JP6287195B2 - Fluorescence measuring device - Google Patents

Description

  The present invention relates to a fluorescence measuring apparatus provided in an automatic analyzer or the like.

  Many of the fluorescence measuring devices provided in an automatic analyzer or the like fix the wavelength bands of excitation light and fluorescence within a specific range according to the measurement target. For selection of the wavelength band, an inexpensive and band-pass optical filter capable of transmitting only a predetermined wavelength is used.

  The fluorescence intensity is proportional to the excitation light intensity. Therefore, in order to accurately measure fluorescence, it is preferable to provide a measurement mechanism having an optical system for measuring the amount of excitation light and a detection circuit. For example, in the fluorescence detection device disclosed in Patent Document 1, a light receiving element (monitor PD) for monitoring the intensity of the excitation LED is provided separately from the fluorescence light receiving unit to perform feedback control of excitation light. However, the aspect requires a space for installing the monitor PD and also requires installation costs.

On the other hand, when measuring the change rate of the fluorescence intensity accompanying the biochemical reaction, the absolute value of the fluorescence intensity is not a problem. For this reason, there is a fluorescence measuring apparatus in which the excitation light measuring mechanism is omitted from the viewpoint of space saving and cost saving. However, the amount of light emitted from the light source (LED or tungsten lamp) deteriorates with use. Therefore, if the fluorescence measuring apparatus is continuously used without knowing the deterioration of the light amount, the S / N ratio is lowered, and even in the measurement of the change rate, it causes an erroneous determination and the reliability of the analysis is lowered. Further, if the excitation light measurement mechanism is omitted, there is a problem that it is impossible to distinguish from a decrease in initial fluorescence intensity caused by denaturation of the measurement reagent. Instead of providing the excitation light measurement mechanism, there is a method of managing the aging deterioration of the light source by the lighting time. However, since the degree of aging deterioration varies depending on the use environment, uniform management is difficult.

  The gene analysis device disclosed in Patent Document 2 disposes a distal end portion in which an emission end face of an excitation light projecting fiber and a light receiving end face of a fluorescence receiving fiber are bundled with respect to a sample container in which a plurality of wells are arranged. A fluorescence measuring device is provided. A solid fluorescent standard sample in which a fluorescent plastic and an ND filter are combined is provided along with the sample well at the movable position of the tip, and an excitation light source and / or a fluorescence detector is obtained by using the standard sample. Monitoring of aging of However, the aspect also requires a space for installing the standard sample.

JP 2012-037355 A JP 2009-014379 A

  An object of the present invention is to provide a fluorescence measuring apparatus capable of monitoring a decrease in excitation light amount (deterioration of a light source) without using an excitation light measurement mechanism or a fluorescence standard sample in a fluorescence measurement apparatus provided in an automatic analyzer or the like. It is in.

  This invention made | formed in view of the said subject includes the following aspects.

That is, the first aspect of the present invention is:
A sample container holder provided with a plurality of holding parts for holding a container containing a sample;
An excitation light irradiating unit that irradiates the container held by the holding unit with excitation light; and a fluorescence detection unit that detects fluorescence emitted from the sample contained in the container; and from the container to the fluorescence detection unit Fluorescence detection means provided with an optical filter in the optical path;
Scanning means for relatively moving and scanning the sample container holder and the fluorescence detection means so that each of the containers held by the holding unit can be irradiated with excitation light;
A fluorescence measuring device comprising:
A scattering member or a reflecting member is further provided at a position where the excitation light can be irradiated in the sample container holder,
The optical filter is capable of transmitting a part of the wavelength of the excitation light;
It is the said fluorescence measuring apparatus.

  In the second aspect of the present invention, when the amount of light detected by the fluorescence detection unit by irradiating the scattering member or the reflection member provided in the sample container holder with excitation light from the excitation light irradiation unit becomes a certain threshold value or less, The fluorescence measuring apparatus according to the first aspect, further comprising means for issuing a warning.

  According to a third aspect of the present invention, the operating current of the excitation light source or the light source detected by the fluorescence detection unit by irradiating the scattering member or the reflection member provided on the sample container holder with excitation light from the excitation light irradiation unit. The fluorescence measurement apparatus according to the first aspect, further comprising means for changing the gain of the fluorescence detection unit.

  Hereinafter, the present invention will be described in detail.

  In the present invention, a container containing a sample means a container containing a sample for measuring fluorescence, and the sample is one before subjecting a target substance to be analyzed to some chemical or physical treatment. It may be after it has been applied.

  The shape of the sample container holder and the arrangement of the holding part are not particularly limited, and may be a rectangular parallelepiped shape in which the holding part is arranged in a straight line, or a disk in which the holding part is arranged in an arc shape or an annular shape. Form may be sufficient. The scanning unit that relatively moves and scans the sample container holder and the fluorescence detection unit includes a linear movement mechanism or a rotation movement mechanism, a sample container holder and / or a fluorescence according to the shape of the sample container holder and the arrangement of the holding unit. Preferably, the scanning means is further provided with a positioning intermittent transfer mechanism that is provided for the detection means and sequentially switches the irradiation position of the excitation light to the plurality of holding portions.

  The fluorescence measurement apparatus of the present invention is characterized in that a scattering member or a reflection member is further provided at a position where excitation light can be irradiated in the sample container holder. For example, for holding parts arranged in a straight line and at equal intervals, a position extended by an equal interval from the position of the end of the arrangement of holding parts, A scattering member or a reflecting member can be arranged at a position extended from the arc by an equal interval.

  When the reflection member is provided, it is necessary to provide the excitation light irradiation unit and the fluorescence detection unit so that the excitation light from the excitation light irradiation unit is reflected on the reflection member to reach the fluorescence detection unit. On the other hand, when the scattering member is provided, the installation angles of the excitation light irradiation unit and the fluorescence detection unit may be determined as appropriate. There is no restriction | limiting in particular in the form of a scattering member or a reflection member, A rigid plate-shaped member may be sufficient, a flexible sealing member may be sufficient, and the form of a coating material may be made. Further, the scattering member or the reflecting member may have an external shape similar to that of the sample container. For example, an empty sample container covered with a light-scattering paint may be held on the holding portion of the sample container holder.

  In the fluorescence measuring apparatus of the present invention, the optical filter (on the fluorescence side) provided in the optical path from the container containing the sample to the fluorescence detection unit can transmit part of the excitation light emitted from the excitation light irradiation unit. It is characterized by that. Usually, the excitation light irradiating unit is provided with an optical filter in the optical path from the excitation light source to the container containing the sample. Therefore, the above characteristics are the transmission wavelength region of the optical filter on the excitation light side and the optical filter on the fluorescence side. In other words, the transmission wavelength region partially overlaps. The degree of overlap may be an overlap that does not push up the background signal of fluorescence measurement, and is preferably 10 nm or less, more preferably about 5 nm.

  Since the fluorescence measuring apparatus of the present invention detects a part of the excitation light emitted from the excitation light irradiation unit by the fluorescence detection unit, the excitation light amount detected by the fluorescence detection unit when the excitation light source deteriorates (the excitation light amount decreases). Also decreases. By utilizing this fact, the deterioration of the excitation light source can be monitored. Specifically, excitation light is irradiated from the excitation light irradiation unit to the scattering member or the reflection member provided in the sample container holder, and the amount of scattered light or the amount of reflection from the member is measured by the fluorescence detection unit. If the measured value decreases with respect to the measured value of the scattered light amount or the reflected light amount when the excitation light source before deterioration is used, it can be understood that the excitation light source is deteriorated. Note that it is preferable to provide a means for issuing a warning when the value of the amount of scattered light or the amount of reflected light is equal to or less than a certain threshold, because it is easy to monitor the deterioration of the excitation light source. On the other hand, a process for reducing deterioration of the excitation light source may be performed by further providing means for changing the operating current of the excitation light source or the gain of the fluorescence detection unit in accordance with the value of the scattered light amount or the reflected light amount.

  The present invention includes a sample container holder provided with a plurality of holding parts for holding a container containing a sample, an excitation light irradiation part for irradiating the container held by the holding part with excitation light, and a sample contained in the container. A fluorescence detection unit having a fluorescence detection unit for detecting emitted fluorescence, and an optical filter provided in an optical path from the container to the fluorescence detection unit, and an excitation light to each of the containers held in the holding unit In the fluorescence measuring apparatus comprising a scanning means for relatively moving and scanning the sample container holder and the fluorescence detecting means, a scattering member or a reflecting member is further provided at a position where the excitation light can be irradiated in the sample container holder, and The optical filter is capable of transmitting a part of the wavelength of the excitation light. According to the present invention, it is possible to monitor a decrease in the amount of excitation light (deterioration of the light source) without further providing an excitation light measurement mechanism or using a special fluorescent standard sample.

  In addition, a means for issuing a warning when the scattering member or the reflecting member provided in the sample container holder is irradiated with excitation light from the excitation light irradiating unit and the amount of light detected by the fluorescence detecting unit falls below a certain threshold value is provided. If the fluorescence measuring apparatus is further provided, erroneous determination due to deterioration of the excitation light quantity can be avoided.

  In addition, the operating current of the excitation light source or the gain of the fluorescence detection unit can be varied according to the amount of light detected by the fluorescence detection unit by irradiating the scattering member or reflection member provided on the sample container holder with excitation light from the excitation light irradiation unit. If the fluorescence measuring apparatus according to the present invention is further provided with a means for performing this, it is possible to suppress the change (decrease) in the amount of excitation light due to deterioration, so that it is possible to reduce the trouble of exchanging excitation light sources.

The figure which showed the one aspect | mode of the fluorescence measuring apparatus of this invention. The figure which showed another aspect of the fluorescence measuring apparatus of this invention. The figure which showed an example of the transmission wavelength band characteristic of the optical filter (excitation light side and fluorescence side) with which the fluorescence measuring apparatus of this invention is equipped. The figure which showed the change of the output detected by the fluorescence detection part at the time of changing LED current.

  Hereinafter, the present invention will be described in more detail with reference to the drawings.

One embodiment of the fluorescence measuring apparatus of the present invention is shown in FIG. The fluorescence measuring apparatus 1 shown in FIG.
It has a sample container holder 10 provided with a plurality of holding parts 11 for holding a sample container 40 containing a sample, an excitation light source 21a and an optical filter 21b, and irradiates the container 40 held by the holding part 11 with excitation light. The excitation light irradiation unit 21, the light receiving element 22a, and the optical filter 22b, the fluorescence detection unit 22 that detects fluorescence emitted from the sample accommodated in the sample container 40, and the excitation light irradiation unit 21 and the fluorescence detection unit 22 are provided. A fluorescence detection means 20 having a support member 23 to support;
Scanning means 30 having a drive shaft 31 that moves and scans the fluorescence detection means 20 via the support member 23 so that each of the sample containers 40 held by the holding unit 11 can be irradiated with excitation light. ,
A scattering member (may be a reflecting member) 12 is provided at a position where the excitation light can be irradiated by the fluorescence detection means 20 and the scanning means 30 in the sample container holder 10.

  In the fluorescence measuring apparatus of FIG. 1, the holding part 11 provided in the sample container holder 10 has a structure having a through hole, and when the sample container 40 is held, the bottom part is exposed. The sample container 40 is made of a material that can transmit the excitation light emitted from the excitation light irradiation unit 21 and the fluorescence emitted from the sample. When measuring the fluorescence of the sample stored in the sample container 40, the bottom of the container 40 is irradiated with excitation light emitted from the excitation light source 21a and limited to a predetermined wavelength by the optical filter 21b, and the container 40 is irradiated with the excitation light. The fluorescence emitted from the sample accommodated in the sample is limited to a predetermined wavelength by the optical filter 22b, and then measured by irradiating the light receiving element 22a.

  Another embodiment of the fluorescence measuring apparatus of the present invention is shown in FIG. The fluorescence measuring apparatus shown in FIG. 2 includes a fluorescence detection means 20 configured by accommodating an excitation light source 21a and an optical filter 21b for excitation light irradiation and a light receiving element 22a and an optical filter 22b for fluorescence detection in one housing. The fluorescence detection means 20 is moved and scanned along the drive shaft 31 so that excitation light can be irradiated to each of the sample containers 40 held on the sample container holder 10 (container 40). Scanning means 30 is provided.

  In the fluorescence measuring apparatus shown in FIG. 2, the sample container 40 is preferably a black container so as to prevent the irregular reflection of the excitation light irradiated from the excitation light source 21a and the fluorescence emitted from the sample. When measuring the fluorescence of the sample accommodated in the sample container 40, the excitation light emitted from the excitation light source 21a and limited to a predetermined wavelength by the optical filter 21b is irradiated from the upper part of the container 40. The fluorescence emitted from the sample accommodated in the container 40 by the excitation light is reflected by the dichroic mirror 24, limited to a predetermined wavelength by the optical filter 22b, and then reaches the light receiving element 22a and is measured. Note that the fluorescence measuring apparatus 1 shown in FIG. 2 also includes a scattering member (or a reflecting member) 12 at a position where the excitation light can be irradiated in the sample container holder 10.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to the said Example.

Example 1
Using the fluorescence detection apparatus 1 of the present invention shown in FIG. 1, it was verified whether fluctuations in the amount of excitation light (that is, deterioration of the excitation light source) can be evaluated. As the scattering member 12 provided in the sample container holder 10, a white light scattering seal was used. As the excitation light source 21a, an LED (current: 20 mA) having a center wavelength of 450 nm was used. As the excitation light side optical filter 21a and the fluorescence side optical filter 22b, interference filters having wavelength band characteristics shown in FIG. 3 were used. As can be seen from FIG. 3, the fluorescence-side optical filter 22b transmits excitation light in the wavelength region from 462 nm to 467 nm.

  An empty sample container 40 or a sample container 40 containing a fluorescent reagent was placed on the holding unit 11, and the amount of light irradiated to the light receiving element 22a was measured. When the empty sample container 40 was placed, 502 was arbitrary. The unit was 25840 when the sample container 40 containing the fluorescent reagent was placed. From this result, the reflection component of the excitation light from the empty sample container 40 is 1/50 when the sample container 40 containing the fluorescent reagent is placed, and the optical filter 22b that transmits a part of the excitation light is used. It was confirmed that even if it was used, it did not significantly affect the fluorescence measurement itself. In addition, when the same measurement was performed with respect to the scattering member 12, it was 38326 arbitrary units.

Example 2
Simulating deterioration of the excitation light source, the amount of light emitted from the scattering member 12 when the current to the excitation light source (LED) 21a was changed was measured with the fluorescence measuring apparatus 1 used in Example 1. The results are shown in FIG. It is confirmed that a linear relationship is obtained between the change in current to the excitation light source 21a and the amount of light, and that the deterioration of the excitation light source can be monitored by measuring the amount of light from the scattering member provided in the fluorescence measuring apparatus of the present invention. did it.

1: Fluorescence measuring device 10: Sample container holder 11: Holding part 12: Scattering member (or reflecting member)
20: Fluorescence detection means 21: Excitation light irradiation part 21a: Excitation light source 21b: Optical filter (excitation light side)
22: Fluorescence detection part 22a: Light receiving element 22b: Optical filter (fluorescence side)
23: Support member 24: Dichroic mirror 30: Scanning means 31: Drive shaft 40: Sample container

Claims (2)

A sample container holder provided with a plurality of holding parts for holding a container containing a sample;
An excitation light irradiating unit that irradiates the container held by the holding unit with excitation light; and a fluorescence detection unit that detects fluorescence emitted from the sample contained in the container; and from the container to the fluorescence detection unit Fluorescence detection means provided with an optical filter in the optical path;
Scanning means for relatively moving and scanning the sample container holder and the fluorescence detection means so that each of the containers held by the holding unit can be irradiated with excitation light;
A fluorescence measuring device comprising:
A scattering member or a reflecting member is further provided at a position where the excitation light can be irradiated in the sample container holder,
Means for varying the operating current of the excitation light source or the gain of the fluorescence detection unit in accordance with the amount of light detected by the fluorescence detection unit by irradiating the scattering member or the reflection member with excitation light from the excitation light irradiation unit; In addition,
The optical filter is capable of transmitting a part of the wavelength of the excitation light;
The fluorescence measuring device. The apparatus further comprises means for issuing a warning when the amount of light detected by the fluorescence detection unit by irradiating the scattering member or the reflection member provided in the sample container holder with the excitation light from the excitation light irradiation unit falls below a certain threshold value. Item 2. The fluorescence measuring apparatus according to Item 1.

Images