CN104345048B - A kind of based on the up-conversion luminescence material detection method of biomolecule and the detecting system implementing it - Google Patents
A kind of based on the up-conversion luminescence material detection method of biomolecule and the detecting system implementing it Download PDFInfo
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention provides a kind of method based on up-conversion luminescence material detection biomolecule, said method comprising the steps of: 1) make biomolecule to be detected be combined with label, described label uses up-conversion luminescence material to prepare, and then removes unconjugated unnecessary label；2) in the reaction system of step 1), agent of dissociating is added, so that described up-conversion luminescence material dissociates to liquid phase；3) detecting step 2) reaction system described in label or the existence of described up-conversion luminescence material or its amount.The method is to be applicable to quantitatively or the multiphoton excitation up-conversion luminescence labelling liquid phase detection technique of qualitative detection biomolecule.Present invention also offers the detecting system for implementing the method.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to the highly sensitive of biomolecule
Spend quantitative or qualitative detection.
Background technology
The biomolecular labeling detection technique as representative with immune detection and gene analysis, as modern biotechnology
The important component part of technology, is not only widely used in current field of biomedical research,
And become in the many departments such as clinical diagnosis, environmental health detection, food safety, forensic identification
For indispensable routine techniques means, play irreplaceable effect.As wherein crucial label and
Its corresponding signal detection means, has been subjected to following main technology developmental stage so far:
1. radioactive label: the middle period occurs and established modern labelling Measurement for Biotechnique on last century
Development foundation, has landmark meaning, thus obtained Nobel Prize.Due to its have highly sensitive,
High accuracy, can in the huge advantage of the intracellular spike of vivo biological tissue, thus be applied to rapidly each
Individual association area, and obtain significant progress.But there is also many obvious shortcomings, such as simultaneously: certain
A little radiosiotope are short due to the half-life, are unfavorable for storage and transport；The pollution of radioactive substance is to work
Health and the environment of making personnel cause certain harm, need special protection and waste reduction and disposal equipment；Some
In application, the sensitivity of the method is the most inadequate etc..Therefore, from the beginning of last century end, its market share
Decline rapidly, essentially dropped out mainstream applications field.
2. revealing label: be divided into enzyme labelling colour developing, gold colloidal, containing several classes such as color microgranules, last century lower half
Leaf occurs, has started the brand-new field of nonradioactive labeling's Measurement for Biotechnique.Have on-radiation, can
Naked-eye observation, without advantages such as half-life restrictions, be therefore of wide application, particularly in bedside health check-up
(POCT) field of survey obtains market widely.Its shortcoming is that sensitivity is slightly worse, accuracy and linear
Scope is not enough, therefore cannot be carried out detection by quantitative accurately, can be only applied in qualitative or half-quantitative detection.
3. fluorescent labeling: the second half in last century occurs, has started on-radiation vivo biological tissue intracellular
The brand-new field of spike.Especially because the discovery of green fluorescent protein (GFP) and application, it is achieved that
Biomolecule and interaction thereof are intracellular at line imaging at vivo biological tissue, therefore obtain this generation
Discipline Nobel Prize just.Its range of application almost covers all biomedical sectors, is currently the most important
One of life science instrument.But owing to various experiment materials and organism itself exist certain background
Fluorescence, adds that shortwave exciting light scatters severe jamming detector, needs exciting and measuring in therefore measuring
Part all must use optical filtering or beam splitting system；And owing to generally using the shortwave exciting lights such as ultraviolet,
Easily lead to fluorescent bleach, cancellation, photodissociation and cell injury, therefore cannot be carried out long-time online sight
Survey.These factors above-mentioned cause that its sensitivity is poor, accuracy and the range of linearity the most inadequate.
4. chemiluminescent labeling: last century end occur, started biomolecule on-radiation high sensitivity,
The uncharted field of wide range of linearity quantitative determination.It not only have simplicity, quickly, safely, effectively the phase long
Feature, and there is the sensitivity exceeding isotope labelling techniques.In the short more than ten years, this technology
Obtain increasingly being widely applied at aspects such as immunology detection, molecule hybridization, and had become as and face
Mainstream applications technology in bed laboratory diagnosis field.But its shortcoming is signal time less stable, it is difficult to
Carry out repeated measure；And in the scope of the intracellular application of vivo biological tissue far away from fluorescent labelling techniques.
5. time-resolved fluorescence: last century end occurs, using rare earth ion complex as label, utilizes
The delayed fluorescence characteristic that it has, by pulse excitation, that delay measurements mode successfully reduces background is glimmering
Optical noise, improves sensitivity, the most also has the advantage of repeatable measurement, thus is applied to rapidly
Clinical Laboratory field, and obtain significant progress.But there is also many obvious shortcomings, such as simultaneously:
Use delay measurements to cause signal attenuation, sacrifice certain sensitivity；Same dvielement in environment and sample
The background caused may interfere with the accuracy of signal.Meanwhile, the flow cell metering system used is easy
Producing cross-contamination and the bubble of interference measurement, flushing process also reduces detection efficiency.It addition, at present
Still may not apply to the intracellular spike of vivo biological tissue.
6. electrochemiluminescence: the second half in last century occurs, utilizes the organic compound conduct of platinum group metal ruthenium
Label, aoxidizes this label and luminous by the electrochemical reaction on electrode.This technology greatly reduces
Background noise, improves sensitivity, and has the advantage of repeatable measurement, thus is also applied to rapidly
Clinical Laboratory field.But its shortcoming is the bubble that flow cell is easily generated cross-contamination and interference measurement, rinse
Process reduces detection efficiency；Also cannot be applied to the intracellular spike of vivo biological tissue simultaneously.
7. multiphoton excitation up-conversion luminescence: the beginning of this century occur, its principle is: some rare earth doped from
The material of son and the asymmetric organic compound of minority can repeatedly absorb long wave by multiple level electron transition
The energy of exciting light, and more higher than exciting light energy (wavelength is shorter) is launched when returning to ground state
Light.This luminescence process has been overturned Stokes' law and (has been thought that material can only be excited by high-energy light and send
The light that the short frequency of low-energy light, i.e. wavelength is high inspires the light that the long frequency of wavelength is low), therefore it is also referred to as
Trans-Stokes (Anti-Stokes) luminescence or up-conversion luminescence.
Due to this detecting system can use the relatively low long wave visible ray even infrared ray of energy as exciting light,
And launch energy higher shortwave light even ultraviolet, its technical advantage is the most notable.First,
Long wave excites the generation that not only prevent fluorescent bleach and fluorescent quenching, contributes to long-time repetition online and sees
Survey, and also eliminate the probability of cell injury, add long wave visible ray and infrared penetration biology group
The ability knitted is higher, is more beneficial in the intracellular application of vivo biological tissue.Secondly as nature
There is not natural up-conversion luminescence material, therefore the background noise of this detecting system is relatively low, adds all kinds of
Photosignal detection components and parts all can considerably beyond long wave for the sensitivity of shortwave light and ultraviolet
See light and infrared ray, therefore substantially improve signal to noise ratio, thus drastically increase detection sensitivity, standard
Really property and the range of linearity, also reduce detecting instrument cost simultaneously.
In the short more than ten years, this technology has obtained increasingly being widely applied in biomedicine, its
Application mainly divides two categories below:
A. photodynamic therapy: can be special with tumor cell by up-conversion luminescence material and specific antibodies medicine etc.
The biomolecule coupling of different combination, thus it is allowed in body tumor tissue rich long-pending, then utilize infrared ray
The ability penetrating biological tissue excites up-conversion luminescence material in vitro so that this material sends ultraviolet and kills
Go out tumor cell.
B. biomolecular labeling: be currently mainly respectively applied to high flux (High Throughput) detection
The microballon coding of middle what is called " liquid chip ", and as in bedside individuality detection (POCT) field
The novel markings thing of immunochromatography (Lateral flow).Although this application overcomes conventional fluorescent element and easily sends out
Raw bleaching and the deficiency of photodissociation, and further increase detection flux, improve spirit the most to a certain extent
Sensitivity and the range of linearity.But owing to exciting light and transmitting light are all existed strong by microgranule and chromatographic film surface
Scattering and Absorption, be not only greatly attenuated signal intensity, and the random variation more causing signal increases；
In addition label high concentration gathering in solid phase is easily caused and excites saturated and concentration quenching so that it is sensitive
Degree, accuracy and the range of linearity are difficult to the raising having essence.And immunochromatography itself as qualitative or
Half-quantitative detection means, its inherent technology feature and defect are also doomed that it cannot really meet high-precision quantitative
The technology requirement of detection.
In sum, biomolecular labeling detection technique, have been subjected to above-mentioned several main development so far
In the stage, its technological progress is very notable, and up-converting phosphor technology therein has complex art advantage, is
The most state-of-the-art technological means.But owing to being not yet fully solved exciting light at present and launching dissipating of light
Penetrate and absorb, and solid phase detection in excite saturated and concentration quenching so that it is sensitivity, accuracy and
The key technical indexes such as the range of linearity still cannot really meet the requirement of high-precision quantitative detection, therefore can only
It is applied to the qualitative or half-quantitative detection means such as immunochromatography.
Therefore, current this area remains a need for developing up-conversion luminescence material in high sensitivity quantitatively or fixed
Property detection biomolecule method.
Summary of the invention
In order to solve above-mentioned technical problem, it is an object of the invention to provide a kind of based on up-conversion luminescence material
The method of detection biomolecule.The method makes full use of the advantage of multiphoton excitation up-conversion luminescence, simultaneously
Avoid the saturated and concentration quenching that excites in solid phase detection, thus improve the spirit utilizing up-conversion luminescence to detect
Sensitivity, accuracy and the range of linearity etc..It is a further object to provide for implementing this detection side
The detecting system of method.
Technical scheme is as follows:
On the one hand, the present invention provides a kind of method based on up-conversion luminescence material detection biomolecule, institute
The method of stating comprises the following steps:
1) make biomolecule to be detected with use up-conversion luminescence material prepare label specific bond with
Form solid-phase complex, then remove unconjugated unnecessary label；
2) to step 1) reaction system in add and dissociate agent, so that described up-conversion luminescence material solution
From to liquid phase；
3) detecting step 2) reaction system in the existence of up-conversion luminescence material or its amount.
At present, up-conversion luminescence material is divided into following two big classes: with Y2O3:Yb3+,Tm3+For the nothing represented
Machine rare earth ion complex, with trans-4-(dimethyl amido)-4`-(diethyl amido) stilbene
(DMAEAS) it is the asymmetric organic compound of representative.Although the two structure and composition is different, but its
Common feature is all to produce up-conversion luminescence, and the present invention is also merely with its up-conversion luminescence, these things
Other character of matter has no effect on the enforcement of this invention.Therefore, the present invention have selected the most respectively
Above two representative substances, to illustrate that the present invention is applicable to all of up-conversion luminescence material.
Preferably, the up-conversion luminescence material that the present invention uses is to change in inorganic rare earth ion complex class
Luminescent substance or asymmetric organic compound species up-conversion luminescence material.Specific embodiment party according to the present invention
Formula, described up-conversion luminescence material is preferably Y2O3:Yb3+,Tm3+Or trans-4-(dimethyl amine
Base)-4`-(diethyl amido) stilbene.
In the detection method that the present invention provides, step 1) be combined in solid phase and carry out, described to be checked
Surveying biomolecule is albumen and derivant, little molecule hormone and medicine, nucleic acid fragment etc..
In the detection method that the present invention provides, step 2) dissociate and carry out in the liquid phase.The institute used
Stating agent of dissociating is the aqueous solution comprising alkali metal hydroxide and organic solvent, wherein alkali metal hydroxide
For one or more in sodium hydroxide, potassium hydroxide, Lithium hydrate, concentration is 0.05N-5.0N,
Preferably 0.1N-1.0N；Organic solvent is in dimethyl sulfoxide, dimethylformamide, ethanol
Planting or multiple, concentration is 10%-95%, preferably 30%-90% (percent by volume).Comprise agent of dissociating
Liquid phase is while making label dissociate, it is provided that one is best suitable for step 3) in signal high efficiency produce
Solution environmental with detection.
In the detection method that the present invention provides, step 3) be detected as that to use excitation source to excite described
Up-conversion luminescence material, then uses photoelectric detector to detect it and launches light.
Wherein, in described step 3) in, described excitation source is single color light emitting devices, and uses simultaneously
Possesses the photomultiplier tube of exciting light insensitive material photocathode.Use single color light emitting devices as exciting light
Source, it is convenient to omit broad spectrum light source and excite end filter or beam splitting system.Use simultaneously and possess exciting light not
The photomultiplier tube of sensitive material photocathode, it is convenient to omit filter or beam splitting system in test side.Described step
3) also include improving detection signal-to-noise ratio by the ratio of optimization blank signal with photomultiplier tube thermal noise.
In step 3) in, described single color light emitting devices is for launching wavelength and up-conversion luminescence material excitation wave
Long identical or close low-power semiconductor laser or light emitting diode.For VISIBLE LIGHT EMISSION upper turn
For changing luminous marker, possesses the photomultiplier tube of exciting light insensitive material photocathode for infrared light
Insensitive double base material photocathode photomultiplier tubes；The up-conversion luminescence label that ultraviolet is launched
For, possess the photomultiplier tube of exciting light insensitive material photocathode for infrared light and visible ray the most not
Sensitive day blind material photocathode photomultiplier tube.
Additionally, the ratio of described optimization blank signal and photomultiplier tube thermal noise is, by adjusting photoelectricity
Comparator threshold after multiplier tube anode output end preamplifier, making blank signal is photomultiplier tube self
1-5 times of thermal noise, preferably 2-3 times.
On the other hand, present invention also offers a kind of for implementing according to the detection system of above-mentioned detection method
System, described detecting system includes: excitation source, photoelectric detector and possess exciting light insensitive material light
The photomultiplier tube of negative electrode.
Wherein, described excitation source is single color light emitting devices, described single color light emitting devices for launch wavelength with
Low-power semiconductor laser that up-conversion luminescence material excitation wavelength is identical or close or light emitting diode；
And
For the up-conversion luminescence label of VISIBLE LIGHT EMISSION, possesses exciting light insensitive
The photomultiplier tube of material photocathode is the double base material photocathode photomultiplier tubes insensitive to infrared light；
For the up-conversion luminescence label that ultraviolet is launched, possesses exciting light insensitive material photocathode
Photomultiplier tube is material photocathode photomultiplier tube blind to the day that infrared light and visible ray are the most insensitive.
The following is the detailed description of the present invention:
At present, in this area, the specific detection means of biomolecule substantially can be divided into solid phase and liquid phase
Detection two big classes.
So-called solid phase detection, refer to by can be the most specific binding a pair molecule in one, such as:
Antibody, antigen, nucleic probe, determinand molecule etc. are fixed to solid matter surface, when treating in sample
After surveying the specific molecular generation association reaction in thing and/or label and solid phase, by technological means such as washings
Remove unconjugated label, finally measure signal produced by the label being deposited in solid phase surface, should
Just there is certain dependency relation with the concentration of determinand molecule in sample in the intensity of signal.This method excellent
Gesture is that signal to noise ratio is high, therefore possesses advantage highly sensitive, that the range of linearity is wide.But when use fluorescence, on
When the labels such as conversion is luminous produce the detection technique of signal the most in position, solid phase surface to exciting light and
Strong scattering and the absorption of launching light have not only been greatly attenuated signal intensity, more cause the random change of signal
Different increase, adds that label high concentration in solid phase assembles to be easily caused and excites saturated and concentration quenching,
Limit the further raising of sensitivity, accuracy and the range of linearity.
So-called Liquid Detection, refer to directly by antibody, antigen, nucleic probe, label etc. can with treat
The molecule surveying thing specific binding (or it is specific binding to affect it) mixes in the solution with sample, makes
It at liquid phase generation association reaction, the most directly measures label in liquid with the determinand molecule in sample
Signal change before and after combining, there is certain with the concentration of determinand molecule in sample in the change of this signal
Dependency relation.The advantage of the method be not only in that simple to operate quickly, and cause the interference of signal fluctuation
Factor is little, and therefore result is stable.But owing to unconjugated label cannot be removed, cause signal to noise ratio remote
Far below solid phase method, its sensitivity and linear measurement range is difficult to meet the requirement of major part quantitative analysis.
To this end, the present invention combines the respective advantage of above two method, develop a kind of brand-new " solid phase
In conjunction with-Liquid Detection " pattern, it may be assumed that first continue to use solid phase method and carry out the combination of biomolecule and label,
And the steps such as uncombined label are removed by washing；Then use a kind of special agent of dissociating by labelling
Thing discharges from solid phase surface, and provides a kind of solution environmental being best suitable for the generation of signal high efficiency；?
After measure the label signal in above-mentioned solution again.This technology not only inherits the excellent of solid phase method high s/n ratio
Gesture, also absorbs the strong point of liquid phase method signal stabilization；More avoid solid phase surface to exciting light and to send out simultaneously
Penetrate the strong scattering of light and absorb, and label high concentration in solid phase assemble exciting of causing saturated and
Concentration quenching.
Obviously, it is achieved the selection that it is critical only that the agent composition that dissociates of " solid phase binding-Liquid Detection " pattern
And preparation.This agent of dissociating must be before not destroying the molecule of label and electronic structure and polymerization state
Put, it is separated from solid phase surface fast and efficiently；The most also to turn on high-quantum efficiency
The generation changing optical signal provides a preferable solution environmental.
The present invention is through research and it is demonstrated experimentally that such agent of dissociating is it is first necessary to contain certain density alkali
Solution, such as: sodium hydroxide (NaOH), potassium hydroxide (KOH), Lithium hydrate (LiOH) etc.,
Its concentration should be between 0.05N-5.0N, and optium concentration is 0.1N-1.0N.Secondly, for ensureing a large amount
The up-conversion luminescence of sub-efficiency, also should contain dimethyl sulfoxide (DMSO), dimethylformamide
(DMF), the organic solvent such as ethanol, its concentration is 10%-95%, and optium concentration is 30%-90% (body
Long-pending percentage ratio).
It addition, in existing signal detector, generally use wide spectral range light source, and at exciting light
Road and signal measurement light path all use the beam splitting systems such as optical filter, grating or prism, scattered to eliminate exciting light
Penetrate and the background fluorescence interference of shuttle and biomaterial itself.But this optical filtering or beam splitting system exist
Result in the decay of signal to a certain extent, also improve manufacturing cost simultaneously, and reduce equipment can
By property and stability.If above-mentioned optical filtering or light splitting system can be simplified by certain particular design or even cancel
System, then be possible not only to be substantially reduced manufacturing cost and improve equipment dependability and stability, moreover it is possible to further
Improve signal to noise ratio, thus promote the key technical indexes such as sensitivity, accuracy and the range of linearity.
It is true that there is not natural up-conversion luminescence material due to nature, and up-conversion luminescence
(i.e. excitation wavelength is far longer than also to have the hugest trans-Stokes (Anti-Stokes) displacement
Wavelength of transmitted light), the sensitivity of the most all kinds of Photoelectric Detection components and parts is all with optical signal
Wavelength increases and reduces rapidly, therefore in the detection of up-conversion luminescence, and exciting light scattering and background noise
Interference be far smaller than fluoroscopic examination.Therefore, as long as selecting appropriate excitation source and photoelectric detector,
It is possible to while cancelling optical filtering or beam splitting system improve signal to noise ratio further.
To this end, the present inventor is through studying for a long period of time and repeatedly testing discovery:
The most directly use and launch the little merit that wavelength is identical or close with up-conversion luminescence label excitation wavelength
The cheap single color light emitting devices such as rate semiconductor laser, light emitting diode is as excitation source, thus omits
Broad spectrum light source and excite end filter or beam splitting system.
2., for the up-conversion luminescence label of VISIBLE LIGHT EMISSION, can directly use infrared light insensitive
Double base material photocathode photomultiplier tubes are to eliminate the interference to photoelectric detector of the infrared excitation light, and omit
Filter or beam splitting system in test side.
3. the up-conversion luminescence label launched for ultraviolet, can be used directly infrared light and visible ray
The most insensitive day, blind material photocathode photomultiplier tube was to eliminate the exciting light interference to photoelectric detector,
And omit test side optical filtering or beam splitting system.
4. for improving signal to noise ratio further, after adjusting photomultiplier tube anode output end preamplifier
Comparator threshold, makes the blank signal (photoelectricity caused merely during n.s under light sealed environment by exciting light
Multiplier tube signal exports) it is that photomultiplier tube self thermal noise is (when closing excitation source under light sealed environment
Photomultiplier tube signal output) 1-5 times, optimum state is 2-3 times.
Compared with existing biomolecule detecting method, the novel detection method that the present invention provides has following excellent
Point: first, is different from existing solid phase or liquid phase detection method, the knot of label in the method for the present invention
Close and complete in solid phase and liquid phase respectively with detection, the most not only there is the advantage of solid phase method high s/n ratio,
Also absorb the strong point of liquid phase method signal stabilization, also avoid solid phase surface simultaneously and to exciting light and launch light
Strong scattering and absorb, and label high concentration in solid phase assemble cause excite saturated and concentration
Cancellation.Additionally, based on this " solid-liquid " pattern, the detection method of the present invention uses and turns
Change stimulative substance as labelling molecule, and excite for its inspection optimization and detect program parameter so that
The detection of this material has obtained further optimization in terms of launching efficiency, detection sensitivity, signal to noise ratio improvement.
Further, utilizing the luminous value of up-conversion luminescence material, the detection method of the present invention can realize to be checked simultaneously
Surveying high-sensitivity detection qualitative and quantitative of biomolecule, therefore range is more extensive.
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 shows that in the calibration object using the method detection of the present invention to obtain in embodiment 1, people promotees first shape
Parathyrine concentration is relative to the linear regression curves of luminous value and equation；
Fig. 2 shows human thyroid in the calibration object using the method detection of the present invention to obtain in embodiment 2
Element concentration is relative to the linear regression curves of luminous value and equation.
Detailed description of the invention
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these
Embodiment is merely to illustrate the present invention, and it limits the scope of the present invention never in any form.
Up-conversion luminescence material is divided into following two big classes: with Y2O3:Yb3+,Tm3+For the inorganic rare earth represented
Ion complex, with trans-4-(dimethyl amido)-4`-(diethyl amido) stilbene (DMAEAS)
For the asymmetric organic compound represented.Although the two structure and composition is different, but its common feature is all
Produce up-conversion luminescence, and the present invention is also merely with its up-conversion luminescence, other character of these materials
Have no effect on the enforcement of this invention.Therefore, following example have selected above two representativeness thing respectively
Matter, to illustrate that the present invention is applicable to all of up-conversion luminescence material.
Experimental technique in following embodiment, if no special instructions, is conventional method.Following enforcement
Medicinal raw material used in example, reagent material etc., if no special instructions, be commercially available purchase product.
Embodiment 1
The up-conversion luminescence labelled immune sandwich assay method launching visible ray using the present invention is quantitatively examined
Survey human serum thyrotropin (protide).
1. anti-human thyrotropin (TSH) monoclonal antibody choosing the two mutual uncontested combinations of strain (is purchased
From Medix biochemica company of Finland).
One strain is used for being coated magnetic particle (polystyrene parcel magnetic particle is purchased from Merck KGaA company), is coated
Condition: 20 μ g/mL antibody add 20mM/L pH9.6 carbonic acid buffer, adds 1% (envelope-bulk to weight ratio)
Magnetic particle 4 DEG C shakes overnight；Discard liquid, add same volume 1% (envelope-bulk to weight ratio) casein room
Temperature concussion 2 hours, abandons liquid standby.
Another strain and up-conversion luminescence material trans-4-(dimethyl amido)-4`-(diethyl amido) two
Styrene (DMAEAS) nano-particle is (according to prolonging Yun Xing etc., asymmetric diphenyl ethylene derivatives
The synthesis of DMAEAS and optical property, applied chemistry magazine, 003,20:2,178-190 synthesize)
In conjunction with forming label: 10mg nano-particle and 1mg antibody add 10ml 200mM/L pH7.2 phosphorus
Acid buffer, concussion addition 0.1% (envelope-bulk to weight ratio) glutaraldehyde overnight, is centrifuged and abandons supernatant with physiology
It is standby that saline cleans particulate matter.
2. take one group of polypropylene transparent reaction cup, be separately added in the step 1 of each 100 μ l prepared being coated
Magnetic particle suspension and label and comprise the calibration object of variable concentrations TSH, this calibration object is by TSH
Sterling is added by variable concentrations to be made without TSH human serum；Separately take a reaction cup, add each 100 μ l
Step 1 in prepare be coated magnetic particle suspension and label and human serum clinical sample (through existing
Commercialization luminescence immunoassay system measurement, its TSH concentration is 2.37mIU/L).By reaction cup in room
The lower concussion of temperature 20 minutes.
3. reaction cup is placed in permanent magnetism bracket upper 30 second, after magnetic particle precipitates completely, pours out liquid.
4. taking off reaction cup from permanent magnetism bracket, every reaction cup adds 500 μ l cleaning mixture (PBS+0.05%
Tween 20), shake 10 seconds.
5. repeat step 3 to after 4 three times, reaction cup is placed in permanent magnetism bracket upper 30 second, treats that magnetic is micro-
Liquid is poured out after grain precipitation completely.
6. in every reaction cup, add agent (0.5N sodium hydroxide+50% dimethyl sulfoxide) of dissociating
100 μ l, room temperature is shaken 5 minutes.
The most again reaction cup is placed in permanent magnetism bracket upper 30 second, in darkroom after magnetic particle precipitates completely
In on following device measure Up-conversion Intensity (each sample determination 1 second).
Excitation source: miniature high energy semiconductor laser AL808T300 (launches wavelength 808 ± 5nm),
Produced by Guangzhou Lv Gao company；
Detector: double alkali photocathode photomultiplier tube R1450 (response wave length 300～650nm), by day
This Bin Song company produces；
Direct-current plate high-voltage value :+1500V；
Blank signal/thermal noise ratio: 2.5.
By the luminous value (RLU) of each calibration object reaction cup that records and TSH dense in respective alignment product
Degree carries out linear regression and obtains working curve and regression equation, then by this regression equation and example reaction
The luminous value of cup extrapolates the concentration of TSH in sample.The concrete data of calibration object and blood serum sample see below
Table 1, working curve and the regression equation of calibration object are shown in Fig. 1.There it can be seen that the present embodiment is not only
There is excellent methodology characteristic, and its measurement result also with existing commercial luminescence immunoassay system
Highly consistent.
Table 1
Embodiment 2
The up-conversion luminescence labelled immune method of analyzing competitiveness launching ultraviolet using the present invention is quantitatively examined
Survey human serum thyroxine (little molecule hormone and medicine).
1. by thyroxine (T4) (Sigma Co., USA's product) and casein covalent bond form multiple
Compound: use EDC method, EDC is purchased from Thermo Fisher company of the U.S., by product description requirement
Operation.Then use this complex to be coated microwell plate, be coated condition: 0.5 μ g/mL complex adds
20mM/L pH9.6 carbonic acid buffer, every hole adds 100 μ l, and 4 DEG C overnight；Discard liquid, add same
Volume 1% (envelope-bulk to weight ratio) casein room temperature is shaken 2 hours, abandons liquid standby.
Up-conversion luminescence material (Y2O3:Yb3+,Tm3+) nanoparticle (foundation Li Tanggang etc., Y2O3:Yb3+,
Tm3+Nano material visible and ultraviolet conversion luminous, Acta Physica Sinica, 2011,60:(7) 073201-6
Synthesis) tie with a strain antithyroidin monoclonal antibody (purchased from Medix biochemica company of Finland)
Close and form label: 10mg nano-particle and 1mg antibody add 10ml 200mM/L pH7.2 phosphoric acid
Buffer, concussion addition 0.1% (envelope-bulk to weight ratio) glutaraldehyde overnight, is centrifuged and abandons supernatant with physiology salt
It is standby that water cleans particulate matter.
2. each hole in described microwell plate is separately added in the step 1 of each 50 μ l the label prepared
And comprise variable concentrations T4Calibration object, this calibration object is by T4Sterling is pressed variable concentrations and is added without T4
Human serum is made；The label and human blood prepared in the step 1 of each 50 μ l is added in other hole
Clear clinical sample is (through existing commercial luminescence immunoassay system measurement, its T4Concentration is respectively 46.3
And 150.9ng/ml).Microwell plate is at room temperature shaken 1 hour.
3., after pouring out above-mentioned reactant liquor, rinse each hole five with cleaning mixture (PBS+0.05% tween 20)
Secondary, dry liquid.
4. agent (0.3N potassium hydroxide+60% dimethylformamide) of dissociating is added to each hole of microwell plate
100 μ l, at room temperature concussion measures Up-conversion Intensity after 5 minutes in darkroom on following device
(each sample determination 1 second).
Excitation source: miniature high energy semiconductor laser AL808T300 (launches wavelength 980 ± 5nm),
Produced by Guangzhou Lv Gao company；
Detector: solar blind light cathode luminous multiplier tube R1080 (response wave length 115～320nm), by day
This Bin Song company produces；
Direct-current plate high-voltage value :+1000V；
Blank signal/thermal noise ratio: 2.3.
By the luminous value (RLU) of each calibration sample wells recorded and T in respective alignment product4Concentration carry out
Linear regression obtains working curve and regression equation, then by this regression equation and the luminous value of sample well
Extrapolate T in sample4Concentration.The concrete data of calibration object and blood serum sample see table 2, calibration object
Working curve and regression equation are shown in Fig. 2.There it can be seen that the present embodiment not only has excellent method
Learn characteristic, and its measurement result is also highly consistent with existing commercial luminescence immunoassay system.
Table 2
Embodiment 3
Use the up-conversion luminescence marking clip core type oligonucleotide molecules hybridization launching ultraviolet of the present invention
Legal detection people's sputum tubercule bacillus 16s RNA (nucleic acid fragment).
1. synthetic two is respectively provided with biotin and the bacillus tuberculosis typus humanus 16s of digoxin latter end labelling
RNA specific oligonucleotide probe 5`-GGT GGA AAG CGC TTT AGC GGT-3`-Biotin and
Dig-5`-GCC CGT ATC GCC CGC ACG CTC ACA-3` (is closed by Sybersyn company of the U.S.
Become).
2. biotin (Sigma Co., USA's product) is coated microwell plate, is coated condition: 20 μ g/mL
Biotin adds 20mM/L pH9.6 carbonic acid buffer, and every hole adds 100 μ l, and 4 DEG C overnight；Discard liquid
Body, adds same volume 1% (envelope-bulk to weight ratio) casein room temperature and shakes 2 hours, abandon liquid standby.
Up-conversion luminescence material (Y2O3:Yb3+,Tm3+) nanoparticle (foundation Li Tanggang etc., Y2O3:Yb3+,
Tm3+Nano material visible and ultraviolet conversion luminous, Acta Physica Sinica, 2011,60:(7) 073201-6
Synthesis) combine formation label: 10mg with a strain anti digoxin antibody (Sigma Co., USA's product)
Nano-particle and 1mg antibody add 10ml 200mM/L pH7.2 phosphate buffer, shake addition 0.1%
(envelope-bulk to weight ratio) glutaraldehyde overnight, centrifugal abandoned supernatant to clean particulate matter with normal saline standby.
Extract normal person and Sputum smears doubtful tubercule bacillus positive patients sputum specimen and various mark the most respectively
The RNA of quasi-bacterial strain (pharmaceutical biological product identification research institute of China provides) (uses U.S. invitrogen
The trizolRNA of company extracts test kit, operates by product description requirement).
4. each hole in described microwell plate is separately added in 30 μ l steps 2 label prepared；30μl
The normal person expectorant RNA prepared in step 3, or smear doubtful tubercule bacillus positive patients sputum specimen
RNA, or various reference culture RNA；The few core prepared in the step 1 of 40 μ l is added again to each hole
Thuja acid probe；Microwell plate is shaken 1 hour at 37 DEG C.
5., after pouring out above-mentioned reactant liquor, rinse each hole five with cleaning mixture (PBS+0.05% tween 20)
Secondary, dry liquid.
6. agent (0.3N Lithium hydrate+40% ethanol) the 100 μ l that dissociate are added to each hole of microwell plate,
In darkroom, on following device, Up-conversion Intensity (each sample is measured after shaking 5 minutes under room temperature
Measure 1 second).
Excitation source: miniature high energy semiconductor laser AL808T300 (launches wavelength 980 ± 5nm),
Produced by Guangzhou Lv Gao company；
Detector: solar blind light cathode luminous multiplier tube R1080 (response wave length 115～320nm), by day
This Bin Song company produces；
Direct-current plate high-voltage value :+1000V；
Blank signal/thermal noise ratio: 2.6.
The luminous value (RLU) in each hole recorded is the positive higher than negative control (normal person) 2.1 times.
Concrete data see table 3.There it can be seen that the present embodiment can not only be from various gene approximation bacterial strain
Accurately tell bacillus tuberculosis typus humanus, and its measurement result also with existing routine clinical inspection technology height one
Cause.
Table 3
Specific description of embodiments of the present invention above is not limiting as the present invention, and those skilled in the art can
To be variously modified according to the present invention or to deform, without departing from the spirit of the present invention, all should be belonged to this
Invention scope of the following claims.
Claims (16)
1. a method based on up-conversion luminescence material detection biomolecule, it is characterised in that described
Method comprises the following steps:
1) make biomolecule to be detected and label specific bond to form solid-phase complex, described labelling
Thing uses up-conversion luminescence material to prepare, and then removes unconjugated unnecessary label；
2) to step 1) reaction system in add and dissociate agent, so that described up-conversion luminescence material solution
From to liquid phase；
3) detecting step 2) reaction system described in the existence of up-conversion luminescence material or its amount.
Method the most according to claim 1, it is characterised in that step 1) be combined in solid phase
Carrying out, described biomolecule to be detected is albumen and derivant, little molecule hormone and medicine or nucleic acid sheet
Section；Wherein, described up-conversion luminescence material be inorganic rare earth ion complex class up-conversion luminescence material or
Asymmetric organic compound species up-conversion luminescence material.
Method the most according to claim 2, it is characterised in that described up-conversion luminescence material is
Y2O3:Yb3+,Tm3+Or trans-4-(dimethyl amido)-4`-(diethyl amido) stilbene.
Method the most according to claim 1 and 2, it is characterised in that step 2) dissociate at liquid
Carry out in mutually, described in agent of dissociating be the aqueous solution comprising alkali metal hydroxide and organic solvent；Wherein,
One or more in sodium hydroxide, potassium hydroxide, Lithium hydrate of described alkali metal hydroxide,
Concentration is 0.05N-5.0N；Described organic solvent is selected from dimethyl sulfoxide, dimethylformamide, ethanol
One or more, with volume percent, concentration is 10%-95%.
Method the most according to claim 4, it is characterised in that described alkali metal hydroxide
Concentration is 0.1N-1.0N.
Method the most according to claim 4, it is characterised in that the concentration of described organic solvent is
30%-90%.
The most according to the method in any one of claims 1 to 3, it is characterised in that described step 3)
Be detected as use excitation source to excite described up-conversion luminescence material, then use photoelectric detector detection
It launches light.
Method the most according to claim 7, it is characterised in that in described step 3) in, described
Excitation source is single color light emitting devices, and uses the photoelectricity possessing exciting light insensitive material photocathode simultaneously
Multiplier tube.
Method the most according to claim 8, it is characterised in that described step 3) also include passing through
The ratio optimizing blank signal and photomultiplier tube thermal noise improves detection signal-to-noise ratio.
Method the most according to claim 8 or claim 9, it is characterised in that described single color light emitting devices
The low-power semiconductor laser identical or close with up-conversion luminescence material excitation wavelength for launching wavelength
Or light emitting diode.
11. methods according to claim 8 or claim 9, it is characterised in that for VISIBLE LIGHT EMISSION
For up-conversion luminescence label, possesses the photomultiplier tube of exciting light insensitive material photocathode for red
Double base material photocathode photomultiplier tubes that outer light is insensitive；Or
For the up-conversion luminescence label that ultraviolet is launched, possesses exciting light insensitive material time
The photomultiplier tube of pole is material photocathode photomultiplier transit blind to the day that infrared light and visible ray are the most insensitive
Pipe.
12. methods according to claim 9, it is characterised in that described optimization blank signal and light
The ratio of electricity multiplier tube thermal noise is, after adjusting photomultiplier tube anode output end preamplifier
The threshold value of comparator, make blank signal be photomultiplier tube self thermal noise 1-5 times.
13. methods according to claim 12, it is characterised in that described optimization blank signal with
The ratio of photomultiplier tube thermal noise is, after adjusting photomultiplier tube anode output end preamplifier
The threshold value of comparator, make blank signal be photomultiplier tube self thermal noise 2-3 times.
14. 1 kinds for implementing the detection system according to the method according to any one of claim 1 to 13
System, it is characterised in that described detecting system includes: excitation source, photoelectric detector and possess exciting light
The photomultiplier tube of insensitive material photocathode.
15. detecting systems according to claim 14, it is characterised in that described excitation source is
Single color light emitting devices, described single color light emitting devices is for launching wavelength and up-conversion luminescence material excitation wave appearance
With or close low-power semiconductor laser or light emitting diode.
16. according to the detecting system described in claims 14 or 15, it is characterised in that for visible ray
For the up-conversion luminescence label launched, possesses the photomultiplier tube of exciting light insensitive material photocathode
For the double base material photocathode photomultiplier tubes insensitive to infrared light；
For the up-conversion luminescence label that ultraviolet is launched, possesses exciting light insensitive material time
The photomultiplier tube of pole is material photocathode photomultiplier transit blind to the day that infrared light and visible ray are the most insensitive
Pipe.
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| CN1877307A (en) * | 2006-06-07 | 2006-12-13 | 中国科学院上海光学精密机械研究所 | One-dimensional multiple detection up-conversion phosphorescence biosensor |
| US7525724B2 (en) * | 2006-03-16 | 2009-04-28 | The University Of Kansas | Laser system for photonic excitation investigation |
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