CN109490259A - Multicolor fluorescence excitation and detection device and its foranalysis of nucleic acids equipment application - Google Patents
Multicolor fluorescence excitation and detection device and its foranalysis of nucleic acids equipment application Download PDFInfo
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- 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
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- 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
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The application discloses a kind of excitation of multicolor fluorescence and detection device, and it includes an at least light emitting module, cassette and an at least detection modules.An at least light emitting module provides the illumination light with particular range of wavelengths.Cassette includes detection chip, and detection chip includes multiple detection slots, around the periphery for being set to detection chip, wherein the corresponding fluorescence sample of accommodating in each detection slot, and each detection slot includes the first wall surface and the second wall surface.Illumination light penetrates the first wall surface with the fluorescence sample in irradiating and detecting slot, and fluorescence sample is excited to penetrate the second wall surface to generate fluorescence signal.An at least detection module receives fluorescence signal, and conversion fluorescence signal is electric signal.
Description
Technical field
The present invention relates to a kind of fluorescence detection device more particularly to a kind of excitation of multicolor fluorescence and detection devices, Yi Jili
With multicolor fluorescence excitation and the foranalysis of nucleic acids equipment of detection device.
Background technique
It flourishes in recent years to obtain the mode of the DNA of a large amount of specific fragments in response to different demands, science
Family endeavour to find out efficient way always to meet its target, and polymerase chain reaction (Polymerase chain
Reaction, PCR) it is one of most economical and quick technology, the specific DNA of 1,000,000,000 copies can be obtained in a short time
Segment.Round pcr can be applicable to multiple fields, such as the selective d NA of Genetic identification separates, analyzes the ancient times in archaeology
The science of law analysis of DNA, genetic test and the medical application of organization type, hospital and research institution infectious disease it is quick and accurate
Diagnosis, food safety environmental hazard examine and genetic fingerprints for investigating criminal etc..Round pcr only need using from
The miniprep dna sample being obtained by extraction in blood or tissue, and fluorescein stain is added in nucleic acid solution, that is, it can pass through fluorescent molecule
Detect the DNA fragmentation of amplification.
Stain and detection technique of fluorescence are a kind of technology being widely used, synchronously to detect and analyse whether mesh
Mark nucleic acid molecules are present in a collection of secondary pollutant sample.Under the light excitation of specific wavelength, when there is fluorescence signal to tie from DNA
When the target nucleic acid molecules of conjunction stain or fluorescence bonding probes issue, this signal is to represent the presence for having target nucleic acid molecules.This
Technology is applied to new-type round pcr, referred to as isothermal duplication mode (isothermal amplification method).Its
In, Optical devices are the indispensable tool of the fluorescence of detection specific nucleic acid fragment sending in qPCR detection technique, this optics
Device must provide light source to excite fluorescence probe in specific wavelength, and detect the fluorescence signal issued from probe simultaneously.
The isothermal duplication mode of another kind substitution is then that need not be controlled using thermal cycle, but be dependent on and utilize internal DNA/
The protein of RNA synthesis mechanism, and dominated by enzyme activity.Therefore, the isothermal system of microminiaturization has design simple and energy
Consume low advantage.There are many complicated degree of analysis (a variety of ferment or introduction), detection sensitivity and specificity are acceptable at present
Isothermal duplication mode is developed, including Nucleic acid sequence amplification technique (nucleic acid sequence-based
Amplification, NASBA), strand displacement amplification (strand displacement amplification, SDA), solution
Revolve enzymatic amplification technology (helicase-dependent amplification, HAD), ring-like isothermal amplification technique (loop-
Mediated isothermal amplification, LAMP), recombinase polymeric enzymatic amplification technology (recombinase
Polymerase amplification, RPA) and nickase amplification technique (nicking enzyme amplification
reaction,NEAR)。
The fluorescence detecting system mature development in multiple fields, such as the application of fluorescence spectrum and fluorescence microscope.Benefit
One group of filter and optical element are mixed with monochromatic source, monochromatic fluorescence probe can be applied to easily.However, most of existing
Fluorescence detecting system all have that volume is big, structure is complicated and defect at high cost.What is more, most of fluorescence detecting systems
The fluorescence signal of generation has lower signal-to-noise ratio (Signal-to-Noise ratio, SNR).Current portable isothermal duplication
The fluorescence detection device design of method still lags behind the development of its biochemical technology.Since purification degrees of the isothermal duplication for sample have
Higher tolerance, most isothermal platform is conceived to the environment created with equilibrium temperature and has middle high throughput in the market
(throughput) detection method.Importantly, the device of most of application isothermal duplications relatively prefers mobile detection ring
In border, so system needs to have highly integrated degree, therefore, although most of existing optical element designs are extensive
With, but it is not appropriate for the device using isothermal duplication.
Summary of the invention
A purpose of the present invention is that a kind of excitation of multicolor fluorescence and detection device are provided, based on non-refractive method to obtain
High s/n ratio is obtained, overall volume and weight are reduced, and makes portable isothermal PCR system that there is preferable property at lower cost
Can, and allow the deviation of detection slot.
Another object of the present invention is to provide a kind of excitation of multicolor fluorescence and detection devices, with close optics knot
Structure has high-effect, robust construction, innovation fluorescence groove body design to avoid aligning and combined difficulty, to avoid light transmission
Caused loss simultaneously keeps high signal-to-noise ratio.
Another object of the present invention is to provide a kind of foranalysis of nucleic acids equipment, it is single device by element set and has
Isothermal duplication function, so that sample purifying, nucleic acid extraction, nucleic acid diffusion and/or the function of detection of nucleic acids can be on single equipments
Immediately it realizes.
A further object of the present invention is to provide a kind of foranalysis of nucleic acids equipment, reach simultaneously using isothermal duplication function
Detect diversified target.
Conception according to the present invention, the present invention provide a kind of excitation of multicolor fluorescence and detection device, and it includes at least one hairs
Optical module, cassette and an at least detection module.Each light emitting module provides the illumination light with particular range of wavelengths.Cassette packet
Containing detection chip, detection chip includes multiple detection slots, around the periphery for being set to detection chip, wherein each detection slot content
Set corresponding fluorescence sample, and each detection slot includes the first wall surface and the second wall surface, wherein illumination light penetrate the first wall surface with
Fluorescence sample in irradiating and detecting slot, and fluorescence sample is excited to penetrate the second wall surface to generate fluorescence signal.At least one detection mould
Block receives the fluorescence signal for being originated from fluorescence sample and penetrating the second wall surface, and conversion fluorescence signal is electric signal.
Conception according to the present invention, the present invention provide a kind of foranalysis of nucleic acids equipment, and it includes multicolor fluorescence excitation and detections
Device, groove body, fluid delivery member, temperature control unit and rotary drive unit.Multicolor fluorescence excitation and detection device include
An at least light emitting module, cassette and an at least detection module.Each light emitting module provides the illumination with particular range of wavelengths
Light.Cassette includes detection chip, and detection chip includes multiple detection slots, around the periphery for being set to detection chip, wherein each
The corresponding fluorescence sample of accommodating in detection slot, and each detection slot includes the first wall surface and the second wall surface, wherein illumination light penetrates
First wall surface excites fluorescence sample to penetrate the second wall surface to generate fluorescence signal with the fluorescence sample in irradiating and detecting slot.Extremely
A few detection module receives the fluorescence signal for being originated from fluorescence sample and penetrating the second wall surface, and conversion fluorescence signal is electric signal.
Groove body receives the cassette to hold.Fluid delivery member is connect with groove body, to convey the fluorescence sample in cassette, to carry out sample
Purifying and/or nucleic acid extraction.Temperature control unit is set in groove body, and provides preset temperature, to carry out nucleic acid amplification.Rotation
Driving unit is connect with groove body, and rotates cassette with a default formula.
Detailed description of the invention
Fig. 1 is one embodiment of the invention using multicolor fluorescence excitation and the structure of the foranalysis of nucleic acids equipment of detection device
Schematic diagram.
Fig. 2 is structural schematic diagram of the foranalysis of nucleic acids equipment shown in FIG. 1 when groove body is opened.
Fig. 3 is locking and the getting loose machine composition of cassette and groove body.
Fig. 4 is the bottom view of cassette.
Fig. 5 is multicolor fluorescence excitation and the structural schematic diagram of detection device of present pre-ferred embodiments.
Fig. 6 is the part enlarged structure schematic diagram of multicolor fluorescence shown in fig. 5 excitation and detection device.
Fig. 7 is the relational graph of the excitation spectrum of four kinds of target fluorescent probes and the passband of four kinds of exciter filters.
Fig. 8 is the relational graph of the emission spectrum of four kinds of target fluorescent probes and the passband of four kinds of transmitting optical filters.
Fig. 9 is the structural schematic diagram of rotary drive unit, light emitting module, cassette and detection module.
Figure 10 is multicolor fluorescence excitation and the structural schematic diagram of detection device of another preferred embodiment of the invention.
Figure 11 is the part enlarged structure schematic diagram of multicolor fluorescence shown in Fig. 10 excitation and detection device.
Description of symbols:
100: foranalysis of nucleic acids equipment
1: groove body
11: top groove body
12: bottom groove body
121: chamber
13: hinge
14: fastener
141: hook part
15: loosening ring
151: protrusion structure
16: loosening actuator
18: accommodating space
2: fluid delivery member
3: temperature control unit
4: rotary drive unit
9: multicolor fluorescence excitation and detection device
6: cassette
60: buckle slot
62: detection chip
625: detection slot
623: the first wall surfaces
621: the second wall surfaces
622: third wall surface
624: the four wall surfaces
63: reagent storage body
632,66: opening
64: first passage
65: second channel
7: light emitting module
71: light source
72: first filter
73: the first pin holes
8: detection module
81: second filter
82: detector
83: the second pin holes
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 can have various variations in different schemes, all without departing from the scope of the present invention, and explanation therein
And attached drawing is illustrated as being used in itself, is not intended to limit the present invention.
The present invention provides a kind of foranalysis of nucleic acids equipment with isothermal duplication application, further explains, and the present invention provides
A kind of foranalysis of nucleic acids equipment has gathered fluid delivery member, temperature control unit, rotary drive unit and multicolor fluorescence excitation
And detection device is on single device, therefore foranalysis of nucleic acids can be carried out immediately on single device to reach sample purifying, nucleic acid extraction
It takes, nucleic acid is spread and the function of detection of nucleic acids.
Fig. 1 is one embodiment of the invention using multicolor fluorescence excitation and the structure of the foranalysis of nucleic acids equipment of detection device
Schematic diagram, Fig. 2 are structural schematic diagram of the foranalysis of nucleic acids equipment shown in FIG. 1 when groove body is opened, and wherein cassette is schematically moved
Foranalysis of nucleic acids equipment out.As shown in Figures 1 and 2, foranalysis of nucleic acids equipment 100 includes groove body 1, fluid delivery member 2, temperature control
Unit 3, rotary drive unit 4 and multicolor fluorescence excitation and detection device 9.Multicolor fluorescence excitation and detection device 9 include cassette
6, an at least light emitting module 7 and at least a detection module 8 (referring to figure 5.).Groove body 1 can be turned on, to install cassette 6 in it
In.Fluid delivery member 2 is connect with groove body 1, and is suitable for conveying the reagent in cassette 6, to carry out sample purifying and/or nucleic acid extraction
It takes.Temperature control unit 3 is set in groove body 1, and is adapted to provide for preset temperature, to carry out nucleic acid amplification.Rotary drive unit 4
It is connect with groove body 1, and formula can be preset and rotate cassette 6 in groove body 1, in one embodiment, rotary drive unit 4 can clamp
Cassette 6.An at least light emitting module 7 and at least a detection module 8 is set on groove body 1, each light emitting module 7 swash comprising at least one
The optical element of light, each detection module 8 include the optical element of an at least detection, are used for detection of nucleic acids or specimen reaction
Detection.
In an embodiment, groove body 1 includes top groove body 11 and bottom groove body 12.Top groove body 11 and bottom groove body 12 are saturating
The connection of hinge (hinge) 13 is crossed, but not limited to this.Bottom groove body 12 has chamber 121, is specifically designed to install cassette 6
In wherein.Top groove body 11 can be turned on, so that cassette 6 can be placed in the chamber 121 of bottom groove body 12.When top groove body 11
After closing, enclosure space is just formed in groove body 1.In one embodiment, the shape of groove body 1 can be but be not limited to it is cylindric, spherical,
Cube, cone or olive-shaped, and groove body 1 can be by (but being not limited to) metal, ceramics, polymer, high-molecular compound, wood
Material, glass or other can provide the material of good thermal isolation to be made.
Bottom groove body 12 is connect through pipe fitting or runner with fluid delivery member 2, when cassette 6 is installed on bottom groove body 12
In, cassette 6 can be locked and be in close contact with fluid delivery member 2, to avoid leakage.For example, cassette 6 can pass through at least
One fixing element is locked in bottom groove body 12, and wherein fixing element may include a fastener (clip), and but not limited to this.
Fig. 3 shows locking and getting loose machine composition between cassette and bottom groove body.As shown in Figures 2 and 3, the column of cassette 6
It include an at least buckle slot 60 on ontology, and bottom groove body 12 includes an at least fastener 14, loosens ring 15 and loosen actuator 16.
The bottom of fastener 14 is fixed, and top has hook part 141.Fastener 14 can be by polymer or flexible sheet metal institute structure
At.When cassette 6 is placed in the chamber 121 of bottom groove body 12, as long as user pushes cassette 6, the card of fastener 14 can be made
The buckle slot 60 of hook part 141 and cassette 6 fastens and locks, and is in close contact cassette 6 and fluid delivery member 2.Pine
Decylization 15 surround the cylindrical body of cassette 6, and is resisted against the bottom surface of hook part 141.The slidably a distance of ring 15 is loosened, and even
It is connected to and loosens actuator 16, such as electromagnetic valve actuator.When cassette 6 to be loosened, as long as triggering loosens actuator 16 and goes to pull
Ring 15 is loosened, can make to loosen the protrusion structure 151 on ring 15 and push against fastener 14, hook part 141 is separated with buckle slot 60,
And then loosen cassette 6.In one embodiment, fastener 14 can be manually operated by user, or be carried out by device according to instruction
Automatic operation.Certainly, locking is not limited to above-mentioned fastener 14 with release mechanism, also can be other cassettes 6 that can lock and loosen
Fixing element.
Fig. 4 shows the bottom view of cassette.Shown in as shown in Figure 2, Figure 3 and Figure 4, cassette 6 includes that detection chip 62 and reagent store
Ontology 63, and detection chip 62 is set to the top of reagent storage body 63.Detection chip 62 is a flat surface type fluid chip, inspection
Surveying chip 62 includes multiple detection slots 625, at least a first passage 64 and an at least second channel 65.An at least first passage
64 via 65 connecting detection slot 625 of an at least second channel.In one embodiment, multiple detection slots 625 surround and are set to the inspection
Survey the periphery of chip 62, and interior sample or the reagent having for nucleic acid amplification and/or detection.For example, detection slot 625 can be coated with
For nucleic acid amplification and/or the sample or reagent of detection, the reagent for example including different fluorescent dyes.The quantity of detection slot 625 is simultaneously
It is unrestricted, and can up to 40 even more slots of slot, so present device can carry out multiplex (multiplexing)
Foranalysis of nucleic acids.In one embodiment, the shape of detection chip 62 is substantially round, so that it is in song that detection chip 62, which has multiple,
The side in face can be arranged in a linear with an at least optical unit 8 and facilitate light focusing.
Reagent storage body 63 includes multiple reagent troughs (not shown), and storage is for sample purifying and/or nucleic acid extraction
Reagent.Reagent storage body 63 also includes multiple runners, is connected to reagent trough for fluid conveying.In one embodiment,
Reagent storage body 63 can be but be not limited to cylindrical body.Reagent storage body 63 further includes being set to 63 bottom of reagent storage body
Multiple openings 632 in face, and opening 632 is connected through runner with reagent trough.The shape of opening 632 can be but be not limited to justify
Shape, line style or other rules or irregular shape.Detection chip 62 further includes at least one opening set on 62 top surface of detection chip
66, and opening 66 is aligned and is connected to an at least reagent trough for reagent storage body 63, for sample to be added in cassette 6.
Fig. 5 is multicolor fluorescence excitation and the structural schematic diagram of detection device of present pre-ferred embodiments, and Fig. 6 is Fig. 5 institute
The part enlarged structure schematic diagram of the multicolor fluorescence excitation and detection device shown.As shown in Figure 1, Figure 2, shown in Fig. 5 and Fig. 6, polychrome is glimmering
Light excitation and detection device 9 include cassette 6, at least a light emitting module 7 and an at least detection module 8.Multicolor fluorescence excitation and
Detection device 9 can be but not be limited to comprising four light emitting modules 7 and four detection modules 8 that each light emitting module 7 is set to bottom
In the accommodating space 18 of groove body 12 (referring to figure 2.), and the illumination light with particular range of wavelengths is provided.
Each detection slot 625 includes the first wall surface 623, the second wall surface 621, third wall surface 622, the 4th wall surface the 624, the 5th
Wall surface and the 6th wall surface (not shown).First wall surface 623 is relative to third wall surface 622, and the second wall surface 621 is relative to the 4th wall surface
624, the 5th wall surface is relative to the 6th wall surface.Second wall surface 621, the 4th wall surface 624, the 5th wall surface and the 6th wall surface are connected simultaneously
It is set between the first wall surface 623 and third wall surface 622.In this present embodiment, the first wall surface 623 is a wall surface, the second wall
Face 621 is a front face, and third wall surface 622 is a upper wall surface, and the 4th wall surface 624 is a rear surface, and the 5th wall surface is the first side
Wall, the 6th wall surface are second sidewall.
First wall surface 623 (that is, wall surface) of the illumination light penetration-detection slot 625 that light emitting module 7 generates, and illuminate inspection
The fluorescence sample in slot 625 is surveyed, and excites fluorescence sample to generate fluorescence signal, fluorescence signal caused by fluorescence sample penetrates
Second wall surface 621 (that is, front face) of detection slot 625.The second wall surface 621 that detection module 8 receives detection slot 625 is transmitted
Fluorescence signal, and fluorescence signal is converted into electric signal.
In one embodiment, the first wall surface 623 of detection slot 625 has a curved surface, and to positioned at an at least light emitting module
7.Second wall surface 621 of detection slot 625 has a curved surface, and during detection of nucleic acids, the second wall surface 621 of detection slot 625
Align an at least detection module 8.First wall surface 623 of detection slot 625 has specific curve, can be but not be limited to round, ellipse
Circle or other shapes, therefore, when the first wall surface 623 of illumination light penetration-detection slot 625, illumination light can be via the first wall
The contraposition of face 623 is located at the fluorescence sample in detection slot 625.Second wall surface 621 of detection slot 625 has specific curve, can be
But it is not limited to round, ellipse or other shapes, therefore, when the second wall surface 621 of illumination light penetration-detection slot 625, fluorescence
Signal can be focused via the second wall surface 621 to positioned at detection module 8.
Referring to Fig. 5 and Fig. 6, light emitting module 7 is disposed adjacent the first wall surface 623 in detection slot 625, and the mould that shines
First wall surface 623 of the optical axis contraposition detection slot 625 of block 7, therefore the first wall surface 623 of detection slot 625 can receive with certain wave
The illumination light of long range.Detection module 8 is disposed adjacent the second wall surface 621 in detection slot 625, and the optical axis pair of detection module 8
Second wall surface 621 of position detection slot 625, therefore detection module 8 can receive the fluorescence letter of the second wall surface 621 of penetration-detection slot 625
Number.
In one embodiment, light emitting module 7 includes light source 71 and first filter 72.Light source 71 can be light emitting diode
(LED) or laser diode (laser diode), and light source 71 is to generation wavelength has wide band illumination light.First filter
Wave device 72 is set between light source 71 and the first wall surface 723, and makes have particular range of wavelengths caused by light source 71
Illumination light passes through, and the illumination light of unwanted wavelength caused by light source 71 is prevented to pass through.
Light emitting module 7 includes also the first pin hole 73, is set between light source 71 and first filter 72, and light emitting module
Illumination light caused by 7 the first pin hole 73 guiding light source 71 is to the first wall surface for being located at first filter 72 and detection slot 625
623, wherein the pore size of the first pin hole 73 between 2.0mm between 3.0mm, but not limited to this.
In some embodiments, first passage 64 is via corresponding 65 connecting detection slot 625 of second channel, wherein first is logical
Road 64 is to send sample to detection slot 625.The sectional area of second channel 65 be less than first passage 64 sectional area be it is preferable,
Therefore second channel 65 has a capillary valve to realize passive flux control.
In one embodiment, the third wall surface 622 of detection slot 625 and the first wall surface 623 are that optical thin film is constituted, the
The thickness of the optical thin film of the thickness of the optical thin film of three wall surfaces 622 and the first wall surface 623 can be between 0.1mm to 0.2mm
Between, but not limited to this.The folding of the optical thin film of the refractive index of the optical thin film of third wall surface 622 and the first wall surface 623
Penetrating rate can be between 1.3 to 1.6, and but not limited to this.
In some embodiments, the volume size of the detection slot 625 of detection chip 62 can between 10 μ L between 50 μ L, but
It is not limited.Detection chip 62 can be by polycarbonate (polycarbonate, PC), polymethyl methacrylate
(polymethyl methacrylate, PMMA) or cyclic olefin copolymer (cyclic olefin copolymer, COC) group
At.The refractive index of the detection slot 625 of detection chip 62 can be between 1.3 to 1.6, and but not limited to this.
Detection module 8 includes second filter 81 and detector 82.Second filter 81 is to receive the of detection slot 625
The fluorescence signal that two wall surfaces 621 are transmitted, and allow the fluorescence signal with particular range of wavelengths to pass through, and prevent unwanted
Wavelength passes through.Detector 82 is converted to receive the fluorescence signal with particular range of wavelengths by second filter 81
Fluorescence signal is electric signal.In an embodiment, detector 82 can be but be not limited to photodiode (photodiode, PD),
Avalanche photodide (avalanche photodiode, APD), photosensitive coupling element (charge coupled device,
) or complementary metal oxide semiconductor (complementary metal-oxide semiconductor, CMOS) CCD.
Detection module 8 also may include the second pin hole 83, the second pin hole 83 be set to detection slot 625 the second wall surface 621 and
Between second filter 81, fluorescence signal caused by the second pin hole 82 guidance fluorescence sample of detection module 8 is to positioned at detection
Module 8, wherein the pore size of the second pin hole 83 can be between 2.0mm between 3.0mm, but not limited to this.
In some embodiments, multicolor fluorescence excitation and detection device 9 include multiple light emitting modules 7 and multiple detection modules
8, such as may include, but are not limited to four light emitting modules 7 and four detection modules 8, plurality of light emitting module 7 provides different face
The illumination light of color is to corresponding detection slot 625, and to carry out fluorescence detection, multiple detection modules 8 receive corresponding fluorescence signal, because
This multiple detection module 8 can detect multiple samples simultaneously and realize that multiplex detects.
In one embodiment, the present invention uses the fluorescein stain of four seed types.Each detection slot 625 is built-in, and there are four types of different
The mixture of fluorescein stain, these stains are standard fluorescence stain, and its title is abbreviated as FAM, HEX, ROX and Cy5.These are glimmering
The excitation spectrum and emission spectrum of light stain are shown in Fig. 7 and Fig. 8.Although present pre-ferred embodiments are with these stains
It presents a demonstration, so system of the invention is not limited to the fluorescein stain of these four types.
Table 1 shows that four kinds of fluorescein stains are applied to the signal-to-noise ratio of multicolor fluorescence excitation and detection device 9 of the invention, wherein
The concentration of four kinds of fluorescein stains is respectively 320nM.It is glimmering that table 1 clearly shows that four kinds of fluorescein stains are applied to polychrome of the invention
Light excitation and detection device 9 signal-to-noise ratio be it is quite high, this represent multicolor fluorescence excitation and detection device 9 sensitivity phase
It is a good.
Table 1
| FAM | HEX | ROX | Cy5 | |
| Bias light (mV) | 4.83 | 11.38 | 0.3 | 4.4 |
| Light source (mV) | 349.1 | 440.2 | 16.33 | 400.7 |
| Signal-to-noise ratio _ 320nM | 72.34 | 38.69 | 53.74 | 91.1 |
Fig. 9 is the structural schematic diagram of rotary drive unit 4, light emitting module 7, cassette 6 and detection module 8.As shown in figure 9,
Rotary drive unit 4 includes also cassette fixture, to clamp and rotate cassette 6.Once the clamped locking of cassette 6, so that cassette 6
It can drive via rotary drive unit 4 and be rotated in groove body 1.There are many form mechanism can according to demand realize clamping and
Loosen cassette 6, wherein cassette fixture also can for calutron, screw, nut, press-fitting component (press fitted parts),
Friction member (frictional parts), grip (grip), pliers (pincer), epoxy resin (epoxy), chemical bonded refractory
(chemical bonding) or other forms, as long as it can clamp cassette 6 according to demand.
In one embodiment, rotary drive unit 4 is installed on the groove body 11 of top.Rotary drive unit 4 can be but unlimited
In motor, and it also can be calutron, manual operation, spring, clockwork or other elements, and can clamp and with preset angle
Degree rotation cassette 6, and sequentially make each detection slot 625 pass through and to be located at each light emitting module 7 and each detection module 8.
In one embodiment, light emitting module 7 is set in the accommodating space 18 of bottom groove body 12, in the process of running, often
One light emitting module 7 is to being located at one of cassette 6 detection slot 625 to provide effective illumination light to detect.Detect mould
Block 8 is set to the outer rim of top groove body 11 to realize optical detection, so that sample can be instant during nucleic acid is spread
It is detected.Once the clamped locking of cassette 6, detection module 8 is in alignment with detection slot 625 one of in cassette 6, therefore nucleic acid
The result of analysis can be read, and cassette 6 rotates so that each detection slot 625 sequentially passes through different light emitting modules 7 and inspection
Survey module 8.In one embodiment, each light emitting module 7 and detection module 8 are capable of providing the illumination light and inspection of unique color
It surveys, fluorescence detection is completed to provide different colours, so that foranalysis of nucleic acids equipment 100 can detect multiple targets simultaneously and realize multiple
Change detection.
In actual operation, when the optical axis of the optical axis of light emitting module 7 or detection module 8 is to possible when being located at detection slot 625
The generation of some deviations is had, and the display of table 2 works as the optical axis of light emitting module 7 or the optical axis of detection module 8 to positioned at detection slot 625
When generating deviation, two kinds of fluorescein stains are applied to the signal-to-noise ratio of multicolor fluorescence excitation and detection device 9 of the invention, and table 2 is clear
Chu shows that two kinds of fluorescein stains are applied to the full width at half maximum of the signal-to-noise ratio of multicolor fluorescence excitation and detection device 9 of the invention
(full-width at half maximum, FWHM) between -2 degree between 2 degree, this represent the optical axis for working as light emitting module 7 or
When the optical axis of detection module 8 is to detection slot 625 is located at, multicolor fluorescence excitation and the production of the receiving of detection device 9 deviation to a certain degree
It is raw.
Table 2
Figure 10 is multicolor fluorescence excitation and the structural schematic diagram of detection device of another preferred embodiment of the present invention, Tu11Wei
The part enlarged structure schematic diagram of multicolor fluorescence excitation shown in Fig. 10 and detection device.In this present embodiment, multicolor fluorescence swashs
Cassette 6, the structure of light emitting module 7 and detection module 8 and the actuation of hair and detection device 9 are similar to respectively shown in Fig. 5 and Fig. 6
Multicolor fluorescence excitation and detection device 9, therefore it is only similar with identical label representative structure and function in this and repeat no more.
In this present embodiment, light emitting module 7 is disposed adjacent in front face, and the optical axis of light emitting module 7 is to being located at front face,
And detection module 8 is disposed adjacent in wall surface, and the optical axis of detection module 8 is to positioned at wall surface, in other words, light emitting module 7
The front face of the illumination light penetration-detection slot 625 of generation with the fluorescence sample in irradiating and detecting slot 625 and inspires fluorescence letter
Number, and the wall surface of fluorescence signal penetration-detection slot 625 caused by fluorescence sample, detection module 8 receive the bottom of detection slot 625
The fluorescence signal that wall surface is transmitted, and further conversion fluorescence signal is electric signal.It is noted that light emitting module 7 and detection
The setting position of module 8 is not limited merely to the setting position of above-described embodiment, and can be adjusted according to different demands.
In conclusion the present invention provides a kind of excitation of multicolor fluorescence and detection device and a kind of foranalysis of nucleic acids equipment, it is more
The excitation of color fluorescence and sets of detection means light emitting module, cassette and detection module are in single device, so that of the invention is more
The excitation of color fluorescence and detection device have the advantage of close structure, lesser volume and lighter weight.In addition, of the invention
Multicolor fluorescence excitation and detection device do not need expensive optical element, so the cost of multicolor fluorescence excitation and detection device
It is lower.Further, due to the configuration of multiple light emitting modules, multiple detection slots and multiple detection modules, the present invention may achieve more
The foranalysis of nucleic acids equipment and multiplex fluorescence detection changed again.In addition, the noise of multicolor fluorescence excitation and detection device of the invention
It is relatively high, and certain deviation is allowed when cassette rotation.
Claims (25)
1. a kind of multicolor fluorescence excitation and detection device, include:
An at least light emitting module provides the illumination light with particular range of wavelengths；
One cassette includes a detection chip, which includes multiple detection slots, surround and is set to the outer of the detection chip
It encloses, wherein the corresponding fluorescence sample of accommodating in each detection slot, and each detection slot includes one first wall surface and one the
Two wall surfaces, the fluorescence sample that wherein illumination light penetrates first wall surface to irradiate in the detection slot, and the fluorescence is excited to try
Sample penetrates second wall surface to generate a fluorescence signal；And
An at least detection module receives the fluorescence signal, and converts the fluorescence signal as an electric signal.
2. multicolor fluorescence as described in claim 1 excitation and detection device, wherein the light emitting module be disposed adjacent in this first
Wall surface, and the optical axis of the light emitting module is disposed adjacent to positioned at first wall surface and the detection module in second wall surface, and
The optical axis of the detection module is to positioned at second wall surface.
3. multicolor fluorescence excitation as described in claim 1 and detection device, wherein first wall surface is a wall surface, this second
Wall surface is a front face.
4. multicolor fluorescence excitation as claimed in claim 3 and detection device, wherein each detection slot also includes a third wall
Face, one the 4th wall surface, one the 5th wall surface and one the 6th wall surface, wherein the third wall surface is relative to first wall surface, second wall
Face relative to the 4th wall surface, the 5th wall surface relative to the 6th wall surface, wherein the third wall surface be a upper wall surface, the 4th
Wall surface is a rear surface, and the 5th wall surface is a first side wall, and the 6th wall surface is a second sidewall.
5. multicolor fluorescence excitation as claimed in claim 4 and detection device, wherein the third wall surface and first wall surface are distinguished
It is made of an optical thin film, wherein the thickness of the optical thin film of the thickness of the optical thin film of the third wall surface and first wall surface
Degree is respectively interposed in 0.1mm between 0.2mm, wherein the refractive index of the optical thin film of the third wall surface and first wall surface should
The refractive index of optical thin film is respectively interposed between 1.3 to 1.6.
6. multicolor fluorescence excitation as described in claim 1 and detection device, wherein first wall surface is a front face, this second
Wall surface is a wall surface.
7. multicolor fluorescence as described in claim 1 excites and detection device, wherein the light emitting module includes:
One light source, generation wavelength have the wide band illumination light；And
One first filter is set between the light source and first wall surface, and makes the illumination with particular range of wavelengths
Light passes through.
8. multicolor fluorescence excitation as claimed in claim 7 and detection device, wherein the light source is that a light emitting diode or one swash
Optical diode.
9. multicolor fluorescence excitation as claimed in claim 7 and detection device, wherein the light emitting module also includes one first pin hole,
It is set between the light source and the first filter, wherein the pore size of first pin hole is between 2.0mm between 3.0mm.
10. multicolor fluorescence excitation as described in claim 1 and detection device, wherein the volume size of the detection slot is between 10 μ L
To between 50 μ L.
11. multicolor fluorescence excitation as described in claim 1 and detection device, wherein the detection chip is by polycarbonate, poly- first
Base methyl acrylate or cyclic olefin copolymer are formed, and wherein the refractive index of the detection slot is between 1.3 to 1.6.
12. multicolor fluorescence as described in claim 1 excites and detection device, wherein the detection module includes:
One second filter receives the fluorescence signal, and the fluorescence signal with particular range of wavelengths is allowed to pass through；And
One detector receives the fluorescence signal with particular range of wavelengths, and converting the fluorescence signal is the electric signal.
13. multicolor fluorescence excitation as claimed in claim 12 and detection device, wherein the detection module also includes one second needle
Hole is set between second wall surface and the second filter, wherein the pore size of second pin hole between 2.0mm extremely
Between 3.0mm.
14. multicolor fluorescence excitation as claimed in claim 12 and detection device, wherein the detector is a photodiode, one
Avalanche photodide, a photosensitive coupling element or a complementary metal oxide semiconductor.
15. multicolor fluorescence as described in claim 1 excites and detection device, wherein multicolor fluorescence excitation and detection device packet
Containing multiple light emitting modules and multiple detection modules, wherein multiple light emitting module provides the illumination lights of different colours to right respectively
The detection slot answered, multiple detection module receive the corresponding fluorescence signal.
16. multicolor fluorescence excitation as described in claim 1 and detection device, wherein the detection chip is a flat surface type fluid core
Piece, and include multiple detection slots, at least a first passage and an at least second channel, wherein an at least first passage is via this
An at least second channel and be connected to multiple detection slot.
17. multicolor fluorescence excitation as described in claim 1 and detection device, wherein the detection chip is circular configuration, and should
First wall surface and second wall surface are respectively provided with specific curve.
18. a kind of foranalysis of nucleic acids equipment, includes:
The excitation of one multicolor fluorescence and detection device include:
An at least light emitting module provides an illumination light of particular range of wavelengths；
One cassette includes a detection chip, which includes multiple detection slots, surround and is set to the outer of the detection chip
It encloses, wherein the corresponding fluorescence sample of setting in each detection slot, and
Each detection slot includes one first wall surface and one second wall surface, and wherein the illumination light penetrates first wall surface to irradiate this
The fluorescence sample in detection slot, and the fluorescence sample is excited to penetrate second wall surface to generate a fluorescence signal；And
An at least detection module receives the fluorescence signal, and converts the fluorescence signal as an electric signal；
One groove body receives the cassette to hold；
One fluid delivery member is connect with the groove body, to convey the fluorescence sample in the cassette carry out sample purifying and/or
Nucleic acid extraction；
One temperature control unit is set in the groove body, and provides a preset temperature, to carry out nucleic acid amplification；And
One rotary drive unit is connect with the groove body, and rotates the cassette with a default formula.
19. foranalysis of nucleic acids equipment as claimed in claim 18, wherein the groove body can be turned on, and including a top groove body and one
Bottom groove body, wherein each light emitting module is set in an accommodating space of the bottom groove body, and each detection module is set
It is placed on the top groove body.
20. foranalysis of nucleic acids equipment as claimed in claim 18, wherein the light emitting module includes:
One light source, generation wavelength have the wide band illumination light；And
The illumination that one first filter is set between the light source and first wall surface, and allows to have particular range of wavelengths
Light passes through.
21. foranalysis of nucleic acids equipment as claimed in claim 20, wherein the light emitting module also includes one first pin hole, it is set to this
Between light source and the first filter, wherein the pore size of first pin hole is between 2.0mm between 3.0mm.
22. foranalysis of nucleic acids equipment as claimed in claim 18, wherein the detection module includes:
One second filter receives the fluorescence signal, and the fluorescence signal with particular range of wavelengths is allowed to pass through；And
One detector receives the fluorescence signal with particular range of wavelengths, and converting the fluorescence signal is the electric signal.
23. foranalysis of nucleic acids equipment as claimed in claim 22, wherein the detection module also includes one second pin hole, it is set to this
Between second wall surface and the second filter, wherein the pore size of second pin hole is between 2.0mm between 3.0mm.
24. foranalysis of nucleic acids equipment as claimed in claim 18, wherein first wall surface is a wall surface, which is one
Front face or first wall surface are a front face, which is a wall surface.
25. foranalysis of nucleic acids equipment as claimed in claim 18, wherein the detection chip is circular configuration, and first wall surface and
Second wall surface is respectively provided with specific curve.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US15/700,791 US10654038B2 (en) | 2016-09-12 | 2017-09-11 | Nucleic acid analysis apparatus |
| US15/700,791 | 2017-09-11 | ||
| SG10201801823UA SG10201801823UA (en) | 2016-09-12 | 2018-03-06 | Multi-color fluorescent excitation and detection device and nucleic acid analysis apparatus employing same |
| SG10201801823U | 2018-03-06 |
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| CN109490259A true CN109490259A (en) | 2019-03-19 |
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| CN201811042086.9A Pending CN109490259A (en) | 2017-09-11 | 2018-09-07 | Multicolor fluorescence excitation and detection device and its foranalysis of nucleic acids equipment application |
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