JP2006258744A - Fluorescence measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of preventing easily an S/N ratio from getting unstable with the lapse of time, without lowering detection sensitivity for fluorescence detection, when measuring a fluorescent substance in a liquid sample using a fluorometric measuring instrument. <P>SOLUTION: This fluorescence measurement method is provided with a means for reducing a concave face curvature radius of a sample liquid face, or for preventing the concave face curvature radius from being changed with the lapse of time, when making excitation light get incident from an upper side of the liquid sample in a measuring container to carrying out the fluorometry, using the fluorometric measuring instrument for detecting fluorescence generated by the excitation light. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for stably detecting and measuring fluorescence from a liquid sample.

  Conventionally, in assays in the biochemical field, fluorescence analysis is known in which quantitative analysis and qualitative analysis are performed by measuring fluorescence generated by reacting a fluorescent reagent. In general, in such a fluorescence analysis, an excitation light such as an excitation lamp or a laser beam is irradiated to a fluorescent substance-containing solution injected into a measurement container, and light emission having a wavelength different from the wavelength of the irradiated excitation light is observed. A fluorescence measurement device that detects a target component in a measurement object by using the phenomenon is used.

  Since it is necessary to detect very weak light in this measurement, the sample to be measured is often placed in a black container so as to suppress the reflected light from the container due to the excitation light, and usually the excitation light is irradiated from above. The fluorescence is detected from above. The influence of the reflected light due to the excitation light is a cause of lowering the detection sensitivity and S / N ratio (the ratio between Signal and Noise) in fluorescence measurement. In response to this, for example, by utilizing the fact that a general fluorescent substance generates a time difference in fluorescence emission with respect to excitation light, the optical path of the detection unit is temporally blocked by a chopper (see Patent Document 1). .

  Fluorescence measurement is also performed when detecting barcodes and characters formed by fluorescent materials on various prepaid cards such as pass tickets and telephone cards. In this case as well, the influence of reflection of excitation light is detected. In spite of this, there is a possibility that it may be mistaken for fluorescence even though fluorescence is not generated. For this problem, for example, a method has been proposed in which only the component having a phase different from that of the excitation light is extracted to remove the influence of the reflected light (see Patent Document 2).

However, a fluorescence measuring apparatus that takes these measures is not only expensive, but also an emergency measure. In addition, the S / N ratio may change over time as time elapses from the injection of the sample to be measured. Therefore, in fluorescence measurement and analysis in the biochemical field, in order to eliminate time-dependent errors in data, a control sample with a known concentration is simultaneously subjected to reaction and separation, and the concentration of an unknown sample is compared and analyzed. It was.
JP 2003-207453 A JP-A-7-302300

  The present invention provides a method capable of easily preventing instability of the S / N ratio over time without reducing the detection sensitivity of fluorescence detection when measuring a fluorescent substance in a liquid sample using a fluorescence measuring apparatus. About providing.

  The present inventors have found that the dispersion of data due to a decrease in detection sensitivity or instability of the S / N ratio over time when fluorescence measurement is performed by entering excitation light from above the liquid sample in the measurement container It was clarified that the reflection of light caused by the shape of the liquid surface is the cause, and it was found that the fluorescence can be stably measured by controlling the shape of the sample liquid surface in the container from the outside.

  That is, according to the present invention, in the case where fluorescence measurement is performed using a fluorescence measurement device that detects excitation fluorescence from the liquid sample in the measurement container and detects the fluorescence generated by the excitation light, the concave curvature radius of the sample liquid surface is set. The present invention relates to a fluorescence measurement method characterized in that a means for preventing the change of the concave curvature radius with time is taken.

  According to the method of the present invention, it is possible to perform stable fluorescence detection with little change in detection of a sample to be measured over time.

The fluorescence measurement method of the present invention is a concave surface of a sample liquid surface in the case where fluorescence measurement is performed using a fluorescence measurement device that makes excitation light incident from above a liquid sample in a measurement container and detects fluorescence generated by the excitation light. Measures are taken to make the radius of curvature smaller or prevent the concave radius of curvature from changing with time.
By making the concave curvature radius of the sample liquid surface smaller or preventing the concave curvature radius from changing with time, the shape of the liquid surface (meniscus) is corrected, and strong reflected light from the excitation light may enter the detection unit. It can be suppressed.

  The fluorescence measuring device used in the fluorescence measuring method of the present invention is not particularly limited as long as it can measure a liquid sample, and generally, a fluorescent compound solution produced by reacting a fluorescent substance or a fluorescent reagent, Fluorescence analyzer that performs quantitative analysis and qualitative analysis by applying excitation light such as ultraviolet rays from above to excite molecular orbital electrons and measure the fluorescence emitted when the electrons transition from the excited state to the reference state. That's fine. For example, a fluorescent device as shown in the schematic configuration diagram of FIG. 1 may be used.

  The measurement container is not particularly limited, but for example, a SBS standardized container such as a microplate or a microtube is used, and any material is used, including those made of resin such as polystyrene and polypropylene. Good. Further, it may be a micro chamber made of PDMS (polydimethylsiloxane) made by microfabrication technology, or a microwell made of silicon or aluminum having good heat conductivity.

Examples of means for reducing the concave curvature radius of the sample liquid surface or preventing the change in the concave curvature radius with time include, for example, 1) increasing the temperature of the sample once, and lowering the temperature after the curvature radius decreases. 2) Adding a surfactant to the solution.
Here, the time-dependent change in the radius of curvature of the concave surface of the sample liquid surface means that the balance of the surface tension at the contact point between the inner wall of the measurement container and the solution that is balanced by a delicate force relationship changes with time. (In some cases, the sample liquid level in the measurement container may have a convex shape). By preventing this, it is possible to prevent the S / N ratio from destabilizing with time, and to suppress variations in data.

1) Temperature adjustment of the sample The concave radius of curvature of the sample liquid surface is determined depending on the contact angle at the contact point between the inner wall of the measurement container and the solution. Generally, this contact angle is determined when the inner wall material of the container is unchanged. Depends on the surface tension of the solution. Therefore, if the surface tension of the solution is lowered, the contact angle between the inner wall of the container and the solution is reduced, and the concave curvature radius of the sample liquid surface is also reduced. When the solution contains water as the main component, the surface tension of water is high following mercury, so when introduced into the measuring vessel, the liquid level is convex or flat, but the solution temperature is increased. And the concave curvature radius becomes smaller. The temperature at which the solution is raised is such that the concave curvature radius decreases in a shorter time as the temperature approaches the boiling point, but is 10 seconds to 10 minutes at 70 ° C. to 100 ° C., preferably 1 minute to 5 minutes at 90 ° C. to 100 ° C.

  In general, a fluorescent substance has high temperature sensitivity, and measurement at a lower temperature is preferable because measurement can be performed with higher sensitivity. The temperature range in which the solution is lowered may be 0 ° C to 40 ° C, preferably 4 ° C to 25 ° C, as long as it can be kept at the same temperature every time measurement is performed. Here, the surface tension of the solution increases as the temperature decreases, but there is a hysteresis effect on the contact angle between the inner wall of the measurement container and the liquid surface (the advancing contact angle and the receding contact angle have hysteresis). For this reason, the concave curvature radius does not increase to the state before the heat treatment. Further, the change in the radius of curvature of the concave surface due to the change in the contact angle does not occur over time.

  In order to realize the above means, a temperature adjustment mechanism for adjusting the temperature of the measurement container may be provided. As the temperature adjustment mechanism, the temperature of the measurement container is increased by adjusting the atmosphere in which the measurement container is placed to a high temperature with hot air, and the temperature of the measurement container is changed to the outside air by letting the hot air out. Although there is a mechanism for lowering to the same extent, a mechanism equipped with a mechanism that can quickly adjust the temperature of the outer wall of the measurement container directly by a temperature adjusting member such as a Peltier element is convenient because the processing time can be shortened.

2) Addition of surfactant Since the surface tension of the solution can be stably reduced by adding the surfactant, the concave curvature radius of the liquid surface can be reduced and its change with time can be prevented.
The surfactant that can be used may be either nonionic or ionic, but a nonionic surfactant is preferred because it has a small effect on the fluorescent properties of the fluorescent material. A polyoxyethylene addition type surfactant having a high hydrophilicity / hydrophobicity ratio (HLB) value which is excellent in reducing ability is particularly preferred. Specific examples include polyoxyethylene (20) sorbitan monolaurate (Tween 20) and polyoxyethylene (10) octyl phenyl ether (Triton X-100). These surfactants can be used alone or in combination.

  The added amount of the surfactant to be used is 1 to 1000 times the critical micelle concentration of the surfactant in the sample solution, and further 5 to 20 times the critical micelle concentration. It is preferable to use it.

  In order to realize the above means, it is only necessary to provide means for introducing a surfactant into the measurement container. As such introduction means, a predetermined amount of the surfactant solution is dispensed at a predetermined measurement container position. A pumping mechanism is provided. As the dispensing pump mechanism, any of a plunger pump system, a peristal pump system, a pressure feed / pinch valve system, etc. may be used, but a plunger pump system capable of precisely controlling the dispensing amount is preferable.

Example 1
1. Method A fluorescent dye Cy3 (Amersham Life Science, absorption maximum wavelength: 550 nm) with a concentration of 0.1 pmol / 100 μL mixed in a PBS buffer was introduced into a microplate, and the fluorescence intensity of this solution was measured with a plate reader. As a control, a PBS buffer (blank) containing no Cy3 was also measured. In order to see the change over time, 10 hours were measured every 5 minutes.

2. Results The fluorescence intensity of the Cy3 solution and the blank increased together with the passage of time, and after 7 hours, the fluorescence intensity decreased and became stable (FIG. 2). From this result, it was considered that the reflected light due to the change in the shape of the liquid surface affects the fluorescence detection.

Example 2
1. Method Next, a solution obtained by mixing 0.1 μmol / 100 μL of Cy3 solution mixed in PBS buffer with a surfactant Tween 20 in a microplate to a concentration of 0.1%, and 95% before measurement. A Cy3 solution having a concentration of 0.1 pmol / 100 μL mixed in a PBS buffer subjected to heat treatment at 25 ° C. for 3 minutes after 3 minutes at 0 ° C. was measured every 5 minutes for 10 hours with a plate reader. As a control, the same measurement was performed on a PBS buffer (blank) containing no Cy3.

2. Results FIG. 3 shows the difference in fluorescence intensity between the Cy3 solution and the blank at each time. In the case where no treatment is performed, the difference in fluorescence intensity greatly changes until 7 hours, whereas the solution mixed with the surfactant and the solution subjected to the heat treatment in advance are stable from the start of the measurement. The difference in fluorescence intensity was measured.

It is a conceptual diagram of a fluorescence measuring device. It is a figure which shows the time-dependent change of the fluorescence intensity of Cy3 solution. It is a figure which shows the time-dependent change of the fluorescence intensity of Cy3 solution which added surfactant, and Cy3 solution which heat-processed before the measurement.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Measurement container holding part 2 Light irradiation part 3 Light detection part 4 Control part 5 Command part 6 Data display part 2a Light source 2b Condensing lens 2c Interference filter 3a Detector 3b Condensing lens 3c Interference filter

Claims (3)

  In the case of performing fluorescence measurement using a fluorescence measuring device that detects excitation light from above the liquid sample in the measurement container and detects fluorescence generated by the excitation light, the concave curvature radius of the sample liquid surface is made smaller or A fluorescence measuring method characterized in that a means for preventing a change with time of a concave curvature radius is taken.   2. The method according to claim 1, wherein the means for reducing the concave radius of curvature raises the temperature of the sample once and lowers the temperature after the radius of curvature is reduced.   The method according to claim 1, wherein the means for reducing the concave radius of curvature is to add a surfactant to the solution.

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