JP4734542B2 - Method and apparatus for measuring membrane transport function of membrane transport molecule using artificial bilayer membrane - Google Patents

Description

  The present invention relates to a function measuring method of a membrane transport molecule using an artificial bilayer membrane (for example, a membrane transport protein such as a drug efflux transporter) and a measurement apparatus thereof, and more particularly to its biomembrane transport function and an inhibitor (inhibition). The response to the drug) is quantitatively measured in detail.

On the cell membrane, there are proteins that are responsible for adjusting the concentration of ions and chemical substances inside and outside the cell, or for signal transmission, and these are called membrane proteins. Among them, the activity of an ion channel or the like that transports ions can be measured by an electrophysiological method (membrane potential / membrane current measurement).
In addition, the following Patent Documents 2 to 4 propose the formation of an artificial lipid bilayer membrane and the application using the same.

  In addition, there is a prior study that images a state in which a cell membrane is directly pressed against a small hole and then crushed and the cytoplasm is eluted, and then the substrate passing through the membrane accumulates in the small hole (Non-Patent Document 1 below). ). In addition, there is a prior study in that an evanescent field of a total reflection microscope is generated only on the bottom surface of a micro (nano) chamber group and high-sensitivity detection is performed at a single molecule level (see Patent Document 1 below).

Similar proposals to the inventors of Non-Patent Document 1 below are reported in Non-Patent Documents 2 to 5 below.
JP 2004-163122 A Japanese Patent Laying-Open No. 2005-083765 Japanese Patent Laid-Open No. 2005-091305 Japanese Patent Laying-Open No. 2005-091308 R. Peters, Annu. Rev. Biophys. Biomol. Struct. , 2003, 32: 47-67. Nikolai I. Kiskin et. al Biophysical Journal Volume 85 October 2003 pp. 2311-2322 M.M. TSCHODRICH-ROTTER & R.T. Peters, Journal of Microscopy, vol. 192, Pt 2, November 1998, pp. 114-125 R. Peters, Traffic 2005; 6: pp. 199-204, Blackwell Munksgaard R. Peters, Traffic 2005; 6: pp. 421-427, Blackwell Munksgaard

  However, in the non-patent document 1 described above, small pores that naturally exist in the polymer sheet are used, the volume is not precisely controlled, and membrane proteins expressed in the living cell membrane are used as they are. Therefore, there are various proteins other than the target protein, and there is a problem that it is impossible to measure at the protein single molecule level.

As described above, there is no reliable quantitative analysis method for membrane transport proteins (transporters) that transport chemical substances and drugs.
Therefore, in view of the above situation, the present invention is an artificial bilayer for quantitatively analyzing the transport efficiency of a membrane protein by fluorescence imaging by combining a microfabricated micro-chamber array and an artificial bilayer membrane incorporating a membrane protein. It is an object of the present invention to provide a method for measuring a membrane transport function of a membrane transport molecule using a molecular membrane and a measurement apparatus therefor.

In order to achieve the above object, the present invention provides
[1] In a method for measuring a membrane transport function of a membrane transport molecule using an artificial bilayer membrane, a minute region in which an artificial bilayer membrane is pressed against a microchamber formed on a glass substrate and the upper surface is closed by the artificial bilayer membrane forming a said artificial bilayer membrane in reconstituted membrane proteins as membrane transport molecules, the addition of a solution of substrate was added to the fluorescent substance on the upper side of the artificial bilayer membrane, the membrane proteins Thus the artificial two the substrate which is transported and passed through a molecular film is accumulated in the small area, high sensitivity and high S / N ratio by the accumulated confocal microscopy by fluorescence imaging the concentrations of the substrate were placed below the glass substrate And measuring the efficiency of transporting the substrate by the membrane protein .

[ 2 ] The method for measuring a membrane transport function of a membrane transport molecule using the artificial bilayer membrane according to [1] above, wherein the microregion is a volume fL (femtoliter) to pL (picoliter). .
[ 3 ] In a membrane transport function measuring apparatus for membrane transport molecules using an artificial bilayer membrane, a microchamber formed on a glass substrate, an artificial bilayer membrane pressed against the microchamber, and an upper surface of the artificial bilayer A microregion closed by a membrane, a membrane protein as a membrane transport molecule reconstituted in the artificial bilayer membrane, a substrate solution to which a fluorescent substance added to the upper side of the artificial bilayer membrane is added, and the glass a confocal microscope is disposed below the substrate, the substrate was transported by passing the artificial bilayer membrane by the membrane protein is accumulated in the small area, said co-concentration of the accumulated substrates It is detected with high sensitivity and high S / N ratio by fluorescence imaging using a focus microscope, and the efficiency of transporting the substrate by the membrane protein is measured .

[ 4 ] In the apparatus for measuring a membrane transport function of a membrane transport molecule using the artificial bilayer membrane according to [ 3 ] above, the microregion is a volume fL (femtoliter) to pL (picoliter). .

  Membrane transport proteins such as transporters are very important for medical and pharmaceutical purposes because they are deeply involved in immune reactions and drug responses in cell membranes. For example, a group of membrane proteins called drug excretion ABC transporters excrete anticancer agents out of the cells, and thus research on inhibitors that inactivate them is necessary. The present invention can contribute to elucidating the functions and properties of these proteins in detail, and the effect is remarkable.

  More specifically, according to the present invention, by combining a microfabricated microchamber array and an artificial bilayer membrane incorporating a membrane protein, the transport efficiency of those membrane proteins is quantitatively analyzed by fluorescent imaging, It is possible to perform ultra-sensitive measurement at the protein single molecule level.

Membrane transport function measuring how the membrane transport molecules using artificial bilayer membrane of the present invention, pressing an artificial bilayer membrane to the micro chamber formed on a glass substrate, the upper surface is closed by the artificial bilayer membrane micro forming a region, the artificial bilayer membrane in reconstituted membrane proteins as membrane transport molecules, the addition of a solution above the substrate by adding a fluorescent substance of the artificial bilayer membrane, thus the artificial to the membrane protein bilayer allowed to pass to accumulate the substrate which is transported into the micro region, high sensitivity and high S / N by the accumulated confocal microscopy by fluorescence imaging the concentrations of the substrate were placed below the glass substrate The ratio is detected and the efficiency with which the membrane protein transports the substrate is measured .

Hereinafter, embodiments of the present invention will be described in detail.
FIG. 1 is a schematic cross-sectional view of an artificial bilayer membrane function measuring apparatus showing an embodiment of the present invention, and FIG. 2 is an enlarged view of part A of FIG.
In these figures, 1 is a glass substrate (cover glass), 2 is a microchamber array made of parylene resin with small holes 2A arranged on the glass substrate 1, and 3A is a substrate stored in the upper chamber 4 Solution (buffer solution), 3B is a substrate solution (buffer solution) stored outside the upper chamber 4, 4 is a funnel-type upper chamber set on the microchamber array 2, and 5 is a funnel-type upper chamber 4. An opening formed in the lower portion, 6 is an artificial bimolecular membrane (artificial lipid bilayer membrane) incorporating a transporter 9 set in the opening 5, 7 is an electrode set in the upper chamber 4, 8 above ground electrodes is set outside the substrate dissolved solution 3 B of the upper chamber 4, transporters incorporated in artificial bilayer membrane 6 9, 10 A fluorescent substance contained in the substrate solution 3A inside the partial chamber 4 , 11 is an amplifier connected between the electrode 7 and the ground electrode 8, 12 is an oscilloscope connected to the amplifier 11, 13 is an objective lens, 14 Is a laser that is irradiated from a laser device (not shown) and generates an evanescent field at the bottom of the microchamber array 2 via the objective lens 13.

Here, the artificial lipid bilayer membrane 6 provided in the lower part of the upper chamber 4 is pressed against the microchamber array 2 formed on the glass substrate 1. Then, the upper surface of the small hole 2A is closed by the artificial lipid bilayer membrane 6, and a micro region (volume fL to about pL) 4A is formed. Since the transporter 9 is reconstituted in the artificial lipid bilayer membrane 6, the substrate fluorescently labeled with the fluorescent substance 10 in the upper chamber 4 passes through the artificial lipid bilayer membrane 6 by the transporter 9 and is transported. Is done. For labeled by transportation fluorescent material 10 substrate accumulated in the bottom of the minute regions 4A, the substrate concentration in the micro-region 4A is rising rapidly, the concentration of the substrate can be detected by fluorescence imaging.

That is, in the present invention, a microchamber array is used to optically detect membrane transport across an artificial planar lipid bilayer through membrane proteins (eg, ion pumps and transporters). Here, the microchamber made of parylene resin is formed on the cover glass using photolithography.
According to the present invention, since the artificial lipid bilayer membrane is used, it is possible to measure the pure function of the target membrane protein at a single molecule level as compared with a method using a membrane protein expressed in a normal living cell membrane. it can. In addition, the substrate transported through the artificial lipid bilayer is usually difficult to detect because it diffuses, but it can be detected with high sensitivity because it accumulates in a small region by using a microchamber. The efficiency with which the membrane protein transports the substrate can be measured in detail as , for example, ATP concentration dependency, substrate concentration dependency, inhibitor effect, and the like.

Until now, there is no experimental system that can measure the function of such a membrane protein in detail. In the present invention, it is possible to use a fluorescence observation technique / device with high sensitivity and high S / N ratio, such as a confocal microscope or a total reflection microscope, as an imaging technique. It becomes.
Figure 3 is a diagram showing a micro-chamber comprising a fabricated parylene resin on a glass substrate (cover glass) showing an embodiment of the present invention, the depth 3μm in diameter 20μm in FIG. 3 (a), 3 ( in b) shows the micro-chamber depth 3μm in diameter 10 [mu] m.

The microchamber is manufactured by patterning parylene deposited on a glass substrate (cover glass) by oxygen plasma ashing.
FIG. 4 is a view showing an upper chamber for forming a planar lipid bilayer membrane of the present invention and its opening, and FIG. 4 (a) shows an upper chamber for forming a planar lipid bilayer membrane as shown in FIG. b) is a view showing an opening formed in the bottom of the upper chamber.

In this embodiment, the microchamber has a diameter of several tens of μm and a depth of several μm. Then, a planar lipid bilayer is formed in an opening of 150 μm formed at the bottom of the upper chamber made of PMMA plastic. The upper chamber is filled with a buffer solution (in this experiment 10 mM MOPS (morpholinopropane sulfate) +100 mM KCl). Furthermore, several μL of a solvent containing PC (phosphatidylcholine: neutral phospholipid) [5 mg PC / 1 mL n-decane (decane: one of C ₁₀ H ₂₂ saturated aliphatic hydrocarbons); hereinafter referred to as lipid solution ] Is added over the openings. Here, a planar lipid bilayer is naturally formed by adjusting the buffer solution level in the upper chamber. Note that the opening of the upper chamber is made lower than the bottom surface of the upper chamber, so that the pressing to the substrate is facilitated (see FIGS. 1 and 4).

  FIG. 5 is a diagram showing a planar lipid bilayer membrane formed in the opening of the upper chamber of the present invention and a state in which it is pressed against the microchamber, and FIG. 5 (a) is a lipid formed in the opening of the upper chamber. FIG. 5 (b) is a diagram showing a state in which the bilayer membrane is pressed against the microchamber. In this example, the lipid bilayer membrane is cracked when pressed directly onto a substrate such as glass or parylene. In this example, the chamber substrate is coated with a hydrophilic phospholipid polymer (2-methacryloyloxyethyl phosphorylcholine polymer or MPC polymer). is doing. When the substrate is covered with such a polymer material, the lipid bilayer membrane is not in direct contact with the substrate, and the molecular structure of the lipid bilayer membrane is not broken even after pressing. Similar effects have been reported for molecules such as agarose, PEG (polyethylene glycol), and dextran.

  As shown in FIG. 5 (a), the planar lipid bilayer membrane is formed at the opening of the upper chamber. The area outside the planar lipid bilayer is a so-called annulus, where a large amount of lipid solution is present to support the planar lipid bilayer. As shown in FIG. 5 (b), the planar lipid bilayer is pressed against the microchamber array by lowering the position of the upper chamber.

FIG. 6 is a diagram showing the locus of the fluorescent beads captured in the microchamber and the single beads observed by the confocal microscope of the present invention.
FIG. 6A shows a 200 nm fluorescent bead captured in the microchamber observed by a confocal microscope, and FIG. 6B shows a single bead trajectory in the microchamber. Since the upper surface of the chamber is covered with a lipid bilayer membrane, it does not leave the chamber even by Brownian motion.

  The present invention ultimately detects molecular transport at the single molecule level. For the imaging of this single fluorescent molecule, a TIRFM (total reflection fluorescence microscope) system is often used. In this system, in order to illuminate several hundred nm from the bottom of the glass substrate, the background radiation is removed and an excellent S / N ratio is obtained. Since the microchamber is made on a normal glass coverslip used for biological observation, the evanescent field (excitation light field on the total reflection surface) is formed only at the bottom of the microchamber. The

FIG. 7 is a view showing a single fluorescent molecule of Cy3 observed by TIRFM, in which images of 200 frames taken at an interval of 100 nsec are superimposed.
This figure is a TIRFM image (200 frames of images are superimposed) when a 10 nM Cy3 fluorescent dye solution is placed on the microchamber of the present invention. As the light reaches the bottom of the microchamber and photobleaches, it appears as a blinking dot in the microchamber.

FIG. 8 is a diagram showing a transmembrane current across a lipid bilayer by reconstitution of nanoporous protein (α-hemolysin) in a voltage-fixed state of 20 mV according to the present invention, and the vertical axis represents current (pA), The horizontal axis indicates time (seconds).
FIG. 9 is a graph showing an increase in fluorescence intensity due to accumulation of rhodamine B transported through nanopores (α-hemolysin) according to the present invention.

  Here, as a model of transporter protein, α-hemolysin known as an antibiotic that forms nanopores (diameter 1.5 nm) in a biological membrane is added to the lipid chamber by adding the solution into the upper chamber. Incorporated into the bilayer. From FIG. 8, it can be seen that when the nanopore is reconstituted in the lipid bilayer, the membrane current increases stepwise. This is because each nanopore has equal conductance. From FIG. 8, the conductance of a single α-hemolysin is calculated to be 0.5 nS with the voltage fixed at 20 mV. Thus, in this system, the number of nanopores is calculated from the total current. Further, the number density of nanopores in a unit area can be calculated in the same manner because the region of the double membrane can be monitored under a microscope as shown in FIG.

After nanopore reconstitution (approximately 10 nanopores / 100 μm ² ), a rhodamine solution (10 μM final concentration) is added to the upper chamber. Since the rhodamine solution is transported through the nanopore, an increase in density in the microchamber is observed as an increase in fluorescence emission intensity, as seen in FIG.
Finally, the detectable concentration range was verified in the transport substrate fluorescence detection system of the present invention. The parylene resin used as a material for forming the microchamber in this example is an optically transparent organic material. Therefore, although it is originally desired to detect only the fluorescence inside the microchamber, both the excitation light and the fluorescence are transmitted through the portion outside the chamber, which causes a problem that the S / N ratio is lowered. When TIRFM is used as a detection method, the excitation light is irradiated with a certain incident angle. At this time, the excitation light leaks from the side surface of the chamber (photo of FIG. 9), and the upper surface of the chamber. However, an evanescent field was generated, and this also had a problem that the S / N ratio decreased. The problem is that if the material of the chamber is made of an opaque substance (for example, metal or silicon), the fluorescence only inside the chamber can be detected, and this is not excluded from the scope of the present invention. However, in this embodiment, the reproducibility is better when the flat film is pressed while observing the state of the flat film. If an opaque material is used, observation with transmitted light becomes impossible.

In order to solve these problems, a sufficiently thin (less than 10 nm) aluminum thin film 21 was deposited between the glass substrate and the parylene resin microchamber as shown in FIG. The aluminum thin film 21 reduces transmitted light by about 10-20%.
FIG. 11 shows fluorescence brightness inside and outside the chamber in confocal microscope observation. The purpose of this experiment was to calibrate the detection system, and the lipid bilayer membrane and membrane protein were not used. A solution containing a fluorescent dye with a specified concentration was dropped on the microchamber and observed.

Here, the apparatus is the same as that shown in FIG. 2 except that the aluminum thin film 21 is formed on the upper surface of the glass substrate (cover glass) 1 excluding the minute region 4A.
When the aluminum thin film 21 is not present, the ratio of the fluorescence brightness inside and outside the chamber is about 1.2, whereas in the chamber where the aluminum thin film 21 is deposited, the ratio increases to about 1.5, and the S / N ratio is improved. . Further, when a 60 × objective lens (Olympus) and a cooled CCD camera (Hamamatsu Photonics, ORCA-ER) were used for detection, it was confirmed that detection was possible in a concentration range of about 100 nM-10 μM.

  FIG. 12 shows the fluorescence brightness inside and outside the chamber in total reflection microscope observation. FIG. 12A shows the case without the aluminum thin film 21. At this time, the exudation of the excitation light from the side wall of the chamber and the generation of the evanescent field on the upper surface of the chamber become obstacles, so that the variation of the dye concentration and the fluorescence luminance from the proportional relationship increases. On the other hand, when the aluminum thin film 21 is deposited (FIG. 12b), since these obstacles can be suppressed, variation is small and good reproducibility is obtained. When the total reflection microscope was used as the detection means, it was confirmed that measurement could be performed in the range of 10 nM-1 μM.

As mentioned above, according to the present invention,
(1) Since the substrate that has passed through the artificial lipid bilayer is accumulated in the microregion, the concentration rapidly increases, and detection is possible with ultra-high sensitivity.
(2) Since an artificial lipid bilayer membrane is used, it is possible to measure the pure effect of only the membrane protein that performs functional analysis. Moreover, the function at the single molecule level can be measured by adjusting the protein concentration.
(3) The artificial lipid bilayer membrane is pressed against the microchamber array to form a microregion. At that time, the lipid bilayer membrane is broken by coating the microchamber surface with a hydrophilic polymer (PEG, MPC polymer, etc.). Can be prevented.
(4) Since the microchamber array is formed on the cover glass, a high-sensitivity and high S / N ratio imaging technique such as a confocal microscope can be used.
(5) Since the micro-chamber array has a uniform shape and volume by being formed by a fine processing technique, data with good reproducibility can be acquired.
(6) Since imaging and electrophysiological techniques can be used simultaneously, membrane transport and accompanying ion and proton movements can be simultaneously measured.

  In addition, this invention is not limited to the said Example, Based on the meaning of this invention, a various deformation | transformation is possible and these are not excluded from the scope of the present invention.

  Membrane transport proteins such as transporters are very important for medical and pharmaceutical purposes because they are deeply involved in immune reactions and drug responses in cell membranes. For example, a group of membrane proteins called drug effluxing ABC transporters excrete anticancer drugs out of the cells, and therefore research on inhibitors that inactivate them is indispensable. The present invention can be a tool for elucidating the functions and properties of these proteins in detail.

It is a cross-sectional schematic diagram of the function measuring apparatus of the membrane transport molecule | numerator which shows the Example of this invention. It is the A section enlarged view of FIG. It is a figure which shows the micro chamber which consists of parylene resin produced on the glass substrate (cover glass) which shows the Example of this invention. It is a figure which shows the upper chamber and its opening for planar lipid bilayer formation of this invention. It is a figure which shows the state which pressed the planar lipid bilayer membrane formed in opening of the upper chamber of this invention, and the micro chamber. It is a figure which shows the locus | trajectory of the fluorescent bead captured in the micro chamber observed with the confocal microscope of this invention, and its single bead. It is a figure which shows the single fluorescence molecule | numerator of Cy3 observed by TIRFM of this invention. It is a figure which shows the transmembrane current which crosses a lipid bilayer membrane by the reconfiguration | reconstruction of a nanopore ((alpha) -hemolysin) in the voltage fixed state of 20 mV based on this invention. It is a figure which shows the increase in the fluorescence intensity by accumulation | storage of rhodamine B transported through nanopore ((alpha) -hemolysin) based on this invention. It is a figure which shows the micro chamber with which S / N ratio improved which shows the Example of this invention. It is a figure which shows the fluorescence brightness of the chamber inner side and outer side in the confocal microscope observation which shows the Example of this invention. It is a figure which shows the fluorescence brightness of the chamber inner side and outer side in the total reflection microscope observation which shows the Example of this invention.

1 Glass substrate (cover glass)
2 Microchamber 2A Small hole 3A, 3B Substrate solution (buffer solution)
4 Funnel-type upper chamber 5 Opening 6 Artificial bilayer membrane (artificial lipid bilayer membrane)
Fluorescent substance 11 amplifier 12 oscilloscope 13 objective lens 14 the laser 21 aluminum thin film contained in the ground electrode 9 transporter 10 Substrate solvent solution in which is set outside the substrate dissolved liquid of the electrode 8 upper chamber which is set to 7 upper chamber

Claims (4)

An artificial bilayer is pressed against a microchamber formed on a glass substrate to form a microregion whose upper surface is closed by the artificial bilayer, and a membrane protein as a membrane transport molecule is reconstituted in the artificial bilayer. , the artificial bilayer membrane solution was added upper side of the substrate by adding a fluorescent substance, the substrate was transported by passing Therefore the artificial bilayer membrane in the membrane protein is accumulated in the small area, the storage The concentration of the obtained substrate is detected with high sensitivity and high S / N ratio by fluorescence imaging with a confocal microscope placed below the glass substrate, and the efficiency of transporting the substrate by the membrane protein is measured. A method for measuring a membrane transport function of a membrane transport molecule using an artificial bilayer membrane.   2. The method for measuring a membrane transport function of a membrane transport molecule using the artificial bilayer membrane according to claim 1, wherein the microregion is a volume fL (femtoliter) to pL (picoliter). Method for measuring membrane transport function of membrane transport molecules using a membrane. (A) a microchamber formed on a glass substrate;
(B) an artificial bilayer film pressed against the microchamber;
(C) a microregion whose upper surface is closed by the artificial bilayer;
(D) a membrane protein as a membrane transport molecule reconstituted into the artificial bilayer ;
(E) a substrate solution to which a fluorescent material added to the upper side of the artificial bilayer is added ;
(F) a confocal microscope disposed below the glass substrate ;
(G) the said substrate which is transported by passing the artificial bilayer membrane by the membrane protein is accumulated in the small area, high sensitivity and high S concentration of the accumulated substrates by fluorescence imaging with the confocal microscope An apparatus for measuring a membrane transport function of a membrane transport molecule using an artificial bilayer membrane, characterized by measuring the efficiency of transporting the substrate by the membrane protein detected by an / N ratio . 4. The apparatus for measuring a membrane transport function of a membrane transport molecule using an artificial bilayer membrane according to claim 3 , wherein the minute region has a volume fL (femtoliter) to pL (picoliter). Membrane transport function measuring device for membrane transport molecules.