JP2005195397A - Electrophoresis detecting apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the collection efficiency for attracting various measurement object materials, increase a concentration of the measurement object material in a detection section and improve the measurement sensitivity. <P>SOLUTION: Electrodes 8-1-8-4 are disposed on a bottom face 6 of a sample cell 2 so as to sequentially narrow a space from an upstream to a downstream of a flow of a sample solution. A metal thin film 10 is disposed downstream of the electrode 8-4. When excitation light enters from outside of the bottom face 6 into the metal thin film 10 through a prism 12 on the condition of total reflection, a surface plasmon resonance is generated, and a change of a refraction index is measured from a surface plasmon resonance angle. When an AC voltage is applied to the electrodes 8-1-8-4, the measurement object material 18 is aggregated on the metal thin film 10 by an electrophoresis, and the measurement object material 18 is condensed and sensitively measured. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a detection apparatus for determining the concentration of a substance to be measured in a sample solution, for example, a microorganism, a protein, a nucleic acid, a sugar, or a mixture in which these are bound.

For the purpose of measuring the substance to be measured in the sample solution with high sensitivity, a substance to be measured such as nucleic acid and protein in the sample cell is provided by providing a pair of electrodes in the measurement cell and applying an alternating electric field between the electrodes. Is collected between electrodes by dielectrophoresis and concentrated, and a method for measuring fluorescence generated by irradiating the concentrated portion with excitation light has been proposed (see Patent Document 1).
According to such dielectrophoresis, since the substance to be measured in the sample cell can be concentrated even if it is thin, the sensitivity can be improved.
JP 2001-13148 A

  Dielectrophoresis is a technique for attracting a substance to be measured by an electric field, more precisely, an electric field gradient. Increasing the electrode spacing generates an electric field gradient far from the electrodes, so that it is possible to attract the substance to be measured far from the electrodes, but conversely, the electric field is not so strong near the electrodes, so The measured substance concentration does not increase so much. On the other hand, if the electrode spacing is reduced, the electric field in the vicinity of the electrode becomes stronger, so that the concentration of the substance to be measured near the electrode surface can be increased. However, the electric field gradient does not reach far from the electrode. Cannot attract the measured substance.

Therefore, in dielectrophoresis, (1) the electrode interval can be widened to collect many substances to be measured, but the local concentration can be reduced, or (2) the electrode interval can be narrowed to increase the local concentration. However, one of the modes with poor collection efficiency must be selected.
Therefore, the object of the present invention is to increase the collection efficiency by attracting a large number of substances to be measured, and to improve the measurement sensitivity by increasing the concentration of the substance to be measured at the detector.

  The dielectrophoresis detection apparatus of the present invention includes a cell in which a sample solution containing a substance to be measured is stored or flowed, and a plurality of electrodes arranged in a row so as to be separated from each other on the inner surface of the cell and to be narrowed. And a detection unit that detects the substance to be measured at a position on an extended line in a direction in which the interval between the electrode arrays is narrowed, and a power supply device that applies an AC voltage to the electrodes.

  An example of the detection unit is to measure the concentration of a substance to be measured from a change in the refractive index of a sample solution, and a metal film disposed on an extension line in a direction in which the interval between the electrode arrays is narrowed, and on the wall surface of the cell A prism disposed at the position of the metal film, an incident optical system that causes light to be incident on the cell under the condition of total reflection that causes surface plasmon resonance to the cell, and the prism reflects the light reflected from the cell. And a light receiving optical system for measuring a refractive index change from a surface plasmon resonance angle.

  Surface plasmon resonance refers to a phenomenon in which when light is incident on a metal surface and evanescent waves having the same wave number (momentum and frequency (energy)) as the surface plasmon overlap, electrons in the vicinity of the metal surface enter a resonance state. Electrons in the metal are collectively oscillated, which is called plasma oscillation or plasma wave. A surface plasmon is a quantum description of a plasma wave on a surface. An evanescent wave is a wave that travels along the surface of a substance, whose energy exponentially decays with distance from the interface, travels along the incident surface in the total reflection region, and Says a wave that does not propagate.

  Another example of the detection unit is to measure the concentration of the substance to be measured from the absorbance of the sample solution, and a prism disposed on the wall surface of the cell at a position on the extended line in the direction in which the interval between the electrode arrays becomes narrower. And an incident optical system that causes light to enter the cell through the prism under conditions of total reflection, and a light receiving optical system that receives the reflected light from the cell via the prism and measures the absorbance. Yes.

The detection unit may measure fluorescence together with a change in refractive index and absorbance, or may measure fluorescence instead of a change in refractive index and absorbance. The detection unit for measuring fluorescence includes a fluorescence light receiving optical system that receives and detects fluorescence generated by excitation of a substance to be measured in the cell.
A preferred example of the cell is a flow cell, and the flow cell is configured so that the sample solution flows in a direction in which the interval between the electrode arrays becomes narrower.
A preferred example of the inner surface on which the electrodes are disposed is the bottom surface of the cell.

  Since the electrodes arranged far from the detection unit have a wide arrangement interval, it is possible to collect substances to be measured far from the electrodes, and the electrode arrangement interval becomes narrower as the detection unit is approached. Therefore, the concentration of the substance to be measured increases as it approaches the detection unit. Furthermore, since an electric field gradient is formed toward the detection unit, the substance to be measured moves so as to be collected toward the detection unit, so that the concentration of the substance to be measured at the detection unit is further increased and the detection sensitivity is increased. .

When the present invention is applied to a flow cell, the flow direction of the sample solution by the flow cell and the direction of the electric field gradient by the electrode arrangement are both directed toward the detection unit, and the concentration effect of the sample concentration at the detection unit is further increased. The measurement sensitivity is further improved.
By forming the electrode array on the bottom surface of the cell, the substance to be measured can be moved onto the bottom surface by both dielectrophoresis and gravity, and the effects of surface plasmon resonance and evanescent waves can be further enhanced.

FIG. 1 schematically shows an embodiment of the present invention. Reference numeral 2 denotes a measurement cell, which is a flow cell in which the sample solution 4 moves rightward in the figure. The height H of the sample cell 2 is, for example, 10 μm.
The bottom surface 6 of the sample cell 2 is made of a transparent material such as quartz glass, and a plurality of electrodes 8-1 to 8-4 separated from each other are provided on the inside of the bottom surface 6. These electrodes 8-1 to 8-4 are strips extending in the direction perpendicular to the paper surface, and are arranged in a line from left to right (in the direction from the upstream side to the downstream side of the flow cell) in the figure. It arrange | positions so that a space | interval may become narrow sequentially along. An example of the electrode spacing is 10 μm between the electrodes 8-1 and 8-2, 5 μm between the electrodes 8-2 and 8-3, and 2 μm between the electrodes 8-3 and 8-4. A metal thin film 10 for causing surface plasmon resonance is formed on the right side of the electrode 8-4 in the figure, and the distance between the electrode 8-4 and the metal thin film 10 is, for example, 1 μm.

  The electrodes 8-1 to 8-4 and the metal thin film 10 do not necessarily have to be made of the same material, but considering the manufacturing process, there is an advantage that the same material can be used to manufacture the electrode 8-1 to 8-4 at the same time. As a material of such electrodes 8-1 to 8-4 and the metal thin film 10, a metal suitable for surface plasmon resonance, for example, gold, silver, copper or aluminum is suitable. The thickness of the electrodes 8-1 to 8-4 and the metal thin film 10 is suitably 30 to 80 nm.

A dielectric thin film may be formed so as to cover the electrodes 8-1 to 8-4 and the metal thin film 10. The dielectric thin film prevents resonance when the electrodes 8-1 to 8-4 and the metal thin film 10 are easily oxidized, such as aluminum, and forms a planar waveguide on the metal thin film 10 to resonate. Further, it serves to prevent the solvent of the sample solution 4 from chemically reacting with the electrodes 8-1 to 8-4 and the metal thin film 10. As such a dielectric thin film, an inorganic dielectric material such as SiO ₂ , Al ₂ O ₃ or TiO ₂ or an organic dielectric material can be used.

A prism 12 for attaching the excitation light to the position facing the metal thin film 10 under the condition of total reflection is attached to the outside of the bottom surface 6. The prism 12 is preferably a hemispherical prism, but a triangular prism, a square prism, or the like can also be used.
Excitation light 14 enters the prism 12 from an incident optical system (not shown), and reflected light 16 from the metal thin film 10 is guided to the light receiving optical system (not shown) via the prism 12.

  The incident optical system includes, for example, a UV excitation light source as a light source unit. As the UV excitation light source, a xenon lamp capable of selecting a wavelength is suitable, but when the wavelength may be fixed, a mercury lamp, a UV laser, or the like may be used. The light receiving optical system includes a UV detector such as a photomultiplier tube, a PD (photodiode), or an APD (avalanche photodiode) in order to receive and detect the reflected light that has passed through the prism 12. The surface plasmon resonance angle can be obtained from the intensity detected by the detector.

A fluorescence light receiving optical system for detecting fluorescence generated from the substance to be measured may be provided outside the upper surface 7 of the cell 2 so as to face the metal thin film 10. Such a fluorescence receiving optical system includes a fluorescence detector such as a photomultiplier tube, PD, APD, PDA (photodiode array). In this case, the material of the upper surface 7 is suitably quartz glass that can transmit fluorescence.
AC power supplies 15-1 to 15-4 are connected to the electrodes 8-1 to 8-4, respectively, and an AC voltage is applied to the electrodes 8-1 to 8-4.

When the sample solution 4 is caused to flow through the sample cell 2 and an AC voltage is applied to the electrodes 8-1 to 8-4, the electric lines of force that the substance 18 to be measured in the sample solution 4 is generated from the electrodes 8-1 to 8-4. In response to both the force due to the dielectrophoresis due to the electric field gradient by 20 and the force due to the flow of the sample solution, it moves in the direction indicated by the dashed arrow in the figure. By making the excitation light 14 incident on the metal thin film 10 via the prism 12, the electric field is enhanced in the region 22 by surface plasmon resonance. By measuring the surface plasmon resonance angle, the refractive index change according to the concentration of the substance to be measured is obtained.
At this time, since the substance to be measured is excited and fluorescence may be generated, the concentration of the substance to be measured can also be obtained by measuring the fluorescence.

  The metal thin film 10 for causing surface plasmon resonance can be omitted from the embodiment of FIG. In that case, the absorbance corresponding to the concentration of the substance to be measured in the sample solution can be measured by the evanescent wave of the light incident by the prism 12.

Although the above embodiment is an application of the present invention to a flow cell, it can also be applied to a microcell of a type containing a solution. In this case, the force due to the flow of the solution does not act, but since the electric field gradient is formed toward the detection unit, the force that moves the substance to be measured far from the bottom surface acts toward the detection unit.
Further, when the present invention is applied to the microcell, by adjusting the phase of the AC voltage applied to the electrodes 8-1 to 8-4, the phase of the electric field gradient is directed toward the detection unit, It becomes possible to effectively collect the substances to be measured in the direction of the detection unit.

  The detection apparatus of the present invention can be used as a detector for an analysis apparatus such as a liquid chromatograph or capillary electrophoresis.

It is a schematic sectional drawing which shows one Example.

Explanation of symbols

2 Measurement cell 4 Sample solution 6 Bottom surface of measurement cell 8-1 to 8-4 Electrode 10 Metal thin film 12 Prism 15-1 to 15-4 AC power supply 18 Measured material 20 Electric field line 22 Electric field enhancement region by surface plasmon resonance

Claims (6)

A cell in which a sample solution containing a substance to be measured is stored or passed;
A plurality of electrodes arranged in a row so as to be separated from each other on one inner surface of the cell, and
A detection unit for detecting the substance to be measured at a position on an extended line in a direction in which the interval between the electrode arrays is narrowed;
A dielectrophoresis detection device comprising: a power supply device that applies an alternating voltage to the electrode.   The detection unit includes a metal film disposed on an extension line in a direction in which the interval between the electrode arrays is narrowed, a prism disposed at the position of the metal film on the wall surface of the cell, and the prism. An incident optical system that allows light to enter under the condition of total reflection that causes surface plasmon resonance to the cell, and light receiving optics that receives reflected light from the cell via the prism and measures a change in refractive index from the surface plasmon resonance angle. The dielectrophoresis detection device according to claim 1, further comprising: a system.   The detector is configured such that a prism disposed on a wall surface of the cell at a position on an extension line in a direction in which the interval between the electrode arrays is narrowed, and light is incident on the cell under the condition of total reflection through the prism. The dielectrophoresis detection device according to claim 1, further comprising: an incident optical system that receives the reflected light from the cell, and a light receiving optical system that receives the reflected light from the cell and measures the absorbance.   4. The dielectrophoresis detection device according to claim 1, wherein the detection unit includes a fluorescence light receiving optical system that receives and detects fluorescence generated by excitation of the substance to be measured in the cell. 5.   5. The dielectrophoresis detection device according to claim 1, wherein the cell is a flow cell, and the sample solution is caused to flow in a direction in which an interval between the electrode arrays becomes narrower. The dielectrophoresis detection device according to claim 1, wherein the inner surface on which the electrode is disposed is a bottom surface of the cell.

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