CN111781143B - Dual-channel fluorescence detection device and detection method thereof - Google Patents
Dual-channel fluorescence detection device and detection method thereof Download PDFInfo
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Abstract
The invention provides a double-channel fluorescence detection device and a detection method thereof, which belong to the technical field of molecular diagnostic instruments, wherein two detection channels and two light path channels are arranged in a base, a light source and a first optical module are arranged in a first light path channel, the first detection channel and the second detection channel are both internally provided with the optical module, one end of the first detection channel is provided with a first converter, the same end of the second detection channel is provided with a second converter, the other end of the second detection channel is close to the second light path channel and is provided with a reaction tank communicated with the second light path channel, and the second converter and the first converter are respectively electrically connected with a processor; each optical module is also positioned at the position communicated with the second light path channel; the light source emits light beams to enter the reaction tank, and after the light beams irradiate the reaction liquid, the first fluorescence and the second fluorescence in the reaction liquid are excited, and signals are converted by the first converter and the second converter and input into the processor. The relative concentration or relative amount of the fluorescent reporter groups is obtained by a method of eliminating crosstalk.
Description
Technical Field
The invention relates to the technical field of molecular diagnostic instruments, in particular to a double-channel fluorescence detection device and a detection method thereof.
Background
In the field of molecular biology experiments and molecular diagnostics, for analytical detection of specific, trace amounts of biomolecules, such as: nucleic acids, proteins, etc., are often fluorescently labeled. The method uses specific fluorescent group molecules to specifically combine with the biological molecules to be detected, then uses light with specific wavelength to excite the fluorescent group molecules, detects fluorescent signals emitted by the fluorescent group molecules at specific wavelength, and finally qualitatively or quantitatively analyzes the information such as concentration, distribution and the like of the specific biological molecules in the detected object through the fluorescent signals. The method has the advantages of high sensitivity, convenient operation, no need of contacting the tested sample and the like, and has wide application.
Because the specific combination between the fluorescent group molecules and the detected biomolecules is generated, it means that if two or more fluorescent group molecules with different fluorescence characteristics are added into the sample at the same time, two or more corresponding biomolecules can be combined at the same time, and the existing dual-channel fluorescence detection device uses the principle for detection.
However, the two existing fluorescence detection channels are mutually independent, the detection can not be performed at the same time only by switching the positions, the detection efficiency is reduced, and the mechanical device for switching the fluorescence detection channels is complex, high in cost and high in failure rate.
Disclosure of Invention
The invention aims to provide a double-channel fluorescence detection device and a detection method thereof, which can simultaneously measure two fluorescence report groups in a reaction tank, greatly improve the detection speed and have low cost.
Embodiments of the present invention are implemented as follows:
An aspect of the embodiment of the invention provides a dual-channel fluorescence detection device, which comprises a base, wherein a first detection channel, a second detection channel, a first light path channel and a second light path channel are arranged in the base, the first detection channel and the second detection channel are respectively positioned at two sides of the first light path channel, and the second light path channel is communicated with the first light path channel, the first detection channel and the second detection channel; the light source and the first optical module arranged along the emergent direction of the light source are arranged in the first light path channel, the second optical module is arranged in the first detection channel, a first converter is arranged at one end of the first detection channel far away from the second light path channel, a third optical module is arranged in the second detection channel, a reaction tank is arranged at one end of the second detection channel close to the second light path channel, a second converter is arranged at the other end of the second detection channel, the reaction tank is communicated with the second light path channel, and the second converter and the first converter are respectively and electrically connected with a processor; the first optical module, the second optical module and the third optical module are also positioned at the communication position of the second optical path channel; the light beam emitted by the light source enters the third optical module through the second optical path channel and then enters the reaction tank, the light beam irradiates the reaction liquid in the reaction tank and then excites first fluorescence and second fluorescence in the reaction liquid, the first fluorescence sequentially passes through the third optical module, the first optical module and the second optical module, sequentially passes through the second optical path channel and the first detection channel, and is converted by the first converter to be input into the processor, and the second fluorescence passes through the third optical module, passes through the second detection channel and is converted by the second converter to be input into the processor.
Optionally, the first optical module includes a first optical filter and a first dichroic mirror that are sequentially disposed along the light source emitting direction, and the first dichroic mirror is located at a communication position between the first light path channel and the second light path channel.
Optionally, the third optical module includes a second dichroic mirror and a second optical filter, where the second optical filter is close to the second converter, and the second dichroic mirror is located at a communication position between the second detection channel and the second optical path channel.
Optionally, two converging lenses are further arranged in the second detection channel, one converging lens is located between the second converter and the second dichroic mirror, and the other converging lens is located between the reaction tank and the second dichroic mirror.
Optionally, the second optical module includes a reflecting mirror, and the reflecting mirror is located at a communication position between the first detection channel and the second optical path channel; and a converging lens is further arranged in the first detection channel, and the converging lens is positioned between the first converter and the reflecting mirror.
Optionally, a third optical filter is disposed in the second optical path, and the third optical filter is located between the first optical module and the second optical module.
Optionally, the first light path channel, the first detection channel and the second detection channel are arranged in parallel, the second light path channel is perpendicular to the first light path channel, the second detection channel and the first detection channel respectively, and an included angle of 45 degrees is formed between the first dichroic mirror and the second light path channel.
Optionally, a cover plate is arranged on the base, the cover plate is positioned at one end of the second detection channel, where the reaction tank is arranged, and a through hole is arranged on the cover plate to be communicated with the first light path channel and the reaction tank; the reaction tank is arranged in the temperature block, and the temperature block is used for heating reaction liquid in the reaction tank; the first converter and the second converter are photoelectric sensors.
Another aspect of the embodiments of the present invention provides a dual-channel fluorescence detection method, which is applied to the dual-channel fluorescence detection device, where the dual-channel fluorescence detection device includes a reaction tank, and a reaction solution in the reaction tank includes a first fluorescent reporter group and a second fluorescent reporter group; the method comprises the following steps: receiving a first fluorescent signal fed back by the first converter and a second fluorescent signal fed back by the second converter respectively; wherein the first converter receives and feeds back a portion of the optical signal reflected by the first fluorescent reporter group, and the second converter receives and feeds back the optical signal reflected by the second fluorescent reporter group and another portion of the optical signal reflected by the first fluorescent reporter group; calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group according to C1-CH 1 and C2-CH 2-k 21CH1/k11; wherein, C1 is the concentration of the first fluorescent reporter group, C2 is the concentration of the second fluorescent reporter group, CH1 is the first fluorescent signal, CH2 is the second fluorescent signal, and k 21/k11 is the crosstalk coefficient.
Optionally, before the calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group according to C1 oc CH1 and C2 oc CH2-k 21CH1/k11, respectively, the method further comprises: according to formula one: ch1=k 11 c1 and formula bich2=k 21C1+k22 c2, wherein k11, k21, k22 are fixed values, and the reaction solution is preset to have only the first fluorescent reporter group, so as to obtain formula three: c2 =0; substituting the formula III into the formula II to obtain a formula IV: ch2=k 21 C1; dividing the formula IV by the formula I to obtain a crosstalk coefficient: k21/k11=ch2/CH 1.
The beneficial effects of the embodiment of the invention include:
The two-channel fluorescence detection device and the detection method thereof provided by the embodiment of the invention are characterized in that a first detection channel, a second detection channel, a first light path channel and a second light path channel are arranged in a base, the first detection channel and the second detection channel are respectively positioned at two sides of the first light path channel, the second light path channel is communicated with the first light path channel, the first light path channel is internally provided with a light source and a first optical module arranged along the emergent direction of the light source, the first detection channel is internally provided with a second optical module, one end of the first detection channel, which is far away from the second light path channel, is provided with a first converter, the second detection channel is internally provided with a third optical module, one end of the second detection channel, which is close to the second light path channel, is provided with a reaction tank, the other end of the second detection channel is communicated with the second light path channel, and the first converter and the second converter are respectively electrically connected with a processor; the first optical module, the second optical module and the third optical module are also positioned at the communication position of the second optical path channel, so that the three optical modules can mutually receive and emit light beams through the common second optical path channel. During detection, light beams emitted by the light source are incident into the third optical module through the first optical module through the second optical path, then are incident into the reaction tank, reaction liquid is arranged in the reaction tank, the reaction liquid contains a first fluorescence reporting group and a second fluorescence reporting group (the fluorescence reporting group is known fluorescein used by an added probe), after the light beams irradiate the reaction liquid, the first fluorescence and the second fluorescence in the reaction liquid are excited, the first fluorescence sequentially passes through the third optical module after being reflected, reaches the first optical module through the second optical path, then enters the first detection path through the second optical module, the first converter converts signals, and the second fluorescence is reflected and then converted into signals by the second converter through the third optical module through the second detection path, and the signals are input into the processor. The processor receives the conversion signals and processes the conversion signals, and detects the corresponding fluorescent report groups, so that two fluorescent report groups in the same reaction tank can be detected by the processor at the same time, the detection speed is greatly improved, the four channels are shared, the optical modules are shared, the mechanical structure is not required to switch the fluorescent detection channels, the volume weight and the cost are reduced, the overall reliability is improved, and the cost of an optical system and the detection cost are reduced. In addition, because the fluorescence reflected by the first fluorescent reporter group reaches the first detection channel after passing through the second detection channel, part of the fluorescence is received by the second converter, and the second detection channel has the condition of fluorescence crosstalk reflected by the two fluorescent reporter groups, the signal received by the first converter corresponds to part of the first fluorescent reporter group, the signal received by the second converter corresponds to the other part of the first fluorescent reporter group and all the second fluorescent reporter groups, and the two-channel fluorescence detection method obtains the relative concentration or the relative quantity of the two fluorescent reporter groups by a method for eliminating the fluorescence crosstalk.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the binding of a fluorophore molecule to a biomolecule to be detected;
FIG. 2 is a schematic diagram of a dual-channel fluorescence detection device according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of one of the optical path diagrams of the dual-channel fluorescence detection device according to the embodiment of the present invention;
FIG. 4 is a second optical path diagram of a dual-channel fluorescence detection device according to an embodiment of the present invention;
FIG. 5 is a fluorescence spectrum chart of a FAM fluorescence reporter group detected by the dual-channel fluorescence detection device provided by the embodiment of the invention;
FIG. 6 is a third optical path diagram of a dual-channel fluorescence detection device according to an embodiment of the present invention;
FIG. 7 is a fluorescence spectrum diagram of HEX fluorescence reporter group detected by the dual-channel fluorescence detection device provided by the embodiment of the invention;
FIG. 8 is a fluorescence spectrum diagram of a second converter of the dual-channel fluorescence detection device provided by the embodiment of the invention for simultaneously detecting HEX and FAM fluorescence report groups;
FIG. 9 is a flowchart of a dual-channel fluorescence detection method according to an embodiment of the present invention;
FIG. 10 is a second flowchart of a dual-channel fluorescence detection method according to an embodiment of the present invention.
Icon: 101-a light source; 102-a first filter; 103-a first dichroic mirror; 104-a second dichroic mirror; 105-a second filter; 106. 110, 109-converging lenses; 107-a third filter; 108-a mirror; 111-cover plate and 112-temperature block; 113-a reaction tank; 114-reaction solution; 115. 116-photo sensor.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the field of molecular biology experiments and molecular diagnostics, for analytical detection of specific, trace amounts of biomolecules, such as: nucleic acids, proteins, etc., are often fluorescently labeled. The method uses specific fluorescent group molecules to specifically combine with the biological molecules to be detected, then uses light with specific wavelength to excite the fluorescent group molecules, detects fluorescent signals emitted by the fluorescent group molecules at specific wavelength, and finally qualitatively or quantitatively analyzes the information such as concentration, distribution and the like of the specific biological molecules in the detected object through the fluorescent signals. The method has the advantages of high sensitivity, convenient operation, no need of contacting the tested sample and the like, and has wide application.
Because the specific combination occurs between the fluorescent group molecules and the detected biomolecules, the fluorescent group molecules with two or more different fluorescent characteristics can be simultaneously combined if two or more fluorescent group molecules with different fluorescent characteristics are simultaneously added into the sample, the aim of simultaneous detection of multiple targets is fulfilled, and the detection efficiency is greatly improved. Then the need for multi-channel fluorescence detection is now raised for fluorescence detection devices.
As shown in FIG. 1, taking real-time fluorescent quantitative PCR (Polymerase Chain Reaction polymerase chain reaction) as an example, the basic reaction step of PCR is thermal denaturation (denaturation) -annealing-extension (extension). The reaction uses Taq polymerase (a DNA polymerase) and a pair of single stranded DNA primers to effect replication of a particular nucleic acid sequence. Meanwhile, a TaqMan probe (probe) is introduced into the reaction system to reflect the amplification condition of nucleic acid in real time. The TaqMan probe body is a single-stranded DNA oligonucleotide complementary to and paired with an amplified nucleic acid sequence, the TaqMan probe has a3 'end and a 5' end, a fluorescence reporting group (R) is marked on the 3 'end, and a fluorescence quenching group (quencher, Q) is marked on the 5' end. When the TaqMan probe has complete structure, the fluorescent reporter group molecule absorbs the excitation photons with specific wavelength and then is converted into a high-energy-level excitation state. When the TaqMan probe is structurally complete, the distance between the fluorescent reporter group and the fluorescent quenching group is only a few nanometers, and a fluorescence resonance energy transfer (Fluorescence Resonance ENERGY TRANSFER, FRET) effect occurs between the two: the fluorescence quenching group absorbs the energy of the fluorescence reporting group in an excited state, the fluorescence reporting group makes a non-radiative transition back to a ground state, and no fluorescence photon is emitted in the process. When Taq polymerase extends downstream along the 3' end of the upstream primer, the Taq polymerase will endohydrolyze the single stranded DNA oligonucleotides of the TaqMan probe bound to the template strand, resulting in separation of the fluorescent reporter group from the fluorescent quenching group. The distance between the fluorescent reporter group and the fluorescent quenching group, which are free in solution, exceeds the radius at which fluorescence resonance energy transfer occurs. At this time, the fluorescent reporter group may emit fluorescent photons under excitation at a specific wavelength. Thus, the fluorescence intensity of the fluorescent reporter group is proportional to the amount or concentration of hydrolysis of the TaqMan probe and also to the amount or concentration of replication to generate template DNA.
Since primers (primers) and TaqMan probes bind to nucleic acid templates via the base complementary pairing principle, there is a high degree of specificity. Thus, two or more nucleic acid sequences can be replicated simultaneously by designing two or more sets of primers with the TaqMan probe. This method is called multiplex PCR (Multiplex PCR). At this time, if the same fluorescent reporter group is used for labeling the TaqMan probe, it is impossible to distinguish which nucleic acid copy the fluorescent signal is generated. It is therefore necessary to use two or more fluorescent reporter groups in the same number as the detection targets.
In the existing two-channel fluorescence detection, one channel is used for detecting fluorescence of FAM fluorescent reporter groups, and the other channel is used for detecting fluorescence of HEX fluorescent reporter groups. The two fluorescence detection channels are mutually independent, and the fluorescence of the two channels of one test tube can be detected only by switching positions, so that the two channels cannot be detected simultaneously. And the mechanical device for switching the fluorescence detection channel is complex, the cost is high, and the failure rate is high. The two fluorescence detection channels are similar in principle, but all optical parts are not shared, so that the cost is high.
In sum, on the basis, the dual-channel fluorescence detection device is provided, and can simultaneously measure the relative concentration or the relative quantity of two fluorescence reporting groups in one target, so that the detection speed is greatly improved; the fluorescent detection channel is not required to be switched by a mechanical structure, so that the volume weight and the cost are reduced, and the overall reliability is improved; in the two or more fluorescence detection channels, optical elements such as a light source and a focusing lens are shared, so that the cost of the optical system is reduced. On the other hand, the fluorescence reflected by the first fluorescent reporter group reaches the first detection channel after passing through the second detection channel, and part of the fluorescence is received by the second converter, and the second detection channel has the condition of fluorescence crosstalk reflected by the two fluorescent reporter groups, so that the signal (first fluorescence signal) received by the first converter corresponds to part of the first fluorescent reporter group, and the signal (second fluorescence signal) received by the second converter corresponds to the other part of the first fluorescent reporter group and all the second fluorescent reporter groups.
Specifically, referring to fig. 2, the present embodiment provides a dual-channel fluorescence detection device, which includes a base, wherein a first detection channel, a second detection channel, a first optical path channel, and a second optical path channel are disposed in the base.
The first detection channel and the second detection channel are respectively positioned at two sides of the first light path channel, and the second light path channel is communicated with the first light path channel, the first detection channel and the second detection channel; a light source 101 and a first optical module arranged along the emergent direction of the light source 101 are arranged in the first light path channel, a second optical module is arranged in the first detection channel, a first converter is arranged at one end of the first detection channel far away from the second light path channel, a third optical module is arranged in the second detection channel, a reaction tank 113 is arranged at one end of the second detection channel near the second light path channel, a second converter is arranged at the other end of the second detection channel, the reaction tank 113 is communicated with the second light path channel, and the first converter and the second converter are respectively electrically connected with a processor; the first optical module, the second optical module and the third optical module are also positioned at the communication position of the second optical path channel.
The light beam emitted by the light source 101 enters the reaction tank 113 after entering the third optical module through the second optical path, and after the light beam irradiates the reaction liquid 114 in the reaction tank 113, the first fluorescence and the second fluorescence in the reaction liquid 114 are excited, the second fluorescence is converted into a signal by the second converter through the third optical module through the second detection channel and is input into the processor, and the first fluorescence sequentially passes through the third optical module, the first optical module and the second optical module, sequentially passes through the second detection channel, the second optical path channel and the first detection channel and is converted into the signal by the first converter and is input into the processor.
The first light path channel, the first detection channel and the second detection channel are arranged in parallel, and the second light path channel is communicated with the first light path channel, the first detection channel and the second detection channel and is perpendicular to the first light path channel, the first detection channel and the second detection channel.
The first light path channel is internally provided with the light source 101 and the first optical module, the second light path channel is close to one side provided with the first optical module, the first detection channel is internally provided with the second optical module, the second detection channel is internally provided with the third optical module, the first optical module, the second optical module and the third optical module are also simultaneously positioned at the position communicated with the second light path channel, and the light emitting parts of the three optical modules are simultaneously positioned in the second light path channel, so that after the three light paths are communicated through the second light path channel, the three optical modules can mutually receive and emit light beams through the common second light path channel, so that the light beams can wander in the first light path channel, the second light path channel, the first detection channel and the second detection channel, and then the light beams enter a specified certain channel through the arrangement of the first optical module, the second optical module and the third optical module.
One end of the first detection channel far away from the second light path channel is provided with a first converter, one end of the second detection channel near the second light path channel is provided with a reaction tank 113, and the other end of the second detection channel near the second light path channel is provided with a second converter.
The first converter and the second converter are both used for converting signals. Specifically, the second converter and the first converter are both photosensors, and the second converter is, for example, a photosensor 115 and the first converter is a photosensor 116. The photoelectric sensor converts the optical signal into an electric signal and feeds the electric signal back to the processor for processing.
The reaction tank 113 is internally provided with a reaction liquid 114, the reaction liquid 114 contains a first fluorescent reporter group and a second fluorescent reporter group, for example, the reaction tank 113 can be internally provided with a PCR reaction liquid 114 containing FAM and HEX fluorescent reporter groups, a light beam emitted by the light source 101 reaches the reaction tank 113, a fluorescent light beam corresponding to fluorescent group molecules is obtained after the reaction liquid 114 is excited, the fluorescent light beam is reflected again and finally received by the first converter and the second converter, an optical signal is converted into other signals, for example, the other signals are converted into electric signals, the converted signals are fed back to the processor, and the processor detects the corresponding fluorescent reporter groups according to the fed back converted signals.
Specifically, the light beam emitted from the light source 101 is incident to the third optical module through the second optical path after passing through the first optical module, and then the third optical module is incident to the reaction liquid 114 in the reaction tank 113, so as to excite the reaction liquid 114 to obtain first fluorescence and second fluorescence, the first fluorescence enters the first detection channel through the second optical path, the second fluorescence enters the second detection channel, the first fluorescence entering the first detection channel is converted by the first converter to be input into the processor, the second fluorescence entering the second detection channel is converted by the second converter to be input into the processor, and the detection of two fluorescence reporting groups in the reaction liquid 114 is completed. This allows two fluorescent reporter groups in the same reaction cell 113 to be detected simultaneously by the processor.
The two-channel fluorescence detection device provided by the embodiment of the invention is characterized in that a first detection channel, a second detection channel, a first light path channel and a second light path channel are arranged in a base, the second detection channel and the first detection channel are respectively positioned at two sides of the first light path channel, the second light path channel is communicated with the first light path channel, the second detection channel and the first detection channel, a light source 101 and a first optical module arranged along the emergent direction of the light source 101 are arranged in the first light path channel, a third optical module is arranged in the second detection channel, a reaction tank 113 is arranged at one end of the second detection channel, which is close to the second light path channel, a second converter is arranged at the other end of the second detection channel, the reaction tank 113 is communicated with the second light path channel, a second optical module is arranged in the first detection channel, a first converter is arranged at one end, which is far away from the second light path channel, and the second converter and the first converter are respectively electrically connected with a processor; the first optical module, the third optical module and the second optical module are also positioned at the communication position of the second optical path channel, so that the three optical modules can mutually receive and emit light beams through the common second optical path channel. The light beam emitted by the light source 101 enters the third optical module through the first optical module through the second optical path, then enters the reaction tank 113 through the third optical module through the second detection path, the reaction liquid 114 is arranged in the reaction tank 113, the reaction liquid 114 contains two kinds of fluorescence reporting groups, the reaction liquid 114 in the reaction tank 113 reacts and excites with the light beam to respectively obtain first fluorescence and second fluorescence, the second fluorescence is converted into a signal by the second converter through the third optical module through the second detection path, the first fluorescence is sequentially converted into the signal by the second detection path, the first optical module and the second optical module through the third optical module, the second optical path and the first detection path through the first converter, the processor receives the converted signal and processes the converted signal, the corresponding fluorescence reporting groups are detected, the two kinds of fluorescence reporting groups in the same reaction tank 113 can be detected by the processor at the same time, the detection speed is greatly improved, the four channels are shared, the optical module is not required to be used, the mechanical structure is not required to switch the fluorescence detection paths, the volume weight and the cost are reduced, the whole reliability is improved, and the optical system cost is reduced.
Specifically, the first optical module includes a first optical filter 102 and a first dichroic mirror 103 sequentially disposed along an emission direction of the light source 101, where the first dichroic mirror 103 is located at a communication position between the first optical path and the second optical path, and changes the emission direction of the light beam emitted from the light source 101 through the first dichroic mirror 103, so that the light beam is emitted from the first optical path, changes the direction, and then is emitted along the second optical path, so as to be directed to the third optical module.
The first filter 102 is used for filtering the light beam emitted by the light beam to obtain the light beam with the required wavelength, so that the light beam is reflected by the first dichroic mirror 103, and the light beam is prevented from being transmitted to the second optical module, but is reflected to the third optical module.
The first light path channel, the second detection channel and the first detection channel are arranged in parallel, the second light path channel is perpendicular to the first light path channel, the second detection channel and the first detection channel respectively, and an included angle of 45 degrees is formed between the first dichroic mirror 103 and the second light path channel.
In this way, after the light beam emitted from the first optical module passes through the first dichroic mirror 103 disposed at an angle of 45 °, the light beam can be changed into a direction perpendicular to the emitting direction of the first optical module, and is directed to the third optical module along the second optical path, so as to change the emitting path of the light beam.
The third optical module comprises a second dichroic mirror 104 and a second optical filter 105, wherein the second optical filter 105 is close to the second converter, and the second dichroic mirror 104 is located at a communication position of the second detection channel and the second optical path channel. The light beam emitted from the first dichroic mirror 103 is directed to the second dichroic mirror 104, an included angle of 45 ° is formed between the second dichroic mirror 104 and the second optical path, and the second dichroic mirror 104 changes the light beam incident from the first dichroic mirror 103 into a light beam perpendicular to the second optical path, and is directed to the reaction cell 113 along the second detection path.
A converging lens 110 is provided between the reaction cell 113 and the second dichroic mirror 104 for condensing light.
Further, both the first dichroic mirror 103 and the second dichroic mirror 104 are long-pass dichroic mirrors.
The light beam excites the reaction liquid 114 in the reaction tank 113 to obtain first fluorescence and second fluorescence, the first fluorescence and the second fluorescence are reflected by the second detection channel, are emitted to the second dichroic mirror 104 through the converging lens 110, and are split at the second dichroic mirror 104, wherein the second fluorescence is transmitted through the second dichroic mirror 104, and is emitted to the second converter through the second detection channel. A converging lens 106 is also provided between the second dichroic mirror 104 and the second converter.
The first fluorescent light is reflected by the second dichroic mirror 104, and then the light beam which is changed to be emitted along the second light path is emitted to the first dichroic mirror 103, and is emitted to the second optical module after being transmitted through the first dichroic mirror 103.
The second optical module comprises a reflecting mirror 108, the reflecting mirror 108 is positioned at the communication position of the first detection channel and the second optical path channel, an included angle of 45 degrees is formed between the reflecting mirror 108 and the second optical path channel, the reflecting mirror 108 receives the first fluorescence, and the emergent direction is changed, so that the first fluorescence is emitted to the first converter along the first detection channel. A converging lens 109 is also arranged between the first transducer and the mirror 108.
In addition, a third optical filter 107 is disposed in the second optical path, and the third optical filter 107 is located between the first optical module and the second optical module, specifically between the first dichroic mirror 103 and the reflecting mirror 108.
The first fluorescent light is transmitted through the first dichroic mirror 103, filtered by the third filter 107, and reflected by the mirror 108 toward the first converter.
Further, a cover plate 111 is disposed on the base, the cover plate 111 is located at one end of the second detection channel where the reaction tank 113 is disposed, and a through hole is disposed on the cover plate 111 to communicate the first light path channel and the reaction tank 113.
The reaction tank 113 is provided with a cover to seal the reaction tank 113, and the cover is provided with a hole which is communicated with the through hole of the cover plate, so that the second detection channel is communicated with the reaction tank 113 through the through hole of the cover plate and the hole of the cover, and the light beam is not blocked.
The cover 111 is provided to press the cover of the reaction tank 113, so as to prevent the cover from being opened by expansion of gas generated by the reaction liquid 114 inside the reaction tank 113 after being heated, so that the reaction liquid 114 volatilizes, and the detection result is affected.
The reaction tank 113 is arranged in the temperature block 112, and the temperature block 112 is used for heating the reaction liquid 114 in the reaction tank 113.
The following specifically describes the detection process by taking FAM fluorescence excitation and HEX fluorescence excitation as examples:
As shown in fig. 3, the vertically placed light source 101 emits a broad spectrum of light downward. The light rays are excited by the first optical filter 102 through the first optical path channel and filtered into 480nm plus or minus 10nm of narrowband excitation light. The excitation light is incident on a first dichroic mirror 103 placed at an angle of 45 deg. to the horizontal. First dichroic mirror 103 allows light having a wavelength less than 500nm to be reflected and light having a wavelength greater than 500nm to be transmitted. Accordingly, the excitation light of 480nm±10nm is reflected by the first dichroic mirror 103, and horizontally enters the second dichroic mirror 104. The second dichroic mirror 104 allows light having a wavelength less than 545nm to be reflected and light having a wavelength greater than 545nm to be transmitted. Therefore, the excitation light of 480nm±10nm is reflected by the second dichroic mirror 104, is converged downward by the converging lens 110, and then sequentially passes through the cover plate 111, and is incident on the reaction tank 113 in the temperature block 112, and the reaction tank 113 may be a transparent reaction tube, and finally irradiates and excites the reaction solution 114 in the PCR reaction tank 113 containing FAM and HEX fluorescent reporter groups.
As shown in fig. 4, FAM fluorescence and HEX fluorescence are obtained after laser, and the fluorescence is randomly generated, wherein the FAM fluorescence is emitted vertically upwards, passes through the transparent reaction tank 113 and the cover plate 111, and enters the second dichroic mirror 104 after entering the converging lens 110. Because the spectral range of fluorescence emitted by the FAM fluorescent reporter is between 490-600 nm, portions of fluorescence having a wavelength less than 545nm are reflected at the second dichroic mirror 104. The fluorescence direction becomes horizontal. The filtered spectral range of the fluorescence passing through the second dichroic mirror 104 becomes between 490 and 545 nm. The fluorescence continues to enter first dichroic mirror 103 horizontally. Since the first dichroic mirror 103 allows transmission of light having a wavelength greater than 495nm, a portion having a fluorescence wavelength greater than 495 is transmitted at the first dichroic mirror 103. The fluorescence direction is unchanged. The fluorescence passes through the first dichroic mirror 103 and the filtered spectral range becomes between 495 and 545 nm. After that, the fluorescence is incident on the second optical path channel third filter 107. The third filter 107 of the second optical path filters the fluorescence into the most energetic narrow band 520nm + -10 nm. The 520 nm.+ -.10 nm fluorescence continues to propagate horizontally, reflecting off a mirror 108 placed at 45 ° to the horizontal, passing vertically upwards through a converging lens 109 and finally focusing on the photosensor 116 of the first detection channel. Through this process, fluorescence emitted from the FAM fluorescent reporter group is converted into an electrical signal by the photosensor 116 of the first detection channel.
The fluorescence spectrum of the device for detecting FAM fluorescence reporter group is shown in figure 5. The left side shadow peak is the normalized excitation spectrum of the FAM fluorescence reporter group, and the right side shadow peak is the normalized emission spectrum of the FAM fluorescence reporter group. The solid line is the excitation filter normalized transmittance spectrum, the one-dot chain line is the first dichroic mirror 103 transmittance spectrum, the two-dot chain line is the second dichroic mirror 104 transmission spectrum, and the broken line is the third filter 107 transmittance spectrum. It is necessary to ensure that 480nm±10nm of the transmission spectrum of the first filter 102 is located on the side where the transmittance of the first dichroic mirror 103 and the second dichroic mirror 104 is low (< 5%), that is, reflection occurs at both the first dichroic mirror 103 and the second dichroic mirror 104. The second filter 105 has a transmission spectrum of 520nm±10nm on the side of the second dichroic mirror 104 where the transmittance is low (< 5%) and on the side of the first dichroic mirror 103 where the transmittance is high (> 95%), that is, the second dichroic mirror 104 reflects and the first dichroic mirror 103 transmits.
As shown in fig. 6, the fluorescence emission directions are random, in which the HEX fluorescence is emitted vertically upwards, sequentially passes through the reaction cell 113 and the cover plate 111, and is incident on the converging lens 110 and then on the second dichroic mirror 104. Because the fluorescence spectrum emitted by the HEX fluorescence reporter group is between 540 and 640nm, most of the fluorescence can be incident on the second filter 105 of the second detection channel through the second dichroic mirror 104. The fluorescence is filtered into a narrow band 580nm + -20 nm with the strongest energy through the second filter 105 of the second detection channel, and finally focused on the photoelectric sensor 115 of the second detection channel through the converging lens 106. Through this process, fluorescence emitted from the HEX fluorescent reporter group is converted into an electrical signal by the photosensor 115 of the second detection channel.
The fluorescence spectrum of the HEX fluorescence reporter group detected by the device is shown in figure 7. The left side shadow peak is HEX fluorescence reporting group normalized excitation spectrum, the right side shadow peak is HEX fluorescence reporting group normalized emission spectrum. The solid line is the excitation filter normalized transmittance spectrum, the one-dot chain line is the first dichroic mirror 103 transmittance spectrum, the two-dot chain line is the second dichroic mirror 104 transmission spectrum, and the broken line is the second filter 105 transmittance spectrum. It is necessary to ensure that 480nm±10nm of the transmission spectrum of the first filter 102 is located on the side where the transmittance of the first dichroic mirror 103 and the second dichroic mirror 104 is low (< 5%), that is, reflection occurs at both the first dichroic mirror 103 and the second dichroic mirror 104. The second filter 105 transmits 580nm±20nm on the side of the second dichroic mirror 104 where the transmittance is high (> 95%), i.e., transmission occurs in the second dichroic mirror 104.
However, the dual-channel fluorescence detection device can detect the fluorescence signal of the FAM fluorescence reporter group on the photoelectric sensor 115 while detecting the HEX fluorescence reporter group. The fluorescence spectrum of this procedure is shown in FIG. 8. The left side shadow peak is the normalized excitation spectrum of the FAM fluorescence reporter group, and the right side shadow peak is the normalized emission spectrum of the FAM fluorescence reporter group. The solid line is the excitation filter normalized transmittance spectrum, the one-dot chain line is the first dichroic mirror 103 transmittance spectrum, the two-dot chain line is the second dichroic mirror 104 transmission spectrum, and the broken line is the second filter 105 transmittance spectrum. Excitation light of 480nm plus or minus 10nm can be absorbed by the FAM fluorescence reporter group, and a fluorescence signal emitted by the FAM fluorescence reporter group can still be received in a detection spectrum range of 580nm plus or minus 20 nm.
Therefore, when the PCR reaction solution contains both the FAM fluorescent reporter group and the HEX fluorescent reporter group, the fluorescence signals of the FAM fluorescent reporter group and the HEX fluorescent reporter group overlap each other on the fluorescence light path and the fluorescence spectrum of the second detection channel, and the signal detected by the photosensor 115 of the second detection channel is a linear superposition of the fluorescence signals of the FAM fluorescent reporter group and the HEX fluorescent reporter group. I.e. the second detection channel has fluorescent cross-talk.
The following method is adopted to eliminate the fluorescence crosstalk of the second detection channel:
as shown in fig. 9 and 10, this embodiment also provides a fluorescence detection method, which is applied to the above-mentioned dual-channel fluorescence detection device, where the dual-channel fluorescence detection device includes a reaction tank 113, and a reaction solution 114 of the reaction tank 113 includes a first fluorescence reporting group and a second fluorescence reporting group; the method comprises the following steps:
S100: the first fluorescent signal fed back by the first converter and the second fluorescent signal fed back by the second converter are respectively received.
Wherein the first converter receives and feeds back a portion of the optical signal reflected by the first fluorescent reporter group, and the second converter receives and feeds back the optical signal reflected by the second fluorescent reporter group and another portion of the optical signal reflected by the first fluorescent reporter group.
Because the fluorescence reflected by the first fluorescent reporter group reaches the first detection channel after passing through the second detection channel, a portion of the fluorescence reflected by the first fluorescent reporter group is received by the photosensor 115 through the second detection channel, and all of the fluorescence reflected by the second fluorescent reporter group is received by the photosensor 115 through the second detection channel, that is, the second fluorescent signal includes a portion of the fluorescence reflected by the first fluorescent reporter group received by the photosensor 115 and all of the fluorescence reflected by the second fluorescent reporter group.
Another portion of the fluorescence reflected by the first fluorescent reporter is received by the first transducer via the first detection channel, that is, the first fluorescent signal includes only another portion of the fluorescence reflected by the first fluorescent reporter received by the photosensor 116.
The part of the first fluorescence and the second fluorescence are coincident on the fluorescence light path and the fluorescence spectrum of the second detection channel.
S110: calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group according to C1-CH 1 and C2-CH 2-k 21CH1/k11; wherein, C1 is the concentration or the quantity of the first fluorescent reporter group, C2 is the concentration or the quantity of the second fluorescent reporter group, CH1 is the first fluorescent signal, CH2 is the second fluorescent signal, and k 21/k11 is the crosstalk coefficient.
The concentration or amount of the first fluorescent reporter group and the concentration or amount of the second fluorescent reporter group can be calculated from C1-CH 1 and C2-CH 2-k 21CH1/k11, respectively.
In the fluorescence detection process, the absolute concentration or absolute quantity of the fluorescence reporter group is not required to be obtained, and only the relative concentration or relative quantity is required to be obtained, so that both C1 and C2 refer to the absolute concentration or absolute quantity, but the absolute concentration or absolute quantity obtained by the formula is approximate to, but not equal to, the channel can be regarded as an approximate value, namely the relative concentration or relative quantity.
From the above formula, after the processor receives the first fluorescent signal and the second fluorescent signal, the processor can obtain the values of CH1 and CH2, and the crosstalk coefficient k 21/k11 is a known fixed value, so as to obtain the relative concentration or the relative quantity of the two fluorescent reporter groups.
The following describes how to obtain the crosstalk coefficient k 21/k11:
Before calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group from C1-CH 1 and C2-CH 2-k 21CH1/k11, respectively, the method further comprises:
S10-1: according to formula one: ch1=k 11 C1 and formula two: ch2=k 21C1+k22 c2, where k11, k21, k22 are fixed values, and the reaction solution 114 is preset to have only the first fluorescent reporter group, so as to obtain formula three: c2 =0;
S10-2: substituting the formula III into the formula II to obtain a formula IV: ch2=k 21 C1;
s10-3: dividing the formula IV by the formula I to obtain a crosstalk coefficient: k21/k11=ch2/CH 1.
The following is a specific example of the excitation of FAM fluorescence and HEX fluorescence:
the relationship between the first fluorescent signal CH1 of the first detection channel and the corresponding FAM fluorescent reporter group concentration or amount C (FAM) is:
CH1＝k11C(FAM)；
wherein the parameter k 11 is the detection efficiency of the FAM fluorescent reporter group of the first optical path channel, and is related to the excitation wavelength, the detection wavelength, the absorptivity, the emissivity, the light source intensity and the response of the photoelectric sensor of the FAM fluorescent reporter group of the first optical path channel. When the wavelength of the first optical module of the first optical path is selected, k 11 is an unknown fixed value.
The relationship between the second fluorescence signal CH2 of the second detection channel and the corresponding FAM fluorescence reporter concentration or amount C (FAM) and HEX fluorescence reporter concentration C (HEX) is:
CH2＝k21C(FAM)+k22C(HEX)
The parameter k 21 is the detection efficiency of the FAM fluorescent reporter group of the second detection channel, and is related to the excitation wavelength, the detection wavelength, the absorptivity, the emissivity, the light source intensity and the response of the photoelectric sensor of the FAM fluorescent reporter group of the second detection channel.
The parameter k 22 is the detection efficiency of the HEX fluorescence reporter group of the second detection channel, and is related to the excitation wavelength, the detection wavelength, the absorption rate, the emissivity, the light source intensity and the response of the photoelectric sensor of the HEX fluorescence reporter group of the second detection channel at the wavelength. When the wavelength of the third optical module in the second detection channel and the photosensor 115 are selected, k 21 and k 22 are unknown fixed values.
Thus, for one PCR reaction solution 114, the two-channel fluorescence detection device can obtain two fluorescence signals, respectively:
CH1＝k11C(FAM)
CH2＝k21C(FAM)+k22C(HEX)
Note that, since the concentration or the amount of the FAM reporter group denoted by C (FAM) is coincident with each other in both the fluorescence light path and the fluorescence spectrum of the second detection channel, CH1 corresponds to the partial fluorescence reflected by only the FAM reporter group received by the photosensor 116, and k 11 C (FAM) represents the calculated amount of the FAM reporter group received by only the photosensor 116, corresponding to CH1, because the coefficient k 11 is provided.
Similarly, CH2 corresponds to another portion of fluorescence reflected by the first fluorescent reporter group received by the photosensor 115 and all fluorescence reflected by the second fluorescent reporter group, but provided with a factor k 21, then k 21 C (FAM) represents the amount of FAM fluorescent reporter group received by the photosensor 115 calculated, plus all HEX fluorescent reporter groups obtained through the second detection channel, corresponding to CH2.
The influence of FAM fluorescent reporter groups can be eliminated by bringing CH1 into CH2, and the following results are obtained:
Since the absolute concentration or the absolute amount of the fluorescent reporter group is not required to be obtained in the fluorescence detection process, only the relative concentration or the relative amount is required to be obtained, so that:
C(FAM)∝CH1
Wherein the method comprises the steps of The fluorescent crosstalk coefficient can be obtained through design experiments. When only FAM fluorescent reporter group, i.e., C (HEX) =0, was added to the PCR reaction solution, two fluorescent signals were obtained:
CH1＝k11C(FAM)
CH2＝k21C(FAM)
Thus can be calculated
Since k 11,k21 and k 22 are both fixed values, the fluorescence-crosstalk coefficient is also a fixed value. After one experiment, the fluorescent probe can be used for detecting the follow-up unknown fluorescent reporter group.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The double-channel fluorescence detection device is characterized by comprising a base, wherein a first detection channel, a second detection channel, a first light path channel and a second light path channel are arranged in the base, the first detection channel and the second detection channel are respectively positioned at two sides of the first light path channel, and the second light path channel is communicated with the first light path channel, the first detection channel and the second detection channel;
The light source and the first optical module arranged along the emergent direction of the light source are arranged in the first light path channel, the second optical module is arranged in the first detection channel, a first converter is arranged at one end of the first detection channel far away from the second light path channel, a third optical module is arranged in the second detection channel, a reaction tank is arranged at one end of the second detection channel close to the second light path channel, a second converter is arranged at the other end of the second detection channel, the reaction tank is communicated with the second light path channel, and the first converter and the second converter are respectively and electrically connected with a processor; the first optical module, the second optical module and the third optical module are also positioned at the communication position of the second optical path channel;
The light beam emitted by the light source enters the third optical module through the second optical path channel and then enters the reaction tank, the light beam irradiates the reaction liquid in the reaction tank and then excites first fluorescence and second fluorescence in the reaction liquid, the first fluorescence sequentially passes through the third optical module, the first optical module and the second optical module and then sequentially passes through the second optical path channel and the first detection channel, signals are converted by the first converter and input into the processor, and the second fluorescence passes through the third optical module and then passes through the second detection channel and then is converted by the second converter and input into the processor.
2. The dual-channel fluorescence detection device of claim 1, wherein the first optical module comprises a first optical filter and a first dichroic mirror sequentially arranged along the light source emitting direction, and the first dichroic mirror is located at a communication position of the first light path channel and the second light path channel.
3. The dual-channel fluorescence detection device of claim 1, wherein the third optical module includes a second dichroic mirror and a second optical filter, wherein the second optical filter is proximate to the second converter, and wherein the second dichroic mirror is positioned in communication with the second detection channel and the second optical path channel.
4. The dual-channel fluorescence detection device of claim 3, wherein two focusing lenses are further disposed in the second detection channel, one focusing lens is disposed between the second converter and the second dichroic mirror, and the other focusing lens is disposed between the reaction cell and the second dichroic mirror.
5. The dual-channel fluorescence detection device of claim 1, wherein the second optical module comprises a mirror positioned in communication with the first detection channel and the second optical path channel; and a converging lens is further arranged in the first detection channel, and the converging lens is positioned between the first converter and the reflecting mirror.
6. The dual-channel fluorescence detection device of claim 1, wherein a third optical filter is disposed in the second optical path, and the third optical filter is located between the first optical module and the second optical module.
7. The dual-channel fluorescence detection device of claim 2, wherein the first light path channel, the first detection channel and the second detection channel are arranged in parallel, the second light path channel is perpendicular to the first light path channel, the second detection channel and the first detection channel, and an included angle of 45 ° is formed between the first dichroic mirror and the second light path channel.
8. The dual-channel fluorescence detection device according to claim 1, wherein a cover plate is arranged on the base and positioned at one end of the second detection channel where the reaction tank is arranged, and a through hole is arranged on the cover plate to communicate the first light path channel and the reaction tank; the reaction tank is arranged in the temperature block, and the temperature block is used for heating reaction liquid in the reaction tank; the first converter and the second converter are photoelectric sensors.
9. A two-channel fluorescence detection method, characterized in that the two-channel fluorescence detection device according to any one of claims 1-8 is used, the two-channel fluorescence detection device comprises a reaction tank, and the reaction liquid of the reaction tank comprises a first fluorescence reporting group and a second fluorescence reporting group; the method comprises the following steps:
receiving a first fluorescent signal fed back by the first converter and a second fluorescent signal fed back by the second converter respectively; wherein the first converter receives and feeds back a portion of the optical signal reflected by the first fluorescent reporter group, and the second converter receives and feeds back the optical signal reflected by the second fluorescent reporter group and another portion of the optical signal reflected by the first fluorescent reporter group;
calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group according to C1-CH 1 and C2-CH 2-k 21CH1/k11; wherein, C1 is the concentration of the first fluorescent reporter group, C2 is the concentration of the second fluorescent reporter group, CH1 is the first fluorescent signal, CH2 is the second fluorescent signal, and k 21/k11 is the crosstalk coefficient.
10. The method according to claim 9, wherein before the calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group according to C1-CH 1 and C2-CH 2-k 21CH1/k11, respectively, the method further comprises:
according to formula one: ch1=k 11 C1 and formula two: ch2=k 21C1+k22 c2, where k 11、 k21、k22 is a fixed value, and presetting that only the first fluorescent reporter group exists in the reaction solution, to obtain formula three: c2 =0;
Substituting the formula III into the formula II to obtain a formula IV: ch2=k 21 C1;
Dividing the formula IV by the formula I to obtain a crosstalk coefficient: k 21/k11 =ch2/CH 1.
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