Disclosure of Invention
An object of the present invention is to provide a fluorescent light source device to solve one of the above problems.
To achieve the above object, a first aspect of the present invention provides a fluorescent light source device, comprising:
at least two fluorescent emission units for emitting excitation light;
And the control unit controls the at least two fluorescent emission units to emit excitation light in different time periods respectively.
Optionally, the fluorescent emission unit includes a light source and an excitation optical fiber, and the excitation optical fiber is used for transmitting the excitation light emitted by the light source.
Optionally, wavelengths of the excitation light emitted by the at least two fluorescent emission units are different from each other.
Another object of the present invention is to provide a PCR detection system, which solves one of the above-mentioned problems.
To achieve the object, the second aspect of the present invention adopts the following technical scheme:
A fluorescence detection light path system comprises the fluorescence light source device and a fluorescence detection unit.
Optionally, the fluorescence detection unit includes at least two fluorescence transmission light paths, and at least two fluorescence emission units are in one-to-one correspondence with at least two fluorescence transmission light paths.
Optionally, each of the fluorescence transmission light paths includes a filter that allows a preset fluorescence signal to pass through.
Optionally, the fluorescence detection unit further includes a detector, and the plurality of fluorescence transmission light paths are all connected to the detector.
Optionally, the number of the detectors is one, and the detectors are electrically connected with the control unit, so as to record the intensity of the fluorescent signal in a time period.
Optionally, the fluorescence detection light path system further includes an optical fiber seat, the fluorescence transmission light path includes a collection optical fiber, the excitation optical fiber of the fluorescence light source device emits one end of the excitation light, and one end of the collection optical fiber emitting fluorescent signals forms an optical fiber group, the optical fiber group is disposed in the optical fiber seat, the optical fibers in the optical fiber seat are arranged along a first direction, and the first direction is radial of the optical fibers, so that the emitting end of the collection optical fiber and the emitting end of the excitation optical fiber are arranged in a flat manner in the optical fiber seat.
Optionally, at least the two fluorescence transmission light paths and the at least two fluorescence emission units form at least two groups of optical fiber groups, and the at least two groups of optical fiber groups are sequentially arranged along the first direction.
Optionally, one of the optical fiber groups includes at least two collecting optical fibers, and at least one collecting optical fiber is disposed on two sides of the excitation optical fiber in the optical fiber group in the first direction.
Optionally, the detector comprises a silicon photomultiplier, a photon detector, or a photomultiplier.
It is still another object of the present invention to provide a PCR detection system to solve one of the above-mentioned problems.
To achieve the object, a third aspect of the present invention adopts the following technical scheme:
The PCR detection system comprises a carrier and the fluorescence detection light path system, wherein the fluorescence detection light path system is used for detecting a reaction sample in the carrier.
Optionally, the carrier includes a first wall and a second wall that are disposed opposite to each other, and a side wall that is disposed between the first wall and the second wall, where the first wall, the second wall, and the side wall form a receiving cavity, the receiving cavity and/or the carrier is in a flat structure, at least a portion of the side wall is transparent, and the fluorescence detection optical path system detects the reaction sample through the transparent side wall.
Optionally, one side or at least two sides of the carrier are provided with the fluorescence emission units.
Optionally, the light-transmitting sidewall material is polydimethylsiloxane, polypropylene, or polycarbonate.
Optionally, the carrier comprises at least one built-in heater, and the first wall and/or the second wall is/are the built-in heater.
Optionally, the built-in heater includes a heating element.
Optionally, the built-in heater comprises at least two independently controlled heating elements.
Optionally, the built-in heater further comprises a temperature calibration part for reflecting the temperature of the heating element.
Optionally, the built-in heater further includes a rapid conduction portion for conducting heat of the heating element to the temperature calibration portion.
Optionally, the PCR detection system may further include a resistance detection member for detecting a resistance of the heating member.
Alternatively, the flat structure means that the dimension of the accommodating chamber or the carrier in the direction perpendicular to the thickness direction thereof is larger than the dimension thereof in the thickness direction.
Optionally, a ratio of a dimension of the receiving cavity or the carrier perpendicular to a thickness direction thereof to a dimension of the carrier perpendicular to the thickness direction thereof is greater than 5:1.
Optionally, the size ratio is 50:1-100:1.
Therefore, according to the technical scheme provided by the invention, the fluorescence emission units are independently arranged, and the excitation light transmitted in the fluorescence emission units is not reflected into the light path for transmitting the fluorescence signal, so that the quantity of the excitation light in the light path of the fluorescence signal is greatly reduced, the background in the fluorescence signal is greatly reduced, and the detection accuracy is improved. Meanwhile, when the reaction sample in the carrier is provided with a plurality of fluorescent probes/dyes, the control unit can control different fluorescent emission units to be sequentially started in different time periods, for example, the first fluorescent emission unit works in the 1 st to 2s of the beginning of detection, the second fluorescent emission unit works in the 2 nd to 3s of the beginning of detection, and so on, only one fluorescent emission unit works in one time period, and the control unit controls the conduction of the fluorescent emission units of all channels to realize millisecond-level switching of excitation light channels, so that the rapid detection of the reaction sample can be realized.
The fluorescence detection unit is used for detecting a fluorescence signal, the fluorescence emission unit is completely separated from the fluorescence detection unit for receiving the excitation signal, the back of the fluorescence signal is reduced, the detection sensitivity of a fluorescence detection light path system is improved, meanwhile, the fluorescence emission unit is completely separated from the fluorescence detection unit, optical elements in the fluorescence detection unit such as a dichroic mirror and the like are reduced, the cost is reduced, and the detection efficiency of fluorescence is improved.
Detailed Description
The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof. It should be further noted that, for convenience of description, only some, but not all of the drawings related to the present invention are shown.
In the present invention, directional terms such as "upper", "lower", "left", "right", "inner" and "outer" are used for convenience of understanding, and thus do not limit the scope of the present invention unless otherwise specified.
In the present invention, unless expressly stated or limited otherwise, a first feature "above" or "below" a second feature may include both the first and second features being in direct contact, as well as the first and second features not being in direct contact but being in contact with each other through additional features therebetween. Moreover, a first feature being "above," "over" and "on" a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicating that the first feature is higher in level than the second feature. The first feature being "under", "below" and "beneath" the second feature includes the first feature being directly under and obliquely below the second feature, or simply means that the first feature is less level than the second feature.
In the description of the present invention, unless explicitly stated or limited otherwise, the terms "connected," "connected," and "fixed" are to be construed broadly, and may, for example, be fixedly connected, detachably connected, or integrally formed, mechanically connected, electrically connected, directly connected, indirectly connected through an intervening medium, or in communication between two elements or in an interaction relationship between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
As shown in fig. 1a, 1b and 2, the present embodiment provides a carrier 4, where the carrier 4 includes a first wall 43 and a second wall 44 disposed opposite to each other, and a side wall 42 disposed between the first wall 43 and the second wall 44, and the first wall 43, the second wall 44 and the side wall 42 form a receiving cavity 41, and the receiving cavity 41 and/or the carrier 4 are of a flat structure, and at least a portion of the side wall 42 is transparent to light.
The flat structure may mean that the dimension in the thickness direction of the accommodation chamber 41 or the carrier 4 (i.e., the direction in which the first wall 43 and the second wall 44 are disposed) is smaller than the dimension in the direction perpendicular to the thickness direction, and as an example, the ratio of the dimension in the direction perpendicular to the thickness direction to the dimension in the thickness direction is greater than 5:1. More preferably, the dimension in the thickness direction of the carrier 4 or the accommodation chamber 41 is much smaller than the dimension in the direction perpendicular to the thickness direction, such as a dimension ratio of 50:1 to 100:1, and as an example, a dimension ratio of 90:1. As an example, the accommodating chamber 41 is a rectangular parallelepiped, and the ratio of the length and the thickness of the rectangular parallelepiped may be greater than 5:1, such as 90:1, for example, the dimension in the thickness direction of the accommodating chamber 41 may be 0.3 to 1.0mm, in this embodiment, the thickness of the accommodating chamber 41 is 0.3 to 0.6mm, and the width and the length of the accommodating chamber 41 are about 10mm and 20mm, respectively, wherein the arrangement direction of the first wall 43 and the second wall 44 in the thickness direction. By way of example, the housing cavity 41 may also be cylindrical in configuration with a diameter to thickness ratio of greater than 5:1, such as a thickness of 0.3-1.0mm and a diameter of 5-20mm. Of course, the cross section of the accommodation chamber 41 may be polygonal or elliptical, etc. Of course, the cross section of the accommodation chamber 41 may be polygonal or elliptical, etc.
The existing PCR detection system usually selects a larger surface for nucleic acid detection, so that the area of a reaction sample for receiving excitation light is large, the generated fluorescent signal is strong, and an accurate detection result is easy to obtain. According to the above-described thinking habit, since the area of the first wall 43 or the second wall 44 in the present embodiment is large, it is often considered that fluorescence detection is performed through the first wall 43 or the second wall 44.
At least one side of the receiving chamber 41 may be heated to heat the reaction sample within the receiving chamber 41. In order to achieve rapid amplification and rapid nucleic acid detection, preferably, the first wall 43 and the second wall 44 of the carrier 4 are respectively provided with an external heater 522 as shown in fig. 8 or an internal heater 45 as shown in fig. 10, and the external heater 522 or the internal heater 45 heats the reaction sample, thereby achieving rapid temperature rise and fall of the reaction sample and rapid nucleic acid detection.
Optionally, at least part of the side wall 42 of the accommodating cavity 41 is transparent, and the fluorescence detection light path system 100 detects the reaction sample through the transparent side wall 42 of the carrier 4, so that the requirement of rapid temperature rise and drop is met, and the detection can be accurately completed.
As shown in fig. 3a, the fluorescence detection light path in the prior art is of an integral structure, that is, the excitation light path 200 and the fluorescence signal path 201 of the fluorescence detection light path are transmitted through one optical fiber, so that the excitation light emitted by the light source 205 enters the fluorescence signal path 201 after being reflected by the optical fiber. The dichroic mirror 203 and the optical filter 204 are arranged on the optical fiber transmission path, and the dichroic mirror 203 and the optical filter 204 both allow the fluorescence signal to pass through and filter the excitation light in the fluorescence signal optical path 201, but since the dichroic mirror 203 and the optical filter 204 cannot filter the excitation light in percentage, the fluorescence signal cannot pass through in percentage, that is, the fluorescence signal is attenuated when passing through the dichroic mirror 203 and the optical filter 204, and part of the filtered excitation light enters the detection circuit 206 for detecting the fluorescence signal. In the prior art, since the area of the reaction sample excited by the excitation light is large, the number of generated fluorescent signals is also large, so that the influence of the excitation light and the attenuated fluorescent signals transmitted through the optical filter 204 on the detection result is small, and a more accurate detection result can be obtained.
However, the thickness of the accommodating cavity 41 in this embodiment is small (0.3-0.6 mm), and the reaction sample capable of receiving excited light is greatly reduced when fluorescence detection is performed through the side wall 42 of the accommodating cavity 41, so that the number of fluorescence signals excited each time is about an order of magnitude smaller. If the existing fluorescence detection light path is used for detection, the signal detected by the detection circuit is low, the excitation light passing through the optical filter 211 can submerge the fluorescence signal, and thus the error generated by detection is large, and even the fluorescence signal cannot be detected by the fluorescence detection circuit.
As shown in fig. 3b, the fluorescent light source device provided in this embodiment includes a control unit and at least two fluorescent light emitting units 1, where the at least two fluorescent light emitting units 1 are configured to emit excitation light, and the excitation light is monochromatic light or polychromatic light, for example, polychromatic light may be white light. The control unit controls at least two fluorescent emission units 1 to emit excitation light in different time periods, respectively, so as to allow the reaction sample to receive only one excitation light at the same time. In this embodiment, the control unit is connected to the fluorescent emission unit 1 by a wire, which allows the control unit to control the fluorescent emission unit 1 without delay. Of course, in other alternative embodiments, the control unit and the fluorescent emission unit 1 may be connected wirelessly by bluetooth or the like. It can be understood that the control unit may be a centralized or distributed control unit, for example, the control unit may be a single-chip microcomputer, or may be a distributed multi-chip microcomputer, where a control program may be run in the single-chip microcomputer, so as to control each component to implement its function.
The fluorescence emission units 1 are independently arranged, excitation light transmitted in the fluorescence emission units 1 cannot be reflected into a light path for transmitting fluorescence signals, so that the quantity of the excitation light in the light path for transmitting the fluorescence signals is greatly reduced, the background in the fluorescence signals is greatly reduced, and the detection accuracy is improved. Meanwhile, when multiple fluorescent probes/dyes are arranged in the reaction sample in the carrier 4, the control unit can control different fluorescent emission units 1 to be sequentially started in different time periods, for example, the first fluorescent emission unit 1 works in the 1 st to 2s of the beginning of detection, the second fluorescent emission unit 1 works in the 2 nd to 3s of the beginning of detection, and so on, only one fluorescent emission unit 1 works in one time period, and the control unit controls the conduction of the fluorescent emission units 1 of all channels to realize millisecond-level switching of excitation light channels, so that the rapid detection of the reaction sample can be realized. Meanwhile, when the excitation light is monochromatic light, only one excitation light needs to be emitted by one fluorescence emission unit 1, so that the wavelength of the excitation light is single in a period of time, the types of fluorescence signals are single, and when the fluorescence signals are detected, noise is not generated by other fluorescence signals, so that the accuracy of detection results can be further improved.
Optionally, the excitation light is monochromatic light, and wavelengths of the excitation light emitted by at least two fluorescent emission units 1 are different from each other, so as to improve the utilization rate of the fluorescent emission units 1. Of course, in order to avoid inaccurate detection results caused by the failure of the fluorescent light emitting units 1, a spare fluorescent light emitting unit 1 may be further provided, for example, in at least two fluorescent light emitting units 1, the wavelengths of the excitation light emitted by the two fluorescent light emitting units 1 are the same.
As shown in fig. 3b, the fluorescent emission unit 1 comprises a light source 12 and an excitation fiber 11, the light source 12 may be an LED or the like, the light source 12 is for emitting excitation light, and the light source 12 is electrically connected to the control unit. The excitation fiber 11 is used to transmit the excitation light emitted from the light source 12 to transmit the excitation light to the reaction sample. The light source 12 and the reaction sample are connected through the excitation optical fiber 11, the excitation optical fiber 11 is convenient for isolating a heat source, so that experimental data are more stable, and the excitation optical fiber 11 is connected with the light source 12 simply, thereby being beneficial to shock resistance. At the same time, the optical fiber itself is small in size, so that the need of the carrier 4 with a flat structure can be satisfied. In addition, a light source 12 corresponds to an excitation optical fiber 11, and each excitation channel is an independent optical fiber, so that the coupling is easy and the light loss is small.
The present embodiment also provides a fluorescence detection light path system 100, which includes the above-described fluorescence light source device and fluorescence detection unit 2. The fluorescence detection unit 2 is used for detecting fluorescence signals, the fluorescence emission unit 1 is completely separated from the fluorescence detection unit 2 for receiving excitation signals, the back of the fluorescence signals is reduced, the detection sensitivity of the fluorescence detection light path system 100 is improved, meanwhile, the fluorescence emission unit 1 is completely separated from the fluorescence detection unit 2, optical elements in the fluorescence detection unit 2 such as a dichroic mirror and the like are reduced, the cost is reduced, and the detection efficiency of fluorescence is improved.
As shown in fig. 4, in the prior art, the fluorescence detection unit 2' includes a detector 22', a fluorescence transmission optical path 21' and a turntable 23', where a plurality of optical filters 211' are disposed on the turntable 23', the fluorescence transmission optical path 21' includes an optical fiber 212', a plurality of fluorescence signals are transmitted to the optical filters 211' through the optical fiber 212', and the plurality of optical filters 211' can respectively pass through one fluorescence signal, and the turntable 23' is rotated to switch channels so that different fluorescence signals pass through and are received by the detector 22 '. However, the turntable 23 'is driven to rotate by a motor or the like, ten milliseconds are required for switching between channels, the detection efficiency is reduced, and vibration is generated when the turntable 23' rotates, so that the detection result is influenced.
In order to solve the above-mentioned technical problem, in the present embodiment, as shown in fig. 3b and 5, it is preferable that the fluorescence detection unit 2 includes at least two fluorescence transmission light paths 21, and at least two fluorescence transmission light paths 21 are in one-to-one correspondence with at least two fluorescence emission units 1, that is, one fluorescence emission unit 1 may correspond to one fluorescence transmission light path 21. The reaction sample at the position opposite to the excitation optical fiber 11 of the fluorescence emission unit 1 can receive stronger excitation light, so that the reaction sample at the position can generate higher fluorescence signals, at least two fluorescence transmission light paths 21 are in one-to-one correspondence with at least two fluorescence emission units 1, the fluorescence transmission light paths 21 can be arranged close to the reaction sample excited by the fluorescence emission units 1 corresponding to the fluorescence transmission light paths, more fluorescence signals are further led into the fluorescence transmission light paths 21, and the accuracy of detection results is further improved.
Each fluorescence transmission optical path 21 includes an optical filter 211 that allows a preset fluorescence signal to pass through, that is, only a specific fluorescence signal can pass through in each fluorescence transmission optical path 21, and compared with the case that only one fluorescence transmission optical path 21 'is set, by mechanically switching different optical filters 211', in this embodiment, when each fluorescence transmission optical path 21 is set with an optical filter 211, the time required for mechanical switching when the fluorescence signal passes through the optical path can be reduced, rapid detection of the fluorescence signal is realized, and meanwhile, the influence of mechanical vibration caused by mechanical switching on the detection result can be eliminated. When the excitation light is polychromatic light, other light such as fluorescence signals other than the preset fluorescence signals cannot pass through the optical filter 211, so that the accuracy of the detection result can be ensured.
As shown in fig. 3b and 5, the fluorescence detection unit 2 further includes a detector 22, and the plurality of fluorescence transmission light paths 21 are connected to the detector 22, where the detector 22 is configured to detect a fluorescence signal.
Preferably, the number of the detectors 22 is one, the detectors 22 are electrically connected with the control unit, so as to record the intensity of the fluorescent signal in a time period, and different fluorescent light transmission light paths 21 are converged on one detector 22 after passing through different optical filters 211. By switching on different fluorescent emission units 1 in time periods through the fluorescent emission units 1, switching of excitation channels is realized, so that different signal fluorescence is excited in time periods, corresponding signal fluorescence can pass through the optical filter 211 corresponding to the signal fluorescence, the fluorescent detector 22 can only detect the intensity of the signal fluorescence and can not detect the type of the signal fluorescence, but can judge what signal fluorescence is according to the receiving time period, and further a reaction sample corresponding to the signal fluorescence is determined. The embodiment judges which signal is fluorescent according to the time period, the equipment cost is low, the equipment structure is simple, no mechanical switching exists, and the detection speed is high.
As shown in fig. 3b, and in combination with fig. 6a, the fluorescence detection optical path system 100 further includes a fiber holder 3, the fluorescence transmission optical path 21 includes a collection fiber 212, one end of the excitation fiber 11 emitting excitation light, and one end of the collection fiber 212 emitting fluorescence signals forms a fiber group 31, the fiber group 31 is disposed in the fiber holder 3, the fibers in the fiber group 31 are arranged along a first direction, the first direction is a radial direction of the fibers (a radial direction of the fibers as indicated by an arrow R in fig. 3 b), so that the emitting end of the collection fiber 212 and the emitting end of the excitation fiber 11 are arranged flat in the fiber holder 3, that is, the fibers (the collection fiber 212 and the excitation fiber 11) are arranged flat near one end of the side wall 42 of the carrier 4, the first direction may be a length direction of the side wall 42, and the fibers in the fiber holder 3 are arranged in a single layer in a thickness direction of the carrier 4, thereby adapting to the carrier 4 with a flat structure. It will be appreciated that the diameter of the optical fiber may be smaller than the thickness of the carrier 4, so that the optical fiber in the optical fiber holder 3 does not protrude from the carrier 4 in the thickness direction of the carrier 4, and therefore, the excitation light emitted from the collection optical fiber 212 can enter the accommodating cavity 41, and the entire excitation optical fiber 11 can collect fluorescence. In the prior art, when the excitation optical fiber and the collection optical fiber are disposed at an included angle, it is often desirable that the excitation light incident point of the laser optical fiber incident on the reaction sample coincides with the fluorescence signal incident point of the laser optical fiber incident on the collection optical fiber, so that the most fluorescence signal enters the collection optical fiber, but more excitation light enters the collection optical fiber, in this embodiment, the excitation optical fiber 11 and the collection optical fiber 212 are arranged in parallel along the radial direction of the optical fiber (the radial direction of the optical fiber is shown by the arrow R in fig. 3 b), so that the amount of the excitation light entering the excitation optical fiber 11 can be reduced, and meanwhile, it can be ensured that as many collection optical fibers 212 accept more fluorescence signals, so that the optimal sensitivity and signal to noise ratio can be obtained. Meanwhile, when the optical fiber group 31 is coupled with the carrier 4, the first direction of the optical fiber arrangement is perpendicular to the thickness direction of the carrier 4, and the optical fiber group 31 and the optical fiber seat 3 are both flat structures, so as to be matched with the carrier 4.
When at least two fluorescence transmission light paths 21 and at least two fluorescence emission units 1 are provided, at least two groups of optical fiber groups 31 are correspondingly formed, at least two groups of optical fiber groups 31 in the optical fiber seat 3 are sequentially arranged along the first direction, that is, when a plurality of optical fiber groups 31 are provided, the plurality of optical fiber groups 31 are also arranged flat, the optical fibers in the optical fiber seat 3 are also arranged in a single layer in the thickness direction of the carrier 4, so that the carrier 4 with a flat structure is adapted, the optical fibers in the optical fiber seat 3 do not protrude from the carrier 4 in the thickness direction of the carrier 4, and therefore, the excitation light emitted by the collection optical fibers 212 can enter the accommodating cavity 41, and the whole excitation optical fibers 11 can collect fluorescence.
Meanwhile, the plurality of optical fiber groups 31 are sequentially arranged, the collecting optical fibers 212 in the same optical fiber group 31 are adjacent to the excitation optical fibers 11, more fluorescent signals can be generated by the reaction sample at the corresponding position of the excitation optical fibers 11, and the collecting optical fibers 212 are close to the corresponding excitation optical fibers 11, so that more fluorescent signals enter the fluorescent transmission optical path 21, and the accuracy of detection results is further improved.
As shown in fig. 3c and 3d, the optical fiber holder 3 includes a holder body 32 and a flat groove 33 provided on one side of the holder body 32, and the optical fiber group 32 is provided in the flat groove 33.
As shown in fig. 6a, an optical fiber group 31 includes at least two collection optical fibers 212, and at least one collection optical fiber 212 is disposed on both sides of the excitation optical fiber 11 in the first direction in the optical fiber group 31. In one optical fiber set 31, the number of the collecting optical fibers 212 is increased to at least two, so that the multi-point detection is performed on the reaction sample, the detection efficiency of fluorescence can be effectively improved, the problem of low signal is solved, and the requirement on the sensitivity of the detector 22 is reduced. Meanwhile, when one of the collection fibers 212 is affected by the bubbles in the reaction sample, correction can be performed by the detection result of the other collection fibers 212.
The detector 22 comprises a silicon photomultiplier (i.e. SiPM), a photon detector (i.e. PD) or a photomultiplier (i.e. PMT), and the fluorescence detector 22 adopting the silicon photomultiplier, the photon detector or the photomultiplier has high sensitivity, so that ultra-fast and high-sensitivity detection of fluorescence signal millisecond level is realized when fluorescence detection is carried out.
As shown in fig. 6a, in a specific embodiment, four fluorescence emitting units 1 are provided, of course, the number of fluorescence emitting units 1 is not limited to four, but may be less than four or more than four, wherein two sides of an excitation fiber 11 are respectively provided with one collection fiber 212, and one fiber group 31 is formed, that is, each fluorescence may be collected by two collection fibers 212, and of course, only one collection fiber 212 or more than two collection fibers 212 may be provided in one fiber group 31.
The present embodiment also provides a PCR detection system, which includes the carrier 4 and the fluorescence detection light path system 100 described above, where the fluorescence detection light path system 100 is used to detect a reaction sample in the carrier 4.
The fluorescence emitting unit 1 may be provided on only one side of the carrier 4 as shown in fig. 6a, or the fluorescence emitting units 1 may be provided on both sides of the carrier 4 as shown in fig. 7, the excitation fibers 11 of one fluorescence emitting unit 1 being provided with the collection fibers 212 correspondingly, each collection fiber 212 on both sides of the carrier 4 preferably being connected to one detector 22, although the collection fibers 212 on each side may be connected to one detector 22, respectively. The two sides of the reaction sample are detected, so that the fluorescence detection efficiency can be effectively improved, the problem of low signal is solved, and the requirement on the sensitivity of the detector 22 is reduced. Also, when the detection result is inaccurate due to the occlusion of one side of the collection fiber 212 by bubbles or the like in the reaction sample, correction can be performed by the detection result of the other side. Of course, it is also possible to detect the position on three or more sides of the carrier 4. Preferably, fluorescence detection is performed through the light-transmitting side walls 42 of the carrier 4. Optionally, the transparent sidewall 42 is made of polydimethylsiloxane (i.e. PDMS), polypropylene (i.e. PP), plexiglass (i.e. PMMA) or polycarbonate (i.e. PC), PMMA, PDMS, PP and PC are optically transparent materials with good biocompatibility, so that the fluorescence detection requirement can be satisfied and the reaction sample is not affected. Fig. 6b is an amplification curve obtained by the fluorescence detection optical path system 100, and the fluorescence detection optical path system 100 can effectively improve the fluorescence detection efficiency, thereby solving the problem of low signal.
Alternatively, the first wall 43 and/or the second wall 44 are/is a film made of a heat conductive material, in particular, the heated side of the carrier 4 is a film made of a heat conductive material. Specifically, the membrane is composed of an aluminum membrane and a separator membrane, and optionally, the separator membrane is a polypropylene membrane (i.e., pp membrane). The isolating film is in direct contact with the reaction liquid, so that the influence of the aluminum film on the reaction sample can be prevented. The thickness of the aluminum film may be several tens of μm, such as 30 μm,60 μm, etc., and the thickness of the aluminum film may be 10 to 30 μm, such as 20 μm, etc., as long as the separation film separates the reaction sample from the aluminum film. Of course, the film may also be an aluminum film, and the thickness of the aluminum film may be several tens of μm, such as 30 μm,60 μm, etc., and the aluminum film of the thickness may be deformed or have a certain strength.
As shown in fig. 8, the first wall 43 and the second wall 44 are heated by external heaters 522, respectively, and the two external heaters 522 may be cooled by cooling units 521, respectively, to achieve rapid temperature rise and fall of the reaction sample. The cooling assembly 521 is used to cool the carrier 4. The cooling unit 521 may be water-cooled or air-cooled, as shown in fig. 9, as a flow channel 5221 for flowing a liquid cooling medium is disposed inside the cooling unit 521, and the cooling medium continuously flows to take away the heat conducted to the cooling unit 521 by the carrier 4 and the external heater 522. Or a cavity is formed in the cooling medium, and a power part such as a pump sprays liquid or gaseous cooling medium into the cavity, wherein the liquid or gaseous cooling medium takes away heat conducted to the cooling component 521 by the carrier 4 and the external heater 522.
As shown in fig. 10, of course, in other alternative embodiments, the external heater 522 may not be provided, the first wall 43 and/or the second wall 44 are not films, and the carrier 4 includes at least one internal heater 45, and the first wall 43 and/or the second wall 44 are internal heaters 45. Specifically, the carrier 4 may include two built-in heaters 45, and the two built-in heaters 45 may be cooled by the cooling assembly 521, respectively, to achieve rapid temperature rise and fall of the reaction sample.
Alternatively, the reaction sample in the accommodation chamber 41 is directly contacted with the built-in heater 45 to improve the heat conduction efficiency between the built-in heater 45 and the reaction sample. The side wall 42 is located between two built-in heaters 45, the side wall 42 is made of a transparent material, and the fluorescence detection light path system 100 performs fluorescence detection through the side wall 42.
As shown in fig. 10, the built-in heater 45 includes a heating member 91. The power supply is connected with the heating element 91, the heating element 91 is a controllable heating source in the built-in heater 45, and can be a resistor, for example, a resistor fine wire structure can be made of copper materials, and heating power is controlled by controlling the magnitude of current flowing through the resistor, so that temperature control is realized. In other alternative embodiments, the heating member 91 may be configured as a coil or be heated by electromagnetic induction using a ferromagnetic material or the like.
As shown in fig. 10, the built-in heater 45 preferably includes at least two independently controlled heating elements 91, and the heating elements 91 may be independently controlled to improve the uniformity of the temperature of the reaction sample. If, for example, the temperature of a heating element 91 does not reach the predetermined temperature (how to detect the temperature of the heating element 91 is described in detail below), the current of the heating element 91 is increased to rapidly raise the reaction sample to the predetermined temperature. In this embodiment, since the built-in heater 45 is in direct contact with the reaction sample, the heat conduction efficiency between the built-in heater 45 and the reaction sample is high, and the temperature of the heating element 91 of the built-in heater 45 can be equal to the temperature of the reaction sample, so that the temperature of each heating element 91 is controlled to reach the preset temperature, and the reaction samples in all places can be at the preset temperature, so that the temperature uniformity of the reaction sample is ensured.
As shown in fig. 10, the built-in heater 45 may further include an upper conductive member 92 and a lower conductive member 95, with the heating member 91 interposed between the upper conductive member 92 and the lower conductive member 95. The upper and lower conductive assemblies 92 and 95 have a conductive heat and insulating effect.
The built-in heater 45 includes a soaking layer 921, and in particular, the upper conductive member 92 may further include a soaking layer 921. The soaking layer 921 is in direct contact with the reaction sample in the accommodating chamber 41, and the soaking layer 921 can ensure uniform conduction of heat in both the longitudinal and transverse directions (i.e., the thickness direction of the reaction sample and the surface perpendicular to the thickness direction), and ensure temperature uniformity of the sample liquid. Optionally, the soaking layer 921 is made of an insulating material, such as a high thermal conductivity ceramic or the like.
The soaking layer 921 is made of an insulating material, and the soaking layer 921 is adjacent to the heating member 91, at this time, the number of layers of the carrier 4 can be reduced, the time for transferring the heat of the heating member 91 to the reaction sample in the carrier 4 can be shortened, and the time required for heat dissipation of the carrier 4 can be shortened.
As shown in fig. 10, the lower conductive assembly 95 also includes an insulating thermal resistance layer 951. The insulating thermal resistance layer 951 has a certain thermal resistance characteristic and an insulating characteristic. The insulating thermal resistance layer 951 may form a longitudinal thermal resistance in addition to insulating the heating member 91. The magnitude of the thermal resistance can be designed by material selection and thickness selection. Typically, the thermal resistance of this layer is much greater than the thermal resistance of the other layers of the structure, so that the thermal edge layer is the primary thermal resistance source for the carrier 4 to dissipate heat and cool down to the cooling assembly 521. The insulating thermal resistance layer 951 is one of the main contributors to the thermal performance of the carrier 4.
Optionally, the lower conductive assembly 95 further includes a thermally conductive layer 952, the thermally conductive layer 952 being located on a side of the insulating thermal resistance layer 951 remote from the heating member 91. Further, the thermally conductive layer 952 is the outermost layer of the lower conductive assembly 95, which is in direct contact with the cooling assembly 521. The thermally conductive layer 952 is made of a metal such as copper or other material having high thermal conductivity. The surface of the lower conductive assembly 95 that contacts the cooling assembly 521 is difficult to avoid point contact due to cost control or tooling limitations. When the outermost layer of the lower conductive element 95 is the heat conductive layer 952, even if the heat conductive layer 952 is in point contact with the cooling element 521, the heat conductive layer 952 can uniformly distribute heat over the entire heat conductive layer 952 due to its good conductivity, thereby uniformly distributing heat from other layers of the lower conductive element 95.
Preferably, the heating element 91 of the present embodiment is a resistor, and there is a specific relationship between the resistor and the temperature thereof, so that the real-time resistance change of the heating element 91 can be measured while heating, and the average temperature of the heating element 91 can be deduced through the temperature coefficient of resistance and the nominal resistance. The temperature shows the current temperature of the carrier 4 in real time without delay, so that the temperature control device can be used for rapidly feedback-controlling the temperature of the carrier 4 and a reaction sample, and can control the temperature of the sample more accurately and improve the overall reaction speed of a temperature control system compared with the prior art.
In order to detect the resistance of the heating member 91, the PCR detection system may optionally further include a resistance detection member for detecting the resistance of the heating member 91 to measure the temperature of the heating member 91 by a resistance temperature measurement method.
However, the resistance temperature measurement method has a disadvantage that, for the same type of resistance, such as copper wire resistance, the nominal resistance value between the resistances and the temperature coefficient of resistance (the resistance value at the nominal temperature is simply referred to as the nominal resistance value), the nominal resistance means that the asserted (or marked) resistance value is true at this temperature, wherein this temperature is the nominal temperature, and the nominal temperature may be arbitrarily selected according to the requirement), the actual temperature coefficient of resistance of the single heating element 91 is slightly different from the nominal resistance value, which may possibly cause a temperature measurement error, so it is preferable that, as shown in fig. 9 and 10, the built-in heater 45 provided in this embodiment further includes a temperature calibration part 93 for reflecting the temperature of the heating element 91, and the temperature calibration part 93 may allow the temperature detection unit 10 to detect the temperature, so that the carrier 4 may be controlled by the resistance temperature measurement method and the dual temperature measurement method for calibrating the temperature of the carrier 4.
Although the temperature calibration part 93 can show the temperature of the heating element 91, the temperature calibration part 93 can be detected by the temperature detection unit 10, but since the temperature of the reaction sample changes rapidly in the amplification stage, when the isothermal detection unit 10 such as the temperature sensor detects the temperature of the carrier 4, a certain time is required for transferring heat from the temperature calibration part 93 to the temperature detection unit 10, so that the detection result measured by the temperature detection unit 10 will have a temperature measurement delay of 1-2s under normal conditions, and the temperature change of the carrier 4 can reach more than 30 ℃ in the rapid temperature rise and fall process, and therefore, it is relatively difficult to control the carrier 4 by the temperature detection unit 10 in the rapid temperature rise and fall process.
The temperature of the carrier 4 is not completely dependent on the temperature value measured by the uncalibrated resistance temperature measurement method, and the carrier 4 is not completely dependent on the temperature control detected by the temperature detection unit 10, but the temperature of the temperature calibration part 93 of the carrier 4 and the temperature of the heating element 91 are measured by the resistance temperature measurement method by combining the two, so that the temperature of the carrier 4 can be rapidly and accurately controlled, the purpose of accurately controlling the temperature is achieved, and the problems of temperature detection delay and large temperature measurement error caused by the common temperature detection method in the prior art are solved.
It is to be understood that when the built-in heater 45 is provided with at least two heating elements 91, each heating element 91 is correspondingly provided with a temperature calibrating portion 93 and a resistance detecting element, so that the temperature of the heating element 91 can be calibrated.
In order to more clearly describe how the temperature calibration section 93 is used to calibrate the temperature detected by the resistance detecting member in the present embodiment, a process of calibrating the resistance thermometry by the temperature detecting unit 10 in one actual detection is shown in conjunction with fig. 11. Before calibrating the temperature values, an initial RT temperature profile, i.e. a temperature preset profile, is preset, and then a small current, e.g. a current which may be less than 1 ma, is applied to the heating element 91 of the carrier 4. Wherein a small current is applied for the purpose of reading the resistance of the heating member 91 without heating the heating member 91.
For the first calibration, the temperature detecting unit 10 detects a first temperature calibration value T1, and the resistance detecting unit detects a first voltage U1 and a first current I1 of the heating element 91 at the temperature of T1, and can obtain a resistance R1 of the heating element 91 at the temperature of T1 according to r=u/I.
Second calibration, the temperature detecting unit 10 detects a second temperature calibration value T2, and the resistance detecting unit detects a second voltage U2 and a second current I2 of the heating element 91 at the temperature of T2, so that the resistance R2 of the heating element 91 at the temperature of T2 can be obtained according to r=u/I.
Finally, according to two sets of binary once equations, namely R1＝R0(1+αΔT1) and R2＝R0(1+αΔT2) (wherein DeltaT1＝T1-T0,ΔT2＝T2-T0,R1 is the resistance value corresponding to the heating element 91 at the temperature T1, R2 is the resistance value corresponding to the heating element 91 at the temperature T2, alpha is the resistance temperature coefficient of the material, T0 is the nominal temperature, and R0 is the nominal resistance value), specific values of R0 and alpha are obtained, namely an accurate R-T curve is obtained, and the temperature of the heating element 91 measured by a resistance temperature measurement method can be used as feedback for accurate temperature control.
The temperature calibration value can be detected in the whole process of nucleic acid amplification, so that the temperature can be calibrated for multiple times in the subsequent process, and the detection precision is further improved.
As shown in fig. 12, to facilitate the measurement of the temperature by the temperature detecting unit 10, the two first contacts of the temperature detecting unit 10 are respectively contacted with the two temperature calibrating portions 93, and the two temperature calibrating portions 93 are not electrically conductive, at this time, optionally, the internal heater 45 may further include an external electrical connecting contact 97 and an electrical connecting lead 98, the number of the external electrical connecting contact 97 and the electrical connecting lead 98 may be two, the two external electrical connecting contacts 97 are respectively located at one side of the two temperature calibrating portions 93 away from each other, one external connecting contact is electrically connected with one temperature calibrating portion 93 through one electrical connecting lead 98, and the other external connecting contact is electrically connected with the other temperature calibrating portion 93 through the other electrical connecting lead 98.
As shown in fig. 10, the soaking layer 921 of the upper conductive component 92 is conducted to the temperature calibration portion 93, the temperature calibration portion 93 is electrically connected with the outside through the electrical connection lead 98 at the external electrical connection contact 97, wherein the diameter of the electrical connection lead 98 is smaller than that of the temperature calibration portion 93 and the external electrical connection contact 97, thereby reducing the heat loss generated by the temperature calibration portion 93 through the electrical connection lead 98, so that the temperature calibration portion 93 can better embody the upper conductive component 92, such as the temperature of the soaking layer 921 of the upper conductive component 92, the temperature detection unit 10 realizes good electrical and thermal contact with the temperature calibration portion 93 through welding spots, when the temperature of the soaking layer 921 of the upper conductive component 92 changes, the temperature detection unit 10 can quickly and accurately sense the temperature change, the temperature change causes the resistance change of the temperature detection unit 10, and the resistance change of the temperature detection unit 10 is detected in real time at the external electrical connection contact 97, so that the real-time temperature detection can be realized.
As shown in fig. 10, optionally, in order to shorten the time when the temperature of the temperature calibration part 93 coincides with the temperature of the heating member 91, the carrier 4 may further include a rapid conduction part 94, the rapid conduction part 94 being for conducting the heat of the heating member 91 to the temperature calibration part 93. Specifically, in the present embodiment, the heat of the heating element 91 is indirectly transferred to the temperature calibration portion 93, for example, the heating element 91 heats the soaking layer 921, and the heat of the soaking layer 921 is transferred to the temperature calibration portion 93 through the quick transfer portion 94, so that the temperature calibration portion 93 accurately represents the temperature of the soaking layer 921, and the temperature detecting unit 10 can accurately measure the temperature of the soaking layer 921. Further, since the thickness of the reaction sample is small, the temperature of the reaction sample is substantially the same as the temperature of the soaking layer 921, and thus the temperature of the reaction sample can be obtained by detecting the temperature of the temperature calibration portion 93.
Preferably, one side of the rapid conduction part 94 is connected to one side of the upper conduction assembly 92 near the heating member 91 or to one side of the lower conduction assembly 95 near the heating member 91, and the other side is connected to the temperature calibration part 93. The lower surface of the upper conductive member 92 and the upper surface of the lower conductive member 95 are closest to the heating member 91, and the temperature thereof is closest to the temperature of the heating member 91 first, so that the rapid conduction portion 94 is disposed in such a manner that the temperature of the rapid conduction portion 94 and the temperature of the heating member 91 are consistent in the shortest time. Alternatively, the rapid conduction portion 94 is made of a material having a high thermal conductivity, such as a metal material of copper or aluminum, or a thermally conductive ceramic, or the like. The thermal conductivity of the fast conducting portion 94 is particularly superior to that of the lower conducting member 95 to rapidly transfer heat to the temperature calibrating portion 93.
The quick-conduction portion 94 includes a patch 941 and one or more guide posts 942, where the patch 941 is attached to a side of the upper conduction assembly 92 close to the heating element 91 or to a side of the lower conduction assembly 95 close to the heating element 91, and one end of the one or more guide posts 942 is connected to the patch 941, and the other end of the one or more guide posts 942 is disposed through the lower conduction assembly 95 and connected to the temperature calibration portion 93. The lower surface of the upper conductive member 92 and the upper surface of the lower conductive member 95 are closest to the heating member 91, and the temperature thereof is closest to the temperature of the heating member 91 first, so that the patches 941 are arranged in such a manner that the temperature of the rapid conductive portion 94 is most rapidly matched with the temperature of the heating member 91. The patch 941 can increase the contact area of the rapid conduction portion 94 with the upper conduction member 92 or the lower conduction member 95, improving the conduction efficiency. The cross-sectional area of the guide post 942 may be smaller than the cross-sectional area of the patch 941, so that the temperature of the patch 941 may be quickly conducted to the temperature calibration portion 93, and the thermal resistance layer is guaranteed to generate a required thermal resistance according to the design. Alternatively, the patches 941 and the pillars 942 are made of a material with high thermal conductivity such as copper, and when the patches 941 and the pillars 942 are required to be made of an insulating material to avoid a short circuit of the carrier 4, the patches 941 or the pillars 942 may be made of a material with high thermal conductivity such as ceramic.
It is understood that the temperature calibration portions 93 may be disposed in one-to-one correspondence with the patches 941, and that two temperature calibration portions 93 may be connected to one patch 941. One temperature calibration part 93 may be connected to one guide post 942, and the temperature calibration part 93 may be connected to a plurality of guide posts 942 to improve temperature uniformity of the temperature calibration part 93.
As shown in fig. 10, in order to obtain the electrical resistance of the heating element 91 and to supply the heating element 91, the outer surface of the carrier 4 is optionally provided with a plurality of second contacts 96, the second contacts 96 being electrically connected to the heating element 91. The current and voltage of the heater 91 can be obtained through the second contact 96, and thus the resistance value of the heater 91 can be obtained. It will be appreciated that when a plurality of heating elements 91 are independently controlled, each heating element 91 is correspondingly provided with a second contact 96 to respectively calibrate the heating element 91 and provide heat to the heating element 91.
In this embodiment, the second contact 96 enables the carrier 4 to realize its own temperature measurement function, and compared with the conventional structure that the temperature can only be measured by the external temperature measurement unit, the embodiment can directly measure the temperature of the carrier 4, so that the temperature measurement is more accurate and rapid, and the accuracy and control speed of the temperature control system can be improved.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.