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Nanoscale electrochemical probes for single cell analysis
Microelectronic Engineering 83 (2006) 1638–1641 www.elsevier.com/locate/mee
Nanoscale electrochemical probes for single cell analysis R.J. Fasching *, S.-J. Bai, T. Fabian, F.B. Prinz Rapid Prototyping Laboratory, Stanford University, Stanford, CA 94305, USA Available online 3 March 2006
Abstract Needle shaped probes with a dual electrode system in submicron size have been developed for electrochemical analyses of living single cells. The probe system is designed for local probing of the cytosolic cell environment and cell organelles using amperometric, potentiometric and impedance spectroscopic methods. Silicon nitride cantilevers with an electrode metal layer system are fabricated on 4 in. wafers using conventional micro fabrication techniques. The probe needle structures with a tip in sub micron scale are patterned using focus ion beam (FIB) technology. A focused ion beam is utilized to write the probe needle shape into the pre-shaped cantilever and, for a dual electrode system, the probe is divided into two parts to create two separate electrodes. Subsequently, the needle structures are released from the supporting bulk silicon during a wet etching step, and a silicon nitride layer is deposited to isolate and embed the electrode metal layer. Finally, FIB milling is used for a precise exposure of the buried metal layer by cutting the top of the tip. Electrochemical characterization of nano-probes showed full functionality of Ag/AgCl as well as of platinum transducer systems. The sharpness of the probe tip with a radius of less than 50 nm and the mechanical robustness of the needle structure allow for a reliable penetration of cell membranes. Initial measurements of cell membrane potentials and cell membrane impedances of rat fibroblast cells using Ag/AgCl transducer probes demonstrate the analytical capability of these probes in biological environments. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Electrochemical nano-probe; Single cell analysis; FIB; AFM; SECM
1. Introduction The motivation for the development of sensing probes for the cytosolic cell space comes from an increasing body of evidence substantiating the role of electron transfer and ion flux mechanism in cell metabolic control. Today, the highest resolution tools investigating cell communication phenomena are cell attached current measurements using patch clamps. The revolutionary insights provided by patch clamp probing made possible a better understanding of transmembrane signaling mechanisms. Problems with patch clamp methods include plasma membrane decoupling from the cytoskeleton, mechanical perturbation of the membrane, cell ballooning and the limited capability to study electrochemical reactions or electron transfers directly [1]. *
Corresponding author. Tel.: +1 650 723 1301; fax: +1 650 723 5034. E-mail address: [email protected] (R.J. Fasching).
0167-9317/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2006.01.262
Direct electrochemical analysis requires the placement of a localized electrode system to the site of interest. Scanning electrochemical microscopy (SECM) methods are based on this concept and have been used for studying biological systems [2–4]. Limitations arising from mechanically non robust and not small enough electrochemical transducer systems do not allow for a controlled penetration of cell membranes and a precise placement inside of a cell [5–7]. In order to overcome these limitations, needle shaped single and dual electrode nano-probes in submicron size have been developed and explored in this study. 2. Experimental 2.1. Fabrication of planar nano-probes The probe fabrication process has been developed on 4 in. wafers and is composed of two main fabrications
R.J. Fasching et al. / Microelectronic Engineering 83 (2006) 1638–1641
Fig. 1. Conventional silicon micro-processing for preparing a silicon platform with a pre-shaped nitride/metal electrode cantilever structure.
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probes are patterned in needle shapes and are divided into two parts to fabricate two separate electrodes on the probe (Fig. 2/7). The average width of the beam is about 300 nm, which defines the minimum distance between the two electrodes. After the FIB patterning step, the needle shaped probes are released by wet etching of the already preetched silicon substrate (Fig. 2/8). Following the wet etching process, another thick silicon nitride layer is deposited using plasma enhanced chemical vapor deposition (PECVD) at 350 °C (Fig. 2/9). The purpose of this layer is to embed and electrically isolate the electrodes. The nominal thickness of the silicon nitride layer is about 200 nm. At last, FIB cutting at the tip of the probe needle structure is done to open ultra micro electrodes (Fig. 2/10). This final process determines the size of the ultra micro electrode (UME) and the sharpness of the probe. 2.2. Electrical and mechanical interface
Fig. 2. Two step FIB processing technique for nano-probe fabrication.
The fabricated nano-probes are attached to a custom made printed circuit board (PCB). The PCB serves as a substrate for mounting the probe on a AFM stage for probe manipulation and accommodates signal pre-amplification and voltage buffering with active guarding. The voltage buffer is formed by a single electrometer-grade op-amp OPA129 (Texas Instruments, Inc.) in a unity amplifier configuration. This configuration allows multipurpose use of the nano-probe for potentiometer as well as amperometric measurements in four-wire mode. A customized AFM nozzle and the PCB in assembled configuration and mounted on the AFM scanner are shown in Fig. 3. 2.3. Apparatus
flows. First, a silicon platform with cantilever shaped electrodes is fabricated using convectional micro-fabrication technologies as shown in Fig. 1. The second fabrication part consists of a two step FIB process and is depicted in Fig. 2. The first process sequence begins with a 4 in. double-side polished silicon wafer. A low stress silicon nitride layer, the main layer of the cantilever, is deposited on both sides of the wafer with a typical thickness of 400 nm using low pressure chemical vapor deposition (LPCVD) (Fig. 1/1). Next, a cantilever structure is pre-shaped in the nitride layer during a reactive ion etch (RIE) step (Fig. 1/2). The silicon nitride on the wafer’s backside is then patterned and a deep RIE process is used to partially release the nitride cantilever (Fig. 1/3). Subsequently, an electrode material, such as platinum or silver, is deposited and patterned on the silicon nitride layer on the front side of the wafer using sputtering or evaporation technique in combination with a lift-off process (Fig. 1/4–6). During the second fabrication sequence a two step FIB milling process is used to pattern the nano-probes. Here
In this study, the nano-probes were mounted an AFM pico plus system (Molecular Imaging, AZ) combined with a confocal laser scanning microscope (CLSM) LSM5 pascal system (Zeiss, Oberkochen, Germany). Immobilization of single cells was achieved using conventional pipette- and planar-patch clamp techniques. Electrochemical measurements were carried out using a Gamry potentiostat (Gamry, PA).
Fig. 3. Photograph of a customized AFM nozzle assembled with the PCB (a); and mounted on the AFM (b).
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R.J. Fasching et al. / Microelectronic Engineering 83 (2006) 1638–1641
3. Results 3.1. Silicon platform and FIB patterning Fig. 4a shows an optical image of a finished silicon platform with two pre-shaped electrode systems ready for FIB processing. In Fig. 4b, a SEM image of a FIB patterned needle structure in a pre-shaped cantilever structure is shown. Due to the re-deposition of sputtered materials during FIB writing, the pattern should be written deep down to the silicon substrate through the nitride layer. Fig. 5 depicts a finished nano-probe with two UMEs embedded in the silicon nitride layer. Platinum or silver are used as UME’s metal, respectively. The metal layer is embedded in a 300 nm thick low stress silicon nitride. Sharpening and cutting the needle tip using a FIB enables a precise opening of UMEs and allows for customizing the shape of the single or dual needle tip (Fig. 5b,c). Defined active area of UMEs down to 0.01 lm2 can be achieved.
Fig. 6. (A) Cyclic voltammogram of a Pt UME; and (B) of a Ag/AgCl UME, the potential is related to a 0.1 M Ag/AgCl reference electrode and the voltage sweep rate is 20 mV/s in both cases.
3.2. Electrochemical characterization of the probe system The electrochemical behavior of the UMEs of the probes has been investigated using cyclic voltammetry (CV). CV was performed in a 10 mM ruthenium hexamine chloride (Ru(NH3)6Cl3)/0.1 M potassium chloride (KCl) solution for Pt UMEs and in 0.1 M potassium chloride solution for Ag/AgCl UME. Fig. 6 shows cyclic voltammograms from Pt and Ag/AgCl UMEs. These probes have an average UME area of 0.1 lm2. Open circuit voltages of the Ag/AgCl UMEs show Nernst behavior above 100 mM chloride concentrations. For lower chloride concentrations the potential becomes undefined and limits the working regime of this electrode for potentiometer measurements.
Fig. 4. (a) Optical image of a silicon platform and electrodes ready for FIB processing; (b) scanning electron microscope (SEM) image of FIB patterned needle structures.
Fig. 7. Single Ag/AgCl nano-probe: (a) before and (b) after insertion in a patch clamped rat fibroblast cell. Diameter of the probed fibroblast cell is 8 lm.
Both the potentiodynamic and potentiostatic electrochemical characterization showed typical characteristics of a UME in the sub-micron size regime and indicate electrochemical functionality of the planar nano-probes. 3.3. Single cell measurement with potential probe An Ag/AgCl single nano-probe was inserted into a rat fibroblast while the cell was being held by a glass pipette. The cell membrane potential was recorded against a reference electrode outside of the cell. The potential data were recorded during a period of more than 10 min. As the working electrode was inserted into the cell a voltage decrease (representing the membrane potential) was recorded. (see Fig. 7.) The voltage kept constant as long as the working electrode remained inside the cell. The constant potential after penetration shows that sealing between the cell membrane and the inserted needle electrode was established. 4. Conclusion
Fig. 5. (a) SEM image of a probe with two UMEs; (b) SEM image of a UME embedded in the silicon nitride on the top of the tip; (c) SEM image of an asymmetrically shaped tip of a dual nano-probe.
Needle shaped probes in submicron size have been developed for electrochemical analyses of living single cells. The sharpness of the probe tips and the mechanical robustness of the needle structures allow for a controlled penetration of cell membranes and electrochemical probing of the cytosolic cell environment. Measurements of cell membrane potentials and cell membrane impedances demonstrated the analytical capability of these probes in biological environments.
R.J. Fasching et al. / Microelectronic Engineering 83 (2006) 1638–1641
References [1] B. Sakmann, E. Neher, Single Channel Recording, second ed., Plenum Press, New York, 1995. [2] J. Bard, F.-R.F. Fan, D.T. Pierce, P.R. Unwin, D.O. Wipf, F. Zhou, Science 254 (1991) 68–74. [3] M.V. Mirkin, Mikrochim. Acta 130 (1999) 127–153. [4] J. Bard, F.-R. Fan, M.V. Mirkin, in: A.J. Bard (Ed.), Electroanal. Chem., vol. 18, Marcel Dekker, NewYork, 1994, pp. 243–273.
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[5] C. Kranz, G. Friedbacher, B. Mizaikoff, A. Lugstein, J. Smoliner, E. Bertagnolli, Integration of an ultramicroelectrode in an AFM cantilever: combined technology for enhanced information, J. Anal. Chem. 73 (2001) 2491–2500. [6] P. Sun, Z. Zhang, J. Guo, Y. Shao, Fabrication of nanometer-sized electrodes and tips for scanning electrochemical microscopy, J. Anal. Chem. 73 (2001) 5346–5351. [7] R. Fasching, Y. Tao, F.B. Prinz, Cantilever tip probe arrays for simultaneous SECM and AFM analysis, Sensor. Actuator. B 108 (2005) 964–972.
Nanoscale electrochemical probes for single cell analysis R.J. Fasching *, S.-J. Bai, T. Fabian, F.B. Prinz Rapid Prototyping Laboratory, Stanford University, Stanford, CA 94305, USA Available online 3 March 2006
Abstract Needle shaped probes with a dual electrode system in submicron size have been developed for electrochemical analyses of living single cells. The probe system is designed for local probing of the cytosolic cell environment and cell organelles using amperometric, potentiometric and impedance spectroscopic methods. Silicon nitride cantilevers with an electrode metal layer system are fabricated on 4 in. wafers using conventional micro fabrication techniques. The probe needle structures with a tip in sub micron scale are patterned using focus ion beam (FIB) technology. A focused ion beam is utilized to write the probe needle shape into the pre-shaped cantilever and, for a dual electrode system, the probe is divided into two parts to create two separate electrodes. Subsequently, the needle structures are released from the supporting bulk silicon during a wet etching step, and a silicon nitride layer is deposited to isolate and embed the electrode metal layer. Finally, FIB milling is used for a precise exposure of the buried metal layer by cutting the top of the tip. Electrochemical characterization of nano-probes showed full functionality of Ag/AgCl as well as of platinum transducer systems. The sharpness of the probe tip with a radius of less than 50 nm and the mechanical robustness of the needle structure allow for a reliable penetration of cell membranes. Initial measurements of cell membrane potentials and cell membrane impedances of rat fibroblast cells using Ag/AgCl transducer probes demonstrate the analytical capability of these probes in biological environments. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Electrochemical nano-probe; Single cell analysis; FIB; AFM; SECM
1. Introduction The motivation for the development of sensing probes for the cytosolic cell space comes from an increasing body of evidence substantiating the role of electron transfer and ion flux mechanism in cell metabolic control. Today, the highest resolution tools investigating cell communication phenomena are cell attached current measurements using patch clamps. The revolutionary insights provided by patch clamp probing made possible a better understanding of transmembrane signaling mechanisms. Problems with patch clamp methods include plasma membrane decoupling from the cytoskeleton, mechanical perturbation of the membrane, cell ballooning and the limited capability to study electrochemical reactions or electron transfers directly [1]. *
Corresponding author. Tel.: +1 650 723 1301; fax: +1 650 723 5034. E-mail address: [email protected] (R.J. Fasching).
0167-9317/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2006.01.262
Direct electrochemical analysis requires the placement of a localized electrode system to the site of interest. Scanning electrochemical microscopy (SECM) methods are based on this concept and have been used for studying biological systems [2–4]. Limitations arising from mechanically non robust and not small enough electrochemical transducer systems do not allow for a controlled penetration of cell membranes and a precise placement inside of a cell [5–7]. In order to overcome these limitations, needle shaped single and dual electrode nano-probes in submicron size have been developed and explored in this study. 2. Experimental 2.1. Fabrication of planar nano-probes The probe fabrication process has been developed on 4 in. wafers and is composed of two main fabrications
R.J. Fasching et al. / Microelectronic Engineering 83 (2006) 1638–1641
Fig. 1. Conventional silicon micro-processing for preparing a silicon platform with a pre-shaped nitride/metal electrode cantilever structure.
1639
probes are patterned in needle shapes and are divided into two parts to fabricate two separate electrodes on the probe (Fig. 2/7). The average width of the beam is about 300 nm, which defines the minimum distance between the two electrodes. After the FIB patterning step, the needle shaped probes are released by wet etching of the already preetched silicon substrate (Fig. 2/8). Following the wet etching process, another thick silicon nitride layer is deposited using plasma enhanced chemical vapor deposition (PECVD) at 350 °C (Fig. 2/9). The purpose of this layer is to embed and electrically isolate the electrodes. The nominal thickness of the silicon nitride layer is about 200 nm. At last, FIB cutting at the tip of the probe needle structure is done to open ultra micro electrodes (Fig. 2/10). This final process determines the size of the ultra micro electrode (UME) and the sharpness of the probe. 2.2. Electrical and mechanical interface
Fig. 2. Two step FIB processing technique for nano-probe fabrication.
The fabricated nano-probes are attached to a custom made printed circuit board (PCB). The PCB serves as a substrate for mounting the probe on a AFM stage for probe manipulation and accommodates signal pre-amplification and voltage buffering with active guarding. The voltage buffer is formed by a single electrometer-grade op-amp OPA129 (Texas Instruments, Inc.) in a unity amplifier configuration. This configuration allows multipurpose use of the nano-probe for potentiometer as well as amperometric measurements in four-wire mode. A customized AFM nozzle and the PCB in assembled configuration and mounted on the AFM scanner are shown in Fig. 3. 2.3. Apparatus
flows. First, a silicon platform with cantilever shaped electrodes is fabricated using convectional micro-fabrication technologies as shown in Fig. 1. The second fabrication part consists of a two step FIB process and is depicted in Fig. 2. The first process sequence begins with a 4 in. double-side polished silicon wafer. A low stress silicon nitride layer, the main layer of the cantilever, is deposited on both sides of the wafer with a typical thickness of 400 nm using low pressure chemical vapor deposition (LPCVD) (Fig. 1/1). Next, a cantilever structure is pre-shaped in the nitride layer during a reactive ion etch (RIE) step (Fig. 1/2). The silicon nitride on the wafer’s backside is then patterned and a deep RIE process is used to partially release the nitride cantilever (Fig. 1/3). Subsequently, an electrode material, such as platinum or silver, is deposited and patterned on the silicon nitride layer on the front side of the wafer using sputtering or evaporation technique in combination with a lift-off process (Fig. 1/4–6). During the second fabrication sequence a two step FIB milling process is used to pattern the nano-probes. Here
In this study, the nano-probes were mounted an AFM pico plus system (Molecular Imaging, AZ) combined with a confocal laser scanning microscope (CLSM) LSM5 pascal system (Zeiss, Oberkochen, Germany). Immobilization of single cells was achieved using conventional pipette- and planar-patch clamp techniques. Electrochemical measurements were carried out using a Gamry potentiostat (Gamry, PA).
Fig. 3. Photograph of a customized AFM nozzle assembled with the PCB (a); and mounted on the AFM (b).
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R.J. Fasching et al. / Microelectronic Engineering 83 (2006) 1638–1641
3. Results 3.1. Silicon platform and FIB patterning Fig. 4a shows an optical image of a finished silicon platform with two pre-shaped electrode systems ready for FIB processing. In Fig. 4b, a SEM image of a FIB patterned needle structure in a pre-shaped cantilever structure is shown. Due to the re-deposition of sputtered materials during FIB writing, the pattern should be written deep down to the silicon substrate through the nitride layer. Fig. 5 depicts a finished nano-probe with two UMEs embedded in the silicon nitride layer. Platinum or silver are used as UME’s metal, respectively. The metal layer is embedded in a 300 nm thick low stress silicon nitride. Sharpening and cutting the needle tip using a FIB enables a precise opening of UMEs and allows for customizing the shape of the single or dual needle tip (Fig. 5b,c). Defined active area of UMEs down to 0.01 lm2 can be achieved.
Fig. 6. (A) Cyclic voltammogram of a Pt UME; and (B) of a Ag/AgCl UME, the potential is related to a 0.1 M Ag/AgCl reference electrode and the voltage sweep rate is 20 mV/s in both cases.
3.2. Electrochemical characterization of the probe system The electrochemical behavior of the UMEs of the probes has been investigated using cyclic voltammetry (CV). CV was performed in a 10 mM ruthenium hexamine chloride (Ru(NH3)6Cl3)/0.1 M potassium chloride (KCl) solution for Pt UMEs and in 0.1 M potassium chloride solution for Ag/AgCl UME. Fig. 6 shows cyclic voltammograms from Pt and Ag/AgCl UMEs. These probes have an average UME area of 0.1 lm2. Open circuit voltages of the Ag/AgCl UMEs show Nernst behavior above 100 mM chloride concentrations. For lower chloride concentrations the potential becomes undefined and limits the working regime of this electrode for potentiometer measurements.
Fig. 4. (a) Optical image of a silicon platform and electrodes ready for FIB processing; (b) scanning electron microscope (SEM) image of FIB patterned needle structures.
Fig. 7. Single Ag/AgCl nano-probe: (a) before and (b) after insertion in a patch clamped rat fibroblast cell. Diameter of the probed fibroblast cell is 8 lm.
Both the potentiodynamic and potentiostatic electrochemical characterization showed typical characteristics of a UME in the sub-micron size regime and indicate electrochemical functionality of the planar nano-probes. 3.3. Single cell measurement with potential probe An Ag/AgCl single nano-probe was inserted into a rat fibroblast while the cell was being held by a glass pipette. The cell membrane potential was recorded against a reference electrode outside of the cell. The potential data were recorded during a period of more than 10 min. As the working electrode was inserted into the cell a voltage decrease (representing the membrane potential) was recorded. (see Fig. 7.) The voltage kept constant as long as the working electrode remained inside the cell. The constant potential after penetration shows that sealing between the cell membrane and the inserted needle electrode was established. 4. Conclusion
Fig. 5. (a) SEM image of a probe with two UMEs; (b) SEM image of a UME embedded in the silicon nitride on the top of the tip; (c) SEM image of an asymmetrically shaped tip of a dual nano-probe.
Needle shaped probes in submicron size have been developed for electrochemical analyses of living single cells. The sharpness of the probe tips and the mechanical robustness of the needle structures allow for a controlled penetration of cell membranes and electrochemical probing of the cytosolic cell environment. Measurements of cell membrane potentials and cell membrane impedances demonstrated the analytical capability of these probes in biological environments.
R.J. Fasching et al. / Microelectronic Engineering 83 (2006) 1638–1641
References [1] B. Sakmann, E. Neher, Single Channel Recording, second ed., Plenum Press, New York, 1995. [2] J. Bard, F.-R.F. Fan, D.T. Pierce, P.R. Unwin, D.O. Wipf, F. Zhou, Science 254 (1991) 68–74. [3] M.V. Mirkin, Mikrochim. Acta 130 (1999) 127–153. [4] J. Bard, F.-R. Fan, M.V. Mirkin, in: A.J. Bard (Ed.), Electroanal. Chem., vol. 18, Marcel Dekker, NewYork, 1994, pp. 243–273.
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[5] C. Kranz, G. Friedbacher, B. Mizaikoff, A. Lugstein, J. Smoliner, E. Bertagnolli, Integration of an ultramicroelectrode in an AFM cantilever: combined technology for enhanced information, J. Anal. Chem. 73 (2001) 2491–2500. [6] P. Sun, Z. Zhang, J. Guo, Y. Shao, Fabrication of nanometer-sized electrodes and tips for scanning electrochemical microscopy, J. Anal. Chem. 73 (2001) 5346–5351. [7] R. Fasching, Y. Tao, F.B. Prinz, Cantilever tip probe arrays for simultaneous SECM and AFM analysis, Sensor. Actuator. B 108 (2005) 964–972.