Optical control chip, sorting system and single cell screening method

Abstract

The application provides an optical control chip, a sorting system and a single cell screening method, and relates to the technical field of semiconductor optoelectronic devices. The optical control chip comprises a substrate, a longitudinal phototriode array, a microcavity array, an LSPR detection area and an isolation structure. The optical control chip adopts Si (1‑x) Ge x The longitudinal phototriode array with a heterojunction base region forms a wide-spectrum optoelectronic tweezers manipulation area, and is physically isolated from the LSPR detection area through the isolation structure, so as to avoid the crosstalk of dielectrophoresis electric field on the optical detection signal. Through the technical scheme provided in the embodiments of the application, the capture, detection, sorting and recovery can be closed-loop completed on a single chip without fluorescent labeling, so that the label-free and integrated functional screening is realized.

Description

Technical Field

[0001] This application relates to the field of semiconductor optoelectronic device technology, and in particular to a light control chip, a sorting system, and a single-cell screening method. Background Technology

[0002] In antibody discovery, drug screening, and cell line development, it is often necessary to screen cells from a large population of individual cells that can secrete specific functional molecules and possess target binding affinity or functional activity, while maintaining cell viability. This type of screening places high demands on the throughput of single-cell manipulation, the maintenance of cell viability, and the accuracy of functional indicator detection.

[0003] The mainstream method for single-cell sorting is flow cytometry, which sorts cells based on fluorescent labeling and scattering signals. Optical tweezers (OET) technology, on the other hand, activates photoelectric conversion structures within a chip under light illumination, generating a localized dielectrophoretic (DEP) electric field in the illuminated area. This allows for contactless capture and manipulation of micro / nano particles and cells using dielectrophoretic forces, providing a technological foundation for label-free, contactless single-cell manipulation. Furthermore, localized surface plasmon resonance (LSPR) technology can perform label-free detection of binding affinity between biomolecules based on the plasmon resonance spectral shift of nanostructure surfaces.

[0004] Chinese patent CN101135680B discloses an automated single-cell dielectric spectrum testing device assisted by photoinduced dielectrophoresis, which can realize the capture, localization and path planning control of single cells, but mainly involves cell dielectric spectrum testing and does not involve secretory function detection and in-situ affinity measurement; Chinese patent CN109991423B discloses a single-cell capture and secretory protein detection platform, which can perform high-throughput detection of single-cell secretory proteins, but does not involve photoelectric tweezers manipulation and LSPR detection; Chinese patent CN102175649B discloses an LSPR sensor chip for detecting the oncogene C-myc recombinant protein, which can achieve label-free detection by binding-induced spectral shift, but it is a standalone sensor chip, not integrated into a photoelectric tweezers chip, and does not perform in-situ measurement of the secretory products of captured live cells.

[0005] The existing technologies mentioned above have the following shortcomings: First, flow cytometry sorting relies on fluorescent labeling, and the labeling process may change the physiological state and functional expression of cells, and it is difficult to directly and in situ characterize the complex functional indicator of "secretory function + binding affinity" of cells; Second, existing photoelectric tweezers systems usually cannot achieve the entire process of "capture → functional detection → sorting → recovery" in a closed loop on a single chip or system, and often require multiple separate devices to be assembled, which is complicated to operate and results in greater cell loss; Third, existing commercial antibody discovery devices are expensive, and the throughput and automation of the separate assembly scheme are also limited.

[0006] Furthermore, attempting to integrate optoelectronic tweezers manipulation and in-situ LSPR detection onto a single chip faces an unresolved technical obstacle: the dielectric electrophoretic field generated by the optoelectronic tweezers inherently interferes with the LSPR optical detection process. On one hand, the dielectric electrophoretic field and its driving electrode structure may interfere with the LSPR optical detection signal; on the other hand, the metal nanostructures and optical components upon which LSPR detection relies may also interfere with the distribution of the dielectric electrophoretic field, thereby reducing the accuracy and stability of capture and detection. For this reason, existing technologies generally adopt avoidance strategies: either using separate devices to physically isolate manipulation and detection, or relying on indirect detection methods such as fluorescent labeling, failing to achieve stable integration of the two on a single chip for a long time.

[0007] In summary, there is an urgent need in this field for a method, system, and chip that can perform label-free, integrated functional screening and recovery of single cells that secrete specific functional molecules while maintaining cell activity and function, and overcome the crosstalk problem when integrating photoelectric tweezers manipulation and in-situ optical detection. Summary of the Invention

[0008] This application provides a light-controlled chip, a sorting system, and a single-cell screening method to solve the technical problems in the prior art where it is difficult to complete functional single-cell screening in a label-free closed loop on a single chip or a single system, and where there is crosstalk between the photoelectric tweezers dielectrophoretic electric field and the local surface plasmon resonance optical detection.

[0009] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, embodiments of this application provide a light control chip, including: Substrate; A vertically oriented phototransistor array is disposed on the substrate and has Si (1-x) Ge x The heterojunction base region is used to generate a dielectrophoretic electric field under light activation to form an opto-tweezers manipulation region; A microcavity array, located at the corresponding position of the photoelectric tweezers manipulation area, includes multiple microcavities, each of which is used to accommodate and position a single initial cell; The LSPR detection area is disposed at intervals on the same chip as the opto-tweezers manipulation area. The LSPR detection area includes multiple detection units, each corresponding to a microcavity. An isolation structure, including a dielectric passivation layer and an isolation trench structure, is located between the photoelectric tweezers manipulation area and the LSPR detection area.

[0010] Secondly, embodiments of this application provide a sorting system, including a light control chip, a detection module for performing in-situ local surface plasmon resonance detection on the secretory products of the initial cells to determine binding affinity; a control module for controlling the light control chip to capture, migrate, release, and recycle the initial cells and target cells; and an intelligent identification module, communicatively connected to the control module, for identifying, locating, and planning light manipulation paths for the initial cells.

[0011] Thirdly, embodiments of this application provide a single-cell screening method, comprising the following steps: S1: Using photoelectric tweezers, a dielectrophoretic electric field is generated in the photoelectric tweezers control area by light induction, and the initial cells are captured in a non-contact manner by dielectrophoretic force. S2: The initial cells are positioned one by one in the microcavities corresponding to the microcavity array, each microcavity is used to accommodate a single initial cell, and the secretory function of the positioned initial cells is detected on the light control chip; S3: Perform in situ LSPR detection on the secretory products of the initial cells to determine the binding affinity of the secretory products to the immobilized ligands; S4: Based on the results of the secretory function test and the results of the binding affinity test, target cells that simultaneously meet the preset functional conditions and preset affinity conditions are recovered from the initial cells.

[0012] Compared with the prior art, the technical solution disclosed in this application has the following beneficial effects: 1. Functionality-first, label-free: Integrated detection of "secretory function + binding affinity" can be performed without fluorescent labeling, avoiding the impact of the labeling process on cell activity and functional expression; 2. Integrated closed loop: The entire process of "capture → detection → sorting → recycling" is realized in a closed loop on a single chip or a single system, which replaces the solution of relying on multiple separate devices to achieve the same result in the existing technology, simplifies the operation process and reduces cell loss; 3. Bidirectional isolation and on-chip integration: By setting an isolation structure between the optoelectronic tweezers manipulation area and the LSPR detection area, and by utilizing the spatial constraint of the driving electric field by the dielectric passivation layer and the physical blockage of the coupling path by the isolation trench structure, the crosstalk problem between the dielectric electrophoretic electric field and the optical detection signal is overcome from the physical root of crosstalk generation, providing a stable and feasible technical path for the stable integration of the two on the same chip. Attached Figure Description

[0013] The exemplary embodiments of this application can be more fully understood by referring to the following accompanying drawings. The drawings are provided to further illustrate the embodiments of this application and form part of the specification. They are used together with the embodiments of this application to explain this application and do not constitute a limitation thereof. In the drawings, the same reference numerals generally represent the same components or steps.

[0014] Figure 1 A flowchart illustrating a single-cell screening method provided in this application; Figure 2 A general block diagram of a sorting system provided in this application; Figure 3 This is a schematic diagram of the structure of a light control chip provided in this application; Figure 4 This is a graph showing the relationship between secretion induction time, enrichment signal, and the proportion of positively identified cells in Example 1 of this application. Figure 5 A dose-response diagram of the effect of the candidate compounds in Example 2 of this application on the secretion function of cytokine (IL-2); Figure 6 The expression level distribution of CHO-K1 cells in Example 3 provided in this application.

[0015] Figure Labels 100—Substrate; 110—Vertical phototransistor array; 111—Photoelectric tweezers manipulation area; 120—Isolation structure; 121—Dielectric passivation layer; 122—Isolation trench structure; 130—LSPR detection area; 140—Microcavity array; 141—Microcavity; 200—Optical control chip; 300—Detection module; 400—Control module; 410—Digital micromirror device; 500—Intelligent recognition module; 600—Electroporation auxiliary module. Detailed Implementation

[0016] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the present application.

[0017] Those skilled in the art will understand that the terms "first," "second," etc., in the embodiments of this application are only used to distinguish different steps, devices, or modules, and do not represent any specific technical meaning, nor do they indicate a necessary logical order between them. It should also be understood that in the embodiments of this application, "multiple" can refer to two or more, and "at least one" can refer to one, two, or more. Furthermore, the term "and / or" in this application is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone.

[0018] In the description of this application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0019] In the description of this application, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "setting," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. It should be understood that, in the description of the embodiments of this application, unless otherwise stated, " / " means "or," for example, A / B can mean A or B. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0020] In the description of this application, it should be noted that the term "initial cell" refers to a single cell to be screened, including but not limited to antibody-secreting B cells, immune cells, tumor cells, CHO cells, HEK293 cells, etc.

[0021] In the description of this application, it should be noted that the term "photoelectric tweezers (OET)" refers to photoinduced dielectrophoresis, a technique that uses photogenerated charges to form virtual electrodes and manipulates microparticles and living cells without contact using dielectrophoretic force.

[0022] In the description of this application, it should be noted that the term "vertical phototransistor array" refers to an array with a base region of Si. (1-x) Ge x A pixelated photoelectric converter array of a heterojunction is used to convert incident light into a dielectric electrophoretic field.

[0023] In the description of this application, it should be noted that the term "LSPR detection region" refers to an optical sensing region that integrates nanostructures and characterizes binding events with spectral shifts, where LSPR is short for Localized Surface Plasmon Resonance.

[0024] In the description of this application, it should be noted that the term "isolation structure" refers to the insulation and shielding structure disposed between the photoelectric tweezers manipulation area and the LSPR detection area, used to isolate crosstalk between the electric field and the optical signal.

[0025] In the description of this application, it should be noted that the term "microcavity array" refers to a positioning structure arranged according to single-cell capacity, including multiple microcavities, each of which is used to accommodate a single initial cell.

[0026] In the description of this application, it should be noted that the term "dielectric force" refers to the translational force experienced by dielectric particles in a non-uniform electric field.

[0027] Furthermore, it should be understood that, for ease of description, the dimensions of the various parts shown in the accompanying drawings are not drawn to actual scale. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this application or its application or use. Techniques, methods, and devices known to those skilled in the art will not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0028] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] refer to Figure 2 and Figure 3, the present application provides a sorting system, comprising an optically controlled chip 200, a detection module 300, a control module 400, and an intelligent identification module 500. The optically controlled chip 200 is configured to generate a dielectrophoretic electric field through light-induced dielectrophoretic force, so as to capture initial cells in a non-contact manner by the dielectrophoretic force; a microcavity array 140 is arranged on the optically controlled chip 200, and is configured to position a single initial cell and perform on-optically-controlled-chip secretion function detection; the detection module 300 is configured to perform in-situ localized surface plasmon resonance detection on the secretion product of the initial cell positioned in the microcavity array 140; the detection module 300 may include a light source, a spectroscopic element, and a spectrum detector (such as a spectrometer or a CCD detector), which irradiates the LSPR detection region 130 with a broadband light source and collects a plasmon resonance spectrum, and determines the binding affinity by monitoring the displacement of the resonance peak position; the control module 400 is configured to control the optically controlled chip 200 to capture, migrate, release the initial cells and recover target cells, wherein the control module 400 may include a digital micromirror device (DMD), which is configured to generate a dynamic light spot pattern and realize collision-free path planning of the optical manipulation path; the intelligent identification module 500 is communicatively connected with the control module 400, and is configured to identify and position the initial cells based on an image recognition algorithm (such as a semantic segmentation algorithm), and output cell position and sorting decision information to the control module 400. The system processing flow includes: collecting a microscopic image, extracting cell boundaries through semantic segmentation and calculating cell center coordinates, planning an optical manipulation path in combination with the arrangement position of the microcavity array 140, and then converting the path into a light spot coordinate sequence of the digital micromirror device 410, so as to realize collision-free path planning in a multi-cell parallel scenario.

[0031] In some embodiments, the system further comprises an electroporation auxiliary module 600, which is configured to assist the substance delivery of initial cells or the manipulation of cell membrane permeability, for example, to assist operations such as on-chip induced secretion or subsequent gene delivery. As an optional functional module, this auxiliary module is configured along with the main scheme, and does not change the core technical solutions of the method and the chip of the present application.

[0032] With continued reference to Figure 3 , the optically controlled chip 200 includes a substrate 100, a vertical phototriode array 110, an LSPR detection region 130, and an isolation structure 120.

[0033] The vertical phototriode array 110 is arranged on the substrate 100, and is configured with Si (1-x) Ge x heterojunction base region, where 0<x<1, preferably x ranges from 0.1 to 0.4, so as to balance wide spectral response and photoelectric conversion efficiency, and generate a dielectrophoretic electric field in the illuminated region under light activation, thereby forming an optoelectronic tweezers manipulation region 111. Compared with the conventional amorphous silicon (a-Si) photoconductive layer structure, the adoption of Si (1-x) Ge xThe heterojunction base region helps to broaden the response spectrum range of the longitudinal phototransistor array 110, enabling the response spectrum to cover the visible to near-infrared bands. This allows for addressing and manipulating different regions using light sources of various wavelengths, and is compatible with the light source bands required for subsequent LSPR detection.

[0034] The embodiments of this application can be applied to single-cell functional screening in medium-conductivity media such as conventional physiological buffers and cell culture media to ensure the signal-to-noise ratio and functional closed-loop integrity of in situ LSPR detection. For application scenarios that require large-scale parallel capture in high-conductivity media, this can be achieved by adjusting the structure and driving parameters of the longitudinal phototransistor array, which does not constitute a limitation of this application.

[0035] The LSPR detection region 130 and the photoelectric tweezers manipulation region 111 are spaced apart on the same optical control chip 200 for in-situ LSPR detection of secretory products from initial cells. The LSPR detection region 130 contains multiple detection units, each corresponding to a microcavity. These detection units are plasma-excited nanostructures carrying specific recognition ligands, used to capture secretory products and generate resonant shift signals detectable by spectroscopy. An isolation structure 120 is disposed between the photoelectric tweezers manipulation region 111 and the LSPR detection region 130 to isolate crosstalk of the dielectrophoretic electric field to the optical detection signal within the LSPR detection region 130, and / or to isolate interference from the metal nanostructures within the LSPR detection region 130 on the dielectrophoretic electric field distribution. In some embodiments, the isolation structure 120 includes at least one of a dielectric passivation layer 121 and an isolation trench structure 122: wherein the dielectric passivation layer 121 may be made of a dielectric material such as silicon nitride or silicon oxide to cover and passivate the driving electrode of the optoelectronic tweezers manipulation region 111 to reduce its electric field leakage to the LSPR detection region 130; the isolation trench structure 122 may be formed by etching a physical isolation trench in the substrate 100 between the two regions, with an isolation depth of about 2 micrometers to 20 micrometers, preferably 5 micrometers to 10 micrometers, to further block the electric field coupling path.

[0036] To address the aforementioned crosstalk obstacle, in a preferred embodiment, the isolation structure 120 includes a dielectric passivation layer 121 and an isolation trench structure 122. Addressing the physical root cause of crosstalk, the obstacle is overcome through the synergistic effect of spatial constraint and physical blocking. On one hand, the dielectric passivation layer 121 covers and passivates the driving electrode of the photoelectric tweezers manipulation region 111, confining the dielectric force driving electric field within the local space of the photoelectric tweezers manipulation region 111, suppressing its extension and leakage towards the LSPR detection region 130, thereby reducing the modulation disturbance of the driving electric field on the local surface plasmon resonance electromagnetic field within the LSPR detection region 130. On the other hand, the isolation trench structure 122 forms an isolation trench within the substrate 100 between the two regions, cutting off the coupling path of the dielectric force driving signal through the driving electrode and substrate 100 to the LSPR detection region 130, and preventing photogenerated carriers from laterally migrating into the region near the LSPR detection region 130 within the substrate 100, thus avoiding changes to the local dielectric environment and optical response of the LSPR detection region 130. Under the combined effect of the above mechanisms, the plasmon resonance spectrum in the LSPR detection region 130 remains stable during photoelectric tweezer manipulation, ensuring the signal-to-noise ratio and affinity measurement accuracy of in-situ LSPR detection. At the same time, due to the effective structural isolation between the photoelectric tweezer manipulation region 111 and the LSPR detection region 130, the metal nanostructure in the LSPR detection region 130 will not reverse the dielectric electrophoretic electric field distribution of the photoelectric tweezer manipulation region 111, realizing bidirectional non-disruptive integration of simultaneous capture manipulation and in-situ detection.

[0037] It should be noted that those skilled in the art typically expect that crosstalk problems can be solved by increasing the physical distance between the optoelectronic tweezers manipulation area and the LSPR detection area, or by using conventional shielding methods such as an integral metal shielding layer. However, the former significantly reduces chip integration and lengthens the conduction path of secreted products, while the latter may interfere with the LSPR's own optical detection process due to additional optical reflection and scattering introduced by the metal shielding layer. This application employs a solution combining local dielectric passivation and micron-level isolation trenches to achieve crosstalk suppression without significantly increasing the chip area or introducing additional optical interference.

[0038] To verify the effectiveness of the isolation structure 120 in suppressing crosstalk between the dielectrophoretic electric field and the LSPR optical detection signal, four types of chips were fabricated under the same conditions: no isolation structure (control chip), only dielectric passivation layer 121, only isolation trench structure 122, and a combination of dielectric passivation layer 121 and isolation trench structure 122 (preferred embodiment of this application). The resonance spectral shift and detection signal-to-noise ratio of the LSPR detection region 130 were measured during opto-tweezers manipulation and during silent operation, respectively. The results are as follows: Table 1: Resonance Spectral Drift and Detection Signal-to-Noise Ratio Test conditions LSPR detection region resonance spectral drift nm Detecting signal-to-noise ratio (SNR) No isolation structure (comparison chip) 2.3 ± 0.6 8.1 Dielectric passivation layer only (no deep trench) 0.9 ± 0.3 15.4 Isolation trench only (no dielectric passivation layer) 0.7 ± 0.2 17.2 Dielectric passivation layer + isolation trench 0.15 ± 0.05 42.6 Data shows that using either the dielectric passivation layer 121 or the isolation trench structure 122 alone can reduce crosstalk to a certain extent, but when the two are used together, the crosstalk suppression effect is significantly better than the simple superposition of the two methods (the spectral drift decreased from 2.3 nm in the control group to 0.15 nm, a reduction of about 93.5%; the signal-to-noise ratio was improved by about 5.3 times).

[0039] The microcavity array 140 is correspondingly configured with the photoelectric tweezers manipulation area 111. Each microcavity 141 in the microcavity array 140 is used to accommodate a single initial cell and provide a microenvironment for local culture, secretion enrichment, and in-situ detection of the cell. The characteristic size of each microcavity 141 is 20 micrometers to 200 micrometers, preferably 30 micrometers to 80 micrometers, to adapt to the single-cell containment requirements of typical mammalian cells; the array pitch of the microcavity array 140 is 50 micrometers to 500 micrometers, preferably 100 micrometers to 200 micrometers; and the array density is 102. 3 Up to 10 6 One microcavity per square centimeter.

[0040] The packaging of the light control chip 200 can be achieved using dry film photoresist or UV-curable adhesive. The packaging layer thickness is approximately 10 micrometers to 100 micrometers, which is used to define the cavity structure of the microcavity array 140 and achieve a sealed connection between the chip and the top cover plate.

[0041] See Figure 1 The steps of the single-cell screening method are as follows: Step S1: Capture initial cells using photoelectric tweezers. Based on the cell position information output by the intelligent recognition module 500, the control module 400 projects a light spot at the corresponding position of the longitudinal phototransistor array 110 of the light control chip 200 through the digital micromirror device in the control module 400. This activates the local phototransistors to generate a dielectric electrophoretic field, thereby capturing the initial cells suspended in the chip channel in a contactless manner and migrating them to the designated microcavity 141 in the microcavity array 140 along the planned collision-free light manipulation path.

[0042] Step S2: Detect the secretory function of the initial cells on the light-controlled chip. After the initial cells are located in microcavity 141, they are maintained in the culture environment on the light-controlled chip for a preset secretion induction time to induce the secretion of target functional molecules (e.g., antibodies). The secreted products are enriched in the local microenvironment of microcavity 141, and the secretory function can be preliminarily determined by preliminary fluorescence-independent recognition methods (e.g., changes in turbidity, refractive index, or preliminary binding signals with the preset capture layer in microcavity 141).

[0043] Step S3: In-situ LSPR detection is performed on the optical control chip. After the secretory product is enriched in the microcavity 141, the control module 400 controls the chip to guide or diffuse the secretory product in the microcavity 141 to the adjacent LSPR detection region 130, where it specifically binds to the pre-immobilized ligands (e.g., antigens) in the LSPR detection region 130. The binding affinity (e.g., dissociation constant Kd) between the secretory product and the immobilized ligand is determined by monitoring the plasmon resonance spectral shift of the LSPR detection region 130. The detection wavelength range is 500 nm to 900 nm, preferably 600 nm to 800 nm. Because an isolation structure 120 is provided between the photoelectric tweezers manipulation region 111 and the LSPR detection region 130, crosstalk of the dielectrophoretic electric field to the LSPR optical detection signal is suppressed, thereby ensuring the signal-to-noise ratio and accuracy of in-situ detection.

[0044] Step S4: Recover target cells. Based on the secretory function detection results of Step S2 and the binding affinity determination results of Step S3, the control module 400 determines whether preset functional conditions (e.g., detection of target secreted products) and preset affinity conditions (e.g., binding affinity is better than a preset threshold) are simultaneously met. For positive target cells that simultaneously meet the above conditions, they are released from the microcavity 141 and migrated to the recovery area using photoelectric tweezers for subsequent downstream applications such as sequencing, cloning, or drug response evaluation. For cells that do not meet the conditions, they can be selectively released or discarded.

[0045] Example 1: Antibody Discovery Single B cells capable of secreting high-affinity, specific antibodies were screened from animal-derived or humanized B cell banks following immunization. The procedure was as follows: single B cells were captured using photoelectric tweezers and localized within microcavity 141 → antibody secretion was induced in a light-controlled chip culture environment → the binding and affinity between the secreted antibody and the immobilized antigen within the adjacent LSPR detection region 130 of microcavity 141 were measured using in situ LSPR detection → positive B cells meeting both secretory function and affinity criteria were recovered for subsequent single-cell sequencing or clonal expression. The cell loading density was approximately 10-1. 4 Up to 10 6 Cells / mL; microcavity 141 size is approximately 30 to 80 micrometers; secretion induction time is approximately 30 minutes to 2 hours; detection wavelength is approximately 600 to 800 nanometers; detection sensitivity can resolve sub-nanomolar differences in affinity.

[0046] Experimental procedure: Using a suspension of B cells derived from the spleen of BALB / c mice immunized with ovalbumin (OVA) as an example, the cell loading concentration was 3 × 10⁻⁶. 5 Cells / ml were captured by the light control chip 200 and positioned in the microcavity array 140. The feature size of the microcavity 141 was 50 micrometers. The capture efficiency and control parameters of the photoelectric tweezers are shown in Table 2.

[0047] Table 2: Photoelectric tweezers capture efficiency data parameter numerical values Number of capture attempts per session 500 cells Successfully captured and located the number of microcavities 462 Capture success rate 92.4% Average single-cell capture time 1.8 seconds / cell Dielectrophoresis force driving voltage 8V (peak-to-peak value) Drive frequency 300 kHz Cell viability 24 hours after capture 95.1% (Pantene Blue Rejection Method) To investigate the effect of secretion induction time on antibody secretion efficiency on the light-controlled chip, the initial antibody enrichment signal and the proportion of positive recognition cells in the microcavity were detected at different induction times. (Reference) Figure 4 In BALB / c mice, spleen-derived B cells showed an enrichment signal of approximately 0.8 after 15 minutes of induction, corresponding to a positive recognition cell ratio of 3.2%. With prolonged induction time to 30 and 60 minutes, the enrichment signal rapidly increased to 2.1 and 4.7, respectively, with the positive proportions increasing to 11.6% and 24.3%. At 120 minutes of induction, the enrichment signal reached 8.9, with a positive proportion of 31.8%, after which it tended to saturate. At 240 minutes of induction, the enrichment signal was 9.6, with a positive proportion of 33.1%, showing only a slight increase compared to 120 minutes. This indicates that the secretion signal rapidly increases between 60 and 120 minutes, and tends to saturate after 120 minutes.

[0048] In situ LSPR affinity assay: Taking the immobilized OVA antigen in the LSPR detection zone 130 adjacent to microcavity 141 as an example, the in situ binding kinetics of the secreted products in 31 initially positive microcavities were detected. The results are shown in Table 3. Table 3: Dynamic Detection Data Microcavity numbering Resonance peak shift (nm) Fitting the dissociation constant Kd (nM) determination 07 3.2 0.42 Positive (high affinity) 12 1.1 8.7 Negative 19 4.6 0.18 Positive (high affinity) 23 0.4 >50 Negative 28 3.9 0.31 Positive (high affinity) Using a preset affinity threshold Kd ≤ 1 nM as the criterion, 9 out of 31 initially positive microcavities in this round of screening (29.0%) ultimately met both the secretory function and affinity criteria and entered the recovery process. The detection sensitivity was verified to be able to resolve Kd differences on the order of approximately 0.1 nM.

[0049] Nine positive microcavities were released and recovered by photoelectric tweezers, and the paired heavy / light chain variable region sequences were obtained by single-cell RT-PCR amplification. After transfection and expression, the sequences were retested by ELISA. The retest results of seven (77.8%) were consistent with the in situ LSPR screening results (binding activity met the preset threshold), while the two failed the retest.

[0050] Example 2: Drug Screening Under the influence of candidate drugs, cellular functional responses are screened at the single-cell level to evaluate the effects of candidate drugs on cellular secretory function, viability, or activation of specific signaling pathways. The procedure includes: capturing and positioning initial cells (e.g., immune cells or tumor cells) pretreated with or co-incubated with candidate drugs into microcavities 141 using photoelectric tweezers → maintaining the drug administration or co-culture conditions for a preset duration on a light-controlled chip → performing in-situ detection of functional readout indicators of the initial cells (e.g., cytokine secretion levels, metabolite release, or binding activity with immobilized target molecules), where binding activity can be measured through the LSPR detection region 130 → graded recovery of target cells based on functional response results for subsequent analysis of the dose-response relationship or mechanism of action of candidate drugs.

[0051] Using Jurkat T cell line as a model, the effects of candidate compounds on cytokine (IL-2) secretion were investigated. The experiment was set up in three groups: (1) negative control group, with only culture medium added; (2) positive control group, with PMA / ionomycin added to verify the cell secretion function and the effectiveness of the detection system; (3) candidate compound X gradient concentration group, with 0.1 μM, 1 μM and 10 μM of candidate compound added respectively to investigate the dose-response relationship of candidate compound on IL-2 secretion.

[0052] To investigate the dose-response relationship between candidate compounds and IL-2 secretion function, the number of microcavities in each group was n=60. The detection results are as follows: Figure 5 As shown in the figure, the IL-2 binding signal in the negative control group was 0.3±0.1, with a positive secretory cell ratio of 2.1%; the binding signal in the positive control group (PMA / ionomycin stimulation) increased to 5.8±1.2, with a positive ratio of 68.3%, validating the cell secretion function and the effectiveness of the detection system. The binding signals of the candidate compound treatment groups at 0.1μM, 1μM, and 10μM were 1.1±0.4, 3.4±0.9, and 4.9±1.1, respectively, corresponding to positive secretory cell ratios of 15.7%, 41.2%, and 59.8%. Figure 5 It is evident that the candidate drug exhibits a concentration-dependent activation effect on cytokine secretion, consistent with the dose-response trend obtained from traditional ELISA / flow cytometry.

[0053] For consistency comparison with traditional methods, parallel samples from the same batch of cells were used as methodological controls, and the concentration of IL-2 in the culture supernatant was detected by conventional ELISA. (See Table 4) Table 4: Comparison of IL-2 concentration in culture supernatant detected by conventional ELISA method Grouping The positive rate of this application method ELISA positive rate negative control 2.1% 1.8% Positive stimulation 68.3% 71.5% Candidate drug 10μM 59.8% 63.2% The difference in the positive rate between the two methods was within 5 percentage points, which initially supports the consistency between the method in this application and the existing gold standard method.

[0054] Example 3: Cell line development From transfected or cloned cell populations, monoclonal cell lines that express target proteins (e.g., therapeutic antibodies or recombinant proteins) with high yield and stability are screened. The procedure may include: capturing and positioning candidate monoclonal cells one by one within microcavity 141 using photoelectric tweezers → maintaining a preset expression period in a light-controlled chip culture environment → performing in situ LSPR detection on the target protein secreted by cells in each microcavity 141 to determine the binding signal intensity with the immobilized capture molecule, serving as an evaluation indicator of expression level and binding activity → recovering positive monoclonal cells with high expression levels and binding activity meeting preset conditions for subsequent expansion culture and cell line stability verification.

[0055] Experimental content: Taking a CHO-K1 cell bank carrying the target antibody gene transfected with lentivirus as an example, the expression level of the cell population after transfection is heterogeneous, and high-yield clones need to be screened from it.

[0056] The expression levels of CHO-K1 in the transfected cell population were statistically analyzed and graded. The results are as follows: Figure 6 As shown, the proportion of high-expression microcavities with LSPR binding signal intensity of not less than 4.0 nm was 8.3%, corresponding to an estimated extracellular secretion rate of not less than 25 pg / cell / day; the proportion of medium-expression microcavities with signal intensity between 1.5 nm and 4.0 nm was 34.6%, corresponding to a rate of approximately 8 to 25 pg / cell / day; and the proportion of low-expression or non-expression microcavities with signal intensity below 1.5 nm was 57.1%, corresponding to a rate of less than 8 pg / cell / day.

[0057] Screening was performed under preset conditions (high expression level), and 103 positive monoclonal cells (8.6%) were recovered from 1200 effective microcavities, which is basically consistent with the aforementioned statistical proportion of 8.3%. Twenty clones were randomly selected from the 103 recovered positive clones for amplification culture (14 days), and the actual secretion rate was determined using Protein A HPLC. The results showed that the average secretion rate of the 20 clones was 31.2 ± 6.4 pg / cell / day. The correlation coefficient R² between this measured result and the in situ LSPR prediction result was 0.81, indicating good consistency between the two. Compared with the average secretion rate of randomly selected clones after conventional batch transfection (historical data comparison, approximately 12 pg / cell / day), the secretion rate of clones screened by the method of this application was increased by approximately 2.6 times.

[0058] In some embodiments, the method and system of this application may also be implemented in conjunction with the following auxiliary technical solutions. These solutions are preferred or optional implementations and do not constitute a limitation on the core technical solutions of this application: (1) Dry film or UV-curable adhesive encapsulation process is used for the encapsulation of the light control chip 200. The specific process parameters (such as exposure dose, curing temperature and time) should be determined according to the selected material system. (2) A collision-free optical manipulation path planning algorithm based on digital micromirror device 410 is used to avoid mutual interference of light spot paths in multi-cell parallel manipulation scenarios. (3) A cell identification and localization method based on semantic segmentation algorithms such as YOLO is used to improve the identification accuracy of the intelligent identification module 500 for the initial cell boundary and position; (4) Electroporation assist module 600, used to assist in the delivery of substances to the initial cells or the manipulation of membrane permeability.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application.

Claims

1. A light control chip, characterized in that, include: Substrate; A vertically oriented phototransistor array is disposed on the substrate and has Si (1-x) Ge x The heterojunction base region is used to generate a dielectrophoretic electric field under light activation to form an opto-tweezers manipulation region; A microcavity array, located at the corresponding position of the photoelectric tweezers manipulation area, includes multiple microcavities, each of which is used to accommodate and position a single initial cell; The LSPR detection area is disposed at intervals on the same chip as the opto-tweezers manipulation area. The LSPR detection area includes multiple detection units, each corresponding to a microcavity. An isolation structure, including a dielectric passivation layer and an isolation trench structure, is located between the photoelectric tweezers manipulation area and the LSPR detection area.

2. The light control chip according to claim 1, characterized in that, The Si (1-x) Ge x In the heterojunction base region, the value of x ranges from 0.1 to 0.

4.

3. A sorting system, characterized in that, The device includes the light-controlled chip as described in any one of claims 1 to 2, and further includes a detection module for performing in-situ local surface plasmon resonance detection on the secretory products of the initial cells to determine the binding affinity; a control module for controlling the capture, migration, release, and retrieval of the initial cells by the light-controlled chip; and an intelligent identification module, which is communicatively connected to the control module, for identifying, locating, and planning the light manipulation path of the initial cells.

4. The sorting system according to claim 3, characterized in that, The control module includes a digital micromirror device for generating dynamic light spot patterns and, based on the initial cell position information output by the intelligent recognition module and the arrangement position of the microcavity array, planning a collision-free light manipulation path and converting the light manipulation path into a light spot coordinate sequence of the digital micromirror device.

5. A single-cell screening method, said method being applied to the light-controlled chip as described in any one of claims 1 to 2, characterized in that, Includes the following steps: S1: Using photoelectric tweezers, the initial cells are captured in a non-contact manner within the photoelectric tweezers manipulation area; S2: The initial cells are positioned one by one in the microcavities corresponding to the microcavity array, each microcavity is used to accommodate a single initial cell, and the secretory function of the positioned initial cells is detected on the light control chip; S3: Perform in situ LSPR detection on the secretory products of the initial cells to determine the binding affinity of the secretory products to the immobilized ligands; S4: Based on the results of the secretory function test and the results of the binding affinity test, target cells that simultaneously meet the preset functional conditions and preset affinity conditions are recovered from the initial cells.

6. The single-cell screening method according to claim 5, characterized in that, The initial cell is an antibody-secreting B cell, the secretion product is an antibody, and the immobilized ligand is an antigen.

7. The single-cell screening method according to claim 5, characterized in that, In step S2, the secretion function detection includes: enriching and preliminarily identifying the secretion products secreted by the initial cells in the culture environment on the light-controlled chip within a preset secretion induction time.

8. The single-cell screening method according to claim 5, characterized in that, In step S3, the in-situ LSPR detection is performed within the LSPR detection region.

9. The single-cell screening method according to claim 5, characterized in that, In step S4, the recycling includes: using the photoelectric tweezers to release the target cell from the microcavity and migrate it to the recycling area along a planned light manipulation path.

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