Single-molecule sensing array module, quantitative analysis chip, and quantitative analysis method

Abstract

The present invention provides a single-molecule sensing array module, a quantitative analysis chip, and a quantitative analysis method. High-throughput parallelized single molecule excitation and sensing can be implemented, and the detection sensitivity limit can reach the single-molecule level. A standard curve can be formed for an optical signal of an ultra-low-concentration target molecule; the problems of uneven optical signal intensity and poor repeatability can be solved by using a digital processing method; accurate quantitative analysis for ultra-low-concentration molecules can be implemented; the detection sensitivity can be improved, and the sensitivity level reaches the single molecule level. The present invention has great application value in the fields of clinical medicine, life sciences, food testing, environmental protection, national defense and public safety, fundamental research, etc. The present invention can also be applied to single-molecule immunoassays to achieve ultra-low-limit-of-detection single-molecule immunoassays.

Description

Single-molecule sensing array module, quantitative analysis chip and quantitative analysis method Technical Field

[0001] This invention relates to the field of single-molecule detection technology, and in particular to a single-molecule sensing array module, a quantitative analysis chip, and a quantitative analysis method. Background Technology

[0002] Single-molecule detection is a disruptive technology in the field of biomarker detection, reaching the limits of molecular detection and representing a long-term goal. It has significant application value in many areas, including the rediscovery of traditional biomarkers, the development of new biomarkers, and new drug development. Currently, single-molecule detection mainly employs atomic force microscopy (AFM) and laser-induced fluorescence (LAF). LAF is the most efficient method for single-molecule detection, particularly suitable for studying single molecules in solution. However, traditional LAF techniques for single-molecule detection often have low throughput, making it difficult to improve detection efficiency and accuracy. High throughput refers to a high level of data or information processed within a given time, which can greatly accelerate data generation and processing, leading to a deeper understanding of many scientific questions and driving technological progress. Furthermore, in unknown and complex backgrounds, accurate quantitative analysis of ultra-low concentration target molecules is difficult to achieve with single-molecule sensitivity, and single-molecule signal intensities are often non-uniform and non-repeatable. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a single-molecule sensing array module, a quantitative analysis chip, and a quantitative analysis method to solve at least one of the following problems in the prior art: low throughput of laser-induced fluorescence technology for single-molecule detection, difficulty in improving detection efficiency and accuracy, and difficulty in achieving accurate quantitative analysis of single-molecule sensitivity, especially for ultra-low concentration target molecules.

[0004] To achieve the above and other related objectives, the present invention provides a single-molecule sensing array module, the single-molecule sensing array module comprising: an optical waveguide, a cascaded array of multimode interferometers, and a waveguide array connected in sequence;

[0005] In the multimode interferometer cascade array, each level of the multimode interferometer splits the excitation light transmitted from the optical waveguide.

[0006] The last level of the multimode interferometer in the multimode interferometer cascade array is connected one-to-one with the waveguides in the waveguide array.

[0007] The waveguide includes a high-refractive-index waveguide layer and a low-refractive-index optical cladding, the optical cladding covering the waveguide layer and forming a surface evanescent wave on the surface of the waveguide layer.

[0008] The waveguide has a plurality of waveguide single-molecule sensing units arranged along its length direction. The minimum distance between the surface of the optical cladding used to support the sample under test and the surface of the waveguide layer in each waveguide single-molecule sensing unit is controlled within the wave field range of the surface evanescent wave.

[0009] Optionally, the minimum distance between the surface of the optical cladding used to support the sample under test and the surface of the waveguide layer in each waveguide monomolecule sensing unit is less than 200 nm.

[0010] Furthermore, the minimum distance between the surface of the optical cladding used to support the sample under test and the surface of the waveguide layer in each waveguide monomolecule sensing unit is 40 nm to 60 nm.

[0011] Optionally, each waveguide single-molecule sensing unit is formed by providing a micropore on the optical cladding, and the minimum distance is the distance between the bottom of the micropore and the surface of the waveguide layer.

[0012] Furthermore, a heterogeneous material layer is disposed on the upper surface of each waveguide single-molecule sensing unit, the heterogeneous material layer being used to prevent non-specific binding of the detected molecules; or the heterogeneous material layer is disposed on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall of the micropore.

[0013] Optionally, each waveguide single-molecule sensing unit is formed by circumferentially disposing of a heterogeneous material layer around the planar optical cladding where the waveguide single-molecule sensing unit is located. The heterogeneous material layer is used to prevent non-specific binding of the detected molecules. The minimum distance is the distance between the surface of the planar optical cladding and the surface of the waveguide layer.

[0014] Furthermore, the heterogeneous material layer is disposed on the surface of the optical cladding or embedded inward from the surface of the optical cladding.

[0015] Furthermore, the material of the heterogeneous material layer is aluminum oxide, hafnium oxide, titanium nitride, or tantalum oxide.

[0016] Furthermore, a gold material layer is disposed on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall of the micropore; or the gold material layer is disposed on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall and bottom wall of the micropore.

[0017] Optionally, the waveguide layer is made of silicon nitride, lithium niobate, tantalum pentoxide, or lithium triborate, and the optical cladding is made of silicon oxide.

[0018] Optionally, the excitation light is coupled into the optical waveguide via end-face coupling.

[0019] Optionally, the single-molecule sensing array module further includes a coupling grating connected to the optical waveguide, the coupling grating being used to couple the excitation light and transmit it to the optical waveguide.

[0020] Furthermore, the coupling grating is a periodic grating, a sector grating, or a subwavelength grating; the excitation light is coupled into the coupling grating via an optical fiber or via spatial light.

[0021] Optionally, the excitation light is a single-wavelength laser or a laser of a preset wavelength band.

[0022] Optionally, the waveguide array is a single-mode waveguide array; in the multimode interferometer cascade array, each level of the multimode interferometer splits the excitation light in a 1:1 ratio; the single-molecule sensing array module further includes several coupling gratings, and each coupling grating is connected to the waveguide in a one-to-one correspondence.

[0023] The present invention also provides a single-molecule quantitative analysis chip, the chip comprising: a single-molecule sensing array module and a microfluidic liquid loading module as described in any one of the above claims;

[0024] The microfluidic liquid loading module is sealed and fixed above the single-molecule sensing array module;

[0025] The microfluidic liquid loading module includes: an inlet, an inlet channel, a fluid containment cavity, an outlet channel, and an outlet, which are sequentially connected in the carrier plate; the fluid containment cavity is located above the single-mode waveguide array in the single-molecule sensing array module, and the fluid containment cavity is a blind cavity extending from bottom to top.

[0026] Optionally, the liquid inlet channel is configured as a single-channel liquid inlet or a multi-layer cascade liquid inlet, wherein in the multi-layer cascade liquid inlet, each layer of the liquid inlet channel divides one main pipeline into two sub-pipelines; the liquid outlet channel is configured as a single-channel liquid outlet or a multi-layer cascade liquid outlet, wherein in the multi-layer cascade liquid outlet, each layer of the liquid outlet channel merges two sub-pipelines into one main pipeline.

[0027] Optionally, the liquid flow direction of the microfluidic liquid loading module is perpendicular to the extension direction of the waveguide array in the single-molecule sensing array module.

[0028] Optionally, the single-molecule sensing array module and the microfluidic liquid loading module are tightly bonded together by a rubber ring and externally sealed and fixed by a mechanical structure; or the single-molecule sensing array module and the microfluidic liquid loading module are sealed and fixed together by an adhesive, wherein the adhesive meets biocompatibility requirements.

[0029] This invention also provides a method for single-molecule quantitative analysis, the method comprising:

[0030] Provide a single-molecule quantitative analysis chip and an optical detection device as described in any one of the above, wherein the optical detection device includes a light signal collection system and a spectral detection system, and the light signal collection system is used to collect the light signal excited in the single-molecule quantitative analysis chip;

[0031] The liquid to be tested is introduced into the inlet of the microfluidic liquid loading module. The liquid to be tested flows into the fluid accommodating cavity through the inlet channel and is dispersed in the waveguide single molecule sensing unit of the waveguide array. It is excited by the surface evanescent wave generated by the waveguide array to generate the optical signal.

[0032] The optical signal collection system collects the optical signal and transmits it to the spectral detection system;

[0033] The spectral detection system performs digital processing on the collected optical signals. The principle of the digital processing is as follows: a threshold is set for the optical signal. When the detected optical signal is not less than the threshold, it is recorded that the waveguide single-molecule sensing unit has detected a target single molecule, and the count is 1. When the detected optical signal is less than the threshold, it is recorded that the waveguide single-molecule sensing unit has not detected a target single molecule, and the count is 0.

[0034] The number of target single molecules appearing in the single-molecule quantitative analysis chip is counted through the digital processing.

[0035] Optionally, before introducing the liquid to be detected into the inlet of the microfluidic liquid loading module, the surface of the waveguide single-molecule sensing unit needs to be sequentially subjected to hydrophilic surface treatment, specific site modification, streptavidin modification, and biotinylated antibody modification.

[0036] Optionally, the optical signal is a fluorescence spectral signal or a Raman spectral signal.

[0037] Furthermore, the optical signal is a spectral pattern signal or a spectral intensity signal.

[0038] As described above, the single-molecule sensing array module, quantitative analysis chip, and quantitative analysis method of this invention can achieve high-throughput parallel excitation and sensing of single molecules, with a detection sensitivity limit down to the single-molecule level. Standard curves can be formed for the light signals of ultra-low concentration target molecules, and digital processing methods can solve the problems of uneven and non-repetitive light signal intensity, enabling accurate quantitative analysis of ultra-low concentration molecules and improving detection sensitivity to the single-molecule level. This has significant application value in clinical medicine, life sciences, food testing, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it can enable the tracking and detection of specific pathological molecules; in life sciences, it can enable the detection of targeted DNA and RNA; and in food testing and environmental protection, it can enable ultra-sensitive quantitative detection of pesticide residues. It can also be applied to single-molecule immunoassay, achieving single-molecule immunoassay with ultra-low detection limits. It is expected to enable early and very early immunodiagnosis, and for various diseases such as neurological diseases, tumors, and infectious diseases, it can achieve more accurate real-time detection and pathological tracking. Attached Figure Description

[0039] Figure 1 shows a schematic diagram of the structure of the single-molecule sensing array module of the present invention.

[0040] Figure 2 shows a schematic cross-sectional view of a single waveguide single-molecule sensing unit in the single-molecule sensing array module of the present invention.

[0041] Figure 3 shows the electric field simulation diagram of a single waveguide single-molecule sensing unit in the single-molecule sensing array module of the present invention.

[0042] Figures 4 to 9 show schematic cross-sectional structures of several different examples of a single waveguide single-molecule sensing unit in the single-molecule sensing array module of the present invention.

[0043] Figure 10 shows a top view of the single-molecule quantitative analysis chip of the present invention.

[0044] Figure 11 shows an exploded view of the separable structure of the single-molecule quantitative analysis chip of the present invention.

[0045] Figure 12 shows a three-dimensional rendering of the single-molecule quantitative analysis chip of the present invention.

[0046] Figure 13 shows a schematic diagram of the detection architecture of the single-molecule quantitative analysis chip of the present invention when performing single-molecule quantitative detection.

[0047] Figures 14 to 16 show the statistical principle diagram of the digital processing of the single-molecule quantitative analysis chip of the present invention when digitally processing optical signals.

[0048] Figure 17 shows the schematic diagram of p-Tau single-molecule immunoassay based on a single-molecule quantitative analysis chip in Experiment Example 1.

[0049] Figure 18 shows a partial microwell fluorescence timing signal diagram of the p-Tau single-molecule immunoassay from Figure 17.

[0050] Figure 19 shows the standard curve of the "1" percentage of the digital processing statistics of p-Tau single-molecule immunoassay in Figure 17.

[0051] Figures 20 to 25 show schematic diagrams of the surface chemical modification preparation of the single-molecule quantitative analysis chip in Experiment Example 1 and the specific connection process of p-Tau protein and fluorescent dye molecules.

[0052] Figure 26 shows the schematic diagram of single-molecule immunomagnetic bead detection based on a single-molecule quantitative analysis chip in Experiment Example 2.

[0053] Figures 27 to 31 show schematic diagrams of the preparation process of surface chemical modification of immunomagnetic beads in Experimental Example 2.

[0054] Figure 32 shows a partial micropore fluorescence timing signal diagram of the single-molecule immunomagnetic bead capture protein from Figure 26.

[0055] Figure 33 shows the standard curve of the "1" percentage of the digital processing statistics for the detection of single-molecule immunomagnetic bead capture proteins, which is the same as that in Figure 26.

[0056] Figure 34 shows a schematic cross-sectional view of the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on surface plasmon resonance enhancement, wherein two gold spheres are contained in the micropores of the waveguide single-molecule sensing unit.

[0057] Figure 35 shows the electric field simulation diagram of the waveguide single-molecule sensing unit in Figure 34.

[0058] Figure 36 shows the electric field simulation diagram of the waveguide single-molecule sensing unit based on surface plasmon resonance enhancement in the single-molecule quantitative analysis chip, in which a gold ball is contained in the micropore of the waveguide single-molecule sensing unit.

[0059] Figure 37 shows a schematic cross-sectional structure of a waveguide single-molecule sensing unit based on surface plasmon resonance enhancement in a single-molecule quantitative analysis chip. The waveguide single-molecule sensing unit has a gold material layer on its surface and two gold spheres contained in the micropores.

[0060] Figure 38 shows the electric field simulation diagram of the waveguide single-molecule sensing unit in Figure 37.

[0061] Figure 39 shows the electric field simulation diagram of the waveguide single-molecule sensing unit based on surface plasmon resonance enhancement in the single-molecule quantitative analysis chip. The waveguide single-molecule sensing unit has a gold material layer formed on its surface and a gold ball is contained in the micropore.

[0062] Figure 40 shows a schematic cross-sectional structure of a waveguide single-molecule sensing unit based on surface plasmon resonance enhancement in a single-molecule quantitative analysis chip, wherein three stacked gold spheres are contained in the micropores of the waveguide single-molecule sensing unit.

[0063] Figures 41 to 46 show schematic diagrams of several distribution patterns of gold spheres of different numbers and sizes in the micropores of the waveguide single-molecule sensing unit based on surface plasmon resonance enhancement in a single-molecule quantitative analysis chip.

[0064] Figures 47 to 50 show schematic diagrams of several shapes of micropores in the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on surface plasmon resonance enhancement.

[0065] Figure 51 shows the standard Raman curve and partial micropore Raman signal diagram of the single-molecule quantitative analysis chip for glucose solution detection based on surface plasmon resonance enhancement in Experiment Example 3.

[0066] Figure 52 shows the standard curve of the "1" proportion of the digital processing statistics of glucose solution detection based on surface plasmon resonance enhancement using the single-molecule quantitative analysis chip in Experiment Example 3.

[0067] Figure 53 shows the partial micropore fluorescence timing signal of Cy3 detection based on surface plasmon resonance enhancement in the single-molecule quantitative analysis chip of Experiment Example 4.

[0068] Figure 54 shows the standard curve of the "1" proportion of the digital processing statistics of Cy3 detection based on surface plasmon resonance enhancement using the single-molecule quantitative analysis chip in Experiment Example 4.

[0069] Figure 55 shows a schematic cross-sectional structure of the waveguide single-molecule sensing unit based on surface plasmon resonance enhancement in the single-molecule quantitative analysis chip of Experiment Example 5.

[0070] Figure 56 shows a schematic diagram of the single-molecule quantitative analysis chip in Experiment Example 5, which is based on the fluorescence-free label-free single-molecule detection principle of surface plasmon resonance enhancement.

[0071] Figure 57 shows the curve of the shift of the plasmon resonance peak in a waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on surface plasmon enhancement in Experiment Example 5.

[0072] Figure 58 shows the standard curve of the "1" proportion of the digital processing statistics of the single-molecule quantitative analysis chip based on surface plasmon resonance enhancement for fluorescence-free label-free single-molecule detection in Experiment Example 5.

[0073] Component Labeling Explanation: 1. Single-molecule quantitative analysis chip; 10. Single-molecule sensing array module; 11. Coupling grating; 12. Multimode interferometer cascade array; 120. Multimode interferometer; 13. Waveguide array; 130. Waveguide; 131. Waveguide layer; 132. Optical cladding; 133. Surface evanescent wave; 134. Micropore; 135. Waveguide single-molecule sensing unit; 136. Heterogeneous material layer; 137. Gold material layer; 14. Coupling grating; 15. Optical waveguide; 16. Optical fiber; 20. Microfluidic liquid loading module; 21. Inlet; 22. Inlet channel; 220. Main pipeline; 221. Sub-pipeline; 23. Fluid containment cavity; 24. Outlet channel; 240. Sub-pipeline; 241. Main pipeline; 25. Outlet; 26. Connecting pipeline; 27. Carrier plate; 30. Optical detection device; 31. Optical signal collection system; 310. Objective lens; 311. Mirror; 312. First lens; 313. 314 Pinhole; 315 Second Lens; 316 Filter; 32 Third Lens; 33 Spectroscopic Detection System; 34 Optical Signal; 35 Immunomagnetic Beads; 36 Gold Spheres; 37 Single Molecule Detailed Implementation

[0074] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0075] Please refer to Figures 1 to 58. It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0076] As shown in Figures 1 and 2, this embodiment provides a single-molecule sensing array module 10, which includes: an optical waveguide 15, a multimode interferometer cascade array 12, and a waveguide array 13 connected in sequence.

[0077] In the multimode interferometer cascade array 12, each level of the multimode interferometer 120 splits the excitation light transmitted from the optical waveguide 15.

[0078] The last level of the multimode interferometer cascade array 12 is connected one-to-one with the waveguide 130 in the waveguide array 13.

[0079] As shown in Figures 1 and 2, the waveguide 130 includes a high-refractive-index waveguide layer 131 and a low-refractive-index optical cladding layer 132. The optical cladding layer 132 covers the waveguide layer 131 inside it and forms a surface evanescent wave 133 on the surface of the waveguide layer 131.

[0080] As shown in Figures 1 and 2, the waveguide 130 is provided with a plurality of waveguide single-molecule sensing units 135 along its length direction. The minimum distance D between the surface of the optical cladding 132 of each waveguide single-molecule sensing unit 135 used to support the sample to be tested and the surface of the waveguide layer 131 is controlled within the wave field range of the surface evanescent wave 133.

[0081] It should be noted that the optical path of the single-molecule sensing array module 10 is connected between all components through optical waveguides.

[0082] The core technology principle of signal detection in the single-molecule sensing array module 10 of this embodiment is shown in Figures 1 and 2. It consists of several waveguide single-molecule sensing units 135 in the waveguide array 13. On the one hand, the minimum distance D between the surface of the optical cladding 132 used to support the sample to be tested and the surface of the waveguide layer 131 in the waveguide single-molecule sensing unit 135 is controlled within the wave field range of the surface evanescent wave 133. The surface evanescent wave is formed on the surface of the waveguide layer when light waves propagating in the high-refractive-index waveguide layer undergo total internal reflection at the interface between the waveguide layer and the low-refractive-index optical cladding layer. The field strength of the surface evanescent wave decreases exponentially with the outward radiation distance (as shown in Figure 3). On the other hand, an appropriate amount of the single-molecule sample to be tested is dispersed on the surface of the waveguide array 13. By controlling the concentration or incubation time, the surface of the optical cladding 132 of each waveguide single-molecule sensing unit 135 in the waveguide array 13 is controlled to have only a few single molecules (e.g., one or two single molecules) to carry the sample, as shown in Figure 2. In this way, the few single molecules on each waveguide single-molecule sensing unit 135 are within the wave field range of the surface evanescent wave 133 generated by the waveguide layer 131 in the waveguide single-molecule sensing unit 135. The surface evanescent wave 133 will excite these single molecules to generate optical signals, so that the detection of single molecules in each waveguide single-molecule sensing unit 135 can be achieved by judging the optical signal information. The high-throughput detection core technology principle of the single-molecule sensing array module 10 in this embodiment is shown in Figure 1. By setting up an optical waveguide 15, a multimode interferometer cascade array 12, and a waveguide array 13, and connecting these structures in sequence, the optical waveguide 15 transmits the excitation light to the single-molecule sensing array module 10. The multimode interferometer cascade array 12 splits the excitation light. After multiple cascades of multimode interferometers 120, the initial excitation light is split into hundreds or even thousands of beams and enters each waveguide 130 of the waveguide array 13. Several (e.g., hundreds or even thousands) waveguide single-molecule sensing units 135 are arranged along the length of each waveguide 130, thereby forming a large number of waveguide single-molecule sensing units 135 in the waveguide array 13. Each waveguide single-molecule sensing unit 135 is similar to a pixel for optical signal detection, that is, a large number of optical signal detection pixels are formed in the waveguide array 13, thereby realizing high-throughput single-molecule detection.

[0083] As an example, since the field strength of the surface evanescent wave 133 decreases exponentially with its radiation distance, its effective range is typically on the order of hundreds of nanometers. Therefore, as shown in Figure 2, the minimum distance D between the surface of the optical cladding used to support the sample under test in each waveguide monomolecule sensing unit 135 and the surface of the waveguide layer can be set to within 200 nm. Alternatively, considering the feasibility of fabrication, this minimum distance D can be set within the range of 40 nm to 60 nm.

[0084] As shown in Figures 2 and 4, the waveguide single-molecule sensing unit 135 can be configured in two structures. The first structure has several micropores 134 on the optical cladding 132 of the waveguide 130. As shown in Figure 2, one micropore 134 is provided on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. The micropore 134 contains the single molecule to be measured. In this case, the minimum distance D refers to the distance between the bottom of the micropore 134 and the surface of the waveguide layer 131. The second structure, as shown in Figure 4, involves setting the surface of the optical cladding 132 of the waveguide 130 as a planar structure. However, to define a single waveguide single-molecule sensing unit 135, a heterogeneous material layer 136 needs to be circumferentially disposed around the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135, as shown in Figure 8. The heteromaterial layer 136 is disposed on the surface of the optical cladding 132 in the circumferential direction of the waveguide single-molecule sensing unit 135, as shown in FIG9. The heteromaterial layer 136 is disposed inside the optical cladding 132 in the circumferential direction of the waveguide single-molecule sensing unit 135, specifically embedded inward from the surface of the optical cladding 132. The heteromaterial layer 136 is used to prevent non-specific binding of the detected molecules, so that the detected molecules can only fall on the surface of the optical cladding 132 outside the heteromaterial layer 136. At this time, the minimum distance D refers to the distance between the surface of the planar optical cladding 132 and the surface of the waveguide layer 131.

[0085] In addition, when the waveguide single-molecule sensing unit 135 is formed by setting micropores 134 on the optical cladding 132, at least a portion of the surface of the optical cladding 132 of the waveguide single-molecule sensing unit 135 may also be provided with the heteromaterial layer 136 to control the binding position and binding mode of the single molecule to be detected in the micropore 134. As shown in FIG5, the heteromaterial layer 136 is formed on the optical cladding 132 outside the micropore 134, so the single molecule to be detected can only be bound in the micropore 134. As shown in FIG6, the heteromaterial layer 136 is formed on the optical cladding 132 outside the micropore 134 and on the inner sidewall of the micropore 134, so the single molecule to be detected can only be bound on the bottom wall of the micropore 134.

[0086] As an example, the material of the heterogeneous material layer 136 may be selected as aluminum oxide, hafnium oxide, titanium nitride, tantalum oxide, or other materials suitable for controlling the binding of biomolecules.

[0087] As an example, the waveguide layer 131 can be made of silicon nitride, lithium niobate, tantalum pentoxide, or lithium triborate, and the optical cladding layer 132 can be made of silicon oxide. In this case, the single-molecule sensing array module can be implemented based on silicon photonics technology, forming an optical cladding layer 132 of silicon oxide on a silicon-based material and forming the waveguide layer 131 within the optical cladding layer 132 of silicon oxide. Silicon photonics technology is a low-cost, high-speed optical communication technology based on silicon photonics. It combines the ultra-large-scale, ultra-high-precision manufacturing characteristics of integrated circuit technology with the ultra-high speed and ultra-low power consumption advantages of photonics technology, making it a disruptive technology to address the failure of Moore's Law. This silicon-based single-molecule sensing array module enables high-throughput analysis, easily achieving detection arrays of millions or even tens of millions of units—a detection throughput almost impossible to achieve with traditional detection technologies. It has enormous application value in fields such as clinical medicine, life sciences, food testing, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it can enable the tracking and detection of specific pathological molecules, and in food testing and environmental protection, it can enable the ultrasensitive quantitative detection of pesticide residue molecules.

[0088] As an example, the excitation light can be directly coupled into the optical waveguide 15 via end-face coupling, and then transmitted to the multimode interferometer cascade array 12 via the optical waveguide 15. As shown in Figure 1, as another example, the excitation light can also be coupled into and transmitted to the optical waveguide 15 via a coupling grating 11. Specifically, the coupling grating 11 is also provided in the single-molecule sensing array module 10, and the coupling grating 11 is connected to the optical waveguide 15. The coupling grating 11 can be selected as a periodic grating, a sector grating, or a subwavelength grating; the light coupling method can be selected as follows: first, as shown in Figure 12, the excitation light is coupled into the coupling grating 11 via optical fiber 16; second, the excitation light is coupled into the coupling grating 11 via spatial light. Furthermore, the excitation light can be coupled into the coupling grating 11 at a preset required angle.

[0089] As an example, the excitation light can be a single-wavelength laser, such as 488nm, 532nm, 633nm, 638nm, etc., or it can be a laser in a preset band, such as 550nm-700nm, etc., depending on the actual needs.

[0090] As a preferred example, the waveguide array 13 is a single-mode waveguide array; the multimode interferometers 120 in each level of the multimode interferometer cascade array 12 split the excitation light in a 1:1 ratio to achieve uniformity of the light output from the waveguide array 13.

[0091] As shown in Figure 1, the single-molecule sensing array module 10 also includes several coupling gratings 14, and the coupling gratings 14 are connected one-to-one with the waveguide 130. The coupling gratings 14 can effectively prevent the excitation light from forming interface reflection and generate standing wave interference effect in the waveguide 130. The excitation light in each waveguide 130 is led out of the module through the coupling gratings 14 to improve the detection accuracy.

[0092] In some specific embodiments, the excitation light can be split into 64, 128, 256, 512, 1024, 2048 or even more waveguides 130. In addition, each waveguide 130 can be provided with 64, 128, 256, 512, 1024, 2048 or even more waveguide single-molecule sensing units 135. The two are combined to form a pixel array containing hundreds, thousands, tens of thousands, hundreds of thousands or even millions of waveguide single-molecule sensing units 135, realizing arrayed single-molecule detection.

[0093] As an example, when the waveguide single-molecule sensing unit 135 is formed by providing micropores 134 on the optical cladding 132, a gold material layer 137 can be disposed on the surface of the optical cladding 132 of the waveguide single-molecule sensing unit 135. This gold material layer 137 is used to generate the surface plasmon resonance enhancement effect. The gold material layer 137 can be disposed on the upper surface of each waveguide single-molecule sensing unit 135 and the inner sidewall of the micropore 134; the gold material layer 137 can also be disposed on the upper surface of each waveguide single-molecule sensing unit 135 and the inner sidewall and inner bottom wall of the micropore 134, as shown in FIG. 7. The surface plasmon resonance effect is a phenomenon in which electromagnetic waves (e.g., surface evanescent waves in this embodiment) excite free charges in a metal and cause collective periodic oscillations. The surface plasmon resonance effect can confine light to the surface of a metal or micro / nanostructure, forming a highly enhanced electric field, thereby effectively enhancing the optical signal of surface-adsorbed molecules. As shown in Figures 34 to 36, gold spheres 35 in the micropores 134 of waveguide single-molecule sensing units 135 generate surface plasmon-enhanced local electric fields on their surfaces, and these local electric fields are confined to the micro / nano structure surfaces (e.g., the gap between two gold spheres and the gap where the gold spheres contact the bottom wall of the micropore). As shown in Figures 37 to 39, when a gold material layer 137 is provided on the surface of the optical cladding 132 of the waveguide single-molecule sensing unit 135 (i.e., the upper surface of each waveguide single-molecule sensing unit 135 and the inner sidewall and inner bottom wall of the micropore 134), a stronger surface plasmon-enhanced local electric field can be generated at the gap where the gold spheres contact the bottom wall of the micropore 134. When using gold spheres 35 for single-molecule detection, there may be multiple gold spheres within the micropore 134. These gold spheres may be stacked and vary in size, with their diameters generally exhibiting a Poisson distribution. Multiple enhanced local electric fields will appear between the gold spheres 35. As long as the molecule to be detected diffuses into the enhanced local electric field of the gold sphere, an enhanced optical signal will be generated, as shown in Figures 41 to 46. There may be 2, 3, 4, 5, 6, 7... gold spheres 35 within the micropore 134, and enhanced local electric fields will appear between them. As long as the molecule to be detected diffuses into the enhanced local electric field range of the gold sphere 35, an enhanced optical signal will be generated. In addition, the shape of the micropore 134 can also be different, as shown in Figures 47 to 50. The shapes of the micropore 134 are triangular, pentagonal, square, and hexagonal, but other shapes are also possible, depending on the actual situation.

[0094] As shown in Figures 10 to 12, this embodiment also provides a single-molecule quantitative analysis chip 1, which includes: the single-molecule sensing array module 10 and the microfluidic liquid loading module 20 described in this embodiment; the specific structure of the single-molecule sensing array module 10 can be referred to the above description, and will not be repeated below;

[0095] The microfluidic liquid loading module 20 is sealed and fixed above the single-molecule sensing array module 10;

[0096] The microfluidic liquid loading module 20 includes: an inlet 21, an inlet channel 22, a fluid accommodating cavity 23, an outlet channel 24, and an outlet 25, which are sequentially connected in the carrier plate 27; the fluid accommodating cavity 23 is disposed above the waveguide array 13 in the single-molecule sensing array module 10, and the fluid accommodating cavity 23 is a blind cavity extending from bottom to top, that is, the opening of the fluid accommodating cavity 23 faces the waveguide array 13 and does not penetrate the carrier plate 27.

[0097] Liquid is injected through the inlet 21 of the microfluidic liquid loading module 20, enters the fluid receiving cavity 23 through the inlet channel 22, and is dispersed in the waveguide single-molecule sensing unit 135 of the waveguide array 13 in the fluid receiving cavity 23. It is excited by the surface evanescent wave 133 generated by the waveguide array 13 to generate an optical signal, thereby realizing the detection of single-molecule signal. The microfluidic liquid loading module 20 controls the flow of liquid, while the single-molecule sensing array module 10 enables high-throughput parallel optical signal excitation and sensing. Sealing and fixing the two together allows for precise fluid control, achieving high-throughput parallel single-molecule excitation and sensing in the detection area. The detection sensitivity limit can reach the single-molecule level, offering significant application value in clinical medicine, life sciences, food testing, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it enables the tracking and detection of specific pathological molecules; in life sciences, it enables targeted detection of DNA and RNA; and in food testing and environmental protection, it enables ultrasensitive quantitative detection of pesticide residues. Similarly, it can be applied to single-molecule immunoassay, achieving ultra-low detection limits. This promises to enable early and very early immunodiagnosis, providing more accurate real-time detection and pathological tracking for various diseases such as neurological diseases, tumors, and infectious diseases.

[0098] As shown in Figure 10, the inlet channel 22 of the microfluidic liquid loading module 20 can be configured as a single-channel inlet or a multi-layer cascade inlet. In the multi-layer cascade inlet, each inlet channel divides a main pipeline 220 into two sub-pipelines 221. Through the multi-layer cascade, the liquid from the initial pipeline can be spread into several pipelines and enter the fluid receiving chamber 23. The outlet channel 24 can also be configured as a single-channel outlet or a multi-layer cascade outlet. In the multi-layer cascade outlet, each outlet channel merges two sub-pipelines 240 into a main pipeline 241. Through the multi-layer cascade, the liquid in the fluid receiving chamber 23 can flow out through several sub-pipelines 240 into a final main pipeline 241. The multi-layered cascaded liquid inlet channel 22 allows the liquid entering from the liquid inlet 21 to flow evenly into the fluid container 23, thereby effectively improving the uniformity of liquid distribution in the detection area and improving detection accuracy. Combined with the multi-layered cascaded liquid outlet channel 24, the liquid in the fluid container 23 can be evenly discharged, further ensuring the uniformity of liquid distribution in the detection area and improving detection accuracy.

[0099] As an example, the liquid can be proportionally dispersed into 1, 2, 4, 8, 16, 32, 64, 128, 256 or even more sub-pipes 221, and finally uniformly dispersed into the fluid receiving chamber 23; in addition, the liquid can be proportionally distributed through 1, 2, 4, 8, 16, 32, 64, 128, 256 or even more sub-pipes 240 and finally converge into a main pipe 241 to flow out.

[0100] Depending on actual needs, the inlet 21 can be connected to the outlet 25 to achieve a liquid circulation path.

[0101] As shown in Figure 11, as an example, the liquid flow direction of the microfluidic liquid loading module 20 can be set perpendicular to the extension direction of the waveguide array 13 in the single-molecule sensing array module 10. This can further improve the uniformity of liquid spreading on the waveguide array 13.

[0102] As an example, the single-molecule sensing array module 10 and the microfluidic liquid loading module 20 can be configured to be detachably sealed and fixed. For example, they can be tightly attached by a rubber ring, externally sealed and fixed by a mechanical structure, or sealed and fixed by using an adhesive that meets biocompatibility requirements. Preferably, adhesives that meet ISO-10993 biocompatibility certification are used for sealing and fixing, such as cured epoxy adhesives.

[0103] As shown in Figures 10 to 13, this embodiment also provides a single-molecule quantitative analysis method, which includes the following steps:

[0104] S1. Provide the single-molecule quantitative analysis chip 1 and optical detection device 30 as described in this embodiment. The optical detection device 30 includes a light signal collection system 31 and a spectral detection system 32. The light signal collection system 31 is used to collect the light signal excited in the single-molecule quantitative analysis chip 1.

[0105] S2. The liquid to be tested is introduced into the inlet 21 of the microfluidic liquid loading module 20. The liquid to be tested flows into the fluid accommodating cavity 23 through the inlet channel 22 and is dispersed in the waveguide single molecule sensing unit 135 of the waveguide array 13. It is excited by the surface evanescent wave generated by the waveguide array 13 to generate the optical signal.

[0106] S3. The optical signal collection system 31 collects the optical signal and transmits it to the spectral detection system 32;

[0107] S4. The spectral detection system 32 performs digital processing on the collected optical signals. The principle of the digital processing is as follows: a threshold is set for the optical signal. When the detected optical signal is not less than the threshold, it is recorded that the waveguide single-molecule sensing unit 135 has detected a target single molecule, and the count is 1. When the detected optical signal is less than the threshold, it is recorded that the waveguide single-molecule sensing unit 135 has not detected a target single molecule, and the count is 0.

[0108] S5. The number of target single molecules appearing in the single-molecule quantitative analysis chip 1 is counted through the digital processing.

[0109] As shown in Figures 14 to 16, the single-molecule quantitative analysis method of this embodiment adopts digital processing when processing the optical signal. A threshold is set for the optical signal. When the detected optical signal is greater than or equal to the threshold, it is determined that the waveguide single-molecule sensing unit 135 has detected a target molecule, and the system count is 1 (as shown in Figure 15). When the detected optical signal is less than the threshold, it is determined that the waveguide single-molecule sensing unit 135 has not detected a target molecule, and the system count is 0 (as shown in Figure 16). By detecting and recording the detection array formed by several waveguide single-molecule sensing units 135 on the single-molecule quantitative analysis chip 1 through this single-molecule counting method, the single-molecule detection array diagram shown in Figure 14 can be obtained. Each 1 or 0 represents the single-molecule detection result of the corresponding waveguide single-molecule sensing unit 135, thereby obtaining the number of target molecules and realizing the quantitative analysis and detection of target molecules. The single-molecule quantitative analysis method in this embodiment can form a standard curve for the optical signal of ultra-low concentration target molecules. By using digital processing methods, the problems of uneven and non-repetitive optical signal intensity can be solved, enabling accurate quantitative analysis of ultra-low concentration molecules, improving detection sensitivity, and achieving a sensitivity level at the single-molecule level.

[0110] As an example, as shown in Figure 13, the optical signal collection system 31 sequentially includes an objective lens 310, a reflector 311, a first lens 312, a pinhole 313, a second lens 314, a filter 315, and a third lens 316 connected by an optical path. The signal light 33 generated by the single-molecule quantitative analysis chip 1 is collected by the objective lens 310, and then passes through the confocal system formed by the reflector 311, the first lens 312, the pinhole 313, the second lens 314, and the third lens 316, and the filter 315 to reduce the interference between the optical signal 33 and the background signal, and finally enters the spectral detection system 32.

[0111] As an example, the optical signal 33 is often selected as a fluorescence spectrum signal or a Raman spectrum signal, specifically determined by the detection method chosen based on the characteristics of the target molecule to be detected. Furthermore, the detection method for the optical signal 33 is often selected as a spectral pattern signal or a spectral intensity signal.

[0112] As an example, depending on the characteristics of different target molecules, the surface of the waveguide single-molecule sensing unit 135 in the single-molecule quantitative analysis chip 1 can be sequentially subjected to hydrophilic surface treatment, specific site modification, streptavidin modification, and biotinylated antibody modification before detection. The light signal 33 obtained after processing the single-molecule quantitative analysis chip 1 is a fluorescence spectrum signal. Hydrophilic surface treatment can be achieved, for example, by plasma treatment. Alternatively, depending on the characteristics of different target molecules, only the surface of the waveguide single-molecule sensing unit 135 in the single-molecule quantitative analysis chip 1 can be subjected to hydrophilic surface treatment before detection, without subsequent specific site modification processes. The specific choice depends on actual needs.

[0113] The single-molecule quantitative analysis method of the present invention will be described in detail below with specific experimental examples. Obviously, the described experimental examples are only a part of the experimental examples of the present invention, and not all of the experimental examples. Based on the experimental examples of the present invention, all other experimental examples obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0114] Experimental Example 1

[0115] This experimental example demonstrates p-Tau single-molecule immunoassay based on a single-molecule quantitative analysis chip.

[0116] Figure 17 illustrates the specific principle of this experimental example, where tau protein is an important characteristic protein of Alzheimer's disease. By chemically modifying the surface of the waveguide single-molecule sensing unit 135 on the single-molecule quantitative analysis chip 1, p-Tau protein (antigen in Figure 17) is attached. Finally, a specific antibody (antibody-Dye in Figure 17) attached to a fluorescent dye molecule is bound to the p-Tau protein. The surface evanescent wave 133 of the waveguide single-molecule sensing unit 135 is used to excite the fluorescent dye molecule Dye, and the fluorescence of the fluorescent dye molecule is digitally recorded.

[0117] Figures 20 to 25 show the surface chemical modification process of single-molecule quantitative analysis chip 1 to prepare for the specific binding of p-Tau protein and fluorescent dye molecules. The specific steps are as follows:

[0118] Surface chemical modification preparation process of single-molecule quantitative analysis chip 1:

[0119] 1. A single-molecule quantitative analysis chip 1 is provided. The waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is shown in Figure 20. Its structure is that the surface of the optical cladding 132 of the waveguide 130 is set as a planar structure, and heterogeneous material layers 136 are provided on both sides of the optical cladding 132 corresponding to the waveguide single-molecule sensing unit 135 along the length direction of the optical cladding 132. First, the single-molecule quantitative analysis chip 1 is ultrasonically cleaned in anhydrous ethanol for 3 min and placed in a vacuum plasma ionizer for O2 plasma for 2 min.

[0120] 2. As shown in Figure 21, the plasma-treated single-molecule quantitative analysis chip 1 was immersed in a 1:100 solution of Silane-PEG / Silane-PEG-Biotin (1% water / ethanol) and incubated at room temperature for 4 hours. After incubation, the surface of the single-molecule quantitative analysis chip 1 was cleaned with a large amount of deionized water.

[0121] 3. As shown in Figure 22, a PBST solution containing 2 mg / ml streptavidin protein (as shown in Figure 17) was applied to the surface of the modified single-molecule quantitative analysis chip 1 and incubated at room temperature for 30 min. After incubation, the surface of the single-molecule quantitative analysis chip 1 was washed multiple times with PBST solution.

[0122] 4. As shown in Figure 23, a PBST solution containing biotinylated p-Tau181 antibody (antibody-biotin in Figure 17) was spread onto the surface of the single-molecule quantitative analysis chip 1 incubated with streptavidin protein. The chip was incubated at room temperature for 2 hours. After the antibody was fully bound to the surface of the single-molecule quantitative analysis chip 1, the surface of the single-molecule quantitative analysis chip 1 was washed multiple times with PBST solution to remove non-specifically adsorbed antibodies.

[0123] Preparation process of single-molecule standard curve of p-Tau:

[0124] 1. A blocking buffer containing 5% BSA was coated onto the surface of the surface-modified single-molecule quantitative analysis chip 1 and incubated at room temperature for 1 hour to block non-specific binding sites;

[0125] 2. The surface of the sealed single-molecule quantitative analysis chip 1 was cleaned multiple times using PBST solution;

[0126] 3. As shown in Figure 24, the p-Tau181 protein standard (antigen in Figure 17) was diluted to a protein solution with a concentration gradient of 1 pg / ml to 100 pg / ml. The solution was then slowly introduced into the inlet channel through the inlet and incubated at room temperature for 1 hour to achieve an immune reaction between the antigen and antibody protein.

[0127] 4. After the reaction is complete, clean the surface of the single-molecule quantitative analysis chip 1 with PBST solution to remove any residual unreacted sample;

[0128] 5. As shown in Figure 25, the Tau protein capture antibody (antibody-Dye in Figure 17) conjugated with the fluorescent dye molecule was diluted with blocking buffer and incubated with the chip surface at room temperature for 1 hour to achieve the binding of the fluorescently labeled antibody to the target protein.

[0129] 6. Clean the surface of the single-molecule quantitative analysis chip 1 multiple times using PBST solution;

[0130] 7. Based on the optical detection device, the real-time fluorescence signal generated in the single-molecule detection area (i.e., the area where the waveguide single-molecule sensing unit 135 is located) of the single-molecule quantitative analysis chip 1 is collected and digitally processed. The proportion of digital "1" is proportional to the concentration of p-Tau181 protein, and a standard curve of p-Tau protein concentration-fluorescence signal can be plotted.

[0131] Figures 18 and 19 show the partial nanopore fluorescence timing signals and standard curves of p-Tau single-molecule immunoassay. Figure 19 shows the R values ​​after three repeated tests. 2 The reproducibility reached 0.98, demonstrating high repeatability. In practical applications, the high reproducibility between analyte concentration and single-molecule events is sufficient to convert the measured percentage of "1" into the actual concentration.

[0132] Experiment Example 2

[0133] This experimental example demonstrates the detection of single-molecule immunomagnetic beads based on a single-molecule quantitative analysis chip.

[0134] Figure 26 illustrates the specific principle of this experimental example. The waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is shown in Figure 26. Its structure consists of a micropore 134 on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. This micropore 134 is used to contain the single molecule to be tested. Immunomagnetic beads 34 are modified with a specific antibody (antibody-biotin in Figure 26) to capture the target protein (antigen in Figure 26). Finally, a specific antibody linked to a fluorescent dye molecule (antibody-Dye in Figure 26) binds to the target protein. The evanescent wave 133 on the surface of the waveguide single-molecule sensing unit 135 excites the fluorescent dye molecule Dye, and the fluorescence of the fluorescent dye molecule is digitally recorded.

[0135] Figures 27 to 31 show the surface modification process of immunomagnetic beads 34. The specific steps are as follows:

[0136] 1. As shown in Figure 27, Silane-PEG-Biotin was modified on the surface of immunomagnetic beads 34. As shown in Figure 28, PBST solution containing 2 mg / mL streptavidin protein (Streptavidin in Figure 26) was incubated with immunomagnetic beads 34 at room temperature for 30 min. The beads were then transferred to a magnetic rack and washed multiple times with PBST solution to remove unbound streptavidin protein.

[0137] 2. As shown in Figure 29, capture antibodies with a biotin tag at one end (antibody-biotin in Figure 26) were co-incubated with immunomagnetic beads at room temperature for 30 min. Then, the beads were transferred to a magnetic rack and resuspended with 5 column volumes (CV) of 1×PBST washing buffer to wash the immunomagnetic beads 5 times, with each washing time being 10 min, to remove non-specifically adsorbed antibodies.

[0138] 3. Incubate the prepared immunomagnetic beads with blocking buffer containing 5% BSA at room temperature for 1 hour to block non-specific binding sites.

[0139] 4. As shown in Figure 30, add test samples at concentration gradients of 1 pg / ml to 100 pg / ml and incubate at room temperature for 2 hours to allow the target protein (antigen in Figure 26) to bind specifically to the capture antibody. After incubation, transfer to a magnetic rack and resuspend and wash the immunomagnetic beads 34 times with PBST solution to remove non-specifically adsorbed impurities.

[0140] 5. As shown in Figure 31, add the labeled antibody with fluorescent molecules (antibody-Dye in Figure 26), incubate at room temperature for 1 hour to achieve specific binding of the fluorescent labeled antibody to the target protein, then transfer to a magnetic rack and wash the immunomagnetic beads 34 multiple times with PBST solution to remove non-specifically bound chromogenic antibodies.

[0141] 6. As shown in Figure 26, the sample resuspended in immunomagnetic beads 34 is added to the surface of the single-molecule quantitative analysis chip 1 and diffuses freely in the solution. When the immunomagnetic beads 34, which are bound to the target protein and labeled antibody, fall into the bottom of the micropore of the waveguide single-molecule sensing unit 135, the fluorescent molecules on the labeled antibody are excited, and the fluorescence signal is collected.

[0142] Figures 32 and 33 show the fluorescence timing signals of some nanopores of the capture protein and the standard curve. Figure 33 shows the R value under three repeated tests. 2 It reached 0.98.

[0143] Experimental Example 3

[0144] This experimental example demonstrates single-molecule Raman detection based on a surface plasmon-enhanced waveguide single-molecule sensing unit 135.

[0145] The waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is shown in Figure 34. Its structure consists of a micropore 134 on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. This micropore 134 is used to accommodate the single molecule to be measured. The diameter of the micropore 134 is 300 nm, and the depth is 200 nm. The spacing between two adjacent micropores 134 is 10 μm. The excitation wavelength is 638 nm, and the excitation power is 100 mW. The acquisition time is 50 ms. The objective lens is a 40x objective lens with a numerical aperture of 0.75. The specific experimental steps are as follows:

[0146] 1. Configure concentration from 10 -14 -10 -10 A series of glucose solutions of M were mixed with gold nanoparticles of 1 nM concentration at a ratio of 1:10 and then ultrasonically dispersed for 5 min.

[0147] 2. Inject the mixed solution into the inlet of the microfluidic liquid loading module and incubate for 1 hour.

[0148] 3. The optical signal collection system (the optical signal collection system used in this experimental example is a confocal system) performs spectral detection and transmits the signal to the spectral detection system. The Raman signal of each waveguide single-molecule sensing unit 135 is obtained by scanning and digitally processed. The proportion of the "1" signal is obtained through the digital results, and a standard curve is obtained by fitting.

[0149] Figure 51 shows the standard Raman curve of glucose solution and the Raman signal of some nanopores. Figure 52 shows the proportion of "1" in the digital statistics, and the R value after three repeated tests. 2 It reached 0.98, demonstrating high repeatability.

[0150] Experiment Example 4

[0151] This experimental example demonstrates single-molecule fluorescence detection based on a surface plasmon-enhanced waveguide single-molecule sensing unit 135.

[0152] The waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is shown in Figure 37. Its structure consists of a micropore 134 on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. This micropore 134 is used to accommodate the single molecule to be measured, and a gold material layer 137 is disposed on the surface of the optical cladding 132 corresponding to the waveguide single-molecule sensing unit 135. The diameter of the micropore 134 is 300 nm, and the depth is 200 nm. The spacing between two adjacent micropores 134 is 10 μm. The excitation wavelength is 638 nm, and the excitation power is 100 mW. The acquisition time is 50 ms. The objective lens is a 40x objective lens with a numerical aperture of 0.75. The specific experimental steps are as follows:

[0153] 1. Configure concentration from 10 -15 -10 -12 A series of Cy3 solutions of M were mixed with gold nanoparticles of 1 nM concentration at a ratio of 1:10 and then ultrasonically dispersed for 5 min.

[0154] 2. Inject the mixed solution into the inlet of the microfluidic liquid loading module and incubate for 1 hour.

[0155] 3. The optical signal collection system (the optical signal collection system used in this experimental example is a confocal system) performs spectral detection and transmits the signal to the spectral detection system. The fluorescence signal of each waveguide single-molecule sensing unit 135 is acquired by scanning and digitally processed. The proportion of the "1" signal is obtained from the digital results, and a standard curve is obtained by fitting.

[0156] Figure 53 shows the fluorescence timing signals of some Cy3 nanopores. Figure 54 shows the standard curve of the "1" proportion in the digital statistics. R1 is shown in the results of three repeated tests. 2 It reached 0.99, demonstrating high repeatability.

[0157] Experimental Example 5

[0158] This experimental example demonstrates the fluorescence-free, label-free single-molecule detection based on the surface plasmon-enhanced waveguide single-molecule sensing unit 135.

[0159] Because surface plasmons in metals exhibit medium-sensitive properties, the refractive index of the surrounding environment affects the position of the plasmon resonance peak. Figure 56 illustrates the basic principle: when a molecule comes into contact with the surface of a metal particle, the position of the plasmon resonance peak shifts. Generally, a larger surrounding refractive index results in a redshift of the resonance peak, while a smaller surrounding refractive index results in a blueshift. This phenomenon enables label-free, fluorescence-free single-molecule detection.

[0160] The waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is shown in Figure 55. Its structure consists of a micropore 134 on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. This micropore 134 is used to accommodate the single molecule to be analyzed. The diameter of the micropore 134 is 300 nm, the depth is 200 nm, and the spacing between two adjacent micropores 134 is 10 μm. The excitation wavelength is 550 nm-700 nm, the excitation power is 100 mW, the acquisition time is 50 ms, the objective lens is a 40x objective lens, and the numerical aperture is 0.75. The specific experimental steps are as follows:

[0161] 1. Configure concentration from 10 -11 -10 -7 A series of solutions of M were mixed with gold nanoparticles of 1 nM concentration at a ratio of 1:10 and then ultrasonically dispersed for 5 min.

[0162] 2. Inject the mixed solution into the inlet of the microfluidic liquid loading module and incubate for 1 hour.

[0163] 3. The optical signal collection system (a confocal optical signal collection system is used in this experimental example) performs spectral detection and transmits the data to the spectral detection system. The scattering spectral signal of each waveguide single-molecule sensing unit 135 is acquired by scanning. When the resonance peak position shifts by 3 nm, it is recorded as the number "1". Finally, the proportion of the "1" signal is counted, and a standard curve is obtained by fitting.

[0164] Figures 57 and 58 show the shift of one of the micropore plasmon resonance peaks and the proportion of "1" values ​​in the digital statistics, as well as the R values ​​after three repeated tests. 2 It reached 0.97, demonstrating high repeatability.

[0165] In summary, this invention provides a single-molecule sensing array module, a quantitative analysis chip, and a quantitative analysis method. These enable high-throughput parallel excitation and sensing of single molecules, achieving a detection sensitivity limit down to the single-molecule level. Standard curves can be formed for the light signals of ultra-low concentration target molecules. Digital processing methods can solve the problems of uneven and non-repetitive light signal intensity, enabling accurate quantitative analysis of ultra-low concentration molecules and improving detection sensitivity to the single-molecule level. This has significant application value in clinical medicine, life sciences, food testing, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it can enable the tracking and detection of specific pathological molecules; in life sciences, it can enable the detection of targeted DNA and RNA; and in food testing and environmental protection, it can achieve ultra-sensitive quantitative detection of pesticide residues. It can also be applied to single-molecule immunoassay, achieving single-molecule immunoassay with ultra-low detection limits. This holds promise for early and very early immunodiagnosis, enabling more accurate real-time detection and pathological tracking for various diseases such as neurological diseases, tumors, and infectious diseases. Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0166] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A single-molecule sensing array module, characterized in that, The single-molecule sensing array module includes: an optical waveguide, a cascaded array of multimode interferometers, and a waveguide array connected in sequence. In the multimode interferometer cascade array, each level of the multimode interferometer splits the excitation light transmitted from the optical waveguide. The last level of the multimode interferometer in the multimode interferometer cascade array is connected one-to-one with the waveguides in the waveguide array. The waveguide includes a high-refractive-index waveguide layer and a low-refractive-index optical cladding, the optical cladding covering the waveguide layer and forming a surface evanescent wave on the surface of the waveguide layer. The waveguide has a plurality of waveguide single-molecule sensing units arranged along its length direction. The minimum distance between the surface of the optical cladding used to support the sample under test and the surface of the waveguide layer in each waveguide single-molecule sensing unit is controlled within the wave field range of the surface evanescent wave.

2. The single-molecule sensing array module according to claim 1, characterized in that: In each of the waveguide monomolecule sensing units, the minimum distance between the surface of the optical cladding used to support the sample under test and the surface of the waveguide layer is less than 200 nm.

3. The single-molecule sensing array module according to claim 2, characterized in that: The minimum distance between the surface of the optical cladding used to support the sample under test and the surface of the waveguide layer in each waveguide monomolecule sensing unit is 40 nm to 60 nm.

4. The single-molecule sensing array module according to claim 1, characterized in that: Each waveguide monomolecule sensing unit is formed by creating a micropore on the optical cladding, and the minimum distance is the distance between the bottom of the micropore and the surface of the waveguide layer.

5. The single-molecule sensing array module according to claim 4, characterized in that: A heterogeneous material layer is disposed on the upper surface of each waveguide single-molecule sensing unit to prevent non-specific binding of the detected molecule; or the heterogeneous material layer is disposed on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall of the micropore.

6. The single-molecule sensing array module according to claim 1, characterized in that: Each waveguide single-molecule sensing unit is formed by circumferentially distributing a heterogeneous material layer around the planar optical cladding where the waveguide single-molecule sensing unit is located. The heterogeneous material layer is used to prevent non-specific binding of the detected molecules. The minimum distance is the distance between the surface of the planar optical cladding and the surface of the waveguide layer.

7. The single-molecule sensing array module according to claim 6, characterized in that: The heterogeneous material layer is disposed on the surface of the optical cladding or embedded inward from the surface of the optical cladding.

8. The single-molecule sensing array module according to any one of claims 5 to 7, characterized in that: The material of the heterogeneous material layer is aluminum oxide, hafnium oxide, titanium nitride, or tantalum oxide.

9. The single-molecule sensing array module according to claim 4, characterized in that: A gold material layer is disposed on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall of the micropore; or the gold material layer is disposed on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall and bottom wall of the micropore.

10. The single-molecule sensing array module according to claim 1, characterized in that: The waveguide layer is made of silicon nitride, lithium niobate, tantalum pentoxide, or lithium triborate, and the optical cladding is made of silicon oxide.

11. The single-molecule sensing array module according to claim 1, characterized in that: The excitation light is coupled into the optical waveguide via end-face coupling.

12. The single-molecule sensing array module according to claim 1, characterized in that: The single-molecule sensing array module also includes a coupling grating connected to the optical waveguide, the coupling grating being used to couple the excitation light and transmit it to the optical waveguide.

13. The single-molecule sensing array module according to claim 12, characterized in that: The coupling grating is a periodic grating, a sector grating, or a subwavelength grating; the excitation light is coupled into the coupling grating via an optical fiber or via spatial light.

14. The single-molecule sensing array module according to claim 1, characterized in that: The excitation light is a single-wavelength laser or a laser in a preset wavelength band.

15. The single-molecule sensing array module according to claim 1, characterized in that: The waveguide array is a single-mode waveguide array; in the multimode interferometer cascade array, each level of the multimode interferometer splits the excitation light in a 1:1 ratio; the single-molecule sensing array module also includes several coupling gratings, and each coupling grating is connected to the waveguide in a one-to-one correspondence.

16. A single-molecule quantitative analysis chip, characterized in that, The chip includes: a single-molecule sensing array module and a microfluidic liquid loading module as described in any one of claims 1 to 15; The microfluidic liquid loading module is sealed and fixed above the single-molecule sensing array module; The microfluidic liquid loading module includes: an inlet, an inlet channel, a fluid containment cavity, an outlet channel, and an outlet, which are sequentially connected in a carrier plate; the fluid containment cavity is located above the waveguide array in the single-molecule sensing array module, and the fluid containment cavity is a blind cavity extending from bottom to top.

17. The single-molecule quantitative analysis chip according to claim 16, characterized in that: The liquid inlet channel is configured as a single-channel liquid inlet or a multi-layer cascade liquid inlet, wherein in the multi-layer cascade liquid inlet, each layer of the liquid inlet channel divides one main pipeline into two sub-pipelines; the liquid outlet channel is configured as a single-channel liquid outlet or a multi-layer cascade liquid outlet, wherein in the multi-layer cascade liquid outlet, each layer of the liquid outlet channel merges two sub-pipelines into one main pipeline.

18. The single-molecule quantitative analysis chip according to claim 16, characterized in that: The liquid flow direction of the microfluidic liquid loading module is perpendicular to the extension direction of the waveguide array in the single-molecule sensing array module.

19. The single-molecule quantitative analysis chip according to claim 16, characterized in that: The single-molecule sensing array module and the microfluidic liquid loading module are tightly attached by a rubber ring and externally sealed and fixed by a mechanical structure; or the single-molecule sensing array module and the microfluidic liquid loading module are sealed and fixed by an adhesive, wherein the adhesive meets biocompatibility requirements.

20. A method for quantitative analysis of a single molecule, characterized in that, The analytical method includes: A single-molecule quantitative analysis chip and an optical detection device as described in any one of claims 16 to 19 are provided, wherein the optical detection device includes a light signal collection system and a spectral detection system, and the light signal collection system is used to collect the light signal excited in the single-molecule quantitative analysis chip. The liquid to be tested is introduced into the inlet of the microfluidic liquid loading module. The liquid to be tested flows into the fluid accommodating cavity through the inlet channel and is dispersed in the waveguide single molecule sensing unit of the waveguide array. It is excited by the surface evanescent wave generated by the waveguide array to generate the optical signal. The optical signal collection system collects the optical signal and transmits it to the spectral detection system; The spectral detection system performs digital processing on the collected optical signals. The principle of the digital processing is as follows: a threshold is set for the optical signal. When the detected optical signal is not less than the threshold, it is recorded that the waveguide single-molecule sensing unit has detected a target single molecule, and the count is 1. When the detected optical signal is less than the threshold, it is recorded that the waveguide single-molecule sensing unit has not detected a target single molecule, and the count is 0. The number of target single molecules appearing in the single-molecule quantitative analysis chip is counted through the digital processing.

21. The single-molecule quantitative analysis method according to claim 20, characterized in that: Before introducing the liquid to be detected into the inlet of the microfluidic liquid loading module, the surface of the waveguide single-molecule sensing unit needs to be sequentially subjected to hydrophilic surface treatment, specific site modification, streptavidin modification, and biotinylated antibody modification.

22. The single-molecule quantitative analysis method according to claim 20, characterized in that: The optical signal is a fluorescence spectrum signal or a Raman spectrum signal.

23. The single-molecule quantitative analysis method according to claim 22, characterized in that: The optical signal is a spectral pattern signal or a spectral intensity signal.

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