CN104749135A - Intermediate infrared spectrometer - Google Patents
Intermediate infrared spectrometer Download PDFInfo
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- CN104749135A CN104749135A CN201510150145.4A CN201510150145A CN104749135A CN 104749135 A CN104749135 A CN 104749135A CN 201510150145 A CN201510150145 A CN 201510150145A CN 104749135 A CN104749135 A CN 104749135A
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Abstract
The invention discloses an intermediate infrared spectrometer for realizing nanosecond time resolution by using pulse warming. The intermediate infrared spectrometer is used for measuring dynamic structure change of protein, and comprises a wideband tunable carbon oxide laser for emitting intermediate infrared detection laser to a to-be-detected protein solution, a nanosecond Q-modulation holmium laser for emitting pumping laser to heat the to-be-detected protein solution, a pumping detection light path system for detecting the strength of the intermediate infrared detection laser penetrating through the to-be-detected protein solution, and a data acquiring system for acquiring the strength data detected by the pumping detection light path system. The intermediate infrared spectrometer disclosed by the invention can be used for accurately measuring instant infrared spectrometry data of protein dynamic structure change and dynamics information of the protein dynamic structure change, the high-precision measurement on the protein secondary protein structure dynamic structure can be realized, and the infrared spectrometry data precision can reach 1*10<-4>OD.
Description
Technical field
The present invention relates to Superfast time resolution middle infrared spectrum fields of measurement, especially relate to a kind of mid-infrared light spectrometer.
Background technology
Protein science be 21 century human sciences with fastest developing speed be also the most popular science.Infrared spectrum technology includes a large amount of structure of matter information due to it, has been widely used in protein science research.At present, the change of the structure of infrared spectrum technology Study on Protein is adopted to mainly contain two fields: the Stable structure change of protein and the dynamic structure change of protein.The Stable structure change of infrared spectrum technology Study on Protein depends on Fourier transform infrared spectrometer (FTIR).It can provide the basic structure information of protein, but cannot provide the dynamic structure information of protein.
In order to the dynamic structure of Study on Protein changes, people have developed reaction-stop method, and the method for temperature-jump method and laser induced chemical reaction is for causing the structure change of protein.Reaction-stop method has the dead time of at least musec order, is difficult to the time resolution reaching nanosecond order.And the method for laser induced chemical reaction, need in protein solution, add the material for laser excitation, have certain damage to protein.And laser pulse temperature-raising method does not almost damage protein because of a coctoprotein solution, become the method compared with tool advantage of induced protein change.Be limited to laser technology, current fast ramp up time differentiates infrared spectrum technology, and the method adopting the 1064nm laser adjusting Q Nd:YAG laser instrument to export to produce the laser of 1900nm through high pressure hydrogen pond produces the impulse source that heavy water heats up more.This method is owing to needing the non-linear process through a Raman frequency shift, and cause the laser of produced 1900nm, optical quality is poor, and spatial jitter is very large, and the long-term working stability of system is also poor.
Summary of the invention
Object of the present invention aims to provide a kind of mid-infrared light spectrometer that temperature-jump can be utilized to realize nanoseconds resolution, can realize the measurement of high-precision secondary protein structure dynamic change.
In order to realize foregoing invention object, the invention provides a kind of mid-infrared light spectrometer, for measuring the dynamic structure change of protein, this mid-infrared light spectrometer comprises: wideband is tunable CO laser, for sending middle infrared acquisition laser to testing protein solution; Nanosecond adjusts Q holmium laser, for sending pumping laser to heat testing protein solution; Pump probe light path system, for detecting the intensity through infrared acquisition laser in after testing protein solution; And data acquisition system (DAS), be used for gathering the data of the intensity detected from pump probe light path system; The pumping laser spot diameter be formed on sample cell that nanosecond adjusts Q holmium laser to send is 2mm.
Further, pump probe light path system comprises the light path part, detector, amplifier and the wave filter that set gradually.
Further, detector is liquid nitrogen refrigerating type high speed mercury-cadmium tellurid detector; Amplifier is DC-100MHz amplifier.
Further, the output wavelength of the tunable CO laser of wideband is 5.2 μm ~ 6.2 μm.
Further, the output pulse width adjusting Q holmium laser is 50ns, and output wavelength is 2080nm.
Further, this mid-infrared light spectrometer also comprises the sample cell for placing testing protein solution, and sample cell has the first area for placing reference heavy water and the second area for placing the heavy aqueous solution containing testing protein solution.
Further, sample cell is rotary structure, and rotates by it pumping laser first area and second area all being received adjust infrared acquisition laser and nanosecond Q holmium laser to send during the tunable CO laser of wideband sends.
Wideband is tunable, and the middle infrared acquisition laser spot diameter be formed on sample cell that CO laser sends is 0.4mm.
Further, data acquisition system (DAS) comprises 8 high-speed collection card, is minimumly distinguished as 0.36mV.
Further, data acquisition system (DAS) is arranged to the down-sampled type collection data of time frame nanosecond to second.
Beneficial effect of the present invention: adopt the nanosecond pulse heating-up time provided by the invention to differentiate mid-infrared light spectrometer, can Measurement accuracy protein dynamic structure change transient state ir data and protein dynamic structure change dynamic information, its ir data precision can reach 1 × 10
-4oD.Wherein, adopt the tunable CO laser of high stability cryogenic flow dynamic formula wideband as detection photodetection Secondary structure information, wide mid-infrared light spectrometry can be realized, cover the absorption of secondary protein structure acid amides I ' completely.Employing nanosecond short pulse adjusts Q holmium laser, directly can carry out the intensification of nanoseconds as pumping laser to the heavy aqueous solution containing protein, has the longitudinal even, evenly horizontal of heavy aqueous solution heating and stablizes the advantages such as heating for a long time.Adopt the pump probe process that pump probe light path system realizes sample, there is easy to operate and system stability, effectively reduce the advantages such as parasitic light.Data acquisition system (DAS) is adopted to realize the high-speed data acquisition of nanoseconds to time second.
According to hereafter by reference to the accompanying drawings to the detailed description of the specific embodiment of the invention, those skilled in the art will understand above-mentioned and other objects, advantage and feature of the present invention more.
Accompanying drawing explanation
Hereinafter describe specific embodiments more of the present invention with reference to the accompanying drawings by way of example, and not by way of limitation in detail.Reference numeral identical in accompanying drawing denotes same or similar parts or part.It should be appreciated by those skilled in the art that these accompanying drawings may not be drawn in proportion.In accompanying drawing:
Fig. 1 differentiates the structured flowchart of mid-infrared light spectrometer for nanosecond pulse heating-up time of adopting in the embodiment of the present invention;
The structural representation of high stability cryogenic flow dynamic formula wideband tunable CO laser of Fig. 2 for adopting in the embodiment of the present invention of the present invention;
Fig. 3 is the structural representation of the optics cavity of the nanosecond short pulse tune Q holmium laser of exemplary embodiments of the present invention;
Fig. 4 is the long-time result schematic diagram adopting down-sampled mode data acquisition in the embodiment of the present invention;
Fig. 5 be when heating up instantaneously in the embodiment of the present invention heavy aqueous solution at 1645cm
-1the absorbance dynamic changing curve schematic diagram at place;
Fig. 6 be when heating up instantaneously in the embodiment of the present invention heavy aqueous solution and containing Cyt C protein heavy aqueous solution at 1645cm
-1the absorbance dynamic changing curve figure at place;
Fig. 7 be in the embodiment of the present invention heavy aqueous solution at 1645cm
-1the dynamic accuracy schematic diagram of the absorbance at place;
Fig. 8 be when heating up instantaneously in the embodiment of the present invention Cyt C protein at 1645cm
-1the absorbance dynamic change schematic diagram at place; And
Fig. 9 is the transient state spectrum schematic diagram of the cromoci heated up instantaneously in the embodiment of the present invention after 2 microseconds.
Embodiment
As shown in Figure 1, the mid-infrared light spectrometer utilizing temperature-jump can realize nanoseconds resolution provided by the invention mainly comprises the tunable CO laser 10 of wideband, nanosecond short pulse tune Q holmium laser 20, pump probe light path system and data acquisition system (DAS) 40.Mid-infrared light spectrometer of the present invention can Measurement accuracy protein dynamic structure change transient state ir data and protein dynamic structure change dynamic information, its ir data precision can reach 1 × 10
-4oD.
Each ingredient of centering infrared spectrometer describes in detail respectively below:
The tunable CO laser of high stability cryogenic flow dynamic formula wideband 10 is for sending middle infrared acquisition laser to testing protein, to detect the secondary structure information of testing protein, wide mid-infrared light spectrometry can be realized, cover the absorption of secondary protein structure completely.Particularly, the mixed gas of the helium containing fixed mixing ratio of CO laser 10 adopts cooled with liquid nitrogen that this wideband is tunable, nitrogen, carbon monoxide and oxygen is as laser medium, and the proportioning of gas regulates according to the difference of required wavelength coverage.As shown in Figure 2, during work, the gas mixed via electronic flow-meter enters quartz ampoule passage.One end of quartz ampoule expanding channels quartz spiral pipe is gas input, and the other end is the outlet side being connected with vacuum pump.Realized the steady flow of gas by electrovalve control and vacuum pump (not shown), during work, air pressure is about 26.5torr.Working gas is cooled by liquid nitrogen.
In detection protein structure change, the infrared laser of detection Protein secondary structure can be directly used in, the quantum cascade laser mainly containing lead salt laser and carbon monoxide (CO) laser instrument and newly go out recently.For lead salt laser and quantum cascade laser, carbon monoxide (CO) laser instrument 10 has wide spectral and exports, can cover completely Secondary structure acid amides I ' with the advantage of infrared indication.
Fig. 2 is the basic structure of the tunable CO laser of high stability cryogenic flow dynamic formula wideband.This wideband is tunable, and CO laser 10 adopts bidirectional high-pressure electric pump as the energisation mode of laser.The concrete structure of high-tension electricity pumping is: laser cavity two ends are anodes, and two anodes share middle negative electrode.Voltage between anode and cathode is about 7KV.One end of laser cavity is the gold-plated concave mirror of r=5m, and one end is Electronic control rotating shutter.Grating combines the gold-plated level crossing placed perpendicular to grating surface and corresponding light path design can be implemented in grating rotation process, and the wavelength variations of Output of laser but the direction of Output of laser do not change.The concave surface gold mirror end mirror of R=5m, for being totally reflected infrared light.This concave surface gold mirror is contained in one can on the mirror holder of electronic fine setting, so that finely tune the optical cavity of laser.
This laser instrument have employed the outgoing mirror of grating as laser instrument dexterously.1 grade of light of grating turns back to laser cavity, and 0 grade of light output, output efficiency is about 10%.In order to make the direction of output light not change, some designs are optically carried out: mirror is vertical places by golden to grating and a plate plane, and the two is fixed on same turntable.Grating and plane gold mirror rotate simultaneously.The deviation in the direction of the light caused by the rotation of grating is compensated by the plane gold mirror therewith rotated, thus ensure that the exit direction of light can not change.In one embodiment of the invention, adopt the beautiful jade steel pipe that can carry out temperature control to ensure long the stablizing of laser chamber.The gas distributing system of laser instrument, grating controls and high voltage control is all realized by labview programmed control electronics element.
Adopt the tunable CO laser 10 of high stability cryogenic flow dynamic formula wideband of the present invention, there is following performance: 1) at 1580cm
-1~ 1980cm
-1export energy in scope and be greater than 10mw; 2) wavelength interval is about 4cm
-1; 3) stability of short time is per mille, illustrates that the Laser output of tunable CO laser 10 is more stable; 4) shake < 20% for a long time, the long-term stability of its working air current and voltage is described.
Nanosecond short pulse tune Q holmium laser 20 can carry out the intensification of nanoseconds to the heavy aqueous solution containing protein as pump light, have and evenly and for a long time the advantages such as heating are stablized to the vertical and horizontal of heavy aqueous solution heating, directly can carry out temperature-jump to heavy water.Fig. 3 is the structural drawing of nanosecond short pulse tune Q Ho laser instrument 20 optics cavity.Laser cavity adopts average cavity configuration, and one end is total reflective mirror 21, to be output efficiency be in one end 20% output coupling mirror 22.Nanosecond, short pulse adjusted the employing of Q holmium laser doped with the Cr of 1.5%Cr, 5.8%Tm, 0.35%Ho element, and Tm, Ho:YAG crystal is as laser crystal 23, and flashlamp 24 is as pumping source.Electro-optic Q-switched crystal is: La
3ga
5siO
14(LGS) crystal 25, adopts and moves back pressure type tune Q mode of operation.The polarizer is a pair aluminium oxide (Al that Brewster angle is placed
2o
3) crystal 26, modulate Depolarization with λ/4 quartz wave-plate 27.The power supply (all without illustrating in the two figure) that flash generator and adjusting Q crystal adopt all derives from business procurement.The high-power water-cooling machine (not shown) that the water-cooled of laser crystal is 0.1 degree by temperature-controlled precision realizes, and during work, water cooling unit water-cooled temperature is 9 DEG C.Adopt water-cooling pattern mainly because laser crystal 23 gain of nanosecond short pulse tune Q Ho laser instrument is lower, pumping that must be larger could realize Laser output, when laser crystal 23 works, temperature is higher, and easily forms heat accumulation, accurately must control its temperature and ensure the stable of laser work.In one embodiment of the invention, the serviceability of nanosecond short pulse tune Q Ho laser instrument 20 is as follows: frequency of operation: 3Hz, output wavelength 2080nm, output pulse width: 50ns, exports energy: 20mj, energy stability: be about 1%.
In an exemplary embodiments of the present invention, also comprise the sample cell 50 for placing testing protein.This sample cell can adopt the structure of the two sample in a pond.That is sample cell can have two regions, for place reference heavy water first area 51 and for placing the second area 52 containing the heavy aqueous solution of testing protein.Sample cell is preferably rotary structure, the pumping laser that first area 51 and second area 52 all can be able to be received adjust infrared acquisition laser and nanosecond Q holmium laser 20 to send during the tunable CO laser 10 of wideband sends by the rotation of sample cell like this.The structure of sample cell 50 is: place a polytetrafluoro thin slice with two circular holes in the middle of the plain film of two blocks of calcium fluoride, the thickness of polytetrafluoro thin slice is 50 microns, thus the thickness realizing sample is 50 microns.
In order to reach higher moment increasing extent of temperature, the laser scioptics being adjusted Q Ho laser instrument 20 to export nanosecond focus on, and controlling the spot size of nanosecond short pulse Q-switch laser at sample place is 2mm.Meanwhile, in order to allow exploring laser light be in completely by within the scope of the sample that heats, and be in the metastable region of temperature, focused on by the short lens of detection light, spot diameter controls at 0.2mm.In a preferred embodiment of the invention, the pumping laser spot diameter be formed on sample cell adjusting Q holmium laser 20 to send is 2mm, and wideband is tunable, and the middle infrared acquisition laser spot diameter be formed on sample cell 50 that CO laser 10 sends is 0.4mm.
Pump probe light path system is for detecting the intensity through infrared acquisition laser in after testing protein solution.Adopt the pump probe process of pump probe light path system realization to protein example to be measured, wherein, the all catoptrons adopted in this pump probe light path system are Jin Jing 32, all lens are calcium fluoride lens 33, adopt wiregrating 31 to be used for regulating the detection luminous energy arriving sample cell 50 place.
In a preferred embodiment of the invention, pump probe light path system comprises the light path part, detector 34, amplifier 35 and the wave filter 36 that set gradually.Wherein, detector 34 is in order to the intensity of detection through infrared acquisition laser in after testing protein.Detector preferred liquid nitrogen refrigerating type high speed mercury-cadmium tellurid detector.Amplifier 35 amplifies in order to the intensity of centering infrared acquisition laser.Amplifier is preferably DC-100MHz amplifier.Wave filter 36 is in order to filter the strength signal of infrared acquisition laser in after amplifying, and the electromagnetic interference (EMI) in filtering appts and space, to obtain the electrical signal of required frequency.
Adopt data acquisition system (DAS) 40 can realize the high-speed data acquisition of nanoseconds to time second.In order to realize the data acquisition of nanosecond to second-time, in a preferred embodiment of the invention, data acquisition system (DAS) 40 have employed exponential down-sampled technology, can ensure the high time resolution precision of short-time dynamics like this, can ensure again long dynamics data collection.The long-time result of down-sampled data acquisition as shown in Figure 4.As can be seen from Figure 4, the acquisition precision of acquisition system within 1 millisecond is higher, detailed dynamic variation information in early stage can be obtained, achieve again the data acquisition range reaching 10 milliseconds simultaneously, long dynamic-change information can be obtained roughly, the smoothness of data acquisition can be ensured again simultaneously.
Particularly, the mode of operation of pump probe light path system and data acquisition system (DAS) 40 is as follows: first the signal that liquid nitrogen refrigerating type high speed mercury-cadmium tellurid detector 34 exports amplifies through the amplifier 35 of the current-voltage of a DC-100MHz, then the filtering through 0 ~ 500MHz low-pass filter 36 is input in the capture card (not shown) of NI5152 type, adopt afterwards labview data acquisition software realize nanosecond-second-time data acquisition and enter in computer and carry out data preservation.
To sum up, adopt the nanosecond pulse heating-up time provided by the present invention to differentiate mid-infrared light spectrometer and there is following effect: wide mid-infrared light spectrometry 1) can be realized, cover the absorption of secondary protein structure completely; 2) can obtain high-precision transient state ir data, measuring accuracy can reach 1 × 10
-4oD; 3) data acquisition of nanosecond to second-time can be realized.
Below in conjunction with the operation measurement of Cyt C protein transient state infrared spectrum being described in detail to mid-infrared light spectrometer.
1) preparation of samples: the two sample sample cell in a pond of sampling, and load onto heavy water as reference in its first area, second area load onto be dissolved with 1% cytochrome c protein heavy aqueous solution as sample.
2) experiment condition: basal temperature 25 DEG C, Ho laser single-pulse energy 17mj, warm skip frame degree is about 11 DEG C, and detection optical beam spot diameter is less than 100 microns, and pump light spot diameter is about 1mm.
3) by the wavelength regulation of tunable CO laser instrument to want measure wavelength place: such as 1645cm
-1wave number place, carries out nanosecond pulse intensification experiment to reference and sample respectively.Now, along with pulse heated up to the moment of heavy water, through the 1645cm of reference and sample
-1laser energy can change, utilize labview capture program call NI data collecting card gather through reference and sample laser energy over time, and be converted into absorption intensity (OD) value.The reference collected and the signal of sample are as shown in figures 5-9.
Wherein, Fig. 5 is that heavy water is at 1645cm
-1the measurement result that the Transient Dynamics signal located is twice.Fig. 6 is that heavy water and sample are at 1645cm
-1in signal schematic representation, as can be seen from Figure 6 the signal of sample is greater than the signal of reference.Fig. 7 is the difference of the dynamic signal of twice heavy water, and as can be seen from Figure 7, the signal to noise ratio (S/N ratio) of mid-infrared light spectrometer of the present invention reaches 10
-4the precision of OD.
4) signal sample signal of the protein obtained in heavy water being deducted heavy water just can obtain 1645cm
-1the change kinetics information of Cyt C protein after temperature raises instantaneously at place, as shown in Figure 8.As can be seen from Figure 8, when temperature raises instantaneously, cromoci is at 1645cm
-1the absorbance at place reduces, and reaches stable after stabilizing the temperature.
5) then change the output wavelength of tunable CO laser instrument, repeat step 3) and 4) just can obtain the protein dynamics information at different wave number place.
6) by step 5) dynamic information at different wave number places that obtains get after heating pulse is come the same time such as 2 microsecond places value mapping (such as, 1645cm
-1place is 2.5 × 10
-3oD), the transient state infrared spectrum of 2 microsecond Cyt C proteins after heating pulse arrives can just be obtained, as shown in Figure 9.As can be seen from Figure 9, after heating up instantaneously, cromoci is at 1620cm
-1-1645cm
-1the absorbance at place reduces, at 1662cm
-1and 1683cm
-1the absorbance at place rises, and means that cromoci is after heating up instantaneously, hydrophilic helical structure (1620cm
-1-1645cm
-1) disappearance and disordered structure (1662cm
-1) and corner (1683cm
-1) generation.
So far, those skilled in the art will recognize that, although multiple exemplary embodiment of the present invention is illustrate and described herein detailed, but, without departing from the spirit and scope of the present invention, still can directly determine or derive other modification many or amendment of meeting the principle of the invention according to content disclosed by the invention.Therefore, scope of the present invention should be understood and regard as and cover all these other modification or amendments.
Claims (10)
1. a mid-infrared light spectrometer, for measuring the dynamic structure change of protein, described mid-infrared light spectrometer comprises:
Wideband is tunable CO laser (10), for sending middle infrared acquisition laser to testing protein solution;
Nanosecond adjusts Q holmium laser (20), for sending pumping laser to heat described testing protein solution;
Pump probe light path system, for detecting the intensity of the described middle infrared acquisition laser after through described testing protein solution; And
Data acquisition system (DAS) (40), for gathering the data of the described intensity detected from described pump probe light path system;
The described pumping laser spot diameter be formed on described sample cell (50) that described nanosecond adjusts Q holmium laser (20) to send is 2mm.
2. mid-infrared light spectrometer according to claim 1, is characterized in that, described pump probe light path system comprises the light path part, detector, amplifier and the wave filter that set gradually.
3. mid-infrared light spectrometer according to claim 2, is characterized in that, described detector is liquid nitrogen refrigerating type high speed mercury-cadmium tellurid detector; Described amplifier is DC-100MHz amplifier.
4. the mid-infrared light spectrometer according to any one of claim 1-3, is characterized in that, the output wavelength of described wideband is tunable CO laser is 5.2 μm ~ 6.2 μm.
5. the mid-infrared light spectrometer according to any one of claim 1-4, is characterized in that, described nanosecond adjusts the output pulse width of Q holmium laser to be 50ns, and output wavelength is 2080nm.
6. the mid-infrared light spectrometer according to any one of claim 1-5, it is characterized in that, also comprise the sample cell (50) for placing described testing protein solution, described sample cell (50) has first area (51) for placing reference heavy water and for placing the second area (52) containing the heavy aqueous solution of described testing protein solution.
7. mid-infrared light spectrometer according to claim 6, it is characterized in that, described sample cell (50) is rotary structure, and makes described first area (51) and described second area (52) all can receive the described middle infrared acquisition laser that the tunable CO laser of described wideband (10) sends and the described pumping laser adjusting Q holmium laser (20) to send described nanosecond by its rotation.
8. the mid-infrared light spectrometer according to any one of claim 6-7, is characterized in that,
Described wideband is tunable, and the described middle infrared acquisition laser spot diameter be formed on described sample cell (50) that CO laser (10) sends is 0.4mm.
9. the mid-infrared light spectrometer according to any one of claim 1-8, is characterized in that, described data acquisition system (DAS) (40) comprises 8 high-speed collection card, is minimumly distinguished as 0.36mV.
10. the mid-infrared light spectrometer according to any one of claim 1-9, is characterized in that, described data acquisition system (DAS) (40) is arranged to the down-sampled type collection data of time frame nanosecond to second.
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