JP3511910B2 - Detector cell - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a detector cell for measuring the absorption of light rays in the ultraviolet or visible region, which is suitable for detecting a trace amount of a component in a liquid sample.

[0002]

2. Description of the Related Art Such a detector cell is used, for example, as a method for accurately and rapidly analyzing a trace amount component in the field of analytical chemistry, for example, in the fields of environmental analysis, clinical practice, pharmaceuticals, etc. It is used as a detector for CE), liquid chromatography (LC), flow injection analysis (FIA) and the like. The detector cell used in these analyzers usually has a sample inlet for introducing a liquid sample to be analyzed, a flow path for the liquid sample and a sample outlet for discharging the liquid sample passing through the flow path. Equipped with a sample chamber in its flow path, which serves as an interaction region between the liquid sample and the detection light in the ultraviolet or visible region, and is used by being connected to the outlet of the analytical column used in the analytical method shown above. To be done. Detection light is applied to the flow path portion serving as the measurement chamber, and the detection light is detected by the measurement optical system after passing through the liquid sample existing in the sample chamber.

In recent years, DJ Harrison et al./Science, Vol.26 has been proposed as a form that can be expected to speed up the analysis and downsize the device in place of the glass capillary which is complicated to handle.
1, p.895-897 (1993), a chip (microchip) formed by bonding two substrates and used for capillary electrophoresis has been proposed. The microchip uses a micromachining technology based on semiconductor manufacturing technology for a flow path for introducing a liquid sample on an electrophoretic member made of a glass substrate and a flow path for separating a liquid sample. It was formed. Compared to conventional capillary electrophoresis devices, electrophoresis devices that use microchips have the advantages of high-speed analysis, extremely low solvent consumption, extremely small amount of sample required, and miniaturization of the device. Have. The feature of this is that it enables on-site (on-side or bed-side) analysis, which was difficult to realize in the conventional analytical equipment in the field of analytical chemistry described above, and also for fields such as DNA analysis. However, from the viewpoint of high-speed analysis, it is considered promising as an advantage for screening.

[0004]

The detector cell used in the capillary electrophoresis apparatus generally has a larger measuring chamber volume than a glass capillary for separating a sample, and when analyzing a trace amount component, In many cases, the separation ability of capillary electrophoresis cannot be utilized. This is because the volume of the measurement chamber is large, so that the separated trace components are remixed or diffused into the medium.

For this problem, for example, Analytical Ch
As described in emistry, Vol.63, p.2835 (1991), an attempt has been made on a laser-excited fluorescence detector using a small cell in which the volume of a measurement chamber is reduced. FIG. 1 shows the laser-excited fluorescence detector. The exit end of the capillary C is inserted into the cell D, and the laser beam from the laser device A is condensed by the lens B and irradiated onto the sample solution emitted from the capillary C. The fluorescence generated from the sample solution in the cell D is condensed by the objective lens E, the excitation light component is removed by the filter CG through the pinhole F, and only the fluorescence component is incident on the photomultiplier tube H and detected. It I is a computer for data processing.

However, even with this laser-excited fluorescence detector, although efforts have been made to reduce the measurement volume, cell D
The inside diameter of Capillary C is smaller than that of Capillary C
Since the wall thickness of B is inevitably large and cannot be downsized sufficiently, there is room for improvement in terms of resolution and accuracy.
On the other hand, a method of using the separation capillary column itself as a detection cell has also been studied. In this case, the sample volume and sample chamber volume required for analysis can be made extremely small, but the detection sensitivity is insufficient due to the short optical path length and the fact that light that does not contribute to detection enters the detector as stray light. Is a problem.

Therefore, the present invention can be used not only as a capillary electrophoresis apparatus but also as a detector cell for the above-described analysis method, or as a detector unit of a microchip. However, it is an object of the present invention to provide a device with improved detection sensitivity.

[0008]

According to the present invention, the volume of the measuring chamber of the detector cell does not impair the separation ability of various separation / analysis means, that is, the cross-sectional area of the flow path is the same as that of the separation capillary column. The detection sensitivity is improved by suppressing the light, which does not interact with the liquid sample to be detected, from among the detected light from entering the detector as stray light. That is, the detector cell of the present invention is provided with a flow passage having a minute cross-sectional area formed inside the transparent plate-like member, and at least a part of the flow passage is used as a measurement chamber so that the detection light is irradiated thereto. And a light-shielding film for the detection light is formed inside the transparent plate-shaped member at least in the region of the measurement chamber on both sides of the flow channel when viewed from the detection light irradiation direction. . Further, the flow path is formed by etching, and the light-shielding film also serves as a protective film at the time of etching and is in contact with the flow path. In the present invention, among the detection light, stray light that does not pass through the liquid sample does not enter the detector, and only light that interacts with the sample, for example, light in which the wavelength corresponding to the sample is absorbed is detected as a signal. , The detection sensitivity is improved.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION This detector cell can be applied as a detector of various analyzers by forming only the detector cell on a transparent plate member of a substrate. In that case, the transparent plate-shaped member in which this detector cell is formed, a sample introduction port for introducing a liquid sample into the flow path constituting the measurement chamber,
A sample discharge port for discharging the liquid sample that has passed through the channel is provided. This detector cell can also be applied as the detector of the microchip shown above. In that case, the transparent plate-shaped member in which the detector cell is formed has an analysis channel and a sample channel intersecting the analysis channel, and one surface of the transparent plate-shaped member is formed. A hole reaching the analysis channel or the sample channel is formed at a position corresponding to the analysis channel and the sample channel, and at least a part of the analysis channel serves as the measurement chamber of the detector cell.

As a material for the transparent plate member on which the detector cell is formed, a resin plate can be used in addition to a glass plate such as quartz or Pyrex. As a light-shielding film,
In addition to the Si (silicon) thin film, a metal thin film such as Cr (chrome) or an organic resin film such as polyimide can be used as long as it has a large absorption coefficient for the detection light.

[0011]

EXAMPLE FIG. 2 shows an example, (a)
Is a top view thereof, (b) is a sectional view taken along the line BB.
(C) is sectional drawing which expands and shows the center part in the AA line position of (a). Glass substrates 1 and 2 made of quartz are bonded together, and a minute channel groove 8 for a liquid sample channel having a width and a depth of several 100 μm or less is formed on the glass substrate 1 side of the bonded surface. Has been done. A sample introduction port 9a is provided at one end of the flow channel 8 so as to penetrate the glass substrate 2, and a sample discharge port 9b is provided at the other end of the flow channel 8 so as to penetrate the glass substrate 2.

On the bonding surface of the glass substrates 1 and 2, a light-shielding film is formed on the glass substrate 1 in order to block the detection light 11 in the ultraviolet or visible region except the channel of the channel groove 8. The light-shielding film constitutes the slit 3 through which the detection light passes only through the flow channel 8. A Si thin film is used here as a light-shielding film forming the slit 3. Both boards 1,
In No. 2, the flow channels 8 are formed by making the surfaces to be bonded face-to-face and closely contacting each other, and then airtightly bonding with a hydrofluoric acid solution by the method shown in FIG.

A part 8a of the channel groove 8 is a measuring chamber 8a for irradiating the detection light and measuring the absorbance. Since a part of the minute channel groove 8 is used as the measuring chamber 8a, it is sufficiently small. A volume measuring chamber can be realized. Further, when the detection light 11 is made incident on the detector cell, the detection light 11 is transmitted only through the measurement chamber 8a which is a part of the flow path groove 8, and the light hitting the light shielding film of the slit 3 is caused by the light shielding film. Since the light is blocked, it is possible to reduce the incidence of stray light on the detector as compared with the related art, and the detection sensitivity is improved.

Next, a method for producing the detector cell of this embodiment will be described with reference to FIG. (A) First, after cleaning the quartz glass substrate 1, a Si film having a thickness of 3000 Å is formed as an etching protection film 5 by a thin film forming device, for example, a sputtering film forming device, and the etching protection film is further formed thereon. As the photoresist 6 for patterning 5, for example, AZ4620 3000r
Spin coat for 40 seconds at a rotation speed of pm. At this time,
The thickness of the resist 6 is about 7 μm. The material and thickness of the photoresist used are not particularly limited,
Any material and thickness that can withstand the subsequent etching step may be used.
Further, the material and thickness of the etching protection film 5 are not particularly limited as long as the material and the thickness can withstand the subsequent substrate etching process. Here, a Si film is used so that the etching protection film 5 also serves as a light shielding film.

(B) Next, the photoresist 6 is exposed using the photomask 7, and then developed. The exposure of the photoresist 6 can be performed using an aligner generally used in semiconductor manufacturing. The developer is not particularly limited as long as it can be used for developing the photoresist used.

(C) Next, the etching protection film 5 is patterned by dry etching using high frequency plasma in SF ₆ gas. Here, the etching gas is not particularly limited as long as Si can be etched without any problem.

(D) Using the patterned etching protection film 5 and the photoresist 6 as a mask, the substrate 1 is etched with, for example, a 46% hydrofluoric acid aqueous solution to form a sample channel groove 8. Also in this case, the etching solution for the substrate 1 is not particularly limited as long as it is a solution capable of etching quartz glass.

(E) After removing the photoresist 6, the surface of the Si film of the etching protection film 5 is oxidized to form a silicon oxide film on the surface of the Si film. (F) On the other hand, on the glass substrate 2, as shown in (f), through holes for the sample inlet 9a and the liquid outlet 9b are formed by a processing method such as a sandblast method. .

(G) Finally, the glass substrate 1 having the slits 3 formed by the light-shielding film also serving as the flow channel 8 and the etching protection film 5 and the glass substrate 2 having the through holes 9a and 9b are superposed on each other. By interposing a 1% hydrofluoric acid aqueous solution at the interface and applying a load of about 1 MPa as needed, the glass substrate 1 and 2 are adhered to form a detector cell by abandoning for 24 hours at room temperature. The etching of the glass substrate 1 in step (d) may be dry etching.

FIG. 4 shows an example of an optical measuring device using the detector cell of this embodiment. 21 is an ultraviolet visible light source,
A deuterium lamp and a tungsten lamp as a light source lamp, an optical system for selecting and extracting light from any one of the lamps, and a spectroscope for selecting light of a predetermined wavelength from the selected light source light are incorporated. There is. Reference numeral 22 denotes a photodetector, which detects light that has passed through the measurement chamber portion of the detector cell 20 and which passes through the photodiode array detector as a photodetection element and the measurement chamber portion of the detector cell 20. And a measuring optical system for guiding the detection light from the light source 21 to the photodiode array detector. Both the light source 21 and the photodetector 22 are generally used in the field of UV-visible measurement.

Reference numeral 23 denotes a stage, and the stage 23
Is provided with a recess 24 in which the detector cell 20 can be positioned. By inserting the detector cell 20 into the recess 24, the inlet channel 25 formed in the stage 23 and the sample introduction port 26 of the detector cell can be brought into close contact with each other, and the stage 23
Outlet channel 27 formed on the substrate and sample outlet 2 of the detector cell
It can be closely attached to 8. As a result, optical measurement becomes possible only by setting the detector cell 20 in the recess 24 of the stage 23.

In the embodiment shown in FIG. 2, the light-shielding film is formed on all surfaces except the flow path portion. However, the light-shielding film is partially formed only on both sides of the flow path in the measurement chamber portion of the glass substrate 1, The slit 3 may be formed only in the measurement chamber portion.

[0023]

FIG. 5 shows an embodiment in which the present invention is applied to a microchip electrophoresis apparatus. A pair of transparent glass substrates 31 and 32 are formed, and a sample flow path 34 and an analysis flow path 35 which intersect each other are formed on the surface of one of the substrates 32, and the flow path 3 is formed on the surface where the flow paths 34 and 35 are formed.
A light shielding film (not shown) is formed on the entire surface except 4, 35. The other substrate 31 is provided with reservoirs 33 as through holes at positions corresponding to both ends of the sample flow path 34 and the analysis flow path 35. The substrates 31 and 32 are (c)
As shown in FIG. 3, the microchannels are formed by laminating the flow paths 34 and 35 and the light shielding film so that they are inward. The microchip of this example can also be manufactured according to the same process as the manufacturing process of FIG.

When using this microchip, the migration buffer is injected into the sample flow path 34 and the analysis flow path 35 from one of the reservoirs 33. After that, after injecting the sample into the reservoir 33 at one end of the sample flow path 34, an electrode is inserted into each reservoir 33, or the electrodes formed in advance in each reservoir 33 are used, A predetermined high voltage is applied to the sample flow path for a predetermined time, and the sample is guided to the intersection 6 between the sample flow path 34 and the analysis flow path 35. Next, a predetermined voltage for migration is applied to both ends of the analysis channel 35, and the sample existing at the intersection 36 is guided into the analysis channel 35 and separated. By disposing the detector cell of the present invention having the slit 3 at the position of the detection portion of the analysis flow path 35, the separated components are detected.

[0026]

According to the detector cell of the present invention, a flow channel having a cross-sectional area approximately equal to that of a separation capillary column is formed with a fine flow channel cross-sectional area having a width and depth formed with high precision by photofabrication technology. Since it can be used as a measurement chamber, it is possible to realize a measurement chamber having a minute volume that does not impair the separation ability of various separation / analysis means. Since the slit formed of the light-shielding film that blocks the incident light other than the light transmitted through the liquid sample in the measurement chamber is provided, the stray light incident on the detector can be reduced and the detection sensitivity can be improved as compared with the conventional case. As an example, FIG. 6 shows a comparison between the embodiment in which the slit 3 made of the light shielding film of the Si film is provided in the detector cell shown in FIG. 2 and the case in which the slit 3 is not provided. The type 1 is the one of the embodiment, and the type 2 is the one without the slit of the light shielding film. In the case of the example, the absorbance increases linearly as the concentration of the sample (uracil) increases,
Moreover, compared with the case where the slit is not provided, the absorbance is large and the detection sensitivity is improved. This is because stray light was suppressed from entering the detector by providing the slit 3. Further, since the detector cell of the present invention can be manufactured by using semiconductor manufacturing technology, the whole detector cell can be processed in a small size and with high accuracy, and further, a plurality of detector cells can be collectively manufactured. It is also possible to reduce the cost.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram showing a conventional laser-excited fluorescence detector.

FIG. 2 is a view showing an embodiment, (a) is a top view,
(B) is sectional drawing in the BB line position, (c) is (a).
It is sectional drawing which expands and shows the center part in the AA line position.

FIG. 3 is a process sectional view showing a method for producing the detector cell of the embodiment of FIG.

FIG. 4 is a schematic sectional view showing an example of an optical measuring device using the detector cell of the embodiment of FIG.

FIG. 5 is a diagram showing an example in which the present invention is applied to a microchip of a microchip electrophoresis apparatus as another example, and (a) and (b) are plan views showing a glass substrate, respectively. c) is a front view showing a state in which they are attached to each other.

FIG. 6 is a diagram showing a comparison of detection sensitivities of a detector cell of one example and a conventional detector cell.

[Explanation of symbols] 1, 2 glass substrate 3 slits 8 channel grooves 8a Measuring room 9a Sample inlet 9b Sample outlet 11 Detection light

─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Hiroaki Nakanishi, No. 1 Kuwabara-cho, Nishinokyo, Nakagyo-ku, Kyoto City, Kyoto Prefecture, Shimadzu Corporation Sanjo Plant (56) Reference JP 10-288580 (JP, A) JP JP 9-288090 (JP, A) JP-A-8-35927 (JP, A) JP-A-5-240774 (JP, A) JP-A-3-108641 (JP, A) Actual development Sho-58-91164 (JP, U) Hiroaki Nakanishi et al., Preparation of quartz and glass electrophoretic chips using micromachining technology and their basic characterization, analysis chemistry, Japan, June 5, 1998, VOL. 47 / NO. 6, PAGE. 361-367 (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 21/00-21/74 JISST file (JOIS)

Claims (2)

(57) [Claims] 1. A detector comprising a transparent plate-shaped member having a flow path of a minute cross-sectional area formed therein, and at least a part of the flow path serving as a measurement chamber, to which detection light is irradiated. The transparent plate-shaped member in which the detector cell is formed is provided with a sample inlet for introducing a liquid sample into the channel and a sample outlet for discharging the liquid sample passing through the channel. A shielding film for the detection light is formed inside the transparent plate member in at least the region of the measurement chamber on both sides of the flow channel when viewed from the detection light irradiation direction, and the flow channel is etched. And a light-shielding film that also serves as a protective film during etching and is in contact with the flow path. 2. A detector comprising a transparent plate-shaped member having a flow passage with a minute cross-sectional area formed therein, and at least a part of the flow passage serving as a measurement chamber, to which detection light is irradiated. The transparent plate-shaped member in which the detector cell is formed has an analysis channel and a sample channel intersecting with the analysis channel as the above-mentioned channels therein. A hole reaching the analysis channel or the sample channel is formed at a position corresponding to the analysis channel and the sample channel on one surface, and at least a part of the analysis channel serves as the measurement chamber. A light-shielding film for the detection light is formed inside the transparent plate-like member at least in the region of the measurement chamber on both sides of the flow channel when viewed from the light irradiation direction, and the flow channel is formed by etching. And the light-shielding film is A detector cell, which also serves as a protective film for etching, and is in contact with the flow path.