Optical system for single molecule detection immunoassay

Abstract

The application discloses a single molecule detection immune analyzer optical system, and relates to the field of single molecule detection immune analyzer optical systems. In the case that the number of main and expensive materials such as lasers and single photon detectors is not increased, a laser beam is divided into three beams with equal intensity through two beam splitters and one attenuator, and the three beams irradiate the object to be detected from three directions, and the fluorescence is collected simultaneously. The optical system comprises a laser light path for generating laser, a forward scattering light detection channel for collecting forward scattering light and a fluorescence detection channel for collecting fluorescence; the laser light path comprises a laser, a beam splitter A, a beam splitter B and an attenuator; the ends of the three fluorescence detection channels are connected to the same optical fiber to output the fluorescence signal outward; the forward scattering light detection channel is symmetrically arranged with one of the laser beam A, the laser beam B or the laser beam C along the flow cell. The effect of improving the positive magnetic bead recognition rate is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical systems of single molecule detection immunoassay. BACKGROUND

[0002] Single molecule detection, which has a detection sensitivity of fg level, is 1000 times that of traditional ELISA.

[0003] The biological principle of detection is the classic immune response-double antibody sandwich method. More than 10*5 capture antibodies are coated on the magnetic beads to capture the antigens in the sample to be tested, and then form a double antibody sandwich structure with the added fluorescent dye labeled detection antibody, i.e. the binding phase. Because the magnetic beads have magnetism, the impurities in the supernatant can be easily removed by magnetic separation.

[0004] One of the commonly used detection methods is to use flow cytometry sheath flow focusing method to detect the forward scattering light signal of the magnetic beads and the fluorescence signal of the fluorescent dye labeled on the antibody excited by the laser. The forward scattering light signal of the magnetic beads is used for counting, and the fluorescence signal is used to determine whether the magnetic beads form a double antibody sandwich binding phase. The double antibody sandwich binding phase is positive magnetic beads, otherwise it is negative magnetic beads.

[0005] When the concentration of antigens in the sample to be tested is at the fg level, only no more than 5% of the magnetic beads can capture antigens to form a double antibody sandwich binding phase. The concentration of antigen protein corresponding to the positive magnetic beads is calculated by using the Poisson distribution theory, realizing the digital fg level ultra-high sensitivity detection.

[0006] When the concentration of antigens in the sample to be tested is high, most of the magnetic beads can capture antigens to form a double antibody sandwich binding phase. At this time, the intensity of the fluorescence signal is positively correlated with the concentration of the sample to be tested, so that a standard curve can be established. By detecting a certain number of magnetic beads, the concentration of the antigen to be tested can be quantitatively measured.

[0007] Using a specially developed magnetic bead reagent system, detection can be performed on existing flow cytometers or flow fluorescence analyzers, and the detection sensitivity is expected to reach the fg level. For example, Connie Wu, Tyler J. Dougan, and David R. Walt et al. published an article entitled "High-Throughput, High-Multiplex Digital Protein Detection with Attomolar Sensitivity", which was detected on a CytoFlex LX flow cytometer produced by Beckman Coulter Company. The sensitivity reached the fg level, realizing single molecule level detection sensitivity.

[0008] But the optical system of flow cytometer or flow fluorescence analyzer, the direction of laser is perpendicular to the direction of fluorescence collection, almost all the flow cytometer optical systems are designed in this way. The reason is that flow cytometer or flow fluorescence analyzer generally needs multiple fluorescence channels, the numerical aperture (NA) of objective lens also requires to be relatively large, and the numerical aperture of 1.2 is a relatively ideal value. The measured object is mainly cell, and the number of cell surface antigens is large, and most of the cells also have certain light transmittance, so the perpendicularity of the laser direction and the fluorescence collection direction of the flow cytometer is beneficial to the optical system design, and if it is other ways such as parallel, the difficulty will be very large, and the difficulty of multi-laser flow cytometer will be increased by one level.

[0009] Such optical system has the following disadvantages when detecting magnetic beads: the magnetic beads used at present are basically not transparent, and at low concentration, each magnetic bead will form a double-antibody sandwich binding phase with a high probability, and two double-antibody sandwich binding phases can be formed with a very low probability. The probability that the fluorescent dye labeled on the antibody is irradiated by the laser is 50%, and the probability that the generated fluorescence is collected by the objective lens is also 50%, so the comprehensive probability of being judged as a positive magnetic bead (both irradiated by the laser and collected by the objective lens) is only 25%. Other cases cannot be guaranteed to be identified as positive magnetic beads.

[0010] At high concentration (1-2 orders of magnitude higher than fg level), each magnetic bead will form at least one double-antibody sandwich binding phase with a high probability, and multiple double-antibody sandwich binding phases may exist, but the number will not be too large. At this time, the basis for establishing the standard curve is that the fluorescence signal intensity is positively correlated with the concentration of the measured object, and the more the double-antibody sandwich binding phases on the surface of the magnetic bead, the greater the fluorescence intensity should be. However, the optical system of the traditional flow cytometer cannot guarantee the uniformity of the laser and the fluorescence intensity. The more the number of double-antibody sandwich binding phases on the surface of the magnetic bead, the better the uniformity.

[0011] Therefore, how to optimize the existing equipment to realize more comprehensive and accurate detection has become a technical problem to be solved by those skilled in the art. SUMMARY

[0012] The present application aims at the above problems, and provides a single molecule detection immune analyzer optical system, which does not increase the number of main and expensive materials such as lasers and single photon detectors (the number is 1), and divides the laser beam into three beams with equal intensity through two beam splitters and one attenuation sheet, and irradiates the measured object (i.e. magnetic beads) from three directions, and collects fluorescence at the same time.

[0013] The technical scheme of the present application is: the optical system comprises a laser light path for generating laser, a forward scattering light detection channel for collecting forward scattering light, and a fluorescence detection channel for collecting fluorescence;

[0014] The laser light path comprises a laser 100, a beam splitter A 211, a beam splitter B 212, and an attenuating sheet 220, the beam splitter A 211 and the beam splitter B 212 are fixedly installed in sequence on one side of the laser 100, the laser emitted by the laser 100 is first split into two by the beam splitter A 211, and then the laser passing through the beam splitter A 211 is again split into two by the beam splitter B 212, so that the laser emitted by the laser 100 forms a laser beam A and a laser beam B after passing through the beam splitter A 211 and the beam splitter B 212, and the laser split by the beam splitter A 211 forms a laser beam C, and the attenuating sheet 220 is fixedly arranged on the path of the laser beam C;

[0015] A dichroic filter A 231 for transmitting laser is fixedly arranged on the path of the laser beam A, a laser reflecting mirror B 202 and a dichroic filter B 232 for reflecting laser are fixedly arranged on the path of the laser beam B, and a laser reflecting mirror C 203 and a dichroic filter C 233 for reflecting laser are fixedly arranged on the path of the laser beam C, so that the laser beam A, the laser beam B, and the laser beam C can irradiate the flow cell 300 from three different directions;

[0016] The dichroic filter A 231 reflects fluorescence while transmitting laser, the dichroic filter B 232 and the dichroic filter C 233 transmit fluorescence while reflecting laser, the fluorescence detection channel has three, three fluorescence detection channels respectively receive fluorescence reflected by the dichroic filter A 231 or transmitted by the dichroic filter B 232 and the dichroic filter C 233, and the ends of the three fluorescence detection channels are connected to the same optical fiber to output the fluorescence signal outward;

[0017] The forward scattering light detection channel is symmetrically arranged with one of the laser beam A, the laser beam B, or the laser beam C along the flow cell 300.

[0018] Among them, between the dichroic filter A 231 and the flow cell 300, there is an achromatic lens A 241 for focusing laser beams and collecting fluorescence;

[0019] Between the dichroic filter B 232 and the flow cell 300, there is an achromatic lens B 242 for focusing laser beams and collecting fluorescence;

[0020] Between the dichroic filter C 233 and the flow cell 300, there is an achromatic lens C 243 for focusing laser beams and collecting fluorescence.

[0021] Further, the forward scattering light detection channel and the laser beam A focused by the achromatic lens A 241 are symmetrically arranged along the flow cell 300;

[0022] The laser beam B and the laser beam C irradiate the flow cell 300 from two different directions after reflection, and the achromatic lens B 242 and the achromatic lens C 243 have an included angle, so that the central beams after passing through the achromatic lens B 242 and the achromatic lens C 243 have an included angle, so that the reverse light path of the laser beam C cannot finally reach the inside of the laser, and at the same time, the reverse light path of the laser beam B after passing through the flow cell 300 cannot finally reach the inside of the laser.

[0023] Further, the beam waists of the laser beam A, the laser beam B and the laser beam C after focusing coincide or have a spacing between the three beam waists.

[0024] Regarding the fluorescence detection channel;

[0025] The three fluorescence detection channels are a fluorescence detection channel A arranged on one side of the dichroic filter A 231, a fluorescence detection channel B arranged on one side of the dichroic filter B 232, and a fluorescence detection channel C arranged on one side of the dichroic filter C 233.

[0026] The fluorescence detection channel A includes, in sequence from the side where the dichroic filter A 231 is located, an on-axis bandpass filter A 521, a fiber coupling lens A 531 and a fiber A 541, so that the fluorescence generated by the flow cell 300 is reflected when it irradiates the dichroic filter A 231, then transmitted through the bandpass filter A 521, and focused by the fiber coupling lens A 531, and finally coupled into the fiber A 541;

[0027] The fluorescence detection channel B includes, in sequence from the side where the dichroic filter B 232 is located, an on-axis bandpass filter B 522, a fiber coupling lens B 532 and a fiber B 542, so that the fluorescence generated by the flow cell 300 is directly transmitted when it irradiates the dichroic filter B 232, then transmitted through the bandpass filter B 522, and focused by the fiber coupling lens B 532, and finally coupled into the fiber B 542;

[0028] The fluorescence detection channel C includes, in sequence from the side where the dichroic filter C 233 is located, an on-axis bandpass filter C 523, a fiber coupling lens C 533 and a fiber C 543, so that the fluorescence generated by the flow cell 300 is directly transmitted when it irradiates the dichroic filter C 233, then transmitted through the bandpass filter C 523, and focused by the fiber coupling lens C 533, and finally coupled into the fiber C 543;

[0029] The optical fiber A 541, the optical fiber B 542, the optical fiber C 543 and another optical fiber D are connected together by high-temperature fusion, and the optical fiber D is connected to the optical fiber interface of the single photon counter.

[0030] Regarding the forward scattering light detection channel;

[0031] The forward scattering light detection channel comprises, in sequence from the side where the flow cell 300 is located, a baffle 410, a convex lens A 420, a convex lens B 430 and a detector 440 arranged on the same optical axis, the convex lens A 420 and the convex lens B 430 are oppositely arranged, the forward scattering light signal emitted by the flow cell 300 is collected through the convex lens A 420 and the light beam is collimated, and the forward scattering light collimated by the convex lens A 420 is converged onto the detector 440 through the convex lens B 430.

[0032] As shown in Figures 1-3 The laser beam emitted by the laser 100 is reflected when passing through the laser reflector A 201, and the direction is turned by 90 degrees. The laser beam passes through the beam splitter A 211 and the beam splitter B 212 in sequence, and the laser beam is divided into three beams.

[0033] The splitting ratio of the beam splitter A 211 and the beam splitter B 212 at the laser wavelength is 50:50, and the attenuation of the attenuation sheet 220 at the laser wavelength is 50%, so that the energy of the three laser beams is basically the same.

[0034] The laser beam that passes through the beam splitter A 211 and the beam splitter B 212 is defined as laser beam A.

[0035] The laser beam that passes through the beam splitter A 211 but is reflected by the beam splitter B 212 is defined as laser beam B.

[0036] The laser beam that is reflected by the beam splitter A 211, reflected by the laser reflector C 203 and attenuated by the attenuation sheet 220 is defined as laser beam C.

[0037] The dichroic filter A 231 is a short-wave pass filter, and the starting response wavelength is between the laser wavelength and the fluorescent wavelength. When the laser beam A passes through the dichroic filter A 231, it will be transmitted, focused by the achromatic lens A 241, and the waist position of the beam coincides with the focal position of the achromatic lens A 241, both of which are located in the flow cell, i.e. the internal flow center position of the observation chamber 300.

[0038] The dichroic filter B 232 is a long-wave pass filter, and the starting response wavelength is between the laser wavelength and the fluorescent wavelength. The laser beam B is first reflected by the laser reflector B 202, then reflected by the dichroic filter B 232, and focused by the achromatic lens B 242, and the waist position of the beam coincides with the focal position of the achromatic lens B 242, both of which are located in the flow cell, i.e. the internal flow center position of the observation chamber 300.

[0039] Dichroic filter C233 is a long-pass filter, the starting response wavelength is between the laser wavelength and the fluorescence wavelength. Laser beam C is reflected by dichroic filter C233 and focused by achromatic lens C243. The waist position of the focused laser beam coincides with the focal point of achromatic lens C243, and both are located in the flow cell, i.e. the inner flow center of observation chamber 300.

[0040] Dichroic filter A231 can also be a long-pass filter, and dichroic filter B232 and dichroic filter C233 can also be short-pass filters.

[0041] Achromatic lens A241, achromatic lens B242 and achromatic lens C243 are used for focusing laser beams and collecting fluorescence signals excited by laser beams. They are made of crown glass and flint glass, and have a focal length of 10-100 mm and a numerical aperture NA of 0.05-0.65. Achromatic lenses can also be replaced by spherical single lenses or aspherical lenses.

[0042] Flow cell, i.e. observation chamber 300, has a square or circular outer shape and a square or circular inner channel shape. The measured object, i.e. magnetic beads 310, passes through the inner channel and generates a forward scattering light signal when passing through the laser beam. The fluorescent substance on the magnetic beads is also excited by the laser to generate fluorescence.

[0043] The forward scattering light signal is used for counting the magnetic beads.

[0044] Convex lens A420 is used to collect the forward scattering light signal generated by laser beam A and collimate the light beam.

[0045] Barrier 410 is located between convex lens A420 and flow cell 300, and is used to block the laser background signal that is not scattered by the magnetic beads.

[0046] Convex lens B430 is used to converge the forward scattering light collimated by convex lens A420 onto detector 440.

[0047] Detector 440 converts the forward scattering light signal into an electrical signal and outputs it to the electronic system.

[0048] Laser beams A, B and C have waist positions after being focused by achromatic lenses, which are coincident in the vertical direction Figure 1 , and can be coincident or have a certain interval in the direction perpendicular to the paper. The interval distance is generally between several tens of microns and several hundred microns. The specific interval distance should ensure that the fluorescence excited by laser beam A ultimately does not reach optical fiber B and optical fiber C, the fluorescence excited by laser beam B ultimately does not reach optical fiber A and optical fiber C, and the fluorescence excited by laser beam C ultimately does not reach optical fiber A and optical fiber B.

[0049] The beam waist positions of the three laser beams can be coincident, which has the advantage of 100% identification of positive magnetic beads, regardless of whether the magnetic beads rotate or not. Moreover, the signals are synchronized without delay. However, the disadvantage is that the magnetic beads are simultaneously irradiated by the three laser beams, the fluorescence background of the magnetic beads is amplified, and the signal-to-noise ratio of the system is reduced.

[0050] The beam waist positions of the three laser beams can also be spaced in the vertical direction, which has the advantage that the fluorescence background of the magnetic beads is not amplified. However, the disadvantage is that three fluorescence signals are generated, and the signals are not synchronized and have a certain delay, which requires an additional step of signal delay calibration. In this case, if the magnetic beads do not rotate, 100% of the positive magnetic beads can be identified. If the magnetic beads do rotate, it is possible that the fluorescent substances on the positive magnetic beads are all on the back side of the laser irradiation when the magnetic beads pass through the beam waist positions of the three laser beams. The probability of this occurrence is theoretically not more than 12.5%, and the detection rate of positive magnetic beads is still more than 87.5%, which is still a great improvement compared to the prior art.

[0051] The central beam after the transmission of the achromatic lens B 242 by the laser beam B is not parallel to the central beam after the transmission of the achromatic lens C 243 by the laser beam C, and there is a small angle between them. This small angle can be achieved in two ways. The first way is that the achromatic lens B 242 and the achromatic lens C 243 have a small angle of inclination relative to the flow cell 300. The second way is that the laser beam has a certain inclination when it enters the achromatic lens B 242 and the achromatic lens C 243. The size of the angle is set such that the laser beam B, after passing through the flow cell 300, cannot finally reach the inside of the laser through the reverse light path of the laser beam C, and the laser beam C, after passing through the flow cell 300, cannot finally reach the inside of the laser through the reverse light path of the laser beam B.

[0052] If the laser beam emitted by the laser is finally transmitted back into the inside of the laser, it may cause instability or other abnormalities of the laser power. Although it is not necessarily abnormal, it should be avoided as much as possible.

[0053] When the magnetic beads 310, which are the objects to be measured, pass through the beam waist position of the laser beam A, the fluorescent substances marked on them are excited by the laser, and the generated fluorescence is emitted in a 360-degree three-dimensional space. Part of the fluorescence is collected and collimated by the achromatic lens A 241, reflected by the dichroic filter A 231, reflected again by the fluorescence mirror A 511, transmitted through the band-pass filter A 521, focused by the fiber coupling lens A 531, and finally coupled into the optical fiber A 541.

[0054] The fluorescence mirror A 511 can not be provided, and the length and width dimensions of the entire optical system may be increased.

[0055] When the to-be-tested substance, i.e. the magnetic beads 310, passes through the waist position of the laser beam B, the fluorescent substance marked thereon is excited again to generate fluorescence by the laser, part of the fluorescence is collected and collimated by the achromatic lens B242, and when the fluorescence is transmitted to the dichroic filter B232, it is transmitted, reflected by the fluorescence mirror B512, transmitted through the band-pass filter B522, focused by the optical fiber coupling lens B532, and finally coupled into the optical fiber B542.

[0056] The fluorescence mirror B512 can not be arranged, and the length and width dimensions of the entire optical system can be increased.

[0057] When the to-be-tested substance, i.e. the magnetic beads 310, passes through the waist position of the laser beam C, the fluorescent substance marked thereon is still excited to generate fluorescence by the laser, part of the fluorescence is collected and collimated by the achromatic lens C243, and when the fluorescence is transmitted to the dichroic filter C233, it is transmitted, reflected by the fluorescence mirror C513, transmitted through the band-pass filter C523, focused by the optical fiber coupling lens C533, and finally coupled into the optical fiber C543.

[0058] The fluorescence mirror C513 can not be arranged, and the length and width dimensions of the entire optical system can be increased.

[0059] The sequence in which the to-be-tested substance, i.e. the magnetic beads 310, passes through the waist position of the laser beam can be arbitrary, and can be any one of A, B, C, A, C, B, B, A, C, B, C, A, C, B, C, A, B, C.

[0060] The optical fiber A541, the optical fiber B542, the optical fiber C543 and another optical fiber D are connected together by high-temperature fusion, realizing the same-wavelength beam combination, and by adjusting the numerical aperture and core diameter of the optical fiber, although there is a certain loss in transmission efficiency, more than 90% of transmission efficiency can still be guaranteed.

[0061] The optical fiber A541, the optical fiber B542 and the optical fiber C543 are input ends, and are used for receiving fluorescence.

[0062] The output end is the optical fiber D, and the optical fiber D is connected to the optical fiber interface of the single-photon counter.

[0063] In the application, there is only one laser and single-photon detector, and under the premise that the main and expensive materials are not increased in quantity, as long as any one laser can irradiate the fluorescent substance on the magnetic beads and collect the generated fluorescence, it can be determined as a positive magnetic bead, in this way, the recognition rate of the positive magnetic bead is greatly improved, the purpose of reducing the coefficient of variation (CV) of the final test result is achieved, and the effect of improving the recognition rate of the positive magnetic bead is realized.

[0064] The application is not only to improve the accuracy of test results by simply increasing the number of fluorescence detection channels, but more importantly, the application also increases the number of laser beams irradiated on the flow cell by optimizing the structure, so that the flow cell can receive laser irradiation from a wider angle, thereby improving the probability of the fluorescent substance on the positive magnetic beads being irradiated by the laser when detecting the positive magnetic beads, and the generated fluorescence can also be collected synchronously. Moreover, the application optimizes the structure so that the laser beam energy irradiated on the flow cell is basically the same, and the reverse light path of the multi-channel laser beam does not return to the laser, thereby achieving the purpose of greatly improving the positive magnetic bead recognition rate with one laser and one single photon detector. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 is a structural schematic diagram of the case,

[0066] Figure 2 is a laser light path diagram of the case,

[0067] Figure 3 is a fluorescence light path diagram of the case;

[0068] 100, laser in the figure;

[0069] 201, laser mirror A; 202, laser mirror B; 203, laser mirror C;

[0070] 211, beam splitter A; 212, beam splitter B;

[0071] 220, attenuator;

[0072] 231, dichroic filter A; 232, dichroic filter B; 233, dichroic filter C;

[0073] 241, achromatic lens A; 242, achromatic lens B; 243, achromatic lens C;

[0074] 300, flow cell, i.e. observation chamber;

[0075] 310, to-be-detected substance, i.e. magnetic beads;

[0076] 410, baffle;

[0077] 420, convex lens A; 430, convex lens B;

[0078] 440, detector;

[0079] 511, fluorescence mirror A; 512, fluorescence mirror B; 513, fluorescence mirror C;

[0080] 521, band-pass filter A; 522, band-pass filter B; 523, band-pass filter C;

[0081] 531, fiber-coupled lens A; 532, fiber-coupled lens B; 533, fiber-coupled lens C;

[0082] 541, fiber A; 542, fiber B; 543, fiber C. Embodiment

[0083] In order to clearly illustrate the technical solutions of the present patent, the present patent will be described in detail below with specific embodiments and with reference to the accompanying drawings.

[0084] As shown in FIG. 1, the laser beam is divided into three beams, i.e., laser beam A, laser beam B and laser beam C. Figure 2 As shown in FIG. 2, the laser beam is divided into three beams, i.e., laser beam A, laser beam B and laser beam C.

[0085] Laser 100, the center wavelength is 488 nm, and the output spot is an elliptical spot. Under a lens with a focal length of 12 mm, the laser beam can be focused to a diameter of 100 um (horizontal direction) and 20 um (vertical direction).

[0086] Laser mirror A 201, laser mirror B 202 and laser mirror C 203 are all 488 nm dielectric film mirrors with reflectivity > 99% @ 488 nm. They are fixed on the mirror frame to facilitate fine adjustment of the pointing of the laser beam, so that the laser is finally focused at the center position of the flow channel in the flow cell (observation chamber) 300.

[0087] Adjusting the mirror frame under the laser mirror A 201 can fine-tune the pointing of the laser beam A.

[0088] Adjusting the mirror frame under the laser mirror B 202 can fine-tune the pointing of the laser beam B.

[0089] Adjusting the mirror frame under the laser mirror C 203 can fine-tune the pointing of the laser beam C.

[0090] Dichroic mirror A 211 and dichroic mirror B 212 have a splitting ratio of 50:50 at 488 nm.

[0091] Attenuation sheet 220 has an attenuation ratio of 50% at 488 nm.

[0092] The laser beam passing through the dichroic mirror A 211 and the dichroic mirror B 212 is defined as the laser beam A.

[0093] The laser beam passing through the dichroic mirror A 211 but reflected by the dichroic mirror B 212 is defined as the laser beam B.

[0094] The laser beam reflected by the dichroic mirror A 211, reflected by the laser mirror C 203 and attenuated by the attenuation sheet 220 is defined as the laser beam C.

[0095] Dichroic filter A 231 is a short wave pass filter, with a starting response wavelength of 503 nm, used to reflect fluorescent light and transmit laser light, with a reflected wavelength range of 515 nm-850 nm and a transmitted wavelength range of 400 nm-491 nm.

[0096] Dichroic filter B 232 and dichroic filter C 233 are long wave pass filters, with a starting response wavelength of 503 nm, used to transmit fluorescent light and reflect laser light, with a transmitted wavelength range of 515 nm-850 nm and a reflected wavelength range of 400 nm-491 nm.

[0097] Achromatic lens A 241, achromatic lens B 242 and achromatic lens C 233, with a focal length of 12 mm, an outer diameter of 9 mm and a numerical aperture of 0.38, are used to focus laser light and collect fluorescent signals generated by magnetic beads 310 in flow cell 300.

[0098] Laser beam A, laser beam B and laser beam C, after being focused by the achromatic lens, have a certain interval in the vertical direction (e.g. Figure 1 , perpendicular to the direction of the paper) of 60 um, which ensures that the fluorescent light excited by laser beam A will not ultimately reach fibers B and C, the fluorescent light excited by laser beam B will not ultimately reach fibers A and C, and the fluorescent light excited by laser beam C will not ultimately reach fibers A and B.

[0099] The angle between the central beam of laser beam B after transmitting through achromatic lens B 242 and the central beam of laser beam A after transmitting through achromatic lens A 241 is 95 degrees.

[0100] The angle between the central beam of laser beam C after transmitting through achromatic lens C 243 and the central beam of laser beam A after transmitting through achromatic lens A 241 is also 95 degrees.

[0101] Flow cell, i.e. observation chamber 300, is square in shape, with an outer dimension of 4 mm*4 mm and a length of 17 mm, and an internal flow shape that is square in shape with a dimension of 0.2 mm*0.2 mm. The magnetic beads are wrapped in a sheath flow, allowing them to pass through the center of the flow channel inside the flow cell stably. At the place where the laser is focused, the magnetic beads are irradiated by the laser, generating forward scattering light signals and fluorescent signals.

[0102] Convex lens A 420, with a focal length of 38.1 mm and an outer diameter of 25.4 mm, is used to collect forward scattering light signals and collimate them into parallel light beams with an angle of ±10 degrees.

[0103] Barrier 410 is located between convex lens A 420 and flow cell 300, and is used to block the background signal of the laser light that is not scattered by the magnetic beads.

[0104] Convex lens B 430, focal length 38.1 mm, outer diameter 25.4 mm, used to focus the forward scattered light collimated by convex lens A 420 onto detector 440.

[0105] Detector 440, converts the forward scattered light signal into an electrical signal, output to the electronics system.

[0106] As Figure 3 is the fluorescence path diagram.

[0107] The definition of the measured object is that the fluorescence generated by the magnetic beads 310 excited by the laser beam A is collected by the achromatic lens A 241, reflected by the dichroic filter A 231, reflected by the fluorescence mirror A 511, transmitted by the band-pass filter A 521, and coupled into the optical fiber A 541 by the optical fiber coupling lens A 531. The fluorescence that is coupled into the optical fiber A 541 is fluorescence A, and the corresponding light path is fluorescence path A. Fluorescence A will also be collected by the other two achromatic lenses, but will not be transmitted into the other two optical fibers.

[0108] The definition of the measured object is that the fluorescence generated by the magnetic beads 310 excited by the laser beam B is collected by the achromatic lens B 242, transmitted by the dichroic filter B 232, reflected by the fluorescence mirror B 512, transmitted by the band-pass filter B 522, and coupled into the optical fiber B 542 by the optical fiber coupling lens B 532. The fluorescence that is coupled into the optical fiber B 542 is fluorescence B, and the corresponding light path is fluorescence path B. Fluorescence B will also be collected by the other two achromatic lenses, but will not be transmitted into the other two optical fibers.

[0109] The definition of the measured object is that the fluorescence generated by the magnetic beads 310 excited by the laser beam C is collected by the achromatic lens C 243, transmitted by the dichroic filter C 233, reflected by the fluorescence mirror C 513, transmitted by the band-pass filter C 523, and coupled into the optical fiber C 543 by the optical fiber coupling lens C 533. The fluorescence that is coupled into the optical fiber C 543 is fluorescence C, and the corresponding light path is fluorescence path C. Fluorescence C will also be collected by the other two achromatic lenses, but will not be transmitted into the other two optical fibers.

[0110] The center wavelength of the band-pass filter A 521, the band-pass filter B 522 and the band-pass filter C 523 is 530 nm, the bandwidth is 30 nm, and the band-pass outside cutoff rate is OD8, that is, no more than 10E-8 light outside the bandwidth is allowed to pass through the filter.

[0111] The optical fiber coupling lens A 531, the optical fiber coupling lens B 532 and the optical fiber coupling lens C 533 are aspherical lenses, model Lightpath354850, diameter 6.325 mm, focal length 22 mm, numerical aperture NA0.13, which can focus the 530 / 30 nm fluorescence to a size less than 50 um in diameter.

[0112] The core diameter of the optical fiber A 541, the optical fiber B 542 and the optical fiber C 543 is 50um, and the numerical aperture NA is 0.12. The core diameter of the optical fiber D is 105um, and the numerical aperture NA is 0.22.

[0113] The optical fiber A 541, the optical fiber B 542, the optical fiber C 543 and another optical fiber D are connected together by high-temperature fusion, realizing the same-wavelength beam combination, and by adjusting the numerical aperture and the core diameter of the input end and the output end optical fiber, the transmission efficiency of more than 90% can be achieved.

[0114] The optical fiber A 541, the optical fiber B 542, the optical fiber C 543 and the optical fiber D constitute a fiber beam combiner, wherein the optical fiber A 541, the optical fiber B 542 and the optical fiber C 543 are input ends for receiving fluorescence, and the optical fiber D is an output end connected to the optical fiber interface of a single photon counter.

[0115] The present application has many specific implementation approaches, and the above description is only the preferred embodiment of the present application. It should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements can be made, and these improvements should also be considered as the protection scope of the present application.

Claims

1. An optical system for a single-molecule detection immunoassay analyzer, characterized in that, The optical system comprises a laser light path for generating laser light, a forward scattering light detection channel for collecting forward scattering light, and a fluorescence detection channel for collecting fluorescence; The laser light path comprises a laser (100), a beam splitter A (211), a beam splitter B (212), and an attenuating sheet (220), the beam splitter A (211) and the beam splitter B (212) are sequentially fixedly installed on one side of the laser (100), the laser light emitted by the laser (100) is first split into two parts by the beam splitter A (211), and then the laser light passing through the beam splitter A (211) is again split into two parts by the beam splitter B (212), so that the laser light emitted by the laser (100) forms laser beams A and B after passing through the beam splitter A (211) and the beam splitter B (212), and the laser light split by the beam splitter A (211) forms a laser beam C, and the attenuating sheet (220) is fixedly arranged on the path of the laser beam C; A dichroic filter A (231) for transmitting laser light is fixedly arranged on the path of the laser beam A, a laser reflecting mirror B (202) and a dichroic filter B (232) for reflecting laser light are fixedly arranged on the path of the laser beam B, and a laser reflecting mirror C (203) and a dichroic filter C (233) for reflecting laser light are fixedly arranged on the path of the laser beam C; The dichroic filter A (231) transmits laser light while reflecting fluorescence, the dichroic filter B (232) and the dichroic filter C (233) reflect laser light while transmitting fluorescence, the fluorescence detection channel has three, three fluorescence detection channels respectively receive the fluorescence reflected by the dichroic filter A (231) or the fluorescence transmitted by the dichroic filter B (232) and the dichroic filter C (233), and the ends of the three fluorescence detection channels are connected to the same optical fiber to output the fluorescence signal outward; The forward scattering light detection channel is symmetrically arranged along one of the laser beam A, the laser beam B or the laser beam C along the flow cell (300).

2. The optical system of a single molecule detection immunoassay analyzer according to claim 1, wherein, A achromatic lens A (241) for focusing laser beams and collecting fluorescence is arranged between the dichroic filter A (231) and the flow cell (300); An achromatic lens B (242) for focusing laser beams and collecting fluorescence is arranged between the dichroic filter B (232) and the flow cell (300); An achromatic lens C (243) for focusing laser beams and collecting fluorescence is arranged between the dichroic filter C (233) and the flow cell (300).

3. The optical system of a single molecule detection immunoassay analyzer according to claim 2, wherein, The forward scattering light detection channel is symmetrically arranged along the laser beam A focused by the achromatic lens A (241) along the flow cell (300); The laser beam B and the laser beam C are irradiated to the flow cell (300) from two different directions after being reflected, and the achromatic lens B (242) and the achromatic lens C (243) have an included angle, so that the central beams of the laser beam B and the laser beam C after passing through the achromatic lens B (242) and the achromatic lens C (243) have an included angle.

4. The optical system of a single molecule detection immunoassay analyzer according to claim 2, wherein, The focused laser beam A, laser beam B and laser beam C are irradiated at the beam waist position of the flow cell (300) or with a spacing between the three beam waist positions.

5. The optical system of a single molecule detection immunoassay analyzer according to any one of claims 1-4, wherein, The three fluorescence detection channels are a fluorescence detection channel A arranged on one side of a dichroic filter A (231), a fluorescence detection channel B arranged on one side of a dichroic filter B (232) and a fluorescence detection channel C arranged on one side of a dichroic filter C (233). The fluorescence detection channel A comprises, from the side where the dichroic filter A (231) is arranged, a same-axial band-pass filter A (521), a fiber coupling lens A (531) and a fiber A (541) in sequence, so that the fluorescence generated by the flow cell (300) is reflected when irradiating the dichroic filter A (231), then transmitted through the band-pass filter A (521), focused by the fiber coupling lens A (531) and finally coupled into the fiber A (541). The fluorescence detection channel B comprises, from the side where the dichroic filter B (232) is arranged, a same-axial band-pass filter B (522), a fiber coupling lens B (532) and a fiber B (542) in sequence, so that the fluorescence generated by the flow cell (300) is directly transmitted when irradiating the dichroic filter B (232), then transmitted through the band-pass filter B (522), focused by the fiber coupling lens B (532) and finally coupled into the fiber B (542). The fluorescence detection channel C comprises, from the side where the dichroic filter C (233) is arranged, a same-axial band-pass filter C (523), a fiber coupling lens C (533) and a fiber C (543) in sequence, so that the fluorescence generated by the flow cell (300) is directly transmitted when irradiating the dichroic filter C (233), then transmitted through the band-pass filter C (523), focused by the fiber coupling lens C (533) and finally coupled into the fiber C (543). The fiber A (541), the fiber B (542), the fiber C (543) and another fiber D are connected together by high-temperature fusion, and the fiber D is connected to the fiber interface of the single photon counter.

6. The optical system of a single molecule detection immunoassay analyzer according to any one of claims 1-4, wherein, The forward scattering light detection channel comprises, from the side where the flow cell (300) is arranged, a same-axial baffle (410), a convex lens A (420), a convex lens B (430) and a detector (440) in sequence, the convex lens A (420) and the convex lens B (430) are oppositely arranged, the forward scattering light signal emitted by the flow cell (300) is collected by the convex lens A (420) and collimated, and the forward scattering light collimated by the convex lens A (420) is converged on the detector (440) by the convex lens B (430).

Images