)Polyimide spin coating and cure to obtain a film with a thickness of ~ 1 µm; g)Aluminum e-gun evaporation (0.5 µm); h)Aluminum patterning (mask 1) to define the tips areas; i)Polyimide removal by O2 plasma etching process. This process leaves the silicon areas exposed to be etched next;
j)Silicon plasma etching with SF6 [8,9] (depths between 30 µm and 50 µm in this work) to define the tip thickness; k)Aluminum patterning (mask 3) to define working electrodes, reference electrodes and bonding pads windows; l)Polyimide removal by O2 plasma etching process; m)Aluminum removal; n)Protection of the front side of the structures with wax; o)KOH backside etching; p)Wax removal; q)Si3N4 sputtering deposition on the backside (~20 nm); r)Platinum sputtering deposition on the backside (~40 nm); s)Wiring bonding to a carrier; t)Selective silver electrodeposition and silver chloride formation by chemical reaction, on the central electrode.
FABRICATION RESULTS Our process sequence revealed to be feasible. Results comparable with those obtained boron etch stop technique can achieved with reasonable thickness control and high aspect ratio.
Figure 4 - Probe shape after the plasma etching step, without removal of the aluminum mask.
Figure 4 displays the shape of a probe tip after the plasma etching step, without removal of the aluminum mask. Little undercutting is observed and the lateral walls, that resulted satisfactorily vertical, are probably covered with a polymer that could not be easily removed. Figure 5 shows details of a released structure. It can be seen that the defined process permits to obtain very thin probe tips, although the lateral walls resulted very rough. As this asperity may limit the probe application in cellular studies, an improvement of the plasma etching is under investigation.
Figure 5 - Probe tip details showing lateral asperity Figure 6 shows SEM images of the final device where the contact pads, and the working and reference electrodes can be seen. After processing, the tips presented a typical width of 60 µm.
Figure 6 - Probe front side.
MECHANICAL TESTS Since we intend to use the probes to puncture cells, a preliminary mechanical test was performed considering the four probe shapes presented in Figure 1. In this case, the plasma etching step was suppressed and the probes were only etched with KOH, leading to samples with a thickness of 300 µm (two inches wafer thickness). An Instron equipment was used to perform a compressive force x displacement experiment. In order to fix the probes vertically, they were first attached to a Plexiglas piece using a strong adhesive. The compressive forces that would fracture the silicon probes were then measured and compared. Figure 7 shows the schematic view for this mechanical assay.
Force
fracture
probe displacement
Instron
Figure 7 - Schematic view of the mechanical assay. Table 1 describes the fracture load on each case, obtained from the force x displacement curve of the four different probe shapes (A to D, Fig. 1). Table 1. - Force x displacement results of the four shapes A-D (see Fig.1). Probe shape A B C D
Fracture load (N) 3.89 4.67 2.34 1.95
A simple comparison shows that samples C and D, which have the sharper designs, need less force to be fractured, about half of the value obtained for probes A and B. This higher compressive stress of samples C and D can be explained due to a smaller cross-sectional area. Structure B presented the maximum force, about 4.67 N. Thus, probe shapes A and B seem to have a better combination of higher fracture force and possibility to place more electrodes close to the tip. These two properties reflect their wider shapes, what could be detrimental in terms of cellular application.
CONCLUSION An array of electrochemical sensors - amperometric detection - was developed using silicon planar technology. The main objective is to detect the activity of free radicals such as nitric oxide (NO) in systems of cellular dimensions. The electrochemical system is composed of: a silver/silver chloride reference electrode, a platinum counter electrode and 10 gold working electrodes, defined on a silicon microprobe with a needle shape. Typical dimensions of the implemented microprobes are: a length of 10 mm, a width of 1 mm, a tip width of 60 µm, and a thickness of 30 mm to 50 µm. The three mask process used plasma etching with SF6 to define the probe thickness. The final structures were released with etching in KOH solution. The complete process revealed to be feasible, although the lateral walls of the probes resulted very rough. This roughness was caused by the plasma etching process, which has to be improved. Such asperity must be overcome or they may limit the microprobe application in cellular studies. Preliminary mechanical tests were performed with four different probe shapes with a thickness of 300 µm (two inches silicon wafer thickness). Probes with wide shapes seem to have a better combination of higher fracture force and possibility to place more electrodes close to the tip. Electrochemical analysis are being performed to investigate the characteristics of the microprobe with and without the use of catalytic films applied on the electrodes. The results will be presented in a further communication.
ACKNOWLEDGMENT The authors wish to acknowledge the support offered by CNPq - Brasilia / Brazil, Mr. Darius Cayetano for his work with mechanical tests at LRSM (UPENN) and all help offered by Mr. Vladmir Dominko at UPENN Microfabrication Laboratory.
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