On-line detection method for endogenous markers of expired gas of human body

Abstract

The invention relates to the technical field of expiration detection, in particular to a human expired gas endogenous marker online detection method, which comprises the following steps: collecting human expired gas, detecting the concentration of a plurality of compounds in the gas, improving an analysis detection method through a soft ionization and direct mass spectrometry mode, and constructing a data model through a human expired gas endogenous VOCs quantitative method. Detection data optimization is achieved, and online data transmission is achieved. Equipment is built by connecting a soft ionization source in series with a small mobile portable mass spectrum, the size of the equipment is reduced, the ionization source, the mass spectrum, a power supply and the like are integrated in a device with the size of a trunk after modification, and expiration detection equipment which is reliable, easy to operate, miniaturized, capable of performing real-time detection and applicable to multiple occasions has greater advantages in the field of instant diagnosis.

Description

Technical Field

[0001] The present application relates to the technical field of breath detection, and in particular to an online detection method for endogenous markers in human exhaled breath. Background Art

[0002] Human breath detection technology is mainly used for non-invasive, rapid and simple online real-time breath detection of the human body, and is applied to human health assessment, disease screening and clinical diagnosis. As an emerging in vitro diagnostic method, the clinical application scope of breath diagnosis is gradually expanding. Currently reported breath analysis methods are mostly static, and quantitative determination is difficult. The quantitative method and accurate determination of endogenous volatile organic compounds (VOCs) in human exhaled breath need further exploration. The current shortcomings of breath diagnosis technology based on gas chromatography are mainly the lack of dynamics and quantitative difficulties. In the current breath detection, the two major steps of breath collection and analysis cannot be completed in a coherent manner, and it takes time for the gas to be collected and sent to the instrument for detection, making it difficult to achieve real-time detection; and most of the research related to breath diagnosis focuses on the search for disease-related characteristic VOCs, and there are few reports on VOCs quantitative methods, and quantitative issues have been ignored for a long time. Summary of the invention

[0003] The purpose of the present invention is to provide an online detection method for endogenous markers in human exhaled breath to solve one or more technical problems existing in the prior art and at least provide a beneficial choice or create conditions.

[0004] A method for online detection of endogenous markers in human exhaled breath, the method comprising the following steps:

[0005] S100: Improved analytical detection methods through soft ionization + direct mass spectrometry;

[0006] S200: Collect human exhaled gas and detect the concentration of several compounds in the gas;

[0007] S300: Building a data model through the quantitative method of endogenous VOCs in human exhaled breath;

[0008] S400: Realize online data transmission by optimizing the detection data.

[0009] Furthermore, in step S100, soft ionization is a mass spectrometry analysis technology that uses relatively low energy to convert molecules into ions or fragments, thereby reducing the generation of molecular fragments, making the peak display in the mass spectrum simpler and easier to parse and identify. The ionization source used in the soft ionization scheme is a flow-through medium passage discharge source, which only utilizes the electrode discharge to momentarily excite and ionize the gaseous chemical substances on the flow path at the inlet end of the mass spectrometer, and identifies the chemical composition and form of the inflowing substance in real time. The ion source using the soft ionization scheme can be used at room temperature and pressure, and can directly and continuously soft-ionize gaseous organic small molecules online, while maintaining complete molecular ion information.

[0010] By connecting the ion source in series with a GC-MS, rapid ionization and analysis of gaseous small molecules can be achieved; known endogenous volatile organic compounds commonly found in human exhaled breath are used as reference substances for high-resolution testing and analysis, instrument parameters are adjusted, and the sensitivity of the instrument is optimized.

[0011] The soft ionization source is connected in series with a small mobile portable mass spectrometer to reduce the size of the equipment. After modification, the ionization source, mass spectrometer, power supply, etc. are integrated into a device the size of a suitcase. The equipment can be directly connected to a laptop or tablet. After commercialization, the equipment can be carried to various application scenarios.

[0012] Preferably, the solution solves the problem of difficulty in quantitative analysis. The detection means of the equipment is based on the carbon dioxide isotope internal standard method, which can realize the function of quantitative analysis, determine the content and proportion of markers in the sample, and qualitatively confirm the molecular ion peak of the compound by high-resolution mass spectrometry;

[0013] The equipment is built by connecting a soft ionization source in series with a small portable mass spectrometer to reduce the size of the equipment. After modification, the ionization source, mass spectrometer, power supply, etc. are integrated into a device the size of a suitcase. The equipment can be directly connected to a laptop or tablet (with data analysis software installed). After commercialization, the equipment can be carried to various application scenarios to achieve non-invasive, fast, and simple online real-time breath detection for the human body. This can solve the problem of the lack of dynamics in the current exhaled breath detection.

[0014] Furthermore, in step S200, the endogenous volatile organic compounds produced by human metabolism in the human exhaled breath are detected and can be used as markers of health status. The endogenous markers of human exhaled breath are defined as VOCs, and the human health index can be determined by the VOCs.

[0015] Endogenous compounds in human exhaled breath, including inorganic gases (such as NO and CO) and VOCs (such as isoprene, ethane, pentane, acetone), are metabolites of human tissues. By analyzing the components and concentrations of endogenous markers, relevant information on human tissue metabolism and health status can be obtained non-invasively, which helps to clarify the corresponding physiological processes, disease occurrence, disease progression, and pharmacological responses.

[0016] Further, in step S300, the VOCs molecular ion peaks are qualitatively confirmed by high-resolution mass spectrometry, and endogenous VOCs closely related to human tissue metabolism and health status are set with a higher priority as specific internal standards; by introducing the collected endogenous VOCs in human exhaled breath, combined with the specific internal standards, a new VOCs quantitative analysis method is established to determine the content and proportion of the introduced and collected endogenous VOCs in human exhaled breath, and in combination with the grouping of the research cohort and the detection results of VOCs in exhaled breath, a data model is established, experimental verification is carried out, and the accuracy and sensitivity of the model are determined;

[0017] Further, in step S400, the method for realizing online data transmission by optimizing the detection data includes the following specific steps:

[0018] S401: The signal transmission efficiency of data transmitted by the mass spectrometer is defined as freqi, freqi = VOCsS / ti, wherein VOCsS is the amount of VOCs data to be transmitted, ti is the time of mapping the current transmission, an empty set freq is established, the empty set freq has a time sequence, the transmission efficiency freqi is added to the empty set freq in a periodic order, freq = [freqi], i is the element number of the set freq, and the information frequency correction value Xfreqi is obtained by calculation, Among them, fred is the maximum value of the set freq and frex is the minimum value of the set freq, and the arithmetic average avg(Xfreqi) of the information frequency correction value is obtained. avg() is a function for obtaining the arithmetic average in the array;

[0019] Define Xfreqi>avg(Xfreqi) as the simple peak information in a transmission information, and record the transmission as jnum, record the number of simple peak information in the transmission information generated during the transmission data process as knum, soft ionization progress coefficient TPO,

[0020]

[0021] exp() is an exponential function, Tk is a transmission period of the transmission information, and during the mapping process, the receiving end reads the simple peak information in the transmission information by recording the number of information change values ​​knum during data transmission within the period, and represents the recorded information of the simple peak information in the successfully transmitted information as ginf. The ginf includes the record information and the sum of the simple peak information in the transmission information. The activation progress coefficient TPO and the record information ginf of successful activation are passed through the function E().

[0022]

[0023] The independent variable of the function E() is the transmission progress coefficient TPO, μ is a constant value, and exp() is an exponential function. If the value of the function E() is ≥1, the transmission information is simple peak data information after successful soft ionization, and the data information is mapped on the screen of the receiving device.

[0024] Furthermore, the method is based on a device for detecting endogenous markers in human exhaled breath, and the device includes: a collection device, a transmission device and a receiving device, the collection device, the transmission device and the receiving device run in a processor, the collection device, the transmission device and the receiving device generate data and store it in a memory, and the collection device, the transmission device, the receiving device, the processor and the memory implement any step of the above-mentioned method for online detection of endogenous markers in human exhaled breath when executing the computer program.

[0025] The beneficial effects of the present invention are: reliable, easy to operate, miniaturized, capable of real-time detection, and applicable to multiple occasions, the breath detection equipment has greater advantages in the field of instant diagnosis. The research and development of small-scale rapid breath detection equipment and the breakthrough of related technical barriers are expected to provide a new means for disease screening and diagnosis, and promote the development and application of breath diagnosis technology. The problem of quantitative analysis difficulties has been solved. The detection method of the equipment is based on the carbon dioxide isotope internal standard method, which can realize the function of quantitative analysis, determine the content and proportion of markers in the sample, and qualitatively confirm the molecular ion peaks of the compound with high-resolution mass spectrometry.

[0026] The equipment is built by connecting a soft ionization source in series with a small portable mass spectrometer to reduce the size of the equipment. After modification, the ionization source, mass spectrometer, power supply, etc. are integrated into a device the size of a suitcase. The equipment can be directly connected to a laptop or tablet (with data analysis software installed). After commercialization, the equipment can be carried to various application scenarios to achieve non-invasive, fast, and simple online real-time breath detection for humans. The full process detection time is expected to be less than 1 minute, which is 5 times more efficient than the InspectIR COVID-19 (detection time is about 3 minutes), and has better high-throughput data collection and disease screening potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other features of the present invention will become more obvious by describing in detail the embodiments shown in the accompanying drawings. The same reference numerals in the accompanying drawings of the present invention represent the same or similar elements. It is obvious that the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0028] In the figure:

[0029] Figure 1 Shown is a flow chart of an online detection method for endogenous markers in human exhaled breath. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments and drawings to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0031] In the description of the present invention, "several" means one or more, "more" means more than two, "greater than", "less than", "exceed" etc. are understood as not including the number itself, and "above", "below", "within" etc. are understood as including the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0032] like Figure 1 As shown, a method for online detection of endogenous markers in human exhaled breath comprises the following steps:

[0033] S100: Improved analytical detection methods through soft ionization + direct mass spectrometry;

[0034] S200: Collect human exhaled gas and detect the concentration of several compounds in the gas;

[0035] S300: Building a data model through the quantitative method of endogenous VOCs in human exhaled breath;

[0036] S400: Realize online data transmission by optimizing the detection data.

[0037] Furthermore, in step S100, soft ionization is a mass spectrometry analysis technology that uses relatively low energy to convert molecules into ions or fragments, thereby reducing the generation of molecular fragments, making the peak display in the mass spectrum simpler and easier to parse and identify. The ionization source used in the soft ionization scheme is a flow-through medium passage discharge source, which only utilizes the electrode discharge to momentarily excite and ionize the gaseous chemical substances on the flow path at the inlet end of the mass spectrometer, and identifies the chemical composition and form of the inflowing substance in real time. The ion source using the soft ionization scheme can be used at room temperature and pressure, and can directly and continuously soft-ionize gaseous organic small molecules online, while maintaining complete molecular ion information.

[0038] By connecting the ion source in series with a GC-MS, rapid ionization and analysis of gaseous small molecules can be achieved; known endogenous volatile organic compounds commonly found in human exhaled breath are used as reference substances for high-resolution testing and analysis, instrument parameters are adjusted, and the sensitivity of the instrument is optimized.

[0039] The soft ionization source is connected in series with a small mobile portable mass spectrometer to reduce the size of the equipment. After modification, the ionization source, mass spectrometer, power supply, etc. are integrated into a device the size of a suitcase. The equipment can be directly connected to a laptop or tablet. After commercialization, the equipment can be carried to various application scenarios.

[0040] Preferably, the solution solves the problem of difficulty in quantitative analysis. The detection means of the equipment is based on the carbon dioxide isotope internal standard method, which can realize the function of quantitative analysis, determine the content and proportion of markers in the sample, and qualitatively confirm the molecular ion peak of the compound by high-resolution mass spectrometry;

[0041] The equipment is built by connecting a soft ionization source in series with a small portable mass spectrometer to reduce the size of the equipment. After modification, the ionization source, mass spectrometer, power supply, etc. are integrated into a device the size of a suitcase. The equipment can be directly connected to a laptop or tablet (with data analysis software installed). After commercialization, the equipment can be carried to various application scenarios to achieve non-invasive, fast, and simple online real-time breath detection for the human body. This can solve the problem of the lack of dynamics in the current exhaled breath detection.

[0042] Furthermore, in step S200, the endogenous volatile organic compounds produced by human metabolism in the human exhaled breath are detected and can be used as markers of health status. The endogenous markers of human exhaled breath are defined as VOCs, and the human health index can be determined by the VOCs.

[0043] Endogenous compounds in human exhaled breath, including inorganic gases (such as NO and CO) and VOCs (such as isoprene, ethane, pentane, acetone), are metabolites of human tissues. By analyzing the components and concentrations of endogenous markers, relevant information on human tissue metabolism and health status can be obtained non-invasively, which helps to clarify the corresponding physiological processes, disease occurrence, disease progression, and pharmacological responses.

[0044] Further, in step S300, the VOCs molecular ion peaks are qualitatively confirmed by high-resolution mass spectrometry, and endogenous VOCs closely related to human tissue metabolism and health status are set with a higher priority as specific internal standards; by introducing the collected endogenous VOCs in human exhaled breath, combined with the specific internal standards, a new VOCs quantitative analysis method is established to determine the content and proportion of the introduced and collected endogenous VOCs in human exhaled breath, and in combination with the grouping of the research cohort and the detection results of VOCs in exhaled breath, a data model is established, experimental verification is carried out, and the accuracy and sensitivity of the model are determined;

[0045] Further, in step S400, the method for realizing online data transmission by optimizing the detection data includes the following specific steps:

[0046] S401: The signal transmission efficiency of data transmitted by the mass spectrometer is defined as freqi, freqi = VOCsS / ti, wherein VOCsS is the amount of VOCs data to be transmitted, ti is the time of mapping the current transmission, an empty set freq is established, the empty set freq has a time sequence, the transmission efficiency freqi is added to the empty set freq in a periodic order, freq = [freqi], i is the element number of the set freq, and the information frequency correction value Xfreqi is obtained by calculation, Among them, fred is the maximum value of the set freq and frex is the minimum value of the set freq, and the arithmetic average avg(Xfreqi) of the information frequency correction value is obtained. avg() is a function for obtaining the arithmetic average in the array;

[0047] Define Xfreqi>avg(Xfreqi) as the simple peak information in a transmission information, and record the transmission as jnum, record the number of simple peak information in the transmission information generated during the transmission data process as knum, soft ionization progress coefficient TPO,

[0048]

[0049] exp() is an exponential function, Tk is a transmission period of the transmission information, and during the mapping process, the receiving end reads the simple peak information in the transmission information by recording the number of information change values ​​knum during data transmission within the period, and represents the recorded information of the simple peak information in the successfully transmitted information as ginf. The ginf includes the record information and the sum of the simple peak information in the transmission information. The activation progress coefficient TPO and the record information ginf of successful activation are passed through the function E().

[0050]

[0051] The independent variable of the function E() is the transmission progress coefficient TPO, μ is a constant value, and exp() is an exponential function. If the value of the function E() is ≥1, the transmission information is simple peak data information after successful soft ionization, and the data information is mapped on the screen of the receiving device.

[0052] Furthermore, the method is based on a device for detecting endogenous markers in human exhaled breath, and the device includes: a collection device, a transmission device and a receiving device, the collection device, the transmission device and the receiving device run in a processor, the collection device, the transmission device and the receiving device generate data and store it in a memory, and the collection device, the transmission device, the receiving device, the processor and the memory implement any step of the above-mentioned method for online detection of endogenous markers in human exhaled breath when executing the computer program.

[0053] The processor may be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field-programmable gate arrays (FPGA) or other programmable logic devices, discrete component gate circuits or transistor logic devices, discrete hardware components, etc. A general-purpose processor may be a microprocessor or any conventional processor, etc. The processor is the control center of the system, and various sub-areas of the entire system are connected using various interfaces and lines.

[0054] The memory can be used to store the computer program and / or module, and the processor realizes various functions of the plant control system based on artificial neural network by running or executing the computer program and / or module stored in the memory, and calling the data stored in the memory. The memory can mainly include a program storage area and a data storage area, wherein the program storage area can store an operating system, an application required for at least one function (such as a sound playback function, an image playback function, etc.), etc.; the data storage area can store data created according to the use of the mobile phone (such as audio data, phone book, etc.), etc. In addition, the memory can include a high-speed random access memory, and can also include a non-volatile memory, such as a hard disk, a memory, a plug-in hard disk, a smart memory card (Smart Media Card, SMC), a secure digital (SecureDigital, SD) card, a flash card (Flash Card), at least one disk storage device, a flash memory device, or other volatile solid-state storage devices.

[0055] Preferably, in the previous research, a method for directly performing qualitative and quantitative analysis on VOCs components collected in breathing bags was successfully established. This method does not require chromatographic separation, has a fast analysis speed (<5min), and uses low-energy EI to achieve qualitative confirmation of high-resolution molecular ion peaks. After improvement, it should be possible to achieve qualitative and quantitative analysis of trace VOCs in breathing gas.

[0056] The project aims to address the current situation that quantitative determination of breath analysis technology is difficult, and the quantitative method and accurate determination of endogenous characteristic VOCs need further research. Based on the clinical demand for non-invasive, rapid, simple and reliable diagnostic methods, the project conducts research and development of breath quantitative analysis methods and rapid testing equipment. Based on mass spectrometry analysis, the breath sample collection method is optimized, and the analysis and detection method is improved by "soft ionization + direct mass spectrometry". This solution connects the soft ionization source with the gas chromatography-mass spectrometer by physical means, and modifies it to reduce the size, producing a new breath detection equipment for non-invasive, rapid and simple online real-time breath detection of the human body.

[0057] The optimization of the breath sample collection method is based on two modes: oral exhalation mode and nasal exhalation mode. Two breath collection devices are built, namely oral exhalation and nasal exhalation. Oral exhalation sampling requires pinching the nose and blowing into a disposable straw with the mouth. For nasal exhalation sampling, a fixed device is required in the mouth, the disposable straw head is placed outside the nostril, and both nostrils are exhaled alternately. Compare the differences between oral exhalation and nasal exhalation, and use the breath collection setting with high detection sensitivity, small external air interference, and good data repeatability in the current environment as needed.

[0058] Use this technology to assess human health and screen for diseases, and improve the ability to detect and prevent diseases early. Place the equipment in the hospital laboratory and wards to collect breath samples from patients with different diseases. By comparing the VOCs data of healthy people with the VOCs of patients with specific diseases (such as viral pneumonia, fungal pneumonia, bacterial pneumonia, tuberculosis, lung cancer, etc.), explore the application potential of breath detectors in disease screening and diagnosis;

[0059] Hospitals can use breath testing equipment for clinical diagnosis, helping doctors to more accurately judge the condition and provide more effective treatment plans;

[0060] Patients can obtain timely health assessments and disease diagnoses through non-invasive and rapid breath testing, and improve their self-health awareness and management capabilities.

[0061] Although the description of the present invention has been quite detailed and has been described in particular with respect to several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, so as to effectively cover the intended scope of the present invention. In addition, the present invention is described above with the embodiments foreseeable by the inventors, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that are not currently foreseen may still represent equivalent changes of the present invention.

Claims

1. A method for online detection of endogenous markers in human exhaled breath, characterized in that: The method comprises the following steps: S100: Improved analytical detection methods through soft ionization + direct mass spectrometry; S200: Collect human exhaled gas and detect the concentration of several compounds in the gas; S300: Building a data model through the quantitative method of endogenous VOCs in human exhaled breath; S400: Realize online data transmission by optimizing the detection data.

2. The method for online detection of endogenous markers in human exhaled breath according to claim 1, characterized in that: In step S100, soft ionization is a mass spectrometry analysis technology that uses relatively low energy to convert molecules into ions or fragments, reducing the generation of molecular fragments, making the peak display in the mass spectrum simpler and easier to analyze and identify. The ionization source used in the soft ionization scheme is a flow-through medium channel discharge source, which only utilizes the electrode discharge to momentarily excite and ionize the gaseous chemical substances on the flow path at the mass spectrometer inlet end, and identifies the chemical composition and form of the inflowing substance in real time. The ion source using the soft ionization scheme can be used at room temperature and pressure, directly and continuously soft-ionizes gaseous organic small molecules online, and maintains complete molecular ion information. By connecting the ion source in series with a GC-MS, rapid ionization and analysis of gaseous small molecules can be achieved; known endogenous volatile organic compounds commonly found in human exhaled breath are used as reference substances for high-resolution testing and analysis, instrument parameters are adjusted, and the sensitivity of the instrument is optimized. The soft ionization source is connected in series with a small mobile portable mass spectrometer to reduce the size of the equipment. After modification, the ionization source, mass spectrometer, power supply, etc. are integrated into a device the size of a suitcase. The equipment can be directly connected to a laptop or tablet. After commercialization, the equipment can be carried to various application scenarios.

3. The method for online detection of endogenous markers in human exhaled breath according to claim 1, characterized in that: In step S200, the endogenous volatile organic compounds produced by several compounds in the human exhaled gas without being metabolized by the human body can be used as markers of health status. The endogenous markers of human exhaled gas are defined as VOCs, and the human health index can be determined by the VOCs.

4. The method for online detection of endogenous markers in human exhaled breath according to claim 1, characterized in that: In step S300, the VOCs molecular ion peaks are qualitatively confirmed by high-resolution mass spectrometry, and endogenous VOCs that are closely related to human tissue metabolism and health status are set with a higher priority as specific internal standards; by introducing the collected endogenous VOCs in human exhaled breath, combined with the specific internal standards, a new VOCs quantitative analysis method is established to determine the content and proportion of the introduced and collected endogenous VOCs in human exhaled breath, and combined with the grouping of the research cohort and the detection results of VOCs in exhaled breath, a data model is established, experimental verification is carried out, and the accuracy and sensitivity of the model are determined.

5. The method for online detection of endogenous markers in human exhaled breath according to claim 1, characterized in that: In step S400, the method for realizing online data transmission by optimizing the detection data includes the following specific steps: S401: The signal transmission efficiency of data transmitted by the mass spectrometer is defined as freqi, freqi = VOCsS / ti, wherein VOCsS is the amount of VOCs data to be transmitted, ti is the time of mapping the current transmission, an empty set freq is established, the empty set freq has a time sequence, the transmission efficiency freqi is added to the empty set freq in a periodic order, freq = [freqi], i is the element number of the set freq, and the information frequency correction value Xfreqi is obtained by calculation, Among them, fred is the maximum value of the set freq and frex is the minimum value of the set freq, and the arithmetic average avg(Xfreqi) of the information frequency correction value is obtained. avg() is a function for obtaining the arithmetic average in the array; Define Xfreqi>avg(Xfreqi) as the simple peak information in a transmission information, and record the transmission as jnum, record the number of simple peak information in the transmission information generated during the transmission data process as knum, soft ionization progress coefficient TPO, exp() is an exponential function, Tk is a transmission period of the transmission information, and during the mapping process, the receiving end reads the simple peak information in the transmission information by recording the number of information change values ​​knum during data transmission within the period, and represents the recorded information of the simple peak information in the successfully transmitted information as ginf. The ginf includes the record information and the sum of the simple peak information in the transmission information. The activation progress coefficient TPO and the record information ginf of successful activation are passed through the function E(). The independent variable of the function E() is the transmission progress coefficient TPO, μ is a constant value, and exp() is an exponential function. If the value of the function E() is ≥1, the transmission information is simple peak data information after successful soft ionization, and the data information is mapped on the screen of the receiving device.

6. The method for online detection of endogenous markers in human exhaled breath according to claim 1, characterized in that: The method is based on a device for detecting endogenous markers in human exhaled breath, and the device comprises: a collection device, a transmission device and a receiving device, wherein the collection device, the transmission device and the receiving device are operated in a processor, and the data generated by the collection device, the transmission device and the receiving device are stored in a memory, and the collection device, the transmission device, the receiving device, the processor and the memory implement the steps of any one of the methods for online detection of endogenous markers in human exhaled breath in claims 1-5 when executing the computer program.

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