CN104977278A - Quantitative analyzing and detecting system based on multispectral fluorescence immunochromatography - Google Patents
Quantitative analyzing and detecting system based on multispectral fluorescence immunochromatography Download PDFInfo
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Abstract
The invention relates to a quantitative analyzing and detecting system based on multispectral fluorescence immunochromatography. The quantitative analyzing and detecting system comprises a multispectral fluorescence collecting unit, a uniform and steady exciting light field, an N-channel excitation optical filter (N is more than or equal to 1), a handheld computer system and an absolute measuring and correcting system for reading five cards simultaneously, wherein an N-channel mosaic optical filter (micro-filter) is tightly combined with a photoelectric conversion unit to form the multispectral fluorescence collecting unit; the multispectral fluorescence collecting unit, the exciting light field and the N-channel excitation optical filter form a detection module for acquiring N stimulated emission fluorescence signals in real time; and, the detection module is connected with the handheld computer to perform semi-quantitative and quantitative high-sensitivity measurement of multi-colour fluorescence immunochromatography simultaneously in real time.
Description
Technical field
The present invention relates to a kind of Portable external fluorescence analysis and detection system, in particular to one based on multispectral fluorescence immune chromatography quantified system analysis, be applicable to side by side judging fast in real time or quantitative measurment of multiple fluorescently-labeled immunochromatography diagnose test paper result.
Background technology
Immunochromatography (immunochromatography) is a kind of quick diagnosis technology of rising nearly ten years, its principle is a certain zone special antibody being first fixed on chromatographic film (as nitrocellulose filter), after chromatographic film one end of this drying drips sample (urine or serum), due to capillary action, sample will move forward along this film, when moving to the region being fixed with antibody, in sample corresponding antigen namely with this antibody generation specific binding, if this region can be made to show certain color with immune colloid gold or Immunoperoxidase Staining, thus realize specific immunodiagnosis.
Immuno-chromatographic test paper strip has accurately and fast and simple operation and other advantages.As colloidal gold immunochromatographimethod technology law, the method is widely applied in clinical examination, its ultimate principle is by colloid gold label thing and analyzes the color (redness) that thing and coated antibody or antigen-reactive form collaurum itself, by visual inspection testing result.But for some antigen or the extremely low sample of antibody content, the very slight color of collaurum and be difficult to the naked eye judged result, sensitivity is low.This is because colloid gold label utilizes Electrostatic Absorption, less stable in the liquid phase, and labeled protein grouping easily comes off; Different materials matrix effect is obvious, and background interference is comparatively large, is difficult to accurate quantitative analysis, causes sensitivity not high.
The fluorescence immune chromatography technology grown up in recent years, combining nano technology, leading mark technology, technology of biological membrane, cross flow technology and detection technique of fluorescence, adopt chemical conjugation methods labelled antibody, possesses the advantage that Traditional immunochromatographic method detects, also overcome the defect that it is intrinsic, have highly sensitive, high specificity, fast, can qualitative and quantitative detection.
Quantitatively detector is detected based on photodiode triode modular immunity test strip detection line color signal as Chinese patent 201010591112.0 " immunochromatographitest test paper strip color signal quantitative detector " proposes one, structure is simple, volume is little, cost is low, various encapsulation test strips color signal can be adapted to detect, improve quantitative detection sensitivity and reliability.But this device needs to scan one by one reagent strip, have impact on the real-time of data acquisition, consumes a large amount of time, is not suitable for batch detection.
In order to realize batch detection, save detection time, the fluorescence immune chromatography analysis and detection technology with area array CCD also progressively develops.Adopt CCD Digital image technology immediately to take detected target as Chinese patent 201110115964.7 " a kind of CCD type quantitative analysis system of colloidal gold immunity chromatography diagnosis test papers " proposes, process and a kind of CCD type quantitative analysis system of colloidal gold immunity chromatography diagnosis test papers of reading and analytical approach.This system adopts to be increased or reduces the time shutter, reduces the noise of image, effectively ensures the stability of reading result, consistance and degree of accuracy, solves subjective bias problem during human eye interpretation effectively.But the generation of CCD photocurrent, amplification and AD conversion, inevitably also will use analog amplify circuit.Mimic channel can, because the factors such as thermonoise, electro-induction, device performance cause noise current, although very low, when brightness is higher, compare normal photocurrent amplitude proportional very little, and the time shutter be very short, so almost can not show out.But this common CCD is to week fluorescent shooting, when accumulating photon signal, long when the exposure of needs, causes CCD to produce more dark current, very large to the quality influence of image, cause the sensitivity of fluorescence signal acquisition extremely low.
Summary of the invention
For avoiding the weak point in background technology, the present invention proposes a kind of based on multispectral fluorescence immune chromatography quantitative test and detection system, comprises the multispectral fluorescence collector unit that N channel mosaic filter (micro-filtration sheet) is combined closely with photoelectric conversion unit; Evenly, stable exciting light light field; N channel exciter filter (N >=1); Comprise palmtop computer system and the same calibration system reading absolute measurement of five cards.What phosphor collection unit, exciting light light field, N channel exciter filter were compact is integrated in a lighttight obturator, form the detection module of real-time collecting N kind stimulated emission fluorescence signal, detection module and palmtop computer system form fluorescence immune chromatography detector system (or being called platform); Palmtop computer system display fluorescence intensity image, and treatment and analysis is carried out to image; Five cards provide the absolute measurement mechanism and the proof that there are fluorescent marker dilution that can read five kinds of variable concentrations with the absolute measurement calibration system read simultaneously.
Wherein, N channel mosaic filter (micro-filtration sheet) is close to the surface of photoelectric conversion unit, or adopt produced by micro processing mode N channel mosaic filter (micro-filtration sheet) to be directly produced on the surface of photoelectric conversion unit, what described N channel mosaic filter (micro-filtration sheet) was aimed at by " pixel for pixel " is pasted onto or is produced on photoelectric conversion unit surface (N >=1).
Wherein, the number of wavelengths that N channel mosaic filter (micro-filtration sheet) passes through is N, comprise multiple optical filtering infinitesimal, each infinitesimal comprises N number of compact arranged passage, namely a passage only allows a kind of light signal of characteristic wavelength to pass through, realize multiple mark fluorescent and side by side collect (N >=1) in real time, this assembling mode makes instrument volume can be very little, real-time collecting multiple tracks (that is: multiple different wave length) stimulated emission fluorescence signal, a machine replaces multimachine, realizes many sick kinds and detects, reduce mechanical component, good stability, freedom from repairs.
Wherein, photoelectric conversion unit is the photoelectric conversion unit low light level to high sensitivity imaging capability, as can be but be not limited to a peacekeeping two-dimensional space resolution, refrigeration, with imageing sensor or the integrated PMT etc. with a peacekeeping two-dimensional space resolution of time integral, the present invention improves image output signal-to-noise ratio by adopting highly sensitive New Image sensor, increases substantially the Monitoring lower-cut of instrument; The fluorescence signal that reagent strip is launched can be collected in real time, the space distribution that kit obtains institute's emitting fluorescence need not be dragged by machinery, there will not be mechanical fault; Synchronous signal exports not by influence of temperature change, instrument stabilizer.
Wherein, the detection module of real-time collecting N kind stimulated emission fluorescence signal that forms of multispectral fluorescence collector unit, exciting light light field, N channel exciter filter and palmtop computer form fluorescence immune chromatography detector system (or being called platform).
Wherein, the multispectral fluorescence collector unit that N channel mosaic filter (micro-filtration sheet) and photoelectric conversion unit are combined closely can carry out time integral and two-dimensional space integration to stimulated emission fluorescence signal; Real-time collects the signal of multiple tracks (that is: multiple different wave length) stimulated emission fluorescence; Signal to noise ratio (S/N ratio) significantly can be promoted by the two-dimensional space integration of camera to the time integral of stimulated emission fluorescence signal and stimulated emission fluorescence signal of weak signal imageing sensor.The fluorescent material lower than 0.005ng/ml can be detected, higher than 20 times of the average sensitivity of the instrument on market.Do not need scanning, export during fructufy, do not have moving-member, fault chance can be accomplished minimum, very convenient and realize simply not in the same time to the collection of stimulated emission fluorescence signal.
Wherein, the multiple tracks of elapsed time anomalous integral two-dimensional space point collection (that is: multiple different wave length) stimulated emission fluorescence signal is uploaded to palmtop computer system by multispectral fluorescence collector unit.Palmtop computer system display fluorescence intensity image, palmtop computer system is open system, the storage of described open system, not by the restriction of number of samples, storage space, there is wireless communication function, the sharing of fluorescence immune chromatography Monitoring Data result, exchanges data and remote monitoring can be realized by wireless Internet in any medical institutions; Described open system is flexible strong, and measuring ability easily adds and revises, and can carry more complicated programmed algorithm; Save signal to amplify, stability maintenance electronic circuit, A/D conversion and other hardware development and engineering process repeatedly, the higher level lanquage of simple application software, program is short, simply, programing work amount is few, do not relate to the interactional programming of software and hardware, the development time significantly shortens significantly reduces with difficulty, and software upgrading is convenient.
Wherein, be provided with one five card with the absolute measurement calibration system read based on multispectral fluorescence immune chromatography quantitative test and detection system, described five cards can read absolute measurement mechanism and the proof of the fluorescent marker dilution of five kinds of variable concentrations simultaneously with the absolute measurement calibration system read; Described absolute measurement mechanism and proof can be calibrated at any time to instrument system, ensure high duplication and the reliability of output detections numerical value.
Wherein, excitation source is driven by constant current source, realizes the stable of light field illumination.
Wherein, excitation source is equipped with dispensing device, realizes the Uniform Illumination of light field.
Wherein, N channel exciter filter is can simultaneously by the multiple tracks narrow band pass filter of the number of wavelengths of needs, and the number of wavelengths passed through is N, excites (N >=1) while realizing multiple sample.
Of the present invention based on multispectral fluorescence immune chromatography quantitative test and detection system, can realize side by side being gathered into picture in real time to different types of fluorescence immune chromatography reagent strip, without the need to scanning one by one reagent strip, data Real-time Obtaining.Not only simplify operating process, and process structure is simple, compact, cost is low, and volume is little, improves fluorescent collecting efficiency simultaneously.The present invention adopts the photoelectric conversion unit having a high sensitivity imaging capability to the low light level to reduce the impact on image quality such as dark current to greatest extent, realize gathering low intensive fluorescence signal, the Monitoring lower-cut of fluorescence immune chromatography system is significantly promoted, is more easily accepted by users.
accompanying drawing explanation
Fig. 1 is fluorescence immune chromatography quantified system analysis schematic diagram.
Fig. 2 is the time integral figure of Image Acquisition.
Fig. 3 to be N channel mosaic filter (micro-filtration sheet) with photoelectric conversion unit combine closely multispectral fluorescence collector unit structural representation formed.
Fig. 4 is the effect schematic diagram (for N=4) of N channel mosaic filter (micro-filtration sheet) in fluorescence immune chromatography detector system.
Fig. 5 is the digital phosphor signal graph that reagent strip detects position output.
Fig. 6 is the effect schematic diagram of multispectral fluorescence collector unit at fluorescence immune chromatography detection system fluorescence signal collection.
Fig. 7 be to fluorescent signals detect time integral and space integral schematic diagram.
concrete case study on implementation
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, coordinate that accompanying drawing is auspicious gives explanation below in conjunction with embodiment.
See also Fig. 1, based on multispectral fluorescence immune chromatography quantified system analysis in the present embodiment, comprise excitation source 1, N channel exciter filter 2, dispensing device 3, low light level photoelectric conversion unit 4, the N channel mosaic filter of combining closely with low light level photoelectric conversion unit (micro-filtration sheet) 5, focusing unit 6, the lighttight compact detection module 10 that reagent strip socket 7 forms.
During system works, the reagent strip the socket 7 first reacted immunochromatography diagnose test paper 9 be placed on PVC end liner 8 being sent into instrument is fixed.Comprise detection zone 92 and quality control band 91 in diagnose test paper 9 in Quasi-quantitative measurement, seizure display can be carried out, by the presence or absence of determinand in the strong and weak show sample of fluorescence signal to the fluorescence of the content of material being not less than sensor detection sensitivity; In quantitatively, in system, record has the typical curve of test substance sample, and software, according to the fluoroscopic image obtained, calculates the respective value of detection zone in test strips.Immunochromatography diagnose test paper 9 is after instrument internal fixtion, and the stable excitation source 1 driven through constant current source forms the excitation wavelength of N number of needs after N channel exciter filter 2, after form uniform illumination by dispensing device 3.What exciting light was even, stable illuminates examination diagnose test paper 9 surface, on the multispectral fluorescence collector unit that emission wavelength line focus unit 6 focal imaging is combined closely at N channel mosaic filter (micro-filtration sheet) 5 and low light level photoelectric conversion unit 4, finally, software according to the situation of the direct Show Color of the digital picture obtained, or calculates the respective value of detection zone of immunochromatography diagnose test paper 9.Palmtop computer system 11 shows fluorescence intensity image, and carries out treatment and analysis to image; Five cards provide absolute measurement mechanism and the proof of the fluorescent marker dilution reading different dilute concentration with the absolute measurement calibration system read.
See also Fig. 2, as shown be the time integral figure of Image Acquisition.Have in the exposure account form of the photoelectric conversion unit of low light level imaging capability, E is the luminous flux projecting photoelectric conversion unit unit area surface, the area of shade is the integration of illuminance E in time t1 ~ t2 that a certain bin in photoelectric conversion unit surface receives, and is formulated as:
H (t) is shaded area, represents the exposure relevant with the time, E(t) represent the time dependent function of luminous flux.And general camera exposure H=E*t.Wherein illumination E is determined by aperture, and the time is by shutter control.Aperture size and shutter length determine exposure number, therefore, general camera exposure is fixing, is difficult to realize shooting to week fluorescent.In the present embodiment, being then adopt the astronomical camera with refrigerating plant, promoting the signal to noise ratio (S/N ratio) of image by reducing dark current.Light signal is simultaneously obtained by the integration of illuminance E in time t1 ~ t2, be conducive to detecting fluorescent signals, thus the sensitivity that General Promotion fluorescence immune chromatography detects.
The multispectral fluorescence collector unit that N channel mosaic filter (micro-filtration sheet) and photoelectric conversion unit are combined closely can carry out time integral and two-dimensional space integration to stimulated emission fluorescence signal; Real-time collects the signal of multiple tracks (that is: multiple different wave length) stimulated emission fluorescence; Signal to noise ratio (S/N ratio) significantly can be promoted by the two-dimensional space integration of multispectral fluorescence collector unit to the time integral of stimulated emission fluorescence signal and stimulated emission fluorescence signal with the imageing sensor of low light level imaging capability.
See also Fig. 3, in the present embodiment, utilizing emitted light is imaged on four-way mosaic filter (for N=4) 5 and has the photoelectric conversion unit 4(of low light level imaging capability as CCD/CMOS imageing sensor or the integrated PMT etc. with spatial resolution after optical image unit imaging) on the multispectral fluorescence collector unit that formed of combining closely.N channel mosaic optical filtering (micro-filtration sheet) comprises multiple optical filtering infinitesimal, the passage 51 containing N number of wavelength in each Microfiltration Unit.
See also Fig. 4, in the present embodiment, N channel mosaic filter (micro-filtration sheet) is at the effect schematic diagram of fluorescence immune chromatography detector system.With the situation of N=4, the effect of N channel mosaic filter (micro-filtration sheet) in fluorescence immune chromatography detector system is described.Number in the figure be 4 parts be the imageing sensor with low light level imaging capability; 5 is micro-filtration sheet; 6 is focusing unit; 9 is reagent strip; 11 is computer system; 13 is data line; 12 is original image; 121,122,123,124 be respectively original Figure 12 be separated after different wave length image; λ
0i(i=1,2,3,4), that is: λ
01, λ
02, λ
03and λ
04for the exciting light of different wave length, λ
i(i=1,2,3,4), that is: λ
1, λ
2, λ
3and λ
4for the excited fluorescence of different wave length.
Given reagent strip 9, the content detected as required, the exciting light required for selection.The exciting light λ of different wave length
01or λ
02or λ
03or λ
04reagent strip 9 is excited, the fluorescence signal λ that (marked certain disease) fluorescent material in reagent strip 9 is launched after being stimulated
1or λ
2or λ
3or λ
4arrive on micro-filtration sheet 5, imageing sensor 4 is connected to computing machine by data line 13, and real-time acquisition original image 12 also splits into corresponding to λ simultaneously
1, λ
2, λ
3and λ
4121,122,123,124 4 width images of different wave length excited fluorescence, only have corresponding to λ in four width images
1or λ
2or λ
3or λ
4image can see bright line, other image then can not show bright line.
See also Fig. 5, as shown be the fluorescence signal that system exports.The present invention can realize the fluorescence signal intensity side by side exporting all detection positions on reagent strip in real time.
See also Fig. 6, it 7 is wherein reagent batten that the detection module that multispectral fluorescence collector unit in the present embodiment, exciting light light field, N channel exciter filter form real-time collecting N kind stimulated emission fluorescence signal and palmtop computer form fluorescence immune chromatography detector system (or being called platform); 9 is reagent strip; 5 is micro-filtration sheet; 4 is weak signal imageing sensor; The 41 phosphor collection unit combined with the imageing sensor 4 with low light level imaging capability for micro-filtration sheet 5; 10 is the detection module of real-time collecting N kind stimulated emission fluorescence signal; 11 is palmtop computer system.
Combine with the imageing sensor 4 with the low light level imaging capability stimulated emission fluorescence signal collection unit 41 that formed of micro-filtration sheet 5 real-time can collect the signal of multiple tracks (that is: multiple different wave length) stimulated emission fluorescence; Signal to noise ratio (S/N ratio) significantly can be promoted by the two-dimensional space integration of camera to the time integral of stimulated emission fluorescence signal and stimulated emission fluorescence signal of weak signal imageing sensor.Detect the fluorescent material lower than 0.005ng/ml, higher than 20 times of the average sensitivity of the instrument on market.
The multiple tracks of elapsed time anomalous integral two-dimensional space point collection (that is: multiple different wave length) stimulated emission fluorescence signal is uploaded to palmtop computer system by multispectral fluorescence collector unit 41; Described palmtop computer system display fluorescence intensity image, described palmtop computer system is open system, the storage of described open system, internal memory is not limit for the quantity detected, more complicated programmed algorithm can be carried, described open system has wireless communication function, can realize the sharing of fluorescence immune chromatography Monitoring Data result, exchanges data and remote monitoring by wireless Internet; Described open system is flexible strong, and measuring ability easily adds and revises; Save signal to amplify, stability maintenance electronic circuit, A/D conversion and other hardware development and engineering process repeatedly, the higher level lanquage of simple application software, program is short, simply, programing work amount is few, does not relate to the interactional programming of software and hardware, and the development time significantly shortens significantly reduces with difficulty.
Based on multispectral fluorescence immune chromatography quantitative test and detection system real-time, do not need scanning, export during fructufy, do not have moving-member, fault chance can be accomplished minimum, very convenient and realize simply not in the same time to the collection of stimulated emission fluorescence signal.
See also Fig. 7, as shown be the time integral to fluorescent signals detection and space integral schematic diagram.Wherein X-Y axial coordinate is two-dimensional space coordinate, Z axis coordinate is time coordinate, the camera that micro-filtration sheet combines with weak signal imageing sensor and the unit of the stimulated emission fluorescence signal formed can carry out time integral and two-dimensional space integration to stimulated emission fluorescence signal, realizes simply not in the same time to the collection of stimulated emission fluorescence signal.Survey fluorescence signal there is temporal resolution and spatial resolution simultaneously.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize instructions of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (9)
1. based on multispectral fluorescence immune chromatography quantitative test and a detection system, comprise: the multispectral fluorescence collector unit that N channel mosaic filter (micro-filtration sheet) and photoelectric conversion unit are combined closely; Evenly, stable exciting light light field; N channel exciter filter (N >=1); Palmtop computer system and five cards are with the absolute measurement calibration system read; It is characterized in that described multispectral fluorescence collector unit, exciting light light field, N channel exciter filter compact be integrated in a lighttight obturator, form the detection module of real-time collecting N kind stimulated emission fluorescence signal; Described detection module and palmtop computer system form fluorescence immune chromatography detector system (or being called platform); Palmtop computer system display fluorescence intensity image, and treatment and analysis is carried out to image; Five cards provide the absolute measurement mechanism and the proof that there are fluorescent marker dilution that can read five kinds of variable concentrations with the absolute measurement calibration system read simultaneously.
2. one according to claim 1 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that described multispectral fluorescence collector unit is close to photoelectric conversion unit surface by N channel mosaic filter (micro-filtration sheet); Or N channel mosaic filter (micro-filtration sheet) direct plating film production is on the surface of photoelectric conversion unit.
3. one according to claim 1 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that described N channel mosaic filter (micro-filtration sheet) comprises multiple optical filtering infinitesimal, each optical filtering infinitesimal comprises N number of compact arranged passage, each passage described only allows a kind of light signal of characteristic wavelength to pass through, and the number of wavelengths that N channel mosaic filter (micro-filtration sheet) passes through simultaneously is N.
4. one according to claim 1 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that the number of wavelengths that the N channel mosaic filter (micro-filtration sheet) of described multispectral fluorescence collector unit passes through simultaneously is N, can realize side by side collecting in real time to the N kind mark fluorescent of the N kind mark fluorescent of a reagent strip or N number of reagent strip.
5. one according to claim 1 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that the photoelectric conversion unit of described multispectral fluorescence collector unit is the photoelectric conversion unit low light level to high sensitivity imaging capability, described photoelectric conversion unit can be but be not limited to be a peacekeeping two-dimensional space resolution, refrigeration, with imageing sensor or the integrated PMT etc. with a peacekeeping two-dimensional space resolution of time integral.
6. one according to claim 5 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that the multispectral fluorescence collector unit that described N channel mosaic filter (micro-filtration sheet) and photoelectric conversion unit are combined closely carries out time integral and two-dimensional space integration imaging to stimulated emission fluorescence signal; The signal of real-time collecting N road (that is: N number of different wave length) stimulated emission fluorescence.
7. one according to claim 6 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that the N road of elapsed time anomalous integral two-dimensional space point collection (that is: N number of different wave length) stimulated emission fluorescence signal is uploaded to palmtop computer system by described multispectral fluorescence collector unit; Described palmtop computer system display fluorescence intensity image, and treatment and analysis is carried out to image.
8. one according to claim 1 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that described Operation system setting has one five absolute measurement calibration system of blocking with reading, described five cards can read absolute measurement mechanism and the proof of the fluorescent marker dilution of five kinds of variable concentrations simultaneously with the absolute measurement calibration system read; Described absolute measurement mechanism and proof can be calibrated at any time to instrument system, ensure high duplication and the reliability of output detections numerical value.
9. one according to claim 1 is based on multispectral fluorescence immune chromatography quantitative test and detection system, it is characterized in that the excitation source that described even, stable exciting light light field is driven by constant current source, N channel exciter filter and dispensing device are formed.
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| CN115932249A (en) * | 2022-12-30 | 2023-04-07 | 杭州纽太生物科技有限公司 | Real-time calibration immunochromatography detection method |
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