CN214584907U

CN214584907U - Ratio type fluorescence detection system

Info

Publication number: CN214584907U
Application number: CN202120452140.8U
Authority: CN
Inventors: 肖文香; 支冬灿
Original assignee: Guilin University of Electronic Technology
Current assignee: Guilin University of Electronic Technology
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2021-11-02
Anticipated expiration: 2031-03-02

Abstract

The utility model discloses a ratio-type fluorescence detection system, which comprises a shading darkroom, and a detection board, a fluorescence detection module, an excitation light source module and an MCU system which are arranged in the shading darkroom; a detection groove for placing a fluorescent probe and a reagent is formed in the detection plate, and the excitation light source is opposite to the detection groove; the fluorescence detection module comprises two groups, wherein each group comprises an optical filter and a photoelectric sensor; the output end of the MCU system is connected with the excitation light source module, the input end of the MCU system is connected with the fluorescence detection module, and the output light of the excitation light source module is emitted into the photoelectric sensor after being filtered by the optical filter after being excited by the fluorescent probe. The utility model discloses a ratio type fluorescence probe has from the reference characteristic, and is no longer harsh to the detection ring border requirement, can effectively avoid coming from the interference of environment, and it is high to detect the precision to but very big degree reduction system error, interference killing feature are extremely strong.

Description

Ratio type fluorescence detection system

Technical Field

The utility model relates to a biological detection technical field, concretely relates to ratio type fluorescence detecting system.

Background

In the current medical field, the detection of substances in the human body is becoming more and more mature according to the principle of fluorescence detection, and most of the detection means are based on the fact that the substances to be detected have fluorescence characteristics or are based on the principle of fluorescence energy resonance transfer. The technique can be described in detail as: the fluorescent substance emits light having a wavelength longer than that of excitation light under irradiation of the excitation light having a specific wavelength, and is called fluorescence; the relationship between the fluorescence intensity (F), the excitation light intensity (I0) and the fluorescent substance concentration (C) is: f2.3 QKI0 ∈ Cl, F KC, Q quantum yield, K fluorescence efficiency, ∈ molar absorption coefficient, and l optical path length. Fluorescence resonance energy transfer is a non-radiative energy transition, and the fluorescence intensity of a donor is reduced by transferring the energy of the excited state of the donor to the excited state of an acceptor through the intermolecular electric dipole interaction, while the acceptor can emit characteristic fluorescence (sensitized fluorescence) stronger than the fluorescence per se or does not emit fluorescence (fluorescence quenching), and a certain linear relation exists between the fluorescence intensity change generated in the process and the concentration of a detected substance.

For example, the patent of the invention in china with the publication number CN107917905B discloses a ratio type photometric analysis device and method based on an intelligent terminal, the device comprises an intelligent terminal and an optical auxiliary device, the optical auxiliary device comprises a light-proof cassette, a row of dual photometric measurement cells are arranged in the light-proof cassette, the outer wall of the measurement cell, which is opposite to a detector, is plated or pasted with a reflection film to enhance optical signals, the row of dual photometric measurement cells receive light emitted by a light source, optical image signals of two channels of a sample solution and a reference solution sequentially pass through a light filtering mechanism and a light focusing mechanism and are collected by the intelligent terminal, the average brightness of three primary colors of red, green and blue in the optical image signals is measured, the brightness ratio of the three primary colors is calculated, and photometric analysis is realized. The detection of the scheme depends on the brightness of light for analysis, the requirement on the excitation light source is harsh, and the detection precision can be influenced due to the problems of ambient light, heat dissipation of the excitation light source and the like.

Disclosure of Invention

The utility model aims to solve the technical problem that a ratio type fluorescence detection system that can effectively avoid coming from the interference of environment, and detect the precision height is provided.

In order to solve the technical problem, the utility model discloses a ratio-type fluorescence detection system, which comprises a shading darkroom, a detection board, a fluorescence detection module, an excitation light source module and an MCU system, wherein the detection board, the fluorescence detection module, the excitation light source module and the MCU system are arranged in the shading darkroom; the detection plate is provided with a detection groove for placing a fluorescent probe and a reagent, and the excitation light source module is opposite to the detection groove; the fluorescence detection module comprises two groups, each group comprises an optical filter and a photoelectric sensor, and 2 optical filters are optical filters with different central wavelengths; the output end of the MCU system is connected with the excitation light source module, the input end of the MCU system is connected with the fluorescence detection module, and the output light of the excitation light source module is emitted into the photoelectric sensor after being filtered by the optical filter after being excited by the fluorescent probe.

In order to facilitate the replacement of the fluorescent probe and the reagent, it is preferable that the detection plate is disposed in a light-shielded darkroom so as to be movable inside and outside.

Preferably, the scheme further comprises a display unit, and the output end of the MCU system is connected with the display unit.

Preferably, the scheme further comprises an upper computer, and the output end of the MCU system is connected with the upper computer.

Preferably, the excitation light source module includes a constant current source, an LED excitation light source, and a coupling unit, which are connected in sequence.

Preferably, the center wavelength of one filter is 435-445nm, and the wavelength of the other filter is 620-630 nm.

Compared with the prior art, the utility model has the advantages of it is following:

1. the utility model discloses a ratio type fluorescence probe has from the reference characteristic, and is no longer harsh to the detection ring border requirement, can effectively avoid coming from the interference of environment, and it is high to detect the precision to but very big degree reduction system error, interference killing feature are extremely strong.

2. The utility model discloses do not need expensive main equipment, detect and become the portable, use the information that low price sensor can acquire fluorescent substance, detect with other ordinary chemical methods and compare rate type fluorescence detection have handle simple, sensitivity is high, the signal is adjustable, harmless, advantage such as real-time.

3. The utility model discloses a ratio type fluorescence detection method, through low-cost alright detection fluorescent substance or the concentration change who takes place reaction material with it.

4. The utility model discloses with detecting system integration in miniature embedded system for examine simple, convenient.

Drawings

FIG. 1 is a schematic diagram of a ratio-based fluorescence detection system according to the present invention;

FIG. 2 is an exploded view of FIG. 1;

FIG. 3 is an exploded view of another directional view of FIG. 1;

FIG. 4 is a block diagram of a ratiometric fluorescence detection system of the present invention;

FIG. 5 shows red/blue₇Emission spectra before and after the action of the gold nanocluster film and serum bilirubin;

FIG. 6 is a graph showing the correlation between the serum bilirubin concentration C and the corresponding ratio F11/F12.

Reference numerals: 1. the device comprises a shading darkroom, 2, a detection plate, 3, a detection groove, 4, an LED excitation light source, 5, a photoelectric sensor, 6 and an MCU system.

Detailed Description

In order to make the objects and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and examples.

The utility model discloses a ratio type fluorescence detecting system, as shown in fig. 1, 2, 3, 4, including shading darkroom 1, pick-up plate 2, fluorescence detection module, excitation light source module and MCU system 6 weld on same circuit board.

The shading darkroom 1 is made of PLA materials and mainly used for reducing the interference of ambient light, the shading darkroom 1 is of a cuboid structure with a hollow interior, and sliding grooves are formed in two side plates along the long side; the detection plate 2 is of a rectangular structure matched with the shading darkroom 1, a detection groove 3 used for placing a fluorescent probe and a reagent is formed in one end of the detection plate 2, the detection plate 2 moves inside and outside along the sliding groove (the shading darkroom 1 can be pushed or pulled), the fluorescent probe and the reagent can be conveniently replaced by pushing and pulling the detection plate 2, and the shading darkroom 1 can be designed to be opened to replace the fluorescent probe and the reagent.

The excitation light source module comprises a constant current source, an LED excitation light source 4 and a coupling unit which are sequentially connected, the LED excitation light source 4 is arranged right opposite to the detection groove 3, and the excitation light source module irradiates the fluorescent probe after being coupled to generate a fluorescent effect. The fluorescence detection modules comprise two groups, each group comprises an optical filter and a photoelectric sensor 5, wherein the detection groove 3 and the photoelectric sensor 5 module form an included angle of 45 degrees, and the photoelectric sensor 5 module converts the detected fluorescence signals into electric signals to be output; the 2 filters are filters with different central wavelengths, wherein the central wavelength of one filter is 435-445nm, and the wavelength of the other filter is 620-630 nm; the optical filter is mainly used for picking out light of a specific waveband for detection and filtering interference of stray light, such as environmental light which is not completely isolated from the outside by the excitation light source module. The output end of the MCU system 6 is connected with the excitation light source module, the input end of the MCU system 6 is connected with the fluorescence detection module, the MCU system 6 controls the working state of the excitation light source module, the output light of the excitation light source module is emitted after being excited by the fluorescent probe, the fluorescence is filtered by the optical filter and then is emitted into the photoelectric sensor 5, the photoelectric sensors 5 transmit the detected signals to the MCU system 6, the MCU system 6 records the fluorescence intensity change of the fluorescent probe, and the frequencies measured by the two photoelectric sensors 5 are proportioned to obtain a value, this value correlates with the degree of fluorescence quenching, and the degree of fluorescence quenching is linear with the reagent concentration, and the MCU system 6 fits a curve relation between the fluorescence quenching degree and the reagent concentration according to the relation by measuring the reagents with different concentrations for multiple times, so that the concentration of the same reagent can be obtained by measuring the frequency ratio.

The utility model discloses still include display element and host computer, MCU system 6's output respectively with display element and host computer connection. The display unit is used for displaying a fluorescence value measured by the photoelectric sensor 5, a linear relation curve graph of fluorescence quenching degree (fluorescence ratio before adding the reagent and after adding the reagent) and reagent concentration, and the like, and the upper computer is used for storing data, a linear relation curve graph of fluorescence quenching degree (fluorescence ratio before adding the reagent and after adding the reagent) and reagent concentration, and the like.

The utility model relates to a detection method of ratio type fluorescence detecting system, including following step: the concentration of the reagent (whose concentration is unknown, referred to as a test agent) is obtained by first fitting a correlation curve of its fluorescence quenching degree and concentration with a reagent of known concentration, and second fitting the fitted correlation curve and the measured fluorescence quenching degree of the reagent (whose concentration is unknown, referred to as a test agent).

Wherein fitting the correlation curve comprises the steps of:

preparing standard solutions (with known concentrations) of the same reagent with different concentration gradients for data fitting.

And secondly, turning on a system main switch to enable other modules of the system to work, and then turning on an excitation light source module switch to wait for the excitation light source to be stable (the numerical value of a display interface tends to be stable).

Thirdly, after the excitation light source is stable, the fluorescent probe is placed in the detection groove 3, then the detection plate 2 is lightly pushed into the shading darkroom 1, at the moment, the detection groove 3 and the photoelectric sensor 5 form an included angle of 45 degrees, and after the data detected by the photoelectric sensor 5 are stable, the initial values F01 and F02 detected by the photoelectric sensor 5 are recorded. Wherein F01 is a value at which the blue fluorescence intensity is detected, and F02 is a value at which the red fluorescence intensity is detected. (F01/F02 here corresponds to a value of 0 for the reagent concentration).

Fourthly, the detection plate 2 is pulled out, 50uL of standard solution is absorbed by a trace pipette gun and dripped on a fluorescent probe to react for 80 to 120 seconds, and then the redundant solution around the film is absorbed by filter paper and dried.

Recording the concentration value C of the currently sucked standard solution and values F11 and F12 detected by optical frequency sensing after the standard solution is dripped.

Sixthly, repeating the step (iv) and the step (v) for multiple times to obtain a plurality of groups of F11 and F12 corresponding to the concentration value C of the standard solution, wherein F11 is the value for detecting the blue fluorescence intensity, and F12 is the value for detecting the red fluorescence intensity. In the present preferred embodiment, it is repeated 5 times;

seventhly, fitting the concentration value C of the standard solution and the corresponding ratio F11/F12 into a curve with correlation to obtain the relation between the concentration value C of the reagent and the corresponding ratio F11/F12, burning the fitted curve formula with the correlation into the MCU system 6, and displaying the curve formula through a display interface;

(2) detection of concentration of to-be-detected liquid of same reagent

Taking the solution to be detected with unknown concentration, repeating the fourth step and the fifth step, and specifically: after an excitation light source is stable, pulling out the detection plate 2, sucking the liquid to be detected by using a micro liquid-transferring gun and dripping the liquid to be detected on the fluorescent probe to react for 80-120s, sucking the redundant solution around the film by using filter paper, after drying, recording values F11 and F12 detected by optical frequency sensing after the liquid to be detected is dripped, then obtaining the concentration of the liquid to be detected by using the correlation curve of the obtained concentration value C and the corresponding ratio F11/F12, and directly viewing the concentration of the liquid to be detected on a display interface to finish detection.

The concentration of the reagent can be detected by replacing different reagents and corresponding fluorescent probes and repeating the steps.

The utility model relates to an application of a ratio type fluorescence detection system in quantitative detection of serum bilirubin. The reagent is serum bilirubin, and the fluorescent probe is a red/blue gold nano-cluster film. In the detection, two photoelectric sensors 5 with different sensitivities are adopted to detect the fluorescence intensity of the quenched red/blue gold nano-cluster film added with serum bilirubin. As shown in FIG. 5, which shows the emission spectra before and after the action of the red/blue gold nanocluster thin film (14 layers) and serum bilirubin, it is clear that the serum bilirubin decreases the intensity of both fluorescence peaks of the thin film. As is clear from FIG. 5, the concentration of the added reagent can be measured by a ratio-type fluorescence detection method since there are peaks of fluorescence intensity at the wavelengths of 440nm and 623nm before the addition of the reagent and fluorescence quenching occurs at both peaks after the addition of the reagent. Table 1 summarizes the fluorescence quenching of serum bilirubin for the red/blue gold nanocluster film using the values measured by the ratiometric fluorescence detection system, and the quenching efficiencies are represented by F01/F11(F01 is the red fluorescence peak intensity of the red gold nanocluster film before serum bilirubin is added, F11 is the red fluorescence peak intensity of the red gold nanocluster film after serum bilirubin is added), F02/F12(F02 is the blue fluorescence peak intensity of the blue gold nanocluster film before serum bilirubin is added, and F12 is the blue fluorescence peak intensity of the blue gold nanocluster film after serum bilirubin is added). The data in table 1 indicate that of the two fluorescence peaks, serum bilirubin quenches the red fluorescence peak at 622nm to a greater extent, indicating that the red fluorescent nanoclusters have a higher sensitivity of response to serum bilirubin. Therefore, in the secondary detection, the red gold nanocluster film mainly serves as a detection unit of serum bilirubin, and the blue gold nanocluster film more serves as a reference unit. Then, the frequency ratio obtained by proportional operation of the frequencies measured by the two photosensors 5 is related to the fluorescence quenching degree, and the fluorescence quenching degree and the serum bilirubin concentration are in a linear relationship, so that the serum bilirubin concentration can be obtained by measuring the frequency ratio, as shown in fig. 6, which is a correlation curve of the serum bilirubin concentration value C and the corresponding ratio F11/F12.

Table 1:

in Table 1, F01/F11 is the fluorescence intensity value of blue light gold nanocluster fluorescence peak (443nm) before (F01) and after (F11) bilirubin is added. F02/F12: fluorescence intensity values of red gold nanocluster fluorescence peaks (622nm) before (F02) and after (F12) bilirubin is added. When F01/F11 and F02/F12 are compared, the change of red fluorescence is more obvious, so that the change of F11/F12 denominator is more obvious, the value is larger and the change gradient is more obvious when F12 (red fluorescence intensity) is used as the denominator.

According to the fact that the fluorescence quenching degree (F0/F) (F0 is the fluorescence intensity value before adding the reagent, F is the fluorescence intensity value after adding the reagent) and the serum bilirubin concentration have corresponding linear relation, the feasibility of the scheme is verified through configuring corresponding standard liquid design experiments. The experimental result shows that the system has better accuracy and stability, the detection result of the system has strong correlation with a standard instrument, the correlation coefficient of the detection result and the standard instrument reaches 0.987, the detection result can be displayed in real time, and the rapid quantitative detection of the serum bilirubin solution can be realized.

The above embodiments are only specific examples for further detailed description of the objects, technical solutions and advantages of the present invention, and the present invention is not limited thereto. Any modification, equivalent replacement, improvement and the like made within the scope of the disclosure of the present invention are all included in the protection scope of the present invention.

Claims

1. A ratio-based fluorescence detection system, comprising: the device comprises a shading darkroom, and a detection board, a fluorescence detection module, an excitation light source module and an MCU system which are arranged in the shading darkroom; the detection plate is provided with a detection groove for placing a fluorescent probe and a reagent, and the excitation light source module is opposite to the detection groove; the fluorescence detection module comprises two groups, each group comprises an optical filter and a photoelectric sensor, and 2 optical filters are optical filters with different central wavelengths; the output end of the MCU system is connected with the excitation light source module, the input end of the MCU system is connected with the fluorescence detection module, and the output light of the excitation light source module is emitted into the photoelectric sensor after being filtered by the optical filter after being excited by the fluorescent probe.

2. A ratio-type fluorescence detection system according to claim 1, wherein: the detection plate can be arranged in the shading darkroom in an inner-outer moving mode.

3. A ratiometric fluorescence detection system according to claim 1 or 2, wherein: the MCU system also comprises a display unit, and the output end of the MCU system is connected with the display unit.

4. A ratiometric fluorescence detection system according to claim 1 or 2, wherein: the MCU system also comprises an upper computer, and the output end of the MCU system is connected with the upper computer.

5. A ratiometric fluorescence detection system according to claim 1 or 2, wherein: the excitation light source module comprises a constant current source, an LED excitation light source and a coupling unit which are connected in sequence.

6. A ratiometric fluorescence detection system according to claim 1 or 2, wherein: the center wavelength of one filter is 435-445nm, and the wavelength of the other filter is 620-630 nm.