A flow aggregate particle analyzer that enhances small particle scattering by tilt angle

Abstract

This invention discloses a flow cytometer for aggregate particle analysis that enhances the scattering of small particles by tilt angle, comprising a fluid flow system, an optical system, and a control system. The optical system includes an excitation optical path, a forward detection optical path, and a lateral detection optical path. The excitation optical path includes a first laser, a second laser, a reflector, and a quartz cup detection cell. The reflector is used to reflect the first laser beam directly emitted by the first laser. The forward detection optical path includes a forward divergence lens group, a forward divergence bandpass filter, and a forward divergence detector arranged in sequence. The lateral detection optical path includes a lateral objective lens, multiple dichroic mirrors, an SSC filter and an SSC detector, and a VSSC filter and a VSSC detector. This invention utilizes tilt angle scattered light signals to extend the detection capability of existing flow cytometers for smaller submicron particles, further advancing the existing 200nm particle size detection limit to the 80nm particle size range.

Description

Technical Field

[0001] This invention relates to the field of particle analysis technology, and in particular to a flow cytometer for aggregate particle analysis that enhances the scattering of small particles by tilting an angle. Background Technology

[0002] Modern biotechnology and biopharmaceutical industries utilize molecular biology, genetic engineering, and large-scale fermentation techniques to produce various biological products, such as antibodies, vaccines, recombinant proteins, and gene therapy vectors. These biological product suspensions are prone to molecular association and aggregation through various pathways during production, purification, dispensing, transportation, and storage, forming impurity particles of varying sizes. They may also contain aggregates of components originating from the host cells themselves, or large amounts of debris from exosomes (30nm-130nm), extracellular vesicles (1μm-10um) secreted by the host cells, or released by dead cells. Additionally, they may contain foreign contaminant particles (such as residual glass microparticles and nanoplastics). These particulate impurities are significant factors affecting the quality of biological products and the safety and efficacy of biopharmaceuticals. In biopharmaceutical formulations, if undetected particulate impurities enter the patient's body, they may cause vascular occlusion, pulmonary embolism, or induce an immune response; even small amounts of protein particles may pose a strong immunogenic risk. The topic of aggregate impurities is crucial for all biopharmaceuticals. Globally, since the late 1990s, aggregated particulate impurities have received widespread attention and have been detected and regulated based on the technological conditions available at the time.

[0003] Biopharmaceutical aggregates are highly heterogeneous, polydisperse, and extremely sensitive to the environment. Their formation pathways vary, resulting in vastly different compositions and physicochemical characteristics, with sizes spanning six orders of magnitude (1 nm to 1 mm). All of these factors contribute to the significant challenges in measuring and characterizing these aggregates. In biopharmaceutical applications, numerous different techniques are required to analyze and characterize a wide range of particulate impurities with diverse characteristics to ensure product safety, quality standards, and regulatory compliance. United States Pharmacopeia (USP) <787> , <788> China has established regulations on the size and concentration limits of particulate matter in injectable formulations, and has clarified the testing procedures and instrument requirements. (USP) <1787> The study recommends methods for analyzing aggregates of different sizes, including novel micro-flow imaging (MFI) and traditional light obscuration (LO).

[0004] Regarding various technologies, those based on overall (or batch) measurement, such as dynamic light scattering (DLS), static light scattering (SLS), optical diffraction, and turbidity determination, can only present the average characteristics of all particles. They can characterize homogeneous, single-peak, and low-dispersion samples, but when performing overall measurements on highly dispersive samples, they suffer from low resolution, data bias, or incomplete descriptions of particle groups and characteristics. Particle separation-based measurement technologies, including differential centrifugal sedimentation (DCS), analytical ultracentrifugation (AUC), filtration techniques, size exclusion chromatography (SEC), and asymmetric flow-field flow fractionation (AF4), separate particles before or during analysis using different principles. They can distinguish aggregates of particles with different sizes and physicochemical characteristics, overcoming the limitations of overall measurement technologies to some extent, and are often used in conjunction with the latter. However, they still do not achieve the ability to detect and analyze single particles.

[0005] Single-particle measurement techniques commonly include microscopy (optical, fluorescence, transmission electron microscopy, atomic force microscopy), Coulter counters, suspended microchannel resonant mass measurement (RMM), and optical obscuration (LO). These methods offer direct quantification capabilities and can generate more accurate and reliable weighted particle size distributions and particle concentration extrapolations. However, they may have limitations such as a narrow applicable concentration range, long processing times, and difficulty in exhausting the analysis of the entire sample. Emerging techniques for aggregate particle analysis, including nanoparticle tracking analysis (NTA), microfluidic imaging (MFI), tunable resistive pulse sensing (TRPS), flow cytometry, and space-and-time-resolved extinction profiling (STEP), also tend towards single-particle measurement. They offer advantages in accuracy, repeatability, resolution, robustness to bias, and throughput, making them more suitable for measuring aggregate particles with multi-peak and polydisperse characteristics.

[0006] Despite the existence of numerous new and old technologies, no single method can meet all detection needs, and none can cover the complete time-size dynamic range required for analyzing aggregates and formation processes in protein suspensions and other products. Currently, batch or group measurement techniques such as DLS, LSL, DCS, and AUC, while capable of detecting microparticles as small as close to 1 nm, provide overall average values ​​and cannot accurately analyze the true distribution and characteristics of highly heterogeneous aggregates. Furthermore, single-particle level detection techniques struggle to accurately detect and count particles smaller than 200 nm, thus creating gaps in relevant standards and regulations.

[0007] In 2011, Mach et al. (Mach, H.; Bhambhani, A.; Meyer, BK; Burek, S.; Davis, H; Blue, JT; Evans, RK. The use of flow cytometry for the detection of subvisible particles in therapeutic protein formulations. J. Pharm. Sci. 2011, 100, 1671–1678.) used flow cytometry to determine the concentration of particles larger than 1 μm in antibody formulations. Filipe et al. (Filipe, V.; Poole, R.; Oladunjoye, O.; Braeckmans, K.; Jiskoot, W. Detection and characterization of subvisible aggregates of monoclonal IgG in serum. Pharm. Res. 2012, 29, 2202–2212) detected particles with a diameter of 200 nm using fluorescent dye labeling. However, adding fluorescent dyes may cause unintended effects such as structural changes due to interactions in the sample, and may introduce additional impurity particles. Furthermore, the staining process has limitations for microparticles. In 2014, Nishi et al. (Nishi, H.; R.; Fürst, R.; Winter, G. Label-free flowcytometry analysis of subvisible aggregates in liquid IgG1 antibody formulations. J. Pharm. Sci. 2014, 103, 90–99.) measured unstained antibody protein aggregates with a particle size greater than 500 nm using flow scattering mode and confirmed a proportional correlation between the results and MFI and LO detection results. In 2018, Hu et al. (Hu Z, Ye C, Mi W, Zhao Y, Quan C, Li WW, Li H, Hang H. Light-scattering detection within the difficult size range of protein particle measurement using flow cytometry. Nanoscale. 2018 Nov 7; 10(41): 19277-19285) modified a flow cytometer for aggregate particle analysis. The instrument has a direct FSC detection channel and a direct lateral SSC in the 488nm laser optical path, and an FSC side-side detector is added through a beam splitter. The FSC side-side detector collects forward scattered light within a solid angle of 15-30° with the laser optical axis. This design reduces the background noise caused by the direct laser and improves the detection sensitivity, enabling the modified device to measure microspheres and protein aggregates above 200nm, further expanding the measurable particle size range of aggregate particles in biological products such as protein suspensions. However, the measurement and analysis of aggregates below 200nm at the single particle level is still not possible, which remains a blank area.

[0008] In fact, using flow cytometry to detect microparticles eliminates the need for sample staining, avoiding a series of problems associated with staining. It can be applied to microparticles of any composition, making it well-suited for analyzing aggregated impurities with different compositions and characteristics. It can directly measure and count individual microparticle impurities, offering advantages such as high throughput and full-sample detection, and can detect extremely low concentrations of rare groups within aggregated impurities. However, considering the flow cytometry methods used for measuring aggregated particles in biological products such as protein suspensions, particles smaller than 200 nm cannot be clearly detected and distinguished from background noise. Possible reasons include: firstly, the intensity of the scattered light signal from microparticles is positively correlated with the sixth power of their size; that is, as the particle size decreases, the scattered light signal generated by the aggregated particles decreases sharply, and the detection difficulty increases exponentially. Secondly, the direct scattering light channel, the side FSC channel, and the VSSC channel in the aforementioned devices may have low efficiency in collecting and transmitting scattered light signals, or the background noise may still be relatively high, or the photoelectric conversion efficiency of the detector may still be relatively low, limiting the sensitivity of submicron particle detection.

[0009] One of the key common characteristic parameters of microparticles is particle size and size distribution. Various related studies have shown that smaller aggregates tend to have higher particle concentrations, and this parameter is associated with suspension homeostasis and in vivo cellular absorption efficiency. According to reports, aggregates of 100-200 nm have an impact on the quality and immunogenicity of biopharmaceuticals. Limits on particle size, distribution, and concentration in drug regulatory regulations are also key indicators. However, current technologies still lack the ability to effectively detect and characterize aggregate impurities smaller than 200 nm at the single-particle level. Therefore, researching relevant technologies to fill this gap in the analysis of microparticles smaller than 200 nm is of great significance. Summary of the Invention

[0010] The purpose of this invention is to provide a flow cytometer for aggregate particle analysis that enhances the scattering of small particles through tilt angle, thereby improving the detection capability of the forward and side-scattering light channels of the flow cytometer for small submicron particles and further advancing the existing 200nm particle size detection (counting) limit to an even smaller particle size range.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] The flow cytometer for aggregate particle analysis of the present invention, which enhances the scattering of small particles by tilt angle, is a technical device that uses the tilt angle scattered light signal to extend the detection capability of existing flow cytometers for smaller submicron particles. It can further advance the existing 200nm particle size detection limit to the 80nm particle size range.

[0013] A flow cytometer for aggregate particle analysis that enhances the scattering of small particles by tilt angle includes a fluid flow system, an optical system, and a control system.

[0014] Typically, in the process of detecting standard microspheres and samples using a flow cytometry aggregate particle analyzer, the fluid flow system is designed to use the principle of laminar flow for fluid dynamic focusing, so that the sampled microparticles flow in the form of a single particle queue, with each particle passing through the laser irradiation area in sequence. Therefore, this particle-by-particle detection method can achieve complete detection at the level of a single particle and can count the detected particles.

[0015] The optical system includes an excitation optical path, a forward detection optical path, and a lateral detection optical path. The excitation optical path includes a first laser (wavelength 488nm) for generating large-angle forward scattered light (FSC) and side scattered light (SSC), a second laser (wavelength 405nm) for generating side scattered light (VSSC), a reflector, and a quartz cup detection cell. The two laser beams are alternately irradiated at different positions in the liquid flow. The reflector is used to reflect the first laser beam directly emitted by the first laser. The forward detection optical path includes a forward divergence lens group, a forward divergence bandpass filter, and a forward divergence detector arranged in sequence. The forward detection optical path is located on the opposite side of the laser incident surface of the quartz cup detection cell. The lateral detection optical path includes a lateral objective lens, and multiple dichroic mirrors are arranged behind the lateral objective lens. One of the dichroic mirrors branches off an optical path containing an SSC filter and an SSC detector, and another dichroic mirror branches off an optical path containing a VSSC filter and a VSSC detector.

[0016] The first laser and the second laser output collimated elongated elliptical laser beams, and the emitted beams are focused over a long distance in the X and Y directions by cylindrical mirrors set in front of their respective lasers. The focal point is located at the axis of the rectangular liquid flow chamber inside the quartz cup detection cell.

[0017] The first laser beam and the second laser beam output by the first laser and the second laser beam, respectively, after passing through the cylindrical mirror, propagate forward in a straight line through the flat lens; before being reflected by the mirror, the propagation direction of the first laser beam is perpendicular to the short side long optical surface of the quartz cup detection cell, and therefore also perpendicular to the flow direction of the microparticle suspension sample in the detection cell; the second laser beam irradiates the quartz cup flow cell at a horizontal lateral offset angle of 20°.

[0018] The angle between the reflector and the direct first laser beam is 22.5°, so that the reflected beam illuminates the vertically upward-flowing single-particle queue at an angle of 45°.

[0019] In current flow cytometry devices, the laser beam typically illuminates the detection cell vertically. A direct forward FSC channel is positioned parallel to the laser incident direction, while a lateral detection channel is positioned vertically in the plane formed by the laser and the liquid flow. This allows for the detection of forward and lateral scattered light and fluorescence generated during laser irradiation of particles. In this detection method, a light-blocking strip and a medium-density attenuating lens must be placed in the forward detection path to block the directly transmitted laser. However, a significant amount of background laser photons inevitably enters the FSC detector, resulting in the detection of forward scattered light occurring against a high-excitation-light background. Consequently, the FSC channel's ability to detect microparticles is limited.

[0020] The first laser in the device provided by this invention propagates in a straight horizontal direction upon beam emission, perpendicular to the short-side optical plane of the rectangular quartz cup detection cell, and thus also perpendicular to the flow direction of the microparticle suspension sample in the detection cell. However, a reflector is placed in the optical path before the first laser beam reaches the quartz cup detection cell, with its reflective surface facing downwards and forming a 22.5° angle with the axis of the straight-propagating first laser beam. This causes the first beam, originally propagating horizontally, to propagate obliquely downwards at a reflection angle of 67.5°, reaching the short-side optical plane of the rectangular quartz cup detection cell, irradiating the aggregated particle queue in the liquid flow at a 45° angle to the vertically upward flowing sample liquid. This design changes the traditional method of vertically irradiating a flowing sample with a laser beam.

[0021] For the SSC detection channel, since the side objective lens is perpendicularly attached to the long side optical surface of the quartz cup detection cell, the SSC detection channel remains in a 90° lateral orientation even after the first laser beam illumination angle changes. For the forward-scattering FSC detection channel, this is equivalent to measuring the forward scattered light of particles at a large tilt angle of 45°. This avoids the high background caused by the high brightness of the straight-transmitted laser beam entering the 45° tilt forward-scattering lens group, significantly improving the sensitivity of the forward-scattering detection channel. The forward-scattering lens group only collects scattered photons, so there is no need to set up additional light-shielding strips and / or medium-density attenuating lenses.

[0022] The second laser of this invention is positioned horizontally at a 20° offset, allowing for independent focusing and collimation paths for both the first and second lasers, without sharing any excitation optical path elements. The second laser beam irradiates the quartz cup flow cell at a 20° horizontal angle. Therefore, for the VSSC side-scattered light generated by the second laser with a wavelength of 405nm (violet), the side objective lens collects and detects the VSSC signal at a 70° side position, rather than a 90° side position.

[0023] like Figure 2As shown, the first laser provides a side-scattered light (SSC) detection channel and two additional fluorescence detection channels (BL1, BL4), while the second laser provides a side-scattered light (VSSC) detection channel and one additional fluorescence detection channel (VL1). The additional fluorescence channels are reserved for detecting microparticles using fluorescence staining methods.

[0024] The first laser emits at a wavelength of 488 nm and a power of 50-100 mW. After focusing, the short axis of the laser beam is parallel to the direction of liquid flow, with a length of 9-15 μm, and the long axis is 70-80 μm, perpendicular to the direction of liquid flow. The second laser emits at a wavelength of 405 nm and a power of 150-180 mW. After focusing, the short axis of the laser beam is 10-18 μm, and the long axis is 95-105 μm, with the short axis parallel to the direction of liquid flow.

[0025] The front divergence lens group consists of two identical aspherical plano-convex lenses arranged in a curved-to-curved manner. The lenses have a diameter of 25 mm, a numerical aperture (NA) > 0.6, a front and rear focal length of 11 mm, and an effective focal length of 20 mm. The front surface of the first lens is 11 mm from the laser illumination point. This design results in a collection half-angle of 36° for the first lens, allowing it to collect scattered light signals within a solid angle range of at least 72°. This significantly improves collection efficiency and plays a crucial role in enhancing detection sensitivity.

[0026] The front diffuser lens group is still located in the conventional direct FSC detection channel position, but because the laser beam irradiates the liquid flow at a 45° angle, the straight-propagating laser beam will not enter the collection range of the front diffuser lens group, and there is more space to design to collect scattered light over a wide angle range, which significantly improves the light collection efficiency and thus improves the sensitivity of microparticle detection.

[0027] The front focal point of the front diverging lens group is designed as the illumination point of the laser beam, and the rear focal point is designed as the photosensitive surface of the front diverging detector. Therefore, after collecting the scattered light, the front diverging lens group focuses it onto the photosensitive surface of the detector. The focusing of light energy can improve the photoelectric conversion efficiency, thereby improving the sensitivity.

[0028] The rear surface of the second lens in the front divergence lens group is 12mm away from the photosensitive surface of the front divergence detector. After collecting the scattered light, the front divergence lens precisely focuses it onto the photosensitive surface of the front divergence detector, with a focused spot size of <1mm. 2 .

[0029] The SSC detector and VSSC detector have a photosensitive surface area of ​​2 mm². 2 APD devices.

[0030] In the forward astigmatism path structure, the first laser beam incident at a 45° angle no longer enters the forward lens group, significantly reducing background light noise. The forward astigmatism lens group, which collects scattered light at a 45° angle to its axis, uses a numerical aperture of NA0.6. This allows it to collect the light signal closer to the quartz cup detection cell, enabling the collection of scattered light signals over a large 72° solid angle range, thus greatly increasing the light signal intensity. The VSSC channel, on the other hand, collects scattered light signals at a 70° lateral angle, covering a 98° solid angle range, further improving the sensitivity of scattered light detection. All channels employ low-noise, high-efficiency ADP detectors to enhance sensitivity.

[0031] The VSSC and SSC signals are collected by the side objective. The side objective is in close contact with the long side optical surface of the quartz cup detection cell, thereby collecting more optical signals. The rear optical fiber transmits the optical signals collected by the side objective and guides them to the corresponding detection optical path.

[0032] Preferably, the side objective has a numerical aperture (NA) > 1.15, the rear fiber has an NA of 0.22, and the rear fiber end face diameter is 400 μm, which can completely collect the light spot focused by the side objective.

[0033] The side objective lens is integrated with the long side optical surface of the rectangular quartz cup detection cell. It collects SSC and VSS side-scattered light signals, as well as fluorescence signals, in a direction perpendicular to the liquid flow and laser plane, and focuses the collected light. With a NA > 1.15, the side objective lens achieves a collection half-angle of 49°, enabling the collection of side-scattered light, including SSC and VSSC, within a solid angle range of 98°. Collecting more side-scattered light signals plays a crucial role in improving sensitivity.

[0034] The front focal point of the side objective lens is positioned at the laser irradiation point in the center of the quartz cup detection cell, and the rear focal point is positioned at the incident end face of the rear optical fiber. The optical signals generated by the first and second lasers are transmitted through the rear optical fiber to each stage of dichroic beam splitters, and the side-scattered light is split and guided to the SSC filter and SSC detector, as well as the VSSC filter and VSSC detector.

[0035] To reduce noise from other wavelengths that may enter the large-angle forward-scattering FSC detector and the VSSC and SSC detectors, a bandpass filter is placed before each detector. The filter specifications depend on the laser wavelength used. This invention uses a 488nm wavelength laser to generate scattered light signals. Preferably, the forward-scattering bandpass filter and the side-scattering SSC bandpass filter have a BP 488 / 10nm specification, allowing only photons in the 483nm-493nm wavelength range to pass through, thereby filtering out secondary laser photons and ionized photon backgrounds from other wavelengths that may originate from the same device. This plays a crucial role in ensuring sensitivity. The VSSC bandpass filter allows only photons in the 400nm-410nm wavelength range to pass through, blocking photons of other wavelengths. The narrow bandpass width greatly avoids photon interference between lasers in the same device and interference from ionized photons of other wavelengths.

[0036] All detectors utilize low-noise, high-efficiency silicon avalanche photodiodes (APDs) to enhance detection sensitivity. The APD detector's operating voltage can be adjusted via the control system, thereby regulating its gain to accommodate varying intensities of scattered light from particles of different sizes. When the APD detector voltage mapping value is set to its maximum of 1000V, it can effectively distinguish between system background noise and standard microspheres as small as 80nm in diameter.

[0037] The fluid flow system includes a sheath fluid unit, a sample injection control unit, and a waste fluid unit;

[0038] The sheath fluid unit comprises a sheath fluid tank, a peristaltic pump, and a sheath fluid filter, connected sequentially via fluid conduits. The peristaltic pump provides the pressure to drive the sheath fluid flow, which in turn drives the fluid into the flow chamber to achieve laminar flow and maintain a constant flow rate. The sheath fluid pressure can be changed by controlling the peristaltic pump speed, thereby altering the sheath fluid flow rate, typically controlled at 2-7 m / s. Lower flow rates increase the time microparticles are irradiated by the laser, resulting in more light signals and improved sensitivity, but reduce fluid focusing ability. Higher flow rates allow for more precise flow dynamics focusing and increase detection throughput, but reduce the time microparticles spend passing through the laser irradiation point, decreasing the total number of photons emitted.

[0039] Preferably, the sheath fluid pressure is set to 28 kPa by default. After the sheath fluid is driven into the flow chamber and focused by hydrodynamics, the average flow velocity is 5 m / s. This flow velocity ensures that the microparticles emit a sufficiently strong light signal when passing through the laser detection point, enabling effective detection of 100 nm microparticles. It also allows the device to perform higher throughput and more precise detection.

[0040] Among them, the highest analysis rate of microparticles can reach about 30,000 particles / s.

[0041] The sheath fluid unit has a capsule-type sheath fluid filter installed in the fluid path before the fluid enters the flow chamber. The filter can have a diameter of 0.1 μm or 0.22 μm. Preferably, for microparticle detection, a 0.1 μm sheath fluid filter is selected to remove interfering particles that may originate from the sheath fluid. Preferably, the sheath fluid is 0.1 μm filtered PBS or ultrapure water.

[0042] The sample introduction control unit includes a sample loading needle and a flow chamber. The outlet of the sample loading needle is located on the central axis of the internal chamber of the flow chamber, and a quartz cup detection cell is located at the top of the flow chamber. The outlet of the sheath fluid filter is connected to the side inlet of the flow chamber via a fluid conduit. The upper part of the flow chamber is a gradually narrowing cone shape. The sample fluid introduced by the sample loading needle will flow forward together with the laminar flow of the sheath fluid. After passing through the gradually narrowing part of the flow chamber, hydrodynamic focusing is achieved, causing the aggregated particles in the sample suspension to form a single-particle queue, flowing along the fluid flow axis at a constant speed, and then entering the quartz cup detection cell. The axis of the quartz cup detection cell is the focusing point of the two laser beams, that is, the irradiation point is the detection point that generates the light signal.

[0043] The sample injection control unit also includes a plunger pump and a sample injection coil connected to each other. The outlet of the sample injection coil is connected to one inlet of a three-way connector. The other inlet of the three-way connector is connected to a sample tube through a sample injection conduit. The outlet of the three-way connector is connected to a sample loading needle through a sample injection conduit. Both sample injection conduits are equipped with solenoid valves. During the sample injection process, the connection direction is switched by controlling the opening and closing of the two solenoid valves to realize the aspiration and injection of sample liquid into the flow chamber.

[0044] The waste liquid unit includes a waste liquid buffer bottle, a diaphragm pump, and a waste liquid tank connected in sequence. The inlet of the waste liquid buffer bottle is connected to the outlet at the upper end of the flow chamber through a conduit to collect the liquid flowing out of the flow chamber.

[0045] This device uses a plunger pump connected to an injection coil for sample suspension aspiration and injection detection. The other end of the injection coil is connected to a three-way valve and a solenoid valve. This three-way valve connects the injection coil and the plunger pump, the injection tubing and a 5mL flow cytometer tube, and the injection tubing and the flow chamber injection needle. During operation, the connection between the plunger pump and the flow cytometer tube or the flow chamber injection needle can be controlled by switching the solenoid valve on and off. The volume of sample aspirated by the plunger pump can be quantitatively set, and the injection speed and volume can also be precisely controlled. The plunger pump aspirates a certain volume of the sample suspension to be tested into the connected injection coil. Then, through the solenoid valve and the three-way valve, it switches to the liquid path connected to the rear end of the coil, causing the plunger pump to push the outflowing sample liquid into the injection needle.

[0046] The plunger pump injection method employed allows for precise control of the sample suspension volume, and combined with the count of detected individual particles, the concentration of aggregated impurity particles can be directly assessed. This provides an optimal method for controlling the diameter of the core sample flow in the flow chamber, the particle detection rate, and for counting and calculating the absolute concentration of detectable microparticles.

[0047] Preferably, the fluid flow system is configured such that both the sheath fluid and the sample suspension are introduced into the flow chamber from bottom to top for hydrodynamic focusing and into the quartz cup detection cell for detection.

[0048] Preferably, the quartz cup detection cell is made of high-purity quartz, which can better propagate photons and reduce light loss. At the same time, the fixed position of the detection cell ensures long-term precise alignment of the excitation and detection optical paths, thereby guaranteeing the stability of detection performance over time.

[0049] The control system includes a signal processing section, electromechanical control circuitry, and a power supply control circuit board. The signal processing section comprises a linear amplifier, threshold trigger circuit, noise reduction circuit, analog-to-digital converter (ADC), and digital signal processing circuitry (DSP), which digitizes and measures the raw analog electrical pulse signals generated by the detector. The electromechanical control circuitry controls the normal operation of the peristaltic pump, thin-film pump, and plunger pump. The power supply control circuitry includes a stable power supply for the first and second lasers and an adjustable voltage power supply for the detector. The laser power supply is kept stable to maintain stable laser output power, while the detector operating voltage can be manually set to suit specific experimental samples.

[0050] The control system supports a particle detection speed of 30,000 particles per second, a signal sampling accuracy of 16 bits, and a sampling frequency of 10 MHz.

[0051] The working method of this flow cytometer for detecting microparticles is as follows: A first laser beam is incident at a 45° angle vertically onto a linearly flowing particulate sample stream in a quartz cup detection cell. A horizontally placed forward-scattering light channel is positioned on the opposite side of the laser incident surface in the detection cell. The forward-scattering light signal emitted by the microparticles under illumination is collected and detected at a 45° angle relative to the laser beam, thus obtaining a light scattering spectrum showing the relationship between the intensity of the large-angle forward-scattering light signal and the size of the microparticles. Furthermore, the side-scattering light signals of the microparticles are collected and measured through the side-scattering light (SSC) and VSSC channels, obtaining a light scattering spectrum showing the relationship between the SSC and VSSC signal intensities and the size of the aggregate particles. The VSSC parameter can be used as a channel for setting thresholds.

[0052] The laser is emitted by a laser device, emitting a beam of light with a specific wavelength and energy. After being focused, it illuminates the sample liquid flow, generating a 360° three-dimensional scattered light signal, including forward scattered light and side scattered light signals. Traditional flow cytometers commonly use 488nm blue lasers and 405nm violet lasers, both common wavelengths. Different laser wavelengths irradiating microparticles result in different sensitivities in detecting microparticles through scattered light; shorter wavelength scattered light is more sensitive to particle size. Therefore, preferably, when the target particle size is any particle size within the 80nm-100nm range, or any particle size, the laser wavelength is 405nm, and the energy is 150-180mW (e.g., 150mW, 160mW, 170mW, 180mW), i.e., violet laser. When the target particle size is within any range of 100nm-1μm, or any particle size, the laser wavelength is 488nm and the energy is 50-100mW (for example, it can be 50mW, 60mW, 70mW, 80mW, 90mW, or 100mW), which is also known as blue laser.

[0053] Among them, the tilt angle FSC channel, tilt angle VSSC channel, and SSC channel can each be at least one independent channel.

[0054] When the target particle size is any particle size within the 80-100nm range, or any particle size in general, preferably, the voltage mapping value of the tilt angle VSSC detector is 1000V, the VSSC threshold is 1000, and the voltage mapping value of the SSC detector is 1000V. When the target particle size is any particle size within the 100nm-1μm range, preferably, the voltage mapping value of the VSSC channel detector is set to 940V; the VSSC threshold is set to 400; the voltage mapping value of the SSC channel detector is set to 935-955V; and the voltage mapping value of the large tilt angle FSC channel detector is 880V-900V.

[0055] When measuring microparticles, especially small-diameter particles such as 80nm and 100nm, in order to determine whether the measured signal is the signal of the real particles or system noise, it is necessary to use sheath fluid as a control sample and set the VSSC channel threshold to 100. This can collect the distribution location of the system background noise. Therefore, when analyzing samples containing microparticles, the signal higher than the system background noise is the real signal from the microparticles.

[0056] After measuring the system background noise level with a VSSC threshold of 100, if the goal is to reduce noise events, the threshold can be set according to the highest boundary of the system noise distribution area in the VSSC channel to exclude the detection of system background noise.

[0057] The standard light scattering spectrum is obtained by adjusting the detector operating voltage according to the intensity range of scattered light signals from standard microspheres of different particle sizes. This voltage can be arbitrarily set within the range of 1-1000V, mapped to the actual operating voltage. The operating voltage setting must ensure that the target particle size distribution in the scattered light spectrum is within the detector's measurement range. Specifically, for particles larger than 1μm, the voltage may need to be reduced to ensure that the largest particle size does not exceed the maximum measurement value; for microparticles in the 100nm-1μm particle size range, a voltage close to the highest level is required to ensure that the signal of 100nm particles in the scattered light spectrum can be clearly distinguished from the background noise in the detection channel without overlap, while also ensuring that the signal of 1μm particles does not exceed the maximum measurement value; for 80nm particles, the maximum operating voltage needs to be set to ensure that the particle signal is separated from the background noise as much as possible.

[0058] The target particle size is defined as any particle size within the 80nm-100nm range, which can be represented by any single point value between 0.1-1μm, such as 80nm and 100nm. Furthermore, the target particle size within the 0.1-1μm range can be any particle size within the range of any two point values ​​between 0.1-1μm, such as, but not limited to, 0.1-0.2μm, 0.1-0.3μm, 0.1-0.9μm, 0.3-0.5μm, 0.5-0.7μm, 0.8-0.9μm, etc. The target particle size is any interval within the range of 0.1-1μm. The particle size can be represented by any single point value between 0.1-1μm, such as 0.1μm, 0.2μm, 0.4μm, 0.5μm, 0.6μm, 0.7μm, 0.8μm, 0.9μm, and 1μm.

[0059] This invention employs a plunger pump for sample loading. During operation, the detection rate of microparticles can be controlled by setting the injection volume and injection speed. Specifically, when detecting the sample at a lower injection volume rate, the sample particles are focused into a smaller core liquid stream. This makes the trajectory of the microparticles flowing through the laser irradiation point more stable and precise, contributing to improved resolution of the scattered light signal intensity from the microparticles. The minimum injection volume rate of this invention is 10 μL / min, at which point the diameter of the focused core sample liquid stream is 5 μm; the maximum injection volume rate is 120 μL / min, at which point the diameter of the focused core sample liquid stream is 20 μm. Preferably, an injection volume rate of 10 μL / min is used for detection to ensure optimal microparticle signal intensity resolution.

[0060] The spacing between microparticles in a flow is random, resulting in microparticles that are very close to each other or even adhere together. These coexisting microparticles, due to insufficient spacing, may be identified as a single microparticle event, leading to errors. Furthermore, as the injection rate increases, the average spacing between microparticles decreases, increasing the probability of adhesion events. Therefore, preferably, the microparticle analysis rate is controlled within the range of 20,000 particles / s to reduce the probability of coexisting events.

[0061] Two-dimensional scatter plots using tilt angle FSC and tilt angle VSSC are fundamental parameters for instrument calibration and particulate impurity measurement. FSC primarily characterizes particle size, while VSSC and SSC primarily characterize particle optical density or internal uniformity. VSSC is generated by a shorter wavelength 405nm second laser, which has better microparticle detection capability than the longer wavelength 488nm first laser. Therefore, a combination of FSC and VSSC is mainly used for microparticle detection. Compared to using a large tilt angle FSC alone, setting appropriate VSSC voltage and threshold makes it easier to locate the target particle in the two-dimensional scatter plot. In practice, noise signals and particle signals may be distinguished by adjusting the voltage and threshold settings of the large tilt angle FSC channel and VSSC channel respectively.

[0062] The large-angle forward scattered light signal is measured by a forward astigmatism lens with a first beam incident at a 45° angle and a collection angle axis of 45°, covering a solid angle of 72°. A side objective lens collects VSSC side scattered light with a 20° tilt angle covering a solid angle of 98°. Besides reflecting particle size and internal particle size as indicated by the traditional direct FSC channel and vertical side VSSC scattered light signal, these signals also include scattered light emission with spatial anisotropy due to differences in microparticle composition, morphology, and other characteristic parameters. Therefore, primarily utilizing the so-called large-angle forward astigmatism channel and tilted-angle VSSC channel, the detection of aggregates of microparticles with different sizes, morphologies, and physicochemical characteristics may reveal more informative and diverse scattered light distribution patterns, thus providing more evidence for the analysis and identification of microparticles.

[0063] Compared with the prior art, the outstanding effect of the flow cytometry aggregate particle analyzer of the present invention, which enhances the scattering of small particles by tilt angle, is as follows:

[0064] (1) The flow cytometer of the present invention uses a 488nm first laser beam at a vertical angle of 45° and a 405nm second laser beam at a horizontal angle of 20° to focus and irradiate a single microparticle array in the flow, thereby collecting FSC scattered light at a forward 45° position and VSSC scattered light at a lateral 70° position. Simultaneously, a forward divergence lens with NA > 0.6 and a lateral lens with NA > 1.15 can collect forward scattered light in a 72° solid angle range and lateral scattered light in a 98° solid angle range, respectively, significantly increasing the number of scattered photons that can be collected and improving sensitivity. Furthermore, by employing a low-noise, high-photoelectric-conversion-efficiency APD detector, the device can clearly distinguish and count system noise from reference standard microspheres with particle sizes as small as 80nm, filling the gap in particle detection and counting in the 80nm-200nm particle size range.

[0065] (2) The flow cytometer of the present invention employs an inclined excitation optical path, which allows for the convenient placement of a forward detection lens group in an orientation where the laser beam exits the short side of the quartz cup detection cell and its axis is perpendicular to both the optical surface and the upward-flowing liquid axis. A side objective lens, aligned with the long side of the quartz cup detection cell, is positioned perpendicular to the plane formed by the first laser beam and the liquid flow. This facilitates the collection and detection of forward and side-scattered light signals from the inclined orientation. The device features a simple structure, higher sensitivity, and the potential to study microparticles with anisotropic scattering patterns. Three fluorescence detection channels are reserved, providing a basis for analyzing the composition and size of microparticles using fluorescent dyes; or, when the microparticles themselves contain highly fluorescent components, the spontaneous fluorescence signal of the microparticles can be detected simultaneously in the fluorescence channels.

[0066] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the flow cytometer for enhancing small particle scattering by tilt angle described in this invention. Attached Figure Description

[0067] Figure 1 This is a schematic diagram of the overall structure of the flow cytometer for enhancing small particle scattering by tilt angle, as described in Example 1.

[0068] Figure 2 This is a schematic diagram of the optical path of the flow cytometer for enhancing small particle scattering by tilt angle, as described in Example 1.

[0069] Figure 3 This is a schematic diagram of the optical path for detecting 45° tilted forward scattered light in Example 1.

[0070] Figure 4 This is a schematic diagram of the optical path for detecting lateral VSSC scattered light at a 70° tilt angle in Example 1.

[0071] Figure 5 This is a schematic diagram of the sheath fluid system of the flow cytometer for enhancing small particle scattering by tilt angle in Example 1.

[0072] Figure 6 This is a schematic diagram of the sample injection control unit and waste liquid unit in Example 1.

[0073] Figure 7 This is the optical path diagram of the forward-spreading lens group in the forward detection optical path of Example 1.

[0074] Figure 8 The scattered light spectra of the sheath fluid of the blank control sample and the 80nm standard microspheres were detected using the flow cytometer of the present invention under the VSSC threshold of 100.

[0075] Figure 9 The scattered light spectra of the sheath fluid of the blank control sample and the 80nm standard microspheres were detected using the flow cytometer of the present invention under the VSSC threshold of 1000.

[0076] Figure 10 The one-dimensional tilt angle VSSC scattering spectra of ThermoFisher standard microspheres of seven particle sizes after dilution were detected using the flow cytometry aggregate particle analyzer of the present invention.

[0077] Figures 11-12 The present invention uses the flow cytometry aggregate particle analyzer to detect one-dimensional SSC scattering spectra of ThermoFisher standard microspheres of seven particle sizes after dilution.

[0078] Figure 13 The flow cytometry aggregate particle analyzer of this invention was used to detect the two-dimensional scattering spectra of diluted ThermoFisher standard microspheres of seven particle sizes at large tilt angles using FSC vs VSSC.

[0079] Among them, 1-fluid flow system, 2-optical system, 3-control system;

[0080] 11-Sheath fluid unit, 12-Sample injection control unit, 111-Sheath fluid tank, 112-Peristaltic pump, 113-Sheath fluid filter, 114-Liquid path conduit, 121-Sample loading needle, 122-Flow chamber, 123-Plunger pump, 124-Sample injection coil, 125-T-connector, 126-Solenoid valve, 127-Sample tube, 131-Waste liquid buffer bottle, 132-Diaphragm pump, 133-Waste liquid tank; 21-Laser optical path, 22-Forward detection optical path, 23-Lateral detection optical path; 211-First laser, 212-Second laser, 2 13-Reflector, 214-Quartz cup detection cell, 215-Cylindrical mirror; 220-Forward divergence collection unit, 221-Forward divergence lens group, 222-Forward divergence bandpass filter, 223-Forward divergence detector; 231-Side objective lens, 232-SSC filter, 233-SSC detector, 234-VSSC filter, 235-VSSC detector; 301-Fiber optic cable, 302-Fiber optic collimator, 303-Bandpass filter, 304-Dialect mirror, 305-Focusing lens, 306-Blue-violet detection module; 401-Planar lens. Detailed Implementation

[0081] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. With regard to numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0082] Example 1

[0083] This embodiment illustrates the basic structure and operating parameters of the flow cytometer for aggregate particle analysis of the present invention for realizing forward large tilt angle scattered light and lateral tilt angle VSSC detection.

[0084] like Figures 1-6 As shown, the flow cytometer for enhancing small particle scattering via tilt angle includes a liquid flow system 1, an optical system 2, and a control system 3. The liquid flow system 1 includes a sheath fluid unit 11, a sample injection control unit 12, and a waste liquid unit. Figure 5 As shown, the sheath fluid unit 11 includes a sheath fluid tank 111, a peristaltic pump 112, and a sheath fluid filter 113 connected in sequence, which are connected via a fluid conduit 114. The sample injection control unit 12 includes a sample loading needle 121 and a flow chamber 122. The outlet of the sample loading needle 121 is located on the central axis of the internal cavity of the flow chamber 122, and a quartz cup detection cell 214 is provided at the top of the flow chamber 122. The outlet of the sheath fluid filter 113 is connected to the side inlet of the flow chamber 122 via the fluid conduit 114. The upper part of the flow chamber 122 is a gradually narrowing cone shape. Figure 6As shown, the sample injection control unit 12 also includes a portion connected below the sample needle 121, comprising a plunger pump 123 and an injection coil 124 connected to each other. The outlet of the injection coil 124 is connected to one inlet of a three-way connector 125, and the other inlet of the three-way connector 125 is connected to the sample tube 127 via an injection conduit. The outlet of the three-way connector 125 is connected to the sample needle 121 via an injection conduit. Both injection conduits are equipped with solenoid valves 126. During the injection process, the connection direction is switched by controlling the opening and closing of the two solenoid valves 126 to achieve the aspiration and injection of sample liquid into the flow chamber.

[0085] The waste liquid unit includes a waste liquid buffer bottle 131, a diaphragm pump 132 and a waste liquid tank 133 connected in sequence. The inlet of the waste liquid buffer bottle 131 is connected to the outlet at the upper end of the flow chamber 122 through a conduit to collect the liquid flowing out of the flow chamber 122.

[0086] The plunger pump 123 and the injection coil 124 can precisely control the volume of sample liquid drawn up to 10-100 μL. The injection rate is precisely controllable and ranges from 10-120 μL / min.

[0087] In the sheath fluid unit 11, a peristaltic pump 112 draws sheath fluid from the sheath fluid tank 111 and provides a constant pressure to drive the flow of the sheath fluid. The rotational speed of the peristaltic pump 112 can be controlled to change the sheath fluid pressure, which determines the average flow velocity of the sheath fluid after hydrodynamic focusing. The pressure is substantially proportional to the flow velocity; for example, a pressure of 14 kPa results in a focused flow velocity of 2.5 m / s, and a pressure of 28 kPa results in a focused flow velocity of 5 m / s. Preferably, the sheath fluid pressure is set to 28 kPa. This will result in a focused flow velocity of 5 m / s in the quartz cup detection cell 214. Higher flow velocities facilitate more accurate and stable hydrodynamic focusing and a higher microparticle detection rate per second.

[0088] The sheath fluid flows into the flow chamber 122, forming a laminar flow that encapsulates the sample liquid exiting through the sample needle 121 in the core region without mixing. The sample liquid then passes through the gradually narrowing conical section at the top of the flow chamber 122. During this process, the microparticles in the sample liquid at the core of the flow are essentially arranged into a sequential flow of individual particles, achieving hydrodynamic focusing. Preferably, the inner diameter of the sample needle 121 is 300 μm. The injection volume velocity of the sample liquid determines the diameter of the core sample liquid flow after hydrodynamic focusing. The injection volume velocity is controlled by the plunger pump 123 of the injection control unit 12. For example, at an injection volume velocity of 10 μL / min, the diameter of the core sample liquid flow after focusing is 5 μm; at a maximum injection velocity of 120 μL / min, the diameter of the core sample liquid flow will reach approximately 20 μm.

[0089] After the sample liquid in the flow chamber 122 is hydrodynamically focused under the sheath fluid, it continues to flow upward into the rectangular quartz cup detection cell 214, where it is irradiated by the focused laser beam, generating various optical signals, including scattered light.

[0090] like Figure 1 As shown, the optical system 2 includes an excitation optical path 21, a forward detection optical path 22, and a lateral detection optical path 23. The excitation optical path 21 includes a first laser 211, a second laser 212, a direct first beam reflector 213, and a rectangular quartz cup detection cell 214. The forward detection optical path 22 includes a forward divergence lens group 221, a forward divergence bandpass filter 222, and a forward divergence detector 223 arranged sequentially. The lateral detection optical path 23 includes a lateral objective lens 231, multiple dichroic mirrors, an SSC filter 232, an SSC detector 233, a VSSC filter 234, and a VSSC detector 235.

[0091] like Figure 2 As shown, the working principle of the lateral detection optical path 23 is as follows: after the light beam passes through the lateral objective lens 231, it propagates through the optical fiber 301, passes through the optical fiber collimating lens 302, then through the dichroic mirror 304, and finally reaches the focusing lens 305 through the bandpass filter 303.

[0092] In the flow chamber 122, microparticles arranged into a single-particle queue by hydrodynamic focusing flow at a constant speed along the central axis of the liquid flow into the quartz cup detection cell 214, and flow sequentially through the two irradiation points of the laser beams emitted from the two lasers, which are also the focal points of the laser beams.

[0093] The first laser 211 and the second laser 212 integrate a shaping optical path, outputting a collimated elongated elliptical laser beam. The beams are focused over long distances in the X and Y directions by two integrated cylindrical mirrors 215 positioned in front of each laser. The focal point is located at the axis of the rectangular liquid flow chamber inside the quartz cup detection cell 214 at the rear end. After passing through the cylindrical mirrors 215, the first and second laser beams propagate forward in a straight line via a thick glass flat lens 401. Before reaching the quartz cup detection cell 214, the propagation direction of the first laser beam is perpendicular to the shorter side of the rectangular quartz cup detection cell 214, and therefore also perpendicular to the flow direction of the microparticle suspension sample in the detection cell. The second laser beam irradiates the quartz cup flow cell 214 at a horizontally offset angle of 20°. Figure 4 As shown. Before the first beam illuminates the quartz cup detection cell 214, a reflector 213 is placed in its optical path to change the propagation direction of the linear illumination beam. See Figure 3The angle between the reflector 213 and the direct first laser beam is 22.5°, so that the reflected beam illuminates the vertically upward-flowing single-particle queue at an angle of 45°.

[0094] The quartz cup detection cell 214 has an inner diameter of 430μm×180μm, and the laser beam is incident from the short side.

[0095] The pressure driving the sheath fluid flow is 28 kPa. After entering the flow chamber 121 and completing the fluid dynamics focusing, the average velocity of the fluid streamline is 5 m / s, which ensures high-throughput detection capability.

[0096] The first laser 211 emits light at a wavelength of 488 nm with a power of 50-100 mW. After focusing, the minor axis of the focused beam is parallel to the liquid flow direction, with a length of 9-15 μm, and the major axis is 70-80 μm, perpendicular to the liquid flow direction. The second laser 212 emits light at a wavelength of 405 nm with a power of 150-180 mW. After focusing, the minor axis of the focused beam is 10-18 μm, and the major axis is 95-105 μm, with the minor axis parallel to the liquid flow direction. Preferably, the first laser has a power of 100 mW. While avoiding direct exposure to high-brightness background photons, a high-power beam can generate stronger scattered light, which is beneficial for improving sensitivity. The second laser has a power of 150 mW.

[0097] The forward detection optical path 22 is located on the opposite side of the laser incident surface of the quartz cup detection cell 214, see Figure 3 As shown. The first laser 211 emits a beam at a 45° angle to irradiate the liquid flow, thus allowing for a convenient large-angle forward-facing detection optical path to be set up in a conventional forward-facing position without increasing structural complexity. The forward detection optical path 22 essentially collects the scattered light signal in a direction at a 45° angle relative to the laser incident direction. Another key aspect of the forward detection optical path 22 is the use of a forward lens group 221 to collect scattered light over a wide angle range. The forward lens group 221 is shown below. Figure 3 As shown, it consists of two identical aspherical plano-convex lenses arranged in a curved-to-curved manner. The lenses have a diameter of 25 mm, a numerical aperture (NA) > 0.6, a front and rear focal length of 11 mm, and an effective focal length of 20 mm. The front surface of the first lens is 11 mm from the laser illumination point, making the laser illumination point also the focal point of the front astigmatic lens group. This results in a collection half-angle of 36° for the first lens, allowing it to collect scattered light signals over a wide range, up to a solid angle of 72°. This significantly improves the scattered light collection efficiency, playing a crucial role in the effective detection of microparticles with diameters as low as 80 nm. The optical path diagram of the front astigmatic lens group is shown below. Figure 7 As shown.

[0098] In this process, the first laser beam irradiates the liquid flow at a 45° tilt angle. Most of the photons in the beam pass directly through the liquid flow without being scattered. These photons, which still propagate in a straight line along the original incident direction, will not enter the collection range of the designed forward divergence lens group 221. This avoids the strong background noise caused by the high-brightness beam and improves the sensitivity of the large-tilt-angle FSC detection channel. This also plays a key role in ensuring the sensitivity of detecting aggregate particles with a particle size of 80nm.

[0099] In this design, the rear surface of the second lens in the front-divergent lens group 221 is 12 mm away from the photosensitive surface of the front-divergent detector 223 (front-divergent APD detector). Therefore, after collecting the scattered light, the front-divergent lens precisely focuses it onto the photosensitive surface of the front-divergent detector 223, resulting in a focused spot size of <1 mm. 2 Preferably, the front diffuser detector 223 has a photosensitive surface area of ​​1 mm². 2 The silicon avalanche photodiode (APD) features high photoelectric conversion efficiency and low background noise. Its small photosensitive surface, when precisely focused and aligned in the optical path, can completely capture all collected and focused light with a size less than 1mm. 2 The reduced scattered light spot size, combined with the smaller photosensitive surface area, also reduces the detection of noise photons. These designs contribute to improved sensitivity / signal-to-noise ratio.

[0100] SSC detector 233 and VSSC detector 235 use a photosensitive surface area of ​​2mm². 2 The APD device of this invention. The actual operating voltage of all APD detectors in the device is typically in the range of approximately 100-200V, and can be set by the control system 3 according to the particle size and other characteristics of the measured microparticle sample to obtain optimized results. Specifically, in the control system 3, the actual operating voltage range of the APD (100-200V) is mapped to a value of 1-1000V, which is a numerical range that can be specified when setting the operating voltage of the APD detector. The bandpass specification of the forward divergence bandpass filter 222 in the forward detection optical path can be determined according to the wavelength of the laser used. In the device of this invention, the laser wavelength is 488nm, the power is 100mW, and the front divergence bandpass filter 222 is BP 488 / 10nm, which can filter out photons outside the wavelength range of 483nm-493nm, thereby removing all photons that may come from other lasers (such as a 405nm laser) and avoiding other noise photons outside the bandpass wavelength range. SSC filter 232 has the same specifications as front divergence bandpass filter 222. VSSC filter 234 has a specification of 405 / 10nm and can filter out photons with wavelengths outside the 400-410nm range.

[0101] Both the forward detection optical path 22 and the lateral detection optical path 23 use high-transmittance lenses, with a combined transmittance of >90%, thereby reducing the loss of light signals during propagation and ensuring the sensitivity of microparticle detection.

[0102] The flow cytometer for aggregate particle analysis based on the tilt angle-enhanced small particle scattering provided by the present invention can also control the first laser beam to illuminate the same position of the single-particle sample stream at a tilt angle other than 45° by controlling the angle and horizontal position of the reflector 213. Furthermore, by cooperating with different specifications of the forward-scattering lens group 221, the forward-scattered light collection angle and range can be controlled, thereby enabling the detection of scattered light at different angles and with different collection ranges using a design different from the optical path specifications of the present invention. This provides a flexible method for analyzing particles with different characteristics using scattered light carrying anisotropic information.

[0103] The control system 3 supports a particle detection rate of 30,000 particles per second, a signal sampling accuracy of 16 bits, and a sampling frequency of 10 MHz. Furthermore, the control system allows for the setting of individual detector voltages, thresholds, and injection speeds.

[0104] In the flow cytometer of this invention, a focused first laser beam illuminates the hydrodynamically focused single-particle sample stream at a 45° angle vertically. This, combined with a large-angle forward-scattering channel (FSC) for collecting scattered light signals, and the use of a high-efficiency silicon avalanche diode (APD) detector, effectively reduces background noise and improves light signal collection and photoelectric detection efficiency, thereby enhancing the sensitivity and resolution of light scattering detection. This design eliminates the traditional direct FSC detection channel, replacing it with a large-angle forward detector to better eliminate direct laser background and more fully utilize the larger range of scattered light signals with greater information content. A second laser beam illuminates the stream at a 20° horizontal angle, thus performing lateral scattering detection (VSSC) at a 70° lateral angle. A side objective with a numerical aperture (NA) > 1.15 is used to collect the large-range light signal. The SSC is provided by a 488nm laser, while the VSSC is provided by a shorter-wavelength 405nm laser. The scattered light generated by the shorter wavelength light source can more sensitively distinguish submicron-sized particles. The test results showed that this flow cytometer, which enhances light by tilt angle scattering, effectively detected standard microspheres with a particle size as small as 80 nm, filling the detection and counting gap in the particle size range of below 200 nm and above 80 nm. This provides a means to promote higher-level product quality control, safety and efficacy evaluation in fields such as biopharmaceuticals.

[0105] Example 2

[0106] The device provided in Example 1 was used to detect standard microspheres with a particle size of 80 nm to verify the device's performance in detecting small-diameter microparticles.

[0107] The particle size reference standard microspheres were detected according to the basic working method of the device and the principles and related parameters described in Example 1.

[0108] 1. Sheath fluid: Use pure water that has been filtered through 0.1μm beforehand.

[0109] II. Detection Methods

[0110] 1) After rinsing the sheath fluid tank three times with 1L of freshly prepared sheath fluid, fill the tank completely, start the device, and run the liquid system and optical system to rinse all pipelines for at least 1 hour, until the particle count is below 100 particles / s and basically stable under the detection conditions described in "3)" below and the VSSC threshold of 1000. During this rinsing process, fresh sheath fluid is used as a sample at a volumetric rate of 120 μL / min to ensure that the entire sample inlet pipeline is rinsed.

[0111] 2) When continuously loading and testing samples, the interval between samples should be 30 seconds.

[0112] 3) Testing conditions:

[0113] A first laser 211 with a wavelength of 488 nm is used; an FSC bandpass filter 222 is used as the first bandpass filter; an SSC bandpass filter 232 is used as the second bandpass filter; a second laser 212 with a wavelength of 405 nm is used; and a VSSC bandpass filter 234 is used as the third bandpass filter. The detector voltage mapping value in the FSC detection channel is set to 910V; the detector voltage mapping value in the SSC detection channel is set to 1000V; and the detector voltage mapping value in the VSSC detection channel is set to 1000V. The VSSC thresholds are set to 100 and 1000. Setting the threshold to 100 allows the distribution of background noise, primarily laser noise, to be observed when using 0.1 μm filtered pure water (i.e., sheath fluid) as a sample, thus delineating the noise region and allowing for observation of whether the detected standard microsphere signal can be distinguished from the noise. Setting the threshold to 1000 essentially eliminates laser noise when detecting standard microspheres. Data from 80 nm microsphere samples were collected at both thresholds.

[0114] The injection rate was set to 10 μL / min, and the sample was loaded and the data was collected.

[0115] III. Standard Microspheres:

[0116] Polypropylene (PS) microspheres from ThermoFisher, Inc. in the United States are traceable particle size reference microspheres that meet NIST standards. In this example, standard microspheres with a specification of 80 nm are used.

[0117] The standard microspheres used were diluted to a suitable concentration with 0.1 μm filtered pure water. Since the concentration of the original standard microspheres was unknown, a stepwise dilution method was adopted: vortex for 30 seconds before use to ensure thorough mixing, then add 2 μL of the stock solution to 4 mL of 0.1 μm filtered pure water for dilution. The sample was loaded using the parameters described in "3) Detection Conditions," but the VSSC threshold was set to 1000, and the loading rate was set to 10 μL / min. The microsphere count was observed. If the microsphere count was higher than 20,000 / s, an appropriate volume of 0.1 μm filtered pure water was added based on the displayed rate, and the sample was loaded again, observing the detection rate. This step was repeated until the microsphere detection rate dropped below 20,000 / s but not lower than 2,000 / s. At this point, no further dilution was performed, and the standard microspheres at this concentration were used for detection and data collection.

[0118] IV. Testing

[0119] The cleaning, startup, and parameter settings were completed according to the steps in "II. Detection Method" of this embodiment. Sheath fluid was used as a blank control sample for testing, and data were collected under VSSC thresholds of 100 and 1000 to determine background noise. Diluted 80nm microspheres were also used for testing, and data were collected under VSSC thresholds of 100 and 1000.

[0120] V. Results

[0121] See Figure 8 , Figure 9 . Figure 8 The results are for detecting sheath fluid blank control and 80nm microspheres under a VSSC threshold of 100. Figure 9 These are the detection results when the VSSC threshold is set to 1000. The measurement results using sheath fluid as a blank control indicate the distribution area of ​​system background noise in the FSC, VSSC, and SSC channels. Under the same conditions, when detecting 80nm standard microspheres, the distribution peak above the noise region represents the signal of the 80nm standard microspheres. It can be seen that in the VSSC channel, the 80nm microsphere signal can be clearly distinguished from the laser noise background region, indicating that the device can effectively detect and count 80nm standard microspheres. The SSC channel can also identify 80nm standard microspheres, but the distinction is slightly lower than that of the VSSC channel. The results show that, compared to existing flow cytometry aggregate impurity analyzers, the device provided by this invention can fill the gap in the 80nm-200nm particle size range.

[0122] Example 3

[0123] This embodiment illustrates a method for detecting various submicron-sized standard microspheres with particle sizes ranging from 100 nm to 1 μm using the device of the present invention, and a method for measuring the degree of signal discrimination between standard microspheres of different particle sizes.

[0124] The method in this embodiment is basically the same as that in Embodiment 2, except that:

[0125] 1) Set the SSC channel voltage mapping value to 955V;

[0126] 2) Set the VSSC channel voltage mapping value to 940V;

[0127] 3) Set the VSSC channel threshold to 100 and 400; the low threshold is also to determine the area where the laser noise is located.

[0128] 4) The standard microspheres used are polypropylene (PS) microspheres from Thermo Fisher Scientific, Inc., which are traceable particle size reference microspheres according to the US NIST standard, including 7 particle sizes: 100nm, 150nm, 200nm, 300nm, 500nm, 800nm, and 1μm. They were serially diluted according to the method described in Example 2, except that 800nm ​​and 1μm microspheres were initially drawn up in batches of 10μL for serial dilution.

[0129] 5) When continuously loading standard microspheres of various particle sizes, use a 2mL tube of fresh sheath solution as the sample between sample tubes to clean the injection tube until the signal from the previous sample tube no longer appears. This avoids contamination from different sample tubes.

[0130] See results Figures 10-13 . Figure 10 To use sheath fluid as a blank control, detection results were obtained under VSSC thresholds of 100 and 400 to determine the region of background noise in the system. In subsequent detection results using standard microsphere samples of different particle sizes, signals above this noise region represent the signals of microspheres of the corresponding particle size. Figures 11-12 Under the condition that the VSSC threshold is set to 400, standard microsphere samples with particle sizes of 100nm, 150nm, 200nm, 300nm, 500nm, 800nm ​​and 1μm are detected, and each particle size is detected separately. Figure 13The results were obtained by detecting a mixture of seven microspheres with particle sizes ranging from 100 nm to 1 μm, with VSSC thresholds set to 100 and 400, respectively. It can be seen that the VSSC and SSC channels can independently and clearly detect 100 nm diameter standard microspheres under the conditions of this embodiment, and the FSC channel can independently and clearly detect at least 150 nm diameter microspheres. Therefore, when the large-angle forward scattering light channel of the device provided by this invention is used independently, compared with existing flow cytometry aggregate impurity analyzers, it advances the detection limit of microparticles with a particle size of 200 nm to at least the level of 150 nm particle size.

[0131] from Figure 13 It can be observed that standard microspheres of different sizes exhibit varying degrees of differentiation in the forward scattered light (FSC) channel, the side scattered light (VSSC) channel, and the conventional SSC channel at tilt angles. This can be measured by calculating the discrimination index (D) between two types of microspheres of adjacent sizes. The formula for calculating this discrimination index is defined as follows:

[0132]

[0133] Median1 and Median2 are the median measurements of the distribution peaks of adjacent microspheres of two different particle sizes, and sd1 and sd2 are the standard deviations of their full peak widths. Since the particle size distribution of standard particles of the same size approximates a normal distribution, when the D value is greater than or equal to 2, the signal distribution regions of the two types of microspheres will be essentially completely distinguishable. Based on the measurement data of Example 3, the discrimination values ​​between pairs of adjacent standard microspheres in the three scattered light channels can be calculated, as shown in the table below.

[0134] Table 1. Discrimination index of measurement signals between a pair of microspheres with adjacent particle sizes.

[0135]

[0136] In the FSC channel, starting with 150nm microspheres, the microsphere signal can be distinguished from the system background noise. Therefore, the discrimination index is calculated pairwise starting with 200nm and 150nm microspheres. It can be observed that the discrimination between two adjacent microspheres in the 100nm–1μm particle size range exhibits different variations across the three scattering light channels at different angles. In the VSSC channel, the discrimination between standard microspheres of different sizes below 300nm is low, but the discrimination index increases significantly starting with the pair of 500nm and 300nm microspheres. In the SSC channel, the variation in the discrimination index between adjacent particle sizes is relatively small. In the FSC channel, the discrimination index generally varies considerably, but the distribution peaks of the 800nm ​​and 500nm microspheres are very close, exhibiting the lowest discrimination. However, this pair of microspheres shows the highest discrimination in both the SSC and VSSC channels. These phenomena are consistent with the intuitive judgment based on visual observation of the distribution patterns. Overall, the distinguishability between two standard microspheres of the same particle size varies significantly across different scattering channels, rather than exhibiting the similar distinguishability expected under isotropic uniform light emission conditions. Furthermore, the difference in these distinguishability indices increases with the size of the microspheres. This indicates that microspheres of different sizes possess anisotropic light emission modes, which can be revealed through detection using the three different scattering channels provided by this invention.

[0137] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A flow cytometer for analyzing aggregate particles by enhancing the scattering of small particles through tilt angle, characterized in that: It includes a fluid flow system (1), an optical system (2), and a control system (3); The optical system (2) includes an excitation optical path (21), a forward detection optical path (22), and a lateral detection optical path (23); the excitation optical path (21) includes a first laser (211), a second laser (212), a reflector (213), and a quartz cup detection cell (214), the reflector (213) being used to reflect the first laser beam directly emitted by the first laser (211); the forward detection optical path (22) includes a forward divergence lens group (221), a forward divergence bandpass filter (222), and a forward divergence lens group (221), arranged in sequence. The scatter detector (223) is located on the opposite side of the laser incident surface of the quartz cup detection cell (214); the lateral detection optical path (23) includes a lateral objective lens (231), and multiple dichroic mirrors are provided behind the lateral objective lens (231). One of the dichroic mirrors is provided with an SSC filter (232) and an SSC detector (233) on the optical path split off from it, and another dichroic mirror is provided with a VSSC filter (234) and a VSSC detector (235) on the optical path split off from it. The first laser (211) and the second laser (212) output collimated elongated elliptical laser beams, and the outgoing beams are focused over long distances in the X and Y directions by cylindrical mirrors (215) set in front of their respective lasers. The focal point is located at the axis of the rectangular liquid flow chamber inside the quartz cup detection cell (214). The first laser beam and the second laser beam output by the first laser (211) and the second laser (212) respectively pass through the cylindrical mirror (215) and then propagate forward in a straight line through the flat lens (401); before being reflected by the mirror (213), the propagation direction of the first laser beam is perpendicular to the short side optical surface of the quartz cup detection cell (214), and therefore also perpendicular to the flow direction of the microparticle suspension sample in the detection cell; the second laser beam illuminates the quartz cup detection cell (214) at a horizontally offset angle of 20°. The angle between the reflector (213) and the direct first laser beam is 22.5°, so that the reflected beam illuminates the vertically upward-flowing single-particle queue at an angle of 45°. The fluid flow system (1) includes a sheath fluid unit (11), a sample injection control unit (12), and a waste fluid unit; The sheath fluid unit (11) includes a sheath fluid tank (111), a peristaltic pump (112), and a sheath fluid filter (113) connected in sequence via a fluid conduit (114); the sample injection control unit (12) includes a sample loading needle (121) and a flow chamber (122), the outlet of the sample loading needle (121) is located on the central axis of the internal cavity of the flow chamber (122), and a quartz cup detection cell (214) is provided at the top of the flow chamber (122); the outlet of the sheath fluid filter (113) is connected to the side inlet of the flow chamber (122) via the fluid conduit (114); the upper part of the flow chamber (122) is a gradually narrowing cone shape; The injection control unit (12) also includes a plunger pump (123) and an injection coil (124) connected to each other. The outlet of the injection coil (124) is connected to one inlet of a three-way connector (125). The other inlet of the three-way connector (125) is connected to the sample tube (127) through an injection conduit. The outlet of the three-way connector (125) is connected to the sample needle (121) through an injection conduit. Both injection conduits are equipped with solenoid valves (126). During the injection process, the connection direction is switched by controlling the opening and closing of the two solenoid valves (126) to realize the aspiration and injection of sample liquid into the flow chamber.

2. The flow cytometer for aggregate particle analysis according to claim 1, characterized in that: The first laser (211) emits at a wavelength of 488 nm and a power of 50-100 mW. After focusing, the short axis of the laser beam is parallel to the direction of liquid flow and has a length of 9-15 μm. The long axis of the laser beam is 70-80 μm and is perpendicular to the direction of liquid flow. The second laser (212) emits at a wavelength of 405 nm and a power of 150-180 mW. After focusing, the short axis of the laser beam is 10-18 μm and the long axis is 95-105 μm. The short axis is parallel to the direction of liquid flow.

3. The flow cytometer for aggregate particle analysis according to claim 2, characterized in that: The front divergence lens group (221) is composed of two identical aspherical plano-convex lenses arranged in a curved-to-curved manner. The lens diameter is 25 mm, the numerical aperture NA is greater than 0.6, the front and rear focal lengths are 11 mm, and the effective focal length is 20 mm. The front surface of the first lens is 11 mm away from the laser irradiation point, so that the collection half angle of the first lens is 36°. The rear surface of the second lens of the pre-divergent lens group (221) is 12 mm away from the photosensitive surface of the pre-divergent detector (223). After collecting the scattered light, the pre-divergent lens precisely focuses it onto the photosensitive surface of the pre-divergent detector (223), and the size of the focused spot is <1 mm. 2 .

4. The flow cytometer for aggregate particle analysis according to claim 3, characterized in that: The SSC detector (233) and VSSC detector (235) have a photosensitive surface area of ​​2 mm². 2 APD devices.

5. The flow cytometer for aggregate particle analysis according to claim 4, characterized in that: The waste liquid unit includes a waste liquid buffer bottle (131), a diaphragm pump (132), and a waste liquid tank (133) connected in sequence. The inlet of the waste liquid buffer bottle (131) is connected to the outlet at the upper end of the flow chamber (122) through a conduit to collect the liquid flowing out from the flow chamber (122).

6. The flow cytometer for aggregate particle analysis according to claim 5, characterized in that: The control system (3) supports a particle detection speed of 30,000 particles / second, a signal sampling accuracy of 16 bits, and a sampling frequency of 10 MHz.

7. The operating method of the flow cytometer for aggregate particle analysis according to any one of claims 1-6, characterized in that: The first laser beam is incident at a 45° angle vertically onto a linearly flowing particulate sample liquid in a quartz cup detection cell. A horizontally placed forward scattering light channel is positioned on the opposite side of the laser incident surface in the detection cell. The forward scattering light signal emitted by the microparticles under illumination is collected and detected at a 45° angle relative to the laser beam, thereby obtaining a light scattering spectrum relating the intensity of the large-angle forward scattering light signal to the size of the microparticles. The side scattering light signals of the microparticles are collected and measured through the side scattering light channels SSC and VSSC, obtaining a light scattering spectrum relating the intensity of the SSC and VSSC signals to the size of the aggregate particles.

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