CN110018139A - Multicolor fluorescence detection device - Google Patents
Multicolor fluorescence detection device Download PDFInfo
- Publication number
- CN110018139A CN110018139A CN201811608963.4A CN201811608963A CN110018139A CN 110018139 A CN110018139 A CN 110018139A CN 201811608963 A CN201811608963 A CN 201811608963A CN 110018139 A CN110018139 A CN 110018139A
- Authority
- CN
- China
- Prior art keywords
- optical filter
- adjustable optical
- detection device
- fluorescence detection
- multicolor fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Spectrometry And Color Measurement (AREA)
Abstract
A kind of multicolor fluorescence detection device includes fluorescence groove body, luminescence unit, detection unit and actuating unit.Fluorescence groove body is to accommodate fluorescent mixture.Luminescence unit includes light source and the first adjustable optical filter, and wherein light source provides broadband illumination, and the first adjustable optical filter allows the light beam of different passbands to pass through based on different location or angle, to excite fluorescent mixture.Detection unit includes the second adjustable optical filter and photodetector, wherein the second adjustable optical filter allows the light beam for providing different passbands from fluorescent mixture transmitting to pass through based on different location or angle, and photodetector receives fluorescence signal and is converted into electronic signal.Actuating unit framework is in first and second adjustable optical filter of driving to different location or angle, to control the passband variation of first and second adjustable optical filter respectively.
Description
Technical field
The present invention relates to a kind of fluorescence detection device, in particular to a kind of multicolor fluorescence detection device.
Background technique
In recent years, the demand for obtaining a large amount of particular patch segment DNAs for different purposes is higher and higher, and in existing DNA sequencing
In technology, polymerase chain reaction (Polymerase Chain Reaction, PCR) is one of most economical and quick
Technology can expand the target DNA fragments of 1,000,000,000 copies in a short time.Round pcr has been widely used, such as reflects for heredity
Appreciation analysis, genetic test and the medical application of tissue typing, hospital of fixed selective d NA separation, archaeology analysis Gu DNA
With research institution to the quick and specific diagnosis of communicable disease, the environmental hazard monitoring of food safety and crime survey
Fingerprint identification etc..Round pcr is only needed using the miniprep dna sample obtained from blood or tissue, and fluorescein stain is added
Enter in nucleic acid solution, the DNA fragmentation of amplification can be detected by fluorescent molecule.
Fluorescein stain detection technique is normally used for synchronous detection and has analysed whether that target nucleic acid molecules are present in a batch
In secondary pollutant sample.After the light source of specific wavelength exposes to target nucleic acid, the fluorescence on DNA combination stain or nucleic acid, which combines, to be visited
Needle can generate reaction and issue fluorescence signal, this fluorescence signal is to represent the presence for having target nucleic acid molecules.This technology is applied
In new-type round pcr, referred to as real-time quantitative PCR (real time quantitative PCR) or qPCR.Compared to normal PCR
Technology, that is, so-called terminal PCR detection (end-point PCR detection), qPCR be with higher sensitivity and
Early stage PCR of preferable accuracy detects (early-phase PCR detection).And Optical devices be qPCR technology to
The indispensable tool of the fluorescence issued from specific nucleic acid section is detected, this Optical devices must provide light source in certain wave
Long excitation fluorescence probe, and the fluorescence signal issued from probe is detected simultaneously.
The fluorescence detecting system mature development in multiple fields, such as the application of fluorescence spectrum and fluorescence microscope.Benefit
With monochromatic source plus the configuration of one group of filter and optical module, specific fluorescent probe can be applied to easily.However, for just
Take the multicolor fluorescence detection device of formula qPCR system because its cost, size and high s/n ratio (signal-to-noise ratio,
SNR), therefore there is the difficulty of exploitation on the market and solution is not yet received.Also, due to filter combination in traditional polychrome qPCR system
Select it is limited, user also therefore be limited to use a few fluorophor.
Therefore, in order to improve the missing of the aforementioned prior art, it is really necessary to provide a kind of fluorescence detection device of improvement, with
QPCR for polychrome is applied.
Summary of the invention
The purpose of the present invention is to provide a kind of multicolor fluorescence detection device, to reduce the size and weight of device, and
It remains to provide excellent properties and the selection of extensive fluorophor in the portable qPCR system of lower cost.
In order to achieve the above object, a broader embodiment of the invention is to provide a kind of multicolor fluorescence detection device, wrap
Contain: a fluorescence groove body, to accommodate a fluorescent mixture；One luminescence unit, it is adjustable comprising an at least light source and at least one first
Formula optical filter, wherein the light source provides a broadband illumination, and the first adjustable optical filter is based on the first adjustable optical filter
Different location or angle and allow the light beam of different passbands to pass through, to excite the fluorescent mixture；One detection unit, comprising extremely
Few one second adjustable optical filter and an at least photodetector, wherein the second adjustable optical filter is based on the second adjustable filter
Different location or the angle of mating plate and allow to emit from the fluorescent mixture and provide the light beams of different passbands and pass through, and the light is examined
Device is surveyed to receive fluorescence signal and the fluorescence signal is converted to electronic signal；And an actuating unit, framework in drive this first
Adjustable optical filter and the second adjustable optical filter are to different location or angle, to control the first adjustable optical filter respectively
And the passband variation of the second adjustable optical filter.
In one embodiment, this of the first adjustable optical filter of the luminescence unit and the detection unit are second adjustable
Optical filter all includes a variable-angle optical filter or a linear variable filters.
In one embodiment, this of the first adjustable optical filter of the luminescence unit and the detection unit are second adjustable
The maximum pass-band of optical filter is 60nm.
In one embodiment, which is a light emitting diode.
In one embodiment, a pin hole is set to the front end of the first adjustable optical filter of the luminescence unit, and the needle
Hole has the aperture of 2 ± 0.5mm.
In one embodiment, which further includes a focus optics, be set to the luminescence unit this first
Between adjustable optical filter and the fluorescence groove body.The position of the focus optics from the light shaft offset 1.3 of the light source ±
0.2mm。
In one embodiment, which has a preceding hole and a metapore.The preceding hole is located at from a direct optical axis with suitable
Clockwise tilts 7 degree of position.
In one embodiment, which also includes a heater, and to provide temperature control, to give this glimmering
Light mixture.The heater includes a thermoelectric cooling heater.
In one embodiment, which also includes a collecting optics component, is set to the fluorescence groove body and the detection
Between the second adjustable optical filter of unit.
In one embodiment, which also includes an imaging optics, be set to the detection unit this second
Between adjustable optical filter and the photodetector.
In one embodiment, which includes silicon photoelectric diode.
In one embodiment, which includes a servo motor, a rotation cause driven by fluid or vacuum pressure
Dynamic device, a piezoelectric actuator, an electronic polymer actuator or an electromagnetic motor.
In one embodiment, which also includes multiple channels linearly arranged, and each channel
With first and second the adjustable optical filter of the respective light source, the fluorescence groove body, the photodetector and one group.
In one embodiment, which also includes multiple channels linearly arranged, and each channel
With the respective light source, the fluorescence groove body and the photodetector, wherein single group first and second adjustable optical filter
It is sequentially displaced into multiple channel.
In one embodiment, this of the first adjustable optical filter of the luminescence unit and the detection unit are second adjustable
Optical filter all includes a variable-angle optical filter.
In one embodiment, actuating unit has multiple default frameworks, to detect multiple fluorescein stains respectively.
Beneficial effects of the present invention: multicolor fluorescence detection device provided by the invention include luminescence unit, heating unit,
Detection unit and actuating unit, luminescence unit includes adjustable exciter filter, and detection unit filters comprising adjustable transmitting
Piece can be made apparatus of the present invention excite and detect a series of wavelength and be fallen into optical filter passband by rotation or mobile spectral filter
Fluorescent mixture, therefore can be applicable to the fluorescent mixture of tool multiple color.Present invention design can be achieved compact in size and reduce
Installation cost, and there is high signal-to-noise ratio and low crosstalk effect.This device has more wide detection bandwidth, and is potential customers
Provide higher stain selection flexibility.
Detailed description of the invention
Fig. 1 shows the multicolor fluorescence detection device schematic diagram of present pre-ferred embodiments.
Fig. 2 shows the sectional view of the multicolor fluorescence detection device of Fig. 1.
Fig. 3 A, Fig. 3 B and Fig. 3 C show the optical path of luminescence unit, wherein the first adjustable optical filter rotate respectively to
0,40 and 75 degree of positions.
Fig. 4 shows the overlapping optical path of Fig. 3 A, Fig. 3 B and Fig. 3 C.
Fig. 5 shows the spectrum of the first adjustable optical filter of luminescence unit.
Fig. 6 A, Fig. 6 B and Fig. 6 C show the optical path of detection unit, wherein the second adjustable optical filter rotate respectively to
0,40 and 75 degree of positions
Fig. 7 shows the overlapping optical path of Fig. 6 A, Fig. 6 B and Fig. 6 C.
Fig. 8 shows available fluorescein stain example.
Fig. 9 A, Fig. 9 B and Fig. 9 C show the optical property that three colour cells of FAM, Alexa 514 and HEX are closed.
Figure 10 A, Figure 10 B and Figure 10 C show the crosstalk effect that three colour cells of FAM, Alexa 514 and HEX are closed.
Description of symbols:
1: luminescence unit
11: light source
12: the first adjustable optical filters
13: focus optics
14: pin hole
15: shell
2: heating unit
21: heater
22: fluorescence groove body
221: preceding hole
222: metapore
3: detection unit
31: the second adjustable optical filters
32: photodetector
33: collecting optics component
34: imaging optics
4: actuating unit
41: the first actuators
42: the second actuators
Specific embodiment
The some exemplary embodiments for embodying feature of present invention and advantage will describe in detail in the explanation of back segment.It should be understood that
It is that the present invention there can be various variations in different modes, neither departs from the scope of the present invention, and explanation therein
And schema inherently is illustrated as being used, rather than to limit the present invention.
The present invention provides a kind of multicolor fluorescence detection device, how glimmering linearly arranges for a kind of irradiation that can sequentially shine
Light sample, and sequentially detect the optical module of the specific fluorescent signal gone out by probe emission.In qPCR amplification procedure, this device
The light beam with different colours is provided by changing the orientation of optical filter to excite different fluorescence probes, and detection by probe
The fluorescence signal launched, therefore the fluorescent mixture that the present invention can be used for having multiple color.
Fig. 1 and Fig. 2 is please referred to, wherein Fig. 1 shows the multicolor fluorescence detection device schematic diagram of present pre-ferred embodiments, figure
The sectional view of the multicolor fluorescence detection device of 2 display Fig. 1.As shown in Figures 1 and 2, multicolor fluorescence detection device mainly includes one
Luminescence unit 1, a heating unit 2, a detection unit 3 and an actuating unit 4.Before luminescence unit 1 is set to heating unit 2
End, detection unit 3 is set to the rear end of heating unit 2, and actuating unit 4 then connects with luminescence unit 1 and detection unit 3 respectively
It connects.
Luminescence unit 1 includes an at least light source 11 and at least one first adjustable optical filter (tunable filter) 12,
Wherein light source 11 provides a broadband illumination (broad band illumination), and the first adjustable optical filter 12 includes a light
Optical filter is learned, different passband (pass can be allowed when light enters the first adjustable optical filter 12 from different location or angle
Band light beam) passes through.It therefore, can be selectively by the first adjustable optical filter 12 of driving to different location or angle
Excite the different fluorescein stains in fluorescent mixture.Heating unit 2 includes a heater 21 and an at least fluorescence groove body 22, wherein
Heater 21 provides the temperature control of qPCR amplification program, and fluorescence groove body 22 then holds to accommodate the liquid with fluorescent mixture
Device.Detection unit 3 includes at least one second adjustable optical filter 31 and an at least photodetector (photo-detector) 32.Together
Sample, when the second adjustable optical filter 31 is rotated into different angle or is moved to different location, the second adjustable optical filter
31, which allow different fluorescein stains from fluorescent mixture to emit, provides the light beams of different passbands and passes through.First adjustable filter
Mating plate 12 and the second adjustable optical filter 31 are that framework is that can correspond to driving, to detect a specific fluorescent stain.Photodetector 32
It then receives fluorescence signal and fluorescence signal is converted into electronic signal.First adjustable filter of the driving luminescence unit 1 of actuating unit 4
Mating plate 12 and the second adjustable optical filter 31 of detection unit 3 are to different location or angle, to control excitation and transmitting respectively
Passband variation, meets the specific wavelength of target fluorescent probe whereby.In one embodiment, actuating unit 4 has multiple default framves
Structure, to detect different fluorescein stains respectively.When actuating unit 4 switches between different frameworks, the first adjustable optical filter 12 and
Both second adjustable optical filters 31 are accordingly driven, and to control the passband variation for exciting and emitting, are carried out whereby not
With the detection of fluorescein stain.
The light that light source 11 is issued is in the broadband of visible wavelength range, to cover target excitation wavelength.One
In embodiment, light source 11 is the light emitting diode (LED) with 10 degree of visual angles.However, light source 11 of the invention is not limited to LED,
Other light sources, such as mercury lamp and the halogen lamp of covering visible wavelength etc., are all applicable to the present invention.
In one embodiment, luminescence unit 1 includes a shell 15, and multiple channels are formed in shell with linear array
In 15.Each channel has respective light source 11, and the pin hole 14 with the aperture 2 ± 0.5mm is set to the first adjustable optical filtering
The front end of the coating surface of piece 12.For example, as shown in Figure 1, multicolor fluorescence detection device includes three linear arrangements altogether
Channel.Certainly, the quantity in channel is not limited to three, can be changed according to different demands.
In one embodiment, the material of the shell 15 of luminescence unit 1 is black ABS (acrylonitrile butadiene
Styrene) resin has the advantages that low thermal conductivity, high thermal resistivity and interior lights scattering reduce.Other materials, such as with low
Reflexive black plastic material, or it is coated with the aluminium for having low reflexive and high-absorbable black anodic coating, all may be used
Suitable for the present invention.
In one embodiment, luminescence unit 1 includes also a focus optics (focusing optics) 13, is set to
Between first adjustable optical filter 12 and fluorescence groove body 22.Light beam after optical filtering can be converged to and is placed in by focus optics 13
Liquid container in fluorescence groove body 22.The position of focus optics 13 offsets downward about 1.3 from the optical axis of light source 11 ±
0.2mm is the optimization design in order to reduce noise.Focus optics 13 are made of BK7 glass, but other kinds of optics
Grade polymer is also applicable to the present invention.
Fig. 3 A, Fig. 3 B and Fig. 3 C show the optical path of luminescence unit, wherein the first adjustable optical filter rotate respectively to
0,40 and 75 degree of positions, Fig. 4 then show the overlapping optical path of Fig. 3 A, Fig. 3 B and Fig. 3 C.As shown in Figures 2 to 4,11 institute of light source
The light beam of offer passes through pin hole 14 first, is then filtered by the first adjustable optical filter 12, the light beam after optical filtering is then by gathering
Burnt optical module 13 converges to the liquid container being placed in fluorescence groove body 22, to excite the fluorescein stain of target-probe.
The adjustable optical filter 12 of the first of luminescence unit 1 is and the first adjustable optical filter 12 as exciter filter
Angle gyrobearing is towards focus optics 13.It can in addition, having the pin hole 14 in the aperture 2 ± 0.5mm that must be set to first
The front end of the coating surface of mode optical filter 12.In one embodiment, the ruler of the first adjustable optical filter 12 in each channel
Very little is 6x 9x 2mm, but its size can change according to different application.The setting of pin hole 14 can prevent the excitation beam of wide-angle
One of generate scattering light and form ambient noise.
Fig. 5 shows the spectrum of the first adjustable optical filter of luminescence unit.As shown in figure 5, when the first adjustable optical filter
When 12 rotation angle increases, the spectrum of the first adjustable optical filter 12 can gradually be displaced to shorter wavelength region.First is adjustable
The maximum pass-band of formula optical filter 12 is about 60nm.By selecting fluorescein stain appropriate, luminescence unit 1 can excite a series of excitations
Wavelength falls into the fluorescent mixture in spectral region, therefore provides higher stain for potential customers and select flexibility.
In one embodiment, the first adjustable optical filter 12 includes a variable-angle optical filter (angle variable
Filter, AVF).The peak wavelength of first adjustable optical filter 12 can be deviated because of rotation up to 60nm.However, other kinds of
Adjustable optical filter is also applicable to the present invention, such as linear variable filters (linear variable filter).It is located at
The linear deflection of this type optical filter of optical channel front end optionally allows the optical signal of specific wavelength to pass through.
Referring again to Fig. 1 to Fig. 2.The liquid container being placed in the fluorescence groove body 22 of heating unit 2 can be a PCR pipe
Part, to accommodate a Biosample during qPCR is expanded and is detected.The front side of fluorescence groove body 22 and rear side all have a circle
Hole is used for transmission excitation and transmitting optical signal.Preceding hole 221 is the entrance as exciting light.The aperture in preceding hole 221 is about 2.5mm,
And preceding hole 221 is located at and tilts 7 degree of position in a clockwise direction from direct optical axis, to realize maximum light intensity.The hole of metapore 222
Diameter is about 3mm, and metapore 222 be located at system axis it is online.
The material of fluorescence groove body 22 is the copper with excellent heat conductivity, but other heat conducting materials such as aluminium are also applicable
In the present invention.The all linear arrangement of each fluorescence groove body 22, carries out batch experiment with benefit.
Fluorescence groove body 22 is mounted on the top of heater 21.In one embodiment, heater 21 may include, but are not limited to heat
Electroluminescent cold (thermoelectric cooling, TEC) heater, can regulate and control circulatingly with the variation of input current
Temperature, and the temperature control of TEC heater is up to the degree of decimal point, therefore can meet the needs of circulating PCR amplification program.Compared to
Other thermal cyclers (thermal cycler), small and exquisite TEC heater facilitates the miniaturization of system, and TEC heats utensil
There is long service life, and easy to maintain.Certainly, other are equally applicable to this via the conventional thermocy method of air or liquid
Invention.
In one embodiment, detection unit 3 also includes a collecting optics component (condensing optics) 33, setting
Between fluorescence groove body 22 and the second adjustable optical filter 31 of detection unit 3.Collecting optics component 33 collects autofluorescence mixing
The fluorescence that object issues, and be refracted as being uniformly distributed on collimated light beam to the second adjustable optical filter 31 of detection unit 3.Converging light
The radius of curvature for learning component 33 is 4mm, and plane is towards fluorescent mixture.Collecting optics component 33 is made of BK7 glass, but its
Glass, optical-grade polymer or the ceramics of his type are also applicable to the present invention.
In one embodiment, detection unit 3 includes also an imaging optics (imaging optics) 34, is set to inspection
It surveys between the second adjustable optical filter 31 and photodetector 32 of unit 3.Imaging optics 34 are by the fluorescent foci after optical filtering
In on the imaging plane of photodetector 32.Imaging optics 34 are made of BK7 glass, and its radius of curvature is about 11mm.So
And other kinds of optical-grade material, such as polymer, glass or ceramics etc., also it is applicable to the present invention.
In one embodiment, photodetector 32 includes silicon photoelectric diode, but other kinds of detector, such as photoelectricity times
Increase pipe (photomultiplier tube, PMT), Charged Coupled Device (charge-coupled device, CCD), complementary gold
Belong to oxidation semiconductor (complementary metal-oxide semiconductor, CMOS) or avalanche photodide
(avalanche photodiode, APD) is also applicable to the present invention.
In one embodiment, detection unit 3 includes also a photodiode amplifier, to convert electrical current into volt, and
Electric signal is amplified to 108 times, so that further data are analyzed and are utilized.
Fig. 6 A, Fig. 6 B and Fig. 6 C show the optical path of detection unit, wherein the second adjustable optical filter rotate respectively to
0,40 and 75 degree of positions, Fig. 7 then show the overlapping optical path of Fig. 6 A, Fig. 6 B and Fig. 6 C.As shown in Fig. 2 and Fig. 6 A to Fig. 7, from
The fluorescence that fluorescent mixture in fluorescence groove body 22 issues first by collecting optics component 33 converge to detection unit 3 it is second adjustable
Formula optical filter 31, the fluorescence after then filtering then are focused on by imaging optics 34 on the imaging plane of photodetector 32.
The adjustable optical filter 31 of the second of detection unit 3 is as transmitting optical filter, and the second adjustable optical filter 31
Angle gyrobearing is towards imaging optics 34.The coating surface of second adjustable optical filter 31 is then towards collecting optics group
Part 33.In one embodiment, the size of the second adjustable optical filter 31 in each channel is 6x 9x 2mm, but its size can
Changed according to different application.
In one embodiment, the second adjustable optical filter 31 includes a variable-angle optical filter.Second adjustable optical filter
31 peak wavelength can be deviated because of rotation up to 60nm.However, other kinds of adjustable optical filter is also applicable to the present invention,
Such as linear variable filters.Certain wave is optionally allowed positioned at the linear deflection of this type optical filter of optical channel front end
Long optical signal passes through.
Similar to the first adjustable optical filter 12 of luminescence unit 1, when the rotation angle of the second adjustable optical filter 31 increases
The spectrum of added-time, the adjustable optical filter 31 of the second of detection unit 3 also can gradually be displaced to shorter wavelength region.Second is adjustable
The maximum pass-band of optical filter 31 is about 60nm.By fluorescein stain appropriate is selected, detection unit 3 can detect launch wavelength and fall into
Fluorescence in spectral region, therefore provide higher stain for potential customers and select flexibility.
Conception according to the present invention, as long as excitation (Ex) wavelength of fluorescein stain and transmitting (Em) wavelength fall into variable-angle
The fluorescein stain of the spectral region of filter set (AVF set) is all applicable to the present invention, wherein variable-angle filter set packet
Containing exciter filter (i.e. the first adjustable optical filter 12 of luminescence unit 1) and transmitting optical filter, (i.e. the second of detection unit 3 can
Mode optical filter 31).In one embodiment, selected variable-angle filter set can excite and detect excitation wavelength between 487-
The fluorescein stain of 547nm and launch wavelength between 500-560nm.Fig. 8 shows available fluorescein stain example.For example,
FAM stain is suitable for wherein one group of variable-angle filter set, and wherein exciter filter rotates 60 degree, and emits optical filter rotation
50 degree.Certainly, rotation angle is not limited to accurately 60 degree and 50 degree, also can be 60 ± 5 degree and 50 ± 5 degree.
In one embodiment, the fluorescein stain quantity for making an addition in PCR pipe part and being detected by fluorescence detection device of the present invention
For at least one.When using single fluorescence, the excitation of nonspecific fluorescein stain and launch wavelength must fall into designed angle
In the passband of variable filter group.
Specifically, fluorescence detection device of the present invention is designed primarily for polychrome (multicolor) or multiple
(multiplexing) fluorescence detection.PCR pipe part may include multiple and different fluorescence probes, to detect different target.Pass through selection
Fluorescein stain appropriate and rotation angle variable filter group in angle appropriate, different targets can in A single sample quilt
Sequentially detect.In this case, the combination of variable-angle filter set and fluorescein stain mixture must be specific, and specific fluorescent
The excitation of stain and launch wavelength must be fallen into the passband of designed variable-angle filter set.
For example, one of variable-angle filter set can be used to detect three comprising FAM, Alexa 514 and HEX
The combination of color stain.Table 1 shows the rotation angle demonstration example of fluorescein stain and variable-angle filter set.Fig. 9 A, Fig. 9 B and Fig. 9 C
Show the optical property that three colour cells of FAM, Alexa 514 and HEX are closed, table 2 shows three colour cells of FAM, Alexa 514 and HEX
The signal-to-noise ratio (signal to noise ratio, SNR) of conjunction.As shown in Fig. 9 A, Fig. 9 B and Fig. 9 C and table 2, fluorescence inspection of the present invention
It surveys device and provides quite excellent performance, and the signal-to-noise ratio of FAM, Alexa 514 and HEX may be up to 60.
Table 1
| Stain | Filter angle phi (°) |
| FAM | Ex(60)&Em(50) |
| Alexa 514 | Ex(40)&Em(30) |
| HEX | Ex(20)&Em(0) |
Table 2
| Stain | SNR@320nM | SNR@20nM |
| FAM | 21.75 | 2.24 |
| Alexa 514 | 58.23 | 4.34 |
| HEX | 60.15 | 4.45 |
Figure 10 A, Figure 10 B and Figure 10 C show the crosstalk effect that three colour cells of FAM, Alexa 514 and HEX are closed
(crosstalk effect).As shown in Figure 10 A, Figure 10 B and Figure 10 C, the fluorescence signal of low concentration intentional stain is even than high
Concentration is also strong adjacent to the fluorescence signal of stain, therefore fluorescence detection device of the present invention closes three colour cells of FAM, Alexa 514 and HEX
Rather low crosstalk effect is presented.Also, signal-to-background ratio (signal to background ratio, SBR) can be by formula: SBR=
The PD voltage of target stain/neighbouring stain PD voltage calculates.As long as signal-to-background ratio is more than or equal to 2, between fluorescent mixture
Crosstalk effect it is i.e. negligible.
Certainly, fluorescein stain of the invention combination is not limited to FAM, Alexa 514 and HEX, and table 3 to table 5 is shown in it
The combination of designed variable-angle filter set and fluorescein stain mixture in his embodiment.
Table 3
| Stain | Filter angle phi (°) |
| FAM | Ex(60)&Em(50) |
| YFP | Ex(40)&Em(30) |
| HEX | Ex(20)&Em(0) |
Table 4
| Stain | Filter angle phi (°) |
| HEX | Ex(75)&Em(55) |
| ATTO 565 | Ex(55)&Em(40) |
| ROX | Ex(35)&Em(0) |
Table 5
| Stain | Filter angle phi (°) |
| HEX | Ex(75)&Em(55) |
| ATTO 565 | Ex(50)&Em(35) |
| Texas Red | Ex(30)&Em(0) |
Referring again to Fig. 1 to Fig. 2.Actuating unit 4 includes one first actuator 41 and one second actuator 42, is difference
It is connect with luminescence unit 1 and detection unit 3, to drive exciter filter (i.e. the first adjustable optical filter 12 of luminescence unit 1)
And transmitting optical filter (i.e. the second adjustable optical filter 31 of detection unit 3), the passband variation of control excitation and transmitting whereby, with
The corresponding specific wavelength for meeting target fluorescent probe.
In one embodiment, the rotation that actuating unit 4 may include, but are not limited to servo motor, be driven by fluid or vacuum pressure
Linear actuator, piezoelectricity (PZT) actuator, electronic polymer (EAP) actuator or electromagnetic motor.In addition, actuating unit 4 can needle
Digital drive is carried out to special angle, or carries out analog-driven in a manner of continuous rotation on demand.
In one embodiment, the number of channels of multicolor fluorescence detection device is at least one.Multicolor fluorescence as shown in Figure 1
Detection device is triple channel fluorescence detection device.In other words, there are three luminous, three fluorescence groove bodies 22 and three detections are logical
Road, and each luminous is mapped across corresponding fluorescence groove body 22 and sense channel.Three channels linearly arrange, to be criticized
Secondary test, and with respective light source 11, fluorescence groove body 22, photodetector 3 and one group, first and second is adjustable in each channel
Optical filter 12,31.Therefore, multicolor fluorescence detection device of the invention can also be used as multichannel fluorescence detection device.Certainly, optics
The quantity in channel is not limited to three, and can be adjusted according to different demands.
In one embodiment, the quantity for exciting and emitting filter set can be one group.In other words, the first adjustable optical filtering
Piece 12 and the second 31 groups of adjustable optical filter need not can configure in each channel, and only single group first and second is adjustable
Optical filter 12,31 is shared on multiple channels.It is more by the way that single group of first and second adjustable optical filter 12,31 to be sequentially displaced into
In a channel, this device can still detected applied to polychrome, and simplify the assembling of multicolor fluorescence detection device more.
In one embodiment, the configuration in channel is not limited to linear array, also can be any configuration according to different application.
In conclusion multicolor fluorescence detection device provided by the invention include luminescence unit, heating unit, detection unit and
Actuating unit.In particular, luminescence unit includes adjustable exciter filter, and detection unit includes adjustable transmitting optical filter.
By rotation or mobile spectral filter, apparatus of the present invention can be made to excite and detect a series of fluorescence that wavelength are fallen into optical filter passband
Mixture, therefore can be applicable to the fluorescent mixture of tool multiple color.Multicolor fluorescence detection device may include multiple linear arrangements
Channel, to carry out batch experiment, therefore can also be used as multichannel fluorescence detection device.Multicolor fluorescence detection device is also single with heating
Member integration, to carry out the temperature control of qPCR application.Present invention design can be achieved compact in size and reduce installation cost.This
Invention device has small and exquisite optical texture, and system overall size is about 85x 30x 23mm.This miniaturization device remains as more
Color application provides excellent properties, and has high signal-to-noise ratio and low crosstalk effect.This device has more wide detection bandwidth, and
Higher stain, which is provided, for potential customers selects flexibility.Therefore, the present invention can reduce the size of multicolor fluorescence detection device
And weight, and remain to provide excellent properties and the selection of extensive fluorophor in the portable qPCR system of lower cost.
Even if the present invention described in detail as above-described embodiment and can as those skilled in the art appoint apply craftsman think and be it is all as
Modification, it is so neither de- as attached claim is intended to Protector.
Claims (20)
1. a kind of multicolor fluorescence detection device, includes:
One fluorescence groove body, to accommodate a fluorescent mixture；
One luminescence unit includes an at least light source and at least one first adjustable optical filter, and wherein the light source provides a broadband and shines
It is bright, and different location or angle of the first adjustable optical filter based on the first adjustable optical filter and allow different passbands
Light beam passes through, to excite the fluorescent mixture；
One detection unit includes at least one second adjustable optical filter and at least a photodetector, the wherein second adjustable filter
Different location or angle of the mating plate based on the second adjustable optical filter and allow to emit from the fluorescent mixture and provide difference
The light beam of passband passes through, and the photodetector receives fluorescence signal and the fluorescence signal is converted to electronic signal；And
One actuating unit, framework in drive the first adjustable optical filter and the second adjustable optical filter to different location or angle
Degree, to control the passband variation of the first adjustable optical filter and the second adjustable optical filter respectively.
2. multicolor fluorescence detection device as described in claim 1, wherein the first adjustable optical filter of the luminescence unit and
The second adjustable optical filter of the detection unit all includes a variable-angle optical filter or a linear variable filters.
3. multicolor fluorescence detection device as described in claim 1, wherein the first adjustable optical filter of the luminescence unit and
The maximum pass-band of the second adjustable optical filter of the detection unit is 60nm.
4. multicolor fluorescence detection device as described in claim 1, wherein the light source is a light emitting diode.
5. multicolor fluorescence detection device as described in claim 1, wherein a pin hole be set to the luminescence unit this first can
The front end of mode optical filter.
6. multicolor fluorescence detection device as claimed in claim 5, wherein the pin hole has the aperture of 2 ± 0.5mm.
7. multicolor fluorescence detection device as described in claim 1, wherein the luminescence unit also includes a focus optics, if
It is placed between the first adjustable optical filter and the fluorescence groove body of the luminescence unit.
8. multicolor fluorescence detection device as claimed in claim 7, wherein light of the position of the focus optics from the light source
1.3 ± 0.2mm of axle offset.
9. multicolor fluorescence detection device as described in claim 1, wherein the fluorescence groove body has a preceding hole and a metapore.
10. multicolor fluorescence detection device as claimed in claim 9, wherein the preceding hole is located at from a direct optical axis with side clockwise
To 7 degree of position of inclination.
11. multicolor fluorescence detection device as described in claim 1 also includes a heater, this is given to provide temperature control
Fluorescent mixture.
12. multicolor fluorescence detection device as claimed in claim 11, wherein the heater includes a thermoelectric cooling heater.
13. multicolor fluorescence detection device as described in claim 1, wherein the detection unit also includes a collecting optics component,
It is set between the fluorescence groove body and the second adjustable optical filter of the detection unit.
14. multicolor fluorescence detection device as described in claim 1, wherein the detection unit also includes an imaging optics,
It is set between the second adjustable optical filter and the photodetector of the detection unit.
15. multicolor fluorescence detection device as described in claim 1, wherein the photodetector includes silicon photoelectric diode.
16. multicolor fluorescence detection device as described in claim 1, wherein the actuating unit includes a servo motor, one by flowing
Body or revolving actuator, a piezoelectric actuator, an electronic polymer actuator or the electromagnetic motor of vacuum pressure driving.
17. multicolor fluorescence detection device as described in claim 1 also includes multiple channels linearly arranged, and each logical
Road has the respective light source, the fluorescence groove body, the photodetector and one group of first and second adjustable optical filter.
18. multicolor fluorescence detection device as described in claim 1 further includes multiple channels linearly arranged, and each logical
Road has the respective light source, the fluorescence groove body and the photodetector, wherein single group first and second adjustable optical filtering
Piece is sequentially displaced into multiple channel.
19. multicolor fluorescence detection device as described in claim 1, wherein the first adjustable optical filter of the luminescence unit and
The second adjustable optical filter of the detection unit all includes a variable-angle optical filter.
20. multicolor fluorescence detection device as described in claim 1, wherein the actuating unit has multiple default frameworks, to divide
Multiple fluorescein stains are not detected.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SG10201800240YA SG10201800240YA (en) | 2018-01-10 | 2018-01-10 | Multi-color fluorescence detection device |
| SG10201800240Y | 2018-01-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110018139A true CN110018139A (en) | 2019-07-16 |
| CN110018139B CN110018139B (en) | 2022-04-26 |
Family
ID=67188690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811608963.4A Active CN110018139B (en) | 2018-01-10 | 2018-12-27 | Multicolor fluorescence detection device |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN110018139B (en) |
| SG (1) | SG10201800240YA (en) |
| TW (1) | TWI693393B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112649365A (en) * | 2019-10-10 | 2021-04-13 | 采钰科技股份有限公司 | Biosensor and light discrimination method |
| US11815445B2 (en) | 2020-01-31 | 2023-11-14 | Delta Electronics, Inc. | Optical calibration tool |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101470075A (en) * | 2007-12-26 | 2009-07-01 | 深圳迈瑞生物医疗电子股份有限公司 | Gas concentration measuring apparatus |
| US20100285490A1 (en) * | 2006-12-29 | 2010-11-11 | Invitrogen Corporation | Detection apparatus |
| US20110018538A1 (en) * | 2005-08-31 | 2011-01-27 | T2 Biosystems, Inc. | Nmr device for detection of analytes |
| CN103134780A (en) * | 2011-12-01 | 2013-06-05 | 中国科学院大连化学物理研究所 | Closely attached type light emitting diode induction fluorescence detector for exciting light path |
| CN105092544A (en) * | 2014-05-12 | 2015-11-25 | 绍兴安尼特微电子科技有限公司 | Optical excitation and detection system of fluorescent quantitative PCR detector |
| CN106019548A (en) * | 2016-06-24 | 2016-10-12 | 宁波舜宇仪器有限公司 | Fluorescent microscope light source and color filter module group linkage device |
| CN107003244A (en) * | 2015-01-27 | 2017-08-01 | 株式会社日立高新技术 | Multicolor fluorescence analysis device |
| CN107817227A (en) * | 2016-09-12 | 2018-03-20 | 台达电子国际（新加坡）私人有限公司 | Fluorescence detection device |
| CN108060068A (en) * | 2016-11-08 | 2018-05-22 | 台达电子国际（新加坡）私人有限公司 | Multichannel fluorescence detection device |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2280902Y (en) * | 1996-10-10 | 1998-05-06 | 青岛市卫生防疫站 | Multifunction blood fluorescent determination instrument |
| EP2605001A1 (en) * | 2011-12-15 | 2013-06-19 | Hain Lifescience GmbH | A device and method for optically measuring fluorescence of nucleic acids in test samples and use of the device and method |
| CN105605679A (en) * | 2016-01-29 | 2016-05-25 | 太仓东能环保设备有限公司 | Electrostatic type indoor air purifier |
-
2018
- 2018-01-10 SG SG10201800240YA patent/SG10201800240YA/en unknown
- 2018-12-27 TW TW107147414A patent/TWI693393B/en active
- 2018-12-27 CN CN201811608963.4A patent/CN110018139B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110018538A1 (en) * | 2005-08-31 | 2011-01-27 | T2 Biosystems, Inc. | Nmr device for detection of analytes |
| US20100285490A1 (en) * | 2006-12-29 | 2010-11-11 | Invitrogen Corporation | Detection apparatus |
| CN101470075A (en) * | 2007-12-26 | 2009-07-01 | 深圳迈瑞生物医疗电子股份有限公司 | Gas concentration measuring apparatus |
| CN103134780A (en) * | 2011-12-01 | 2013-06-05 | 中国科学院大连化学物理研究所 | Closely attached type light emitting diode induction fluorescence detector for exciting light path |
| CN105092544A (en) * | 2014-05-12 | 2015-11-25 | 绍兴安尼特微电子科技有限公司 | Optical excitation and detection system of fluorescent quantitative PCR detector |
| CN107003244A (en) * | 2015-01-27 | 2017-08-01 | 株式会社日立高新技术 | Multicolor fluorescence analysis device |
| CN106019548A (en) * | 2016-06-24 | 2016-10-12 | 宁波舜宇仪器有限公司 | Fluorescent microscope light source and color filter module group linkage device |
| CN107817227A (en) * | 2016-09-12 | 2018-03-20 | 台达电子国际（新加坡）私人有限公司 | Fluorescence detection device |
| CN108060068A (en) * | 2016-11-08 | 2018-05-22 | 台达电子国际（新加坡）私人有限公司 | Multichannel fluorescence detection device |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112649365A (en) * | 2019-10-10 | 2021-04-13 | 采钰科技股份有限公司 | Biosensor and light discrimination method |
| US11815445B2 (en) | 2020-01-31 | 2023-11-14 | Delta Electronics, Inc. | Optical calibration tool |
Also Published As
| Publication number | Publication date |
|---|---|
| TWI693393B (en) | 2020-05-11 |
| SG10201800240YA (en) | 2019-08-27 |
| CN110018139B (en) | 2022-04-26 |
| TW201930853A (en) | 2019-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110161003B (en) | Optical detection device and real-time fluorescence quantitative nucleic acid amplification detection system | |
| EP2825865B1 (en) | Portable device for detecting molecule(s) | |
| JP5021614B2 (en) | Multiple fluorescence detector with removable optical module | |
| CN105008878B (en) | Optical interrogation device | |
| TWI734929B (en) | Multi-color fluorescent excitation and detection device and nucleic acid analysis apparatus employing same | |
| CN110220872B (en) | Portable multi-color fluorescence detection device | |
| US10620123B2 (en) | Fluorescence detection instrument | |
| JP2008534966A (en) | Multiple fluorescence detector with a fiber bundle coupling multiple optical modules to a common detector | |
| CN107817227A (en) | Fluorescence detection device | |
| CN211227169U (en) | Multichannel fluorescence quantitative PCR detection system | |
| CN110018139A (en) | Multicolor fluorescence detection device | |
| US20180252646A1 (en) | Optical structure and optical light detection system | |
| US10175171B2 (en) | Compact multi-UV-LED probe system and methods of use thereof | |
| TWI636248B (en) | Fluorescence detection device | |
| WO2023069651A1 (en) | Fluorescence detection system | |
| CN118076443A (en) | Screening system for identifying pathogens or genetic differences | |
| CN212989140U (en) | Optical device of quantitative fluorescence PCR instrument | |
| US20230137550A1 (en) | Optical signal detection device | |
| TWI735023B (en) | Multi-color fluorescent excitation and detection device | |
| US20230125059A1 (en) | Fluorescence detection system | |
| WO2023164712A2 (en) | Wide-spectrum analysis system | |
| WO2015189298A1 (en) | An optical system for detecting fluorescent or luminescent signals of at least two samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |