CN212321416U - Double-channel fluorescence detection device - Google Patents
Double-channel fluorescence detection device Download PDFInfo
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- CN212321416U CN212321416U CN202021496618.9U CN202021496618U CN212321416U CN 212321416 U CN212321416 U CN 212321416U CN 202021496618 U CN202021496618 U CN 202021496618U CN 212321416 U CN212321416 U CN 212321416U
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Abstract
The utility model provides a binary channels fluorescence detection device, belong to molecular diagnostic instrument technical field, be equipped with two detection passageways and two light path passageways in the base, there are light source and first optical module in the first light path passageway, all be equipped with optical module in first detection passageway, the second detection passageway, first detection passageway one end has first converter, the same end of second detection passageway is equipped with the second converter, the other end has the reaction tank who communicates with the second light path passageway near the second light path passageway, second converter and first converter electricity respectively connect the treater; each optical module is also positioned at the position communicated with the second light path channel; the light source emits light beams to the reaction tank, after the light beams irradiate the reaction liquid, the first fluorescence and the second fluorescence in the reaction liquid are excited, and the signals are converted by the first converter and the second converter respectively and are input into the processor. The relative concentration or relative amount of the fluorescent reporter group is obtained by a method of eliminating crosstalk.
Description
Technical Field
The utility model relates to a molecular diagnostic instrument technical field particularly, relates to a binary channels fluorescence detection device.
Background
In the field of molecular biological experiments and molecular diagnostics, specific, trace amounts of biomolecules are detected for analysis, such as: nucleic acids, proteins, etc., are often labeled using fluorescent means. The method uses specific fluorescent group molecules to specifically combine with biomolecules 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 the specific wavelength, and finally qualitatively or quantitatively analyzes information such as concentration, distribution and the like of the specific biomolecules in a detected object through the fluorescent signals. The method has the advantages of high sensitivity, convenient operation, no need of contacting with a sample to be detected and the like, and is widely applied.
Because the fluorophore molecules are specifically combined with the biomolecules to be detected, if two or more fluorophore molecules with different fluorescence characteristics are added into a sample at the same time, the two or more corresponding biomolecules can be combined at the same time, and the conventional dual-channel fluorescence detection device utilizes the principle to detect.
However, the two existing fluorescence detection channels are independent from each other, and can only be detected by switching positions, and cannot be detected simultaneously, so that the detection efficiency is reduced, and a mechanical device for switching the fluorescence detection channels is complex, high in cost and high in failure rate.
SUMMERY OF THE UTILITY MODEL
An object of the utility model is to provide a binary channels fluorescence detection device can two kinds of fluorescence report groups in the simultaneous measurement reaction tank, promotes detection speed by a wide margin, and with low costs.
The embodiment of the utility model is realized like this:
the embodiment of the utility model provides a binary channels fluorescence detection device, it includes the base, be equipped with first detection channel, second detection channel, first light path passageway and second light path passageway in the base, first detection with the second detection channel is located the both sides of first light path passageway respectively, the second light path passageway communicates first light path passageway, first detection channel and the second detection channel; a light source and a first optical module arranged along the emergent direction of the light source 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, away from the second light path channel, of the first detection channel, a third optical module is arranged in the second detection channel, a reaction tank is arranged at one end, close to the second light path channel, of the second detection 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 position communicated with the second optical path channel; and light beams emitted by the light source are incident into the reaction tank through the first optical module and the second optical module through the second light path channel, after the light beams irradiate reaction liquid in the reaction tank, first fluorescence and second fluorescence in the reaction liquid are excited, the first fluorescence sequentially passes through the third optical module, the first optical module and the second optical module sequentially through the second light path channel and the first detection channel and is converted by the first converter into signals to be input into the processor, and the second fluorescence passes through the third optical module and passes through the second detection channel and is converted by the second converter into signals to be input into the processor.
Optionally, the first optical module includes 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 between the first optical path channel and the second optical 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 disposed 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 cell and the second dichroic mirror.
Optionally, the second optical module includes a mirror, and the mirror is located at a communication position between the first detection channel and the second optical path channel.
Optionally, a converging lens is further disposed in the first detection channel, and the converging lens is located between the first converter and the reflector.
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, and the first dichroic mirror and the second light path channel are arranged at an included angle of 45 degrees.
Optionally, a cover plate is arranged on the base, the cover plate is located at one end, provided with the reaction tank, of the second detection channel, and a through hole is formed in the cover plate to communicate the first light path channel with the reaction tank.
Optionally, the reaction device further comprises a temperature block, wherein 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 both photosensors.
The utility model discloses beneficial effect includes:
the embodiment of the utility model provides a double-channel fluorescence detection device, 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 arranged at two sides of the first light path channel, the second light path channel is communicated with the first light path channel, the first detection channel and the second detection channel, the detection device comprises a first light path channel, a second light path channel, a first optical module, a second optical module, a third optical module, a reaction pool and a second converter, wherein the light source and the first optical module are arranged in the first light path channel along the emergent direction of the light source; the first optical module, the second optical module and the third optical module are also positioned at the position communicated with the second light path channel, so that the three optical modules can mutually receive and emit light beams through the common second light path channel. During detection, light beams emitted by a light source are incident into a third optical module through a second light path channel through a first optical module and then are incident into a reaction tank, reaction liquid is contained 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, first fluorescence and second fluorescence in the reaction liquid are excited, the first fluorescence is reflected and then sequentially passes through the third optical module, reaches the first optical module through the second light path channel, is incident into a first detection channel through the second optical module, is converted into signals by a first converter and then is input into a processor, and after the second fluorescence is reflected, the signals are converted by the third optical module through a second detection channel and then are input into the processor through a second converter. The processor receives the conversion signal and then processes the conversion signal, detects corresponding fluorescence report radicals, realizes that two fluorescence report radicals in the same reaction tank can be detected by the processor at the same time, greatly improves the detection speed, is communicated through four channels, and shares an optical module, does not need a mechanical structure to switch fluorescence detection channels, reduces the volume weight and the cost, improves the overall reliability, and reduces the cost of an optical system and the detection cost.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the binding of fluorophore molecules to biomolecules to be detected;
FIG. 2 is a schematic structural view of a dual-channel fluorescence detection device according to an embodiment of the present invention;
fig. 3 is one of optical path diagrams of a dual-channel fluorescence detection device provided by an embodiment of the present invention;
fig. 4 is a second optical path diagram of the dual-channel fluorescence detection device according to the embodiment of the present invention;
FIG. 5 is a fluorescence spectrum diagram of FAM fluorescence reporter detected by the dual-channel fluorescence detection apparatus provided by the embodiment of the present invention;
fig. 6 is a third optical path diagram of the dual-channel fluorescence detection device according to the embodiment of the present invention;
fig. 7 is a fluorescence spectrum diagram of the two-channel fluorescence detection apparatus provided by the embodiment of the present invention for detecting HEX fluorescence reporter group;
FIG. 8 is a fluorescence spectrum diagram of a second converter of the dual-channel fluorescence detection apparatus for simultaneously detecting HEX and FAM fluorescence reporter groups according to an embodiment of the present invention;
FIG. 9 is a flow chart of a dual-channel fluorescence detection method according to an embodiment of the present invention;
fig. 10 is a second flowchart of the dual-channel fluorescence detection method according to the embodiment of the present invention.
Icon: 101-a light source; 102-a first optical filter; 103-a first dichroic mirror; 104-a second dichroic mirror; 105-a second filter; 106. 110, 109-a converging lens; 107-third filter; 108-a mirror; 111-cover plate, 112-temperature block; 113-a reaction tank; 114-a reaction solution; 115. 116-a photosensor.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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 obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention, as 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 present invention, presented in the accompanying drawings, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative efforts belong to the protection scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
In the field of molecular biological experiments and molecular diagnostics, for the analytical detection of specific, minute quantities of biomolecules, such as: nucleic acids, proteins, etc., are often labeled using fluorescent means. The method uses specific fluorescent group molecules to specifically combine with biomolecules 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 the specific wavelength, and finally qualitatively or quantitatively analyzes information such as concentration, distribution and the like of the specific biomolecules in a detected object through the fluorescent signals. The method has the advantages of high sensitivity, convenient operation, no need of contacting with a sample to be detected and the like, and is widely applied.
Because the specific combination occurs between the fluorescent group molecules and the tested biological molecules, the fluorescent group molecules with two or more different fluorescent characteristics can be simultaneously combined with the corresponding two or more biological molecules if the two or more fluorescent group molecules with different fluorescent characteristics are added into a sample at the same time, the aim of simultaneously detecting multiple targets is fulfilled, and the detection efficiency is greatly improved. The need for multi-channel fluorescence detection is now present for fluorescence detection devices.
As shown in FIG. 1, the PCR basic Reaction steps are thermal denaturation annealing extension, taking real-time fluorescence quantitative PCR (Polymerase Chain Reaction) as an example. This 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 a reaction system to reflect the condition of nucleic acid amplification in real time. The TaqMan probe main body is a single-stranded DNA oligonucleotide which is complementarily paired with an amplified nucleic acid sequence, the TaqMan probe is provided with a 3 'end and a 5' end, the 3 'end is marked with a fluorescence reporter group (reporter for short) and the 5' end is marked with a fluorescence quenching group (quencher for short Q). When the TaqMan probe is complete in structure, the fluorescent reporter group molecule absorbs excitation photons with specific wavelengths and then is converted into a high-energy-level excitation state. When the TaqMan probe is complete in structure, the distance between the Fluorescence reporter group and the Fluorescence quencher group is only a few nanometers, and a Fluorescence Resonance Energy Transfer (FRET) effect occurs between the Fluorescence reporter group and the Fluorescence quencher group: the fluorescence quenching group absorbs the energy of the fluorescence reporter group in an excited state, the fluorescence reporter group generates radiationless transition and returns to a ground state, and no fluorescence photon is emitted in the process. When the Taq polymerase extends along the 3' end of the upstream primer to the downstream, the Taq polymerase can cleave and hydrolyze the single-stranded DNA oligonucleotide of the TaqMan probe bound on the template strand, so that the fluorescent reporter group is separated from the fluorescent quenching group. The distance between the fluorescence reporter group and the fluorescence quencher group free in solution exceeds the radius at which fluorescence resonance energy transfer occurs. At this time, the fluorescent reporter group can emit a fluorescent photon under excitation of a specific wavelength. Thus, the fluorescence intensity of the fluorescent reporter is proportional to the amount or concentration of TaqMan probe hydrolysis and also to the amount or concentration of template DNA produced by replication.
Since the primer (primer) and TaqMan probe bind to the nucleic acid template by the base complementary pairing principle, there is a high degree of specificity. Thus, two or more nucleic acid sequences can be simultaneously replicated by designing two or more sets of primers and TaqMan probes. This method is called multiplex PCR (multiplex PCR). In this case, if the TaqMan probe is labeled with the same fluorescent reporter group, it is impossible to distinguish which nucleic acid is replicated and the fluorescent signal is generated. It is therefore necessary to use the same number of two or more fluorescent reporter groups as the number of detection targets.
In the existing two-channel fluorescence detection, one channel is used for detecting the fluorescence of a FAM fluorescence reporter group, and the other channel is used for detecting the fluorescence of a HEX fluorescence reporter group. Two fluorescence detection passageways are mutually independent, need the switching position just can detect the fluorescence of two passageways of a test tube, can't detect simultaneously. In addition, the mechanical device for switching the fluorescence detection channel is complex, high in cost and high in failure rate. The principle of the two fluorescence detection channels is similar, but all optical parts are not shared, and the cost is high.
In conclusion, on the basis, the double-channel fluorescence detection device is provided, the relative concentration or the relative quantity of two fluorescence reporter groups in one target can be measured simultaneously, and the detection speed is greatly improved; a fluorescence detection channel does not need to be switched by a mechanical structure, so that the volume, weight and cost are reduced, and the overall reliability is improved; in two or more fluorescence detection channels, optical elements such as a light source, a focusing lens and the like are shared, so that the cost of an optical system is reduced. On the other hand, the fluorescence reflected by the first fluorescence reporter group reaches the first detection channel after passing through the second detection channel, and some fluorescence is received by the second converter, and the second detection channel has the situation of fluorescence crosstalk reflected by two fluorescence reporter groups, so that a signal (a first fluorescence signal) received by the first converter corresponds to a part of the first fluorescence reporter groups, and a signal (a second fluorescence signal) received by the second converter corresponds to another part of the first fluorescence reporter groups and all the second fluorescence reporter groups.
Specifically, referring to fig. 2, the present embodiment provides a dual-channel fluorescence detection apparatus, which includes a base, and a first detection channel, a second detection channel, a first optical channel, and a second optical 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, a second optical module is arranged in the first detection path, a first converter is arranged at one end of the first detection path far away from the second light path, a third optical module is arranged in the second detection path, a reaction pool 113 is arranged at one end of the second detection path close to the second light path, a second converter is arranged at the other end of the second detection path, the reaction pool 113 is communicated with the second light path, 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 position communicated with the second optical path channel.
Light beams emitted by the light source 101 are incident into the third optical module through the second light path channel and then are incident into the reaction tank 113, after the light beams irradiate the reaction liquid 114 in the reaction tank 113, first fluorescence and second fluorescence in the reaction liquid 114 are excited, the second fluorescence passes through the third optical module and passes through the second detection channel, signals are converted by the second converter through the second converter, the signals are input into the processor, and the first fluorescence passes through the third optical module, the first optical module and the second optical module sequentially, passes through the second detection channel, the second light path channel and the first detection channel, and is converted by the first converter and 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 arranged perpendicular to the first light path channel, the first detection channel and the second detection channel.
Establish light source 101 and first optical module in the first light path passageway, the second light path passageway is close to the one side that is equipped with first optical module, establish second optical module in the first detection passageway, establish third optical module in the second detection passageway, first optical module, second optical module and third optical module still are located with second light path passageway intercommunication department simultaneously, the light-emitting portion of these three optical module also is located the second light path passageway simultaneously, after through the three passageway of second light path passageway intercommunication, three optical module can receive and emit the light beam each other through the second light path passageway that passes through altogether, make the light beam can be at first light path passageway, the second light path passageway, first detection passageway and second detection passageway wander wantonly, rethread first optical module, the setting of second optical module and third optical module, make the light beam get into certain appointed passageway.
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 cell 113, and the other end 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, illustratively, the second converter is the photosensor 115, and the first converter is the photosensor 116. And converting the optical signal into an electric signal through the photoelectric sensor, and feeding the electric signal back to the processor for processing.
The reaction liquid 114 is in the reaction cell 113, the reaction liquid 114 contains a first fluorescence reporter group and a second fluorescence reporter group, for example, the reaction cell 113 can be a PCR reaction liquid 114 containing FAM and HEX fluorescence reporter groups, a light beam emitted from the light source 101 reaches the reaction cell 113, a fluorescence light beam corresponding to a fluorescence molecule is obtained after exciting the reaction liquid 114, the fluorescence 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, an electrical signal, the converted signal is fed back to the processor, and the processor detects the corresponding fluorescence reporter group according to the fed back converted signal.
Specifically, light beams emitted by the light source 101 pass through the first optical module and then enter the third optical module through the second light path channel, and then enter the third optical module into the reaction solution 114 in the reaction tank 113 to excite the reaction solution 114 to obtain first fluorescence and second fluorescence, the first fluorescence enters the first detection channel through the second light path channel, the second fluorescence enters the second detection channel, the first fluorescence entering the first detection channel is converted by the first converter into a signal and is input into the processor, and the second fluorescence entering the second detection channel is converted by the second converter into a signal and is input into the processor, so that detection of two fluorescence reporter groups in the reaction solution 114 is completed. This allows two fluorescent reporter groups in the same well 113 to be detected by the processor at the same time.
The double-channel fluorescence detection device provided by the embodiment of the utility model is provided with a first detection channel, a second detection channel, a first light path channel and a second light path channel in the base, wherein the second detection channel and the first detection channel are respectively arranged 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 a first light path channel, a third optical module is arranged in a second detection channel, one end, close to the second light path channel, of the second detection channel is provided with a reaction tank 113, the other end of the second detection channel is provided with a second converter, the reaction tank 113 is communicated with the second light path channel, the second optical module is arranged in the first detection channel, one end, far away from the second light path channel, of the first detection channel is provided with a first converter, and the second converter and the first converter are respectively and electrically connected with a processor; the first optical module, the third optical module and the second optical module are also positioned at the position communicated with the second light path channel, so that the three optical modules can mutually receive and emit light beams through the common second light path channel. The light beam emitted by the light source 101 is incident into the third optical module through the second light path channel via the first optical module, and then is incident into the reaction tank 113 through the third optical module via the second detection channel, the reaction liquid 114 in the reaction tank 113 contains two fluorescence reporter groups, the reaction liquid 114 in the reaction tank 113 reacts with the light beam to excite and respectively obtain the first fluorescence and the second fluorescence, the second fluorescence is converted into a signal by the second converter via the second detection channel via the third optical module and the second detection channel and is input into the processor, the first fluorescence is converted into a signal by the first converter via the third optical module, the first optical module and the second optical module sequentially via the second detection channel, the second light path channel and the first detection channel and is input into the processor, the processor receives the converted signal and then processes the signal to detect the corresponding fluorescence reporter groups, thereby realizing that the two fluorescence reporter groups in the same reaction tank 113 can be detected by the processor at the same time, promote detection speed by a wide margin, through four passageways commons, and sharing optical module, need not mechanical structure and switch fluorescence detection passageway, reduced volume weight and cost, promoted holistic reliability to optical system cost and detection cost have been reduced.
Specifically, the first optical module includes a first optical filter 102 and a first dichroic mirror 103 that are sequentially arranged along the emitting direction of the light source 101, the first dichroic mirror 103 is located at the communication position of the first optical path channel and the second optical path channel, and the emitting direction of the light beam emitted from the light source 101 is changed by the first dichroic mirror 103, so that the direction of the light beam is changed after the light beam is emitted from the first optical path channel, and the light beam is emitted along the second optical path channel to irradiate the third optical module.
The first optical filter 102 is used for filtering the light beam emitted from the light beam to obtain a light beam with a desired wavelength, so that the light beam is reflected by the first dichroic mirror 103 to prevent the light beam from transmitting to the second optical module, and the light beam is reflected to the third optical module.
First light path passageway, second detection channel and first detection channel parallel arrangement, second light path passageway respectively with first light path passageway, second detection channel, first detection channel perpendicular, be 45 contained angles setting between first dichroic mirror 103 and the second light path passageway.
Thus, after the light beam emitted from the first optical module passes through the first dichroic mirror 103 with an included angle of 45 °, the light beam can be changed into a direction perpendicular to the emitting direction of the first optical module and emitted 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 emitted to the second dichroic mirror 104, an included angle of 45 degrees is formed between the second dichroic mirror 104 and the second light path channel, and the light beam emitted from the first dichroic mirror 103 is changed into a light beam perpendicular to the second light path channel by the second dichroic mirror 104 and emitted to the reaction cell 113 along the second detection channel.
A converging lens 110 is disposed between the reaction cell 113 and the second dichroic mirror 104 for converging light.
Further, the first dichroic mirror 103 and the second dichroic mirror 104 are both long-pass dichroic mirrors.
The light beam excites the reaction solution 114 in the reaction cell 113 to obtain a first fluorescence and a second fluorescence, the first fluorescence and the second fluorescence are reflected by the second detection channel, and are emitted to the second dichroic mirror 104 through the converging lens 110, and are branched 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 becomes a light beam emitted along the second optical path, and the light beam is emitted to the first dichroic mirror 103, and is emitted to the second optical module after transmitting the first dichroic mirror 103.
The second optical module comprises a reflector 108, the reflector 108 is located at a communication position of the first detection channel and the second optical channel, an included angle of 45 degrees is formed between the reflector 108 and the second optical channel, and the reflector 108 receives the first fluorescence and changes the emitting direction to enable the first fluorescence to emit to the first converter along the first detection channel. A converging lens 109 is also provided between the first converter and the mirror 108.
In addition, a third optical filter 107 is arranged 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 fluorescence transmits through the first dichroic mirror 103, is filtered by the third filter 107, and is reflected by the mirror 108 toward the first converter.
Further, a cover plate 111 is arranged on the base, the cover plate 111 is located at one end, provided with the reaction tank 113, of the second detection channel, and a through hole is formed in the cover plate 111 to communicate the first light path channel with the reaction tank 113.
The reaction cell 113 is provided with a cover for closing the reaction cell 113, 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 cell 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 lid of the reaction chamber 113, so as to prevent the gas generated by the internal reaction solution 114 from expanding to push the lid open after the reaction chamber 113 is heated, so that the reaction solution 114 volatilizes and the detection result is affected.
The device also comprises a temperature block 112, 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 description will be made specifically for the detection process by taking the excitation of FAM fluorescence and HEX fluorescence as examples:
as shown in fig. 3, a vertically disposed light source 101 emits broad spectrum light downward. The light is filtered into narrow-band excitation light 480nm +/-10 nm by exciting the first optical filter 102 through the first optical path channel. The excitation light is incident on a first dichroic mirror 103 placed at an angle of 45 ° to the horizontal. The first dichroic mirror 103 allows light having a wavelength of less than 500nm to be reflected and light having a wavelength of more than 500nm to be transmitted. Therefore, excitation light of 480nm ± 10nm is emitted and reflected at the first dichroic mirror 103, and horizontally enters the second dichroic mirror 104. The second dichroic mirror 104 allows reflection of light with a wavelength of less than 545nm and transmission of light with a wavelength of more than 545 nm. Therefore, the excitation light of 480nm ± 10nm is reflected by the second dichroic mirror 104, is downwardly converged by the converging lens 110, passes through the cover plate 111 in sequence, and enters the reaction cell 113 in the thermoblock 112, the reaction cell 113 can be a transparent reaction tube, and finally irradiates and excites the reaction solution 114 in the PCR reaction cell 113 containing FAM and HEX fluorescent reporter groups.
As shown in fig. 4, FAM fluorescence and HEX fluorescence are obtained after laser light, and the fluorescence generation directions are random, wherein the FAM fluorescence is emitted vertically upward, passes through the transparent reaction cell 113 and the cover plate 111, enters the collecting lens 110, and then enters the second dichroic mirror 104. Because the spectrum range of the fluorescence emitted by the FAM fluorescence reporter group is 490-600 nm, the part of the fluorescence with the wavelength less than 545nm is reflected by the second dichroic mirror 104. The direction of fluorescence becomes horizontal. The filtering spectral range of the fluorescence passing through the second dichroic mirror 104 is changed to 490-545 nm. The fluorescent light continues to horizontally enter the first dichroic mirror 103. Since first dichroic mirror 103 allows light having a wavelength of more than 495nm to transmit, a portion of fluorescent light having a wavelength of more than 495 is transmitted at first dichroic mirror 103. The fluorescence direction was unchanged. The filtering spectral range of the fluorescence through the first dichroic mirror 103 is changed to be 495-545 nm. Thereafter, 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 a narrow band with the strongest energy of 520nm +/-10 nm. The 520nm + -10 nm fluorescence continues to propagate horizontally, is reflected at the mirror 108 placed at an angle of 45 deg. to the horizontal, passes vertically upward through the converging lens 109 and is finally focused on the photosensor 116 of the first detection channel. Through this process, the fluorescence emitted by the FAM fluorescent reporter is converted into an electrical signal by the photosensor 116 of the first detection channel.
The fluorescence spectrum of the device for detecting FAM fluorescent reporter group is shown in FIG. 5. The shaded peak on the left is the normalized excitation spectrum of the FAM fluorescence reporter group, and the shaded peak on the right is the normalized emission spectrum of the FAM fluorescence reporter group. The solid line is the excitation filter normalized transmittance spectrum, the single-dot chain line is the first dichroic mirror 103 transmittance spectrum, the double-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 the 480nm ± 10nm transmission spectrum band of the first optical filter 102 is located at the side with low transmittance (< 5%) of the first dichroic mirror 103 and the second dichroic mirror 104, i.e. the first dichroic mirror 103 and the second dichroic mirror 104 are both reflected. The second optical filter 105 has a transmission spectrum band of 520nm ± 10nm on the side where the transmittance of the second dichroic mirror 104 is low (< 5%) and on the side where the transmittance of the first dichroic mirror 103 is high (> 95%), i.e., the second dichroic mirror 104 reflects and the first dichroic mirror 103 transmits.
As shown in fig. 6, the fluorescence emission direction is random, wherein the HEX fluorescence is emitted vertically upward, and then enters the second dichroic mirror 104 after passing through the reaction cell 113 and the cover plate 111 and entering the collecting lens 110. Since the spectrum of the fluorescence emitted by the HEX fluorescent reporter group is between 540 and 640nm, most of the fluorescence can be incident on the second optical filter 105 of the second detection channel through the second dichroic mirror 104. The fluorescence is filtered to a narrow band 580nm +/-20 nm with the strongest energy by a second filter 105 of the second detection channel, and is finally focused on a photoelectric sensor 115 of the second detection channel by a converging lens 106. Through this process, the fluorescence emitted by the HEX fluorescent reporter is converted into an electrical signal by the photosensor 115 of the second detection channel.
The fluorescence spectrum of the device for detecting the HEX fluorescent reporter group is shown in figure 7. The left shaded peak is the HEX fluorescence reporter group normalized excitation spectrum, and the right shaded peak is the HEX fluorescence reporter group normalized emission spectrum. The solid line is the excitation filter normalized transmittance spectrum, the single-dot chain line is the first dichroic mirror 103 transmittance spectrum, the double-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 the 480nm ± 10nm transmission spectrum band of the first optical filter 102 is located at the side with low transmittance (< 5%) of the first dichroic mirror 103 and the second dichroic mirror 104, i.e. the first dichroic mirror 103 and the second dichroic mirror 104 are both reflected. The second filter 105 transmission band 580nm ± 20nm is located on the side of the second dichroic mirror 104 where the transmittance is high (> 95%), i.e. transmission occurs at the second dichroic mirror 104.
However, the dual-channel fluorescence detection device can detect the fluorescent signal of the FAM fluorescent reporter on the photoelectric sensor 115 while detecting the HEX fluorescent reporter. The fluorescence spectrum of this process is shown in FIG. 8. The shaded peak on the left is the normalized excitation spectrum of the FAM fluorescence reporter group, and the shaded peak on the right is the normalized emission spectrum of the FAM fluorescence reporter group. The solid line is the excitation filter normalized transmittance spectrum, the single-dot chain line is the first dichroic mirror 103 transmittance spectrum, the double-dot chain line is the second dichroic mirror 104 transmission spectrum, and the broken line is the second filter 105 transmittance spectrum. The excitation light of 480nm +/-10 nm can be absorbed by the FAM fluorescent reporter group, and the fluorescence signal emitted by the FAM fluorescent reporter group can still be received in the detection spectral range of 580nm +/-20 nm.
Therefore, when the PCR reaction solution contains both FAM and HEX fluorescent reporter groups, the fluorescent signals of both are overlapped on the fluorescent light path and the fluorescent 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 fluorescent signals of FAM and HEX fluorescent reporter groups. I.e. the second detection channel is in the presence of fluorescence crosstalk.
The fluorescence crosstalk of the second detection channel is eliminated by adopting the following method:
as shown in fig. 9 and fig. 10, this embodiment further provides a fluorescence detection method, which applies the above-mentioned dual-channel fluorescence detection apparatus, where the dual-channel fluorescence detection apparatus includes a reaction cell 113, and a reaction solution 114 of the reaction cell 113 includes a first fluorescence reporter group and a second fluorescence reporter 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, part 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 fluorescence signal includes part of the fluorescence reflected by the first fluorescent reporter group and all of the fluorescence reflected by the second fluorescent reporter group received by the photosensor 115.
Another portion of the fluorescent light 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 fluorescent light reflected by the first fluorescent reporter received by the photosensor 116.
The part of the first fluorescence and the second fluorescence are overlapped on a fluorescence light path and a fluorescence spectrum of the second detection channel.
S110: according to C1 ℃. alpha.CH 1 and C2 ℃. alpha.CH 2-k21CH1/k11Calculating the concentration of the first fluorescent reporter group and the concentration of the second fluorescent reporter group respectively; wherein C1 is the concentration or amount of the first fluorescent reporter group, C2 is the concentration or amount of the second fluorescent reporter group, CH1 is the first fluorescent signal, CH2 is the second fluorescent signal, k21/k11Is the crosstalk coefficient.
According to C1 ℃. alpha.CH 1 and C2 ℃. alpha.CH 2-k21CH1/k11The concentration or amount of the first fluorescent reporter group and the concentration or amount of the second fluorescent reporter group can be calculated separately.
The absolute concentration or absolute amount of the fluorescent reporter group is not required to be obtained in the fluorescence detection process, but only a relative concentration or relative amount is required to be obtained, so that both C1 and C2 refer to the absolute concentration or absolute amount, but because the above formula is approximate, not equal, the absolute concentration or absolute amount obtained by the above formula of the channel can be regarded as an approximate value, namely, the relative concentration or relative amount.
From the above formula, when the processor receives the first fluorescence signal and the second fluorescence signal, the values of CH1 and CH2, and the crosstalk coefficient k can be obtained21/k11The relative concentrations or relative amounts of the two fluorescent reporter groups can be obtained for a known fixed value.
The following describes in detail how to obtain the crosstalk coefficient k21/k11：
According to C1 ℃. alpha.CH 1 and C2 ℃. alpha.CH 2-k21CH1/k11Prior to calculating the concentration of the first fluorescent reporter and the concentration of the second fluorescent reporter, respectively, the method further comprises:
s10-1: according to the formula one: CH1 ═ k11C1 and formula two: CH2 ═ k21C1+k22C2, wherein k11, k21 and k22 are fixed values, and only the first fluorescent reporter group is preset in the reaction solution 114, thereby obtaining the formula three: c2 ═ 0;
s10-2: substituting the formula three into the formula two to obtain a formula four: CH2 ═ k21C1；
S10-3: and dividing the formula four by the formula one to obtain the crosstalk coefficient: K21/K11 ═ CH2/CH 1.
The following is further illustrated by the examples of stimulated FAM fluorescence and HEX fluorescence:
the first fluorescence signal CH1 of the first detection channel is related to the corresponding FAM fluorescence reporter concentration or amount c (FAM):
CH1＝k11C(FAM)；
wherein the parameter k11The efficiency of detection of the FAM fluorescent reporter for the first optical path is related to the excitation wavelength, the detection wavelength, the absorbance of the FAM fluorescent reporter at this wavelength, the emissivity, the intensity of the light source, and the response of the photosensor of the first optical path. K after the wavelength of the first optical module of the first optical path channel is selected11As an unknown fixed value.
The second fluorescence signal CH2 of the second detection channel is related to the corresponding FAM fluorescence reporter concentration or amount c (FAM) and HEX fluorescence reporter concentration c (HEX):
CH2＝k21C(FAM)+k22C(HEX)
wherein the parameter k21The efficiency of detection of the FAM fluorescent reporter for the second detection channel is related to the excitation wavelength, the detection wavelength, the absorbance of the FAM fluorescent reporter at this wavelength, the emissivity, the intensity of the light source, and the response of the photosensor for the second detection channel.
Parameter k22The efficiency of detecting the HEX fluorescent reporter group for the second detection channel is related to the excitation wavelength, the detection wavelength, the absorptivity of the HEX fluorescent reporter group at the wavelength, the emissivity, the light source intensity and the response of the photoelectric sensor of the second detection channel. K after the wavelength of the third optical module in the second detection channel and the photosensor 115 are selected21And k22As an unknown fixed value.
Therefore, for one PCR reaction solution 114, the two-channel fluorescence detection device can obtain two fluorescence signals, which are:
CH1＝k11C(FAM)
CH2＝k21C(FAM)+k22C(HEX)
it should be noted that, the total concentration or amount of FAM reporter group represented by c (FAM) is because the two fluorescence signals overlap on the fluorescence light path and the fluorescence spectrum of the second detection channel, so CH1 corresponds to the partial fluorescence reflected by FAM fluorescence reporter group received by the photosensor 116, and the coefficient k is set11Then k is11C (FAM) represents the calculated amount of FAM fluorescent reporter received by photosensor 116 alone, corresponding to CH 1.
Similarly, CH2 corresponds to the other portion of the fluorescence reflected from the first fluorescent reporter and the total fluorescence reflected from the second fluorescent reporter received by the photosensor 115, but with a factor k21Then k is21C (FAM) represents the amount of FAM fluorescent reporter group calculated to be received by the photosensor 115, plus all HEX fluorescent reporter groups obtained through the second detection channel, corresponding to CH 2.
The influence of FAM fluorescent reporter groups can be eliminated by bringing CH1 into CH2, and the following results are obtained:
since it is not required to obtain the absolute concentration or amount of the fluorescent reporter group during the fluorescence detection process, only the relative concentration or amount is obtained, therefore:
C(FAM)∝CH1
wherein
The crosstalk coefficient of fluorescence can be obtained by designing experiments. When only FAM fluorescent reporter, i.e., c (hex) ═ 0, was added to the PCR reaction solution, two fluorescent signals were obtained:
CH1＝k11C(FAM)
CH2＝k21C(FAM)
thus, it can calculate
Because k is11，k21And k22Are all fixed values, so the fluorescence crosstalk coefficient is also a fixed value. After one experiment is obtained, the method can be used for detecting the subsequent unknown fluorescent reporter group.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A 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;
a light source and a first optical module arranged along the emergent direction of the light source 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, away from the second light path channel, of the first detection channel, a third optical module is arranged in the second detection channel, a reaction tank is arranged at one end, close to the second light path channel, of the second detection 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 position communicated with the second optical path channel;
and light beams emitted by the light source are incident into the reaction tank through the first optical module and the second optical module through the second light path channel, after the light beams irradiate reaction liquid in the reaction tank, first fluorescence and second fluorescence in the reaction liquid are excited, the first fluorescence sequentially passes through the third optical module, the first optical module and the second optical module sequentially through the second light path channel and the first detection channel and is converted by the first converter into signals to be input into the processor, and the second fluorescence passes through the third optical module and passes through the second detection channel and is converted by the second converter into signals to be 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 position where the first optical path and the second optical path are communicated.
3. The dual-channel fluorescence detection device of claim 1, wherein the third optical module comprises a second dichroic mirror and a second optical filter, wherein the second optical filter is located proximate to the second switch, and the second dichroic mirror is located at a position where the second detection channel communicates with the second optical path channel.
4. The dual-channel fluorescence detection device of claim 3, wherein two converging lenses are further disposed in the second detection channel, one converging lens being disposed between the second converter and the second dichroic mirror, and the other converging lens being 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 includes a mirror positioned to communicate the first detection channel with the second optical channel.
6. The dual channel fluorescence detection device of claim 5, wherein a converging lens is further disposed within the first detection channel, the converging lens being positioned between the first transducer and the mirror.
7. The dual channel fluorescence detection device of claim 1, wherein a third optical filter is disposed within the second optical channel, the third optical filter being positioned between the first optical module and the second optical module.
8. The dual-channel fluorescence detection device of claim 2, wherein the first optical path, the first detection channel and the second detection channel are disposed in parallel, the second optical path is perpendicular to the first optical path, the second detection channel and the first detection channel, respectively, and the first dichroic mirror is disposed at an angle of 45 ° with respect to the second optical path.
9. The dual-channel fluorescence detection device of claim 1, wherein a cover plate is disposed on the base, the cover plate is located at an end of the second detection channel where the reaction chamber is located, and a through hole is disposed on the cover plate to communicate the first light path channel and the reaction chamber.
10. The dual-channel fluorescence detection device of claim 1, further comprising a temperature block, wherein the reaction tank is disposed in the temperature block, and the temperature block is used for heating the reaction solution in the reaction tank; the first converter and the second converter are both photosensors.
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