KR101306930B1 - Simultaneous spectrophotometer for fluorescence and absorption measurement - Google Patents

Abstract

PURPOSE: A fluorescence measuring spectrophotometer is provided to measure even a little amount of samples after controlling the length of optical paths of the samples, thereby improving measurement sensitivity and accuracy. CONSTITUTION: A fluorescence measuring spectrophotometer (100) includes a spectrophotometer body (102), a light source (106), a light transmission unit (112), a total reflection window (104a), and a multichannel detecting unit (120). The light source is mounted inside the body. The light transmission unit controls progressing paths of lights emitted by the light source. The total reflection window is arranged on a sample placed on the top of the light transmission unit. The multichannel detecting unit detects the lights emitted after being reflected by the total reflection window and penetrated through the light transmission unit. The light transmission unit forms optical progressing paths of which lengths are different. The multichannel detecting unit distributes lights coming into the light transmission unit after being penetrating through the optical progressing paths to other independent optical progressing paths at the same time.

Description

Simultaneous Spectrophotometer for Fluorescence and Absorption measurement

The present invention relates to a spectrophotometer for simultaneous simultaneous measurement of fluorescence absorption, and more particularly, to a spectrophotometer for simultaneous simultaneous measurement of multi-channel detection fluorescence absorption for analyzing trace amounts of biosamples.

Measuring the content of protein contained in the sample is called protein quantification, and protein quantification includes spectroscopic method, burette method, Lowry method and Bradford method. In the case of bio samples including protein samples, fast measurement time and trace measurement reduction to prevent denaturation are very important.

However, in the case of general equipment, micro-cells used to measure trace bio-samples are very expensive, sampling and cumbersome due to infusion and washing steps, and bubbles may occur inside the cells, and the cells should be dried after washing. There is a problem.

According to another general technique, the bio sample is compressed and relaxed to closely adhere the sample to the upper and lower fibers using surface tension of water, and then irradiate with light to measure the optical characteristics thereof to analyze the sample. In general, according to Lambert-Beer Law, for samples of the same concentration, absorbance is proportional to the light path.

However, in the case of using the surface tension method, the optical path that can be secured is limited, and accordingly, the optical path is short and thus the absorbance is low, resulting in a low measurement sensitivity.

SUMMARY OF THE INVENTION An object of the present invention is to provide a spectrophotometer for fluorescence absorption simultaneous measurement that can solve a problem as described above to analyze trace bio-samples on the order of several microliters without additional pretreatment.

Another object of the present invention is to provide a spectrophotometer for fluorescence absorption simultaneous measurement which improves measurement sensitivity by simultaneously detecting and spectroscopically analyzing the light passing through the elongated light path and the relatively short light path as needed.

Another object of the present invention is to provide a spectrophotometer for fluorescence absorption simultaneous measurement that can implement a separate rotation lattice optical path for each channel, that is, for each optical path.

In order to achieve the object as described above, the spectrophotometer for simultaneous simultaneous measurement of fluorescence absorption according to an embodiment of the present invention is a photometer body light source mounted in the main body light transmission unit for controlling the traveling path of the light generated from the light source A total reflection window disposed on an upper sample placed on an upper side of the sample; and a multi-channel detector detecting light emitted from the total reflection window after passing through the light transfer unit, wherein the light transfer unit has a plurality of light paths having different lengths of travel paths. Forming a path, the multi-channel detector for simultaneously spectroscopic light entering the light path after passing through the traveling paths of different lengths in different independent light paths.

Here, the multi-channel detection unit, a plurality of slits each of which a plurality of light is introduced into a plurality of parallel light concave mirror for reflecting a plurality of light passing through the slit to be parallel light a plurality of light reflected from the concave mirror for parallel light A grating portion having a diffraction grating formed to diffract a plurality of concave mirrors for condensing a plurality of light spectroscopy from the grating portion, and a plurality of detectors respectively detecting the light collected from the condensing mirror.

On the other hand, the light transfer unit has an outer shape coupled to the two square pyramids facing each other, the side having a parallel and flat top and bottom, the light incident surface formed on the inclined side facing the light incident surface It has a light exit surface formed on the side of the picture.

The light source is oriented to irradiate light toward the light incident surface.

In addition, the spectrophotometer for simultaneous simultaneous measurement of fluorescence absorption according to an embodiment of the present invention further includes an optical fiber set for guiding the light emitted from the light transporter through a path having a different length to the multi-channel detector.

The optical fiber set includes a first optical fiber and a second optical fiber, wherein the first optical fiber is disposed to correspond to the lower surface of the light transfer unit, and the second optical fiber is disposed to correspond to the light exit surface of the light transfer unit. do.

Here, the number of the plurality of slits is two, the first optical fiber transmits light to one of the two slits, the second optical fiber transmits light to the remaining slits.

In addition, the two slits are arranged up and down, and two parallel light concave mirrors and two condensing concave mirrors corresponding to two lights passing through the respective slits and traveling inside the multi-channel detector are also correspondingly disposed up and down. Thus, the two lights traveling inside the multi-channel detector do not cross each other and travel along an independent path to reach the two detectors, respectively.

The spectrophotometer for simultaneous simultaneous absorption measurement of fluorescence according to an embodiment of the present invention further includes a collimator between the light source and the light transfer unit.

In addition, the spectrophotometer for simultaneous simultaneous measurement of fluorescence absorption according to an embodiment of the present invention further includes an excitation filter between the collimator and the light transfer unit.

According to the spectrophotometer for fluorescence absorption simultaneous measurement according to an embodiment of the present invention,

First, even a small amount of sample can be measured after controlling the length of the optical path differently, thereby improving measurement sensitivity and accuracy.

Secondly, the absorption / fluorescence analysis of a small amount of sample can be performed with one device,

Third, the spectral spectrum can be obtained without a separate sample vessel,

Fourth, it can shorten the washing and drying time of the cell,

Fifth, absorbance / fluorescence analysis can be performed simultaneously.

By connecting the plurality of light exiting the light transport unit controlling the sixth optical path through the optical fiber to the multi-channel detection unit, the usability of the internal space of the spectrophotometer for simultaneous measurement of fluorescence absorption is improved, and the compact size can be realized.

Seventh, absorption spectra in two or more wavelength bands having different absorption rates can be measured with appropriate path lengths for each wavelength band, thereby improving measurement accuracy.

Eighth, since the absorbance analysis and the fluorescence analysis do not interfere with each other, accurate measurement is possible.

1 is a cross-sectional view schematically showing the overall configuration of the cover of the operating state of the spectrophotometer for fluorescence absorption simultaneous measurement according to an embodiment of the present invention.
FIG. 2 is a perspective view illustrating a traveling path of light in the light transfer part of FIG. 1.
3 is a cross-sectional view taken along the line III-III of FIG. 2.
4 is a schematic perspective view of the inside of the multi-channel detector of FIG. 1.
FIG. 5 is a schematic plan view of the interior of the multi-channel detector of FIG. 1.

The invention is explained in detail below with reference to the accompanying drawings.

In the present invention, spectrophotometry was used to measure bio samples such as protein samples. Spectrophotometry is a method of measuring the intensity of light. In order to investigate the distribution of light energy for each wavelength using the light spectrum, the light is divided into monochromatic light using a spectrometer and the intensity of the sample is measured. Chemical experimental values can be obtained.

Here, the spectrometer measures the spectrum of light emitted or absorbed by the material.

1 is a cross-sectional view schematically showing the overall configuration of the cover of the operating state of the spectrophotometer for simultaneous measurement of fluorescence absorption according to an embodiment of the present invention.

Referring to FIG. 1, the spectrophotometer 100 for simultaneous simultaneous fluorescence absorption measurement according to an embodiment of the present invention includes a photometer body 102 and a light source 106 mounted in the photometer body 102 and the light source 106. A light transfer unit 112 for controlling a path of the light generated by the light source, a total reflection window 104a disposed on a sample placed on the upper side of the lens, and detecting the light emitted through the light transfer unit after being reflected from the total reflection window; The multi-channel detector 120 is provided.

Here, the light transporter 112 forms a plurality of light travel paths having different lengths of travel paths, and the multi-channel detector 120 passes through the travel paths having different lengths from the light transporter 112, respectively. Incoming light is simultaneously spectroscopically in different independent light paths.

The multi-channel detector 120 includes a detector case 121 that functions as an optical shielding structure so that an optical element disposed therein is not affected by external light, and includes an optical element inside the detector case 121. do.

Meanwhile, a collimator 108 is formed between the light source 106 and the light transfer part 112 to form parallel rays.

In addition, an excitation filter 110 is installed between the collimator 108 and the light transfer unit 112 for the purpose of measuring fluorescence. The excitation filter 110 may be equipped with a plurality of filters to form an excitation filter assembly that rotates in a rotary manner.

FIG. 2 is a perspective view illustrating a traveling path of light in the light transfer part of FIG. 1.

The light transporting unit 112 has a geometrical shape in which two quadrangular pyramids are coupled to face each other with their bottom surfaces, but the top and bottom surfaces of the entire structure are parallel to each other.

That is, the upper side of the light transmitting part 112 has four inclined side surfaces, and four inclined side surfaces are formed also under the light transmitting part 112.

Although the drawings show that four inclined surfaces are formed on the upper side and the lower side of the light transmitting unit 112, respectively, but are not necessarily limited thereto, and basically two surfaces inclined at positions facing the lower side of the light transmitting unit 112. After the formation, the remaining two surfaces of the lower side of the light transfer part 112 may be formed to be inclined or may be vertically formed without being inclined.

Similarly, four inclined surfaces are not necessarily formed on the upper side of the light transfer part 112, but only two opposing surfaces may be inclined and the remaining two surfaces may be vertically formed.

As shown in FIGS. 1 and 2, the light generated by the light source 106 is incident on the light incident surface formed on the lower inclined side of the light transfer part 112 (indicated by a thick arrow to the right in FIG. 2). Then, a light exit surface is formed on the inclined side surface of the light transmitting unit 112 to face the light incident surface (indicated by a thick left arrow in FIG. 2).

As shown in FIG. 1, the light source 106 is oriented to irradiate light toward the light incident surface. The light source 106 is controlled on / off by a light source control device (not shown). In addition, the property of the light of the light source 106 may also be controlled by the light source control device.

As shown in FIG. 1, the spectrophotometer for simultaneous simultaneous fluorescence absorption measurement according to an embodiment of the present invention includes a set of optical fibers 118 and 119 for guiding the light emitted from the light transporter 112 to the detector 120. It is provided.

The optical fiber sets 118 and 119 include a first optical fiber 119 and a second optical fiber 118, and one end of the first optical fiber 119 is an exit surface of the light transfer part 112. The other end of the first optical fiber 119 is disposed to correspond to the first slit 122a formed on, for example, a side surface of the multi-channel detector 120.

One end of the second optical fiber 118 is disposed to correspond to the bottom surface of the light transfer unit 112, and the other end of the second optical fiber 118 is disposed at, for example, a side surface of the multi-channel detection unit 120. Is connected to the second slit 122b.

The first optical fiber 119 and the second optical fiber 118 are connected to the side surface of the detector main body 121 of the multi-channel detector 120 spaced apart from each other at predetermined intervals.

A collimator 114 is disposed between one end of the first optical fiber 119 and the light exit surface of the light transfer part 112. Meanwhile, the collimator 116 is disposed between one end of the second optical fiber 118 and the lower surface of the light transfer part 112.

Referring to FIG. 1, a cover 104 is rotatably disposed on a portion of the photometer body 102, for example, above the photometer body 102. When the cover 104 closes the photometer body 102, the total reflection window 104a is installed in the variegated light removal trap 104b mounted inside the cover 104.

As shown in FIG. 1 and FIG. 2, the total reflection window 104a is arranged in alignment with an upper surface of the light transfer part 112.

On the other hand, the light transfer part 112 includes silicon dioxide, for example.

3 is a cross-sectional view taken along the line III-III of FIG. 2.

2 to 3, the sample S to be analyzed is disposed between the top surface of the light transfer part 112 and the total reflection window 104a.

Light incident inclined at a predetermined angle to the light incident surface of the light transporting part 112 passes through the upper surface of the light transporting part 112 and the boundary surface of the sample S, and then passes through the sample S to enter the total reflection window 104a. do.

3 shows the path of light propagation in the light transfer section, the sample, and the total reflection window.

As the light generated from the light source passes through the light transfer unit 112, the sample S, and the total reflection window 104a, the light transfer unit 112, the sample S, and the total reflection window 104a are applied by Snell's law. When the light propagation path is bent due to the difference in refractive index of), when the light incidence angle is greater than or equal to the angle at which total internal reflection occurs, total reflection occurs at the total reflection window 104a, and the light is reflected at the same angle as the incidence angle to emit light. In the process of emitting light, the path of the light is broken by the difference in the refractive index of the medium.

The light flowing into the light incident surface of the light transfer unit 112 and exiting through the exit surface after total reflection is guided to the multi-channel detection unit 120 by the first optical fiber 119 as shown in FIG. 1.

As the light passes through the light transfer part 112, the sample S, and the total reflection window 104a, the path of the light passing through the sample S becomes longer than the thickness of the sample S itself.

In this case, according to Lambert-Beer Law, the absorbance is proportional to the light path in the case of the sample of the same concentration, so that the absorbance increases as the light path becomes longer and eventually the simultaneous measurement of fluorescence absorption The sensitivity of the spectrophotometer is raised.

Separately, the light traveling vertically downward from the upper surface of the light transmitting part 112 is emitted through the lower surface of the light transmitting part 112, as shown in FIGS. 1 and 3, and the lower surface of the light transmitting part 112. The light emitted through is guided to the multi-channel detector 120 by the second optical fiber 118 via the collimator 116.

2 and 3, the traveling path of the light inclined to the light transmitting part 112 and exited inclinedly through the light exiting surface is longer than the traveling path of the light emitted downward of the light transmitting part 112.

Referring to FIG. 4, the multi-channel detector 120 includes a plurality of slits 122a and 112b through which a plurality of lights flow into the detector case 121, and two passes through the slits 122a and 122b, respectively. A parallel light concave mirror portion 124 provided with two parallel light concave mirrors 124a and 124b for reflecting two light beams into parallel light, and two light reflected from the concave mirrors 124a and 124b for parallel light. A diffraction grating portion 126 having a diffraction grating pattern formed thereon, and two condensing mirror portions 128 provided with two condensing mirrors 128a and 128b for condensing two light spectroscopy from the diffraction grating portion 126. ) And two detectors 129a and 129b respectively detecting the light collected by the condensing mirrors 128a and 128b.

The slit is provided to pass only the transmitted light condensed at the focal position near the focal position of the transmitted light.

The surface 127 on which the diffraction grating pattern is formed in the diffraction grating portion 126 may have a planar shape. The surface on which the diffraction grating pattern is formed spectroscopy two lights traveling inside the multi-channel detection unit 120 on the surface on which one diffraction grating pattern is formed.

Referring back to FIG. 1, the first optical fiber 119 guides light to the first slit 122a, which is one of the two slits, and the second optical fiber 118 is the remaining slit. Light is guided to the two slits 122b.

Since the first optical fiber 119 and the second optical fiber 118 are flexible, light can be guided regardless of the shape of the installation space in which the optical fiber is disposed.

4 and 5, the first slit 122a and the second slit 122b are disposed up and down, and correspond to two lights passing through the respective slits and traveling inside the multi-channel detector 120. The first parallel concave mirror 124a and the second parallel concave mirror 124b and the first condensed concave mirror 128a and the second condensed concave mirror 128b are also disposed up and down correspondingly. The two lights traveling inside the channel detector 120 do not cross each other and travel along an independent path to reach the first detector 129a and the second detector 129b, respectively.

Although not shown in the drawing, the first detector 129a and the second detector 129b are provided with an arithmetic unit (not shown) through which an optical intensity signal from the detector is input through an amplifier (not shown), an AD converter (not shown), and the like. ) Is connected.

Such a calculation unit converts the light intensity signal into an absorption spectrum or a fluorescence spectrum, and calculates a concentration value of the multicomponent of the sample based on the spectrum. A display unit (not shown) for displaying the concentration value of the component obtained by the calculation unit is connected to the calculation unit.

Here, the number of the slit, the concave mirror for parallel light, and the concave mirror for condensing may be two by way of example, but is not necessarily limited thereto. The number of the optical fiber, the slit, the concave mirror for parallel light, and the concave mirror for condensing may be considered as the number of light emitted from the light transfer part 112.

The slit, the concave mirror for parallel light, the diffraction grating portion, the concave mirror for condensing light, and the detector are arranged at predetermined intervals to form a mutual path to form a path through which light is reflected.

On the other hand, when white light emitted from the continuous light source is passed through a dilution solution or vapor of a material to be analyzed with a spectrophotometer, some black lines appear between the continuous spectra of the white light.

This dark absorption line appears when light corresponding to the wavelength of an element of the material is absorbed by a low temperature gas atom.

This spectrum is the absorption spectrum, and black absorption lines appear at the same position as the emission line wavelength of the discontinuous line spectrum observed in gas discharge.

The absorption and emission lines of the element are nearly identical, but not all of the emission lines appear in the absorption spectrum. The difference between the absorption spectrum and the emission spectrum is generally complex and depends on the temperature of the absorbing vapor.

On the other hand, the fluorescence spectrum is referred to as the emission spectrum or the emission spectrum in order to distinguish the absorption spectrum from the emission electromagnetic spectrum when atoms, molecules or aggregates thereof transition from a high energy level to a low energy level.

Since each atom has a constant quantized energy value, the value of the electromagnetic wave resulting from the energy difference is different from atom to atom.

In FIG. 1, light detected by the multi-channel detector 120 after being guided through the first optical fiber 119 is detected as an absorption spectrum, and multi-channel detector 120 after being guided through the second optical fiber 118. The light detected at is detected by the fluorescence spectrum.

According to the spectrophotometer for simultaneous simultaneous measurement of fluorescence absorption in one embodiment of the above-described structure, the absorption spectrum and the fluorescence spectrum of a small amount of sample can be simultaneously measured.

The foregoing is only illustrative of the embodiments of the present invention and should not be construed as limiting. Various modifications are possible without departing from the scope of the invention.

100: spectrophotometer for simultaneous measurement of fluorescence absorption
102: photometer body
104: cover 104a: total reflection window
106: light source S: sample
108: collimator 110: excitation filter
112: light transfer part 114: collimator
116: collimator 118: second optical fiber
119: first optical fiber 120: multi-channel detection unit
122a: first slit 122b: second slit
121: detector case 124: concave mirror portion for parallel light
124a: concave mirror for first parallel light 124b: concave mirror for second parallel light
126: diffraction grating portion 128: light concave mirror portion
128a: first concave mirror for condensing 128b: second concave mirror for condensing
129a: first detector 129b: second detector

Claims (10)

Photometer body
A light source mounted in the main body
Light transfer unit for controlling the traveling path of the light generated by the light source
A total reflection window disposed on the sample placed above the light transfer part;
A multi-channel detector configured to detect light emitted from the total reflection window and passed through the light transfer unit,
The light transfer part forms a traveling path of a plurality of light having different lengths of traveling paths,
The multi-channel detection unit spectrophotometer for simultaneous simultaneous absorption of fluorescence absorption characterized in that the light transmission after passing through the different paths of different lengths and simultaneously spectroscopic light in different independent light paths. The method of claim 1,
The multi channel detector,
A plurality of slits into which a plurality of lights respectively enter
A plurality of concave mirrors for parallel light reflecting the plurality of light passing through the slit to be parallel light
A diffraction grating portion in which a diffraction grating pattern is formed to spectroscopic a plurality of light reflected by the concave mirror for parallel light
A plurality of concave mirrors for condensing a plurality of light spectroscopy in the diffraction grating portion
And a plurality of detectors respectively detecting the light collected by the concave mirror for collecting light. 3. The method of claim 2,
The light transfer part has an outer shape in which two square pyramids face each other with a bottom surface, and has a parallel and flat top and bottom surfaces, and a light incident surface formed on an inclined side surface and an inclined side facing the light incident surface. A spectrophotometer for simultaneous measurement of fluorescence absorption, comprising a light exit surface formed on the substrate. The method of claim 3, wherein
And the light source is oriented to irradiate light toward the light incident surface. 5. The method of claim 4,
And a set of optical fibers for guiding the light emitted from the light transfer part through the paths having different lengths to the multi-channel detector. The method of claim 5, wherein
The optical fiber set includes a first optical fiber and a second optical fiber, wherein the first optical fiber is disposed to correspond to the lower surface of the light transfer unit, and the second optical fiber is disposed to correspond to the light exit surface of the light transfer unit. Spectrophotometer for simultaneous measurement of fluorescence absorption, characterized in that The method according to claim 6,
The number of the plurality of slits is two,
And the first optical fiber transmits light to one of the two slits, and the second optical fiber transmits light to the remaining slits. The method of claim 7, wherein
The two slits are arranged up and down, and two parallel light concave mirrors and two condensing concave mirrors corresponding to two lights passing through the respective slits and traveling inside the multi-channel detection unit are also correspondingly disposed up and down, The spectrophotometer for fluorescence absorption simultaneous measurement, characterized in that the two light traveling in the multi-channel detection unit does not cross each other and proceed along an independent path to reach each of the two detectors. The method of claim 1,
A spectrophotometer for fluorescence absorption simultaneous measurement, further comprising a collimator between the light source and the light transfer unit. The method of claim 9,
A spectrophotometer for simultaneous measurement of fluorescence absorption, further comprising an excitation filter between the collimator and the light transfer unit.

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