MICRO ELECTROCHEMICAL SENSOR

Related Field of The Invention

This invention relates to a sensor for detecting a variety of target analytes, including cells, viruses, macromolecules and ionic molecules and relates to the methods of making the sensors.

Background of The Invention (Prior Art)

In the last decades, there has been a great effort on developing sensors for detection of biological analytes or materials such as cells and bacteria, especially for the diagnosis of cancer, infectious diseases, and sexually transmitted diseases. Conventional methods for identification of these biological materials are generally depend on culturing, at which it is necessary to incubate biological samples at least 24-72 hours to get accurate results. These methods are time consuming and they necessitate optical devices and experts for evaluation of the results. However, early diagnosis and initiation of appropriate treatment are necessary to decrease morbidity and mortality rates. In this aspect, rapid and accurate detection of biological materials at the point of care is necessary.

Miniaturization of the detection systems with high specificity and high sensitivity has led to the development of portable systems at which biological detection is combined with signal transduction. There are mainly four detection methods as optical, magnetic, mechanical, and electrical. Among them, electrical detection provides a big advantage for scaling down detection systems, which has not only high sensitivity and high specificity but also low-power requirements and high compatibility with micro- fabrication technologies (J. Wang, Trac-Trends in Analytical Chemistry, 2002, 21, 226-232).

In the miniaturization of sensors, micro-electro-mechanical systems (MEMS) offer tremendous promise due to their ability to integration with electronics and its suitability to mass production in batches. The fabrication of microelectrodes and/or ultra microelectrodes by MEMS fabrication processes enables miniaturization of the electrochemical sensors for detection of biological analytes or materials. These devices can be combined with microfluidics to increase probability of adsorption of biological analytes or materials on sensor surfaces and with integrated circuit (IC) to miniaturize potentiostat, which is an electronic hardware required to control potentials during the measurements to develop hand held systems for point of care diagnosis.

In the electrochemical sensors, the introduction of reagents and/or solutions containing biological analytes or materials over the electrodes is necessary for surface activation, rinsing, and measurement steps. Although immersing the electrodes into the reagent and/or solution containing biological analytes or materials can be used in the macro-scale electrochemical sensors, it is not practical in the miniaturized electrochemical sensors due to their small size. In micro-scale electrochemical sensors, the preferred method to introduce the reagent and/or solution containing biological analytes or materials is to make a droplet of the related reagent over the electrodes (A.S. Blawas and W.M.Reichert, Biomaterials, 1998, 19, 595-609). However, the most apparent drawback of using droplets for miniaturized electrochemical sensors is evaporation of micro-volume droplet, when it is open to air and when the time for adsorption of biological analytes takes a long time. The solution to this problem is the use of channels and reservoirs to direct the sample to the electrodes and limit the evaporation.

Another problematic issue is nonspecific binding of biological analytes or materials on the surface of substrate of micro electrochemical sensor. Exemplary conventional suggestion for preventing the nonspecific binding of biological analytes or materials to the surface of substrate includes silanization of the surface. In silanization, an exact reaction scheme including the parameters like humidity, solvent type, reaction time, and temperature is necessary to have reproducible homogeneous silane-monolayers. Moreover, the stability of silane-monolayers is limited and they tend to form multilayer coverage or incomplete surface coverage, which can result in an increase in nonspecific binding (A.S. Blawas and W.M.Reichert, Biomaterials, 1998, 19, 595-609; F. Luderer and U. Walschus, Immobilisation of DNA on Chips I., 2005, 260, 37-56). The solution to this problem is deposition of a type of polymer on the surface of substrate (US20090018032 Al). In this case, Parylene C, which reduces nonspecific binding to the surface of substrate, is preferred due to its inertness and compatibility with MEMS fabrication processes. Parylene C is also used in the formation of channels and reservoirs to have uniform surface properties on each surface of channels, reservoirs, and a detection chamber except the electrodes because of its low water permeability that limits the evaporation of reagent and/or solution containing biological analytes or materials (W. Ehrfeld, et al., Ullmann's Encyclopedia of Industrial Chemistry, 2000, Wiley-VCH).

The other issue is to enhance binding of biological analytes or materials on the working electrode to increase specificity and sensitivity when the small amounts of reagent and/or solution containing biological analytes or materials are used. For this purpose, two methods are used in the proposed sensor: (1) The area of working electrode is increased by growing conductive micro pillars on the electrode, (2) The use of micro-channels and micro-reservoirs with height smaller than 50 pm can make the mass transport diffusion dominant with short diffusion paths since surface area to volume ratio (volume of reagent and/or solution containing biological analyte or material) is high (W. Ehrfeld, et al., Ullmann's Encyclopedia of Industrial Chemistry, 2000, Wiley-VCH).

Therefore, there is a need for an improved micro electrochemical sensor with a substrate that can overcome with the above-mentioned problems. Brief Description of The Invention

The objective of the present invention is to provide a sensor, which combines microfluidics with a three-electrode electrochemical sensor for the detection of biological entities and ionic molecules.

The present invention comprises micro-reservoirs, micro-channels, and a detection chamber, which have at least one electrochemical sensor with a working electrode, an auxiliary (counter) electrode, and a reference electrode.

The first aspect of the present invention is to provide a sensor incorporated on a single substrate (e.g. silicon, glass, Pyrex, or quartz) by using surface micromachining processes. The electrodes are produced from pure metals while the micro-reservoirs and the micro-channels are made out of Parylene C whose unique properties such as biocompatibility, conformal coating, pinhole-free, and moisture barrier are advantageously used. An advantageous embodiment of the device is that the whole system is compatible with IC and MEMS fabrication processes. The embodiment makes the present invention suitable for portable and hand-held systems at point of care diagnosis.

In another advantageous embodiment of the sensor, reagent and/or solution containing biological analytes or materials are directed to the detection chamber by using micro-channels and are enclosed in detection chamber and in the micro-channels during adsorption of target analytes on the working electrode. It is especially advantageous to limit the evaporation of the reagent and/or solution when small amounts of reagent and/or solution containing biological analytes or materials are used.

In a three-electrode electrochemical sensor, a fixed potential difference is applied between the reference electrode and the working electrode. The potential triggers the electrochemical reaction, which occurs on the surface of the working electrode. The reaction results to a change in current, which is opposite to the direction of current at the auxiliary (counter) electrode. The potential applied to the working electrode is measured against to the reference electrode. Various electrode materials can be used for embodying the working, reference, and auxiliary (counter) electrodes. However, it is important to select the materials so that they fulfill the requirements of design criteria so that fabricated device can have the same performance as the electrochemical sensors at macro scale. In another advantageous embodiment of the present invention, platinum (Pt) is used as auxiliary (counter) electrode due to its stability and inertness, silver (Ag) is used as reference electrode due to its high sheet resistance, stable electrode potential, and gold (Au) is used as working electrode due to its high electrical conductivity, long- term stability, and biocompatibility. In an electrochemical sensor, the surface of the working electrode is the place where reactions occur. Therefore, it plays an important role in the detection of target analyte. It is important to have specific binding mostly on the working electrode. In the present invention, the materials, that reagent/solution encounters, are Parylene C, Au, Ag, and Pt, not silicon which has affinity to biological materials. Since Parylene C is hydrophobic and chemically inert (CP. Tan and H. G. Craighead, Mater., 2010, 3, 1803-1832), the nonspecific adsorption can be eliminated. Moreover, the adsorption of biological particles to Pt is much lower than the one on Au surface (T. Laiho et al., Surface Science, 2005, 584, 83-89). The enhancement of specific binding of target analytes on the working electrode improves the sensitivity of the device.

In another advantageous embodiment of the sensor, the height of channels and the detection chamber is less than 50 pm so that the analytes are very close to the electrodes. Therefore, there is no need of mixing for enhancing adsorption, and diffusion of analytes can only be used. In another advantageous embodiment of the sensor, the area of working electrode is increased by growing micro pillars with 10 pm in height on the working electrode (Table 1). The increase in the area of working electrode increases the adsorption sites; this also increases the sensitivity of the sensor.

Table 1.

The present invention will be explained in detail based on the embodiments. The novelty of the invention will be pointed out with particular emphasis of its features especially in the claims annexed to and forming a part of this disclosure.

Definition of The Figures

In the figures:

FIG. 1. is a schematic view of an electrochemical sensor on a substrate according to the present invention;

FIG. 2. is a schematic sectional view of detection chamber;

FIG. 3. is a diagram showing the first step of how the micro electrochemical sensor can be fabricated;

FIG. 4. is a diagram showing the second step of how the micro electrochemical sensor can be fabricated;

FIG. 5. is a diagram showing the third step of how the micro electrochemical sensor can be fabricated;

FIG. 6. is a diagram showing the fourth step of how the micro electrochemical sensor can be fabricated;

FIG. 7. is a diagram showing the fifth step of how the micro electrochemical sensor can be fabricated;

FIG. 8. is a diagram showing the sixth step of how the micro electrochemical sensor can be fabricated;

FIG. 9. is a diagram showing the seventh step of how the micro electrochemical sensor can be fabricated;

FIG. 10. is a diagram showing the eighth step of how the micro electrochemical sensor can be fabricated;

FIG. 11. is a diagram showing the ninth step of how the micro electrochemical sensor can be fabricated;

FIG. 12. is a diagram showing the first step of how the micro pillars can be fabricated on the working electrode;

FIG. 13. is a diagram showing the second step of how the micro pillars can be fabricated on the working electrode;

Description of The Components And Parts Of The Invention

The components shown in the figures prepared for a better explanation of the micro electrochemical sensor are numbered separately and explanation of each number is given below.

1. Micro-reservoirs

2. Micro-channels

3. Bonding pads

4. Detection chamber

5. Wall structure

6. Substrate

7. Reference electrode

8. Auxiliary (counter) electrode

9. Working electrode

100. Wafer

110. Silicon nitride

120. Titanium

130. Platinum

140. Gold

150. Photo resist layers

150-1. 1. Photo resist layer

150-2. 2. Photo resist layer

150-3. 3. Photo resist layer

150-4. 4. Photo resist layer

150-5. 5. Photo resist layer

150-6. 6. Photo resist layer

150-7. 7. Photo resist layer

150-8. 8. Photo resist layer

160. Masks

160-1. 1. Mask

160-2. 2. Mask

160-3. 3. Mask

160-4. 4. Mask

160-5. 5. Mask

160-6. 6. Mask

160-7. 7. Mask 170. Parylene C

170-1. 1. Parylene C

170-2. 2. Parylene C

180. Silver

190. Micro pillars

Detailed Description of The Invention

Referring to the drawings in particular, the embodiment of FIG. 1 is a sensor, which combines micro-flu id ics with a three-electrode electrochemical sensor according to the present invention comprising micro-reservoirs (1) as inlet and outlet, micro-channels (2), bonding pads (3) and a detection chamber (4), on a substrate (6). FIG. 2 shows a sectional view of the detection chamber (4) with its wall structure (5), in which a reference electrode (7), an auxiliary (counter) electrode (8), and a working electrode (9) are located. Preferably, the reference electrode (7), the auxiliary (counter) electrode (8), and the working electrode (9) are made out of silver, platinum, and gold, respectively. The reference electrode (7) and the auxiliary (counter) electrode (8) are symmetric and in the shape of block arc. Their width size is 150 pm. A symmetrical geometry is used for counter electrode (8) and reference electrode (7) to have a valid assumption of equivalent current paths on the working electrode (9), which minimizes the potential drop due to cell resistance (Wang, Analytical Electrochemistry, 2006, Wiley-VCH). The working electrode (9) is in disc -shaped and its size changes from 100 pm up to 500 μιη radius. The disk- shaped working electrode (9) is used to decrease the background current induced by isotropic diffusion and increase signal to noise ratio (Lorenz and Plieth, Electrochemical Nanotechnology: In Situ Local Probe Techniques at Electrochemical Interfaces, 1998, Wiley-VCH). The remaining parts of the detection chamber (4) are made out of Parylene C.

The fabrication of the present invention using standard mass production techniques will be easy and inexpensive. The compatibility of the present invention with MEMS fabrication processes and IC enables volume production of high quality devices. FIG. 3 shows the first step of fabrication of the present invention. A layer of silicon nitride (Si ₃ N ₄ , 2000A) (110) is deposited on a bare wafer (100) (e.g., silicon, glass, Pyrex, or quartz) used as substrate (6). However, materials for the substrate (6) are not limited to these materials. The silicon nitride (110) is used as insulation layer between the electrodes (7, 8, and 9) and the substrate (6). Then, titanium (120) (Ti, 250 A) is sputtered on silicon nitride (110) layer to enhance adhesion between platinum (130) and silicon nitride (110) layers as shown in FIG. 4. Next, platinum (130) (Pt, 2000A) and gold (140) (Au, 1800A) are sputtered, respectively. Next, referring to FIG. 5, photo resist layer (150-1), which is durable to wet chemical etch processes, is spin coated and patterned by mask (160-1). The gold layer (140) is etched by using a gold etchant based on potassium iodide and iodine chemistry. Referring to FIG. 6, the photo resist (150-1) is stripped. Then, photo resist layer (150-2), which is durable to plasma etch processes, is spin coated and patterned by mask (160-2). The platinum layer (130) is etched by using chlorine-based gases in an inductively coupled plasma. Referring to FIG. 7, the photo resist (150-2) is stripped by using oxygen plasma process. Photo resist (150-3), which is durable to plasma etch processes, is spin coated and patterned by mask (160-3). The titanium layer (120) is etched in HBr/Ar based plasma. As shown in FIG. 8, the photo resist (150-3) is stripped by using oxygen plasma. Next, Parylene C (170-1) is deposited for a thickness of one pm by using A-174 Silane (>98% Gamma-Methacryloxypropltrimethoxysilane) as adhesion promoter. Negative resist (150-4), which is durable to plasma etch processes, is spin coated and patterned with mask (160-3) by using reverse image process since mask (160-3) has the patterns for all metal layers. The Parylene C (170-1) layer is etched by using O2/CF4 inductively coupled plasma. Referring to FIG. 9, the photo resist (150-4) is stripped by dissolving in acetone. Silver (180) (Ag, 3200A) is sputtered. Photo resist (150-5), which is durable to wet chemical etch processes, is spin coated and patterned by mask (160-4). The silver layer (180) is etched in aqueous nitric acid solution. Referring to FIG. 10, the photo resist (150-5) is stripped. Then, photo resist (150-6) is spin coated and patterned by mask (160-5) to form the micro-channels (2), micro-reservoirs (1), and the detection chamber (4) with a height of 15 pm. Lastly, Parylene C (170-2) is deposited for a thickness of 15 pm. Photo resist (150-7), which is durable to plasma etch processes, is spin coated and patterned by mask (160-6). The Parylene C (170-2) layer is etched by using O2/CF4 inductively coupled plasma (FIG. 11). The processed wafer is diced and individual dies are immersed into acetone to dissolve the photo resist (150-6) and the photo resist (150-7).

The fabrication of the sensor with micro pillars has a difference with one-step when it is compared with the process flow of the sensor without micro pillars. The process flow is same up to the gold etch process. As it is shown in FIG. 12, a photo resist (150-8), which is durable to electroplating processes, is spin coated and patterned by mask (160-6) to form openings for growing micro pillars (190). Micro pillars (190) are grown on the working electrode (9) by electroplating. Then, referring to FIG. 13, the photo resist (150-8) is stripped by dissolving in acetone. The remaining processes are same with the process flow of the sensor without micro pillars from the FIG. 5.

In electrochemical detection, it is necessary to use the reagent and/or solution containing biological analytes or materials. The introduction of the reagent and/or solution containing biological analytes or materials can be performed in three ways: (1) immersing the electrode to the reagent and/or solution containing biological analytes or materials, (2) leaving a droplet of the reagent and/or solution containing biological analytes or materials over the electrodes (US 8,062,491 Bl), (3) directing the flow of the reagent and/or solution containing biological analytes or materials over the electrodes. All of the methods necessitate an enough time for adsorption of biological analytes or materials to the working (measuring) electrodes. Evaporation of reagent/solution containing biological analytes or materials is ignored in the use of open to air macro scale electrochemical sensors. However, the evaporation of reagent and/or solution containing biological analytes or materials is important in micro scale electrochemical sensors since it can change the concentration of reagent and/or solution containing biological analytes or materials. Therefore, third method can be a useful solution to limit the evaporation of the reagent and/or solution containing biological analytes or materials. In addition, the material that is used in the fabrication of channels for directing flow has to limit the evaporation of the reagent and/or solution containing biological analytes or materials. In the present invention, micro-channels (2), detection chamber (4) and micro-reservoirs (1), which are made out of Parylene C, are used to enclose the flow over the electrodes (7, 8, and 9) (FIG. 11). Parylene C has low permeability to water and gases and it is possible to form highly conformal pinhole free coatings.

In another advantageous embodiment of the sensor, the height of micro-channels (2) and the detection chamber (4) is less than 50 m so that the analytes are very close to the electrodes (7, 8, and 9). Therefore, there is no need of mixing for enhancing adsorption, and diffusion of analytes can only be used.

In another advantageous embodiment of the sensor, Ag reference electrode (7), Pt auxiliary (counter) electrode (8), and Au working electrode (9) are preferred in the selection of electrically conductive layers for the electrodes of the sensor. Ag is preferred for the fabrication of reference electrode (7) to apply a stable electrode potential. The use of Ag as working electrode (9) has led to some problems such as silver oxidation and desorption of biological analytes from surface, which results in a decrease for the sensitivity of the sensor (Cataldi et al., 2005, 827 (2), 224-231). Pt is preferred for the fabrication of auxiliary (counter) electrode (8) due to its stability and high sheet resistance, which provides better current signal in the measurements. Au is preferred for the fabrication of working electrode (9) due to its compatibility with biological materials.

Another embodiment of the present invention is the elimination of nonspecific adsorption of biological analytes or materials on the substrate (6) by depositing a polymer such as Parylene C. The biological analytes or materials on the surface of Parylene C can be physisorbed when the reagent containing biological analytes or materials are introduced to the sensor. However, these materials are removed at washing steps since Parylene C does not have active surface chemistry for the chemisorption of biological analytes or materials.

In another embodiment of the present invention, the multiplex detection can be performed at the same time. For this purpose, there are three ways to change the structure of the sensor: 1. at least two electrochemical sensor units, which comprises a reference electrode (7), an auxiliary (counter) electrode (8), and a working electrode (9), are placed in a detection chamber (4); 2. at least two sensors, which comprises micro-reservoirs (1), micro-channels (2), bonding pads (3), and a detection chamber (4), are combined with a major inlet and a major outlet reservoirs; 3. at least two sensors, which comprises micro-reservoirs (1), micro-channels (2), bonding pads (3), and a detection chamber (4), in which at least two electrochemical sensor units which comprises a reference electrode (7), an auxiliary (counter) electrode (8), and a working electrode (9), are placed, are combined with a major inlet and a major outlet reservoirs.

Another embodiment of the sensor is the use of micro pillars to increase the area of working electrode, which increases the sensitivity of the sensor. The micro pillars (190) on the working electrode (9) are 10 pm in height, and with 8-10 pm intervals. For a working electrode (9) with 500 pm radius and with micro pillars (190) on it, the area of adsorption increases about 94% compared to flat working electrode area while for a working electrode (9) with 100 pm radius and with micro pillars (190) on it, the area of adsorption increases about 110% compared to flat working electrode area (Table 1).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

References

1. Trac-Trends in Analytical Chemistry, 21, 2002, 226-232 J. Wang, "Portable electrochemical systems".

2. Biomaterials, 19, 1998, 595-609 A. S. Blawas and W. M. Reichert, "Protein patterning".

3. Immobilisation of DNA on Chips I., 260, 2005, 37-56 F. Luderer and U. Walschus, "Immobilization of oligonucleotides for biochemical sensing by self-assembled monolayers: Tniol-organic bonding on gold and silanization on silica surfaces".

4. Mater., 3, 2010, 1803-1832 C. P. Tan and H.G. Craighead, "Surface Engineering and Patterning Using Parylene for Biological Applications".

5. Ullmann's Encyclopedia of Industrial Chemistry, 2000, Wiley-VCH, W. Ehrfeld, et al., "Microreactors".

6. Surface Science, 584, 2005, 83-89 T. Laiho, et al. "Chemisorption of alkyl thiols and S-alkyl thiosulfates on Pt and polycrsytaline platinum surfaces".

7. Analytical Electrochemistry, 2006, J. Wang,Wiley-VCH

8. Electrochemical Nanotechnology: In Situ Local Probe Techniques at Electrochemical Interfaces, 1998, Wiley-VCH, W. J. Lorenz and W. Plieth, "Beyond the Landscapes: Imaging the Invisible".

9. Journal of Chromatography B, 827, 2, 2005, 224-231, T. R.I. Cataldi, A. Rubino, M. Carmela Laviola, R. Ciriello. "Comparison of silver, gold and modified platinum electrodes for the electrochemical detection of iodide in urine samples following ion chromatography".