CN110487731B - Helicobacter pylori detection device and method based on CN free radical isotope spectrum - Google Patents
Helicobacter pylori detection device and method based on CN free radical isotope spectrum Download PDFInfo
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- 238000001228 spectrum Methods 0.000 title claims abstract description 77
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 26
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 26
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- 238000012545 processing Methods 0.000 claims abstract description 18
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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Abstract
The invention relates to a helicobacter pylori detection device and method based on CN free radical isotope spectrum, the detection process is as follows: the method comprises the steps of discharging the exhaled gas of a tested person in a pulse form into a detection box, ablating the exhaled gas into plasma by a focused laser beam, combining CO 2 in the exhaled gas with N 2 in the environment to generate CN free radical molecules, collecting and obtaining CN free radical molecular isotope spectrum data by a multichannel spectrometer, transmitting the CN free radical molecular isotope spectrum data to a data processing computer for data processing to obtain frequency displacement and spectral line intensity of CN free radical molecular spectral lines, and further obtaining abundance information of carbon element isotopes in exhaled gas of the tested person. The invention can avoid complex sample pretreatment, does not need to detect in a vacuum environment, can shorten the detection time, improve the detection efficiency and reduce the cost of the detection device. In addition, the emission spectrum of CN free radical molecules is not interfered by atomic spectrum lines, and mass spectrum detection is difficult to calibrate the material spectrum lines with similar mass numbers, so that the detection precision of the invention is higher.
Description
Technical Field
The invention belongs to the field of isotope online detection, relates to measurement of carbon isotope abundance and molecular emission spectrum, and particularly relates to a helicobacter pylori detection device and method based on CN free radical isotope spectrum.
Background
Helicobacter pylori is one of the most common chronic infectious pathogenic bacteria in the world and is the only species of microorganism currently known to survive in the human stomach. Research shows that helicobacter pylori infection can cause chronic gastritis, gastric and duodenal ulcers, lymphoid tissue lymphoma related to digestive gastric mucosa, gastric adenocarcinoma and other diseases. Thus, it is important to find helicobacter pylori infected persons early and to kill helicobacter pylori effectively by antibiotics.
There are various methods for detecting helicobacter pylori infection, such as biopsy, isolated culture of helicobacter pylori, rapid urease test, urea expiration test, urinary ammonia excretion test, serological test, and polymerase chain reaction. Currently, the mainstream detection method adopted in hospitals is the urea [ 13 C ] exhalation detection method, which is to orally take urea [ 13 C ] capsules by patients, if helicobacter pylori exists in the stomach of the patients, the bacteria can secrete urease to hydrolyze urea, carbon dioxide is formed after the urea is hydrolyzed to enter the lung along with blood and is discharged through respiration, and then 13 C isotopes can be detected in the exhaled air of the patients. The detection method is safe, accurate, convenient, painless, noninvasive and cross-infection free, and is recognized as one of effective methods for detecting helicobacter pylori.
In the urea [ 13 C ] exhalation detection method, the most critical step is the accurate detection of the carbon isotope (13 C) content. The existing isotope detection means mainly rely on mass spectrum detection, although the accuracy of mass spectrum detection is higher, the pretreatment process of a sample is complicated, the exhaled gas of a patient needs to be mixed with carrier gas in advance and then enters a mass spectrum detection box, and meanwhile, the mass spectrum detection needs to be carried out in vacuum, so that the requirement on the detection environment is extremely high. Thus, this method is relatively long for analysis of isotopic abundance. In addition, since most of mass spectrometry detection equipment is imported from abroad, the cost is high, and the cost of the exhalation detection method based on mass spectrometry detection is high.
Disclosure of Invention
The invention provides a helicobacter pylori detection device and a helicobacter pylori detection method based on CN free radical isotope spectrum, which have high efficiency, low cost and lower requirements on detection environment, and the rapid on-line detection of 13 C in a urea expiration method of helicobacter pylori is realized.
The technical scheme adopted by the invention is as follows:
helicobacter pylori detection device based on CN free radical isotope spectrum includes: detection case, gas conduit, time schedule controller, multichannel spectrum appearance, data processing computer set up pulse valve, nd in the detection case: YAG pulse laser, focusing lens and optical fiber probe;
The gas conduit extends into the detection box, the pulse valve is arranged at the end part of the gas conduit, and a blowing nozzle is arranged at the end part of the opposite end of the gas conduit; the Nd: the laser beam emitted by the YAG pulse laser is focused to the outlet position of the pulse valve by the focusing lens, and the fiber probe faces the outlet position of the pulse valve; the timing controller is respectively connected with the Nd: YAG pulse laser with multichannel spectrum appearance links to each other, fiber probe with multichannel spectrum appearance links to each other, multichannel spectrum appearance with data processing computer links to each other.
Further, the timing controller is respectively connected with the Nd: YAG pulse laser with multichannel spectrum appearance passes through fiber connection, fiber probe with multichannel spectrum appearance passes through fiber connection, multichannel spectrum appearance with data processing computer passes through fiber connection, it has a plurality of through-holes that supply optic fibre to pass to open on the detection case.
Further, a filter for absorbing moisture in the detected gas is also arranged on the gas conduit.
The helicobacter pylori detection method based on CN free radical isotope spectrum comprises the following steps:
Step one, a blowing nozzle receives gas exhaled by a tested person after taking urea 13 C capsules, and the gas is discharged into a detection box in a pulse mode at a pulse valve after water is absorbed by a filter along a gas guide pipe;
step two, the expired gas molecules discharged into the detection box are subjected to Nd: the laser beam of 1064nm emitted by the YAG pulse laser and focused by the focusing lens is ablated into plasma, CO 2 molecules in the exhaled gas are combined with N 2 molecules in the environment in the detection box to generate CN free radical molecules, the multichannel spectrometer collects the isotope spectrum data of the CN free radical molecules through the optical fiber probe, and in the process, the time sequence controller is adjusted to eliminate the background noise of the spectrum data;
Step three, the CN free radical molecular isotope spectrum data collected by the multichannel spectrometer are transmitted to a data processing computer for data processing, so that the frequency shift and spectral line intensity of the CN free radical molecular spectrum in the isotope spectrum are obtained, and further the abundance information of the carbon element isotope in the expired breath of the detected person is obtained;
the frequency shift Deltav formula of the CN free radical molecular spectrum is as follows:
In the formula (1), Mu is the approximate mass number of CN free radical molecules, i represents 13 C and other carbon isotopes, omega e and chi e are spectral constants, v is the number of vibration quanta, A 'represents an upper energy level parameter, and A' represents a lower energy level parameter;
The spectral line intensity of the CN free radical molecular spectrum is the characteristic peak area obtained by integrating the frequency spectral line corresponding to the CN free radical molecule;
The method specifically comprises the following steps:
Step 3-1, firstly, sequentially carrying out normalization and wavelet transformation noise reduction treatment on CN free radical molecular isotope spectrum data, then extracting a frequency spectrum line corresponding to 12 CN free radical molecules and a frequency spectrum line corresponding to 13 CN free radical molecules in a noise-reduced spectrum under energy state transition of B 2∑+(v＝0)→X2∑+ (v=1), and respectively obtaining characteristic peak areas of 12 CN and 13 CN free radical molecular frequency spectrum lines, namely spectrum line intensities through integration;
Step 3-2, calculating the vibration level information of 12 CN and 13 CN free radical molecules, and obtaining a characteristic spectral line frequency theoretical value from E=hν, wherein E is vibration level energy, h is Planck constant, and ν is the vibration frequency of the molecules; performing experimental measurement on a CO 2 isotope sample with a known abundance ratio, collecting a CN free radical molecular spectrum of the sample, extracting spectral line intensity at a theoretical value of a characteristic spectral line frequency, and determining a calibration curve between the CN free radical spectral line intensity and isotope abundance by using a least square method;
And 3-3, substituting the spectral line intensity obtained in the step 3-1 into the calibration curve of the step 3-2 for comparison, inverting the abundance of the isotope, and determining the content of 13 C isotope.
Further, in step 3-1, the CN radical adopts a transition of the B 2∑+(v＝0)→X2∑+ (v=1) energy state, and the 12 CN radical molecule corresponds to the line frequency v 1＝8.3624×1014Hz,13 CN radical molecule corresponds to the line frequency v 2＝8.3521×1014 Hz of the transition.
The invention has the beneficial effects that:
(1) According to the invention, the spectrum detection is adopted to replace the existing mass spectrum technology to carry out rapid and accurate detection on the carbon isotope, the exhaled air can directly enter the detection box along the gas conduit to carry out detection, so that complicated sample pretreatment steps are avoided, the detection time for helicobacter pylori can be greatly shortened, and meanwhile, the detection efficiency is effectively improved.
(2) The invention is based on the emission spectrum of CN free radical molecules to realize detection of 13 C isotope abundance, and because most spectral lines in the laser spectrum are atomic spectral lines and the difference between the molecular spectral lines and the atomic spectral lines is large in the line shape and the distribution characteristics, the emission spectral lines of CN free radical molecules cannot be interfered, so that the difficulty of calibrating the spectral lines of substances with similar mass numbers in the mass spectrum technology is avoided, and the detection precision of 13 C isotope abundance, namely the detection precision of helicobacter pylori content is improved.
(3) The mass spectrum technology adopted by the existing detection system needs to detect the sample in a vacuum environment, and the CN free radical isotope spectrum technology adopted by the invention can be directly carried out in an atmospheric environment, so that the experimental environment requirement is greatly reduced, the detection flow is simplified, and the portability of the detection device is facilitated; meanwhile, the cost of the detection device is reduced, and the commercialization of products is facilitated.
Drawings
FIG. 1 is a schematic diagram of the structure of a helicobacter pylori detection device based on CN free radical isotope spectrum of the present invention;
FIG. 2 is a flow chart of a method for detecting helicobacter pylori based on CN free radical isotope spectrum of the invention;
FIG. 3 is an isotope spectrum of CN free radicals (12 CN and 13 CN free radical molecules);
Reference numerals: 1-detection box, 2-mouthpiece, 3-filter, 4-gas conduit, 5-pulse valve, 6-Nd: YAG pulse laser, 7-focusing lens, 8-laser beam, 9-optical fiber probe, 10-optical fiber, 11-time schedule controller, 12-multichannel spectrometer and 13-data processing computer.
Detailed Description
The invention mainly generates CN free radical isotope molecular emission spectrum by the action of laser and gas substances, and realizes accurate online detection of carbon isotopes by measuring the frequency displacement of the isotope molecular emission spectrum.
The apparatus and method for detecting helicobacter pylori based on CN free radical isotope spectrum of the present invention will be further described with reference to the accompanying drawings and the detailed description.
The helicobacter pylori detection device based on the CN free radical isotope spectrum as shown in figure 1 comprises: detection box 1, gas conduit 4, pulse valve 5, nd: YAG pulse laser 6, focusing lens 7, fiber optic probe 9, timing controller 11, multichannel spectrometer 12, and data processing computer 13. Wherein, pulse valve 5, nd: the YAG pulse laser 6, the focusing lens 7, and the optical fiber probe 9 are disposed in the inspection box 1, and the timing controller 11, the multichannel spectrometer 12, and the data processing computer 13 are located outside the inspection box 1.
The gas conduit 4 stretches into the detection box 1, the pulse valve 5 is installed at the tip of the gas conduit 4, the tip of the other end opposite to the gas conduit 4 is provided with the blowing nozzle 2, the gas conduit 4 is also provided with the filter 3, the filter 3 can remove the moisture in the detected gas, a better detection effect is obtained, and in the embodiment, the filter 3 is a water removal filter element. Nd: the laser beam 8 emitted by the YAG pulse laser 6 is focused by the focusing lens 7 to the position of the outlet of the pulse valve 5, and the optical fiber probe 9 faces the position of the outlet of the pulse valve 5. The timing controller 11 is respectively connected with Nd: the YAG pulse laser 6 is connected with the multichannel spectrometer 12 through an optical fiber 10, the optical fiber probe 9 is connected with the multichannel spectrometer 12 through the optical fiber 10, the multichannel spectrometer 12 is connected with the data processing computer 13 through the optical fiber 10, and a plurality of through holes for the optical fiber 10 to pass through are formed in the detection box 1.
As shown in fig. 2, the method for detecting helicobacter pylori based on the CN free radical isotope spectrum adopts the detection device and comprises the following steps:
Step one, the mouthpiece 2 receives the gas exhaled by the person taking the urea 13 C capsule, and the gas is discharged into the detection box 1 in a pulse form at the pulse valve 5 after absorbing moisture along the gas conduit 4 and through the filter 3.
Step two, the expired gas molecules discharged into the detection box 1 are subjected to Nd: the 1064nm laser beam 8 emitted by the YAG pulse laser 6 and focused by the focusing lens 7 is ablated into plasma (laser ablation), polyatomic molecules CO 2 (mainly comprising 12 C and 13 C) in the gas exhaled by the edge part of the high-temperature plasma are combined with N 2 molecules in the environment in the detection box 1 to generate CN free radical molecules, and CN free radical molecular spectrum is radiated (the edge temperature of the high-temperature plasma is lower than the center temperature of the high-temperature plasma, the excessive CN free radical molecules cannot exist, and the temperature condition for generating CN free radicals is only provided at the edge of the plasma with lower temperature). The multichannel spectrometer 12 collects the spectrum data of the CN free radical molecular isotopes through the optical fiber probe 9, and in the process, the time sequence controller 11 is regulated, and the proper delay time is set to eliminate the background noise of the spectrum data, and the specific delay time is determined by debugging.
And thirdly, transmitting the CN free radical molecular isotope spectrum data collected by the multichannel spectrometer 12 to a data processing computer 13 for data processing to obtain the frequency shift and spectral line intensity of the CN free radical molecular spectrum in the isotope spectrum data, and further obtaining the abundance information of the carbon element isotope in the expired breath of the detected person.
The frequency shift Deltav formula of the CN free radical molecular spectrum is as follows:
In the formula (1), Mu is the approximate mass number of CN free radical molecules, i represents 13 C and other carbon isotopes, omega e and chi e are spectral constants, v is the number of vibration quanta, A 'represents an upper energy level parameter, and A' represents a lower energy level parameter;
The spectral line intensity of the CN free radical molecular spectrum is the characteristic peak area obtained by integrating the frequency spectral line corresponding to the CN free radical molecule.
Specifically, the third step includes:
Step 3-1, firstly, sequentially carrying out normalization and wavelet transformation noise reduction treatment on the CN free radical molecular isotope spectrum data, then extracting a frequency spectrum line corresponding to 12 CN free radical molecules and a frequency spectrum line corresponding to 13 CN free radical molecules in the energy state transition of B 2∑+(v＝0)→X2∑+ (v=1) in the spectrum after noise reduction, and respectively obtaining the characteristic peak areas of the 12 CN and 13 CN free radical molecular frequency spectrum lines, namely the spectrum line intensity through integration.
In this embodiment, the CN radical adopts transition of B 2∑+(v＝0)→X2∑+ (v=1) energy state, the 12 CN radical molecule corresponds to spectral line frequency v 1＝8.3624×1014Hz,13 CN radical molecule corresponds to spectral line frequency v 2＝8.3521×1014 Hz of the transition, as shown in fig. 3, the spectral line corresponding to the transition of the CN radical molecule spectrum generates frequency shift due to different carbon isotopes, and the carbon isotopes can be distinguished by frequency shift Δv.
Step 3-2, calculating to obtain vibration level information of 12 CN and 13 CN free radical molecules through Gaussian 09 software, firstly constructing a 12 CN free radical molecular structure in the software, then optimizing the molecular structure by using a B3PW91/6-31+G (d) group, and outputting optimized molecular structure information, wherein a calculation result shows that the vibration frequency of 12 CN free radical molecules is 2167.96/cm -1. Then, a 13 CN free radical molecular structure is constructed in software, the same basic group is adopted to calculate and obtain the 13 CN molecular vibration frequency as 2122.44/cm -1, and the vibration energy level difference between 12 CN and 13 CN is 0.0056eV, wherein E is vibration energy level, h is Planck constant, and v is the molecular vibration frequency. And subtracting the theoretical vibration energy level difference from the energy corresponding to the 12 CN free radical molecular spectral line to obtain the energy corresponding to the 13 CN free radical molecular characteristic spectral line and the frequency theoretical value thereof. And then, carrying out experimental measurement on a CO 2 isotope sample with a known abundance ratio, collecting a CN free radical molecular spectrum, extracting spectral line intensity at a theoretical value of a characteristic spectral line frequency, and determining a calibration curve between the CN free radical spectral line intensity and the isotope abundance by using a least square method. The established calibration curve is a relationship of abundance ratio to line intensity information, i.e. once line intensity is known, the abundance ratio of carbon isotopes can be known.
And 3-3, substituting the spectral line intensity obtained in the step 3-1 into the calibration curve of the step 3-2 for comparison, and inverting the abundance of the isotope to obtain the content of 13 C isotope.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any alternatives or modifications, which are easily conceivable by those skilled in the art within the scope of the present invention, should be included in the scope of the present invention.
Claims (4)
1. The helicobacter pylori detection method based on CN free radical isotope spectrum is characterized in that the device applied to the method comprises: the device comprises a detection box (1), a gas conduit (4), a time sequence controller (11), a multichannel spectrometer (12), a data processing computer (13), and pulse valves (5) and Nd) arranged in the detection box (1): a YAG pulse laser (6), a focusing lens (7) and an optical fiber probe (9);
The gas conduit (4) stretches into the detection box (1), the pulse valve (5) is arranged at the end part of the gas conduit (4), and the blowing nozzle (2) is arranged at the end part of the other end opposite to the gas conduit (4); nd: a laser beam (8) emitted by the YAG pulse laser (6) is focused to the outlet position of the pulse valve (5) by a focusing lens (7), and an optical fiber probe (9) faces to the outlet position of the pulse valve (5); the time schedule controller (11) is respectively connected with Nd: the YAG pulse laser (6) is connected with the multichannel spectrometer (12), the optical fiber probe (9) is connected with the multichannel spectrometer (12), and the multichannel spectrometer (12) is connected with the data processing computer (13);
the method comprises the following steps:
Step one, the blowing nozzle (2) receives the gas exhaled by the testee after taking urea 13 C capsules, and the gas is discharged into the detection box (1) in a pulse form at the pulse valve (5) after absorbing water through the filter (3) along the gas conduit (4);
Step two, the expired gas molecules discharged into the detection box (1) are subjected to Nd: the laser beam (8) of 1064nm emitted by the YAG pulse laser (6) and focused by the focusing lens (7) is ablated into plasma, CO 2 molecules in the exhaled gas are combined with N 2 molecules in the environment in the detection box (1) to generate CN free radical molecules, the multichannel spectrometer (12) collects and obtains CN free radical molecular isotope spectrum data through the optical fiber probe (9), and in the process, the time sequence controller (11) is regulated to eliminate spectrum data background noise;
Step three, the CN free radical molecular isotope spectrum data collected by the multichannel spectrometer (12) are transmitted to a data processing computer (13) for data processing, so that the frequency shift and spectral line intensity of the CN free radical molecular spectrum in the isotope spectrum are obtained, and further the abundance information of the carbon element isotope in the expiration of a detected person is obtained;
the frequency shift Deltav formula of the CN free radical molecular spectrum is as follows:
In the formula (1), Mu is the approximate mass number of CN free radical molecules, i represents 13 C and other carbon isotopes, omega e and chi e are spectral constants, v is the number of vibration quanta, A 'represents an upper energy level parameter, and A' represents a lower energy level parameter;
The spectral line intensity of the CN free radical molecular spectrum is the characteristic peak area obtained by integrating the frequency spectral line corresponding to the CN free radical molecule;
The method specifically comprises the following steps:
Step 3-1, firstly, sequentially carrying out normalization and wavelet transformation noise reduction treatment on CN free radical molecular isotope spectrum data, then extracting a frequency spectrum line corresponding to 12 CN free radical molecules and a frequency spectrum line corresponding to 13 CN free radical molecules in a noise-reduced spectrum under energy state transition of B 2∑+(v＝0)→X2∑+ (v=1), and respectively obtaining characteristic peak areas of 12 CN and 13 CN free radical molecular frequency spectrum lines, namely spectrum line intensities through integration;
Step 3-2, calculating the vibration level information of 12 CN and 13 CN free radical molecules, and obtaining a characteristic spectral line frequency theoretical value from E=hν, wherein E is vibration level energy, h is Planck constant, and ν is the vibration frequency of the molecules; performing experimental measurement on a CO 2 isotope sample with a known abundance ratio, collecting a CN free radical molecular spectrum of the sample, extracting spectral line intensity at a theoretical value of a characteristic spectral line frequency, and determining a calibration curve between the CN free radical spectral line intensity and isotope abundance by using a least square method;
And 3-3, substituting the spectral line intensity obtained in the step 3-1 into the calibration curve of the step 3-2 for comparison, inverting the abundance of the isotope, and determining the content of 13 C isotope.
2. The method for detecting helicobacter pylori based on CN free radical isotope spectrum according to claim 1, wherein the timing controller (11) is respectively connected with Nd: YAG pulse laser (6) and multichannel spectrum appearance (12) are connected through optic fibre (10), and fiber probe (9) are connected through optic fibre (10) with multichannel spectrum appearance (12), and multichannel spectrum appearance (12) are connected through optic fibre (10) with data processing computer (13), open on detection case (1) and have a plurality of through-holes that supply optic fibre (10) to pass.
3. The method for detecting helicobacter pylori based on CN free radical isotope spectrum according to claim 1 or 2, characterized in that a filter (3) for absorbing moisture in the detected gas is further provided on the gas conduit (4).
4. The method for detecting helicobacter pylori based on the isotope spectrum of CN free radicals according to claim 1, wherein in step 3-1, CN free radicals adopts transition of energy state of B 2∑+(v＝0)→X2∑+ (v=1), 12 CN free radical molecule corresponds to spectral line frequency v 1＝8.3624×1014Hz,13 CN free radical molecule corresponds to spectral line frequency v 2＝8.3521×1014 Hz of the transition.
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