A single-molecule fluorescence detection system and a method for preparing tapered capillary microtubes

Abstract

The present application proposes a single-molecule fluorescence detection system, including a laser light source, a tapered capillary, a fluorescence collection device and a data processing device; the tapered capillary includes a first cone and a second cone, the first cone and the second cone are connected, and the connection between the first cone and the second cone is the thinnest part of the tapered capillary; the output end of the laser light source and the input end of the fluorescence collection device are directly opposite to the thinnest part of the tapered capillary; the excitation light emitted by the laser light source is irradiated to the thinnest part of the tapered capillary, and the fluorescent marker is excited by the excitation light and emits fluorescence; the fluorescence collection device collects the fluorescence and transmits the collected fluorescence to the data processing device; the data processing device converts the fluorescence input by the fluorescence collection device into an electrical signal to determine the number of fluorescent markers passing through the thinnest part of the tapered capillary per unit time, and determines the antigen concentration of the target solution based on the number of fluorescent markers.

Description

Technical Field

[0001] The present application relates to the field of biological detection, and in particular to a single-molecule fluorescence detection system and a method for preparing a tapered capillary microtube. Background Art

[0002] Single-molecule fluorescence detection technology has ultra-high sensitivity that surpasses chemiluminescence immunoassays and is widely used in the field of biological detection. Currently, there are two mainstream single-molecule fluorescence detection technologies. One is to use semiconductor technology to make a chip with a microwell array, label the antigen and divide it in the microwells, and use an imaging system to observe the luminescence of each microwell to obtain the analyte concentration. However, this method has high process requirements for the microwell array and is difficult to reuse, which greatly increases the measurement cost. The other method uses the principle of confocal microscopy to excite and observe the fluorescence intensity within the optical focus range to achieve the purpose of cutting the sample volume. However, the confocal microscope system in this method limits the fluorescence excitation and collection area. The optical system is expensive and difficult to maintain, which also increases the detection cost. Summary of the Invention

[0003] The present application provides a single-molecule fluorescence detection system and a method for preparing a tapered capillary microtube to solve the technical problems of the existing fluorescence detection technology in that the detection device is difficult to reuse and is expensive.

[0004] In a first aspect, a single-molecule fluorescence detection system is provided, the system comprising a laser light source, a tapered capillary microtube, a fluorescence collection device, and a data processing device;

[0005] The tapered capillary comprises a first tapered portion and a second tapered portion, the first tapered portion being connected to the second tapered portion, and the connection between the first tapered portion and the second tapered portion being the thinnest point of the tapered capillary; the output end of the laser light source and the input end of the fluorescence collection device are aligned with the thinnest point of the tapered capillary; the data processing device is connected to the fluorescence collection device;

[0006] The tapered capillary is used to transfer a target solution containing a fluorescently labeled antigen. The excitation light emitted by the laser light source is irradiated onto the thinnest part of the tapered capillary, and the fluorescent marker is excited by the excitation light to emit fluorescence. The fluorescence collection device collects the fluorescence and transmits the collected fluorescence to the data processing device. The data processing device converts the fluorescence input by the fluorescence collection device into an electrical signal to determine the number of fluorescent markers passing through the thinnest part of the tapered capillary per unit time, and determines the antigen concentration of the target solution based on the number of fluorescent markers.

[0007] In combination with the first aspect, in one achievable manner, the tapered capillary tube, the output end of the laser light source, and the input end of the fluorescence collection device are arranged at 90 degrees to each other.

[0008] In combination with the first aspect, in one achievable manner, the inner diameter of the tapered capillary tube at its thinnest point is 1 to 50 micrometers.

[0009] In combination with the first aspect, in one achievable manner, the fluorescence collection device includes a microscope objective lens and a bandpass filter, the microscope objective lens is used to collect fluorescence, and the bandpass filter is used to filter out stray light in the fluorescence collected by the microscope objective lens.

[0010] In combination with the first aspect, in one achievable manner, the fluorescence collection device further includes a coupling device, wherein the coupling device is used to couple the fluorescence passing through the bandpass filter into an optical fiber to transmit the fluorescence to the data processing device.

[0011] In combination with the first aspect, in one feasible manner, the data processing device includes a single-photon detector, a single-photon counter, and a computing unit; the single-photon detector converts the fluorescence signal into an electrical signal and transmits the electrical signal to the single-photon counter; the single-photon counter is used to determine the number of fluorescent markers passing through the thinnest part of the tapered capillary per unit time; and the computing unit is used to calculate the antigen concentration of the target solution.

[0012] In a second aspect, a method for preparing a tapered capillary microtube is provided, the method comprising:

[0013] Determining the inner diameter and outer diameter of the glass capillary microtube;

[0014] Determining the inner diameter and the outer diameter of the tapered region according to the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary; the tapered region includes a first tapered portion and a second tapered portion;

[0015] Based on the inner diameter of the tapered region and the outer diameter of the tapered region, the glass capillary microtube is stretched using a flame heating stretching technique to obtain the tapered capillary microtube.

[0016] In conjunction with the second aspect, in one achievable manner, the inner diameter of the glass capillary tube, the outer diameter of the glass capillary tube, the inner diameter of the tapered region, and the outer diameter of the tapered region satisfy the following relationship:

[0017]

[0018] Wherein, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, r0 is the inner diameter of the thinnest part of the tapered region, and R0 is the outer diameter of the thinnest part of the tapered region.

[0019] In conjunction with the second aspect, in one achievable manner, determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary includes: determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0020]

[0021] Wherein, D(x) is the outer diameter of the conical area, x represents the coordinate of the conical area, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0022] In conjunction with the second aspect, in one achievable manner, determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary includes: determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0023]

[0024] Wherein, d(x) is the inner diameter of the tapered region, D(x) is the outer diameter of the tapered region, x represents the coordinate of the tapered region, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, d s is the inner diameter shrinkage, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0025] This application can achieve the following beneficial effects: It uses tapered capillaries to deliver target solutions, exploiting the rapidly changing inner diameter of tapered capillaries to create microfluidics, effectively creating volumetric partitioning of the measured sample at the narrowest point of the tapered capillary. The tapered capillaries used for sample partitioning in this application are reusable, effectively reducing testing costs. Furthermore, this method eliminates the need for expensive sample partitioning equipment, further reducing testing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of the structure of a tapered capillary microtube used in a single-molecule fluorescence detection system proposed in this application;

[0027] Figure 2 A schematic diagram of target solution transmission in a single-molecule fluorescence detection system proposed in this application;

[0028] Figure 3 This is a schematic diagram of the structure of a single-molecule fluorescence detection system proposed in this application;

[0029] Figure 4 This is a schematic diagram of the structure of a single-molecule fluorescence detection system proposed in this application;

[0030] Figure 5 A schematic flow chart of a method for preparing a tapered capillary microtube provided in an embodiment of the present application;

[0031] Figure 6 A schematic diagram of parameters of a tapered capillary microtube provided in an embodiment of the present application;

[0032] Figure 7 A schematic diagram of parameters of a tapered capillary microtube provided in an embodiment of the present application;

[0033] Figure 8 A schematic structural diagram of a tapered capillary microtube preparation device provided in an embodiment of the present application;

[0034] Figure 9 A schematic diagram of the structure of a computer device provided in an embodiment of the present application. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application.

[0036] The technical solution of the present application can be applied to various scenarios of immunoassays. Specifically, the technical solution of the present application can be applied to calculate antigen concentration through fluorescence detection in immunoassay scenarios. In practical applications, the sample can be segmented using a device with a sample segmentation function, and then fluorescence detection can be performed after stimulating a fluorescent marker with excitation light to determine the antigen concentration in the sample.

[0037] In order to facilitate understanding of the technical solution of this application, the tapered capillary microtube in this application is first introduced. Figure 1 As shown, Figure 1 This is a schematic diagram of the structure of a tapered capillary microtube used in a single-molecule fluorescence detection system proposed in this application. The tapered capillary microtube 20 includes a first tapered portion 201 and a second tapered portion 202. The connection between the first tapered portion 201 and the second tapered portion 202 is the thinnest part of the tapered capillary microtube 20. The antigens in the solution are labeled with a fluorescent marker, and the labeled solution is used as the target solution, and the target solution is transferred to the tapered capillary microtube 20. After the target solution flows into the tapered capillary microtube 20 with a rapidly changing inner diameter, the antigens in the target solution are dispersed in the tapered portion and effectively form a volume segmentation of the measurement sample at the thinnest part of the tapered capillary microtube. After being irradiated by the excitation light irradiated on the thinnest part of the tapered capillary microtube, the antigens pass through the thinnest part of the tapered capillary microtube in a temporally discrete manner.

[0038] The tapered capillary microtube 20 is a fused-cone capillary microtube, which is prepared by melting a glass capillary microtube through flame heating and stretching technology.

[0039] In one embodiment, Figure 2 As shown, Figure 2 This is a schematic diagram of the target solution transmission in a single-molecule fluorescence detection system proposed in this application. During fluorescence detection, a vacuum pump provides negative pressure to the tapered capillary 20, causing the target solution to enter the tapered capillary 20 from one side of the tapered capillary 20, pass through the thinnest part of the tapered capillary 20, and then flow out from the other side of the tapered capillary 20 into the waste liquid pool. The flow rate of the target solution in the tapered capillary 20 is controlled by the vacuum pump. The stronger the negative pressure provided by the vacuum pump, the faster the flow rate of the target solution. The flow rate of the target solution is fastest at the thinnest part of the tapered capillary, ranging from 0.1 mm / s to 10 m / s.

[0040] The tapered capillary microtube 20 in the present application has the characteristic of rapid change in inner diameter, which can effectively form volume segmentation of the sample; at the same time, the overall inner diameter of the tapered capillary microtube 20 is large, which can avoid excessive fluid resistance. Moreover, when the target solution is transmitted, the liquid channel through which the target solution flows is completely closed, which can withstand the high pressure of the fluid and thus achieve a high flow rate.

[0041] In one embodiment, Figure 3 As shown, Figure 3 This is a schematic diagram of the structure of a single-molecule fluorescence detection system proposed in this application. The system includes a laser light source 10, a tapered capillary 20, a fluorescence collection device 30, and a data processing device 40; the tapered capillary 20 includes a first cone 201 and a second cone 202, the first cone 201 and the second cone 202 are connected, and the connection between the first cone 201 and the second cone 202 is the thinnest part of the tapered capillary 20; the output end of the laser light source 10 and the input end of the fluorescence collection device 30 are opposite to the thinnest part of the tapered capillary; the data processing device 40 is connected to the fluorescence collection device 30; the tapered capillary 20 is used to transmit A target solution containing a fluorescently labeled antigen; excitation light emitted by a laser light source 10 is irradiated onto the thinnest portion of the tapered capillary, and the fluorescent marker is excited by the excitation light and emits fluorescence; a fluorescence collection device 30 collects the fluorescence and transmits the collected fluorescence to a data processing device 40; the data processing device 40 converts the fluorescence input by the fluorescence collection device 30 into an electrical signal to determine the number of fluorescent markers passing through the thinnest portion of the tapered capillary per unit time, and determines the antigen concentration of the target solution based on the number of fluorescent markers.

[0042] The laser light source 10 can illuminate the tapered capillary 20 at its narrowest point using either side illumination or spot illumination. Side illumination refers to illuminating the tapered capillary 20 with excitation light from the side, while spot illumination refers to illuminating the tapered capillary 20 with excitation light from above.

[0043] The excitation light emitted by the laser light source 10 can be transmitted to the tapered capillary 20 using an optical fiber, or focused on the tapered capillary 20 using a microscope objective. The laser light source 10 has a laser power of 1 mW to 500 mW, and the excitation light spot diameter is 1 to 50 microns, covering the inner diameter of the tapered capillary. When the fluorescently labeled antigen passes through the narrowest part of the tapered capillary, it is excited by the excitation light and produces fluorescence. This fluorescence is collected by a fluorescence acquisition device 30 and transmitted to a data processing device 40.

[0044] In one embodiment, the tapered capillary microtube 20, the output end of the laser light source 10, and the input end of the laser collection device are arranged at 90 degrees to each other.

[0045] Among them, setting the tapered capillary microtube 20, the output end of the laser light source 10 and the input end of the laser collection device at 90 degrees to each other can effectively prevent the excitation light from being collected by the fluorescence collection device 30, thereby reducing the stray light collected by the fluorescence collection device 30 and improving the accuracy of fluorescence detection.

[0046] In one embodiment, the inner diameter of the tapered capillary tube at its thinnest point is 1 to 50 micrometers.

[0047] In one embodiment, Figure 4 As shown, Figure 4 The structure diagram of a single-molecule fluorescence detection system proposed in this application is shown in FIG. The fluorescence collection device 30 includes a microscope objective 301 and a bandpass filter 302 . The microscope objective 301 is used to collect fluorescence, and the bandpass filter 302 is used to filter out stray light in the fluorescence collected by the microscope objective 301 .

[0048] The fluorescence is collected by the microscope objective lens 301 and filtered by the bandpass filter 302 to remove the scattered excitation light.

[0049] In one embodiment, the fluorescence collection device 30 further includes a coupling device, which is used to couple the fluorescence passing through the bandpass filter into an optical fiber, so as to transmit the fluorescence to the data processing device 40 .

[0050] The coupling device may be a space mirror or other device with a coupling function, through which the filtered fluorescence is coupled into the optical fiber, and the fluorescence is transmitted to the data processing device 40 through the optical fiber.

[0051] In one embodiment, the data processing device 40 includes a single-photon detector 401, a single-photon counter 402, and a calculation unit 403; the single-photon detector 401 converts the fluorescent signal into an electrical signal and transmits the electrical signal to the single-photon counter 402; the single-photon counter 402 is used to determine the number of fluorescent markers passing through the thinnest part of the tapered capillary per unit time; the calculation unit 403 is used to calculate the antigen concentration of the target solution.

[0052] Among them, the single-photon counting function is realized by the single-photon detector 401 and the single-photon counter 402. Since the analyte molecules pass through the tapered capillary microtube 20 discretely in time, their fluorescence signals are displayed as pulsed light intensity envelopes in time. The single-photon detector 401 converts each received photon into a TTL electrical signal and sends it to the time-resolved single-photon counter 402, records the time of arrival of the photon, and then restores it to the change of light intensity over time. The single-photon counter 402 counts the pulse envelopes in the light intensity and determines the number of fluorescent markers (antigens) passing through the thinnest part of the tapered capillary microtube per unit time. The calculation unit 403 calculates the concentration of the target solution. The calculation formula is: c=N / Q, where c is the antigen concentration and N is the number of light intensity envelopes measured per unit time, that is, the number of fluorescent markers. Q is the flow rate, and the flow rate Q can be measured by a flowmeter.

[0053] The single photon detector 401 may be a photon counting PMT, a silicon photomultiplier tube, a single photon counting APD or an MPPC, etc. The single photon counter 402 may be implemented by programming a high-speed FPGA or using a time-to-digital converter card.

[0054] This application uses a tapered capillary tube 20 to deliver the target solution. This rapidly changing inner diameter of the tapered capillary tube 20 can be exploited to create a microfluidic channel, effectively segmenting the sample volume at the narrowest point. The tapered capillary tube 20 used for sample segmentation in this application is reusable, effectively reducing testing costs. Furthermore, this method eliminates the need for expensive sample segmentation equipment, further reducing testing costs.

[0055] In one embodiment, Figure 5 As shown, Figure 5 A schematic flow chart of a method for preparing a tapered capillary microtube provided in an embodiment of the present application. The method comprises:

[0056] Step 501: Determine the inner diameter and outer diameter of a glass capillary tube.

[0057] The present application utilizes flame heating and stretching technology to melt glass capillary tubes, causing them to deform and thereby produce tapered capillary tubes. The inner diameter of the glass capillary tube refers to the inner diameter of the glass capillary tube before deformation, and the outer diameter of the glass capillary tube refers to the outer diameter of the glass capillary tube before deformation. In a tapered capillary tube, the inner diameter at each end of the tapered capillary tube is the inner diameter of the glass capillary tube, and the outer diameter at each end of the tapered capillary tube is the outer diameter of the glass capillary tube.

[0058] Step 502: Determine the inner diameter and outer diameter of the tapered region according to the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary; the tapered region includes a first tapered portion and a second tapered portion.

[0059] Specifically, such as Figure 6 and Figure 7 As shown, Figure 6 、 Figure 7 This is a schematic diagram of parameters of a tapered capillary microtube provided in an embodiment of the present application. The inner diameter of the glass capillary microtube, the outer diameter of the glass capillary microtube, the inner diameter of the tapered region, and the outer diameter of the tapered region satisfy the following relationship:

[0060]

[0061] Wherein, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, r0 is the inner diameter of the thinnest point of the tapered region, i.e., the minimum inner diameter, and R0 is the outer diameter of the thinnest point of the tapered region, i.e., the minimum outer diameter.

[0062] Specifically, determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0063]

[0064] Wherein, D(x) is the outer diameter of the conical area, x represents the coordinate of the conical area, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0065] Wherein, determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0066]

[0067] Wherein, d(x) is the inner diameter of the tapered region, D(x) is the outer diameter of the tapered region, x represents the coordinate of the tapered region, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, and L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .d s is the inner diameter shrinkage.

[0068] Among them, d s It is the amount of inner diameter shrinkage, which is related to factors such as the inner-outer diameter ratio, material, and stretching area.

[0069] Step 503 : Based on the inner diameter and the outer diameter of the tapered region, the glass capillary is stretched using a flame heating stretching technique to obtain the tapered capillary.

[0070] The glass capillary microtube can be stretched according to the inner diameter change formula of the tapered region and the outer diameter change formula of the tapered region, thereby obtaining a tapered capillary microtube.

[0071] The tapered capillary microtubules in the present application have the characteristic of rapid changes in inner diameter, which can effectively form volumetric segmentation of the sample; at the same time, the overall inner diameter of the tapered capillary microtubules is large, which can avoid excessive fluid resistance, and when the target solution is transmitted, the liquid channel through which the target solution flows is completely closed, which can withstand high fluid pressure and thus achieve a high flow rate. The present application uses tapered capillary microtubules to deliver the target solution, and can utilize the rapid change in the inner diameter of the tapered capillary microtubules to prepare microchannels, effectively forming a volumetric segmentation of the measured sample at the thinnest point of the tapered capillary microtubules. The tapered capillary microtubules used for sample segmentation in the present application can be reused, which can effectively reduce the cost of detection; and the present application uses tapered capillary microtubules to perform sample segmentation, which does not require expensive sample segmentation instruments, and can further reduce the cost of detection.

[0072] In one embodiment, Figure 8 As shown, Figure 8 This is a schematic diagram of a device for preparing tapered capillary microtubes provided in an embodiment of the present application. The device comprises:

[0073] A first parameter determination module 801 is configured to determine the inner diameter and the outer diameter of the glass capillary microtube;

[0074] A second parameter determination module 802 is configured to determine the inner diameter and the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary; the tapered region includes a first tapered portion and a second tapered portion;

[0075] The microtube stretching module 803 is configured to stretch the glass capillary microtube using a flame heating stretching technique based on the inner diameter and the outer diameter of the tapered region to obtain the tapered capillary microtube.

[0076] like Figure 9 As shown in FIG. 1 , in one embodiment, it is an internal structure diagram of a computer device. The computer device may be a tapered capillary microtube preparation device, or a terminal or server connected to a tapered capillary microtube preparation device. Figure 9As shown, the computer device includes a processor, a memory, and a network interface connected via a system bus. The memory includes a non-volatile storage medium and an internal memory. The non-volatile storage medium of the computer device stores an operating system and may also store a computer program. When the computer program is executed by the processor, the processor may implement a method for preparing a tapered capillary microtube. The internal memory may also store a computer program. When the computer program is executed by the processor, the processor may implement a method for preparing a tapered capillary microtube. The network interface is used to communicate with an external device. Those skilled in the art will understand that Figure 9 The structure shown in the figure is only a block diagram of a part of the structure related to the solution of the present application, and does not constitute a limitation on the computer device to which the solution of the present application is applied. The specific computer device may include more or fewer components than shown in the figure, or combine certain components, or have a different component arrangement.

[0077] In one embodiment, the method for preparing a tapered capillary microtube provided by the present application can be implemented in the form of a computer program. The computer program can be used in Figure 9 The computer device is shown in FIG. A memory of the computer device may store various program templates constituting the age prediction device, such as a first parameter determination module 801, a second parameter determination module 802, and a microtubule stretching module 803.

[0078] A computer device includes a memory and a processor. The memory stores a computer program. When the computer program is executed by the processor, the processor performs the following steps: determining the inner diameter and the outer diameter of a glass capillary microtube; determining the inner diameter and the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary microtube, the inner diameter of the glass capillary microtube, and the outer diameter of the glass capillary microtube; the tapered region includes a first tapered portion and a second tapered portion; and based on the inner diameter and the outer diameter of the tapered region, using a flame heating stretching technique to stretch the glass capillary microtube to obtain the tapered capillary microtube.

[0079] In one embodiment, the inner diameter of the glass capillary microtube, the outer diameter of the glass capillary microtube, the inner diameter of the tapered region, and the outer diameter of the tapered region satisfy the following relationship:

[0080]

[0081] Wherein, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, r0 is the inner diameter of the thinnest part of the tapered region, and R0 is the outer diameter of the thinnest part of the tapered region.

[0082] In one embodiment, determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0083]

[0084] Wherein, D(x) is the outer diameter of the conical area, x represents the coordinate of the conical area, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0085] In one embodiment, determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0086]

[0087] Wherein, d(x) is the inner diameter of the tapered region, D(x) is the outer diameter of the tapered region, x represents the coordinate of the tapered region, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, d s is the inner diameter shrinkage, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0088] In one embodiment, when the computer program is executed by the processor, the processor further performs the following steps: determining the inner diameter of the glass capillary microtube and the outer diameter of the glass capillary microtube; determining the inner diameter of the tapered region and the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary microtube, the inner diameter of the glass capillary microtube and the outer diameter of the glass capillary microtube; the tapered region includes a first tapered portion and a second tapered portion; based on the inner diameter of the tapered region and the outer diameter of the tapered region, the glass capillary microtube is stretched using a flame heating stretching technique to obtain the tapered capillary microtube.

[0089] In one embodiment, the inner diameter of the glass capillary microtube, the outer diameter of the glass capillary microtube, the inner diameter of the tapered region, and the outer diameter of the tapered region satisfy the following relationship:

[0090]

[0091] Wherein, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, r0 is the inner diameter of the thinnest part of the tapered region, and R0 is the outer diameter of the thinnest part of the tapered region.

[0092] In one embodiment, determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: determining the outer diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0093]

[0094] Wherein, D(x) is the outer diameter of the conical area, x represents the coordinate of the conical area, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0095] In one embodiment, determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: determining the inner diameter of the tapered region based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula:

[0096]

[0097] Wherein, d(x) is the inner diameter of the tapered region, D(x) is the outer diameter of the tapered region, x represents the coordinate of the tapered region, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, d s is the inner diameter shrinkage, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

[0098] Those skilled in the art will appreciate that all or part of the processes in the above-described method embodiments can be implemented by instructing related hardware through a computer program. The program can be stored in a computer-readable storage medium, and when executed, the program can include the processes in the above-described method embodiments. The storage medium can be a magnetic disk, an optical disk, a read-only memory (ROM), or a random access memory (RAM).

[0099] The above disclosure is only a preferred embodiment of the present application, and certainly cannot be used to limit the scope of rights of the present application. Therefore, equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A method for preparing a tapered capillary microtube, characterized in that: The method comprises: Determining the inner diameter and outer diameter of the glass capillary microtube; Determining the inner diameter and the outer diameter of the tapered region according to the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary; the tapered region includes a first tapered portion and a second tapered portion; Based on the inner diameter of the tapered region and the outer diameter of the tapered region, the glass capillary microtube is stretched using a flame heating stretching technique to obtain the tapered capillary microtube; The step of determining the outer diameter of the tapered region according to the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, and the outer diameter of the glass capillary comprises: The outer diameter of the tapered region is determined based on the length of the tapered region of the tapered capillary microtubule, the inner diameter of the glass capillary microtubule, the outer diameter of the glass capillary microtubule, and the following formula: Wherein, D(x) is the outer diameter of the tapered region, R is the outer diameter of the glass capillary microtube, x represents the coordinate of the tapered region, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

2. The method according to claim 1, characterized in that The inner diameter of the glass capillary microtube, the outer diameter of the glass capillary microtube, the inner diameter of the tapered region, and the outer diameter of the tapered region satisfy the following relationship: Wherein, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, r0 is the inner diameter of the thinnest part of the tapered region, and R0 is the outer diameter of the thinnest part of the tapered region.

3. The method according to claim 1, characterized in that Determining the inner diameter of the tapered region according to the length of the tapered region of the tapered capillary microtube, the inner diameter of the glass capillary microtube, and the outer diameter of the glass capillary microtube comprises: The inner diameter of the tapered region is determined based on the length of the tapered region of the tapered capillary, the inner diameter of the glass capillary, the outer diameter of the glass capillary, and the following formula: Wherein, d(x) is the inner diameter of the tapered region, D(x) is the outer diameter of the tapered region, x represents the coordinate of the tapered region, r is the inner diameter of the glass capillary microtube, R is the outer diameter of the glass capillary microtube, d s is the inner diameter shrinkage, L s is the length of the stretching zone, L f0 is the length of the initial melting zone, L t is the length of the tapered region, d(L s ) is the collapse factor, satisfying: L t =L s +L f0 .

4. A single molecule fluorescence detection system, characterized in that: The system comprises a laser light source, a tapered capillary microtube prepared by the method of any one of claims 1 to 3, a fluorescence collection device, and a data processing device; The tapered capillary comprises a first tapered portion and a second tapered portion, the first tapered portion being connected to the second tapered portion, and the connection between the first tapered portion and the second tapered portion being the thinnest point of the tapered capillary; the output end of the laser light source and the input end of the fluorescence collection device are aligned with the thinnest point of the tapered capillary; the data processing device is connected to the fluorescence collection device; The tapered capillary microtube is used to transfer a target solution containing an antigen labeled with a fluorescent marker; the excitation light emitted by the laser light source is irradiated onto the thinnest part of the tapered capillary microtube, and the fluorescent marker is excited by the excitation light and emits fluorescence; The fluorescence collecting device collects the fluorescence and transmits the collected fluorescence to the data processing device; The data processing device converts the fluorescence input by the fluorescence collection device into an electrical signal to determine the number of fluorescent markers passing through the thinnest part of the tapered capillary per unit time, and determines the antigen concentration of the target solution according to the number of fluorescent markers.

5. The system according to claim 4, characterized in that The tapered capillary microtube, the output end of the laser light source, and the input end of the fluorescence collection device are arranged at 90 degrees to each other.

6. The system according to claim 4, characterized in that The inner diameter of the tapered capillary at the thinnest point is 1 to 50 microns.

7. The system according to claim 4, wherein: The fluorescence collection device includes a microscope objective lens and a bandpass filter. The microscope objective lens is used to collect fluorescence, and the bandpass filter is used to filter out stray light in the fluorescence collected by the microscope objective lens.

8. The system according to claim 7, characterized in that The fluorescence collection device further comprises a coupling device, which is used to couple the fluorescence passing through the bandpass filter into an optical fiber, so as to transmit the fluorescence to the data processing device.

9. The system according to claim 4, wherein: The data processing device includes a single photon detector, a single photon counter and a calculation unit; The single-photon detector converts the fluorescence signal into an electrical signal, and transmits the electrical signal to the single-photon counter; The single photon counter is used to determine the number of fluorescent markers passing through the thinnest part of the tapered capillary per unit time; The calculation unit is used to calculate the antigen concentration of the target solution.

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