CN106525807A - Method and device for measuring fluid delayed luminescence - Google Patents
Method and device for measuring fluid delayed luminescence Download PDFInfo
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- CN106525807A CN106525807A CN201710042628.1A CN201710042628A CN106525807A CN 106525807 A CN106525807 A CN 106525807A CN 201710042628 A CN201710042628 A CN 201710042628A CN 106525807 A CN106525807 A CN 106525807A
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- 238000003996 delayed luminescence Methods 0.000 title claims abstract description 55
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- 238000001514 detection method Methods 0.000 claims abstract description 64
- 238000005259 measurement Methods 0.000 claims abstract description 39
- 230000005284 excitation Effects 0.000 claims abstract description 37
- 238000001228 spectrum Methods 0.000 claims description 7
- 239000006185 dispersion Substances 0.000 claims description 6
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- 238000011144 upstream manufacturing Methods 0.000 claims description 5
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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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- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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Abstract
The invention provides a method and device for measuring delayed luminescence. An excitation light path and a detection light path which do not intersect with each other are established, a sample is firstly located on the excitation light path and then transferred on the detection light path, and thus delayed luminescence signals of the sample can be detected. Compared with traditional time resolution detection, the method does not need pulses, delay and shutter control, TTL control, a pulse generator, a chopper and other equipment are omitted, it is achieved that a steady state light source detects the delayed luminescence signals, apparatuses are simplified, and cost is reduced. The method is suitable for time resolution luminescence measurement of the fluid sample and can be used for micro-fluidic chip detection and delayed luminescence detection of a flow cytometry. The method can be matched with a high-power near-infrared laser and is used for time resolution detection of an upconversion material or a near-infrared transmitting material.
Description
Technical field
The present invention relates to the measuring method of luminescence generated by light, the method can be used for the design and manufacture of the equipment and instrument of correlation
In, can be used for the measurement and imaging of delayed luminescence.Belong to optical instrument manufacture field.
Background technology
In luminescence generated by light phenomenon, molecule is excited by illumination and transits to excited state, and the molecule of excited state can be by discharging light
Son returns to the fluorescence or phosphorescence of ground state, i.e. molecule.The excited state of molecule has the molecule of life-span, i.e. excited state will be when crossing one section
Between after just discharge photon and return to ground state, and the lifetime of excited state of different molecular is each different, and the molecular-excited state life-span is longer, its
Lighting time interval is longer.Under normal circumstances, the fluorescence lifetime of molecule is in nanosecond, phosphorescent lifetime can reach Microsecond grade with
On.Delayed fluorescence molecule, its life-span can also reach Microsecond grade even Millisecond.Some phosphorescent molecules for developing in recent years, longevity
Life can reach second level.In addition some inorganic nano materials, its life-span is more than Millisecond.These long-life lighting are referred to as
Delayed luminescence, also referred to as aura.
In the measurement of fluorescence and phosphorescence, two light paths of structure are generally required：Light path one is excitation light path, i.e., exciting light shines
The light path of sample is penetrated, its objective is to make the molecule in sample to be excited by illumination；Light path two is that detection light path, i.e. sample are luminous to be reached
The light path of detector, its objective is to detect the information such as the luminous spectrum of sample, intensity.
Two light path intersects at same point, and sample is placed on the intersection point, to detect the fluorescence and phosphorescence of sample.
In the measurement process of stable state, light path one and light path two are opened simultaneously, i.e. the condition of exciting light irradiating sample always
Lower measurement, this mode can obtain the information of sample luminescent spectrum and intensity, but can not obtain the sample excitation state life-span
Information.
For the signal of measuring samples delayed luminescence, it usually needs detection light path is opened after excitation light path is closed, i.e.,
After stopping exciting, measuring samples it is luminous.Based on such thinking, people developed it is many measurement delayed luminescences methods and
Instrument.（Referenced patent prospectus CN201310392018.6, CN200510092520.0, CN201180017387.6,
CN201110005032.7 etc..）Wherein the more commonly used method is to adopt light-pulse generator, and in a cycle, light application time is very short,
The luminous signal of remaining time is delayed luminescence signal；It is luminous strong using the detectable sample of the single photon counter of time correlation
Spend time dependent curve.Another conventional method is also to adopt light-pulse generator, with the CCD detection with gate control function
Delayed luminescence signal.
Also a kind of conventional method, controls the switch time of wherein one or two light path using chopper, to reach
The purpose of detection delay luminous signal.（List of references Biophysical Journal 1994,67,957-965,
Biophysical Journal 1998, 74, 2210–2222、Anal. Chem. 2011, 83, 2294-2300、
CN205484048U etc..）As the luminescent lifetime of many samples is below millisecond, thus two light path switch are when needing accurate
Between control, for this purpose, many Instantaneous Spectrometers and time-resolved imaging device are using TTL modulation, pulse generator, time delay generation
Device etc. controls the switch opportunity of two light paths.Additionally, in order to avoid interference of stray light, in addition it is also necessary to which the pulse of light source, detector are fast
Door, chopper control etc. all have higher temporal resolution.
The addition of above-mentioned various parts increased the complexity of instrument, in addition the parts valency of many high time resolutions
Lattice are very high, such as picosecond laser, high speed camera, single photon counter, high-precision TTL controllers etc..
In order to simplify instrument reduces cost, patent US6839134B2, CN205080051U and CN106066317A are disclosed
The method that excitation light path and detection light path are controlled simultaneously using chopper, does not use laser instrument, high speed camera, TTL controls
System can also realize the detection of delayed luminescence.The method has certain suitability, but in order to obtain high temporal resolution,
The light hole of chopper is as far as possible narrow, this directly reduces the luminous flux of exciting light, also just reduces luminous signal.Additionally,
For some special samples or device, also need to change the spy that light path could realize delayed luminescence while increasing chopper
Survey.
The fluorescence measurement of fluid sample has important application in fields such as chromatograph, micro-fluidic chip, flow cytometers.
In conventional microchannel sample measurement, excitation light path and detection light path all focus on the same point of microchannel, are mainly used in
The fluorescence measurement of non-delayed（Patent CN105917211A）, in order to measure the delayed luminescence of sample, need to carry out excitation light path
Pulse Width Control, carries out delay control to detection（Patent US20100032584A1, CN106053786A）, such method is not only
The complexity of instrument is increased, measurement efficiency is also reduced.
The content of the invention
In order to further simplify instrument, with the signal for more easily measuring delayed luminescence, the present invention is constructed and is different from
The measuring method of conventional thought.In conventional scheme, excitation light path and detection light path intersect or focus on same point, and sample is needed
It is placed on the intersection point and measures, this mode must controls excitation light path and detection light path ability respectively using pulse, shutter
The delayed luminescence of measuring samples.
In order to omit pulse and shutter control, the method that the present invention is adopted is：
Excitation light path and detection light path are built, makes two light paths non-intersect, so, when sample is on excitation light path, its
Light not in detection light path；When sample is when detecting in light path, exciting light is unable to irradiating sample；
In the measurement of delayed luminescence, mobile example makes sample first be located on excitation light path, is then transferred to detect in light path, this
Sample can just detect the delayed luminescence signal of sample.
Wherein, excitation light path and detection light path refer to existing technique construction, and excitation light path includes light source, detects light path
Comprising detector, slit, diaphragm, prism, grating, filter element, lens, optical fiber can also be set as needed in light path
Deng optical element.
The principle of the method measurement delayed luminescence is apparent：When sample is located on excitation light path, the fluorescence or phosphorescence quilt of sample
Excite, due to excitation light path and detection light path it is non-intersect, thus, the luminous of non-delayed will not be detected；Again sample is shifted
To in detection light path, as transfer needs experience a period of time, thus detection is the luminous signal for postponing；Wherein：The method
In transfer refer to change sample relative to light path position, both can be transfer sample, or transfer light path；This turn
The time for moving past Cheng Jingli is exactly time delay, and the length of time delay is relevant with mobile relative distance and relative speed.
The luminous advantage of the method detection delay is apparent：Pulse Width Control need not be carried out to light source, eliminate TTL controls,
The equipment such as pulse generator, chopper, the light source of any stable state can serve as excitation source；When need not carry out to detector
Between, shutter control, eliminate the adnexaes such as chopper, shutter；In some cases, it is not necessary to periodically excite, postpone, measuring
Process, as long as repeating mobile example or light path, increasing time of exposure can enhancing detectable signal.
This method is applied to the measurement of fluid sample.Fluid sample includes：Liquid, gas and plasma sample.Wherein liquid-like
This includes pure substance liquid, solution, suspension, colloid, and gas sample includes homogeneous gaseous state sample, steam sample, aerosol
Sample etc..
By taking liquid sample as an example, in concrete measurement, liquid sample at the uniform velocity passes through certain pipeline, and excitation light path is gathered in pipeline
Upstream somewhere, makes the sample at this be excited, and detection light path focuses on pipe downstream somewhere, and making that sample at this is luminous can be by
Detector detection, during such sample flow to the downstream of pipeline from the upstream of pipeline, have passed through and excite, and postpone, measurement
Process, thus the delayed luminescence of the detectable fluid sample of the method.
Relative to the fluid measurement instrument that other time differentiates, the method does not need the modulation of pulse shutter, it is not necessary to extra to add
Any adnexa, simplifies instrument, and reduces cost.Additionally, can be changed by coutroi velocity or the position for changing light path focusing
Become time delay, relative to regulation and control shutter, this method is simply easy to implement.
In such fluid test device, containing pipeline, excitation light path and detection light path, wherein excitation light path focuses on pipeline
Upper 1 point of A, detection light path focus on other 1 point of B on pipeline, and sample flows through pipeline, first passes through point A, then passing point B, then this is
System can detect the delayed luminescence of fluid sample in pipeline.
Preferably, the device also includes a pump, by fluid sample with constant rate of speed flow in pipes, controls fluid by pump
Speed can adjust time delay.
Preferably, fluid sample can be circulated flow in pipes, the time of exposure for extending detection can strengthen detection signal
Intensity.
Preferably, in the device, the position that excitation light path and detection light path are focused on can be moved or be adjusted, by mobile or
The position for adjusting excitation light path and detection light path focusing can adjust time delay.
Preferably, the device contains two or more detection light path, and each detection light path is focused on pipeline
Difference, sample flow through each point successively, and each light path can detect the luminous signal of different delays time simultaneously.
In above-mentioned all schemes, if detection light path includes dispersion element and detector, wherein dispersion element be selected from prism or
Grating, detector are selected from line array CCD or cmos sensor, then the device can be used for the luminous spectrum of detection delay.
In the method, sample from A points to the time of B points be time delay, i.e., stream of the distance passed through from A to B divided by sample
The value of speed.It it is being scalable time delay through in the case of constant, adjusting flow velocity.It is horizontal by pipeline in the unit interval
In the case that the sample volume in section is constant, flow velocity and the cross-sectional area of sample are inversely proportional to, when liquid with 1 microlitre/second by pipe
During road, if cross-section of pipeline is the square of 10 microns of the length of side, the flow velocity of sample is 10 meter per seconds, if passed through from A to B
Distance is 1 centimetre, then time delay is 1 millisecond, and the system can measure the delayed luminescence signal of millisecond.If further improved
Flow velocity, and shorten the distance of AB, then can shorten to Microsecond grade time delay, most of phosphorescence, delayed fluorescence material can be met
The detection of material.
In above-mentioned all schemes, fluid line is not limited to orthoscopic, can be dioptric type, bending-type.
Actual fluid sample is often heterogeneous, such as contains cell in blood sample, contains solid particle in air, river,
Thus the delayed luminescence of fluid sample may come from solute or particle in sample, by above-mentioned microfluidic test systems, can
To measure the delayed luminescence of these solutes or particle.
Additionally, the fluorescence and phosphorescence of many organic compound or nano material are surveyed in generally requiring to dissolve or be suspended in liquid
Amount, the delayed luminescence of these compounds or nano material can also be measured in above-mentioned micro-fluidic system.
The fluorescence measurement of fluid sample has important application in fields such as chromatograph, micro-fluidic chip, flow cytometers, thus should
Method has positive effect for above-mentioned field.This method makes full use of the mobility of fluid, on conventional measurement devices basis
On, it is only necessary to the position for changing light path is capable of achieving the measurement of delayed luminescence.
For example, in the application of flow cytometer, excitation light path and detection light path is made to focus on flow chamber microflow channels respectively
The difference of the upstream and downstream in road, you can to realize that the high speed to cell delayed luminescence is detected.
Detection process approximately as：Every time by a cell in microchannel, cell is into after pipeline in ducts upstream
Excited by illumination, when the cell stream is to downstream, have passed through one section of time delay, then the luminous signal collected by detector
As delayed luminescence signal.
Wherein, cell is directly related with time delay in the speed of pipeline.
The speed of existing most of business flow cytomery cells can reach 1000/second, have it is per second can
Up to ten thousand cells of detection, according to same separation velocity, individual cells flow through the time of pipeline less than 1 millisecond, thus this
Method can detect luminous signal of the time delay in Millisecond even more short delaing time.
Knowable to the principle of the test, can arrange in pairs or groups arbitrary stable light source of the method is used, such as mercury lamp, xenon lamp etc., relative to
Using the pulse laser of single wavelength, its excitation wavelength can be adjusted using grating, and scope covers wide, can test different sharp
The material of wavelength is sent out, cost is greatly reduced.The method can also be arranged in pairs or groups powerful near infrared laser, such as 980nm,
808nm etc., can be used for the time-resolved fluid detection of up-conversion or near infrared emission material.
Description of the drawings
Fig. 1 is the schematic diagram for measuring fluid delayed luminescence, and 101 is pipeline, and 102 is fluid sample, and 103 is lens, and 104 are
Lens, 105 is detector.
Fig. 2 is the schematic diagram for measuring fluid delayed luminescence spectrum, and 201 is pipeline, and 202 is fluid sample, and 203 is lens,
204 is lens, and 205 is reflecting grating, and 206 is detector.
Fig. 3 is the schematic diagram for measuring fluid delayed luminescence, and 301 is pipeline, and 302 is fluid sample, and 303 is lens, and 304 are
Lens, 305 is illuminator, and 306 is lens, and 307 is detector.
Fig. 4 is the schematic diagram with optical fiber measurement stream body delayed luminescence, and 401 is pipeline, and 402 is fluid sample, 403,404,
405th, 406 is optical fiber, and 407,408,409 is detector.
Fig. 5 is to measure the luminous schematic diagram of fluid in zigzag, and 501 is pipeline, and 502,503,504 is optical fiber.
Fig. 6 is flow cytometer flow chamber light path schematic diagram, and 601 is pipeline, and 602 is fluid sample, and 603 is laser instrument,
604th, 606,613 be lens, 605,610,611,612,617,618,619 be detector, 607,608,609,614,615,616
For dichroic mirror.
Specific embodiment
In order to illustrate the principle and its advantage of the present invention, the present invention is made further below by specific embodiment
It is bright, its object is to help and be better understood from present disclosure, but these specific embodiments limit this never in any form
The protection domain of invention.In actual applications, most suitable scheme can be implemented as the case may be.
Embodiment 1, the measurement of fluid delayed luminescence.
As shown in figure 1, the fluid sample 102 in pipeline 101 at the uniform velocity flows, exciting light converges at pipeline through lens 103
On 101 at A1, the sample at this is excited, the sample at B1 is luminous downstream converges in detector 105 through lens 104.Its
Do not have exciting light to pass through at middle B1, thus the sample at this lights and comes from the delay passed through after sample is stimulated at A1 at B1
Luminous, time delay is time of the sample from A1 to B1.
The method uses stable light source, it is not necessary to pulse and shutter control, with low cost, as long as fluid sample is always
At the uniform velocity through pipeline 101, by the time of exposure for increasing excitation light intensity and detector, detection signal intensity can be improved.
Embodiment 2, the measurement of fluid delayed luminescence spectrum.
As shown in Fig. 2 the fluid sample 202 in pipeline 201 at the uniform velocity flows, exciting light converges at pipeline through lens 203
On 201 at A2, the sample at this is excited, the sample at B2 is luminous downstream switchs to directional light after lens 204, through grating
Dispersion after 205 reflections is the light of different wave length, is radiated on detector 206.In the principle and Fig. 1 of the measurement device delayed luminescence
It is identical, except for the difference that, employ dispersion element, it is preferred that detector 206 is line array CCD or cmos sensor, can detect and prolong
The spectrum of tardy light.
Embodiment 3, time delay adjustable fluid measurement instrument.
As shown in figure 3, the fluid sample 302 in pipeline 301 at the uniform velocity flows, exciting light converges at pipeline through lens 303
On 301 at A3, the sample at this is excited, the sample at B3 is luminous downstream switchs to directional light after lens 304, through reflective
Mirror 305 reflects, then converges in detector 307 through lens 306.The principle of the measurement device delayed luminescence is identical with Fig. 1,
Wherein, lens 304 and illuminator 305 can be moved in parallel along 301 direction of pipeline, can make detection light path by this movement
Any one place on pipeline 301 is focused on, in the case where flow velocity is constant, the luminous signal of diverse location has different delays
Time.
Embodiment 4, with optical fiber measurement stream body delayed luminescence.
As shown in figure 4, the fluid sample 402 in pipeline 401 at the uniform velocity flows, exciting light is irradiated in pipeline through optical fiber 403
On 401 at A4, the sample at this is excited, sample delay downstream is luminous to arrive separately at detection through optical fiber 404,405,406
Device 407,408,409.
Relative to lens, optical fiber small volume, multiple spot detection is facilitated implementation, multiple detectors separately detect diverse location in figure
Luminous signal, due to time delay it is related to position linearity, thus, the light intensity signal of the multiple points of comparison can obtain luminous strong
Spend the life-span that information, i.e. the method over time can measure delayed luminescence.
Knowable to principle, the method is not limited to three detection optical fibers in figure, increases the point of fiber-optic probe, it is possible to obtain more
Many delayed luminescence information.
Embodiment 5, the delayed luminescence detection of zigzag fluid.
As shown in figure 5, fluid sample at the uniform velocity flows in pipeline 501, exciting light is irradiated in pipeline 501 through optical fiber 502
At upper A5, its transmitted light can be detected by detector through optical fiber 502, it is possible to obtain the absorption spectrum information of sample, pipe downstream
B5 at sample is luminous can be detected by detector through optical fiber 503, it is possible to obtain delayed luminescence information.
Tortuous microchannel is wide in actual application, by pipe shape reasonable in design, can reduce fluid
The interference of scattered light.By the principle of the present invention, the method can be used in other nonlinear pipeline fluid measurements.
This method is applied to the delayed luminescence of liquid chromatograph and detects.
Embodiment 6, the detection of delayed luminescence in flow cytometer.
The method is can apply in flow cytometer, detects the delayed luminescence signal of granule.
As shown in fig. 6, the fluid sample 602 in microchannel 601 flows downward, laser instrument 603 is radiated in pipeline 601
A points, constitute excitation light path；Sample is excited by illumination in A points, and its back-scattering light converges at detector 605 through lens 604
In, it is the first detection light path；The fluorescence that A points send converges at the direction of dichroic mirror 607, wherein dichroic through lens 606
Mirror 607,608,609 is that long wave leads to dichroic mirror, and cutoff wavelength increases successively, and the light below cutoff wavelength is divided by each dichroic mirror
Do not reflex in detector 610,611,612, constitute second and detect light path；The light that B points below A points send is through lens
613, the direction of dichroic mirror 614 is converged at, wherein dichroic mirror 614,615,616 is that long wave leads to dichroic mirror, cutoff wavelength
Increase successively, each dichroic mirror reflexes to the light below cutoff wavelength in detector 617,618,619 respectively, constitute the 3rd and visit
Light-metering road.
In the device, excitation light path and first and second detects the light channel structure that light path is classical flow cytometer flow chamber, uses
In the detection of granule steady generation.3rd excitation light path focus on excitation light irradiation less than B points, the delay of fluid can be detected
Luminous, principle is identical with example above.
Knowable to principle, the device can detect simultaneously sample non-delayed is luminous and delayed luminescence, it is not necessary to pulse, prolong
Control late.Flow velocity is faster, and time delay is shorter, is more conducive to detecting delayed luminescence signal, while improve the efficiency of separation.
Preferably, the detector in the device is photomultiplier tube.
If being intended merely to measure delayed luminescence, it is convenient to omit first and second detection light path.
The device is used for into flow cytometer, the interference of cellular autofluorescence can be avoided.Relative to existing time resolution
Measuring method, the method are with low cost, simple to operate.
Additionally, in all devices of the example 1 in example 6, thus it is possible to vary the detection direction of delayed luminescence, delay can also be increased
Luminous detection light path, to obtain more delayed luminescence information.Can also according to specific needs, in light path add slit,
Diaphragm, grating, lens, optical filter, light splitting piece, attenuator etc., dichroic mirror, prism, illuminator or optical fiber can also be increased with
Change the direction of light path, to strengthen the signal to noise ratio of detection.
Claims (10)
1. it is a kind of measurement delayed luminescence method, it is characterised in that：
Excitation light path and detection light path are built, make two light paths non-intersect,
In the measurement of delayed luminescence, mobile example makes sample first be located on excitation light path, is then transferred to detect in light path, this
Sample can just detect the delayed luminescence signal of sample.
2. the method for claim 1 is used to build fluid measurement instrument, it is characterised in that：
Containing pipeline, excitation light path and detection light path in device, wherein excitation light path focuses on 1 point of A on pipeline, detects light path
Other 1 point of B on pipeline is focused on, sample flows through pipeline, first passes through point A, then passing point B, then the system can be detected in pipeline
The delayed luminescence of fluid sample.
3. fluid measurement instrument as claimed in claim 2, it is characterised in that：The device also includes a pump, by fluid sample
With constant rate of speed flow in pipes, the speed for controlling fluid by pump can adjust time delay.
4. fluid measurement instrument as claimed in claim 3, it is characterised in that：Fluid sample can circulate flow in pipes, extend
The time of exposure of detection can strengthen detection signal intensity.
5. fluid measurement instrument as claimed in claim 3, it is characterised in that：The position that excitation light path and detection light path are focused on can
With mobile or regulation, time delay can be adjusted by position that is mobile or adjusting excitation light path and detection light path focusing.
6. fluid measurement instrument as claimed in claim 2, it is characterised in that：The device contains two or more detection
Light path, each detection light path focus on the difference on pipeline, and sample flows through each point successively, and each light path can be detected not simultaneously
With the luminous signal of time delay.
7. fluid measurement instrument as claimed in claim 2, it is characterised in that：Detection light path includes dispersion element and detector,
Wherein dispersion element is selected from prism or grating, and detector is selected from line array CCD or cmos sensor, and the device can be used for detection delay
Luminous spectrum.
8. fluid measurement instrument as claimed in claim 2, it is characterised in that：Include some optical elements, these light in light path
Learn element and be selected from slit, diaphragm, grating, lens, optical filter, light splitting piece, attenuator, dichroic mirror, prism, illuminator or light
It is fine.
9. fluid measurement instrument as claimed in claim 2, it is characterised in that：Fluid line is selected from dioptric type or bending-type.
10. fluid measurement instrument as claimed in claim 2 is applied in flow cytometer, it is characterised in that：Excitation light path and
Detection light path focuses on the difference of the upstream and downstream of flow chamber microchannel respectively, you can to realize the spy to cell delayed luminescence
Survey.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710042628.1A CN106525807A (en) | 2017-01-20 | 2017-01-20 | Method and device for measuring fluid delayed luminescence |
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| CN201710042628.1A CN106525807A (en) | 2017-01-20 | 2017-01-20 | Method and device for measuring fluid delayed luminescence |
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| CN201710042628.1A Pending CN106525807A (en) | 2017-01-20 | 2017-01-20 | Method and device for measuring fluid delayed luminescence |
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