JP4864388B2 - Microchip and analysis method and apparatus using the same - Google Patents

Description

  The present invention relates to a microchip having a flow path provided in a substrate, and a method and apparatus for performing analysis using the microchip.

  In recent years, research on μTAS (Micro Total Systems) has become active in the field of analytical chemistry. The microchip performs various chemical processes such as mixing, chemical reaction, separation, and extraction in a small flow path formed in a small chip, for example, several centimeters square. When the chemical process is performed at the micro size, the interface area per unit volume where the chemical reaction occurs is larger than when the chemical process is performed at the macro size, thus reducing the size of the chemical device, high-speed processing, and reducing the amount of reagents and their disposal. Expected to shorten measurement time, increase efficiency and save energy.

The sample solution processed by the microchip has been detected by a visible / ultraviolet absorption method or laser spectroscopy.
However, since these methods are not methods for quantitatively determining a plurality of elements at the same time, it is difficult to perform a microanalysis of the plurality of elements using a microchip.

On the other hand, a fluorescent X-ray analysis method is known as a method for simultaneously quantifying a plurality of elements. Among them, the total reflection X-ray fluorescence analysis method in which primary X-rays are incident on the sample under the condition of total reflection can measure the X-ray fluorescence from the sample surface or impurities thereon with high sensitivity. In the total reflection X-ray fluorescence analysis method, since the incident angle of primary X-rays is very small, reflected X-rays and scattered X-rays are difficult to enter the X-ray detector, and the fluorescence X detected by the X-ray detector The continuous X-ray background noise is small compared to the line output level, and there is an advantage that the S / B (signal / background) ratio can be increased (see Patent Document 1).
JP 2003-202306 A

However, since the total reflection X-ray fluorescence analysis method totally reflects primary X-rays on a flat solid surface, X-rays are incident at a small incident angle that satisfies the total reflection condition on the sample flowing in the microchannel. The idea itself of using a total reflection X-ray fluorescence analysis method for detection with a microchip has not been made so far.
Accordingly, an object of the present invention is to provide an analysis method and apparatus using a microchip as a method for simultaneously analyzing a plurality of elements with high sensitivity.

The microchip of the present invention comprises a substrate, a channel formed inside the substrate, and a part of a flat surface of the substrate, and an outlet of the channel is formed as an opening, and overflows from the opening. And an analysis unit that the measurement target liquid remains on the flat surface of the substrate and becomes an analysis sample.
A preferred example of the analysis unit is one in which primary X-rays are incident under the condition of total reflection, and fluorescent X-rays generated from an analysis sample in the analysis unit are detected by an X-ray detector provided separately. .

The analysis method of the present invention uses the microchip and includes the following steps (A) to (C).
(A) Using this microchip, a step of causing the measurement target liquid to overflow as an analysis sample from the flow path through the opening to the analysis unit;
(B) A step of causing primary X-rays to be incident on the analysis sample overflowing the analysis unit under a condition of total reflection, and (C) a step of detecting fluorescent X-rays generated from the analysis sample.

When the analysis sample is an aqueous solution, water absorbs X-rays. Therefore, when the X-rays are absorbed by the liquid sample, the S / B ratio becomes small. Therefore, between steps (A) and (B) It is preferable to include a step of drying the analysis sample of the analysis unit.
The flow path performs, for example, liquid feeding, chemical reaction, or separation.

  The microchip analyzer of the present invention uses this microchip as a measurement medium, and the condition of total reflection of the primary X-rays to the X-ray source that generates primary X-rays and the analysis part of the microchip. And an X-ray detector that detects fluorescent X-rays generated from an analysis sample in the analysis unit, and is arranged to face the analysis unit of the microchip. .

  Until now, there has been no method that uses a microchip to detect multiple elements at the same time. However, according to the analysis method using the microchip of the present invention, the total reflection X-ray fluorescence analysis method can be applied to the detection. It becomes possible to analyze a plurality of elements simultaneously with high sensitivity under non-destructive and atmospheric pressure.

When the analysis sample in the analysis unit is dried, the S / B ratio of fluorescent X-rays is improved and the measurement sensitivity is improved.
In addition, the total reflection fluorescent X-ray measurement apparatus can be miniaturized and has good compatibility with a microchip that is originally small, so that it is possible to construct a portable analysis system as a whole.

An embodiment of the present invention will be described below.
FIG. 1 is an embodiment of a microchip, (A) is a perspective view, (B) is a sectional view along a flow path from a liquid inlet 9a to a liquid outlet 9c, (C) is a top view, and (D). Is an example of a cross-sectional view of a microchannel.

  The microchip 1 includes a substrate 30, a channel 23 formed inside the substrate 30, and a part of a flat surface of the substrate 30, and an outlet of the channel 23 is formed as an opening 9c. The measurement target liquid that overflows stays on the flat surface of the substrate 30 and includes an analysis unit 10 that becomes an analysis sample.

  The substrate 30 is a laminate of two flat substrates 30a and 30b. A groove 23 serving as a flow path is formed in one substrate 30a, and the other substrate 30b is bonded to cover the groove 23. Through holes 9 a to 9 c are formed in the substrate 30 b at the end portion of the flow path 23. When the substrates 30a and 30b are bonded to form the microchip 1, the holes 9a to 9c appear as openings on the flat surface of the substrate 30b. The holes 9a and 9b are liquid inlets for the reaction solution and the hole 9c is a liquid outlet.

  The size of the microchip 1 is, for example, length x = 70 mm, width y = 30 mm, and the thicknesses of the substrates 30a and 30b are each 0.7 mm. The cross section 36 of the flow path 23 has, for example, a depth a = 40 μm and a width b = 100 μm. The inner diameters of the through holes 9a and 9b are 0.5 mm, and the inner diameter of the through hole 9c is 0.2 mm. However, the shape and size of the microchip and the channel of the present invention are not limited to those described above.

FIG. 2 is a schematic front view of an analyzer using the microchip.
Reference numeral 13 denotes a sample table on which the microchip 1 is placed. A primary X-ray irradiating unit 2 is provided that allows primary X-rays to enter the analysis unit on the surface of the microchip 1 placed on the sample stage 13 at an incident angle causing total reflection. The primary X-ray irradiation unit 2 includes an X-ray source 3 for generating X-rays, a multilayer film substrate 5 for monochromaticizing the X-rays, and a slit 7a for irradiating only the monochromatic X-rays to the sample. 7b.
In order to detect fluorescent X-rays generated from the sample of the analysis part of the microchip 1, an X-ray detector 11 is arranged facing the analysis part of the microchip 1 placed on the sample stage 13.

  The sample stage 13 includes adjustment mechanisms 13a and 13b, and the height direction (Z direction) of the surface of the microchip 1 placed on the sample stage 13 by the adjustment mechanisms 13a and 13b and the microchip for incident X-rays. The tilt direction (θ) of the surface of 1 can be adjusted. The adjustment mechanisms 13a and 13b include a stepping motor 13a that performs adjustment in the height direction, and an inclination motor 13b that is disposed below the stepping motor 13a and adjusts the inclination of the surface of the microchip 1 with respect to incident X-rays. It is out.

A commercially available X-ray tube is used as the X-ray source 3, and an X-ray transmitting material such as beryllium, boron nitride, or graphite is used for the X-ray emission window of the X-ray source 3. Between the X-ray emission window of the X-ray source 3 and the multilayer film substrate 5, in order to prevent the X-ray derived from the tube from affecting the fluorescent X-ray measurement, the measurement target element such as zirconium, aluminum, brass, etc. A suitable filter may be provided accordingly.
Here, as the X-ray source 3, an X-ray tube having molybdenum as a target and a beryllium X-ray emission window is used, and a zirconium filter is disposed between the X-ray emission window of the X-ray source 3 and the multilayer substrate 5. did.

  The X-ray detector 11 includes a type equipped with a wavelength dispersive spectrometer and a type equipped with an energy dispersive X-ray spectrometer. A wavelength dispersive spectrometer uses the diffraction phenomenon of a curved crystal. The energy dispersive X-ray spectrometer takes in all X-rays generated from a sample simultaneously into a detector, and electrically selects X-ray energy. The energy dispersive X-ray spectrometer has the advantage of being able to measure the entire energy range at the same time because the energy resolution is inferior to that of the wavelength dispersive spectrometer, but the measurement time is short. On the other hand, the wavelength dispersive spectrometer has the advantage that the measurement time is longer than that of the energy dispersive X-ray spectrometer, but the energy resolution is excellent. In elemental analysis, any of these two types of spectrometers having different resolutions may be used, or they may be used in combination. Here, an energy dispersive X-ray spectrometer is used to shorten the measurement time.

  When the liquid sample overflowing into the analysis unit 10 of the microchip 1 is an aqueous solution, it is preferably dried and concentrated to form a solid sample in order to avoid absorption of X-rays by water. Drying may be done by natural drying, but heating and forcing to dry will take less time. Therefore, a heating mechanism may be provided on the sample stage 13.

Next, an example of the manufacturing method of the microchip 1 is shown.
A quartz glass substrate is used as the substrates 30a and 30b. First, a photoresist is applied to one glass substrate 30a and prebaked, and the photoresist is exposed to UV (ultraviolet) light through a photomask. Thereafter, the photoresist is developed and patterned, post-baked, and then the substrate 30a is etched using the photoresist pattern as a mask to form a groove serving as the flow path 23. Thereafter, the photoresist is removed. Etching may be dry etching or wet etching. For example, wet etching is performed using a hydrofluoric acid aqueous solution as an etching solution.

Through holes 9a to 9c are opened in the other substrate 30b by a sandblasting method or the like.
As described above, the two substrates 30a and 30b in which the flow channel groove 23 and the through holes 9a to 9c are formed are superposed on the surface of the substrate 30a on which the groove 23 is formed, and heating and pressurization are performed. Join liquid-tight.

  As a material of the substrate, in addition to the quartz glass substrate, other glass substrates, silicon substrates, and resin substrates can be used. In any case, the groove and the through hole can be formed by a chemical or mechanical means, or by various means such as laser irradiation or ion etching.

  In this example, a microchip made of quartz glass was manufactured. However, since quartz glass is inherently hydrophobic, the surface of the flow path is hydrophilic before joining the two substrates in order to make the aqueous solution sample easier to handle. It is preferable to perform a chemical treatment that makes it more effective.

An example of the hydrophilic treatment is shown below in the order of steps.
(1) Immerse in hydrochloric acid to dissolve and remove impurity metal components.
(2) Wash with water.
(3) Immerse in ethanol to connect aqueous and organic solvents.
(4) Soak in acetone to remove organic matter.
(5) Immerse in ethanol again to connect the aqueous and organic solvents.
(6) Wash with water.
(7) Immerse in a NaOH solution for about 30 minutes to modify OH groups on the surface.
(8) Wash with water to wash away NaOH.
By passing through such a process, the flow path formed in the microchip 1 becomes hydrophilic.

Next, the operation of this embodiment will be described.
FIG. 3 is a schematic diagram of an experimental operation when a sample solution is injected into the microchip 1 for analysis. The reaction solution is injected into the liquid inlet 9 a of the microchip 1 using the microsyringe 21. Similarly, the other reaction liquid is injected into the liquid inlet 9b.
Both reaction liquids react in the flow path 23, overflow from the flow path 23 to the analysis section on the substrate 30 b through the liquid outlet 9 c and spread.

FIG. 4 is an image showing a state in which the flowed-out solution spreads around the liquid outlet 9c, and is a solution in which a substantially circular region indicated by reference numeral 25 spreads to the analysis section.
The solvent of this solution sample is removed by drying, and then elemental analysis of the sample is performed by total reflection X-ray fluorescence analysis by irradiating primary X-rays under total reflection conditions.

  FIG. 5 shows an example of a fluorescent X-ray spectrum measured using the microchip of this example. In this case, the channel 23 was not used for the reaction, but only for the flow of the sample solution. Therefore, one liquid inlet 9b was used in a closed state. A Zn standard solution (1.017 mg / mL) was measured as a sample solution. 0.08 mL of the sample solution was supplied from the liquid inlet 9 a by the syringe 21 at a flow rate of 0.2 mL / h, overflowed into the analysis part around the liquid outlet 9 c and developed. After the developed sample solution was dried, total reflection X-ray fluorescence analysis was performed.

For the measurement, the Mo target of the X-ray source 3 was operated at 30 KeV and 20 mA, and the fluorescent X-ray was detected for 60 seconds with an energy dispersive detector.
In the fluorescent X-ray spectrum of FIG. 5, the horizontal axis represents energy, and the vertical axis represents fluorescent X-ray intensity (count / second). Of the fluorescent X-ray spectrum in the energy range of 1 to 20 keV, the portion surrounded by the frame of 10 to 15 keV is shown enlarged in the upper right. The peak near 1.8 keV is a fluorescent X-ray generated from Si in quartz glass as a microchip substrate, and the peaks near 8.5 keV and 9.5 keV are fluorescent X-rays generated from Zn in a Zn standard sample, The peak in the vicinity of 10.5 keV is fluorescent X-rays generated from Pb, and a lead plate used for shielding X-rays is considered to be the generation source. The peak in the vicinity of 17.4 keV is the MoKa line that is the excitation X-ray.
From this result, it was observed that noise was small as a whole signal, and that the analysis method using the microchip of the present invention had a good S / B ratio.

FIG. 6 shows the Zn concentration (ppm) and the intensity of the fluorescent X-ray spectrum peak of ZnKα rays (the count value for 60 seconds) for a Zn standard sample prepared by changing the concentration of Zn ions to 4 types in the range of 0 to 1000 ppm. The integrated value is the fluorescent X-ray intensity.) The multiple correlation coefficient R ² is 0.9959, indicating good linearity, indicating that this data can serve as a calibration curve when measuring the concentration of an unknown sample.

  FIG. 7 shows that the X-ray fluorescence spectrum shown in FIG. 5 was measured for a sample solution containing Fe, Cu and Zn using the above-mentioned microchip in order to show that simultaneous detection of a plurality of components is possible. It is a fluorescent X-ray spectrum when it measures on the same conditions. The horizontal axis represents energy, and the vertical axis represents detected X-ray intensity (count / second). The sample solution has 330 ppm of Fe, 500 ppm of Cu, and 167 ppm of Zn, and the composition ratio is Fe: Cu: Zn = 2: 3: 1.

Of the total X-ray fluorescence spectrum in the energy range of 1 to 20 keV, the portion surrounded by the 6 to 11 keV frame is shown enlarged in the upper right. A peak near 6.5 keV is a fluorescent X-ray of Fe, a peak near 8 keV is a fluorescent X-ray of Cu, and a peak near 9 keV is a fluorescent X-ray of Zn.
Since these peaks are clearly separated from each other, it was confirmed that a plurality of elements can be quantified simultaneously.

  The invention is not limited to the embodiments described above but can be practiced within the scope of the claims. For example, in the above embodiment, the stepping motor 13a and the tilt motor 13b are used as the adjustment mechanism for adjusting the incident angle of the primary X-ray. However, the primary X-ray is adjusted on the microchip by adjusting the X-ray irradiation unit 2. May be adjusted so as to satisfy the total reflection condition, or the primary X-ray may be adjusted so as to satisfy the total reflection condition on the microchip by both the X-ray irradiation unit 2 and the stepping motor 13a and the tilting motor 13b. Good.

  INDUSTRIAL APPLICABILITY The present invention can be used in an instrument analyzer that handles the reaction or separation of a small amount of solution using a microchip and detects the result.

It is a figure which shows one Example of a microchip, (A) is a perspective view, (B) is sectional drawing along the flow path from the liquid inlet 9a of (A) to the liquid outlet 9b, (C) is a top view. (D) is a cross-sectional view of the microchannel. It is a schematic front view which shows one Example of an analyzer. It is a schematic perspective view of experiment operation when inject | pouring a sample solution into a microchip and analyzing. It is an image which shows the state by which the liquid for samples which flowed out to the analysis part was developed. It is a fluorescent X-ray spectrum when measuring a Zn standard sample. It is a graph which shows the relationship between the fluorescence X-ray spectrum peak intensity about a standard sample, and a density | concentration. It is a fluorescent X-ray spectrum when measuring the sample solution containing Fe, Cu, and Zn.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microchip 2 X-ray irradiation part 3 X-ray source 5 Reflector 7a, 7b Slit 9a, 9b, Liquid inlet 9c Liquid outlet 10 Analysis part 11 Detector 13 Sample stand 13a Stepping motor 13b Inclination motor 21 Micro syringe 23 Channel 30a 30b Substrate 36 Flow path cross section

Claims (5)

A substrate,
A flow path formed inside the substrate;
The substrate comprises a partial area of the flat surface of the substrate, and includes an outlet of the flow path. The outlet is formed as an opening in the flat surface of the substrate, and the liquid to be measured overflowing from the opening is the substrate. An analysis part that remains on the flat surface of the sample and serves as an analysis sample ,
The analysis unit has a flat region with a size that allows primary X-rays to be incident under total reflection conditions, and
The said analysis part is a microchip for total reflection X-ray fluorescence analysis in which the fluorescence X-rays generated from the analysis sample in the analysis part are detected by the X-ray detector separately provided . An analysis method comprising the following steps (A) to (C) using the microchip for total reflection X-ray fluorescence analysis according to claim 1 .
(A) using the microchip, causing the analysis target liquid to overflow as an analysis sample from the flow path through the opening to the analysis unit;
(B) A step of causing primary X-rays to be incident on the analysis sample overflowing the analysis unit under a condition of total reflection, and (C) a step of detecting fluorescent X-rays generated from the analysis sample. The analysis method according to claim 2 , further comprising a step of drying the analysis sample of the analysis unit between the steps (A) and (B). The analysis method according to claim 2 or 3 , wherein at least one of liquid feeding, chemical reaction, and separation is performed in the flow path. The microchip for total reflection X-ray fluorescence analysis according to claim 1 is used as a measurement medium,
An X-ray source that generates primary X-rays;
A primary X-ray incidence adjusting mechanism for making the primary X-ray incident on the analysis unit of the microchip under conditions of total reflection;
An X-ray detector that is arranged opposite to the analysis part of the microchip and detects fluorescent X-rays generated from an analysis sample in the analysis part;
A microchip analyzer equipped with