CN110146475A - A kind of enhanced light Emitting Diode-Induced Fluorescence detection system of Capillary Electrophoresis and its detection method - Google Patents
A kind of enhanced light Emitting Diode-Induced Fluorescence detection system of Capillary Electrophoresis and its detection method Download PDFInfo
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- 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
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Abstract
A kind of enhanced light Emitting Diode-Induced Fluorescence detection system of Capillary Electrophoresis and its detection method are mainly made of components such as capillary column, excitation light source, convex lens, conduction optical fiber, silver-plated concave mirror and photoelectricity times pipe PMT；Silver-plated concave mirror is arranged in capillary detection beneath window；Photomultiplier tube PMT is arranged above capillary detection window, positioned at the surface of silver-plated concave mirror, detects to the transmitting light by the convergence of silver-plated concave mirror, enhanced sample；The output signal that record system receives and handles photomultiplier tube PMT is detected, and recording and displaying is carried out to obtained testing result.It is intended to the light for propagating the transmitting light-of sample caused by excitation light source on different directions by the convergence of silver-plated concave mirror, is converged and is reflected, to enhance the intensity and brightness of reflected light, finally greatly improve detection sensitivity.Under the conditions of same other, the sensitivity of this detection method is 30 times of commonsense method.
Description
Technical field
The present invention relates to trace samplings analysis and detection device, especially enhanced light Emitting Diode-Induced Fluorescence detection system
And its related detecting method.
Background technique
Capillary Electrophoresis (CE) is a kind of efficient separate analytical technique.The separation of analyte by its charge-mass ratio difference height
Piezoelectric field driving.In recent years, Capillary Electrophoresis LED induced fluorescence detection system, at low cost because its is small in size, stability is high, wave
The advantages that long range is big and attracted more and more researchers.It is compared with the traditional method, can be used as testing (ribonucleic acid)
The diversified sample test such as RNA.
CE can match a variety of detectors, and Chinese patent literature is disclosed directly below:
Document 1 " a kind of detector of fluorescence induced by light-emitting diode in high brightness " (CN 200410071057.7): it is by exciting
Light source, lens group, beam splitter, optical filter, electrooptical device and detection cell composition, the detector using phosphor collection optical path and
Ipsilateral and coaxial optical texture of the excitation light path in detection cell.
A kind of document 2 " array light-emitting diode induced fluorescent tester " (CN200510046861.4): the fluorescence detection
Device is mainly by array light-emitting diode, excitation fiber, detection cell, collection optical fiber, optical filter, photoelectric conversion device and signal processing
Circuit composition, detection cell are coupled by optical fiber and other optical components, which uses N number of identical light emitting diode
For excitation light source；Each excitation light source is irradiated in detection cell by 1 excitation fiber, is shared N number of optical fiber while being irradiated to inspection
It surveys in pond；Excitation fiber is fixed in the conplane different loci of detection cell, excitation fiber, collect optical fiber directly and liquid
Flow path contact, liquid flow path, collection optical fiber and excitation fiber three grip intersection altogether.
Document 3 " LED-induced minisize fluorescence detector " (CN200410081011.3): it is by excitation light source, school
The composition such as quasi- assistance platform, optical filter, photoelectric conversion device and detection cell, the detector is using optical fiber as phosphor collection and transmission matchmaker
It is situated between；Excitation light path, optical fiber and capillary detection pond three are mutually perpendicular to, and realize nature calibration by calibration assistance platform.
Document 4 " a kind of detector of fluorescence induced by light-emitting diode of close contact excitation light path "
(CN201110393217.X): it uses close contact structure, abandons making light-emitting diodes using any condenser lens and excitation fiber
The outer wall of pipe exciter filter, limit light beam aperture and capillary column protective layer is successively snugly into each other.Light emitting diode and detection window
The distance between it is smaller, higher light intensity can be obtained.
Above-mentioned document 1~4 inherently belongs to CE-LEDIF detection system, and still, it is to be improved that there are excitation intensities,
The lower problem of detection sensitivity.
Summary of the invention
In order to solve the problems in the prior art, the purpose of the present invention is to and provide a kind of Capillary Electrophoresis it is enhanced shine two
Pole pipe induced fluorescence detection system, it is intended to by the high-accuracy efficient convergence effect for calculating silver-plated concave mirror of optics, by exciting light
The transmitting light for the sample that source is excited efficiently is collected, is converged, strength reflects, thus enhance the intensity and brightness of reflected light,
Finally greatly improve detection sensitivity.
The object of the present invention is achieved like this: a kind of enhanced light Emitting Diode-Induced Fluorescence detection system of Capillary Electrophoresis
System is mainly made of capillary column, excitation light source, convex lens, conduction optical fiber, silver-plated concave mirror and photoelectricity times pipe PMT；
Capillary column: as the fluorescent material or fluorescence labeling material for injecting detection to be analyzed, outer wall is equipped with detection
Window；
Excitation light source: it is made of light emitting diode (LED), alternatively, by laser constitution；The maximum wavelength of its exciting light
Selection is determined by the labeling dye of tested substance or the excitation-emission attribute of probe；
Convex lens: receive by excitation light source come exciting light be focused, matching low pass filter wavelength be 500nm；
Conduction optical fiber: it outside capillary column or protrudes into capillary column, the exciting light after convex lens is focused leads to hair
The detection window of capillary column；
Silver-plated concave mirror: it is arranged in capillary detection beneath window, the top surface of silver-plated concave mirror is parallel to capillary axle center
Line, and the focus of silver-plated concave mirror is located on capillary axis；
Photomultiplier tube PMT: it is arranged above capillary detection window, positioned at the surface of silver-plated concave mirror, to by plating
The transmitting light for the sample that silver-colored concave mirror comes is detected；
It detects record system: receiving and handle the output signal of photomultiplier tube PMT, and obtained testing result is carried out
Recording and displaying.
The diameter of the silver-plated concave mirror: 4mm；Thickness: 3mm；Radius of curvature: 2.46mm；Focal length: 1.23mm；Finish:
60。
The conduction optical fiber is to the excitation light path of capillary detection window and the phosphor collection axial line of capillary column at 30 °
~45 ° of inclination angles, but it is not limited to the angle.
The fluorescent material or fluorescence labeling material sample introduction height of the detection to be analyzed are 20cm, and sample injection time is 7 seconds, but
It is not limited to the input mode.
Compared with prior art, the invention has the characteristics that and advantage:
1. the present invention cleverly devises one piece of silver-plated concave mirror at capillary detection the window's position of face PMT.When
When the analyte particles of fluorescent marker push detection window to by electroosmotic flow, emit under the excitation of exciting light.At this point, analyte can
It is considered as illuminating source point.When phosphor dot is moved to the focus of concave mirror, which, which shines, is collected by concave mirror, reflects together
After Shi Zengqiang transmitting, then detected by PMT, so that CELEDIF detection sensitivity be greatly enhanced.
2. the radius of curvature R that silver-plated concave mirror has been determined through theory deduction and repetition test is 2.46nm, focal length is
1.23nm and other specific sizes.Using silver-plated concave mirror, makes to focus and the ability of reflected light is far longer than direct reflection
Or plane mirror radiation pattern.
3. due to the reflectivity of silverskin in silver-plated concave mirror influenced by incidence angle it is smaller, compared to golden film, copper film and
For aluminium film, silverskin has highest reflectivity in far ultraviolet and visible light region, and reflectivity is up to 97%.
4. a simple Experiment of Analytical Chemistry is arranged, two kinds of tumor markers are detected, for verifying the invention patent institute
The enhanced LEDIF sensitivity detected referred to, it is other it is consistent in the case where.
(low pass is matched as excitation light source, and using 510nm as the LED of the 480nm wavelength used selected in detection
Optical filter wavelength focuses convex lens for 500mm) and carrys out filter background light, is intended merely to that noise is effectively reduced, effectively reduces external
Interference of the factor to detection.
The optimization of CE separation condition
The best deposition condition of the L-Leu and Valine with FITC label has been determined through many experiments are as follows: by
The borate buffer solution of the PH9.2 of 12%v/vACN (acetonitrile), 20mM β-CD (beta-cyclodextrin) and 20mM sodium tetraborate composition,
The separation voltage of ACN is 12kV, gravity sample introduction height 20cm, continues 7s.
The present invention cleverly devises one piece of silver-plated concave mirror at capillary detection the window's position of face PMT.When glimmering
When the analyte particles of signal push detection window to by electroosmotic flow, emit under the excitation of exciting light.At this point, analyte is visual
For illuminating source point.When phosphor dot is moved to the focus of concave mirror, which, which shines, is collected by concave mirror, reflects simultaneously
After enhancing transmitting, then detected by PMT, to improve detection sensitivity.Light source of the present invention is induced using simplest LED
Fluorescence detecting system (LEDIF).Result of study shows that using the sensitivity of present invention enhancing detection method be common detecting methods
30 times.In addition, the present invention cannot be only used for the research and development of the enhanced LEDIF detection system of Capillary Electrophoresis, in capillary
Research, the exploitations of novel high-sensitivities detector such as the other fluorescence detection methods of electrophoresis tube etc. all have potential significance.
Detailed description of the invention
Fig. 1 is present system composition schematic diagram.
Fig. 1 a is the enlarged drawing of K portion shown in Fig. 1.
Fig. 2 is the fluorescence excitation and emission spectra of FITC-L-leu and FITC-L-val.
Fig. 2 a be silver-plated concave mirror cross-sectional view (Fig. 2 a is cross-sectional view, shows concave mirror thickness capillary tube center distance concave surface
The span of mirror upper end, concave mirror diameter etc.)；
Fig. 2 b shows the focal length and radius of curvature of concave mirror；Fig. 2 c is concave mirror light optical path mechanism figure；Fig. 2 d is recessed
Face mirror top view.
Fig. 3 is the polarization measurement data for plating silver coating.
(it is 20mM borate that A represents running buffer to the electrophoretogram for the influence that Fig. 4 is separated in pairs for the group of back-ground electolyte
Solution, it is the 20mM borate solution containing 12% (v/v) ACN that B, which represents running buffer, C represent running buffer be containing
The 20mM borate solution of 12% (v/v) ACN and 20mM β-CD, other deposition conditions: pH9.2, apply voltage 12kV, gravity into
Sample is in 20cm height sample introduction 7s).
Fig. 5 a, Fig. 5 b are respectively electrophoretogram (wherein, Fig. 5 a, that is, A use of two kinds of tumor cell markers of FITC label
CSM-LEDIF, B use LEDIF, and deposition condition is equal are as follows: in the 20mM borate buffer solution of pH 9.2 containing 20mM β-CD and
The voltage of 12% (v/v) ACN is 12kV, gravity sample introduction height 20cm, continues 7s).
Specific embodiment
Referring to Fig. 1, Fig. 1 a, a kind of enhanced light Emitting Diode-Induced Fluorescence detection system of Capillary Electrophoresis, mainly by hair
Capillary column, excitation light source, convex lens, conduction optical fiber, silver-plated concave mirror and photoelectricity times pipe PMT composition；
Design concave surface silver mirror construction: it is accurately calculated by optical theory, sets and be suitble to the optimal recessed of Detection wavelength range
The radius of curvature of face mirror designs the concave mirror for being suitble to capillary column detection window to match thickness；Then concave mirror material is selected, is plated
Silver.
Concave surface silver mirror and capillary matched design, high-accuracy modularization assembling: outer diameter, internal diameter in conjunction with capillary, and
It is coupled at the focus (calculating design by accurate) of concave mirror with capillary central axis precision.
Silver-plated concave mirror: it is arranged in capillary detection beneath window, the top surface of silver-plated concave mirror is parallel to capillary axle center
Line, and the focus of silver-plated concave mirror is located on capillary axis line；
Capillary column: as the fluorescent material or fluorescence labeling material for separating detection to be analyzed, outer wall is equipped with detection
Window；
Excitation light source: it is made of light emitting diode (LED), alternatively, by laser constitution；The maximum wavelength of its exciting light
Selection is determined by the labeling dye of tested substance or the excitation-emission attribute of probe；
Convex lens: receive by excitation light source come exciting light be focused, matching low pass filter wavelength be 500nm；
Conduction optical fiber: it outside capillary column or protrudes into capillary column, the exciting light after convex lens is focused leads to hair
The detection window of capillary column；
Silver-plated concave mirror: it is arranged in capillary detection beneath window, the top surface of silver-plated concave mirror is parallel to capillary axle center
Line, and the focus of silver-plated concave mirror is located on capillary axis；
Photomultiplier tube PMT: it is arranged above capillary detection window, positioned at the surface of silver-plated concave mirror, to by plating
The transmitting light for the sample that silver-colored concave mirror comes is detected；
It detects record system: receiving and handle the output signal of photomultiplier tube PMT, and obtained testing result is carried out
Recording and displaying.
But according to different detection system support constructions, for other thickness parameters, radius of curvature parameter, focal length parameter
With claim.It can be used for detecting trace, the fluorescent material of ultra trace or fluorescence labeling material, do not limit target detected
Object.
Optical fiber is conducted to the excitation light path of capillary detection window and the phosphor collection circuit axial line of capillary column into 30 °
~45 ° of inclination angles.
Focus point of the present invention is matched concave surface silver mirror at detection window, the i.e. silver-plated detection window in concave surface and column infrared optical fiber
Light Emitting Diode-Induced Fluorescence detects (CSM-LEDIF).When analyte is moved to the focus of concave mirror, analyte is excited
Light excitation.The light of transmitting is collected by concave mirror immediately, is reflected while enhancing transmitting, is then detected by PMT, to mention
High detection sensitivity.As far as we know.The optics mechanism of present invention research raising detection sensitivity；Additionally set up one group it is simple
The experiment of contrast sensitivity degree, by the detection sensitivity of the tumor markers of two kinds of FITC label use respectively new detection system and
Most traditional orthogonal light path detection method compares.Experiment show it is contemplated that feasibility.
2. experimental section
2.1. chemicals and material
Analyte L-Leu (L-leu, 99%) and Valine (L-val, 99%) purchased from change summer reagent (China at
All).FITC (> 98%), beta-cyclodextrin (β-CD, 98%) and anhydrous sodium tetraborate are purchased from source leaf biology Co., Ltd (in China
Sea).Acetonitrile (ACN, 99.9%) is purchased from Aladdin reagent (Chinese Shanghai).Every other chemicals used in this work and have
Solvent is analysis level or more.Use borate buffer solution (20mM) hydrochloric acid (1M HCl) and the hydroxide of preceding preparation
Sodium (1M NaOH) adjusts pH to 9.2.In entire work using the Milli-Q water of resistivity 18M Ω/cm (Millipore,
Bedford, MA, USA).Before injection separates capillary, all solution are passed through into 0.45 μm of membrane filter filtering.
Concave mirror has parameter: diameter: 4mm made to order；Thickness: 3mm；Radius of curvature: 2.46mm；Focal length: 1.23mm；Finish:
60；It is eccentric: < 3'；Effective aperture: > 90%；Reflectivity: > 99.5%；Thickness deviation: ± 0.1mm；Coating: silver-plated.
2.2. instrument
The design philosophy of the experiment is to compare detection sensitivity by two test boards.There is no the detection device of concave mirror such as
Shown in background technology part document report:
F.Huo,R.Guijt,D.Xiao,M.C.Breadmore,Dual wavelength excitation
fluorescence detector for capillary electrophoresis using a pulsed bi-colour
light emitting diode,The Analyst 136(2011) 2234-2241.
F.Huo,H.Yuan,M.C.Breadmore,D.Xiao,Multi-wavelength light emitting
diode array as an excitation source for light emitting diode-induced
fluorescence detection in capillary electrophoresis,Electrophoresis 31(2010)
2589-2595.
F.Huo,H.Yuan,X.Yang,M.C.Breadmore,D.Xiao,Pulsed multi-wavelength
excitation using fiber-in-capillary light emitting diode induced fluorescence
detection in capillary electrophoresis,Talanta 83(2010)521-526.
There is the present invention detection device of concave surface silver mirror to be shown in FIG. 1.Here, our purposes be explore it is same other
Under part, only compares installation concave surface silver mirror and concave mirror, the comparative study of detection sensitivity are not installed.As schemed shown in c, the experiment
It is not converged closely using the accurate coupling of such as self-focusing lens etc, but uses simple LED (480nm) directly logical
Cross then inspection that exciting light is introduced capillary outer wall by common convex lens convergence (d is walked in Fig. 1) by optical fiber (e is walked in Fig. 1)
Survey window.Exciting light passes through capillary detection window and excites sample, and convergence is simultaneously by concave mirror (a is walked in Fig. 1) for the fluorescence of transmitting
Then strong reflection is received by PMT (portion g in Fig. 1), to obtain highly sensitive detection (h is walked in Fig. 1).In fig 1 a, b is recessed
The convergence of face silver mirror a, the enlarged drawing for shining, reflecting signal of reflected sample.
Fig. 1 is shown, the enhanced Capillary Electrophoresis LEDIF detecting system schematic diagram of the present invention.A is concave mirror in figure；C is
LED；D is common convex lens；E is optical fiber；G is photomultiplier tube PMT；H is highly sensitive detection record system (prior art).
2.3. the derivatization of standard
The stock solution of L-leu and L-val (9.6mM) are prepared in 0.1M HCl, and FITC is prepared in ACN
(4.3mM, i.e. 4.3m mol/l, molar concentration, similarly hereinafter).Then they are stored in refrigerator (- 20 DEG C), and are using preceding use
Runtime buffer solution is further diluted to required concentration.
Before derivatization, by FITC, the dilution in 20mM borate buffer solution (pH 9.2), obtains 2.15mM solution.It will
Above-mentioned solution is mixed with the standard stock solution of 10 μ L L-leu and 10 μ L L-val to the liquor capacity of 1mL.By the mixture black
It incubates 15 hours at room temperature in the dark.That is, standard solution dilutes 100 times respectively.The FITC solution in bottle is added
The concentration of (in ACN) is greater than 20 times of L-leu and L-val, to obtain complete and thorough reaction, but generates simultaneously less
By-product.Next, the AAs of label is maintained in 4 DEG C of dark using preceding.Work is diluted with running buffer before the injection
Make solution, and fresh preparation daily.
2.4.CE process
By continuously use 1.0M NaOH rinse 30 minutes, 0.1M NaOH 30 minutes, water 30 minutes, finally for the first time
New capillary is activated using preceding running buffer 10 minutes.In daily start, the effective 1M NaOH pretreatment 30 of capillary
Minute, deionized water pre-processes 30 minutes, and running buffer pre-processes 10 minutes.Between continuous injection twice, 1M is successively used
NaOH, water and running buffer rinse capillary 5 minutes.It is injected a sample into capillary by fluid dynamics injection,
It is carried out 7 seconds at the height of 20cm, but is not limited to the input mode.Blue led excitation wavelength is the fluorescence of 488nm.Using by containing
There are the 20mM of 12%ACN (v/v) and 20mM β-CD, the operation borate buffer solution of 9.2 borate of pH composition is under 12kV voltage
It is separated.All CE programs carry out at room temperature.
3. results and discussion
3.1. LED is selected
Suitable excitation light source plays a crucial role the detection sensitivity of LED-IFD detector.Fig. 1 is shown
The fluorescence excitation and emission spectra of L-Leu derived from FITC (FITC-L-leu) and Valine (FITC-L-val).
FITC-L-leu and FITC-L-val shows hyperfluorescence, and excitation maximum is about 498nm, emission maximum 528nm,
Stokes shift is 30nm.If using the LED with broader spectrum bandwidth and maximum emission wavelength 498nm, fluorescence hair
Penetrating will be Chong Die with about 35% exciting light, this will lead to strong background noise.We select to use 480nm wave in entire experiment
Long LED carrys out filter background light as excitaton source, and using 500nm optical filter (i.e. focus lens group) to reduce noise.LED
Half-wavelength be only several nanometers, therefore will not interference experiment.
3.2 explore the Optical Mechanism of enhancing detection sensitivity
Because concave mirror has convergent effect to light, if point light source is located at the focal point of concave mirror, what light source issued
Light can be used as directional light reflection.For example, the headlight high beam filament of automobile is exactly the focus positioned at its concave mirror.The convergence of concave mirror is imitated
The light propagated on different directions should can be concentrated on, so that it propagates in the same direction, emits in parallel, and brightness increases
By force, irradiate it on the object at far place, therefore intensity of reflected light much larger than no mirror surface or uses the reflection of plane mirror
Luminous intensity.
In addition, in order to reach the optimum efficiency of concave mirror convergence reflected light, according to the outer diameter and inner diameter of capillary used, with
And design thickness and the parametric synthesis such as height of detection plate, it is assumed that analyte fluorescent emission point is located at the center of capillary inner diameter,
The radius of curvature of concave mirror can be accurately calculated:
1/u+1/v=1/f=2/R (1)
(1) formula is concave mirror imaging formula.U, from object to the distance on concave mirror vertex；V, from imaging to concave surface vertex
The distance of point；F, the focal length of concave mirror；R, the radius of curvature of concave mirror, the relationship between the focal length and radius of curvature of concave mirror are
F=R/2.If be overlapped by the picture that the object in experiment is formed with object, v=u, and formula (1) becomes 2/v=1/fv
=2f, to obtain v=u=2f=R.
(2) when measuring image distance (or object distance) at this time, radius of curvature R is obtained, therefore focal length f can be calculated.It is this
Method is known as object and as coincidence method.In general, inconsistent with object by the picture that object is formed, it is clear to obtain to change object distance and image distance
Image, i.e. focal length can be obtained by (1).
As shown in Fig. 2 (a-d), best design: radius of curvature R 2.46mm, the focal length of concave mirror are 1.23mm.Root
Upper theory accordingly, other than LEDIF detection method, other optical detecting methods of Capillary Electrophoresis, as long as in the same terms
Under, using concave mirror, focuses and the ability of reflected light is all far longer than directly transmitting light or plane mirror reflection mode.
The size of the customized concave mirror of Fig. 2, texture map: a is concave mirror thickness, cross-sectional view, on capillary tube center distance concave mirror
The span at end, concave mirror diameter etc.；The focal length and radius of curvature of b concave mirror；C is concave mirror light optical path mechanism figure；D is recessed
Face mirror is gold-plated, top view.
According to the emission spectrum range of sample, the position for combining luminous point in the shape feature and capillary of capillary is considered
It sets.Since the reflectivity of silverskin reflective concave surface mirror is influenced by incidence angle smaller, silverskin is in far ultraviolet and visible light region
With highest reflectivity.In order to reach the best reflection efficiency of concave mirror, we are gold-plated using the silver-plated replacement of concave mirror inner membrance,
It copper facing and aluminizes.The polarization effect of silver coating material with high reflectance (reflectivity ≈ 97%) is as shown in figure 3, it is perpendicular to
The reflectivity (S-polarization) of the polarised light of film deposition plane is parallel to the polarised light (P polarization) and non-polarized light of film deposition plane
The reflectivity of (unpolarized).Within the scope of the launch wavelength needed for test experience sample, polarization measurement under these three modes
Reflection efficiency is 94.6-97.8%, and variation range is small, to obtain optimal concave surface silver mirror reflecting effect.
The optimization of 3.3 CE separation conditions
Potential systematic error is introduced peak area reading by the peak integral that part is total to migration analysis object, therefore allows all mesh
It is indispensable for marking the separation condition of the baseline separation of compound.Isolated purpose be (i) all target compounds (L-leu,
L-val and FITC) baseline separation, and (ii) separation have good stability.Based on above-mentioned purpose, briefly has studied and use FITC
The separation condition of two kinds of amino acid of label.FITC shows hyperfluorescence under alkaline condition, therefore in our current research, for it
His condition, pH value are fixed as 9.2.It is contemplated that increasing separation voltage as much as possible under suitable electric current to reduce Joule heat simultaneously
Shorten transit time, and determines that separation voltage is 12kV.With this condition, the composition of back-ground electolyte (BGE) is explored.We
20mM borate buffer solution is used not reach baseline separation as BGE, the L-leu and L-val of FITC label, such as scheme first
Shown in 4 (A).Then it uses the 20mM borate containing 12%ACN to be separated, is not reached yet although being improved separating effect
To baseline separation, as shown in Fig. 4 (B).As shown in Fig. 4 (C), when in the BGE composition in 20mM borate include 12%ACN
When with 20mM β-CD, the L-leu and L-val of FITC, FITC label are kept completely separate.This can be by the fact that explain: one
Aspect, ACN can reduce EOF, while reduce the viscosity and dielectric constant of BGE.On the other hand, FITC-L-leu and FITC-L-
Val can occupy β-CD based on their space structure, electrostatic interaction and Van der Waals force and β-CD to varying degrees and dredge
Water cavity, to promote their separation.In short, the best CE condition of two amino acid for separating FITC label is 12%v/
The application voltage of the BGE and 12kV of v ACN, 20mM β-CD and 20mM borate (pH 9.2).
The electrophoretogram for the influence that the group of Fig. 4 back-ground electolyte separates in pairs.(A) running buffer is that 20mM borate is molten
Liquid, (B) running buffer are the 20mM sodium tetraborate decahydrate solution containing 12% (v/v) ACN, (C) running buffer be containing
The 20mM sodium tetraborate decahydrate solution of 12% (v/v) ACN and 20mM β-CD, other deposition conditions are identical: pH9.2, capillary light beam both ends
Apply voltage (i.e. electrophoretic voltage) 12kV, gravity is infused in 20cm height sample introduction 7s.
3.4 with the remolding sensitivity of ECFO-LED-IFD-CE compared with
Under the above-mentioned condition obtained by simple optimization, we are passed through respectively with the derivatization sample of three kinds of various concentrations
CSM-LEDIF system and LED-IFD system are detected, the electrophoretogram 5A-5B of acquisition.And the result of acquisition is compared.
For two kinds of tumor markers, limited using the detection that the CSM-LEDIF system that concentration is 96 μM reaches software.In same concentrations
Under, detection of the LED-IF system to the detection of two kinds of tumor markers far below software limits.Obviously, newly developed using us
The sensitivity of CSM-LEDIF system is significantly larger than routine LED-IFD, and transit time and resolution ratio have almost no change.Through
Calculating is crossed, the peak height signal difference between two systems is about 30 times, this shows that concave surface silver mirror can be by undetected transmitting
Light enhances and is reflected into PMT, greatly improves detection sensitivity.
The electrophoretogram of two kinds of tumor cell markers of Fig. 5 .FITC label uses (A) CSM-LEDIF (B) LEDIF respectively,
Deposition condition: the voltage in the 20mM borate buffer solution of pH 9.2 containing 20mM β-CD and 12% (v/v) ACN is 12kV, weight
Power sample introduction height 20cm continues 7s.
3.4 analysis performance characteristics
Have checked the linear of two systems, detection limit (LOD) etc., further to study their detection performance.Passing through will
Corresponding peak area and the function of analyte normal concentration establish regression equation.LOD and LOQ is the letter for generating 3 and 10 respectively
The minimum analyte concentration made an uproar than (S/N).Meanwhile comparing the detection performance of two systems.It is being listed in table 1 the result shows that
CSM-LEDIF-CE has and LOQ LOD more lower than LEDIF-CE.As a result further prove silver mirror prevent it is undetected glimmering
Light penetration-detection window simultaneously plays a decisive role fluorescent reflection into PMT to be detected.A bit of words are deleted.In addition,
The reproducibility of CSM-LEDIF-CE system and LEDIF-CE system is judged by transit time and corresponding peak height.The experiment is daily
Six times are repeated to carry out withinday precision test, and carries out precision in several days test for three days on end.Using transit time and accordingly
The percentage of the relative standard deviation (RSD%) of peak height characterizes precision.From table 1 it follows that with CSM-LEDIF-CE and
The transit time and corresponding peak height that LEDIF-CE is obtained are respectively smaller than 1% and 3.3%.It should be the result shows that two kinds of systems be all available
In two kinds of AA of detection, and CSM-LEDIF-CE system ratio LEDIF-CE system is even better.
The recurrence side for the FITC marked tumor marker that table 1 is obtained by CSM-LEDIF-CE system and LEDIF-CE system
Journey and characteristic parameter
Y: the peak area (μ V*s) of corresponding amino acid, x: corresponding amino acid concentration (μM).
A) six repetitions are continuously measured in one day.
B) it is repeated three times in three days.
Finally, if use pulse LED instead, and after the focusing of tight self-focusing lens, using highly sensitive LEDIF or LIF,
The sensitivity that whole detection system may be improved, rather than just 30 times.
4. conclusion
In short, we are that LED induced fluorescence capillary electrophoresis (CSM-LEDIF) system is set outside column from physioptial angle
A concave surface silver mirror is counted.The system has sensitivity more higher than LED induced fluorescence outside common column, because concave surface silver mirror converges
And enhance reflection.Enhanced completely by the transmitting light of the sample from 360 DEG C of directions of excitation, is then reflected into
PMT.In addition, by the analysis to physical optics theory and a simple Experiment of Analytical Chemistry is established, test FITC label
Two tumor markers, new detection system is sensitiveer than common detection system to be higher by more than 30 times, sufficiently demonstrates advanced system
Superiority.
Claims (4)
1. a kind of enhanced light Emitting Diode-Induced Fluorescence detection system of Capillary Electrophoresis, which is characterized in that mainly by capillary
Column, excitation light source, convex lens, conduction optical fiber, silver-plated concave mirror and photoelectricity times pipe PMT composition；
Capillary column: as the fluorescent material or fluorescence labeling material for injecting detection to be analyzed, outer wall is equipped with detection window；
Excitation light source: it is made of light emitting diode (LED), alternatively, by laser constitution；The choosing of the maximum wavelength of its exciting light
It selects and is determined by the labeling dye of tested substance or the excitation-emission attribute of probe；
Convex lens: receive by excitation light source come exciting light be focused, matching low pass filter wavelength be 500nm；
Conduction optical fiber: it outside capillary column or protrudes into capillary column, the exciting light after convex lens is focused leads to capillary
The detection window of column；
Silver-plated concave mirror: being arranged in capillary detection beneath window, and the top surface of silver-plated concave mirror is parallel to capillary axial line, and
The focus of silver-plated concave mirror is located on capillary axis；
Photomultiplier tube PMT: it is arranged above capillary detection window, positioned at the surface of silver-plated concave mirror, to by silver-plated recessed
The transmitting light for the sample that face mirror comes is detected；
It detects record system: receiving and handle the output signal of photomultiplier tube PMT, and obtained testing result is recorded
And display.
2. the enhanced light Emitting Diode-Induced Fluorescence detection system of a kind of Capillary Electrophoresis according to claim 1, special
Sign is, the diameter of the silver-plated concave mirror: 4mm;Thickness: 3mm;Radius of curvature: 2.46mm;Focal length: 1.23mm；It is bright and clean
Degree: 60.
3. the enhanced light Emitting Diode-Induced Fluorescence detection system of a kind of Capillary Electrophoresis according to claim 1, special
Sign is that the conduction optical fiber is to the excitation light path of capillary detection window and the phosphor collection axial line of capillary column at 30 °
~45 ° of inclination angles, but it is not limited to the angle.
4. the enhanced light Emitting Diode-Induced Fluorescence detection system of a kind of Capillary Electrophoresis according to claim 1, special
Sign is that the fluorescent material or fluorescence labeling material sample introduction height of the detection to be analyzed are 20cm, and sample injection time is 7 seconds, but
It is not limited to the input mode.
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