CN107576802A - A kind of detection means and detection method of fluorescence micro albumen - Google Patents
A kind of detection means and detection method of fluorescence micro albumen Download PDFInfo
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- CN107576802A CN107576802A CN201710749306.0A CN201710749306A CN107576802A CN 107576802 A CN107576802 A CN 107576802A CN 201710749306 A CN201710749306 A CN 201710749306A CN 107576802 A CN107576802 A CN 107576802A
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Abstract
A kind of detection means and detection method of fluorescence micro albumen, belong to technical field of biological, it is characterized in that:Excitation source is located at sample cell both sides, excites photo detecting unit between excitation source and sample cell, and fluorescence detecting unit A, fluorescence detecting unit B are vertically positioned at the both sides of sample cell and exciting light light path.Transmission line is closed transmission line.Optical filter A, optical filter B are respectively between excitation source A, excitation source B and sample cell, in transmission line.Detection method is:First, by fluorometric reagent and phosphate buffer with 1:198 200 mix;2nd, take two various concentrations albumen and phosphate buffer with 1:18 20 mix;3rd, the bovine serum albumin solution of various concentrations is put into device, draws standard curve;4th, after obtaining standard curve, take testing sample with phosphate buffer with 1 20:180 199 ratios mix;5th, the protein concentration corresponding with sample fluorescence intensity is read.Beneficial effect is:Fluorescence signal can just be launched when fluorescent dye is only combined with albumen target molecule, it is sensitiveer compared to traditional ultraviolet light absorption method, more accurate.
Description
Technical field
The invention belongs to technical field of biological.
Background technology
The analysing content that the quantitative analysis of protein is biochemistry and other life sciences are most often related to, it is clinically to examine
Disconnected disease and the important indicator for checking rehabilitation situation, and many biological products, medicine, the important indicator of food quality detection.
In Bioexperiment, accurately and reliably quantitative analysis is carried out to the protein in sample, is that one often carried out is extremely important
Work.Protein is a kind of highly important large biological molecule:Its species is a lot, and structure heterogeneity, molecular weight differs again
It is very big, Various Functions, thus carry out many tools to the method generation for establishing a preferable and general Protein quantitative analysis
The difficulty of body.The method of measure protein content has many kinds at present, and one established according to protein heterogeneity is listed below
A little method of protein measurement:Physical property:Ultraviolet spectrophotometry.Chemical property:Kjeldahl's method, biuret method, Lowry
Method, BCA methods, colloidal gold method.Dyeing property:Coomassie light blue decoration method, argentation.Other properties:Fluorescence method protein determination
Method it is a lot, but every kind of method has its feature and a limitation, thus needs on the basis of various methods are understood according to not
Appropriate method is selected with situation, to meet different requirements.Such as Kjeldahl's method result is most accurate, but complex operation, use
It is then less qualified in the test of batch samples;Biuret method is simple to operate, and linear relationship is good, but poor sensitivity, and sample needs
Amount is big, and measurement range is narrow, therefore the application in scientific research is restricted;And phenol reagent process the shortcomings that compensate for it, thus in section
It is widely adopted in grinding, but its disturbing factor is more;The bright blue decoration method of Coomassie attracts attention again because of its simplicity beginning;
For BCA methods again with its stable reagent, antijamming capability is stronger, as a result stablizes, high sensitivity and receive an acclaim, but testing cost compared with
It is high.
The content of the invention
The purpose of the present invention is:A kind of detection means and detection method of fluorescence micro albumen are provided, it is contaminated using fluorescence
Material is combined with specific target molecule, greatly promotes sensitivity and the accuracy of sample detection.
The technical scheme is that:The present apparatus includes:Complete machine housing, sample groove lid, touch display screen, hardware circuit
Plate, fluorescence detection optical path.The fluorescence detection optical path includes:Sample cell, excitation source, excite photo detecting unit, fluorescence detection
Unit, bandpass filter, transmission line, radome.
The technical scheme is that:
The present apparatus includes excitation source A, excitation source B, excites photo detecting unit A, excites photo detecting unit B, fluorescence to visit
Survey unit A, fluorescence detecting unit B, optical filter A, optical filter B, optical filter C, optical filter D, sample cell, transmission line, signal
Detection unit, main control unit, excite light control unit, liquid crystal display, circuit board, housing, sample groove lid, radome.
Excitation source A (1) is located on the left of sample cell (11), and excitation source B (2) is located on the right side of sample cell (11), welds respectively
It is connected on circuit board (17), in transmission line (12), for exciting the fluorescent material in sample.
Excite photo detecting unit A (3) to be located between excitation source A (1) and sample cell (11), excite photo detecting unit B (4)
Between excitation source B (2) and sample cell (11), photo detecting unit B (4) is excited highly to be below exciting with sample cell (11)
Light source B (2), photo detecting unit B (4) is excited to be welded on sample cell (11) on circuit board (17), embedded in transmission line (12)
It is interior, ensure that exciting light can be irradiated to sample cell (11) interior sample, monitored in real time for the state to exciting light, confirm to swash
It is luminous that whether normal work, luminous intensity are stablized.
Fluorescence detecting unit A (5), fluorescence detecting unit B (6) are respectively perpendicular positioned at sample cell (11) and exciting light light path
Both sides, it is welded on circuit board (17), in transmission line (12).Receive and detect by sample slot (11) interior sample to be tested
The fluorescence signal intensity sent, using high accuracy, highly sensitive photodiode, fluoroscopic examination precision can be improved, is measured
Scope is 300-1,000nm.Fluorescence signal can just be launched when these fluorescent dyes are only combined with these target molecules, even in dense
When degree is very low, the inaccurate repeated work for measuring and bringing is avoided.Transmission channel:- the 580nm of the green glow 510, -720nm of feux rouges 665.Choosing
When selecting blue light and exciting, the fluorescence of green or far infrared passage is read.When exciting light is feux rouges, the glimmering of far infrared passage is only read
Light.
Transmission line (12) is closed transmission line, and bottom is fixed on circuit board (17) by expansion bolt,
The each component of light path has been subjected to effectively protection, excitation source A (1), excitation source B (2) transmitting light has been collected, passes through respectively
Optical filter A (7), optical filter B (8) reach sample cell (11), and collect the fluorescence signal excited, by optical filter C (9), filter
Piece D (10) reaches fluorescence detecting unit A (5), fluorescence detecting unit B (6).The interference of external stray light can be effectively eliminated, and
Pollution of the dust to optical path component can be prevented, makes optical transport cleaner, substantially increases accuracy and the sensitivity of detection.
Optical filter A (7), optical filter B (8) respectively positioned at excitation source A (1), excitation source B (2) and sample cell (11) it
Between, in transmission line (12), for filtering out the veiling glare outside exciting light;The optical filter C (9), optical filter D (10)
Respectively between sample cell (11) and fluorescence detecting unit A (5), fluorescence detecting unit B (6), for filtering in addition to fluorescence
Veiling glare.
Detecting signal unit (13) is welded on circuit board (17), for by fluorescence detecting unit A (5), fluorescence detection list
The electric signal of first B (6) conversion, carries out preposition amplification, demodulation, filtering, collection.
Main control unit (14) is welded on circuit board (17), the dual core processor of use, rapidly and accurately quantitative in 5 seconds
DNA, RNA and albumen.Result data is shown to LCDs (16), and stored.
Light control unit (15) is excited to be welded on circuit board (17), for controlling excitation source A (1), excitation source B
(2) output light intensity.
Liquid crystal display (16) is embedded in housing (18) upper surface, is connected by communication cable with circuit board (17), uses
In man-machine interaction, function selection, data storage.
Sample groove lid (19) is located on sample cell (11), is made using light-proof material, is covered tightly during detection, is used for
Reduce influence of the veiling glare in environment to experimental result.
Radome (20) cover using thin aluminum, disturbs on transmission line (12) for shielding electromagnetic signal,
Signal to noise ratio can be effectively improved.
Detection method is:
Step 1:By fluorometric reagent pico orange and phosphate buffer with 1:198-200 is mixed;
Step 2:Take the bovine serum albumin(BSA)s of two various concentrations with phosphate buffer with 1 respectively:18-20 is mixed;
Step 3:The bovine serum albumin solution of mixed various concentrations is put into device respectively, incubation at room temperature 15
Minute, two-point calibration is carried out, draws standard curve (protein concentration-fluorescence intensity);
Step 4:After obtaining standard curve, take testing sample with phosphate buffer with 1-20:180-199 ratios mix;
Step 5:It is put into sample cell, is incubated at room temperature 15 minutes, it is dense reads the albumen corresponding with sample fluorescence intensity
Degree.
The beneficial effects of the invention are as follows:These fluorescent dyes can just launch fluorescence when only being combined with these albumen target molecules
Signal, fluorescence signal even can be also sent when concentration is very low.It is sensitiveer compared to traditional ultraviolet light absorption method, more accurate,
Especially overcome the interference of DNA nucleic acid UV absorptions.Therefore apparatus of the present invention are especially suitable for situations below:When sample is very dilute
Have and be difficult to handle;Protein content is seldom in sample;Therefore the inventive method is used, fluorescent dye is specifically tied with protein molecular
Close, sensitivity and the accuracy of sample detection will be greatly promoted.
Brief description of the drawings
Fig. 1 is outward appearance overall structure figure of the present invention.
Fig. 2 is overall structure partial cutaway view of the present invention.
Fig. 3 is structure and working principle schematic diagram of the present invention.
Fig. 4 fluorescence intensities of the present invention-protein concentration canonical plotting.
Embodiment
Embodiment 1
Patent of the present invention is described in further details with reference to the accompanying drawings and examples.
As illustrated, 1 it is excitation source A, 2 is excitation source B, 3 is that to excite photo detecting unit A, 4 be to excite optical detection list
First B, 5 be fluorescence detecting unit A, 6 be fluorescence detecting unit B, 7 be optical filter A, 8 be optical filter B, 9 be optical filter C, 10 be filter
Mating plate D, 11 be sample cell, 12 transmission lines, 13 be detecting signal unit, 14 be main control unit, 15 be to excite photocontrol list
Member, 16 be liquid crystal display, 17 be circuit board, 18 be housing, 19 be sample groove lid, 20 be radome.
The excitation source A (1) is located on the left of sample cell (11), and excitation source B (2) is located on the right side of sample cell (11), point
It is not welded on circuit board (17), in transmission line (12), for exciting the fluorescent material in sample.
It is described to excite photo detecting unit A (3) to be located between excitation source A (1) and sample cell (11), excite photo detecting unit
B (4) is located between excitation source B (2) and sample cell (11), and both are highly below excitation source, is welded in circuit board (17)
On, in transmission line (12), ensure that exciting light can be irradiated to sample cell (11) interior sample, for the shape to exciting light
State is monitored in real time, confirms whether exciting light whether stablize by normal work, luminous intensity;
The fluorescence detecting unit A (5), fluorescence detecting unit B (6) are located at sample cell (11) for intersection point respectively, with swashing
The vertical both sides of luminous light path, are welded on circuit board (17), in transmission line (12).Receive and detect by sample slot
(11) fluorescence signal intensity that interior sample to be tested is sent, using high accuracy, highly sensitive photodiode, fluorescence can be improved
Accuracy of detection, measurement range 300-1,000nm.Fluorescence can just be launched when these fluorescent dyes are only combined with these target molecules
Signal, when concentration is very low, avoid the inaccurate repeated work for measuring and bringing.Transmission channel:It is-the 580nm of green glow 510, red
- the 720nm of light 665.When selection blue light excites, the fluorescence of green or far infrared passage is read.When exciting light is feux rouges, only read
The fluorescence of far infrared passage.
The transmission line (12), it is closed transmission line, circuit board is fixed in bottom by expansion bolt
(17) on, each component of light path has been subjected to effectively protection, has collected excitation source A (1), excitation source B (2) transmitting light,
Sample cell (11) is reached by optical filter A (7), optical filter B (8) respectively, and collects the fluorescence signal excited, passes through optical filter C
(9), optical filter D (10) reaches fluorescence detecting unit A (5), fluorescence detecting unit B (6).The dry of external stray light can effectively be eliminated
Disturb, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase accuracy and the spirit of detection
Sensitivity.
The optical filter A (7), optical filter B (8) are located at excitation source A (1), excitation source B (2) and sample cell respectively
(11) between, in transmission line (12), for filtering out the veiling glare outside exciting light;The optical filter C (9), filter
Piece D (10) is respectively between sample cell (11) and fluorescence detecting unit A (5), fluorescence detecting unit B (6), for filtering out fluorescence
Outside veiling glare.
The detecting signal unit (13) is welded on circuit board (17), for fluorescence detecting unit A (5), fluorescence to be visited
The electric signal of unit B (6) conversion is surveyed, carries out preposition amplification, demodulation, filtering, collection.
The main control unit (14) is welded on circuit board (17), the dual core processor of use, rapidly and accurately fixed in 5 seconds
Measure DNA, RNA and albumen.Result data is shown to LCDs (16), and stored.
It is described to excite light control unit (15) to be welded on circuit board (17), for controlling excitation source A (1), exciting light
Source B (2) output light intensity.
The liquid crystal display (16) is embedded in housing (18) upper surface, is connected by communication cable and circuit board (17)
Connect, for man-machine interaction, function selection, data storage.
The sample groove lid (19) is located on sample cell (11), is made using light-proof material, is covered tightly during detection,
For reducing influence of the veiling glare in environment to experimental result.
The radome (20) is covered on transmission line (12), using thin aluminum, is done for shielding electromagnetic signal
Disturb, signal to noise ratio can be effectively improved.
In this example, bidifly light emitting source includes blue-ray LED and red-light LED, can manually select as needed.When selection is blue
When light excites, the device reads the fluorescence of green or far infrared passage.When exciting light is feux rouges, far infrared passage is only read
Fluorescence.
It is above-mentioned to excite photo detecting unit in this example, the state of exciting light can monitor in real time and defeated by laser
Go out unit to compensate.When not detecting exciting light, then prompting is alarmed on the display screen;When deviation be present in excitating light strength,
Then carry out exciting light compensation.
In this example, each component of light path has been carried out effectively protection by transmission line, and can effectively eliminate the external world
The interference of veiling glare, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase detection
Accuracy and sensitivity.
In this example, bandpass filter is directed to two kinds of excitation sources, and holmium glass and didymium glass material has been respectively adopted
Optical filter, improve the degree of accuracy of excitation light wave.
In this example, fluorescence detecting unit improves fluoroscopic examination essence using high accuracy, highly sensitive photodiode
Degree, by detecting signal unit by the electric signal that photodiode change be sent to main control unit carry out data processing, and show and
On display screen, and data can preserve.
In this example, each component of light path has been carried out effectively protection by transmission line, and can effectively eliminate the external world
The interference of veiling glare, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase detection
Accuracy and sensitivity.
In this example, each component of light path has been carried out effectively protection by transmission line, and can effectively eliminate the external world
The interference of veiling glare, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase detection
Accuracy and sensitivity.
In this example, the detection means of fluorescence micro albumen, its application method comprises the following steps:
Step 1:1ul fluorometric reagent pico orange and phosphate buffer 1 99ul are mixed;
Step 2:190 μ L phosphate buffers are taken, are separately added into 10 μ L 0.1mg/ml bovine serum albumin(BSA)s, 1mg/ml oxen
Seralbumin mixes;
Step 3:Mixed 0.1mg/ml bovine serum albumin(BSA)s and 1mg/ml bovine serum albumin(BSA)s are put into device respectively
In, it is incubated at room temperature 15 minutes, carries out two-point calibration, draws standard curve (protein concentration-fluorescence intensity);
Step 4:After obtaining standard curve, the μ L of testing sample 10 and 190 μ L phosphate buffers are taken to mix;
Step 5:It is put into apparatus of the present invention sample cell, is incubated at room temperature 15 minutes, reads corresponding with sample fluorescence intensity
Protein concentration.
Claims (3)
1. a kind of detection means of fluorescence micro albumen, it is characterized in that:
Excitation source A is located on the left of sample cell, and excitation source B is located on the right side of sample cell, is respectively welded on circuit boards, embedded in light
In transmission channel, for exciting the fluorescent material in sample;Photo detecting unit A is excited between excitation source A and sample cell,
Photo detecting unit B is excited to excite photo detecting unit B to be below swashing with sample groove height between excitation source B and sample cell
Light emitting source B, photo detecting unit B is excited to be welded on circuit boards with sample cell, in transmission line, guarantee excites luminous energy to shine
It is mapped to sample in sample cell;
Fluorescence detecting unit A, fluorescence detecting unit B are respectively perpendicular the both sides positioned at sample cell and exciting light light path, are welded on electricity
On the plate of road, in transmission line;
Transmission line is closed transmission line, and bottom is fixed on circuit board by expansion bolt;
Optical filter A, optical filter B are respectively between excitation source A, excitation source B and sample cell, in transmission line;
Detecting signal unit welds on circuit boards, for the electric signal for changing fluorescence detecting unit A, fluorescence detecting unit B,
Carry out preposition amplification, demodulation, filtering, collection;
Main control unit welds on circuit boards;
Light control unit is excited to be welded on circuit board;
Liquid crystal display is embedded in housing upper surface, is connected by communication cable with circuit board.
2. a kind of rapid fluorescence proportioning device as claimed in claim 1, it is characterized in that:
Fluorescence detecting unit A, fluorescence detecting unit B are respectively perpendicular the both sides positioned at sample cell and exciting light light path, are welded in electricity
On the plate of road, in transmission line;Measurement range is 300-1,000nm;Transmission channel is the -580nm of green glow 510, feux rouges
665–720nm;When selection blue light excites, the fluorescence of green or far infrared passage is read;When exciting light is feux rouges, only read remote
The fluorescence of infrared channel.
3. a kind of detection method of fluorescence micro albumen, its method are:
Step 1, by fluorometric reagent pico orange and phosphate buffer with 1:198-200 is mixed;
Step 2, take the bovine serum albumin(BSA)s of two various concentrations with phosphate buffer with 1 respectively:18-20 is mixed;
Step 3, the bovine serum albumin solution of mixed various concentrations is put into device respectively, is incubated at room temperature 15 minutes,
Two-point calibration is carried out, draws standard curve;
Step 4:After obtaining standard curve, take testing sample with phosphate buffer with 1-20:180-199 ratios mix;
Step 5:It is put into sample cell, is incubated at room temperature 15 minutes, reads the protein concentration corresponding with sample fluorescence intensity.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN108088816A (en) * | 2018-01-23 | 2018-05-29 | 深圳市国赛生物技术有限公司 | Small-sized specific protein analyzer |
CN112414984A (en) * | 2020-11-06 | 2021-02-26 | 中国计量大学 | Portable milk powder protein content detection box |
CN112833959A (en) * | 2021-01-27 | 2021-05-25 | 中南民族大学 | O-shaped catalyst2-CO2-T three-parameter real-time monitoring system |
CN116577311B (en) * | 2023-06-01 | 2024-05-31 | 北京贝泰科技有限公司 | Protein determination analysis method |
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CN105203518A (en) * | 2015-10-08 | 2015-12-30 | 三峡大学 | Fluorescent reagent and preparation method and application thereof |
CN106645065A (en) * | 2016-12-13 | 2017-05-10 | 三峡大学 | Synthesis method of fluorescent reagent for identifying specificity and sensitively detecting human albumin and application |
CN107389644A (en) * | 2017-08-11 | 2017-11-24 | 长春理工大学 | A kind of rapid fluorescence proportioning device |
CN107421935A (en) * | 2017-08-11 | 2017-12-01 | 长春理工大学 | A kind of fluorescence micro RNA detection means and detection method |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102618060A (en) * | 2012-03-17 | 2012-08-01 | 江南大学 | Method for preparing asymmetrical cyanine dye and method for detecting bovine serum albumin by asymmetrical cyanine dye |
CN105203518A (en) * | 2015-10-08 | 2015-12-30 | 三峡大学 | Fluorescent reagent and preparation method and application thereof |
CN106645065A (en) * | 2016-12-13 | 2017-05-10 | 三峡大学 | Synthesis method of fluorescent reagent for identifying specificity and sensitively detecting human albumin and application |
CN107389644A (en) * | 2017-08-11 | 2017-11-24 | 长春理工大学 | A kind of rapid fluorescence proportioning device |
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CN108088816A (en) * | 2018-01-23 | 2018-05-29 | 深圳市国赛生物技术有限公司 | Small-sized specific protein analyzer |
CN112414984A (en) * | 2020-11-06 | 2021-02-26 | 中国计量大学 | Portable milk powder protein content detection box |
CN112833959A (en) * | 2021-01-27 | 2021-05-25 | 中南民族大学 | O-shaped catalyst2-CO2-T three-parameter real-time monitoring system |
CN116577311B (en) * | 2023-06-01 | 2024-05-31 | 北京贝泰科技有限公司 | Protein determination analysis method |
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