CN109655437A - A kind of two-parameter Dynamic Fluorescence detection system and method - Google Patents
A kind of two-parameter Dynamic Fluorescence detection system and method Download PDFInfo
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- CN109655437A CN109655437A CN201811550497.9A CN201811550497A CN109655437A CN 109655437 A CN109655437 A CN 109655437A CN 201811550497 A CN201811550497 A CN 201811550497A CN 109655437 A CN109655437 A CN 109655437A
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- 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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Abstract
The present invention provides a kind of two-parameter Dynamic Fluorescence detection system and method, including, light source, emit optical filter, cuvette, first photomultiplier tube, second photomultiplier tube, first receives optical filter, second receives optical filter, data collector, light source, emit optical filter and cuvette forms fluorescent exciting road, after monochromatic light stimulus, quantum dot inspires the different two kinds of fluorescence signals of wavelength from sensitive materials respectively, pass through the first photomultiplier tube, second photomultiplier tube detects the fluorescence intensity of two kinds of different wave lengths respectively, so as to realize the simultaneously and rapidly measurement of two kinds of parameters.The device of the invention is small in size, light-weight.
Description
Technical field
The present invention relates to fluorescence detecting systems and method, and in particular, to a kind of two-parameter Dynamic Fluorescence detection system and
Method.
Background technique
In optical detecting method, the innate detection high sensitivity of fluorescence method, required sample size is few, and detection limit is usually compared with it
His method is low, while including a variety of measurements such as fluorescence intensity, fluorescence lifetime, quantum yield, excitation peak wavelength, emission peak wavelength
Parameter is available, therefore application is relatively broad.Currently, fluorescence method has been widely used for biochemistry, chemistry, Pharmaceutical Analysis, food
In the fields such as product examine tests, geography, metallurgy, medicine, environmental science, life science.
Mainly there are fluorescence analyser and sepectrophotofluorometer currently based on the instrument of fluorescence method, can measure fluorescence
The analysis of the property and content of matter.Fluorescence analyser is primarily directed to may contain nuisance in production process and in raw material
The product of matter carries out a kind of tool of environmental protection tests, is generally used for factory and customs's detection.Sepectrophotofluorometer mainly includes
Hand-held, automatic recording type and Microcomputer formula several types, for measurement accuracy, first two type much not as good as the latter, and
Three types volume is bigger than normal, is not easy to flexibly use.
But existing fluorescence detection equipment has the following disadvantages:
First, existing fluorescence detection equipment sample bin is to belong to closing mode, it can only be in steady in sample
Feeding sample bin is detected after determining state, and not only operating process is complicated, time-consuming, but also is unable to measure sample in reaction zero
The variation at moment, there are the check frequencies of initial stage.This can not accomplish reality for the biochemical reaction that many moments occur
When, dynamic, quick dynamic detection.
Second, existing fluorescence detection equipment is spectrum-type detecting instrument, modulated excitation wavelength can detect transmitting
Wave spectrum.Although being applicable to a variety of measured matter ingredients, each detection process can only measure single parameter, and system is multiple
It is miscellaneous, at high cost.
Summary of the invention
Aiming at the shortcomings in the prior art, the object of the present invention is to provide a kind of two-parameter Dynamic Fluorescence detection system and inspections
Survey method is, it can be achieved that two-parameter simultaneously and rapidly detect.
According to the first aspect of the invention, a kind of two-parameter Dynamic Fluorescence detection system is provided, the system comprises: light
Source, transmitting optical filter, cuvette, the first photomultiplier tube, the second photomultiplier tube, first receive optical filter, the second reception filter
Mating plate, data collector, in which:
The light source is single broad spectrum light source；
The transmitting optical filter is used to generate the monochromatic light of required wavelength, is generated not by the transmitting optical filter of different wave length
Fluorescent excitation with the approximate monochromatic light of narrowband wavelength, for different material ingredient；
The cuvette is the mixing of quantum dot buffer Yu solution to be measured for containing detection solution, the detection solution
Solution, the quantum dot surface modified to material composition to be measured have specificity sensitive materials, the sensitive materials with to
The designated substance for surveying solution chemically reacts；
The light source, the transmitting optical filter and the cuvette form fluorescent exciting road, when the light source issues
After monochromatic light stimulus, the cuvette is reached after transmitting optical filter optical filtering, makes to detect solution in the cuvette
The quantum dot and the sensitive materials inspire two kinds of fluorescence signals of different wave length respectively；Two kinds of fluorescence signals pass through respectively
The first reception optical filter, described second arrive first photomultiplier tube, second photomultiplier transit after receiving optical filter
Pipe；
First photomultiplier tube and first receives filter set into the first fluorescence reception optical path, first photoelectricity times
Increase pipe and receive the fluorescence signal for receiving optical filter phase co-wavelength with described first, the wavelength of the first reception optical filter corresponds to it
A kind of wavelength of middle fluorescence signal；
Second photomultiplier tube and second receives filter set into the second fluorescence reception optical path, for receive with it is described
Second receives the fluorescence signal of optical filter phase co-wavelength, and the described second wavelength for receiving optical filter corresponds to another fluorescence
The wavelength of signal；
The data collector is separately connected the output end of the first photomultiplier tube and the output end of the second photomultiplier tube,
The fluorescence signal of the different wave length of first fluorescence reception optical path and the second fluorescence reception optical output described in synchronous acquisition is realized double
Parameter synchronization detection.
Preferably, the first photomultiplier tube, the second photomultiplier tube are symmetrically distributed in the two sides of the cuvette, the first light
The axis of electric multiplier tube and the second photomultiplier tube is in 90 ° of angles with fluorescent exciting road, can be glimmering with synchro measure two-way
Optical signal, or as reference signal, measurement pattern can be with unrestricted choice for fluorescence signal and another way all the way for measurement.
In the present invention, the detection solution contained in the cuvette is that quantum dot buffer and the mixing of solution to be measured are molten
Liquid will be inspired the fluorescence signal of two kinds of different wave lengths after the light that is excited irradiates, and then two-parameter synchronous inspection may be implemented
It surveys.
In the present invention, the transmitting optical filter can be according to the material composition of solution to be measured and the different needs of parameter to be measured
It is reasonably selected and is replaced, to measure the fluorescence intensity signals of different material ingredient or different parameters.
Preferably, the system also includes cabinets, wherein the light source, the transmitting optical filter, the cuvette, institute
State the first photomultiplier tube, second photomultiplier tube, the first reception optical filter, the second reception optical filter, institute
State power supply, the data collector is set in the cabinet, the cabinet is divided into first chamber and second by first partition
Chamber, the first partition have the first light hole, and first light hole is used for light passing, and the light source is set to described first
Chamber, the transmitting optical filter are set in the first partition, and are located at first light hole, for filtering out light source hair
Penetrate the stray light of other wavelength in light；
The light source front end needs to be adjacent to first partition, and the first light hole being aligned in first partition；The transmitting filter
In the first partition that mating plate is mounted on, and the first light hole being aligned in first partition.
The cuvette, first photomultiplier tube, second photomultiplier tube, it is described first receive optical filter,
The second reception optical filter is set to the second chamber, and the second chamber distinguishes shape by second partition and third partition
At three chambers, the cuvette is set to intermediate cavity, and the position of the cuvette corresponds to the position of the light source, makes described
Light source forms fluorescent exciting road by transmitting optical filter, first light hole and the cuvette；
First photomultiplier tube, second photomultiplier tube are symmetrically disposed on two side cavities of the cuvette,
The second light hole is provided on the second partition, the first reception optical filter is set on the second partition, and is located at
At second light hole, for extracting the fluorescence for receiving optical filter phase co-wavelength with described first, make first photoelectricity times
Increase pipe and receives optical filter and second light hole the first fluorescence reception optical path of formation by first；It is arranged on the third partition
There is third light hole, the second reception optical filter is set on the third partition, and is located at the third light hole, is used
In extracting the fluorescence for receiving optical filter phase co-wavelength with described second, receives second photomultiplier tube by second and filter
Piece and the third light hole form the second fluorescence reception optical path.
The power supply is the light source, the data collector, first photomultiplier tube, second photomultiplier transit
Pipe power supply.The power supply is high stability integrated regulated power supply, is the light source, the data collector, first photoelectricity
Multiplier tube, second photomultiplier tube provide corresponding voltage, and are first photomultiplier tube, second photoelectricity times
Increase pipe and accurate adjustable control voltage is provided, provides adjustable voltage for the light source.
The second light hole that optical filter is aligned on second partition is received by described first, receives optical filter pair for described second
Third light hole on quasi- third partition.
By first photomultiplier tube, second photomultiplier tube difference face described in chamber where cuvette
Room, closely second partition and third partition, and the third on the second light hole and third partition being respectively aligned on second partition
Light hole；First photomultiplier tube, the model of second photomultiplier tube are completely the same, use same power supply power supply；
Preferably, first photomultiplier tube and second photomultiplier tube are symmetrically distributed in cuvette two sides, peace
For dress apart from identical, parameter is identical, the axis of first photomultiplier tube and second photomultiplier tube respectively with it is described glimmering
Phot-luminescence road is in 90 ° of angles.
First photomultiplier tube and second photomultiplier tube at the beginning of solution to be measured is put into the cuvette just
Start to sample fluorescence signal, therefore can obtain and chemically react initial moment and later fluorescence signal variation overall process, thus
Realize the most quickly detection of Dynamic Fluorescence signal.
Preferably, the power supply and the data acquisition device are set in the first chamber, and the first chamber is logical
The 4th partition, the 5th partition three chambers of formation are crossed, the light source is set between the power supply and the data acquisition device
Chamber.
Preferably, the cabinet is sealed cabinet, and the cabinet has instrument lid, and the instrument lid is described for covering
Square hole is arranged on the instrument lid for cabinet, and the position of the square hole corresponds to the cuvette, and the size of the square hole is greater than described
The size of cuvette, for manually operating to the cuvette, shading cover is arranged in the top of the square hole.The instrument lid
It can be close to completely with cabinet, each chamber is isolated；The square hole size is more bigger than the cuvette, the positive contrastive colours of square hole
Ware set-down location, convenient for manually being operated to the cuvette；The instrument lid is provided with a small shading cover, can directly cover
Above the cuvette, for blocking the interference of environment light.
Preferably, the inner surface of the cabinet and the instrument lid is set as black matt face, for reducing stray light
Interference.It can be sticked for black matte spray painting, black black-out cloth.
Preferably, the cuvette is the quartz colorimetric utensil of four sides light passing.It needs when installation by the quartz colorimetric utensil
Chamber where light source described in the face of slit side.
Preferably, the power supply is integrated regulated power supply, first photomultiplier tube and second photomultiplier tube
Control voltage fine adjustment directly can be carried out by the precision potentiator on the integrated regulated power supply, the size for controlling voltage determines
The voltage amplification factor for having determined photomultiplier tube can critically adjust the size of output signal by adjusting this voltage.
Preferably, the analog voltage signal of acquisition is converted to digital signal by the data collector, is transmitted to host computer.
Preferably, the data that the host computer processing data collecting card transmits, are obtained by calculation required parameter.
Preferably, the light source is ultraviolet lamp, and the wavelength of the ultraviolet lamp is 300nm~500nm.The light source be with
The ultraviolet lamp of certain wavelength width, brightness and irradiation time can be adjusted, to adjust the fluorescence intensity signals and photograph of output
The time is penetrated, reasonable exciting light condition is reached.
Preferably, it described first receives optical filter and described second and receives optical filter, be respectively provided with different wavelength, it can be with
It is reasonably selected and is replaced according to the central wavelength of the material composition of solution to be measured and the excitation fluorescence of parameter to be measured, to survey
Measure the fluorescence signal of different material ingredient or different parameters.
Preferably, the data collector is two-way high-speed synchronous data acquiring device, to the first light described in synchronous acquisition
The fluorescence signal of electric multiplier tube and the different wave length of second photomultiplier tube output realizes two-parameter synchronous detection.
The working principle of above system of the present invention is as follows: the quantum dot buffer Jing Guo surface modification being put into colorimetric first
In ware, light source is opened, waits one section of pre- heat-stable time；Then prepare liquid is put among cuvette, the exciting light from light source
The liquid being irradiated in cuvette, quantum dot will be inspired a kind of fluorescence of wavelength, and the modifier of quantum dot surface and quilt
The designated substance for surveying solution chemically reacts, and inspires the fluorescence of another different wave length.The fluorescence of two kinds of different wave lengths
Signal penetrates the first reception optical filter and second respectively and receives optical filter later respectively by the first photomultiplier tube and the second photoelectricity
Multiplier tube receives, and the intensity of the first photomultiplier tube and the second photomultiplier measurement fluorescence simultaneously exports corresponding analog signal,
Two-way analog signal is by same data collector synchronous acquisition and switchs to digital signal, and simultaneous transmission is subsequent to host computer progress
Processing, may finally obtain the measurement result of two parameters simultaneously.
According to the second aspect of the invention, a kind of two-parameter Dynamic Fluorescence detection method is provided, which comprises
According to the material composition of solution to be measured and parameter property to be measured, the wavelength of selective exitation light, and cardiac wave in selection
Long matched transmitting optical filter；
According to solution error percentage to be measured and parameter property to be measured, quanta point material is selected, buffer is configured to, is filled into
Among cuvette；
It is special according to solution error percentage to be measured and parameter property to be measured and the excitation wavelength and quanta point material
Property, select corresponding with two kinds of wavelength of fluorescence of excitation first to receive optical filter, the second reception optical filter；
The fluorescence of the first photomultiplier tube acquired by data collector continuous synchronization and the output of the second photomultiplier tube
Strength signal, and it is averaged F respectivelya0And Fb0Initial value as two-way fluorescence signal；
Solution to be measured is filled among the cuvette again, shading cover is covered, described the first of continuous synchronization acquisition
The fluorescence intensity signals of photomultiplier tube and second photomultiplier tube output, and it is averaged F respectivelyaAnd FbAs two-way
The measured value of fluorescence signal；
Two measured values are obtained into F compared with respective initial value respectivelya/Fa0And Fb/Fb0, finally obtain two parameters
Measurement result.
Compared with prior art, the present invention have it is following the utility model has the advantages that
The present invention can be achieved it is two-parameter simultaneously and rapidly detect, detectable response initial stage and later Dynamic Fluorescence change full mistake
Journey, the biochemical reaction occurred for many moments can accomplish real-time, dynamic, quick dynamic detection, overcome existing skill
Fluorescence detection equipment can only be sent into sample bin after sample is in stable state and be detected in art, not only operating process
Complexity, time-consuming, and is unable to measure sample in the variation of reaction zero moment, and there are the check frequencies of initial stage.
Two-parameter synchronous detection is realized through the invention, is detected every time compared to existing existing fluorescence detection equipment
Journey is only capable of measuring single parameter, and system complex, at high cost；And the device of the invention is small in size, it is light-weight.
The present invention is used for biochemistry detection process, facilitates the situation of change for analyzing cell metabolism overall process, can also be with
For other biochemistry detection process.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is fluorescence detecting system plane composition schematic diagram in one embodiment of the present invention；
Fig. 2 is box structure diagram in one embodiment of the present invention；
Fig. 3 is fluorescence detecting system interior layout schematic three dimensional views in one embodiment of the present invention；
Fig. 4 is fluorescence detecting system instrument lid schematic diagram in one embodiment of the present invention；
Figure acceptance of the bid note is expressed as: cabinet 1, light source 2, transmitting optical filter 3, cuvette 4, the first photomultiplier tube 5a,
Second photomultiplier tube 5b, first receive optical filter 6a, second receive optical filter 6b, power supply 7, data collector 8, instrument lid 9,
Shading cover 10, first partition 11, the first light hole 12, second partition 13, the second light hole 14, third partition 15, third light passing
Hole 16.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment:
If Fig. 1-3 is the structural schematic diagram of a preferred embodiment in a kind of two-parameter Dynamic Fluorescence detection system of the present invention,
As shown in Figure 1, including in figure: cabinet 1, light source 2, transmitting optical filter 3, cuvette 4, the first photomultiplier tube 5a, the second light
Electric multiplier tube 5b, first receive optical filter 6a, the second reception optical filter 6b, power supply 7, data collector 8, instrument lid 9, shading cover
10。
As shown in Figure 1, Figure 3, a kind of structural schematic diagram of two-parameter Dynamic Fluorescence detection device of the present invention, as shown in Fig. 2,
Cabinet 1 is sealed cabinet, and the inside of cabinet 1 is divided into six chambers, and the chamber of center upper portion is used to install light source 2, top
The two chambers of the left and right sides are respectively intended to installation power supply 7 and data collector 8, and the chamber of lower central is used to install cuvette
4, the two chambers at left and right sides of lower part are symmetrically installed the first photomultiplier tube 5a and the second photomultiplier transit of two same sizes
Pipe 5b, production has the light hole of specification shape on the partition between each chamber, is used for light passing.
As shown in Fig. 2, 2 front end of light source needs to be adjacent to the first partition 11 between upper and lower central bore, and be aligned first every
The first light hole 12 on plate 11；
As shown in figure 3, cuvette 4 is the quartz colorimetric utensil of four sides light passing, when installation, needs the slit side of cuvette 4
Chamber where face light source 2.
As shown in Figure 1-3, the first photomultiplier tube 5a and the second photomultiplier tube 5b, is separately mounted to 4 two sides of cuvette
Two chambers in, the chamber where the face cuvette of the first photomultiplier tube 5a 4 closely second partition 13 and is directed at second
Light hole 14, the chamber where the face cuvette 4 of the second photomultiplier tube 5b closely third partition 15 and are directed at third light passing
Hole 16, the first photomultiplier tube 5a and the second photomultiplier tube 5b model are completely the same, are powered using same power supply 7.
As shown in Figure 1, transmitting optical filter 3 be set to 2 place chamber of light source and cuvette 4 between the chambers first every
On plate 11, and the first light hole 12 being aligned in first partition 11, for filtering out the spuious of other wavelength in the transmitting light of light source 2
Light.
First reception optical filter 6a be set to 4 place chamber of cuvette and the first photomultiplier tube 5a between the chambers
In first partition 11, and the first light hole 12 being aligned in first partition 11, for extracting the fluorescence signal of certain wavelength；Second
It receives optical filter 6b to be set on the third partition 15 of 4 place chamber of cuvette and the second photomultiplier tube 5b, and is directed at third
Third light hole 16 on partition 15, for extracting the fluorescence signal of certain wavelength.
Power supply 7 is high stability integrated regulated power supply, is light source 2, data collector 8, the first photomultiplier tube 5a and the
Two photomultiplier tube 5b provide corresponding voltage, and providing precision for the first photomultiplier tube 5a and the second photomultiplier tube 5b can
The control voltage of tune provides adjustable voltage for light source 2.
Data collector 8 acquires the analog voltage signal of the first photomultiplier tube 5a and the second photomultiplier tube 5b output,
And digital signal is converted to, it is transmitted to host computer and carries out data processing and other operations.
As shown in Figure 4: instrument lid 9 can cover entire cabinet 1, and instrument lid 9 can be close to completely with cabinet 1, every
From each chamber；Instrument lid 9 is equipped with a square hole, and square hole size is more bigger than cuvette 4, the placement of square hole face cuvette 4
Place, operates, instrument lid 9 can directly cover on cuvette 4 equipped with a small shading cover 10 convenient for artificial contrast's color ware 4
Side, for blocking the interference of environment light.
The working principle of fluorescence detecting system of the invention is as follows: first putting the quantum dot buffer Jing Guo surface modification
Enter in cuvette 4, then prepare liquid is put among cuvette 4, the exciting light from light source 2 is irradiated to the liquid in cuvette 4
Body, quantum dot buffer will be inspired a kind of fluorescence of wavelength, and the modifier of quantum dot surface and detected solution is specified
Substance chemically reacts, and inspires the fluorescence of another different wave length.The fluorescence signal of two kinds of different wave lengths penetrates respectively
First, which receives optical filter 6a and second, receives optical filter 6b later respectively by the first photomultiplier tube 5a and the second photomultiplier tube
5b is received, and the intensity of the first photomultiplier tube 5a and the second photomultiplier tube 5b measurement two-way fluorescence signal simultaneously correspondingly exports two
Road analog signal, this two-way analog signal is by same 8 synchronous acquisition of data collector and switchs to digital signal, and simultaneous transmission is supreme
Position machine carries out subsequent processing, may finally obtain the measurement result of two parameters simultaneously.
For example, can be repaired using cumarin to measure the pH value and two parameters of dissolved oxygen concentration of cell metabolism generation
The CdSe quantum dot colloidal solution of decorations is detected.295 μ L detected solutions can be taken, the CdSe of the cumarin modification of 5 μ L is added
Quantum dot colloidal solution obtains final mixed solution, is packed among the cuvette 4 of 400 μ L and is detected.Exciting light is
365nm, the fluorescence centre wavelength that quantum dot inspires are about 625nm, and amplitude size can characterize the size of pH value；Cumarin
The fluorescence centre wavelength of excitation is about 485nm, and amplitude size can characterize the height of dissolved oxygen concentration；Utilize the first photoelectricity times
Increase pipe 5a and the second photomultiplier tube 5b and measure 625nm and 485nm fluorescence signal respectively, can be obtained respectively by data processing
Two parameters of pH value and dissolved oxygen.
The inner surface of cabinet 1 and instrument lid 9 carries out black matt processing, such as black matte spray painting, black black-out cloth
It sticks, reduces the interference of stray light.
Light source 2 is the ultraviolet lamp with certain wavelength width, such as 300nm~500nm；When the brightness and irradiation of light source 2
Between can be adjusted, with adjust output fluorescence intensity signals and irradiation time, reach reasonable exciting light condition.
The solution contained in cuvette 4 is the mixed solution of quantum dot buffer Yu solution to be measured, when the light irradiation that is excited
It will be inspired the fluorescence signal of two kinds of different wave lengths afterwards, and then two-parameter synchronous detection may be implemented；400 μ L as escribed above
The CdSe quantum dot colloid mixed solution of detected solution and cumarin modification, under the excitation of wavelength 365nm light, quantum dot excitation
Fluorescence centre wavelength out is about 625nm, and the fluorescence centre wavelength of cumarin excitation is about 485nm, utilizes two photomultiplier transits
Pipe 5a and 5b measure 625nm and 485nm fluorescence signal respectively, can obtain pH value and dissolved oxygen two respectively by data processing
Parameter.
The control voltage of first photomultiplier tube 5a and the second photomultiplier tube 5b can be directly by the accurate current potential on power supply 7
Device carries out fine adjustment.The size of this control voltage determines the voltage amplification factor of photomultiplier tube 5a and 5b, passes through adjusting
This voltage can critically adjust the size of output signal.
First photomultiplier tube 5a and the second photomultiplier tube 5b is symmetrically distributed in the two sides of cuvette 4, mounting distance phase
Together, parameter is identical, and the axis of the first photomultiplier tube 5a and the second photomultiplier tube 5b are in 90 ° of angles with the optical path of light source 2, can
With synchro measure two-way fluorescence signal, or as reference signal, measurement pattern can be with for fluorescence signal and another way all the way for measurement
Unrestricted choice.
For the present invention during specific implementation, transmitting optical filter 3 can be according to the material composition of solution to be measured and to be measured
The difference of parameter is reasonably selected and is replaced, to measure the fluorescence intensity letter of different material ingredient or different parameters
Number.
Such as: for above-mentioned mixed solution to be measured, the central wavelength for swashing method light is 365nm, then emits the center of optical filter 3
Wavelength is taken as 365nm.
First receive optical filter 6a and second receive optical filter 6b, be respectively provided with different central wavelength, can according to
The central wavelength for surveying the material composition of solution and the excitation fluorescence of parameter to be measured is reasonably selected and is replaced, to measure not jljl
The fluorescence intensity signals of matter ingredient or different parameters.Such as: for above-mentioned mixed solution, under the excitation of wavelength 365nm light,
The two kinds of fluorescence centre wavelength inspired are respectively 625nm and 485nm, therefore first receives the reception optical filtering of optical filter 6a and second
The central wavelength of piece 6b is taken as 625nm and 485nm respectively, is respectively used to two parameters of pH value determination and dissolved oxygen.
First photomultiplier tube 5a and the second photomultiplier tube 5b just start to sample at the beginning of solution to be measured is put into cuvette 4
Fluorescence signal, therefore can obtain and chemically react initial moment and later fluorescence signal variation overall process, to realize dynamic
The most quickly detection of state fluorescence signal.
Data collector 8 is two-way high-speed synchronous data acquiring device, can be with synchronous acquisition the first photomultiplier tube 5a and the
The fluorescence signal of the different wave length of two photomultiplier tube 5b output, and then realize two-parameter synchronous detection.
The present invention provides the design and processing method of a kind of fluorescence detecting system, can apply fluorescence detecting system, method
Include:
(1) it according to the material composition of solution to be measured and parameter property to be measured, the central wavelength of selective exitation light, and selects
Transmitting optical filter 3 is installed to 2 place chamber of light source and cuvette 4 by central wavelength suitable narrow emission optical filter 3 therewith
In first partition 11 between the chambers, and the first light hole 12 being aligned in first partition 11.Such as: above-mentioned mixing is molten
The central wavelength of liquid, a length of 365nm of selective exitation light wave, transmitting optical filter 3 is also 365nm；
(2) according to solution error percentage to be measured and parameter property to be measured, suitable quanta point material is selected, concentration is configured to
Suitable buffer, using being filled among cuvette 4.Such as: above-mentioned 400 μ L mixed solution is 395 μ L detected solutions and 5
The CdSe quantum dot colloid of μ L cumarin modification mixes；
(3) excitation wavelength and quantum selected according to solution error percentage to be measured and parameter property to be measured and front
Point material property selects corresponding with two kinds of wavelength of fluorescence first to receive optical filter 6a and second and receives optical filter 6b, installs respectively
4 place chamber of cuvette and the first photomultiplier tube 5a and the second photomultiplier tube 5b second partition 13 between the chambers
On third partition 15, and it is respectively aligned to the second light hole 14, third light hole 16.Such as: above-mentioned mixed solution, first receives
The central wavelength 625nm of optical filter 6a and second receives the central wavelength 485nm of optical filter 6b；
(4) switch of power supply 7 is opened, detection system powers on, then the preheated one-section time, so that detection system tends to be steady
It is fixed；
(5) data collector 8 is opened, continuous synchronization acquires a series of first photomultiplier tube 5a and the second photomultiplier transit
The fluorescence intensity signals of pipe 5b output, and it is averaged F respectivelya0And Fb0Initial value as two-way fluorescence signal；
(6) solution to be measured is filled among cuvette 4, shading cover 10 is covered, continuous synchronization acquisition a series of the
The fluorescence intensity signals of one photomultiplier tube 5a and the second photomultiplier tube 5b output, and it is averaged F respectivelyaAnd FbAs two
The measured value of road fluorescence signal；
(7) respectively by two measured values compared with respective initial value, F is obtaineda/Fa0And Fb/Fb0, as two parameter pH
The measurement result of value and dissolved oxygen.
Using single broad spectrum light source as exciting light, the high-performance optical filter by replacing different wave length produces the system
The approximate monochromatic light of raw different narrowband wavelengths, meets the fluorescent excitation demand of different material ingredient.Using quantum dot solution conduct
Buffer, quantum dot surface have modified the sensitive materials for having specificity to material composition to be measured.After monochromatic light stimulus, amount
Son point inspires the different two kinds of fluorescence of wavelength from sensitive materials respectively.Two are detected respectively by two independent photomultiplier tubes
The fluorescence intensity of kind different wave length, so as to realize the synchro measure of two kinds of parameters.Since biochemical reaction is to buffer
It is carried out in liquid, two photomultiplier tubes can excite the overall process of fluorescence with real-time detection, it is hereby achieved that two parameters pair
The fluorescence dynamic rule answered, and then the measurement result of two parameters can be obtained within the shortest time, without waiting
Fluorescence signal is measuring after stablizing, detection speed with efficiency is available significantly improves.
Compared with prior art, income effect of the invention is: the device volume is small, light-weight, it can be achieved that two-parameter same
Step quickly detection, detectable response initial stage and later Dynamic Fluorescence change overall process.It is complete that this will be helpful to analysis cell metabolism
The situation of change of process, and can be used for other biochemistry detection process.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (10)
1. a kind of two-parameter Dynamic Fluorescence detection system, it is characterised in that: the system comprises: light source, transmitting optical filter, colorimetric
Ware, the first photomultiplier tube, the second photomultiplier tube, first receive optical filter, the second reception optical filter, data collector,
In:
The light source is single broad spectrum light source；
The transmitting optical filter is used to generate the monochromatic light of required wavelength, is generated not by the transmitting optical filter of different wave length
Fluorescent excitation with the approximate monochromatic light of narrowband wavelength, for different material ingredient；
For the cuvette for containing detection solution, the detection solution is that quantum dot buffer and the mixing of solution to be measured are molten
Liquid, the quantum dot surface modified to material composition to be measured have specificity sensitive materials, the sensitive materials with it is to be measured
The designated substance of solution chemically reacts；
The light source, the transmitting optical filter and the cuvette form fluorescent exciting road, when the light source issues monochrome
After light stimulus, the cuvette is reached after transmitting optical filter optical filtering, makes to detect the described of solution in the cuvette
Quantum dot and the sensitive materials inspire two kinds of fluorescence signals of different wave length respectively；Two kinds of fluorescence signals pass through described respectively
First reception optical filter, described second arrive first photomultiplier tube, second photomultiplier tube after receiving optical filter；
First photomultiplier tube and described first receives filter set into the first fluorescence reception optical path, first photoelectricity times
Increase pipe and receive the fluorescence signal for receiving optical filter phase co-wavelength with described first, the wavelength of the first reception optical filter corresponds to it
A kind of wavelength of middle fluorescence signal；
Second photomultiplier tube and second receives filter set at the second fluorescence reception optical path, for receiving and described second
The fluorescence signal of optical filter phase co-wavelength is received, the described second wavelength for receiving optical filter corresponds to another fluorescence signal
Wavelength；
The data collector be separately connected first photomultiplier tube output end and second photomultiplier tube it is defeated
Outlet, the fluorescence signal of the different wave length of the first fluorescence reception optical path and the second fluorescence reception optical output described in synchronous acquisition,
Realize two-parameter synchronous detection.
2. a kind of two-parameter Dynamic Fluorescence detection system according to claim 1, it is characterised in that: first photomultiplier transit
Pipe, second photomultiplier tube are symmetrically distributed in the two sides of the cuvette, first photomultiplier tube and described second
The axis of photomultiplier tube is in 90 ° of angles with the fluorescent exciting road, is used for synchro measure two-way fluorescence signal.
3. a kind of two-parameter Dynamic Fluorescence detection system according to claim 1, it is characterised in that: the system also includes
Cabinet, wherein the light source, the transmitting optical filter, the cuvette, first photomultiplier tube, second photoelectricity
Multiplier tube, the first reception optical filter, the second reception optical filter, the power supply, the data collector are set to institute
It states in cabinet, the cabinet is divided into first chamber and second chamber by first partition, and the first partition has the first light passing
Hole, first light hole are used for light passing, and the light source is set to the first chamber, and the transmitting optical filter is set to described
In first partition, and it is located at first light hole, for filtering out the stray light of other wavelength in the source emissioning light；
The cuvette, first photomultiplier tube, second photomultiplier tube, described first receive optical filter, described
Second reception optical filter is set to the second chamber, and the second chamber is respectively formed three by second partition and third partition
A chamber, the cuvette are set to intermediate cavity, and the position of the cuvette corresponds to the position of the light source, make the light source
Fluorescent exciting road is formed by the transmitting optical filter, first light hole and the cuvette；
First photomultiplier tube, second photomultiplier tube are symmetrically disposed on two side cavities of the cuvette, described
The second light hole is provided on second partition, the first reception optical filter is set on the second partition, and is located at described
At second light hole, for extracting the fluorescence for receiving optical filter phase co-wavelength with described first, make first photomultiplier tube
Optical filter is received by described first and second light hole forms the first fluorescence reception optical path；It is arranged on the third partition
There is third light hole, the second reception optical filter is set on the third partition, and is located at the third light hole, is used
In extracting the fluorescence for receiving optical filter phase co-wavelength with described second, receive second photomultiplier tube by described second
Optical filter and the third light hole form the second fluorescence reception optical path.
4. a kind of two-parameter Dynamic Fluorescence detection system according to claim 3, it is characterised in that: first photoelectricity times
The axis for increasing pipe and second photomultiplier tube is in respectively 90 ° of angles with the fluorescent exciting road.
5. a kind of two-parameter Dynamic Fluorescence detection system according to claim 3, it is characterised in that: the system comprises electricity
Source, the power supply and the data acquisition device are set in the first chamber, and the first chamber passes through the 4th partition, the
Five partitions form three chambers, and the light source is set to chamber between the power supply and the data acquisition device.
6. a kind of two-parameter Dynamic Fluorescence detection system according to claim 5, it is characterised in that: the power supply is integrated
The control voltage of regulated power supply, first photomultiplier tube and second photomultiplier tube can be directly by the integrated voltage-stabilized
Precision potentiator on power supply carries out fine adjustment, and the size for controlling voltage determines the voltage amplification factor of photomultiplier tube,
The size of output signal can be critically adjusted by adjusting this voltage.
7. a kind of two-parameter Dynamic Fluorescence detection system according to claim 3, it is characterised in that: the cabinet is sealing
Formula cabinet, the cabinet have instrument lid, and square hole, institute is arranged for covering the cabinet in the instrument lid on the instrument lid
The position for stating square hole corresponds to the cuvette, and the size of the square hole is greater than the size of the cuvette, for manually to described
Cuvette is operated, and shading cover is arranged in the top of the square hole.
8. a kind of two-parameter Dynamic Fluorescence detection system according to claim 3, it is characterised in that: it is specific following a kind of or
Various features:
The inner surface of the cabinet is set as black matt face, for reducing the interference of stray light；
The cuvette is the quartz colorimetric utensil of four sides light passing；
The light source is ultraviolet lamp, and the wavelength of the ultraviolet lamp is 300nm~500nm.
9. a kind of two-parameter Dynamic Fluorescence detection system according to claim 1-8, it is characterised in that: the number
It is two-way high-speed synchronous data acquiring device according to collector, to the first photomultiplier tube described in synchronous acquisition and second photoelectricity
The fluorescence signal of the different wave length of multiplier tube output realizes two-parameter synchronous detection.
10. a kind of two-parameter Dynamic Fluorescence detection method, which is characterized in that the described method includes:
According to the material composition of solution to be measured and parameter property to be measured, the wavelength of selective exitation light, and select central wavelength with
The transmitting optical filter to match；
According to solution error percentage to be measured and parameter property to be measured, quanta point material is selected, buffer is configured to, is filled into colorimetric
Among ware；
According to solution error percentage to be measured and parameter property to be measured and the excitation wavelength and quanta point material characteristic, choosing
It selects corresponding with two kinds of wavelength of fluorescence of excitation first and receives optical filter, the second reception optical filter；
The fluorescence intensity of the first photomultiplier tube acquired by data collector continuous synchronization and the output of the second photomultiplier tube
Signal, and it is averaged F respectivelya0And Fb0Initial value as two-way fluorescence signal；
Solution to be measured is filled among the cuvette again, shading cover is covered, first photoelectricity of continuous synchronization acquisition
The fluorescence intensity signals of multiplier tube and second photomultiplier tube output, and it is averaged F respectivelyaAnd FbAs two-way fluorescence
The measured value of signal；
Two measured values are obtained into F compared with respective initial value respectivelya/Fa0And Fb/Fb0, finally obtain the measurement of two parameters
As a result.
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