Based on the multisample scentometer of cavity ring-down spectroscopyTechnical field
The present invention relates to field of biomedicine technology, particularly relate to a kind of multisample scentometer based on cavity ring-down spectroscopy.
Background technology
Health is the basis of people's development in an all-round way, the happiness affected the livelihood of every family.Along with air quality problems highlights day by day in recent years, it obtains global attention to the long lasting effect of health.On Dec 19th, 2013, the article of air pollution and European crowd's mortality ratio correlativity delivered by The Lancet magazine, and in display air, PM2.5 concentration increases by 10 μ g/m3, and the mortality ratio that a variety of causes can be made to cause increases by 6%, and heart death rate increases by 9%.In China, urban air pollution about can cause 2,500 ten thousand laborer's disabilities every year, and its health hazard brought accounts for 31% of total Disease Spectrum.And as one of most important vital movement of the mankind, breathe and both comprised human body and extraneous gas exchanges, air inclusion transports exchange in vivo again, therefore the information that comprises of breathing gas and body metabolism, disease and environment have close relationship, relevant research not only scientific meaning is given prominence to, and also has important social effect to fitness-for-all and social harmonious development.
Associated pathology investigation and research show, after the internal organs of human body or tissue damage or pathology, its change functionally correspondingly can cause the change of corresponding metabolic product, these metabolic products enter into blood will cause relatively increasing of some metabolite content, just can be diagnosed out the degree of injury of internal organs by the concentration detecting metabolic product.Therefore, diagnosed the illness by some special component in analyzing blood and become a kind of conventional means of modern medical diagnosis.And the metabolic product in blood can enter lung by ABB, and then cause the change of expiratory air component, some specific gas concentration excreted is raised, thus can as the biomarker of some diseases.
In 1971, two Nobel laureate Linus Pauling utilized vapor-phase chromatography (GC) to find to contain 250 various features materials in tidal air, have started this brand-new research field of modern analysis of respiratory gas.It is a research field relating to medical science, chemistry, physics and statistics etc.Along with modern analytical technique and biomedicine.Development, particularly laser spectrum tech, electrochemical sensing technology and molecular biological development, the research of analysis of respiratory gas has become a fast-developing new disciplines [12].Research subsequently finds, breath is a kind of mixed gas of complexity, comprises the VOCs of more than 2000 kind of low concentration in everyone breath.The breath of normal person is except comprising H2o, CO2, O2. outside the air Small molecular that equal size is relatively very high, most of trace gas and volatile organic matter (as ammonia, acetone, propyl alcohol and hydro carbons etc.) equal size are at ppm (10-6) or ppb (10-9) magnitude, what have is even low to moderate ppt magnitude (10-12).In table 1 some low content gas and volatility is organic has been confirmed to be and disease, or body metabolism is abnormal, or the impact of the environment of outside is correlated with, and namely becomes and breathes biomarker (Breath Biomarker).
Containing thousands of kinds of VOCs in human body respiration gas, concentration is low, and gas componant is complicated, except stable molecule also has free radical.Measure the sensitivity of absolute concentration to measuring technique of some breathing gas compositions, selectivity and time response all there is very high requirement.At present for respiratory gas analysis mainly based on MS technology, Electronic Nose and laser spectrum method.GC-MS is a current techique of trace gas analysis, has high sensitivity and high selectivity, and selectively can measure multiple compounds simultaneously.Adopt GC-MS technology to carry out trace gas analysis, need first to carry out low temperature enrichment (or other enrichment method) to gaseous sample, adopt GC technology to be separated, then detected by MS, 1 hours is needed usually to the measurement of single sample.Expect the absolute concentration of this sample, also need to carry out calibrating (external standard method, internal standard method or normalization method).Collection and the low temperature enrichment processing procedure of sample can bring analytical error.Therefore, although the method for GC-MS has high sensitivity and high selectivity, cannot meet real-time online, need not the analysis of respiratory gas requirement of sample pre-treatments.At present, also also have other methods based on mass-spectrometric technique to be used for gas analysis, as proton transfer reactions mass spectrum (PTR-MS) and selectivity ion flight pipe mass spectrum (SIFT-MS), but these technology still under study for action.Another kind of breast rail method is electronic sensor, and its advantage is that volume is little, and cost is low, but this kind of method sensitivity is lower, and needs to calibrate frequently, easily baseline wander and spurious alarm occurs.Under Chemical Background complicated situation, selectivity is bad.Electronic Nose and novel nano-material combine and may address these problems, but at present still under study for action.In a word, the current analytical technology based on MS and Electronic transducer technology all can not be used for real-time online high sensitivity and the high-selectivity analysis of breathing gas.Therefore need to research and develop new instrument and equipment to satisfy the demands.
Mainly there is two problems in the development of the research field of current analysis of respiratory gas:
(1) to the breathing biomarker determined at present, quantitative correlation is lacked with the state of people itself;
(2) contain thousands of kinds of VOCs in human body respiration gas, only found 30 various respiratory biomarkers at present, also a large amount of VOCs requires study as breathing biomarker, contacting of the aspects such as foundation and disease, metabolic disorder and environmental impact.
Solve this two greatly challenge be two important scientific methods: (1) is to confirmed breathing biomarker, obtain a large amount of breathing gas data, find itself and the quantitative correlation with abnormal (disease, health, the external environment influence) Diagnostic parameters of human body; (2) need to upgrade more advanced respiratory gas analysis and instrument and equipment, the specific crowd chosen is carried out effectively to the analysis of respiratory gas of (" effectively " refers in real time, online, low cost, high sensitivity, high selectivity, high-precision mass data obtain), and then find new breathing biomarker.Just because of this, various countries researchist is seeking various means, solves this two large important scientific issues in analysis of respiratory gas.If research and develop the analysis of respiratory gas system of a set of highly sensitive, high precision, real-time online, realize effectively analyzing fast of breathing gas sample, to the correlative study of existing breathing biomarker and human body diseases and metabolism, study simultaneously and do not determine a large amount of VOCs, set up and the contacting of the aspect such as disease, metabolic disorder and environmental impact, and then it is significant to carry out setting up of human body diseases mechanism, environmental medicine and biomedical research and healthy large data platform.
Summary of the invention
The object of the invention is for the defect existing for GC-MS in prior art and Electronic Nose, and provide a kind of have high precision, response in real time, volume is little and without the need to the multisample scentometer based on cavity ring-down spectroscopy of complicated pre-treatment.
The technical scheme adopted for realizing object of the present invention is: based on the multisample scentometer of cavity ring-down spectroscopy, it is characterized in that comprising:
(1) multiwavelength laser output system
This subsystem as light source by the accurate single-mode laser of a series of all solid state small size, can be worked simultaneously, export the laser of multiple wavelength (263nm, 266nm, 355nm), and this is the optionally key core technology strengthening Fingerprint;
(2) Stress control sample chamber system
Sample chamber system adopts automatic control system to realize pressure from main regulation and voltage stabilizing, and this is the key strengthening the selectivity of Fingerprint and the high precision of measurement and repeatability further.Sample chamber system connects sampling system, vacuum pump and pressure gauge respectively by the pipeline of Teflon material, by solenoid control gas path on-off, thus stability contorting cavity pressure;
(3) optics cavity mirror
Adopt metal washer to fix high reflection mirror, the micro-displacement eliminating system chamber mirror may be caused declining swinging signal distortion; High reflection mirror two sides adopts spherical pad and corrugated gasket clamping plated film high reflective mirror respectively, spherical pad adds one deck annular shim again seal for cavity, simultaneously cavity outermost loads one piece of piece of stainless steel with optical window and seals, and adopts M6 stud to lock.Due to rigidity and the sealing of metal, this structure can be applicable to work in the vibration environment such as ocean, aviation, in breast rail process, thus have stability very reliably.In addition, the inside surface of sample chamber adopts special plated film technology, can effectively prevent the VOCs in breathing gas or air or other molecules from remaining, and can ensure that the long-term stability of cavity is run.
(4) chamber mirror life system
1) high pure nitrogen or dry air enter sample chamber by the air intake opening near chamber mirror, produce airflow scouring effect, constantly extracted out by intracavity gas by vacuum pump near cavity mirror face.The flow velocity that gas enters cavity is controlled by reduction valve.
2) meanwhile, the ring-type heating plate being attached to cavity two ends heats cavity mirror end (small size place), promotes the volatilization of the VOCs molecule of cavity mirror face adventitious deposit, is finally washed air-flow and takes cavity out of.Control system will ensure that heating-up temperature is no more than 60 DEG C.This functional device, without the need to frequent unlatching, only needs the maintenance carrying out increasing the service life when instrument long-time running.
(5) data Collection & Processing System
Intensity is I0pulse laser incide in an optical resonator be made up of a pair high reflectance eyeglass, wherein resonator is long is d, and lens reflecting rate is R1=R2=R (more than 99.9%), so the transmissivity T=1-R<<1 (scattering, the diffraction loss of eyeglass are ignored) of mirror.Such light pulse meeting roundtrip between two minute surfaces, by M at every turn after coming and going2export photomultiplier (PMT) to.The loss of whole resonator cavity is relevant with the absorption of sample in the emissivity of mirror and chamber, and its light intensity can be expressed as over time:
I＝I0et/τ (1)
The timeconstantτ of the decay in formula (1) can be expressed as:
Wherein c is the light velocity, and σ (ν) is the absorption cross section relevant to optical maser wavelength, i.e. the characteristic fingerprint of molecule, and n is the concentration of testing sample.When sample in chamber to laser without absorption or not at absorption peak time, formula (2) can be expressed as:
Above-mentioned two formula derived sample absorptivities:
For 266nm wavelength, the absorbance of healthy population breath is defined as by we:
Formula (8)
Same, the corresponding parameter of diabetes patient is defined as:
Formula (9)
When it is measured, breathing gas is all full of whole declining and swings in chamber.The two subtracts each other and is:
Formula (10)
(6) sampling system
The breast rail method that GC-MS etc. are traditional needs complicated preparation of samples and pre-treatment step, and therefore the present invention adopts direct injected mode, with the error avoiding complicated sample preparation to bring.For different sampling situations, implement online measurement and off-line type is measured;
Online sampling device refers to that experimenter directly will breathe in incoming call Sample Buffer chamber, then enters sample chamber by solenoid control sample.Middle without the need to any preparation of samples and pretreating device;
Off-line type sampling device is by among human body respiration sample collection to breathing sampler bag, within 3h, then introduces sample chamber measure;
(7) instrumentation control system
Comprise multiwavelength laser control system, voltage-controlled chamber control system, data acquisition and data handling system four part.
Compared with prior art, the invention has the beneficial effects as follows:
(1) by high-precision detection method, the present invention can realize the detection of trace VOCs in human body respiration gas with quantitative
(2) spectral characteristic that can realize biomarker based on the CRDS in voltage-controlled chamber is separated, thus improves the resolution and specificity analyzed, solves the specificity issues existing for Electronic Nose.
(3) pass through close to real-time analysis mode, the present invention can realize the collection of a large amount of clinical data, thus carries out clinical case measurement widely, for the relation found between known breath biomarker and disease provides foundation
(4) by multi-wavelength CRDS technology, detect while not only can realizing multiple VOCs, also can improve specificity and the precision of same VOCs detection.In addition, also by multi-wavelength, exploratory development is carried out to the biomarker of the unknown.
Accompanying drawing explanation
Figure 1 shows that the multisample breast rail instrument apparatus schematic diagram based on cavity ring-down spectroscopy.
embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment:
Respiration measurement process:
(1) breathing gas is gathered
For off-line type metering system, breath sample gathers by breathing sampler bag.Collection capacity is about about 1L, after experimenter has a suck of gas deeply, by pulmonary gases by disposable blow gun incoming call sampler bag.Then close sampler bag outlet valve, be stored in portable heat-insulation box, prevent too high or too low for temperature, avoid strong illumination.After preserving a period of time, in the suitable time, take out sampler bag and measure.The general storage time is no more than 6h, is causing VOCs concentration to reduce to prevent breath sample due to chemical reaction or other factors.
For online metering system, experimenter is by once to blow gun, and by pulmonary gases incoming call breath sample cushion chamber, breath sample is introduced sample chamber and measured by solenoid valve then by automatically controlling at once.
(2) sample introduction is measured
Measuring principle is based on background deduction method.Measure the content of a certain VOC in breath sample, must with air as a setting, therefore, measure start before to measure the actual absorption of air.Also introduce sample chamber by air, measure ring-down time.Average after measuring 3 times, calculate the absorption of air, as a setting.Then sample measurement is carried out.After each measurement breath sample, vacuum pump being utilized to extract sample out sample chamber, for preventing remaining, also needing to use high pure nitrogen or dry air to clean.
(3) data handling procedure
According to CRDS ultimate principle, after calculating the sample absorbance measured for each time, background deduction method need be adopted, draw the actual absorption of tested VOC.Then tested VOC concentration is determined according to pressure when measuring and molecule absorpting section.
Be illustrated in figure 1 the multisample breast rail instrument apparatus schematic diagram based on cavity ring-down spectroscopy, comprise:
(1) multiwavelength laser output system
This subsystem as light source by the accurate single-mode laser of a series of all solid state small size, can be worked simultaneously, export the laser of multiple wavelength (263nm, 266nm, 355nm), and this is the optionally key core technology strengthening Fingerprint;
(2) Stress control sample chamber system
Sample chamber system adopts automatic control system to realize pressure from main regulation and voltage stabilizing, and this is the key strengthening the selectivity of Fingerprint and the high precision of measurement and repeatability further.Sample chamber system connects sampling system, vacuum pump and pressure gauge respectively by the pipeline of Teflon material, by solenoid control gas path on-off, thus stability contorting cavity pressure;
(3) optics cavity mirror
Adopt metal washer to fix high reflection mirror, the micro-displacement eliminating system chamber mirror may be caused declining swinging signal distortion; High reflection mirror two sides adopts spherical pad and corrugated gasket clamping plated film high reflective mirror respectively, spherical pad adds one deck annular shim again seal for cavity, simultaneously cavity outermost loads one piece of piece of stainless steel with optical window and seals, and adopts M6 stud to lock.Due to rigidity and the sealing of metal, this structure can be applicable to work in the vibration environment such as ocean, aviation, in breast rail process, thus have stability very reliably.In addition, the inside surface of sample chamber adopts special plated film technology, can effectively prevent the VOCs in breathing gas or air or other molecules from remaining, and can ensure that the long-term stability of cavity is run.
(4) chamber mirror life system
1) high pure nitrogen or dry air enter sample chamber by the air intake opening near chamber mirror, produce airflow scouring effect, constantly extracted out by intracavity gas by vacuum pump near cavity mirror face.The flow velocity that gas enters cavity is controlled by reduction valve.
2) meanwhile, the ring-type heating plate being attached to cavity two ends heats cavity mirror end (small size place), promotes the volatilization of the VOCs molecule of cavity mirror face adventitious deposit, is finally washed air-flow and takes cavity out of.Control system will ensure that heating-up temperature is no more than 60 DEG C.This functional device, without the need to frequent unlatching, only needs the maintenance carrying out increasing the service life when instrument long-time running.
(5) data Collection & Processing System
Intensity is I0pulse laser incide in an optical resonator be made up of a pair high reflectance eyeglass, wherein resonator is long is d, and lens reflecting rate is R1=R2=R (more than 99.9%), so the transmissivity T=1-R<<1 (scattering, the diffraction loss of eyeglass are ignored) of mirror.Such light pulse meeting roundtrip between two minute surfaces, by M at every turn after coming and going2export photomultiplier (PMT) to.The loss of whole resonator cavity is relevant with the absorption of sample in the emissivity of mirror and chamber, and its light intensity can be expressed as over time:
I＝I0et/τ (1)
The timeconstantτ of the decay in formula (1) can be expressed as:
Wherein c is the light velocity, and σ (ν) is the absorption cross section relevant to optical maser wavelength, i.e. the characteristic fingerprint of molecule, and n is the concentration of testing sample.When sample in chamber to laser without absorption or not at absorption peak time, formula (2) can be expressed as:
Above-mentioned two formula derived sample absorptivities:
For 266nm wavelength, the absorbance of healthy population breath is defined as by we:
Formula (8)
Same, the corresponding parameter of diabetes patient is defined as:
Formula (9)
When it is measured, breathing gas is all full of whole declining and swings in chamber.The two subtracts each other and is:
Formula (10)
(6) sampling system
The breast rail method that GC-MS etc. are traditional needs complicated preparation of samples and pre-treatment step, and therefore the present invention adopts direct injected mode, with the error avoiding complicated sample preparation to bring.For different sampling situations, implement online measurement and off-line type is measured;
Online sampling device refers to that experimenter directly will breathe in incoming call Sample Buffer chamber, then enters sample chamber by solenoid control sample.Middle without the need to any preparation of samples and pretreating device;
Off-line type sampling device is by among human body respiration sample collection to breathing sampler bag, within 3h, then introduces sample chamber measure;
(7) instrumentation control system
Comprise multiwavelength laser control system, voltage-controlled chamber control system, data acquisition and data handling system four part;
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.