Membrane electrochemical signal detection system

Abstract

The present invention is related to a membrane electrochemical signal detection system, which comprises a detection platform and a probe, wherein the detection platform comprises a substrate having a cavity; a hydrogel layer disposed in the cavity of the substrate; and a carrier film disposed above the substrate and the hydrogel layer with at least one through hole corresponding to the cavity of the substrate as a sample slot. The surface of the probe is covered by an insulating layer and a metal for detection is exposed at a tip portion of the probe.

Claims

1. A membrane electrochemical signal detection system, comprising a detecting platform and a probe; Wherein, the detecting platform comprises: a substrate having a cavity; a hydrogel layer disposed in the cavity of the substrate; a carrier film disposed above the substrate and the hydrogel layer and having at least one through hole corresponding to the cavity of the substrate as a sample slot; a gap between the carrier film and the hydrogel layer, wherein the gap is 50 nm˜1 μm; and the probe is covered by an insulating layer and a metal for detection is exposed at a tip portion of the probe.

2. The membrane electrochemical signal detection system as claimed in claim 1, wherein the system further comprises an atomic force microscope.

3. The membrane electrochemical signal detection system as claimed in claim 1, further comprising a power supply, which provides a current signal or a voltage signal to the probe.

4. The membrane electrochemical signal detection system as claimed in claim 3, further comprising an electrode, which is disposed beneath the hydrogel layer.

5. The membrane electrochemical signal detection system as claimed in claim 1, wherein a thickness of the carrier film is 20 nm˜500 nm.

6. The membrane electrochemical signal detection system as claimed in claim 1, wherein a volume of a sample slot is 0.1 nL˜10 nL.

7. The membrane electrochemical signal detection system as claimed in claim 1, wherein a thickness of the insulating layer is 10 nm˜100 nm.

8. The membrane electrochemical signal detection system as claimed in claim 1, wherein the metal for detection is selected from the group consisting of platinum, iridium, cobalt, palladium, rhodium, and alloys thereof.

9. The membrane electrochemical signal detection system as claimed in claim 1, wherein an exposed area of the metal for detection is 100 nm.sup.2˜2 μm.sup.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 to 8 are schematic diagrams showing the preparation method of the detection platform in preparation example 1;

(2) FIGS. 9 to 11 are schematic diagrams showing the preparation method of the probe in preparation example 2;

(3) FIG. 12 is a schematic diagram showing the method for positioning the membrane protein in a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Preparation Example 1—Preparation Method of the Detection Platform

(4) First, as shown in FIG. 1, a silicon substrate 12 is provided with its upper and lower surfaces coated with two low stress silicon nitride layers (SiN.sub.4) 11 and 13 each with a thickness of 50 nm˜200 nm. The silicon nitride layer 11 is served as a carrier film. Second, as shown in FIG. 2, the silicon nitride layer 13 beneath the silicon substrate 12 is dry etched using reactive ion beam etching (RIE). Portions of the silicon substrate 12 are exposed. Third, as shown in FIG. 3, the silicon substrate 12 is immersed in a wet-etching solution (KOH). A cavity 15 was formed by wet etching the exposed portions of the silicon substrate 12. The area A of the cavity 15 may be 50 μm.sup.2˜2 mm.sup.2, wherein the area of the cavity 15 is the area of the bottom side (opening) of the cavity 15. Fourth, as shown in FIG. 4, one or more nano/micro scale through holes corresponding to the cavity 15 are formed as sample slots 16 across the silicon nitride layer 11 above the silicon substrate 12 using focused ion beam lithography. The size and shape of the through holes can be controlled by electron beam lithography. As shown in FIG. 5, which is the top view of FIG. 4, the shape of the sample slot 16 is not particularly limited; however, circular shape is preferred. The volume of the sample slot 16 may be 0.1 nL˜5 nL. Fifth, as shown in FIG. 6, the silicon substrate 12 below the carrier film 11 is immersed in a wet etching solution (KOH). The portions of the silicon substrate 12 below the carrier film 11 are removed by etching. Sixth, as shown in FIG. 7, a polymer hydrogel is injected into the cavity 15 from the bottom of the silicon substrate 12. A hydrogel layer 17 is formed by curing the polymer hydrogel with UV light irradiation. A gap a between the carrier film 11 and hydrogel layer 17 may be 50 nm˜1 μm. The resulting detection platform 100 is shown in FIG. 7.

(5) In addition, an electrode 18 may be disposed under the detection platform 100 shown in FIG. 7. The resulting detection platform 101 is shown in FIG. 8. The electrode 18 receives the current signals or voltage signals from the samples in the sample slots to analyze the electro-physiologic signals at the inner and outer sides of the membrane proteins.

Preparation Example 2—Preparation Method for the Probe

(6) First, as shown in FIG. 9, a metal layer 22 is coated on the surface of a probe 21 of an atomic force microscope. The probe 21 may be any probes of any atomic force microscopes known in the art and the material of the probe 21 is not particularly limited. The metal layer 22 may be selected from the group consisting of platinum, iridium, cobalt, palladium, rhodium, and alloys thereof. The thickness of the metal layer 22 may be 5 nm˜20 nm. Next, as shown in FIG. 10, an insulating layer 23 is deposited on the surface of the metal layer 22. The insulating layer 23 may be selected from the group consisting of SiO.sub.2, Si.sub.3N.sub.4, HfO.sub.2, and other insulating materials known in the art. The thickness of the insulating layer 23 may be 10 nm˜30 nm. In the present preparation example, the insulating layer 23 may be deposited on the metal layer 22 by atomic layer deposition (ALD). However, in other embodiments, other methods such as plasma enhanced CVD (PECVD) or physical vapor deposition (PVD) may be applied to deposit the insulating layer 23 on the metal layer 22. Then, the probe is installed in an atomic force microscope. A scanning process is executed repeatedly on a hard material where the tip of the probe and the hard material are in repeated scuffing against each other. The insulating layer 23 on the tip portion of the probe is rubbed off until an appropriate area 24 of the metal layer 22 beneath the insulating layer 23 is exposed at the tip portion of the probe. The exposed portion 24 of the metal layer 22 at the tip portion of the probe may have an area of 500 nm.sup.2˜1 μm.sup.2. The resulting probe 200 is shown in FIG. 11.

Example 1

(7) The present example demonstrates a method for detecting the electro-physiological signals at the inner and outer sides of the membrane proteins by using the detection platform and the probe from the preparation examples 1 and 2, respectively.

(8) In the present example, lipid molecules 322 self-assembled into a lipid bilayer 32 in the sample slot of the detection platform 101 by using a Langmuir-Blodgett Trough. The protein tested is a proton-pumping pyrophosphatase (H.sup.+-PPase) which is a membrane protein channel 321 formed across the lipid bilayer 32. Hydrogen ions are capable of passing in and out of the lipid bilayer 32 through this membrane protein channel 321.

(9) The probe 200 is then installed in an atomic force microscope. A conductive electrical wire (not shown) connects the back end of the probe 200 to a power supply (not shown) and an oscilloscope (not shown).

(10) As shown in FIG. 12, the lipid bilayer 32 is scanned by an atomic force microscope. The membrane protein channel 321 located across the lipid bilayer 32 is still active, so hydrogen ions (H.sup.+) 31 are being transported. When the hydrogen ions 31 are transported by the membrane protein channel 321, the hydrogen ions 31 are concentrated near the membrane protein channel 321. The density of the hydrogen ions is higher at position (2) on the lipid bilayer 32. When the probe 200 scans from position (1) to position (2) and then to position (3), a protruding portion at position (2) can be detected. The position of the membrane protein channel 321 can then be inferred by the higher density of the hydrogen ions.

(11) After the position of the membrane protein channel 321 across the lipid bilayer 32 is accurately determined according to the above detection method, the power supply provides an additional current signal through the probe 200 to the electrode probe to detect the electro-physiological signals at the inner and outer sides of the membrane at this specific area.

(12) Overall, the detection platform of the present invention provides a platform for lipid bilayer formation where the structure of the lipid bilayer formed can be supported. Since the stability of the lipid bilayer is improved; thus, the lipid bilayer can be scanned by an atomic force microscope. The probe of the present invention has a nano scale metal tip portion. When detecting the electro-physiological signals at the inner and outer sides of the membrane, the area for detection may be narrowed to a specific small area. The noises from the other areas of the membrane can also be reduced effectively as well.

(13) Accordingly, the membrane electrochemical signal detection system provided by the present invention can solve the difficulties in the positioning of the protein transport channels and the confirmation of their structures. The detection system can be further combined with structural analysis techniques, such as X-ray crystallography, to analyze the position and structure of the protein transport channel. For example, according to an embodiment of the present invention, this novel detection system is able to help researchers to understand H.sup.+-PPase. The physiological mechanism of the hydrogen ions transportation, the structure of the transport channel, and other issues such as the titration of transport of the H.sup.+-PPase can be studied.

(14) Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.