Single-molecule fluorescence detection system

Abstract

The present invention discloses a single-molecule fluorescence detection system, which includes a fluorescence excitation unit, a unit of the liquid to be measured, a fluorescence collection unit, and a signal processing unit; the fluorescence excitation unit includes a laser, a conductive optical fiber, and a first gradient-index lens. The laser beam generated by the laser is conducted through the conductive optical fiber to the first gradient-index lens to obtain an excitation beam; the unit of the liquid to be measured includes a microchannel that allows single molecules to pass through. The liquid to be measured passes through the microchannel, and the excitation beam irradiates the microchannel to excite the single-molecule fluorescent label located in the microchannel to emit fluorescent photons; the fluorescence collection unit includes a second gradient-index lens and a detection optical fiber. The second gradient-index lens collects the fluorescent photons in dark field and conducts them to the detection optical fiber; the detection optical fiber is connected to the signal processing unit, and the signal processing unit processes the fluorescent photons passing through the detection optical fiber to obtain information about the fluorescent label. The present invention provides a single-molecule fluorescence detection system that is miniature, low-cost, and high-efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of single-molecule fluorescence detection, and more specifically, to a single-molecule fluorescence detection system. Background Art

[0002] Fluorescence detection mainly utilizes the detection of special proteins with fluorescent labels. By detecting their concentrations, it can be used for the diagnosis of diseases such as respiratory tract and autoimmune diseases, and it is also one of the most common detection methods in in vitro diagnosis. Currently, with the continuous development of medicine and biology, and the continuous improvement of the requirements for speed, accuracy, and reliability, traditional detection methods (including currently popular chemiluminescence methods, ELISA, immunoprecipitation methods, etc.) can no longer meet the current needs. The emergence of single-molecule fluorescence detection has filled the demand for advantages such as rapidity, high sensitivity, and high reliability, and is also known as the next-generation fluorescence detection technology. Single-molecule fluorescence detection can optimize the lowest detection limit to 100 times or even more than 1000 times.

[0003] Currently, there are two types of single-molecule fluorescence detection devices on the market. One is developed by Quanterix. It uses semiconductor processes to prepare a chip structure with a microporous array, confines the analyte in each micropore, and then uses an imaging system to detect whether there is an analyte in each micropore, thereby obtaining the content information of the analyte. However, the processing technology requirements for the microporous array are high, which increases the cost of the chip. The other is developed by Singulex. It uses a confocal microscope to observe within the focal range, excites the area with a laser, and then collects the fluorescence intensity of the area to analyze the content of the analyte in the area. However, the confocal microscope has a complex structure, large volume, high price, and is not easy to maintain.

[0004] In addition, the above two single-molecule fluorescence detection devices both perform spatial segmentation on the analyte, dividing the analyte into smaller units or individuals for observation. Affected by high background noise, each single step requires long-term cumulative exposure to reach the observable threshold of the receiver, so that the final measurement time is much longer than that of the chemiluminescence method.

[0005] Therefore, single-molecule fluorescence detection devices are affected by problems such as cost, measurement efficiency, and instrument volume, and currently have a low market share and are difficult to promote. Summary of the Invention

[0006] The object of the present invention is to overcome the above-mentioned defects existing in the prior art and provide a miniaturized, low-cost, and high-efficiency single-molecule fluorescence detection system.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A single-molecule fluorescence detection system includes a fluorescence excitation unit, a unit of the liquid to be measured, a fluorescence collection unit, and a signal processing unit;

[0009] The fluorescence excitation unit includes a laser, a conducting optical fiber, and a first gradient-index lens. The laser beam generated by the laser is conducted to the first gradient-index lens through the conducting optical fiber, and an excitation beam for exciting the liquid to be measured to generate fluorescence is obtained after being focused by the first gradient-index lens;

[0010] The unit of the liquid to be measured includes a microchannel that allows single molecules to pass through. The liquid to be measured passes through the microchannel, and the excitation beam irradiates the microchannel to excite a single-molecule fluorescent marker located in the microchannel to emit fluorescent photons;

[0011] The fluorescence collection unit includes a second gradient-index lens and a detection optical fiber. The second gradient-index lens is arranged on the optical path other than the excitation beam. The second gradient-index lens collects the fluorescent photons in dark field, and the fluorescent photons are conducted to the detection optical fiber after being focused by the second gradient-index lens;

[0012] The detection optical fiber is connected to the signal processing unit, and the signal processing unit processes the fluorescent photons passing through the detection optical fiber to obtain information of the fluorescent marker.

[0013] Implementing the embodiments of the present invention will have the following beneficial effects:

[0014] In the embodiment of the present invention, by providing a first gradient refractive index lens and a second gradient refractive index lens to replace the complex optical lens focusing structure composed of multiple curved lenses in the prior art, the spatial size of the optical path propagation is effectively reduced. Moreover, since the volume of the gradient refractive index lens can be controlled within the millimeter level, the volume and structural complexity of the single-molecule fluorescence detection system are greatly reduced in the present invention. By arranging the second gradient refractive index lens on the optical path of the non-excitation beam to collect fluorescence photons in dark field, it is avoided that the light of the excitation beam is collected by the second gradient refractive index lens, which may form a large background noise and easily submerge some weak single-molecule fluorescence signals, thereby improving the detection speed and accuracy of the single-molecule fluorescence signal. By providing the second gradient refractive index lens and the detection optical fiber, the front end of the second gradient refractive index lens collects the generated fluorescence photons. After being refracted and focused by the second gradient refractive index lens, the fluorescence photons are coupled into the detection optical fiber. The limitation of the fiber aperture of the detection optical fiber plays a role of diaphragm denoising, which is equivalent to the confocal pinhole in the existing confocal microscope. The present application realizes the confocal function of the existing confocal microscope by using the combination of the second gradient refractive index lens and the detection optical fiber, which can significantly reduce the volume and cost of the single-molecule fluorescence detection system. By adopting the method of spatial confinement of the microchannel, only one analyte molecule exists in the sample volume for each detection, and then information such as the concentration and content of the fluorescently labeled analyte molecules in the sample to be measured is obtained through statistics, avoiding spatial segmentation of the sample to be measured and prolonging the detection time.

[0015] In summary, on the premise of ensuring the advantages of ultra-sensitivity and high efficiency of the single-molecule fluorescence detection technology, the present invention uses the gradient refractive index lens to extremely effectively simplify the hypersensitive fluorescence detection system and obtain a micro single-molecule fluorescence detection system. This micro system has an all-solid structure, is stable, easy to integrate with other functional modules, and the manufacturing cost is also much lower than that of a large-scale microscopic system, thus overcoming the disadvantages of the existing single-molecule fluorescence detection device such as complexity, large design difficulty, difficult maintenance and high cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Among them:

[0018] Figure 1 is a schematic structural diagram of a single-molecule fluorescence detection system according to a specific embodiment of the present invention.

[0019] Figure 2It is a curve relationship diagram of the refractive index N(r) at any point on the radial radius r of the first / second refractive index gradient lens in a specific embodiment of the present invention.

[0020] Figure 3 It is a schematic diagram of the light transmission of the first / second refractive index gradient lens in a specific embodiment of the present invention.

[0021] Figure 4 It is a schematic structural diagram of the unit to be measured solution in a specific embodiment of the present invention.

[0022] Figure 5 It is a schematic structural diagram of the micro pipeline in a specific embodiment of the present invention. Specific embodiments

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] Reference Figure 1 The present invention discloses a single molecule fluorescence detection system, including a fluorescence excitation unit 10, a unit to be measured solution 20, a fluorescence collection unit 30, and a signal processing unit 40.

[0025] The fluorescence excitation unit 10 includes a laser, a conduction optical fiber 11, and a first refractive index gradient lens 12. The laser beam generated by the laser is conducted to the first refractive index gradient lens 12 through the conduction optical fiber 11, and after being focused by the first refractive index gradient lens 12, an excitation beam for exciting the unit to be measured solution to generate fluorescence is obtained.

[0026] The unit to be measured solution 20 includes a micro pipeline 21 which allows single molecules to pass through. The unit to be measured solution passes through the micro pipeline 21, and the excitation beam irradiates the micro pipeline 21 to excite the single molecule fluorescent marker located in the micro pipeline 21 to emit fluorescent photons.

[0027] The fluorescence collection unit 30 includes a second refractive index gradient lens 31 and a detection optical fiber 32. The second refractive index gradient lens 31 is arranged on the optical path of the non-excitation beam to collect fluorescent photons in dark field. The fluorescent photons are focused by the second refractive index gradient lens 31 and then conducted to the detection optical fiber 32.

[0028] The detection optical fiber 32 is connected to the signal processing unit 40, and the signal processing unit 40 processes the fluorescent photons passing through the detection optical fiber 32 to obtain information of the fluorescent marker.

[0029] In the above embodiments, by providing the first gradient refractive index lens 12 and the second gradient refractive index lens 31 to replace the complex optical lens focusing structure composed of multiple curved lenses in the prior art, the spatial size of the optical path propagation is effectively reduced. Moreover, since the volume of the gradient refractive index lens can be controlled within the millimeter level, the volume and structural complexity of the single-molecule fluorescence detection system are greatly reduced in the present invention. By disposing the second gradient refractive index lens 31 on the optical path of the non-excitation beam to collect fluorescence photons in dark field, it is avoided that the light of the excitation beam is collected by the second gradient refractive index lens 31, which may form a large background noise and easily submerge some relatively weak single-molecule fluorescence signals, thereby improving the detection speed and accuracy of the single-molecule fluorescence signals. By providing the second gradient refractive index lens 31 and the detection optical fiber 32, the front end of the second gradient refractive index lens 31 collects the generated fluorescence photons. After being refracted and focused by the second gradient refractive index lens 31, the fluorescence photons are coupled to the detection optical fiber 32. The limitation of the fiber aperture of the detection optical fiber 32 plays a role of diaphragm noise reduction, which is equivalent to the function of the confocal pinhole in the existing confocal microscope. The present application realizes the confocal function of the existing confocal microscope by using the combination of the second gradient refractive index lens 31 and the detection optical fiber 32, which can significantly reduce the volume and cost of the single-molecule fluorescence detection system. By adopting the spatial limitation method of the microchannel, only one analyte molecule exists in the sample volume for each detection, and then information such as the concentration and content of the fluorescently labeled analyte molecules in the test solution is obtained by statistics, avoiding the spatial segmentation of the test solution in the prior art and avoiding the extension of the detection time.

[0030] In summary, on the premise of ensuring the advantages of ultra-sensitivity and high efficiency of the single-molecule fluorescence detection technology, the present invention extremely effectively simplifies the hypersensitive fluorescence detection system by using the gradient refractive index lens, and obtains a micro single-molecule fluorescence detection system. This micro system has an all-solid structure, is stable, is easy to be integrated with other functional modules, and the manufacturing cost is also much lower than that of the large-scale microscopic system, thereby overcoming the disadvantages of the existing single-molecule fluorescence detection device, such as complexity, large design difficulty, difficult maintenance and high cost.

[0031] In the above embodiments, both the first gradient refractive index lens 12 and the second gradient refractive index lens 31 are cylindrical structures with two planar end faces, and the center of the cylindrical structure has a high refractive index, and the refractive index gradually decreases from the center of the cylindrical structure along the radial direction outward.

[0032] Specifically, the formula for the refractive index N(r) in the radial direction of the first or second gradient refractive index lens 31 is:

[0033] N(r) = (0)(1 - r 2 / 2)

[0034] Wherein, N(0) is the refractive index at the center of the refractive index gradient lens, r is the radius of the refractive index gradient lens, A is the square of the refractive index distribution constant of the refractive index gradient lens, and the curve of the refractive index N(r) at any point on the radial radius r is for reference Figure 2 The parameters of the first refractive index gradient lens 12 and the parameters of the second refractive index gradient lens 31 may be the same or different.

[0035] The principle of self-focusing of the above refractive index gradient lens is as follows: Due to the change in the refractive index of the lens medium, the light entering the lens bends regularly, and then the light can be converged to a point. The severity of the change in the propagation direction of the light inside the refractive index gradient lens can be adjusted by adjusting the change gradient of the radial refractive index of the refractive index gradient lens, and then the length of one cycle of change of a beam of light inside the refractive index gradient lens and the maximum diameter of the radial change are determined.

[0036] In a specific embodiment, the length of the first refractive index gradient lens 12 is an integer multiple of a quarter pitch. The length of one sine wave cycle change of the light in the refractive index gradient lens is the pitch, and the pitch P satisfies the following relationship:

[0037]

[0038] Wherein, Z is the length of the first refractive index gradient lens 12. The first refractive index gradient lens 12 focuses the parallel light transmitted through the transmission optical fiber 11 onto the central main axis of the first refractive index gradient lens 12 for output, as Figure 3 shown.

[0039] In a specific embodiment, the length of the second refractive index gradient lens 31 is also an integer multiple of a quarter pitch, and P also satisfies the above relationship. The fluorescent photons are incident on the second refractive index gradient lens 31 in parallel, and after being refracted by the second refractive index gradient lens 31, they are output from the central main axis of the second refractive index gradient lens 31 to the detection optical fiber 32.

[0040] In a specific embodiment, the signal processing unit 40 includes a single photon detector, a single photon calculator, and a data processing module. The single photon detector is connected to the detection optical fiber 32. The single photon detector converts a single fluorescent photon passing through the detection optical fiber 32 into a TTL electrical signal and sends it to the single photon counter. The single photon calculator records the arrival time of a single fluorescent photon, and the data processing module processes the TTL electrical signal and the arrival time of a single fluorescent photon into information on the change of light intensity over time, and calculates the information of the fluorescent label passing through the microchannel 21 according to the information on the change of light intensity over time. In the present invention, since single molecules pass through the microchannel 21 discretely in time, the TTL electrical signal is a TTL level pulse signal, which shows a pulse light intensity envelope in time.

[0041] In a specific embodiment, calculating the information of the fluorescent label passing through the microtube 21 according to the information of the change of light intensity over time includes the following process:

[0042] 1) Calculate the number of light intensity envelopes obtained per unit time according to the information of the change of light intensity over time. The number of light intensity envelopes represents the number of fluorescent labels.

[0043] 2) Calculate the concentration of the fluorescent label in the liquid to be measured according to the number of light intensity envelopes obtained per unit time. The calculation formula for the concentration c of the fluorescent label in the liquid to be measured is: c = N / Q, where N is the number of light intensity envelopes measured per unit time, and Q is the flow rate. The flow rate Q can be measured by a flow meter or calculated by observing the volume of a certain volume of liquid passing through the microtube 21.

[0044] Specifically, the single-photon detector can use a photon-counting PMT, a silicon photomultiplier tube, a single-photon-counting APD, or a single-photon-counting MPPC, etc. The single-photon counter can be implemented by using a high-speed FPGA real-time data transmission program module or a time-to-digital conversion card.

[0045] Reference Figure 4 , in a specific embodiment, the liquid to be measured unit 20 includes a microtube 21, a waste liquid pool, and a pump. The pump drives the liquid to be measured into the microtube 21 and flows into the waste liquid pool through the microtube 21. All the liquid to be measured passes through the microtube 21, so that all the fluorescent label numbers in the liquid to be measured can be obtained.

[0046] Reference Figure 5 , in a specific embodiment, the microtube 21 is a capillary tube. The diameter of the capillary tube can be determined according to the size of the fluorescent label to be detected actually. The capillary tube is easier to obtain and has a lower cost compared with a chip. Of course, the microtube 21 can also be other various types and materials of micro-sized channels.

[0047] Continuing to refer to Figure 5 , further, the liquid to be measured unit 20 includes a transparent substrate 22. A microtube 21 is arranged in the transparent substrate 22. The liquid pipeline is connected to the microtube 21 through a connector 23.

[0048] Further, in a specific embodiment, the material of the transparent substrate 22 is PDMS material. Of course, it can also be other light-transmitting materials, such as silica, quartz, etc.

[0049] In a specific embodiment, the diameter of the microtube 21 is 1 μm to 50 μm, and fluorescent detections with diameters in the range of 5 nm to 10 μm can be detected. In a specific embodiment, the microtube 21 is a square pipeline with a side length of 4 μm to 10 μm, which can enable PS fluorescent microspheres with a diameter of about 20 nm to pass through the microtube 21 individually under the action of pressure.

[0050] In a specific embodiment, a long-pass filter film is provided on the end face of the second refractive index gradient lens 31 close to the detection optical fiber 32. The long-pass filter film is used to transmit light longer than the cut-off wavelength to remove the noise light rays of the incident excitation light and improve the signal-to-noise ratio.

[0051] In a specific embodiment, the fluorescence excitation unit 10 further includes a reflector 13, and a band-pass filter film is provided on the exit surface of the reflector 13. The reflector 13 is disposed at one end of the first refractive index gradient lens 12 far from the conduction optical fiber 11. The reflector 13 is used to bend the excitation light beam emitted from the first refractive index gradient lens 12 so that the optical path direction of the excitation light beam is along the flowing direction of the liquid to be measured. The band-pass filter film is used to filter the stray light in the excitation light beam, and the stray light is mainly generated by the components through which the laser passes.

[0052] In a specific embodiment, the conduction optical fiber 11 is a single-mode optical fiber, which mainly transmits an excitation light beam of a single wavelength.

[0053] In a specific embodiment, the detection optical fiber 32 is a multi-mode optical fiber. The fluorescence photons generated by the excitation light beam exciting the fluorescent marker are multi-wavelength fluorescence photons. The multi-mode optical fiber can be used to collect the fluorescence photons within a certain wavelength range. The fiber aperture of the detection optical fiber 32 can be determined according to the size of the confocal field of view.

[0054] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A single-molecule fluorescence detection system, characterized in that, It includes a fluorescence excitation unit, a unit of the liquid to be measured, a fluorescence collection unit and a signal processing unit; The fluorescence excitation unit includes a laser, a conductive optical fiber and a first gradient refractive index lens. The laser beam generated by the laser is conducted to the first gradient refractive index lens through the conductive optical fiber, and after being focused by the first gradient refractive index lens, an excitation beam for exciting the liquid to be measured to generate fluorescence is obtained; The unit of the liquid to be measured includes a microchannel that allows single molecules to pass through. The liquid to be measured passes through the microchannel, and the excitation beam irradiates the microchannel to excite the single molecule fluorescent marker located in the microchannel to emit fluorescent photons; The fluorescence collection unit includes a second gradient refractive index lens and a detection optical fiber. The second gradient refractive index lens is arranged on the optical path other than the excitation beam to collect the fluorescent photons in dark field. The fluorescent photons are focused by the second gradient refractive index lens and then conducted to the detection optical fiber; The detection optical fiber is connected to the signal processing unit, and the signal processing unit processes the fluorescent photons passing through the detection optical fiber to obtain information of the fluorescent marker.

2. The single-molecule fluorescence detection system according to claim 1, wherein, The signal processing unit includes a single photon detector, a single photon counter and a data processing module. The single photon detector is connected to the detection optical fiber. The single photon detector converts a single fluorescent photon passing through the detection optical fiber into a TTL electrical signal and sends it to the single photon counter. The single photon counter records the arrival time of the single fluorescent photon. The data processing module processes the TTL electrical signal and the arrival time of the single fluorescent photon into information about the change of light intensity over time, and calculates the information of the fluorescent marker passing through the microchannel according to the information about the change of light intensity over time.

3. The single-molecule fluorescence detection system according to claim 2, wherein The single photon detector is a photon counting PMT, a silicon photomultiplier, a single photon counting APD or a single photon counting MPPC; The single photon counter is a high-speed FPGA real-time data transmission program module or a time-to-digital conversion card.

4. The single-molecule fluorescence detection system according to claim 1, characterized in that, The microchannel is a capillary tube.

5. The single-molecule fluorescence detection system according to claim 1 or 4, characterized in that, The unit of the liquid to be measured further includes a pump and a waste liquid pool. The pump drives the liquid to be measured into the microchannel and flows into the waste liquid pool through the microchannel.

6. The single-molecule fluorescence detection system according to claim 1, wherein The unit of the liquid to be measured includes a transparent substrate, and the microchannel is arranged in the transparent substrate.

7. The single-molecule fluorescence detection system according to claim 1, wherein A long-pass filter film is arranged on the end face of the second gradient refractive index lens close to the detection optical fiber.

8. The single-molecule fluorescence detection system according to claim 7, wherein The fluorescence excitation unit further includes a reflector, and a band-pass filter film is arranged on the exit surface of the reflector. The reflector is arranged at one end of the first gradient refractive index lens far from the conductive optical fiber. The reflector is used to bend the excitation beam emitted from the first gradient refractive index lens, and the band-pass filter film is used to filter the stray light in the excitation beam.

9. The single-molecule fluorescence detection system according to claim 1, wherein The conductive optical fiber is a single-mode optical fiber.

10. The single-molecule fluorescence detection system according to claim 1, wherein The detection optical fiber is a multi-mode optical fiber.

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