[go: up one dir, main page]

WO2020253518A1 - Procédé d'immunoessai de filtrage de spectre de fluorescence à excitation ultraviolette continue - Google Patents

Procédé d'immunoessai de filtrage de spectre de fluorescence à excitation ultraviolette continue Download PDF

Info

Publication number
WO2020253518A1
WO2020253518A1 PCT/CN2020/093941 CN2020093941W WO2020253518A1 WO 2020253518 A1 WO2020253518 A1 WO 2020253518A1 CN 2020093941 W CN2020093941 W CN 2020093941W WO 2020253518 A1 WO2020253518 A1 WO 2020253518A1
Authority
WO
WIPO (PCT)
Prior art keywords
light source
ultraviolet
excitation light
signal
processing module
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/093941
Other languages
English (en)
Chinese (zh)
Inventor
焦迪
李建国
应继伟
汪邦运
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Future Biotechnology Co Ltd
Original Assignee
Shanghai Future Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Future Biotechnology Co Ltd filed Critical Shanghai Future Biotechnology Co Ltd
Publication of WO2020253518A1 publication Critical patent/WO2020253518A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the present invention relates to the technical field of immunochromatography, in particular to a multifunctional immunological detection method and system.
  • Time-resolved fluorescence immunoassay is a fluorescence immunoassay method that uses complexes of rare earth ions as markers and delay measurement time to eliminate background to improve detection sensitivity.
  • the luminescence mechanism of ionic complexes is characterized by Eu3+, Tb3+, Sm3+ and Dy3+ ions and some ligands (such as ⁇ -diketones, aromatic amines and other ligands) forming complexes that absorb ultraviolet light and emit very Strong metal ion characteristic fluorescence.
  • some ligands such as ⁇ -diketones, aromatic amines and other ligands
  • the excitation energy from the triplet state is higher than the energy level of the rare earth ion , And then transfer energy to rare earth ions.
  • the rare earth ions are excited to the resonance energy level after receiving the transferred energy, and emit fluorescence during the transition from the resonance energy level to the ground state, which is the characteristic fluorescence of the rare earth ions.
  • This luminescence is produced by the energy transfer from the ligand to the central ion in the complex. Based on this luminescence mechanism, the fluorescence luminescence of the rare earth complex has the following characteristics compared to ordinary organic fluorescent markers:
  • the fluorescence emission of rare earth ions has an extremely narrow bandwidth, usually 20 to 30 nm.
  • the background fluorescence noise mainly comes from scattered light and non-specific fluorescence produced by various coexisting substances in the tested sample, and the lifetime is usually between 1-10 ns (nanoseconds). Since the fluorescence lifetime of rare earth markers is usually more than 100 ⁇ s, it lays a physical foundation for the realization of time-resolved fluorescence measurement technology: the fluorescence emitted by long-lived fluorescent substances is measured after the background noise fluorescence is quenched, so as to avoid background fluorescence noise and improve Determination of sensitivity.
  • the time-resolved fluorescence detection method based on the above physical mechanism has the advantages of high sensitivity, strong specificity, and no radiation pollution.
  • As a promising label immunoassay ultra-micro biochemical quantitative analysis method it is widely used in various on-site real-time detection occasions. Enumerate the following applications:
  • Tumor markers refer to a class of substances produced by tumor tissues that are present in tumor tissues or secreted into blood or other body fluids or are produced by host cells due to tumor tissue stimulation and whose content is significantly higher than the normal reference value. By determining its presence or content in the body, it can help diagnose tumors, analyze the course of the disease, guide treatment, monitor recurrence or metastasis, and judge prognosis.
  • the sites for detecting tumor markers include liver, lung, gastrointestinal tract, pancreas, prostate, bladder, ovary, breast, etc. Common test indicators are: alpha-fetoprotein, carcinoembryonic antigen, prostate specific antigen, transferrin, calprotectin, gastric Helicobacter pylori antigen antibody and so on.
  • myocardial damage protein markers has improved the specificity and sensitivity of acute coronary syndrome (ACS) diagnosis, and can reflect minor cardiomyopathy and unstable angina pectoris, as well as evaluate the treatment effect, determine the prognosis of the disease, and ACS risk is stratified.
  • Common detection indicators are: cardiac troponin I, myoglobin, creatine kinase isoenzyme, heart-shaped fatty acid binding protein, N-terminal pro-brain natriuretic peptide, C-reactive protein, procalcitonin, etc.
  • Infectious diseases are diseases that are caused by pathogenic microorganisms and can be spread to others.
  • the analyzer can be used for the detection of various infectious diseases. Common test indicators are: A, B, C, D, hepatitis E virus, encephalitis virus, respiratory syncytial virus, rotavirus, adenovirus, hemorrhagic fever virus, syphilis, cytomegalovirus, rubella virus, HIV, etc.
  • Diabetes is a chronic and systemic metabolic disease caused by the long-term combined action of genetic factors and environmental factors. If diabetes is not well controlled for a long time, it can also cause damage to multiple systems, leading to chronic complications of tissues and organs such as eyes, kidneys, nerves, blood vessels, and heart. Common detection indicators are: microalbumin, glycosylated hemoglobin, insulin, C peptide, cystatin C, ⁇ 2 microglobulin, neutrophil gelatinase-related lipocalin, alfa1 microglobulin, etc.
  • test indicators are: human chorionic gonadotropin, luteinizing hormone, follicle stimulating hormone, fetal fibronectin, insulin-like growth factor binding protein, sperm acrosomal protein 10, Neisseria gonorrhoeae, Trichomonas vaginalis , Chlamydia trachomatis, Candida albicans, etc.
  • the thyroid is an important organ indispensable for normal human survival. Diseases of the thyroid gland can cause various metabolic disorders.
  • test indicators are: thyroxine, free thyroxine, total triiodothyronine, total thyroxine, free triiodothyronine, free thyroxine and so on.
  • drugs refer to opium, heroin, methamphetamine (meth), morphine, marijuana, cocaine, and other narcotic drugs and psychotropic drugs that can cause addiction to people under the control of the state.
  • methamphetamine meth
  • morphine morphine
  • marijuana morphine
  • cocaine and other narcotic drugs and psychotropic drugs that can cause addiction to people under the control of the state.
  • control of drug abuse has become an important task of government security agencies.
  • Common test indicators are: opium, heroin, amphetamine, methamphetamine (meth), morphine, cannabis, cocaine, 3,4-methylenedioxy-N-methamphetamine (ecstasy), phencyclidine , Methadone, ketamine, hymeninone, propoxyphene, tricyclic antidepressants, benzodiazepines, barbiturates, etc.
  • pet and animal disease detection include: canine influenza virus, canine adenovirus, canine coronavirus, canine parvovirus, rabies virus, feline leukemia antigen, feline caliciform antigen, cat fever virus, swine fever antigen antibody, etc.
  • the common indicators of autoantibody detection are: anti-nuclear antibody, anti-ENA antibody, anti-cardiolipin antibody, anti-ds-DNA antibody, anti-Sm antibody, anti-SS-DNA antibody, etc.
  • the pulsed ultraviolet light source is used as the excitation signal during the detection process, and the intensity of the pulsed excitation light source is difficult to achieve real-time control, which is bound to affect The fluorescence spectrum intensity as the output response; 2).
  • the pulsed ultraviolet light source device In the pulse working mode, the pulsed ultraviolet light source device has been subjected to the shock response of the electric pulse, which will cause its service life to be greatly shortened, and the luminous intensity will also be sharply attenuated, 3).
  • the time difference method is used to separate the response fluorescence spectrum in the dynamic decay process, that is, the fluorescence spectrum in the dynamic decay period.
  • the dynamic light intensity decay rate is related to the intensity of the excitation signal, the concentration of the marker and other factors, which will affect the detection accuracy .
  • the purpose of the present invention is to provide a multifunctional immunoassay method and system that effectively solves the insufficient detection accuracy of the existing time-resolved fluorescence immunoassay and is compatible with the detection of fluorescent quantum dots and colloidal gold test paper.
  • a continuous excitation multifunctional immune detection method including:
  • the ultraviolet spectrum in the response spectrum signal is filtered by a color filter placed in the reflection light receiving area.
  • the sample to be tested is a fluorescent test paper
  • the fluorescence detection result is obtained according to the quality control C line and the detection T line in the response spectrum signal.
  • the spectral signal receiving device is a CCD sensor or a photomultiplier tube.
  • the step of controlling the stable output of the ultraviolet excitation light source is further included:
  • a light intensity control signal is generated according to the light signal, and then the ultraviolet excitation light source is controlled to continuously and stably output by the light intensity control signal.
  • the method further includes: receiving a control instruction and switching the excitation light source according to the test mode selected in the control instruction:
  • the ultraviolet excitation light source is controlled to continuously illuminate the sample to be tested, and at the same time, the color filter is controlled to move to the reflected light receiving area.
  • the method includes the following steps:
  • the response spectrum signal is received by the spectrum signal receiving device, and the colloidal gold detection result is obtained for the quality control C line and the detection T line in the response spectrum signal.
  • test mode selected in the received control instruction is the initialization calibration mode
  • it also includes using preselected standard fluorescent test paper to calibrate the light intensity of the ultraviolet excitation light source one by one and/or use preselected
  • the standard colloidal gold test paper is the step of correcting the light intensity of the broad-spectrum excitation light source one by one.
  • the invention also provides a continuous excitation multifunctional immune detection system, including:
  • At least one ultraviolet excitation light source arranged relative to the detection area and connected to the processing module, for continuously irradiating the sample to be tested placed in the detection area under the control of the processing module;
  • Ultraviolet filter device placed in the reflected light receiving area, used to filter the ultraviolet spectrum in the response spectrum signal;
  • the spectral signal receiving device is placed in the reflected light receiving area and connected to the processing module for receiving the response spectral signal after ultraviolet filtering by the ultraviolet filtering device, and sending it to the processing module for processing to obtain the fluorescence detection result.
  • the ultraviolet filter device is a color filter, which filters the ultraviolet spectrum in the response spectrum signal in the reflected light receiving area.
  • the sample to be detected is a fluorescent test paper
  • the spectral signal receiving device is a CCD sensor or a photomultiplier tube
  • the spectrum signal receiving device receives the response spectrum signal after ultraviolet filtering and sends it to the processing module, and the processing module obtains the fluorescence detection result according to the quality control C line and the detection T line in the response spectrum signal.
  • the multifunctional immune detection system further includes a light receiving device connected to the processing module and placed around the ultraviolet excitation light source for receiving the light signal emitted by the ultraviolet excitation light source and sending it to the processing module;
  • the processing module After the processing module receives the optical signal, it compares the received optical signal intensity with a preset threshold; when the received optical signal intensity deviates from the preset threshold, a light intensity control signal is generated according to the optical signal, and then the light intensity is passed
  • the control signal controls the UV excitation light source to continuously and stably output.
  • the multifunctional immune detection system further includes at least one broad-spectrum excitation light source, and the processing module is further configured to receive a control instruction and switch the excitation light source according to the test mode selected in the control instruction; the ultraviolet filter device Connect with the processing module through a transmission device;
  • the processing module controls the ultraviolet excitation light source to continuously irradiate the sample to be tested, and at the same time controls the transmission device to move the ultraviolet filter device to the reflected light receiving area.
  • the processing module controls the broad-spectrum excitation light source to continuously irradiate the sample to be tested, and remove the ultraviolet filter device, the sample to be tested is a colloidal gold test paper;
  • the spectral signal receiving device receives the response spectral signal and sends it to the processing module.
  • the processing module obtains the colloidal gold detection result for the quality control C line and the detection T line in the response spectral signal.
  • test mode selected in the received control instruction is the initialization correction mode
  • the processing module controls the ultraviolet excitation light source to irradiate the pre-selected standard fluorescent test paper one by one, and corrects the light intensity of the ultraviolet excitation light source according to the intensity of the response spectrum signal after ultraviolet filtering;
  • the processing module controls the broad-spectrum excitation light source to irradiate the pre-selected standard colloidal gold test paper one by one, and corrects the light intensity of the broad-spectrum excitation light source according to the intensity of the response spectrum signal.
  • the present invention discards the pulsed ultraviolet or broad-spectrum light source excitation mode with unstable light intensity, adopts pulse width modulation and low-pass filtering technology to control the luminous intensity of the light source, and uses a continuous light source with constant luminous intensity as the excitation light source (including ultraviolet excitation light source) And broad-spectrum excitation light source), which effectively solves the defect that the luminous intensity of the excitation light source in the system changes with the number of times of use and affects the intensity of the response spectrum signal, and at the same time avoids the technical problem that the excitation light source is affected by frequent electrical surges affecting the service life , Which greatly extends the life of the excitation light source in the system.
  • the ultraviolet excitation light source continuously irradiates the sample to be tested (fluorescence test paper), and the fluorescence spectrum in the response spectrum signal is always in a stable state without dynamic attenuation.
  • the optical filter is used to filter out the background noise, and the fluorescence spectrum in a stable state is screened out, and then the fluorescence detection is performed, which effectively avoids the time-resolved fluorescence immunoassay method and the time difference method in the dynamic attenuation.
  • the situation of the fluorescence spectrum in the process greatly improves the detection accuracy.
  • the present invention uses a CCD sensor or a photomultiplier tube to detect the quality control C line and T line in the fluorescence spectrum of the fluorescent test paper to achieve fluorescence detection.
  • the use of a high-sensitivity CCD sensor or photomultiplier tube effectively overcomes the problems in the prior art
  • the technical problem of insufficient photosensitive sensitivity when the photosensitive receiving tube is used for detection further improves the detection accuracy.
  • the device provided by the present invention can simultaneously realize high-precision measurement of fluorescence (including fluorescent quantum dots) and colloidal gold modes, and can realize self-correction of system detection accuracy at any time, which greatly expands the application range.
  • Fig. 1 is a schematic diagram of an embodiment of the continuous excitation multifunctional immune detection method in the present invention
  • Figure 2 is a response spectrum diagram of the present invention after being filtered by a color filter
  • Figure 3 is a schematic structural diagram of an embodiment of the continuous excitation multifunctional immune detection system in the present invention.
  • Figure 4 is a schematic structural diagram of an example of the multifunctional immune detection system of the present invention.
  • the sample to be tested 20-UV excitation light source, 30-UV filter device, 40-spectral signal receiving device, 1-test paper cassette, 2-first micro stepping motor, 3-excitation light source, 4-photosensitive receiving tube , 5-color filter, 6-second micro stepping motor, 7-CCD sensor, 8-processing module, 9-closed black body cavity composition.
  • the present invention uses rare earth materials to generate fluorescence with a large Stokes shift, uses electronic processing technology to generate continuous and stable luminous intensity ultraviolet light source excitation and uses narrow-band spectral filtering to separate effectively Fluorescence spectroscopy provides a new method for biochemical and quantitative detection of ultra-micro amounts of labeling immunoassay.
  • the continuous excitation multifunctional immune detection method includes:
  • S20 controls at least one ultraviolet excitation light source set relative to the detection area to continuously irradiate the sample to be detected;
  • S30 receives the response spectrum signal after ultraviolet filtering, and obtains the fluorescence detection result according to the received response spectrum signal.
  • the ultraviolet excitation light source is controlled (the ultraviolet excitation light source is always in working state during the entire fluorescence detection process) to irradiate the sample to be tested, and the fluorescence spectrum generated by the sample to be tested is mixed with the reflected ultraviolet spectrum to form a response spectrum signal into the reflected light receiving area to Therefore, after filtering the ultraviolet spectrum in the response spectrum signal in the reflected light receiving area, the fluorescence spectrum can be obtained, and the fluorescence detection of the sample to be detected is completed.
  • the entire detection process is carried out in a closed black body cavity to shield external stray light interference.
  • the ultraviolet excitation light source is a continuous ultraviolet light source, and it is in a constant light intensity state under the control of a light intensity control signal (specifically a pulse width modulation signal, referred to as a PWM signal) sent from the outside, so as to control the light intensity of the ultraviolet excitation light source.
  • a light intensity control signal specifically a pulse width modulation signal, referred to as a PWM signal
  • PWM signal pulse width modulation signal
  • the number of ultraviolet excitation light sources can be set according to actual application requirements. For example, configure 1 ultraviolet excitation light source, configure 2 ultraviolet excitation light sources, 3 ultraviolet excitation light sources or more, as long as the purpose of the invention can be achieved, continue to stimulate the pending detection
  • the sample can excite the fluorescence spectrum.
  • multiple ultraviolet excitation light sources the arrangement can also be adjusted according to actual applications. For example, when two ultraviolet excitation light sources are configured, the two ultraviolet excitation light sources are placed symmetrically with respect to the sample to be tested; when six ultraviolet excitation light sources are configured , Set it around the sample to be tested for uniform irradiation.
  • the filtering of the ultraviolet spectrum in the response spectrum signal is specifically realized by a color filter placed in the reflected light receiving area, for example, in an example, as shown in Figure 2 (the abscissa is the wavelength and the unit is nm; the ordinate is the color filter The spectral transmittance of the plate, in %).
  • the color filter filters out the spectral signals with a wavelength below 500nm (nanometers) in the response spectral signal, and retains the spectral signals with a wavelength above 510nm (the wavelength of the fluorescence spectrum is between 580 and 620nm). Between), to achieve ultraviolet filtering in the response spectrum signal.
  • the sample to be detected is a fluorescent test paper (including quantum dot fluorescence detection), and a spectral signal receiving device is used to receive the response spectral signal after ultraviolet filtering.
  • the spectral signal receiving device is a CCD sensor or a photomultiplier tube.
  • the ultraviolet excitation light source is then controlled to irradiate the fluorescent test paper.
  • the response spectrum signal reaches the photosensitive lens of the CCD sensor (the color filter is placed before the photosensitive lens of the CCD sensor) after being filtered by the ultraviolet filter of the color filter, and the fluorescence detection result is obtained according to the spectrum signal obtained by the CCD sensor.
  • the fluorescence test of the fluorescent test paper is completed according to the quality control C line and the detection T line of the fluorescent test paper.
  • the quality control C line is used to test the validity of the fluorescent test paper under the effect of storage, transportation, or other factors, and the T line is tested Used to characterize the results of fluorescence detection.
  • the quality control C line is valid, if there is no detection T line related to the fluorescence detection content, the result is characterized as "negative”; otherwise, the result is characterized as "positive” and the fluorescence intensity of the detection T line is excited. The degree of "positive" of the detected content.
  • the above-mentioned embodiment is improved to obtain a new embodiment.
  • the light intensity of the ultraviolet excitation light source is monitored in real time through a feedback mechanism.
  • the light receiving device (such as photosensitive receiving tube) around the light source receives the light signal emitted by the ultraviolet excitation light source and converts it into an electrical signal, and then obtains the light intensity of the ultraviolet excitation light source according to the electrical signal, and compares it with a preset threshold to determine Whether the UV excitation light source is in a stable output state.
  • a light intensity control signal (adjusting the duty cycle in the signal) is generated according to the light signal, and the output of the ultraviolet excitation light source is controlled by the light intensity control signal to ensure ultraviolet
  • the excitation light source outputs stably with a preset light intensity.
  • the placement position of the light receiving device is not specifically limited here, and it is only necessary to establish in advance the correlation between the intensity of the light signal received by the light receiving device and the light intensity of the ultraviolet excitation light source.
  • the light receiving device is arranged around one of the ultraviolet excitation light sources for monitoring, and the luminous intensity of each ultraviolet excitation light source is adjusted according to the monitoring results.
  • multiple light receiving devices may be provided, or even one light receiving device may be configured for each ultraviolet excitation light source, so as to realize accurate monitoring of each ultraviolet excitation light source.
  • At least one ultraviolet excitation light source in addition to at least one ultraviolet excitation light source, at least one broad-spectrum excitation light source (ordinary light source) is also configured in the system. Specifically, after receiving the control instruction sent by the inspector, the corresponding excitation light source is switched according to the test mode selected in the control instruction. The control command can be received in any existing way.
  • test mode selected in the received control instruction is the initialization calibration mode
  • use the preselected standard fluorescent test paper to calibrate the light intensity of the ultraviolet excitation light source one by one and/or use the preselected standard colloidal gold test paper
  • the light intensity of the broad-spectrum excitation light source is calibrated one by one, wherein the standard fluorescent test paper and the standard colloidal gold test paper are both test papers pre-selected as calibration standards.
  • the ultraviolet excitation light source is controlled to continuously illuminate the sample to be tested, and the color filter is driven to the reflected light receiving area by controlling the stepping motor (when the system is in standby, the color filter is The film is in the retracted state).
  • the spectral signal receiving device receives the response spectral signal through the ultraviolet filter, the fluorescence detection result is obtained according to the quality control C line and the detection T line therein.
  • the broad-spectrum excitation light source is controlled to continuously illuminate the sample to be tested.
  • the sample to be tested is colloidal gold test paper; the response spectrum signal of the colloidal gold test paper enters the reflected light receiving area And enter the spectral signal receiving device, and then obtain the colloidal gold detection result according to the quality control C line and the detection T line in the detected spectral signal.
  • the light intensity of the broad-spectrum excitation light source is also in a constant light intensity state under the control of the light control signal (PWM signal) sent from the outside during the working process.
  • the light intensity of the broad-spectrum excitation light source is controlled by a feedback mechanism.
  • the monitoring is carried out. Specifically, a light receiving device (such as a photosensitive receiving tube) placed around the broad-spectrum excitation light source receives the light signal emitted by the broad-spectrum excitation light source and converts it into an electrical signal, and then obtains the broad-spectrum excitation light source according to the electrical signal.
  • the light intensity is compared with a preset threshold to determine whether the broad-spectrum excitation light source is in a stable output state.
  • a light intensity control signal is generated according to the light signal, and the output of the broad-spectrum excitation light source is controlled by the light intensity control signal to ensure that the broad-spectrum excitation light source is preset Stable light output.
  • the system is configured with both an ultraviolet excitation light source and a broad-spectrum excitation light source, and switches according to the test mode selected by the inspector, so that the system can simultaneously achieve high-precision measurement of fluorescence and colloidal gold modes.
  • multiple ultraviolet excitation light sources and multiple spectral excitation light sources are configured in the system at the same time, they can be arranged in a spaced manner or in other ways without affecting the irradiation of the sample to be tested.
  • the location of the light receiving device is also not specifically limited here, as long as it is fixed around the ultraviolet excitation light source and the broad-spectrum excitation light source, the intensity of the light signal received by the light receiving device and the light intensity emitted by the excitation light source are measured in advance. The relationship between the two.
  • multiple ultraviolet excitation light sources and spectral excitation light sources are configured in the system at the same time, and they are arranged in a circle around the detection area at intervals, the sample to be tested is placed in the center position, and the photosensitive receiving tube is set in A group of excitation light sources (an ultraviolet excitation light source and a spectral excitation light source are a group) to realize real-time monitoring of the light intensity of the excitation light source; the CCD sensor is set at the center of the excitation light source to receive the maximum response spectrum signal.
  • the ultraviolet excitation light source is controlled to simultaneously irradiate the sample to be tested; when the inspector selects the colloidal gold test mode, the broad-spectrum excitation light source is controlled to simultaneously irradiate the sample to be tested.
  • the fluorescence detection system includes: a processing module (not shown in the figure), at least one ultraviolet excitation light source 20.
  • the ultraviolet filter device 30 and the spectral signal receiving device 40 wherein the ultraviolet excitation light source 20 and the spectral signal receiving device 40 are respectively connected to the processing module, and the ultraviolet excitation light source 20 continuously irradiates the to-be-detected area placed in the detection area under the control of the processing module Sample 10; the ultraviolet filter device 20 filters the ultraviolet spectrum in the response spectrum signal in the reflected light receiving area; the spectrum signal receiving device 40 obtains the response spectrum signal after ultraviolet filtering by the ultraviolet filter device, and sends it to the processing module for processing to obtain the fluorescence Test results.
  • the processing module controls the ultraviolet excitation light source (the ultraviolet excitation light source is always working during the fluorescence detection process) to irradiate the sample to be tested, and the fluorescence spectrum generated by the sample to be tested is mixed with the reflected ultraviolet spectrum to form a response spectrum signal into the reflected light receiving area
  • the ultraviolet filter device filters the ultraviolet spectrum in the response spectrum signal in the reflected light receiving area to obtain a fluorescence spectrum
  • the spectral signal receiving device receives the fluorescence spectrum to realize fluorescence detection of the sample to be detected.
  • the entire detection process is carried out in a closed black body cavity to shield external stray light interference.
  • the ultraviolet excitation light source is a continuous ultraviolet light source, and is in a constant light intensity state under the control of the light intensity control signal (PWM signal) sent by the processing module, so as to effectively control the light intensity of the ultraviolet excitation light source.
  • PWM signal light intensity control signal
  • the randomness of the excitation light source intensity in the pulsed excitation method is avoided, and the fluorescence detection by testing the fluorescence spectrum in the dynamic decay period is avoided, which greatly improves the accuracy of fluorescence detection.
  • the number of ultraviolet excitation light sources is not specifically limited here, and it can be set according to actual application requirements. For example, one ultraviolet excitation light source can be configured, or two ultraviolet excitation light sources, three ultraviolet excitation light sources or more can be configured.
  • the sample to be tested can be continuously excited to excite the fluorescence spectrum.
  • the arrangement can also be adjusted according to actual applications. For example, when two ultraviolet excitation light sources are configured, the two ultraviolet excitation light sources are placed symmetrically with respect to the sample to be tested; when six ultraviolet excitation light sources are configured , Set it around the sample to be tested for uniform irradiation.
  • the ultraviolet filtering device is specifically a color filter, and when performing ultraviolet filtering on the response spectrum signal, the ultraviolet spectrum in the response spectrum signal is filtered through the color filter placed in the reflection light receiving area.
  • the filtering wavelength of the color filter can be selected according to the actual application. For example, the color filter filters out the spectral signals with a wavelength below 500 nm in the response spectral signal, and retains the spectral signals with a wavelength above 510 nm.
  • the sample to be detected is a fluorescent test paper, and a spectral signal receiving device is used to receive the response spectral signal after ultraviolet filtering.
  • the spectral signal receiving device is a CCD sensor or a photomultiplier tube.
  • the processing module controls the ultraviolet excitation light source to illuminate the fluorescence test paper.
  • the response spectrum signal reaches the photosensitive lens of the CCD sensor (the color filter is placed before the photosensitive lens of the CCD sensor) after being UV filtered by the color filter, and the fluorescence detection result is obtained according to the spectrum signal obtained by the CCD sensor.
  • the fluorescence test of the fluorescent test paper is completed according to the quality control C line and the detection T line of the fluorescent test paper.
  • the quality control C line is used to test the validity of the fluorescent test paper under the effect of storage, transportation, or other factors, and the T line is tested Used to characterize the results of fluorescence detection.
  • the quality control C line is valid, if there is no detection T line related to the fluorescence detection content, the result is characterized as "negative”; otherwise, the result is characterized as "positive” and the fluorescence intensity of the detection T line is excited. The degree of "positive" of the detected content.
  • the fluorescence detection system includes a processing module, an ultraviolet excitation light source, an ultraviolet filter device, and a spectral signal receiving device, as well as a device arranged around the ultraviolet excitation light source.
  • Light receiving device (such as photosensitive receiving tube).
  • the light receiving device monitors the light intensity of the ultraviolet excitation light source in real time. Specifically, the light receiving device receives the light signal emitted by the ultraviolet excitation light source and converts it into an electrical signal and sends it to the processing module.
  • the light intensity of the ultraviolet excitation light source is obtained according to the electrical signal, and it is compared with a preset threshold to determine whether the ultraviolet excitation light source is in a stable output state.
  • a light intensity control signal is generated according to the light signal, and the output of the ultraviolet excitation light source is controlled by the light intensity control signal (PWM signal) to ensure that the ultraviolet excitation light source is preset Stable output at a certain light intensity.
  • PWM signal light intensity control signal
  • the placement position of the light receiving device is not specifically limited here, and it is only necessary to establish in advance the correlation between the intensity of the light signal received by the light receiving device and the light intensity of the ultraviolet excitation light source.
  • the light receiving device is arranged around one of the ultraviolet excitation light sources for monitoring, and the luminous intensity of each ultraviolet excitation light source is adjusted according to the monitoring result.
  • multiple light receiving devices can also be provided, or even one light receiving device for each ultraviolet excitation light source, so as to realize accurate monitoring of each ultraviolet excitation light source.
  • the fluorescence detection system includes a processing module, an ultraviolet excitation light source, an ultraviolet filter device, a spectral signal receiving device, and a light receiving device, as well as at least one broad spectrum Excitation light source and a transmission device, the processing module switches the excitation light source according to the test mode selected in the received control instruction; the ultraviolet filter device is connected to the processing module through a transmission device, and the processing module drives the ultraviolet filter device through the transmission device to move to the preset Fixed location.
  • the preselected standard fluorescent test paper is used to calibrate the light intensity of the ultraviolet excitation light source one by one and/or use the preselected
  • the standard colloidal gold test paper is used to calibrate the light intensity of the broad-spectrum excitation light source one by one.
  • the standard fluorescent test paper and the standard colloidal gold test paper are both test papers pre-selected as calibration standards.
  • the processing module controls an ultraviolet excitation light source to be calibrated to continuously illuminate the standard fluorescent test paper; after receiving the ultraviolet filtered response spectrum signal, it is judged whether the intensity of the response spectrum signal reaches the preset light intensity If not, generate a light intensity control signal to control the ultraviolet excitation light source to output at a preset light intensity. Repeat this cycle until all the UV excitation light sources in the system are calibrated.
  • the processing module controls a broad-spectrum excitation light source to be corrected to continuously illuminate the standard colloidal gold test paper; after receiving the response spectrum signal, it is judged whether the intensity of the response spectrum signal reaches the preset light intensity, If not, generate a light intensity control signal to control the broad-spectrum excitation light source to output at a preset light intensity. In this cycle, until all the spectral excitation light source correction in the system is completed.
  • the processing module controls the ultraviolet excitation light source to continuously irradiate the fluorescent test paper, and at the same time controls the transmission device to move the ultraviolet filter device to the reflected light receiving area.
  • the fluorescence spectrum generated by the sample to be tested is mixed with the reflected ultraviolet spectrum to form a response spectrum signal and enters the reflected light receiving area.
  • the ultraviolet filter device filters the ultraviolet spectrum in the response spectrum signal in the reflected light receiving area to obtain the fluorescence spectrum, which is received by the spectral signal receiving device
  • the fluorescence spectrum realizes the fluorescence detection of the sample to be tested.
  • the processing module controls the broad-spectrum excitation light source to continuously irradiate the colloidal gold test paper, and the spectral signal receiving device obtains the response spectrum signal and sends it to the processing module for processing to obtain the fluorescence detection result.
  • the light intensity of the broad-spectrum excitation light source is also in the processing module of the light
  • a feedback mechanism is used to monitor the light intensity of the broad-spectrum excitation light source.
  • a light receiving device such as a photosensitive receiving tube placed around the broad-spectrum excitation light source receives the light signal emitted by the broad-spectrum excitation light source and converts it into electricity. Signal, and then obtain the light intensity of the broad-spectrum excitation light source according to the electrical signal, and compare it with a preset threshold to determine whether the broad-spectrum excitation light source is in a stable output state.
  • a light intensity control signal is generated according to the light signal, and the output of the broad-spectrum excitation light source is controlled by the light intensity control signal to ensure that the broad-spectrum excitation light source is preset Stable light output.
  • the system is configured with both an ultraviolet excitation light source and a broad-spectrum excitation light source, and switches according to the test mode selected by the inspector, so that the system can simultaneously achieve high-precision measurement of fluorescence and colloidal gold modes.
  • multiple ultraviolet excitation light sources and multiple spectral excitation light sources are configured in the system at the same time, they can be arranged in a spaced manner or in other ways without affecting the irradiation of the sample to be tested.
  • the location of the light receiving device is also not specifically limited here, as long as it is fixed around the ultraviolet excitation light source and the broad-spectrum excitation light source, the intensity of the light signal received by the light receiving device and the light intensity emitted by the excitation light source are measured in advance. The relationship between the two.
  • the multifunctional immune detection system is also equipped with an LCD touch screen, and the inspector sends control instructions including test mode information through the LCD touch screen; and after the processing module receives the response spectrum signal, the processing result is displayed on the LCD In the touch screen, in this way, the inspector obtains the detection result (including whether the corresponding peak exists, the intensity of the peak, etc.) according to the peaks of the C line and T line displayed on the LCD touch screen.
  • the multifunctional immune detection system is also equipped with a communication module, and the multifunctional immune detection system sends the processing results of the processing module.
  • the communication method can be selected according to the needs, such as through the network port, WiFi (wireless security) True) mode, Bluetooth mode, etc.
  • the fluorescence detection system consists of a test paper cartridge 1, a first micro stepping motor 2, an excitation light source 3, a photosensitive receiving tube 4, a color filter 5, a second micro stepping motor 6, CCD sensor 7, processing module 8 and closed black body cavity 9, in which, test paper cartridge 1, first micro stepping motor 2, excitation light source 3, photosensitive receiving tube 4, color filter 5, second micro stepping motor 6.
  • the CCD sensor 7, the processing module 8 are placed in the closed black body cavity 9 to shield external stray light interference; the first micro stepping motor 2, the excitation light source 3, the photosensitive receiving tube 4, the second micro stepping motor 6 and
  • the CCD sensor 7 is connected to the processing module 8 respectively.
  • the excitation light source 3 includes an ultraviolet excitation light source and a broad-spectrum excitation light source, which are used as excitation light sources for the fluorescence test mode and the colloidal gold test mode, respectively, and are switched under the control of the processing module 8.
  • the figure includes two groups of excitation light sources.
  • the black body cavity 9 is provided with a drawer-type test paper holding member that is connected to the first micro stepping motor 2 in transmission. The test paper holding tray is opened/closed under the driving of the first micro stepping motor 2, and the test paper cassette 1 to Accurate detection area.
  • the color filter 5 is transmitted to the photosensitive lens of the CCD sensor 7 to filter out the spectral signal with a wavelength below 500nm, and realize the switch between the fluorescence test mode and the colloidal gold test mode.
  • the inspector inputs control instructions through an LCD touch screen.
  • the test paper tray is opened under the transmission of the first micro stepping motor 2; after the inspector inserts the test paper cassette 1 into the test paper tray, further input instructions through the LCD touch screen , Control the first micro stepping motor 2 to drive and close the test paper tray, and guide the test paper (fluorescent test paper or colloidal gold test paper) to the accurate detection area.
  • the inspector selects a test mode (fluorescence test mode or colloidal gold test mode) on the LCD touch screen according to the loaded test test paper type and sends a control command to the processing module 8, and the system starts to test in the selected test mode.
  • the processing module 8 selects the ultraviolet excitation light source as the excitation to irradiate the fluorescent test paper, and at the same time controls the second micro stepping motor 6 to drive the color filter 6 to and Before the light-sensitive lens of the CCD sensor 7, which is closely matched, filters out the spectral signal with a wavelength below 500 nm in the response spectrum signal, so that the spectral signal with a wavelength above 510 nm reaches the light-sensitive lens of the CCD sensor 7.
  • the ultraviolet excitation light source is always in working condition, and the light intensity is in a constant state under the control of the PWM signal of the processing module; at the same time, the ultraviolet excitation light source is continuously monitored through the photosensitive receiving tube 4, and the light intensity is continuously monitored according to the light received by the photosensitive receiving tube 4.
  • the optical signal is feedback controlled by the processing module 8 to realize the stability of the light intensity of the ultraviolet excitation light source.
  • the processing module 8 selects a broad-spectrum excitation light source as the excitation to irradiate the colloidal gold test paper, which is received by the CCD sensor 7 photosensitive lens in the reflected light receiving area Respond to the spectrum signal.
  • the broad-spectrum excitation light source is always in working condition, and the light intensity is in a constant state under the control of the PWM signal of the processing module; at the same time, the broad-spectrum excitation light source is continuously monitored through the photosensitive receiving tube 4, according to the photosensitive receiving tube 4
  • the received light signal is feedback controlled by the processing module 8 to realize the stability of the light intensity of the broad-spectrum excitation light source.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé et un système d'immunoessai multifonctionnel à excitation continue, le procédé d'immunoessai multifonctionnel à excitation continue consistant : à placer un échantillon à détecter dans une région de détection (S10); à commander à au moins une source de lumière d'excitation ultraviolette disposée en face de la région de détection d'irradier en continu l'échantillon à détecter (S20); et à recevoir un signal de spectre de réponse après filtrage ultraviolet, et à obtenir un résultat de détection de fluorescence pour le signal de spectre de réponse reçu (S30). Une source de lumière continue présentant une intensité lumineuse constante est utilisée en tant que source de lumière d'excitation, ce qui permet de prolonger la durée de vie de la source de lumière d'excitation et d'améliorer la précision de détection.
PCT/CN2020/093941 2019-06-19 2020-06-02 Procédé d'immunoessai de filtrage de spectre de fluorescence à excitation ultraviolette continue Ceased WO2020253518A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910529313.9 2019-06-19
CN201910529313.9A CN110161228A (zh) 2019-06-19 2019-06-19 连续紫外激励荧光光谱滤色法免疫检测方法

Publications (1)

Publication Number Publication Date
WO2020253518A1 true WO2020253518A1 (fr) 2020-12-24

Family

ID=67626017

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/093941 Ceased WO2020253518A1 (fr) 2019-06-19 2020-06-02 Procédé d'immunoessai de filtrage de spectre de fluorescence à excitation ultraviolette continue

Country Status (2)

Country Link
CN (1) CN110161228A (fr)
WO (1) WO2020253518A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110161228A (zh) * 2019-06-19 2019-08-23 上海菲伽生物科技有限公司 连续紫外激励荧光光谱滤色法免疫检测方法
CN117859051A (zh) * 2022-08-05 2024-04-09 柯正浩 筛检试纸读取量测方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050208593A1 (en) * 2004-03-19 2005-09-22 Arizona Board Of Regents, Acting For And On Behalf Of Northern Arizona University Lateral flow diagnostic assay reader with radial cassette
US20090155921A1 (en) * 2007-12-12 2009-06-18 Arbor Vita Corporation Method and apparatus for reading test strips
WO2013131057A1 (fr) * 2012-03-01 2013-09-06 Quidel Corporation Système et appareil de diagnostics sur site des soins
CN107255712A (zh) * 2017-05-03 2017-10-17 上海交通大学 一种基于智能手机的免疫层析芯片定量分析系统
CN207964840U (zh) * 2018-01-12 2018-10-12 广东星创众谱仪器有限公司 可实现荧光免疫层析定量检查的食品安全综合分析装置
CN109297592A (zh) * 2018-11-14 2019-02-01 北京敏视达雷达有限公司 一种稳定光源的方法及相关装置
CN110161228A (zh) * 2019-06-19 2019-08-23 上海菲伽生物科技有限公司 连续紫外激励荧光光谱滤色法免疫检测方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100494988C (zh) * 2007-02-14 2009-06-03 上海新波生物技术有限公司 层析试条扫描检测方法及其扫描检测仪
CN201107254Y (zh) * 2007-09-26 2008-08-27 中国人民解放军军事医学科学院微生物流行病研究所 气溶胶颗粒分析仪中的紫外激光能量监控和受激荧光补偿系统
CN105259151B (zh) * 2015-11-02 2018-07-31 深圳市锦瑞生物科技有限公司 一种荧光检测系统及仪器
CN105259148B (zh) * 2015-11-02 2019-05-14 深圳市锦瑞生物科技有限公司 一种荧光免疫定量分析仪
WO2017206143A1 (fr) * 2016-06-02 2017-12-07 Henkel (China) Investment Co., Ltd. Détecteur portatif d'intensité de fluorescence excitée par les ultraviolets
CN105866090A (zh) * 2016-06-03 2016-08-17 苏州百源基因技术有限公司 紫外可见光荧光检测系统
CN106290289A (zh) * 2016-09-30 2017-01-04 广州蓝勃生物科技有限公司 一种时间分辨荧光免疫检测仪和检测方法
WO2018173210A1 (fr) * 2017-03-23 2018-09-27 株式会社ヴィーネックス Dispositif de détection de couleur fluorescente ultraviolette et procédé de détection de couleur fluorescente ultraviolette
CN208297338U (zh) * 2018-06-21 2018-12-28 荣捷生物工程（苏州）有限公司 Led紫外光源吸光度检测器
CN109297905B (zh) * 2018-10-09 2020-08-04 中国科学院半导体研究所 双门控相关荧光成像装置及成像方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050208593A1 (en) * 2004-03-19 2005-09-22 Arizona Board Of Regents, Acting For And On Behalf Of Northern Arizona University Lateral flow diagnostic assay reader with radial cassette
US20090155921A1 (en) * 2007-12-12 2009-06-18 Arbor Vita Corporation Method and apparatus for reading test strips
WO2013131057A1 (fr) * 2012-03-01 2013-09-06 Quidel Corporation Système et appareil de diagnostics sur site des soins
CN107255712A (zh) * 2017-05-03 2017-10-17 上海交通大学 一种基于智能手机的免疫层析芯片定量分析系统
CN207964840U (zh) * 2018-01-12 2018-10-12 广东星创众谱仪器有限公司 可实现荧光免疫层析定量检查的食品安全综合分析装置
CN109297592A (zh) * 2018-11-14 2019-02-01 北京敏视达雷达有限公司 一种稳定光源的方法及相关装置
CN110161228A (zh) * 2019-06-19 2019-08-23 上海菲伽生物科技有限公司 连续紫外激励荧光光谱滤色法免疫检测方法

Also Published As

Publication number Publication date
CN110161228A (zh) 2019-08-23

Similar Documents

Publication Publication Date Title
CN109975557B (zh) Il-6/pct联合检测时间分辨检测试剂盒及方法
JP6231999B2 (ja) 調時機構を有する相互作用的試験デバイスおよび装置
CN1157602C (zh) 荧光偏振法检测样品的方法及其中使用的试剂
CN106872420B (zh) 一种时间分辨荧光定量检测尿微量白蛋白的试剂盒及方法
US20140170674A1 (en) Membraine-Based Assay Devices Utilizing Time-Resolved Up-Converting Luminescence
Zhang et al. A CCD-based reader combined quantum dots-labeled lateral flow strips for ultrasensitive quantitative detection of anti-HBs antibody
CN105247348A (zh) 用于多路化组织学的数字增强的显微镜检查
CN114761517B (zh) 长余辉发光有机微球、其制备方法和应用
CN110702893A (zh) 一种aie免疫层析试纸
CN115389760B (zh) 用于免疫分析试条的检测试剂
WO2020253518A1 (fr) Procédé d'immunoessai de filtrage de spectre de fluorescence à excitation ultraviolette continue
US20110306065A1 (en) Use of an antibody and a rare-earth based crystal
BR112021005771A2 (pt) quantificação de analito
Wang et al. Direct fluorimetric determination of γ-globulin in human serum with organic nanoparticle biosensor
US12007327B2 (en) Optical bio-sensing device for bio-material analysis using upconversion nanoparticles, and bio-material quantitative analysis using the same
CN112858699B (zh) 便携式压力与精神疾病客观指标评估试纸条及方法
CN108291908A (zh) 用免疫和化学方法快速简便测定s-腺苷蛋氨酸，s-腺苷同型半胱氨酸和同型半胱氨酸
CN112823278B (zh) 一种在生物液体中检测人血清白蛋白的方法
CN107525937A (zh) 基于上转换发光标记物、蛋白芯片及检测方法
US11320425B2 (en) Enhanced infrared ray absorbing/emitting nanoparticles and on-site diagnosis kit using same
Zhao et al. Quantitative detection of morphine based on an up-conversion luminescent system
CN118067987A (zh) 一种点阵式免疫层析检测卡、制备方法及其应用
CN112924686B (zh) 用于检测血清淀粉样蛋白a的免疫层析试纸条及其制备与检测方法
EP4610653A1 (fr) Procédé de détection d'analyse dans un échantillon et bandelette réactive pour dosage immunologique
Xu et al. Evaluation of the bioconjugation efficiency of different quantum dots as probes for immunostaining tumor-marker proteins

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20827864

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20827864

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20827864

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20827864

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 02/08/2022)