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Continuous differential impedance spectroscopy of single cells.

Malleo D, Nevill JT, Lee LP, Morgan H - Microfluid Nanofluidics (2009)

Bottom Line: Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell.We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

ABSTRACT
A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.

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Fabrication steps: The device was formed by assembling two microfabricated substrates. a On the bottom substrate (glass), Ti/Pt was deposited using an electron beam evaporator. A 1-μm layer of SU8 was used to insulate the leads of the working electrodes, such that only the active areas of the metal are exposed to the cell solution. A second SU8 layer (25 μm thick) was patterned on top to form both the walls of the fluidic channels as well as the U-shaped cell traps. b On the top substrate (ITO-coated glass), Ti/Pt metal pads were evaporated and a 3-μm layer of SU8 was patterned to create a gap separating the top of the traps on the bottom substrate from the top ITO substrate. c The device was assembled by aligning the two substrates with the aid of a stereoscope, clamping them and bonding them with UV-curable glue
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Fig2: Fabrication steps: The device was formed by assembling two microfabricated substrates. a On the bottom substrate (glass), Ti/Pt was deposited using an electron beam evaporator. A 1-μm layer of SU8 was used to insulate the leads of the working electrodes, such that only the active areas of the metal are exposed to the cell solution. A second SU8 layer (25 μm thick) was patterned on top to form both the walls of the fluidic channels as well as the U-shaped cell traps. b On the top substrate (ITO-coated glass), Ti/Pt metal pads were evaporated and a 3-μm layer of SU8 was patterned to create a gap separating the top of the traps on the bottom substrate from the top ITO substrate. c The device was assembled by aligning the two substrates with the aid of a stereoscope, clamping them and bonding them with UV-curable glue

Mentions: A large area optically transparent electrode is used for the lid of the device. Each cell trap is bounded by an SU8 structure that almost completely encloses the driven small electrode. The device was made by bonding two microfabricated substrates, aligning the cell traps to the measurement electrodes. The general scheme for fabrication is shown in Fig. 2. A lift-off process was used to pattern metal electrodes (70 nm Pt with a 25 nm Ti adhesion layer) onto 4″ Pyrex wafers, for the bottom substrate and onto an Indium Tin Oxide- (ITO) coated Pyrex wafer for the top substrate. The metal on the bottom substrate serves as working electrodes for the impedance measurements. The metal on the top substrate serves to make low-resistance connections between the ITO surface and electronics. The bottom substrate was patterned with a 1 μm thick layer of SU8 (SU8 2001, Microchem) which insulated the connections to the sensing electrodes. A second 25-μm thick SU8 layer (SU8 2025) was patterned on top of this layer to form both the walls of the fluidic channels and the U-shaped cell traps. The top (ITO) substrate was patterned with a 3-μm layer of SU8 (SU8 2005), to create a 3-μm separation between the top of the traps and the top substrate, in addition to insulating the upper electrode outside the sensing areas (see Electronic Supplementary Materials for autoCAD files of the lithography masks). This gap is integral to the hydrodynamic trapping process. Similar traps have been fabricated using PDMS (Sylgard 184, Dow Corning) (Di Carlo et al. 2006b); however, the flexible nature of PDMS makes it prohibitively difficult to reliably align the traps with the metal electrodes on the base without significant and costly engineering. Each pair of wafers had 12 devices. Wafers were diced with an MP500 free shape-cutting machine (MDI Schott, Germany). After drilling access ports, the substrates were aligned and bonded using a UV-curable glue (Norland Optical Adhesive 74).Fig. 2


Continuous differential impedance spectroscopy of single cells.

Malleo D, Nevill JT, Lee LP, Morgan H - Microfluid Nanofluidics (2009)

Fabrication steps: The device was formed by assembling two microfabricated substrates. a On the bottom substrate (glass), Ti/Pt was deposited using an electron beam evaporator. A 1-μm layer of SU8 was used to insulate the leads of the working electrodes, such that only the active areas of the metal are exposed to the cell solution. A second SU8 layer (25 μm thick) was patterned on top to form both the walls of the fluidic channels as well as the U-shaped cell traps. b On the top substrate (ITO-coated glass), Ti/Pt metal pads were evaporated and a 3-μm layer of SU8 was patterned to create a gap separating the top of the traps on the bottom substrate from the top ITO substrate. c The device was assembled by aligning the two substrates with the aid of a stereoscope, clamping them and bonding them with UV-curable glue
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2944380&req=5

Fig2: Fabrication steps: The device was formed by assembling two microfabricated substrates. a On the bottom substrate (glass), Ti/Pt was deposited using an electron beam evaporator. A 1-μm layer of SU8 was used to insulate the leads of the working electrodes, such that only the active areas of the metal are exposed to the cell solution. A second SU8 layer (25 μm thick) was patterned on top to form both the walls of the fluidic channels as well as the U-shaped cell traps. b On the top substrate (ITO-coated glass), Ti/Pt metal pads were evaporated and a 3-μm layer of SU8 was patterned to create a gap separating the top of the traps on the bottom substrate from the top ITO substrate. c The device was assembled by aligning the two substrates with the aid of a stereoscope, clamping them and bonding them with UV-curable glue
Mentions: A large area optically transparent electrode is used for the lid of the device. Each cell trap is bounded by an SU8 structure that almost completely encloses the driven small electrode. The device was made by bonding two microfabricated substrates, aligning the cell traps to the measurement electrodes. The general scheme for fabrication is shown in Fig. 2. A lift-off process was used to pattern metal electrodes (70 nm Pt with a 25 nm Ti adhesion layer) onto 4″ Pyrex wafers, for the bottom substrate and onto an Indium Tin Oxide- (ITO) coated Pyrex wafer for the top substrate. The metal on the bottom substrate serves as working electrodes for the impedance measurements. The metal on the top substrate serves to make low-resistance connections between the ITO surface and electronics. The bottom substrate was patterned with a 1 μm thick layer of SU8 (SU8 2001, Microchem) which insulated the connections to the sensing electrodes. A second 25-μm thick SU8 layer (SU8 2025) was patterned on top of this layer to form both the walls of the fluidic channels and the U-shaped cell traps. The top (ITO) substrate was patterned with a 3-μm layer of SU8 (SU8 2005), to create a 3-μm separation between the top of the traps and the top substrate, in addition to insulating the upper electrode outside the sensing areas (see Electronic Supplementary Materials for autoCAD files of the lithography masks). This gap is integral to the hydrodynamic trapping process. Similar traps have been fabricated using PDMS (Sylgard 184, Dow Corning) (Di Carlo et al. 2006b); however, the flexible nature of PDMS makes it prohibitively difficult to reliably align the traps with the metal electrodes on the base without significant and costly engineering. Each pair of wafers had 12 devices. Wafers were diced with an MP500 free shape-cutting machine (MDI Schott, Germany). After drilling access ports, the substrates were aligned and bonded using a UV-curable glue (Norland Optical Adhesive 74).Fig. 2

Bottom Line: Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell.We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

ABSTRACT
A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus