CN219142597U - Single molecule detection immunoassay appearance optical system - Google Patents

Abstract

The utility model discloses an optical system of a single-molecule detection immunoassay analyzer, and relates to the field of optical systems of single-molecule detection immunoassay analyzers. Under the condition that the use quantity of main and expensive materials such as lasers and single photon detectors is not increased, the laser beams are divided into 3 beams with equal intensity through 2 spectroscopes and 1 attenuation sheet, and the objects to be detected are respectively irradiated from 3 directions, and fluorescence is collected. The optical system comprises a laser light path for generating laser light, a forward scattered light detection channel for collecting forward scattered light and a fluorescence detection channel for collecting fluorescence; the laser light path comprises a laser, a spectroscope A, a spectroscope B and an attenuation sheet; the tail ends of the three fluorescence detection channels are connected with the same optical fiber to output fluorescence signals outwards; the forward scattered light detection channel and one of the laser beam A, the laser beam B or the laser beam C are symmetrically arranged along the flow cell. The effect of improving the identification rate of the positive magnetic beads is achieved.

Description

Single molecule detection immunoassay appearance optical system

Technical Field

The utility model relates to the field of optical systems of single-molecule detection immunity analyzers.

Background

The detection sensitivity of single molecule detection can reach fg grade, which is 1000 times of that of traditional ELISA.

The biological principle of detection is a classical immunoreaction-double antibody sandwich method, a capture antibody of which the magnitude exceeds 10 x 5 is coated on magnetic beads, the capture antibody captures an antigen in a sample to be detected, and then a double antibody sandwich structure, namely a binding phase, is formed by the capture antibody and the detection antibody marked by the added fluorescent dye. Because the magnetic beads have magnetism, the impurities in the supernatant can be conveniently removed by utilizing a magnetic separation mode.

Detection by using a flow cytometry sheath flow focusing method is one of the common detection methods, and detects forward scattered light signals of magnetic beads and fluorescent signals generated by excitation of fluorescent dyes marked on antibodies by laser. The forward scattered light signals of the magnetic beads are used for counting, the fluorescent signals are used for judging whether the magnetic beads form a double-antibody sandwich binding phase, and the double-antibody sandwich binding phase is positive magnetic beads, or else, the magnetic beads are negative magnetic beads.

When the antigen concentration in the sample to be detected is fg, only no more than 5% of the magnetic beads can capture the antigen to form a double-antibody sandwich binding phase. And calculating an antigen protein concentration value corresponding to the positive magnetic beads by using a poisson distribution theory, and realizing the ultra-high sensitivity detection of the digital fg level.

When the concentration of the antigen in the sample to be detected is high, most of the magnetic beads can capture the antigen to form a double-antibody sandwich binding phase. At this time, the intensity of the fluorescent signal and the concentration of the analyte are positively correlated, so that a standard curve can be established. By detecting a certain number of magnetic beads, the concentration of the antigen to be detected can be quantitatively measured.

The detection is carried out on the existing flow cytometry or flow fluorescence analyzer by utilizing a specially developed magnetic bead reagent system, and the detection sensitivity is expected to reach fg level. For example, connieWu, tylerJ.Dougan, and DavidR, walt et al, entitled "High-Throughput, high-Multiplex DigitalProtein Detection with Attomolar Sensitivity," are performed on a CytoFlexLX flow cytometer, manufactured by Beckmann Coulter, to achieve a sensitivity of fg level and a single-molecule level of detection sensitivity.

However, the direction of the laser light of the optical system of the flow cytometer or the flow fluorescence analyzer is generally perpendicular to the direction of fluorescence collection, and almost all the optical systems of the flow cytometer are designed in this way. Since flow cytometry or flow fluorescence analyzers generally require a plurality of fluorescence channels, the Numerical Aperture (NA) of the objective lens is also required to be relatively large, and it is preferable that the numerical aperture reaches 1.2. The object to be measured is mainly cells, the surface antigens of the cells are numerous, and most cells have certain light transmittance, so that the laser direction of the flow cytometer is perpendicular to the fluorescence collection direction, which is beneficial to the design of an optical system, and if other modes are parallel, the difficulty is very high, and the difficulty of the multi-laser flow cytometer is increased by one level.

Such an optical system has the following drawbacks when detecting magnetic beads: because the magnetic beads used at present are basically opaque, only one double-antibody sandwich binding phase can be formed on each magnetic bead with high probability at low concentration, 2 fluorescent dyes marked on detection antibodies can be formed with extremely low probability, the probability of being irradiated by laser is 50%, the probability of being collected by an objective lens is also 50%, and only 25% of the probability is comprehensively determined as positive magnetic beads (being irradiated by the laser and collected by the objective lens). Other situations cannot guarantee that a positive magnetic bead must be identified.

At high concentrations (1-2 orders of magnitude higher than fg levels), there may be multiple, but not too many, binding phases forming at least one double antibody sandwich per bead with a high probability. The basis for establishing the standard curve is that the intensity of the fluorescence signal and the concentration of the object to be detected are in positive correlation, and the more the combination phase is, the larger the fluorescence intensity is, but the uniformity of the fluorescence intensity of the laser energy cannot be ensured by the optical system of the traditional flow cytometer. The greater the number of binding phases of the double antibody sandwich on the surface of the magnetic bead, the better the uniformity.

This results in a relatively large Coefficient of Variation (CV) of the final test results, both at low and high concentrations. Therefore, how to optimize the existing equipment and realize more comprehensive and more accurate detection becomes a technical problem to be solved urgently by those skilled in the art.

Disclosure of Invention

The present utility model proposes an optical system for a single-molecule detection immunoassay analyzer, in which, in the case that the number of main and expensive materials such as lasers and single-photon detectors is not increased (the number is 1), the laser beam is divided into 3 beams with equal intensity by 2 spectroscopes and 1 attenuation sheet, and the objects to be measured (i.e., magnetic beads) are irradiated from 3 directions, respectively, while fluorescence is collected.

The technical scheme of the utility model is as follows: the optical system comprises a laser light path for generating laser light, a forward scattered light detection channel for collecting forward scattered light and a fluorescence detection channel for collecting fluorescence;

the laser path includes a laser 100, a beam splitter a211, a beam splitter B212, and an attenuation sheet 220, where the beam splitter a211 and the beam splitter B212 are sequentially and fixedly installed on one side of the laser 100, the laser emitted by the laser 100 is first split into two parts by the beam splitter a211, and then the passed laser is split into two parts by the beam splitter B212 again, 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 a211 and the beam splitter B212, and the laser beam C is formed by the laser beam split by the beam splitter a211, and the attenuation sheet 220 is fixedly arranged on the path of the laser beam C;

a dichroic filter a231 for transmitting laser light is fixedly provided on the path of the laser beam a, a laser mirror B202 and a dichroic filter B232 for reflecting laser light are fixedly provided on the path of the laser beam B, and a laser mirror C203 and a dichroic filter C233 for reflecting laser light are fixedly provided 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;

the dichroic filter A231 transmits laser and simultaneously reflects fluorescence, the dichroic filter B232 and the dichroic filter C233 reflect laser and simultaneously transmit fluorescence, the three fluorescence detection channels respectively receive the fluorescence reflected by the dichroic filter A231 or transmitted by the dichroic filter B232 and the dichroic filter C233, and the tail ends of the three fluorescence detection channels are connected with the same optical fiber to output fluorescence signals outwards;

the forward scattered light detection channel is symmetrically arranged along the flow cell 300 with one of the laser beam a, the laser beam B or the laser beam C.

Wherein an achromatic lens A241 for focusing a laser beam and collecting fluorescence is provided between the dichroic filter A231 and the flow cell 300;

an achromat B242 for focusing laser beam and collecting fluorescence is disposed between the dichroic filter B232 and the flow cell 300;

an achromatic lens C243 for focusing a laser beam and collecting fluorescence is provided between the dichroic filter C233 and the flow cell 300.

Further, the forward scattered light detection channel and the laser beam a focused by the achromat a241 are symmetrically arranged along the flow cell 300;

the laser beams B and C are reflected to irradiate the flow cell 300 from two different directions, and an included angle is formed between the achromat B242 and the achromat C243, so that an included angle is formed between the center beams of the laser beams B and C after passing through the achromat B242 and the achromat C243, respectively, so that the reverse optical path of the laser beam C cannot reach the interior of the laser finally, and meanwhile, the reverse optical path of the laser beam B cannot reach the interior of the laser finally after passing through the flow cell 300.

Further, the beam waist positions of the laser beam a, the laser beam B, and the laser beam C, which are focused, are overlapped or spaced apart from each other.

Regarding the fluorescence detection channel;

the three fluorescence detection channels are respectively a fluorescence detection channel A arranged on one side of a dichroic filter A231, a fluorescence detection channel B arranged on one side of a dichroic filter B232 and a fluorescence detection channel C arranged on one side of a dichroic filter C233;

the fluorescence detection channel A sequentially comprises a bandpass filter A521, an optical fiber coupling lens A531 and an optical fiber A541 which are coaxial from the side where the dichroic filter A231 is positioned, so that fluorescence generated by the flow cell 300 is reflected when irradiated on the dichroic filter A231, then transmitted through the bandpass filter A521, focused by the optical fiber coupling lens A531 and finally coupled into the optical fiber A541;

the fluorescence detection channel B sequentially comprises a bandpass filter B522, an optical fiber coupling lens B532 and an optical fiber B542 which have the same optical axis from the side where the dichroic filter B232 is positioned, so that fluorescence generated by the flow cell 300 is directly transmitted when the dichroic filter B232 is irradiated, then transmitted through the bandpass filter B522, focused through the optical fiber coupling lens B532 and finally coupled into the optical fiber B542;

the fluorescence detection channel C sequentially comprises a bandpass filter C523, an optical fiber coupling lens C533 and an optical fiber C543 which have the same optical axis from the side where the dichroic filter C233 is positioned, so that fluorescence generated by the flow cell 300 is directly transmitted when the dichroic filter C233 is irradiated, then transmitted through the bandpass filter C523, focused through the optical fiber coupling lens C533 and finally coupled into the optical fiber C543;

the optical fiber A541, the optical fiber B542, the optical fiber C543 and the other 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.

A forward scattered light detection channel;

the forward scattered light detection channel sequentially comprises a baffle 410, a convex lens A420, a convex lens B430 and a detector 440, which are arranged on the same optical axis from the side of the flow cell 300, wherein the convex lens A420 and the convex lens B430 are arranged oppositely, forward scattered light signals emitted by the flow cell 300 are collected through the convex lens A420 and collimated, and forward scattered light collimated by the convex lens A420 is converged on the detector 440 through the convex lens B430.

As shown in fig. 1-3, the laser beam emitted from the laser 100 is reflected by the laser mirror a201, and the direction is turned 90 degrees. The laser beam passes through a beam splitter A211 and a beam splitter B212 in this order, and the laser beam is split into 3 beams.

The split ratio of beam splitter A211 to beam splitter B212 at the laser wavelength is 50:50, and the attenuation of the attenuator 220 at the laser wavelength is 50%, so that the energy of the 3 laser beams is substantially the same.

The laser beams transmitted through the beam splitters a211 and B212 are defined as laser beams a.

The laser beam transmitted through the beam splitter a211 but reflected by the beam splitter B212 is defined as the laser beam B.

The laser beam reflected by the beam splitter a211, reflected by the laser mirror C203, and attenuated by the attenuation sheet 220 is defined as the laser beam C.

The dichroic filter a231 is a short-wave pass filter, the initial response wavelength is between the laser wavelength and the fluorescence wavelength, the laser beam a is transmitted when passing through the dichroic filter a231 and focused by the achromatic lens a241, and the beam waist position coincides with the focal position of the achromatic lens a241, and is located in the flow cell, that is, the inner flow center position of the observation chamber 300.

The dichroic filter B232 is a long-pass filter, the initial response wavelength is between the laser wavelength and the fluorescence wavelength, the laser beam B is reflected by the laser mirror B202, and also reflected by the dichroic filter B232, and focused by the achromatic lens B242, and the beam waist position coincides with the focal position of the achromatic lens B242, and is located in the flow cell, that is, the inner flow center position of the observation chamber 300.

The dichroic filter C233 is a long-pass filter, the initial response wavelength is between the laser wavelength and the fluorescence wavelength, the laser beam C is reflected when passing through the dichroic filter C233, and is focused by the achromat C243, and the beam waist position coincides with the focal position of the achromat C243, and is located in the flow cell, that is, the inner flow center position of the observation chamber 300.

The dichroic filter a231 may be a long-wavelength filter, and the dichroic filter B232 and the dichroic filter C233 may be short-wavelength filters.

The achromat A241, the achromat B242 and the achromat C243 are shared by a laser light path and a fluorescence light path, are formed by bonding crown glass and flint glass, and are used for focusing laser beams and collecting fluorescence signals excited by the laser beams. The focal length is between 10mm and 100mm, and the numerical aperture NA is between 0.05 and 0.65. The acromatic lenses may also be replaced with spherical single lenses or aspherical lenses.

The flow cell, i.e., the viewing chamber 300, has a square or circular external shape and a directional or circular internal orifice shape. The analyte, i.e., the magnetic beads 310, passes through the internal channels thereof, and generates forward scattered light signals when passing through the laser beam, and fluorescent substances thereon are excited by the laser to generate fluorescence.

The forward scattered light signal is used to count the magnetic beads.

Convex lens a420 for collecting the forward scattered light signal generated by laser beam a and collimating the beam.

The baffle 410 is located between the convex lens a420 and the flow cell 300, and is used for shielding the laser background signal which is not scattered by the magnetic beads.

And a convex lens B430 for converging the forward scattered light collimated by the convex lens a420 onto the detector 440.

The detector 440 converts the forward scattered light signal into an electrical signal and outputs the electrical signal to an electronic system.

The beam waist positions of the laser beam A, the laser beam B and the laser beam C after being focused by the achromatic lens can be coincident in the vertical direction as shown in fig. 1, and can be in a certain interval, wherein the interval distance is generally between tens of micrometers and hundreds of micrometers, the specific interval distance can ensure that fluorescence excited by the laser beam A does not finally reach the optical fiber B and the optical fiber C, the fluorescence excited by the laser beam B does not finally reach the optical fiber A and the optical fiber C, and the fluorescence excited by the laser beam C does not finally reach the optical fiber A and the optical fiber B.

The beam waist positions of the 3 lasers can be coincident, and the advantage of this approach is that 100% of positive magnetic beads can be identified, regardless of whether the beads are rotated or not. And the signals are synchronous and have no delay, and the disadvantage is that the magnetic beads are irradiated by 3 beams of laser simultaneously, the fluorescent background of the magnetic beads is amplified, and the signal to noise ratio of the system is reduced.

The beam waist positions of the 3 laser beams can be spaced at a certain interval in the vertical direction, and the method has the advantages that the fluorescent background of the magnetic beads cannot be amplified, and the method has the disadvantages that 3 fluorescent signals are generated, the signals are asynchronous and have certain delay, and the step of signal delay calibration is needed to be added. In this case, if the magnetic beads themselves are not rotated, 100% of the positive magnetic beads can be recognized. If the beads themselves are rotating, it is possible that the positive beads are passing through the beam waist of the 3 laser beams, with the fluorescent substance on them all on the back of the laser irradiation. The probability of occurrence of the situation is calculated to be not more than 12.5% theoretically, the detection rate of the positive magnetic beads is still more than 87.5%, and the detection rate is still greatly improved compared with the prior art.

The central beam after the laser beam B transmits the achromat B242 is not parallel to the central beam after the laser beam C transmits the achromat C243, and has a small included angle, which can be achieved in two ways, namely: acromatic lenses B242 and C243 have a small angle of inclination with respect to the flow cell 300; mode two: the laser beam is incident on achromat B242 and achromat C243 at a certain inclination angle. The size of this angle is set as: the laser beam B after passing through the flow cell 300 cannot finally reach the inside of the laser through the reverse optical path of the laser beam C, and at the same time, the laser beam C after passing through the flow cell 300 cannot finally reach the inside of the laser through the reverse optical path of the laser beam B.

The laser beam emitted by the laser, if finally transmitted back into the laser, may cause unstable laser power or other anomalies, and should be avoided as much as possible, although anomalies are not necessarily generated.

When the object to be measured, i.e. the magnetic bead 310 passes through the beam waist position of the laser beam a, the fluorescent substance marked on the magnetic bead is excited by the laser, the generated fluorescence is emitted to the 360-degree stereoscopic space, part of the fluorescence is collected and collimated by the achromatic lens a241, reflected when the fluorescence is transmitted to the dichroic filter a231, reflected again by the fluorescent reflector a511, transmitted through the bandpass filter a521, focused by the optical fiber coupling lens a531, and finally coupled into the optical fiber a541.

The fluorescent mirror a511 may not be provided, and the length-width dimension of the entire optical system may become large.

When the object to be measured, i.e. the magnetic bead 310 passes through the beam waist position of the laser beam B, the fluorescent substance marked on the magnetic bead is excited again by the laser to generate fluorescence, part of the fluorescence is collected and collimated by the achromatic lens B242, the fluorescence is transmitted to the dichroic filter B232, reflected by the fluorescent mirror B512, transmitted through the bandpass filter B522, focused by the optical fiber coupling lens B532, and finally coupled into the optical fiber B542.

Fluorescent mirror B512 may not be provided, and the length-width dimension of the entire optical system may become large.

When the object to be measured, i.e. the magnetic bead 310 passes through the beam waist position of the laser beam C, the fluorescent substance marked thereon is still excited by the laser to generate fluorescence, part of the fluorescence is collected and collimated by the achromatic lens C243, the fluorescence is transmitted through the dichroic filter C233, reflected by the fluorescent mirror C513, transmitted through the bandpass filter C523, focused by the optical fiber coupling lens C533, and finally coupled into the optical fiber C543.

The fluorescent mirror C513 may not be provided, and the length-width dimension of the entire optical system may become large.

The order of the positions of the magnetic beads 310 passing through the beam waist of the laser beam can be any of A, B, C, A, C, B, B, A, C, B, C, A, C, A, B and C, B, A.

The optical fiber A541, the optical fiber B542, the optical fiber C543 and the other optical fiber D are connected together in a high-temperature welding mode, so that the same-wavelength beam combination is realized, and the transmission efficiency can still be ensured to be more than 90% through adjusting the numerical aperture and the fiber core diameter of the optical fibers, although the transmission efficiency is still lost to a certain extent.

The optical fibers a541, B542, and C543 are input ends for receiving fluorescence.

The output end is an optical fiber D which is connected to an optical fiber interface of the single photon counter.

According to the utility model, only one laser and one single photon detector are provided, and on the premise that the number of main and expensive materials is not increased, the positive magnetic beads can be judged as long as any laser can irradiate fluorescent substances on the magnetic beads and collect generated fluorescence, so that the identification rate of the positive magnetic beads is greatly improved, the purpose of reducing the variation Coefficient (CV) of the final test result is achieved, and the effect of improving the identification rate of the positive magnetic beads is realized.

The utility model not only improves the accuracy of the test result by simply increasing the number of fluorescence detection channels, but also increases the number of laser beams irradiated on the flow cell by optimizing the structure, so that the flow cell can receive the irradiation of laser from a wider angle, the probability of irradiating fluorescent substances on positive magnetic beads by the laser can be improved when the positive magnetic beads are detected, and simultaneously, the generated fluorescence can be synchronously collected. In addition, the utility model ensures that the energy of laser beams irradiated on the flow cell is basically the same through structural optimization, and the reverse light paths of multiple laser beams can not return to the laser, thereby realizing the purpose that the identification rate of positive magnetic beads can be greatly improved by one laser and one single photon detector.

Drawings

Figure 1 is a schematic view of the structure of the present case,

figure 2 is a diagram of the laser path of the present case,

FIG. 3 is a fluorescent light path diagram of the present case;

100, a laser;

201, a laser mirror a;202, a laser mirror B;203, a laser mirror C;

211, spectroscope a;212, beam splitter B;

220, an attenuation sheet;

231, dichroic filter a;232, a dichroic filter B;233, a dichroic filter C;

241, acromatic lens a;242, achromat B;243, achromat C;

300, flow cell, i.e. viewing chamber;

310, the object to be detected, i.e. the magnetic beads;

410, a baffle;

420, convex lens a;430, convex lens B;

440, a detector;

511, fluorescent mirror a;512, fluorescent mirror B;513, fluorescent mirror C;

521, a bandpass filter a;522, bandpass filter B;523, bandpass filter C;

531, fiber coupling lens a;532, fiber coupling lens B;533, a fiber coupling lens C;

541, optical fiber a;542, fiber B;543, fiber C.

Detailed Description

In order to clearly illustrate the technical features of the present patent, the following detailed description will make reference to the accompanying drawings.

Fig. 2 is a laser light path diagram.

The laser 100, with a centre wavelength of 488nm, outputs an elliptical spot, and focuses the laser beam to a diameter of 100um (horizontal direction) and 20um (vertical direction) with a lens with a focal length of 12 mm.

The laser reflector A201, the laser reflector B202 and the laser reflector C203 are 488nm dielectric film reflectors, the reflectivity is more than 99% @488nm, and the reflectors are fixed on the reflector frame, so that the direction of the laser beam is convenient to be finely adjusted, and the laser is finally focused at the center position of the middle flow channel of the flow cell (observation chamber) 300.

The direction of the laser beam a can be finely adjusted by adjusting the mirror holder under the laser mirror a 201.

The orientation of the laser beam B can be fine-tuned by adjusting the mirror mount under the laser mirror B202.

The direction of the laser beam C can be finely adjusted by adjusting the mirror holder under the laser mirror C203.

Spectroscope A211 and spectroscope B212, which have a 50:50 ratio of the light at 488 nm.

An attenuation sheet 220 having an attenuation ratio of 50% at 488 nm.

The laser beams transmitted through the beam splitters a211 and B212 are defined as laser beams a.

The laser beam transmitted through the beam splitter a211 but reflected by the beam splitter B212 is defined as the laser beam B.

The laser beam reflected by the beam splitter a211, reflected by the laser mirror C203, and attenuated by the attenuation sheet 220 is defined as the laser beam C.

The dichroic filter A231 is a short-wave pass filter, has an initial response wavelength of 503nm, is used for reflecting fluorescence and transmitting laser, has a reflection wavelength range of 515nm-850nm, and has a transmission wavelength range of 400nm-491nm.

The dichroic filter B232 and the dichroic filter C233 are long-wave pass filters with an initial response wavelength of 503nm and are used for transmitting fluorescence and reflecting laser, the transmission wavelength ranges from 515nm to 850nm, and the reflection wavelength ranges from 400nm to 491nm.

Acromatic lenses A241, B242 and C233 have a focal length of 12mm, an outer diameter of 9mm and a numerical aperture of 0.38 for focusing the laser light and collecting the fluorescent signals generated by the magnetic beads 310 in the flow cell 300.

The beam waist positions of the laser beam A, the laser beam B and the laser beam C after being focused by the achromatic lens are spaced at a certain interval in the vertical direction (as shown in fig. 1 and perpendicular to the paper surface), the spacing distance is 60um, the spacing distance can ensure that fluorescence excited by the laser beam A does not finally reach the optical fiber B and the optical fiber C, fluorescence excited by the laser beam B does not finally reach the optical fiber A and the optical fiber C, and fluorescence excited by the laser beam C does not finally reach the optical fiber A and the optical fiber B.

The angle between the central beam after laser beam B transmitted through achromat B242 and the central beam after laser beam a transmitted through achromat a241 is 95 degrees.

The angle between the central beam after laser beam C transmitted through achromat C243 and the central beam after laser beam a transmitted through achromat a241 is also 95 degrees.

The flow cell, i.e. the observation chamber 300, is square, with external dimensions of 4mm by 4mm, length of 17mm, internal flow shape of square, dimensions of 0.2mm by 0.2mm, and magnetic beads passing through the center of the flow channel inside the flow cell stably under the wrapping of sheath flow. Is irradiated by the laser light where the laser light is focused, and generates a forward scattered light signal and a fluorescence signal.

The convex lens A420 has a focal length of 38.1mm and an outer diameter of 25.4mm, and is used for collecting forward scattered light signals, collimating the forward scattered light signals into parallel light beams, and collecting the light beams at an angle of +/-10 degrees.

The baffle 410 is located between the convex lens a420 and the flow cell 300, and is used for shielding the laser background signal which is not scattered by the magnetic beads.

Convex lens B430, focal length 38.1mm, outer diameter 25.4mm, is used to focus the forward scattered light collimated by convex lens A420 onto detector 440.

The detector 440 converts the forward scattered light signal into an electrical signal and outputs the electrical signal to an electronic system.

Fig. 3 is a fluorescence light path diagram.

The fluorescence generated by excitation of the magnetic bead 310 by the laser beam a is collected by the achromatic lens a241, reflected by the dichroic filter a231, reflected by the fluorescent mirror a511, transmitted by the bandpass filter a521, and coupled into the optical fiber a541 by the optical fiber coupling lens a531 is defined as fluorescence a, and the corresponding optical path is fluorescence optical path a. Fluorescence a will also be collected by the other two achromats but will not be transmitted into the other two fibers.

The fluorescence generated by excitation of the magnetic beads 310 by the laser beam B is collected by the achromatic lens B242, transmitted by the dichroic filter B232, reflected by the fluorescent mirror B512, transmitted by the bandpass filter B522, coupled into the optical fiber B542 by the optical fiber coupling lens B532, and the corresponding optical path is the fluorescent optical path B. Fluorescence B will also be collected by the other two achromats but will not be transmitted into the other two fibers.

The fluorescence generated by excitation of the magnetic beads 310 by the laser beam C is collected by the achromatic lens C243, transmitted by the dichroic filter C233, reflected by the fluorescent mirror C513, transmitted by the bandpass filter C523, coupled into the optical fiber C543 by the optical fiber coupling lens C533, and the corresponding optical path is the fluorescent optical path C. Fluorescence C will also be collected by the other two achromats but will not be transmitted into the other two fibers.

The center wavelength of the band-pass filter A521, the band-pass filter B522 and the band-pass filter C523 is 530nm, the bandwidth is 30nm, and the band-pass external cutoff rate is OD8, namely, the light with the bandwidth exceeding 10E-8 is not allowed to pass through the filter.

The optical fiber coupling lens A531, the optical fiber coupling lens B532 and the optical fiber coupling lens C533 are aspheric lenses, the model is Lightpath354850, the diameter is 6.325mm, the focal length is 22mm, the numerical aperture NA is 0.13, and the fluorescent light with the diameter of 530/30nm can be focused to the size with the diameter of less than 50 um.

The core diameters of the optical fibers a541, B542, and C543 were 50um, and the numerical aperture NA was 0.12. The core diameter of the fiber D was 105um and the numerical aperture NA was 0.22.

The optical fiber A541, the optical fiber B542, the optical fiber C543 and the other optical fiber D are connected together in a high-temperature welding mode, so that the same-wavelength beam combination is realized, and the transmission efficiency of more than 90% can be achieved by adjusting the numerical aperture and the fiber core diameter of the optical fibers at the input end and the output end.

The optical fiber A541, the optical fiber B542, the optical fiber C543 and the optical fiber D form an optical fiber combiner, wherein the optical fiber A541, the optical fiber B542 and the optical fiber C543 are input ends and are used for receiving fluorescence, and the optical fiber D is output end and is connected to an optical fiber interface of the single photon counter.

While there have been described what are believed to be the preferred embodiments of the present utility model, it will be apparent to those skilled in the art that many more modifications are possible without departing from the principles of the utility model.

Claims (6)

1. An optical system of a single-molecule detection immunoassay analyzer, characterized in that the optical system comprises a laser light path for generating laser light, a forward scattered light detection channel for collecting forward scattered light, and a fluorescence detection channel for collecting fluorescence;

the laser light path comprises a laser (100), a spectroscope A (211), a spectroscope B (212) and an attenuation sheet (220), wherein the spectroscope A (211) and the spectroscope B (212) are sequentially and fixedly arranged on one side of the laser (100), laser emitted by the laser (100) is firstly divided into two parts through the spectroscope A (211), and then the passed laser is divided into two parts through the spectroscope B (212), so that laser emitted by the laser (100) forms a laser beam A and a laser beam B after passing through the spectroscope A (211) and the spectroscope B (212), laser beams C are formed by laser beams separated through the spectroscope A (211), and the attenuation 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 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 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 and simultaneously reflects fluorescence, the dichroic filter B (232) and the dichroic filter C (233) reflect laser and simultaneously transmit fluorescence, the three fluorescence detection channels are respectively used for receiving fluorescence reflected by the dichroic filter A (231) or transmitted by the dichroic filter B (232) and the dichroic filter C (233), and the tail ends of the three fluorescence detection channels are connected with the same optical fiber to output fluorescence signals outwards;

the forward scattered light detection channel and one of the laser beam A, the laser beam B or the laser beam C are symmetrically arranged along the flow cell (300).

2. The single molecule detection immunoassay analyzer optical system of claim 1, wherein an achromatic lens a (241) for focusing a laser beam and collecting fluorescence is provided 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 a laser beam and collecting fluorescence is provided between the dichroic filter C (233) and the flow cell (300).

3. The optical system of a single molecule detection immunoassay analyzer of claim 2, wherein the forward scattered light detection passageway is symmetrically disposed along the flow cell (300) with the laser beam a focused by the acromatic lens a (241);

the laser beams B and C irradiate the flow cell (300) from two different directions after being reflected, and an included angle is formed between the achromat B (242) and the achromat C (243), so that an included angle is formed between central beams after the laser beams B and C respectively pass through the achromat B (242) and the achromat C (243).

4. The optical system of claim 2, wherein the beam waist positions of the laser beam a, the laser beam B, and the laser beam C are coincident or spaced apart from each other.

5. The optical system of any one of claims 1 to 4, wherein the three fluorescence detection channels are a fluorescence detection channel a provided on a side of a dichroic filter a (231), a fluorescence detection channel B provided on a side of a dichroic filter B (232), and a fluorescence detection channel C provided on a side of a dichroic filter C (233), respectively;

the fluorescence detection channel A sequentially comprises a bandpass filter A (521), an optical fiber coupling lens A (531) and an optical fiber A (541) which are coaxial from the side where the dichroic filter A (231) is positioned, so that fluorescence generated by the flow cell (300) is reflected when irradiated on the dichroic filter A (231), then transmitted through the bandpass filter A (521), focused by the optical fiber coupling lens A (531) and finally coupled into the optical fiber A (541);

the fluorescence detection channel B sequentially comprises a bandpass filter B (522), an optical fiber coupling lens B (532) and an optical fiber B (542) which are coaxial from the side where the dichroic filter B (232) is positioned, so that fluorescence generated by the flow cell (300) directly transmits when the dichroic filter B (232) is irradiated, then passes through the bandpass filter B (522), is focused through the optical fiber coupling lens B (532), and finally is coupled into the optical fiber B (542);

the fluorescence detection channel C sequentially comprises a bandpass filter C (523), an optical fiber coupling lens C (533) and an optical fiber C (543) which are coaxial from the side where the dichroic filter C (233) is positioned, so that fluorescence generated by the flow cell (300) directly transmits when the dichroic filter C (233) is irradiated, then passes through the bandpass filter C (523), is focused through the optical fiber coupling lens C (533), and finally is coupled into the optical fiber C (543);

the optical fiber A (541), the optical fiber B (542), the optical fiber C (543) and the other optical fiber D are connected together in a high-temperature welding mode, and the optical fiber D is connected to an optical fiber interface of the single photon counter.

6. The optical system of any one of claims 1 to 4, wherein the forward scattered light detection channel sequentially comprises a baffle (410), a convex lens a (420), a convex lens B (430) and a detector (440) which are arranged on the same optical axis from the side where the flow cell (300) is located, the convex lens a (420) and the convex lens B (430) are oppositely arranged, forward scattered light signals emitted by the flow cell (300) are collected through the convex lens a (420) and collimated, and the forward scattered light collimated by the convex lens a (420) is converged on the detector (440) through the convex lens B (430).

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