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

Abstract

The invention provides a single molecule sensing array module, a quantitative analysis chip and a quantitative analysis method, which can realize excitation and sensing of high-flux parallel single molecules, and the detection sensitivity limit can reach a single molecule level; a standard curve can be formed for optical signals of ultralow-concentration target molecules, the problem that the intensity of the optical signals is not uniform and not repeated is solved through a digital processing method, accurate quantitative analysis of the ultralow-concentration molecules can be achieved, the detection sensitivity is improved, and the sensitivity grade reaches the single-molecule grade. The method has great application value in the fields of clinical medicine, life science, food detection, environmental protection, national defense, public safety, basic research and the like. The method can also be applied to single-molecule immunodetection, and single-molecule immunodetection with ultralow detection limit is realized.

Description

Technical Field

[0001] The present invention relates to the field of single molecule detection technology, and in particular to a single molecule sensor array module, a quantitative analysis chip and a quantitative analysis method. Background Art

[0002] Single molecule detection is a disruptive technology in the field of biomarker detection. Reaching the limit of molecular detection has been a long-term goal pursued by people. It has important application value in many fields such as "rediscovery of traditional markers", "development of new markers", and "development of new drugs". At present, the detection of single molecules mainly adopts atomic force microscopy and laser induced fluorescence technology. Among them, laser induced fluorescence is the most effective method for single molecule detection, which is particularly suitable for the study of single molecules in solution. However, the traditional laser induced fluorescence technology for single molecule detection often has low throughput, and it is difficult to improve the efficiency and accuracy of detection. High throughput means that the amount of data or information processed in a certain period of time has reached a high level. It can greatly accelerate the generation and processing of data, thereby more deeply understanding many scientific problems and promoting the progress of human science and technology. In addition, in an unknown and complex background, it is difficult to achieve accurate quantitative analysis of single molecule sensitivity for ultra-low concentration target molecules, and the signal intensity of single molecules is often uneven and non-repetitive. Summary of the invention

[0003] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a single-molecule sensor array module, a quantitative analysis chip and a quantitative analysis method, which are used to solve at least one of the problems in the prior art that the single-molecule detection flux of laser induced fluorescence technology is low, the efficiency and accuracy of detection are difficult to improve, and the single-molecule sensitivity, especially the single-molecule sensitivity for ultra-low concentration target molecules, is difficult to achieve accurate quantitative analysis.

[0004] To achieve the above-mentioned object and other related objects, the present invention provides a single-molecule sensor array module, which comprises: an optical waveguide, a multi-mode interferometer cascade array and a waveguide array connected in sequence;

[0005] The multi-mode interferometer at each level in the multi-mode interferometer cascade array splits the excitation light transmitted from the optical waveguide;

[0006] The multi-mode interferometers of the last level in the multi-mode interferometer cascade array are connected to the waveguides in the waveguide array in a one-to-one correspondence;

[0007] The waveguide includes a waveguide layer with a high refractive index and an optical cladding layer with a low refractive index, wherein the optical cladding layer covers the waveguide layer and forms a surface evanescent wave on the surface of the waveguide layer;

[0008] The waveguide is provided with a plurality of waveguide single-molecule sensing units along its length direction, and the minimum distance between the surface of the optical cladding for carrying the sample to be measured 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, in each of the waveguide single-molecule sensing units, a minimum distance between a surface of the optical cladding layer used to carry a sample to be measured and a surface of the waveguide layer is less than 200 nm.

[0010] Furthermore, in each of the waveguide single-molecule sensing units, the minimum distance between the surface of the optical cladding layer used to carry the sample to be measured and the surface of the waveguide layer is 40 nm to 60 nm.

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

[0012] Furthermore, a heterogeneous material layer is arranged on the upper surface of each waveguide single-molecule sensing unit, and the heterogeneous material layer is used to prevent nonspecific binding of the detected molecules; or the heterogeneous material layer is arranged on the upper surface of each waveguide single-molecule sensing unit and the inner side wall of the micropore.

[0013] Optionally, each of the waveguide single-molecule sensing units is formed by arranging a heterogeneous material layer in a circumferential direction around the optical cladding with a planar structure where the waveguide single-molecule sensing unit is located, the heterogeneous material layer is used to prevent nonspecific binding of the detected molecules, and the minimum distance is the distance between the surface of the optical cladding with a planar structure and the surface of the waveguide layer.

[0014] Furthermore, the heterogeneous material layer is disposed on the surface of the optical cladding or embedded inwardly 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 provided on the upper surface of each waveguide single-molecule sensing unit and the inner side wall of the micropore; or a gold material layer is provided on the upper surface of each waveguide single-molecule sensing unit and the inner side wall and inner bottom wall of the micropore.

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

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

[0019] Optionally, the single-molecule sensor array module further comprises a coupling-in grating connected to the optical waveguide, and the coupling-in grating is used to couple the excitation light and transmit it to the optical waveguide.

[0020] Furthermore, the coupling-in grating is a periodic grating, a fan-shaped grating or a sub-wavelength grating; the excitation light is coupled into the coupling-in grating through an optical fiber or the excitation light is coupled into the coupling-in grating through a spatial light mode.

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

[0022] Optionally, the waveguide array is a single-mode waveguide array; the multimode interferometer at each level in the multimode interferometer cascade array splits the excitation light in a 1:1 ratio; the single-molecule sensor array module also includes a plurality of outcoupling gratings, and the outcoupling gratings are connected to the waveguides in a one-to-one correspondence.

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

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

[0025] The microfluidic liquid loading module comprises: a liquid inlet, a liquid inlet channel, a fluid containing cavity, a liquid outlet channel and a liquid outlet which are arranged in sequence and connected in a carrier plate; the fluid containing cavity is arranged above the single-mode waveguide array in the single-molecule sensor array module, and the fluid containing cavity is a blind cavity extending from bottom to top.

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

[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 sensor array module.

[0028] Optionally, the single-molecule sensor array module and the microfluidic liquid loading module are tightly attached via a rubber ring and sealed and fixed externally with a mechanical structure; or the single-molecule sensor array module and the microfluidic liquid loading module are sealed and fixed via an adhesive, and the adhesive meets biocompatibility.

[0029] The present invention also provides a single molecule quantitative analysis method, the analysis method comprising:

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

[0031] The liquid to be detected is introduced into the liquid inlet of the microfluidic liquid loading module, and the liquid to be detected flows into the fluid containing cavity through the liquid inlet channel and is dispersed in the waveguide single molecule sensing unit of the waveguide array and 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 spectrum detection system;

[0033] The spectrum detection system performs digital processing on the collected optical signal, and the principle of the digital processing is: setting an optical signal threshold, when the detected optical signal is not less than the threshold, it is recorded as the waveguide single molecule sensing unit detecting a target single molecule, and the count is 1; when the detected optical signal is less than the threshold, it is recorded as the waveguide single molecule sensing unit not detecting the 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 the liquid to be detected is introduced into the liquid inlet of the microfluidic liquid loading module, the surface of the waveguide single-molecule sensing unit needs to be subjected to hydrophilic surface treatment, specific site modification, streptavidin modification and biotinylated antibody modification in sequence.

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

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

[0038] As described above, the single-molecule sensor array module, quantitative analysis chip and quantitative analysis method of the present invention can realize high-throughput parallel excitation and sensing of single molecules, and the detection sensitivity limit can reach the single-molecule level; the optical signal of the ultra-low concentration target molecule can form a standard curve, and the problem of uneven and non-repetitive optical signal intensity can be solved by using a digital processing method, so that accurate quantitative analysis of ultra-low concentration molecules can be achieved, and the detection sensitivity can be improved, and the sensitivity level can reach the single-molecule level. It has great application value in the fields of clinical medicine, life sciences, food testing, environmental protection, national defense and public security, and basic research. For example, in clinical medicine, it can realize the tracking detection of specific pathological molecules, in the field of life sciences, it can realize the detection of targeted DNA and RNA, and in food testing and environmental protection, it can realize ultra-sensitive quantitative detection of pesticide residue molecules. It can also be applied to single-molecule immunoassays to achieve single-molecule immunoassays with ultra-low detection limits. It is expected to achieve early and very early immune diagnosis, and more accurate real-time detection and pathological tracking can be achieved for various diseases such as neurological disease detection, tumor detection, and infectious disease detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Shown is a schematic structural diagram of a single-molecule sensor array module of the present invention.

[0040] Figure 2 Shown is a schematic diagram of the cross-sectional structure of a single waveguide single molecule sensing unit in the single molecule sensing array module of the present invention.

[0041] Figure 3 Shown is an 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 Shown are 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] Fig.10 Shown is a top view of the single-molecule quantitative analysis chip of the present invention.

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

[0045] Fig.12 Shown is a three-dimensional rendering of the single-molecule quantitative analysis chip of the present invention.

[0046] Fig.13 It is a schematic diagram of the detection structure of the single molecule quantitative analysis chip of the present invention when performing single molecule quantitative detection.

[0047] Figures 14 to 16It shows the statistical principle diagram of digital processing when the single-molecule quantitative analysis chip of the present invention performs digital processing on the optical signal.

[0048] Fig.17 Shown is a schematic diagram of the principle of single-molecule immunoassay for p-Tau based on a single-molecule quantitative analysis chip in Experimental Example 1.

[0049] Fig.18 Display as Fig.17 Part of the microwell fluorescence time series signal diagram of p-Tau single molecule immunoassay.

[0050] Fig.19 Display as Fig.17 The standard curve of the “1” ratio of the digital processing statistics of p-Tau single molecule immunoassay.

[0051] Figure 20 to Figure 25 Shown is a schematic diagram of the surface chemical modification preparation of the single-molecule quantitative analysis chip in Experimental Example 1 and the specific connection process of p-Tau protein and fluorescent dye molecules.

[0052] Fig.26 Shown is a schematic diagram of the principle of single-molecule immunomagnetic bead detection based on a single-molecule quantitative analysis chip in Experimental Example 2.

[0053] Figures 27 to 31 Shown is a schematic diagram of the preparation process of surface chemical modification of immunomagnetic beads in Experimental Example 2.

[0054] Fig.32 Display as Fig.26 Part of the microwell fluorescence time series signal diagram of single-molecule immunomagnetic beads capturing proteins.

[0055] Fig.33 Display as Fig.26 The standard curve of the “1” ratio of the digital processing statistics of single-molecule immunomagnetic bead captured protein detection.

[0056] Fig.34 Shown is a schematic diagram of the cross-sectional structure of a waveguide single-molecule sensing unit of a single-molecule quantitative analysis chip based on surface plasmon enhancement, wherein two gold balls are accommodated in the micropores of the waveguide single-molecule sensing unit.

[0057] Fig.35 Display as Fig.34 Electric field simulation diagram of the waveguide single-molecule sensing unit.

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

[0059] Fig.37 Shown is a schematic diagram of the cross-sectional structure of a waveguide single-molecule sensing unit of a single-molecule quantitative analysis chip based on surface plasmon enhancement, wherein a gold material layer is formed on the surface of the waveguide single-molecule sensing unit and two gold balls are accommodated in the microwells.

[0060] Fig.38 Display as Fig.37 Electric field simulation diagram of the waveguide single-molecule sensing unit.

[0061] Fig.39 It shows the electric field simulation diagram of the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on the surface plasmon enhancement method. A gold material layer is formed on the surface of the waveguide single-molecule sensing unit and a gold ball is accommodated in the micropore.

[0062] Fig.40 Shown is a schematic diagram of the cross-sectional structure of a waveguide single-molecule sensing unit of a single-molecule quantitative analysis chip based on surface plasmon enhancement, wherein three stacked gold balls are accommodated in the microwells of the waveguide single-molecule sensing unit.

[0063] Figure 41 to Figure 46 Shown is a schematic diagram of several distribution patterns of gold spheres of different numbers and sizes in the microwells of the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on surface plasmon enhancement.

[0064] Figures 47 to 50 Schematic diagram showing several shapes of microholes in the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on surface plasmon enhancement.

[0065] Fig.51 Shown are the standard Raman curve and partial micropore Raman signal diagram of the single-molecule quantitative analysis chip of Experimental Example 3 for glucose solution detection based on the surface plasmon enhancement method.

[0066] Fig.52 The figure shows the standard curve of the “1” ratio of the digital processing statistics of glucose solution detection performed by the single-molecule quantitative analysis chip of Experimental Example 3 based on the surface plasmon enhancement method.

[0067] Fig.53 Shown is a partial microwell fluorescence timing signal diagram of the single-molecule quantitative analysis chip of Experimental Example 4 for Cy3 detection based on surface plasmon enhancement.

[0068] Fig.54 The figure shows the standard curve of the “1” ratio of the digital processing statistics of Cy3 detection performed by the single-molecule quantitative analysis chip of Experimental Example 4 based on the surface plasmon enhancement method.

[0069] Fig.55Shown is a schematic diagram of the cross-sectional structure of the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip of Experimental Example 5 based on the surface plasmon enhancement method.

[0070] Fig.56 Shown is a schematic diagram of the principle of fluorescence-free and label-free single-molecule detection based on the single-molecule quantitative analysis chip of Experimental Example 5 using surface plasmon enhancement.

[0071] Fig.57 The graph shows the shift of the plasmon resonance peak in a waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip of Experimental Example 5 based on the surface plasmon enhancement method.

[0072] Fig.58 The figure shows the standard curve of the “1” ratio of the digital processing statistics of the non-fluorescence and label-free single-molecule detection based on the single-molecule quantitative analysis chip of Experimental Example 5 using the surface plasmon enhancement method.

[0073] Component number description

[0074] 1 Single-molecule quantitative analysis chip

[0075] 10 Single-molecule sensor array module

[0076] 11 Incoupling grating

[0077] 12 Multimode Interferometer Cascade Array

[0078] 120 Multimode Interferometer

[0079] 13 Waveguide Array

[0080] 130 Waveguide

[0081] 131 waveguide layer

[0082] 132 Optical Cladding

[0083] 133 Surface evanescent waves

[0084] 134 Micropores

[0085] 135 Waveguide Single-Molecule Sensing Unit

[0086] 136 Heterogeneous Material Layer

[0087] 137 Gold material layer

[0088] 14 Outcoupling grating

[0089] 15 Optical waveguide

[0090] 16 Fiber Optic

[0091] 20 Microfluidic Liquid Loading Module

[0092] 21 Liquid inlet

[0093] 22 Liquid inlet channel

[0094] 220 Main Road

[0095] 221 Sub-pipeline

[0096] 23 Fluid receiving chamber

[0097] 24 Liquid outlet channel

[0098] 240 Sub-pipeline

[0099] 241 Main Road

[0100] 25 liquid outlet

[0101] 26 Connecting pipes

[0102] 27 Carrier Board

[0103] 30 Optical detection device

[0104] 31. Optical signal collection system

[0105] 310 Objective

[0106] 311 Reflector

[0107] 312 First lens

[0108] 313 Pinhole

[0109] 314 Second lens

[0110] 315 filter

[0111] 316 Third lens

[0112] 32 Spectral detection system

[0113] 33 light signal

[0114] 34 Immunomagnetic beads

[0115] 35 Golden Globe

[0116] 36 Single molecule DETAILED DESCRIPTION

[0117] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0118] See also Figures 1 to 58 It should be noted that the illustrations provided in this embodiment are only used to illustrate the basic concept of the present invention in a schematic manner, and the illustrations only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0119] like Figure 1 and Figure 2 As shown, this embodiment provides a single molecule sensor array module 10, and the single molecule sensor array module 10 includes: an optical waveguide 15, a multi-mode interferometer cascade array 12 and a waveguide array 13 connected in sequence;

[0120] The multi-mode interferometer 120 at each level in the multi-mode interferometer cascade array 12 splits the excitation light transmitted from the optical waveguide 15;

[0121] The multi-mode interferometers 120 of the last level in the multi-mode interferometer cascade array 12 are connected to the waveguides 130 in the waveguide array 13 in a one-to-one correspondence;

[0122] like Figure 1 and Figure 2 As shown, the waveguide 130 includes a waveguide layer 131 with a high refractive index and an optical cladding 132 with a low refractive index. The optical cladding 132 covers the waveguide layer 131 inside thereof and forms a surface evanescent wave 133 on the surface of the waveguide layer 131.

[0123] like Figure 1 and Figure 2 As shown, the waveguide 130 is provided with a plurality of waveguide single-molecule sensing units 135 along its length direction, and the minimum distance D between the surface of the optical cladding 132 for carrying the sample to be tested and the surface of the waveguide layer 131 in each of the waveguide single-molecule sensing units 135 is controlled within the wave field range of the surface evanescent wave 133.

[0124] It should be noted here that in the entire optical path of the single-molecule sensor array module 10, optical path connectivity between all component structures is achieved through optical waveguides.

[0125] The core technical principle of signal detection of the single molecule sensor array module 10 of this embodiment is as follows: Figure 1 and Figure 2As shown in FIG. 1 , there are 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 in the waveguide single-molecule sensing unit 135 for carrying the sample to be measured and the surface of the waveguide layer 131 is controlled within the wave field range of the surface evanescent wave 133. The surface evanescent wave is a light wave propagating in the waveguide layer with a high refractive index. At the interface between the waveguide layer and the optical cladding with a low refractive index, the surface evanescent wave 133 is formed on the surface of the waveguide layer. The field intensity of the surface evanescent wave decays exponentially with the outward radiation distance (e.g., Figure 3 On the other hand, a proper amount of the single-molecule sample to be detected is dispersed on the surface of the waveguide array 13, and the concentration or incubation time is controlled to control that each waveguide single-molecule sensing unit 135 in the waveguide array 13 has only a few single molecules (e.g., one or two single molecules) on the surface of the optical cladding 132 for carrying the sample to be detected, as shown in FIG. Figure 2 As shown, in this way, several single molecules on each waveguide single molecule sensor 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 sensor unit 135, and the surface evanescent wave 133 will excite these single molecules to generate light signals, so that the detection of single molecules in each waveguide single molecule sensor unit 135 can be achieved by judging the light signal information. The core technical principle of high-throughput detection of the single molecule sensor array module 10 of this embodiment is as follows Figure 1 As shown, by setting an optical waveguide 15, a multi-mode 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 sensor array module 10, the multi-mode interferometer cascade array 12 splits the excitation light, and the multi-mode interferometer 120 cascaded through multiple stages splits the initial beam of excitation light into hundreds or even thousands of beams required and enters each waveguide 130 of the waveguide array 13 respectively, and a plurality of (e.g., hundreds or even thousands) waveguide single-molecule sensor units 135 are set along the length direction of each waveguide 130, so that countless waveguide single-molecule sensor units 135 are formed in the waveguide array 13 on a large scale, and each waveguide single-molecule sensor unit 135 is similar to a pixel for light signal detection, that is, countless light signal detection pixels are formed in the waveguide array 13, so that high-throughput single-molecule detection can be achieved.

[0126] As an example, since the field intensity of the surface evanescent wave 133 decays exponentially with its radiation distance, its effective range is usually in the order of hundreds of nanometers. Figure 2 As shown, the minimum distance D between the surface of the optical cladding used to carry the sample to be measured and the surface of the waveguide layer in each waveguide single molecule sensing unit 135 can be set to be within 200 nm. In addition, considering the feasibility of process implementation, the minimum distance D can be set within the range of 40 nm to 60 nm.

[0127] like Figure 2 and Figure 4 As shown, the waveguide single molecule sensing unit 135 can be configured into two structures. The first structure is to configure a plurality of microholes 134 on the optical cladding 132 of the waveguide 130, such as Figure 2 In the embodiment, a microhole 134 is provided on the optical cladding 132 corresponding to each waveguide single molecule sensing unit 135, and a single molecule to be detected is placed in the microhole 134. At this time, the minimum distance D refers to the distance between the bottom of the microhole 134 and the surface of the waveguide layer 131; the second is as follows Figure 4 The surface of the optical cladding 132 of the waveguide 130 is set as a planar structure, but in order to define a single waveguide single molecule sensing unit 135, a heterogeneous material layer 136 needs to be set in the circumferential direction of the optical cladding 132 corresponding to each waveguide single molecule sensing unit 135, such as Figure 8 In the embodiment, the heterogeneous material layer 136 is disposed on the surface of the optical cladding 132 in the outer circumference of the optical cladding 132 of the waveguide single molecule sensing unit 135 where the heterogeneous material layer 136 is located. Fig. 9 In the embodiment, the heterogeneous material layer 136 is arranged inside the optical cladding 132 in the peripheral direction of the optical cladding 132 of the waveguide single molecule sensing unit 135 where it is located, specifically, embedded inwardly from the surface of the optical cladding 132. The heterogeneous material layer 136 is used to prevent non-specific binding of the molecules to be detected, so that the molecules to be detected can only fall on the surface of the optical cladding 132 outside the heterogeneous material layer 136. At this time, the minimum distance D refers to the distance between the surface of the optical cladding 132 in a planar structure and the surface of the waveguide layer 131.

[0128] In addition, when the waveguide single molecule sensing unit 135 is formed by setting the micropore 134 on the optical cladding 132, the heterogeneous material layer 136 may also be set on at least part of the surface of the optical cladding 132 of the waveguide single molecule sensing unit 135 to control the binding position and binding mode of the single molecule to be detected in the micropore 134, such as Figure 5 The heterogeneous material 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, such as Figure 6 The heterogeneous material layer 136 is formed on the optical cladding 132 outside the microwell 134 and on the inner wall of the microwell 134 , so the single molecule to be detected can only be bound to the bottom wall of the microwell 134 .

[0129] As an example, the material of the heterogeneous material layer 136 may be aluminum oxide, hafnium oxide, titanium nitride, tantalum oxide, or other materials suitable for controlling the inability of biological molecules to bind.

[0130] As an example, the material of the waveguide layer 131 can be selected from one of silicon nitride, lithium niobate, tantalum pentoxide, and lithium triborate, and the material of the optical cladding 132 is silicon oxide. At this time, the single-molecule sensor array module can be implemented based on silicon photonics technology, forming an optical cladding 132 of silicon oxide material on a silicon-based material and forming a waveguide layer 131 in the optical cladding 132 of silicon oxide material. Silicon photonics is a low-cost, high-speed optical communication technology based on silicon photonics. It combines the ultra-large-scale and ultra-high-precision manufacturing characteristics of integrated circuit technology with the advantages of ultra-high speed and ultra-low power consumption of photonic technology. It is a disruptive technology to cope with the failure of Moore's Law. With the help of this silicon-based single-molecule sensor array module, high-throughput analysis can be achieved, and millions or even tens of millions of detection arrays can be easily realized. This is a detection flux that is almost impossible to achieve with traditional detection technology. It has great application value in clinical medicine, life sciences, food testing, environmental protection, national defense and public security, and basic research. For example, in clinical medicine, it can realize the tracking detection of specific pathological molecules, and in food testing and environmental protection, it can realize ultra-sensitive quantitative detection of pesticide residue molecules.

[0131] As an example, the excitation light can be directly coupled into the optical waveguide 15 by end-face coupling, and then the excitation light is transmitted to the multi-mode interferometer cascade array 12 through the optical waveguide 15. Figure 1 As shown, as another example, the excitation light can also be coupled into and transmitted to the optical waveguide 15 through the coupling grating 11. Specifically, the coupling grating 11 is further provided in the single molecule sensor 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 fan-shaped grating or a sub-wavelength grating; the light coupling method can be selected as follows: the first method, such as Fig.12 As shown, the excitation light is coupled into the coupling grating 11 through the optical fiber 16; secondly, the excitation light is coupled into the coupling grating 11 through spatial light. In addition, the excitation light can be coupled into the coupling grating 11 at a preset desired angle.

[0132] As an example, the excitation light may be a single-wavelength laser, such as 488nm, 532nm, 633nm, 638nm, etc., or a laser of a preset wavelength band, such as 550nm-700nm, etc., which may be selected according to actual needs.

[0133] As a preferred example, the waveguide array 13 is a single-mode waveguide array; the multimode interferometer 120 at each level in the multimode interferometer cascade array 12 splits the excitation light in a 1:1 ratio to achieve uniform light output from the waveguide array 13.

[0134] like Figure 1As shown, the single-molecule sensor array module 10 also includes a plurality of outcoupling gratings 14, and the outcoupling gratings 14 are connected to the waveguides 130 one by one. The outcoupling gratings 14 can effectively prevent the excitation light from forming interface reflection, generate a standing wave interference effect in the waveguide 130, and export the excitation light in each waveguide 130 out of the module through the outcoupling gratings 14, thereby improving the detection accuracy.

[0135] In some specific embodiments, the excitation light can be split into 64, 128, 256, 512, 1024, 2048 or even more waveguides 130. In addition, 64, 128, 256, 512, 1024, 2048 or even more waveguide single-molecule sensing units 135 can be set on each waveguide 130. The combination of the two forms a pixel array containing hundreds, thousands, tens of thousands, hundreds of thousands, or even millions of waveguide single-molecule sensing units 135 to achieve arrayed single-molecule detection.

[0136] As an example, when the waveguide single molecule sensing unit 135 is formed by setting a micropore 134 on the optical cladding 132, a gold material layer 137 may be set on the surface of the optical cladding 132 of the waveguide single molecule sensing unit 135, and the gold material layer 137 is used to generate a surface plasmon enhancement effect. The gold material layer 137 may be set on the upper surface of each of the waveguide single molecule sensing units 135 and the inner side wall of the micropore 134; the gold material layer 137 may also be set as Figure 7 As shown, it is arranged on the upper surface of each waveguide single molecule sensing unit 135 and the inner side wall and inner bottom wall of the microhole 134. The surface plasmon effect is a phenomenon in which an electromagnetic wave (such as the surface evanescent wave of this embodiment) excites free charges in a metal and causes collective periodic oscillations. The surface plasmon effect can confine light to the surface of a metal or micro-nano structure, forming a highly enhanced electric field, thereby effectively enhancing the optical signal of the surface adsorbed molecules. Figure 34 to Figure 36 As shown, the gold ball 35 is in the micropore 134 of the waveguide single molecule sensor unit 135, and the surface plasmon is excited on the surface of the gold ball 35 to enhance the local electric field, and the local electric field is confined to the surface of the micro-nano structure (for example, the gap between the two gold balls and the gap where the gold ball contacts the bottom wall of the micropore), as shown in FIG. Figure 37 to Figure 39As shown, after 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 of the waveguide single-molecule sensing units 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 ball contacts the bottom wall of the micropore 134. When the gold ball 35 is used for single-molecule detection, there may be multiple gold balls in the micropore 134, and the multiple gold balls may be stacked and have different sizes. The diameter distribution thereof is generally Poisson distribution. Multiple enhanced local electric fields will appear between the gold balls 35. As long as the molecules to be detected diffuse into the enhanced local electric field of the gold balls, an enhanced optical signal will be generated, such as Figure 41 to Figure 46 As shown, there may be 2, 3, 4, 5, 6, 7, ... gold balls 35 in the micropore 134, and 2, 3, 4, 5, 6, 7, ... enhanced local electric fields will appear between the gold balls 35. As long as the molecules to be detected diffuse into the enhanced local electric field range of the gold balls 35, an enhanced light signal will be generated. In addition, the shape of the micropore 134 can also be different, such as Figures 47 to 50 As shown, the shapes of the micropores 134 are triangles, pentagons, squares, and hexagons, but may also be other shapes, depending on actual conditions.

[0137] like Figures 10 to 12 As shown, this embodiment further provides a single molecule quantitative analysis chip 1, and the single molecule quantitative analysis chip 1 comprises: the single molecule sensor array module 10 and the microfluidic liquid loading module 20 described in this embodiment; the specific structure of the single molecule sensor array module 10 can refer to the above description, and will not be repeated below;

[0138] The microfluidic liquid loading module 20 is sealed and fixed above the single molecule sensor array module 10;

[0139] The microfluidic liquid loading module 20 includes: a liquid inlet 21, a liquid inlet channel 22, a fluid accommodating cavity 23, a liquid outlet channel 24 and a liquid outlet 25 which are arranged in sequence and connected in a carrier plate 27; the fluid accommodating cavity 23 is arranged above the waveguide array 13 in the single-molecule sensor 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 pass through the carrier plate 27.

[0140] The liquid is injected through the liquid inlet 21 of the microfluidic liquid loading module 20, enters the fluid containing chamber 23 through the liquid inlet channel 22, and is dispersed in the waveguide single-molecule sensing unit 135 of the waveguide array 13 in the fluid containing chamber 23, and is excited by the surface evanescent wave 133 generated by the waveguide array 13 to generate a light signal, thereby realizing the detection of a single-molecule signal. The microfluidic liquid loading module 20 realizes the circulation control of the liquid, and the single-molecule sensor array module 10 realizes the excitation and sensing of high-throughput parallel optical signals. The two are sealed and fixed together to realize the precise control of the fluid, and the high-throughput parallel excitation and sensing of single molecules are realized in the detection area. The detection sensitivity limit can reach the single-molecule level, which has great application value in clinical medicine, life sciences, food testing, environmental protection, national defense and public security, and basic research. For example, in clinical medicine, it can realize the tracking detection of specific pathological molecules, in the field of life sciences, it can realize the detection of targeted DNA and RNA, and in food testing and environmental protection, it can realize ultra-sensitive quantitative detection of pesticide residue molecules, etc.; it can also be applied to single-molecule immunoassays to realize single-molecule immunoassays with ultra-low detection limits. It is expected to realize early and very early immune diagnosis, and more accurate real-time detection and pathological tracking can be realized for various diseases such as neurological disease detection, tumor detection, and infectious disease detection.

[0141] like Fig.10 As shown, the liquid inlet channel 22 of the microfluidic liquid loading module 20 can be set to a single-channel liquid inlet mode or a multi-layer cascade liquid inlet mode, wherein in the multi-layer cascade liquid inlet mode, each layer of the liquid inlet channel divides a main line 220 into two sub-lines 221, and the liquid in the initial line can be flattened and divided into several lines to enter the fluid containing chamber 23 through the multi-layer cascade mode; the liquid outlet channel 24 can also be set to a single-channel liquid outlet mode or a multi-layer cascade liquid outlet mode, wherein in the multi-layer cascade liquid outlet mode, each layer of the liquid outlet channel merges two sub-lines 240 into one main line 241, and the liquid in the fluid containing chamber 23 can be merged into a final main line 241 through the multi-layer cascade mode to flow out through several sub-lines 240. The multi-layer cascade liquid inlet channel 22 setting can evenly circulate the liquid entering from the liquid inlet 21 to the fluid containing chamber 23, thereby effectively improving the uniformity of the liquid in the detection area and improving the detection accuracy. Combined with the multi-layer cascade liquid outlet channel 24 setting, the liquid in the fluid containing chamber 23 can be evenly discharged, further ensuring the uniformity of the liquid in the detection area and improving the detection accuracy.

[0142] As an example, the liquid can be distributed in equal proportion to 1, 2, 4, 8, 16, 32, 64, 128, 256 or even more sub-pipelines 221, and finally evenly distributed in the fluid containing chamber 23; in addition, the liquid can be distributed in equal proportion to 1, 2, 4, 8, 16, 32, 64, 128, 256 or even more sub-pipelines 240, and finally converge into a main pipeline 241 to flow out.

[0143] According to actual needs, the liquid inlet 21 and the liquid outlet 25 can also be connected to realize a circulation path of the liquid.

[0144] like Fig.11 As shown, as an example, the liquid flow direction of the microfluidic liquid loading module 20 can be set to be perpendicular to the extension direction of the waveguide array 13 in the single molecule sensor array module 10, so as to further improve the paving uniformity of the liquid on the waveguide array 13.

[0145] As an example, the single-molecule sensing array module 10 and the microfluidic liquid loading module 20 can be set to a detachable sealing and fixing mode, for example, they can be tightly attached by a rubber ring, sealed and fixed externally with a mechanical structure, or sealed and fixed by using an adhesive that meets biocompatibility requirements, preferably using glue that complies with ISO-10993 biocompatibility certification, such as a cured epoxy adhesive.

[0146] like Figures 10 to 13 As shown, this embodiment also provides a single molecule quantitative analysis method, which includes the following steps:

[0147] S1. Provide the single molecule quantitative analysis chip 1 and the optical detection device 30 described in this embodiment. The optical detection device 30 includes an optical signal collection system 31 and a spectrum detection system 32. The optical signal collection system 31 is used to collect the optical signal excited in the single molecule quantitative analysis chip 1.

[0148] S2, the liquid to be detected is introduced into the liquid inlet 21 of the microfluidic liquid loading module 20, the liquid to be detected flows into the fluid containing cavity 23 through the liquid inlet channel 22 and is dispersed in the waveguide single molecule sensing unit 135 of the waveguide array 13 and is excited by the surface evanescent wave generated by the waveguide array 13 to generate the optical signal;

[0149] S3, the optical signal collection system 31 collects the optical signal and transmits it to the spectrum detection system 32;

[0150] S4, the spectrum detection system 32 performs digital processing on the collected optical signal, and the principle of the digital processing is: setting an optical signal threshold, when the detected optical signal is not less than the threshold, it is recorded as the waveguide single molecule sensing unit 135 detecting a target single molecule, and the count is 1; when the detected optical signal is less than the threshold, it is recorded as the waveguide single molecule sensing unit 135 not detecting the target single molecule, and the count is 0;

[0151] S5. Counting the number of target single molecules appearing in the single-molecule quantitative analysis chip 1 through the digital processing.

[0152] like Figures 14 to 16 As shown, the single molecule quantitative analysis method of this embodiment uses a digital processing method when processing the optical signal, sets the optical signal threshold, and when the detected optical signal is greater than or equal to the threshold, it is determined that the waveguide single molecule sensor unit 135 has detected a target molecule, and the system count is 1 (such as Fig.15 When the detected light signal is less than the threshold, it is determined that the waveguide single molecule sensing unit 135 has not detected the target molecule, and the system count is 0 (as shown in FIG. Fig.16 As shown), by detecting and recording the detection array formed by several waveguide single molecule sensing units 135 on the single molecule quantitative analysis chip 1 by the single molecule counting method, the following can be obtained: Fig.14 , wherein 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 quantitative analysis and detection of target molecules. The single molecule quantitative analysis method of this embodiment can form a standard curve for the optical signal of ultra-low concentration target molecules, and the problem of uneven and non-repetitive optical signal intensity can be solved by using a digital processing method, so as to realize accurate quantitative analysis of ultra-low concentration molecules, improve detection sensitivity, and achieve a sensitivity level of single molecule level.

[0153] As an example, Fig.13 As shown, the optical signal collection system 31 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 in an optical path in sequence. 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.

[0154] As an example, the optical signal 33 is often selected as a fluorescence spectrum signal or a Raman spectrum signal, which is determined by the detection method selected according to the characteristics of the target molecule that needs to be detected. In addition, the detection method of the optical signal 33 is often selected as a spectrum type signal or a spectrum intensity signal.

[0155] As an example, according to the characteristics of different target molecules, the surface of the waveguide single molecule sensor unit 135 in the single molecule quantitative analysis chip 1 can be subjected to hydrophilic surface treatment, specific site modification, streptavidin modification and biotinylated antibody modification in sequence before detection, and the light signal 33 obtained after the single molecule quantitative analysis chip 1 is treated is a fluorescence spectrum signal. The hydrophilic surface treatment can be achieved, for example, by plasma treatment. In addition, according to the characteristics of different target molecules, only the surface of the waveguide single molecule sensor unit 135 in the single molecule quantitative analysis chip 1 can be subjected to hydrophilic surface treatment before detection, without the need for subsequent specific site modification and other processes. The specific selection is made according to actual needs.

[0156] The single molecule quantitative analysis method of the present invention is described in detail below in conjunction with specific experimental examples. Obviously, the experimental examples described are only part of the experimental examples of the present invention, not all of the experimental examples. Based on the experimental examples of the present invention, all other experimental examples obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0157] Experimental Example 1

[0158] This experimental example is a p-Tau single molecule immunoassay based on a single molecule quantitative analysis chip.

[0159] Fig.17 The specific principle of this experimental example is shown, in which 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, the p-Tau protein ( Fig.17 The antigen in the sample is then linked to a specific antibody ( Fig.17 The antibody-Dye in the waveguide single-molecule sensing unit 135 is combined with the p-Tau protein, and 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.

[0160] Figure 20 to Figure 25 The figure shows the surface chemical modification preparation of the single molecule quantitative analysis chip 1 and the specific connection process between the p-Tau protein and the fluorescent dye molecule. The specific steps are as follows:

[0161] Surface chemical modification preparation process of single molecule quantitative analysis chip 1:

[0162] 1. Provide a single molecule quantitative analysis chip 1, wherein the waveguide single molecule sensor unit 135 of the single molecule quantitative analysis chip 1 is as follows: Fig. 20 As shown, the structure is that the surface of the optical cladding 132 of the waveguide 130 is set to a planar structure, and a heterogeneous material layer 136 is set 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 minutes, and placed in a vacuum plasma O2 plasma for 2 minutes;

[0163] 2. If Fig.21 As shown, the single-molecule quantitative analysis chip 1 after plasma was immersed in a 1:100 Silane-PEG / Silane-PEG-Biotin solution (1% water / ethanol), incubated at room temperature for 4 hours, and after the incubation was completed, the surface of the single-molecule quantitative analysis chip 1 was cleaned with a large amount of deionized water;

[0164] 3. Such as Fig. 22 As shown, a 2 mg / ml streptavidin protein (such as Fig.17 The PBST solution containing Streptavidin in the mixture was incubated at room temperature for 30 min. After the incubation, the surface of the single-molecule quantitative analysis chip 1 was washed several times with the PBST solution.

[0165] 4. Such as Fig.23 As shown, the surface of the single-molecule quantitative analysis chip 1 incubated with streptavidin protein is coated with a biotinylated p-Tau181 antibody (such as Fig.17 The antibody-biotin) in the PBST solution was incubated at room temperature for 2 hours. After the antibody was fully combined with the surface of the single-molecule quantitative analysis chip 1, the surface of the single-molecule quantitative analysis chip 1 was washed several times with the PBST solution to remove the non-specifically adsorbed antibody;

[0166] Preparation process of single molecule standard curve of p-Tau:

[0167] 1. Apply a blocking buffer containing 5% BSA to the surface of the single-molecule quantitative analysis chip 1 after surface chemical modification, and incubate at room temperature for 1 hour to block non-specific binding sites;

[0168] 2. Use PBST solution to wash the surface of the blocked single molecule quantitative analysis chip 1 multiple times;

[0169] 3. Such as Fig.24 As shown, p-Tau181 protein (as Fig.17The standard sample (antigen in the sample) is diluted to a protein solution with a concentration gradient of 1pg / ml-100pg / ml, and slowly flows into the liquid inlet channel from the liquid inlet. It is incubated at room temperature for 1 hour to achieve an immune reaction between the antigen and the antibody protein.

[0170] 4. After the reaction is completed, the surface of the single molecule quantitative analysis chip 1 is cleaned with PBST solution to remove the residual unreacted sample;

[0171] 5. As Fig.25 As shown, a Tau protein capture antibody (such as Fig.17 Antibody-Dye in the sample was diluted with blocking buffer and incubated on the chip surface at room temperature for 1 hour to achieve the binding of the fluorescently labeled antibody to the protein to be tested;

[0172] 6. Use PBST solution to wash the surface of the single molecule quantitative analysis chip 1 multiple times;

[0173] 7. Based on the optical detection device, the real-time fluorescence signal generated in the single molecule detection area of ​​the single molecule quantitative analysis chip 1 (i.e., the area where the waveguide single molecule sensing unit 135 is located) is collected and digitized. The proportion of digitized "1" is proportional to the p-Tau181 protein concentration, and a standard curve of p-Tau protein concentration-fluorescence signal can be drawn.

[0174] Fig.18 and Fig.19 Some nanopore fluorescence time series signals and standard curves of p-Tau single molecule immunoassay are shown, Fig.19 The results of the three repeated tests are shown in R 2 The reproducibility reached 0.98, showing a high degree of repeatability. In practical applications, the high reproducibility between analyte concentration and single-molecule events is sufficient to convert the measured "1" fraction into actual concentration.

[0175] Experimental Example 2

[0176] This experimental example is a single molecule immunomagnetic bead detection based on a single molecule quantitative analysis chip.

[0177] Fig.26 The specific principle of this experimental example is shown, wherein the waveguide single molecule sensing unit 135 of the single molecule quantitative analysis chip 1 is as shown in FIG. Fig.26 As shown, the structure is that a micropore 134 is provided on the optical cladding 132 corresponding to each waveguide single molecule sensing unit 135, and the micropore 134 is used to accommodate the single molecule to be detected. Fig.26 The antibody-biotin in the Fig.26 The antigen in the sample is then linked to a specific antibody (e.g. Fig.26The antibody-Dye in the waveguide single molecule sensing unit 135 is combined with the protein to be tested, and 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.

[0178] Figures 27 to 31 The surface modification process of the immunomagnetic beads 34 is shown, and the specific steps are as follows:

[0179] 1. As Fig. 27 As shown, Silane-PEG-Biotin is modified on the surface of immunomagnetic beads 34, such as Fig.28 As shown, 2 mg / mL streptavidin protein (such as Fig.26 The PBST solution containing Streptavidin in the solution was incubated with the immunomagnetic beads at room temperature for 30 min, transferred to a magnetic rack, and washed several times with PBST solution to remove unbound streptavidin protein.

[0180] 2. If Fig.29 As shown, a capture antibody with a biotin tag at one end (such as Fig.26 The antibody-biotin in the solution was incubated with the immunomagnetic beads at room temperature for 30 min, then transferred to a magnetic rack and resuspended in 5 column volumes (CV) of 1×PBST washing solution and washed 5 times, each washing time was 10 min, to remove non-specifically adsorbed antibodies.

[0181] 3. Incubate the prepared immunomagnetic beads with blocking buffer containing 5% BSA at room temperature for 1 h to block nonspecific binding sites.

[0182] 4. Such as Fig.30 As shown, the sample to be tested was added in a concentration gradient of 1pg / ml-100pg / ml and incubated at room temperature for 2h to make the target protein (such as Fig.26 The antigen in the beads is specifically bound to the capture antibody. After the incubation, the beads are transferred to a magnetic rack and the immunomagnetic beads are resuspended and washed several times with PBST solution to remove non-specifically adsorbed impurities.

[0183] 5. As Fig.31 As shown, add labeled antibodies with fluorescent molecules (such as Fig.26 The beads were incubated at room temperature for 1 h to achieve specific binding of the fluorescently labeled antibody to the target protein, and then transferred to a magnetic stand and resuspended and washed with PBST solution for multiple times to remove non-specifically bound color-developing antibodies.

[0184] 6. Such as Fig.26As shown, the resuspended immunomagnetic beads 34 sample is added to the surface of the single molecule quantitative analysis chip 1 and diffuses freely in the solution. When the immunomagnetic beads 34 combined with the target protein and the 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 fluorescent signal is collected.

[0185] Fig.32 and Fig.33 Some nanopore fluorescence time series signals of captured proteins and standard curves are shown, e.g. Fig.33 The results of the three repeated tests are shown in R 2 Reached 0.98.

[0186] Experimental Example 3

[0187] This experimental example is based on single-molecule Raman detection of the waveguide single-molecule sensing unit 135 enhanced by surface plasmons.

[0188] The waveguide single molecule sensing unit 135 of the single molecule quantitative analysis chip 1 is as follows: Fig.34 As shown, its structure is that a microhole 134 is set on the optical cladding 132 corresponding to each waveguide single molecule sensor unit 135, and the microhole 134 is used to accommodate the single molecule to be measured; the diameter of the microhole 134 is 300nm, and the hole depth is 200nm; the spacing between two adjacent microholes 134 is 10μm; the excitation light wavelength is 638nm, the excitation light power is 100mW; the acquisition time is 50ms; the objective lens is a 40x objective lens, and the numerical aperture is 0.75. The specific experimental steps are as follows:

[0189] 1.Configuration concentration from 10 -14 -10 -10 A series of glucose solutions with a concentration of 1 nM were mixed with gold nanoparticles with a concentration of 1 nM at a ratio of 1:10 and then dispersed by ultrasonication for 5 min.

[0190] 2. Inject the mixed solution into the liquid inlet of the microfluidic liquid loading module and incubate for 1 hour.

[0191] 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 it to the spectral detection system. The Raman signal of each waveguide single-molecule sensing unit 135 is acquired by scanning and digitized. The proportion of the "1" signal is obtained through the digitized result, and the standard curve is obtained by fitting.

[0192] Fig.51 The standard Raman curve of glucose solution and some nanopore Raman signals are shown. Fig.52 The digital statistics of the "1" ratio are shown. The R 2 It reached 0.98, showing a high degree of repeatability.

[0193] Experimental Example 4

[0194] This experimental example is based on single-molecule fluorescence detection of the waveguide single-molecule sensing unit 135 enhanced by surface plasmons.

[0195] The waveguide single molecule sensing unit 135 of the single molecule quantitative analysis chip 1 is as follows: Fig.37 As shown, its structure is that a microhole 134 is set on the optical cladding 132 corresponding to each waveguide single molecule sensor unit 135, and the microhole 134 is used to accommodate the single molecule to be measured, and a gold material layer 137 is set on the surface of the optical cladding 132 corresponding to the waveguide single molecule sensor unit 135; the diameter of the microhole 134 is 300nm, and the hole depth is 200nm; the spacing between two adjacent microholes 134 is 10μm; the excitation light wavelength is 638nm, the excitation light power is 100mW; the acquisition time is 50ms; the objective lens is a 40x objective lens, and the numerical aperture is 0.75. The specific experimental steps are as follows:

[0196] 1.Configuration concentration from 10 -15 -10 -12 A series of Cy3 solutions with a concentration of 1 nM were mixed with gold nanoparticles with a concentration of 1 nM at a ratio of 1:10 and then dispersed by ultrasonication for 5 min.

[0197] 2. Inject the mixed solution into the liquid inlet of the microfluidic liquid loading module and incubate for 1 hour.

[0198] 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 it to the spectral detection system. The fluorescence signal of each waveguide single-molecule sensing unit 135 is obtained by scanning and digitized. The digitized result obtains the proportion of the "1" signal and is fitted to obtain a standard curve.

[0199] Fig.53 Part of the nanopore fluorescence timing signal of Cy3 is shown. Fig.54 The standard curve of the percentage of "1" in digital statistics is shown. 2 It reached 0.99, showing a high degree of repeatability.

[0200] Experimental Example 5

[0201] This experimental example is based on the fluorescence-free and label-free single-molecule detection of the waveguide single-molecule sensing unit 135 enhanced by the surface plasmon.

[0202] Since the surface plasmons of metals exhibit medium-sensitive characteristics, the refractive index of the surrounding environment will affect the position of the plasmon resonance peak. Fig.56The basic principle is demonstrated. When a molecule contacts the surface of a metal particle, the position of the plasmon resonance peak will shift. Generally speaking, when the surrounding refractive index increases, the resonance peak position will redshift; when the surrounding refractive index decreases, the resonance peak position will blueshift. This phenomenon can achieve label-free and fluorescence-free single-molecule detection.

[0203] The waveguide single molecule sensing unit 135 of the single molecule quantitative analysis chip 1 is as follows: Fig.55 As shown, its structure is that a microhole 134 is set on the optical cladding 132 corresponding to each waveguide single molecule sensor unit 135, and the microhole 134 is used to accommodate the single molecule to be measured; the diameter of the microhole 134 is 300nm, the hole depth is 200nm, and the spacing between two adjacent microholes 134 is 10μm; the excitation light wavelength is 550nm-700nm, the excitation light power is 100mW; the acquisition time is 50ms, the objective lens is a 40x objective lens, and the numerical aperture is 0.75. The specific experimental steps are as follows:

[0204] 1.Configuration concentration from 10 -11 -10 -7 A series of solutions of 1 nM and 1 nM gold nanoparticles were mixed at a ratio of 1:10 and then dispersed by ultrasonication for 5 min.

[0205] 2. Inject the mixed solution into the liquid inlet of the microfluidic liquid loading module and incubate for 1 hour.

[0206] 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 it to the spectral detection system. The scattered spectrum signal of each waveguide single molecule sensor unit 135 is obtained by scanning. When the resonance peak position is offset by 3nm, it is recorded as a number "1". Finally, the proportion of the "1" signal is counted and fitted to obtain a standard curve.

[0207] Fig.57 and Fig.58 The shift of one of the micropore plasmon resonance peaks and the digital statistical "1" ratio are shown. The R 2 It reached 0.97, showing a high degree of repeatability.

[0208] In summary, the present invention provides a single-molecule sensor array module, a quantitative analysis chip and a quantitative analysis method, which can realize high-throughput parallel excitation and sensing of single molecules, and the detection sensitivity limit can reach the single-molecule level; the optical signal of the ultra-low concentration target molecule can form a standard curve, and the problem of uneven and non-repetitive optical signal intensity can be solved by using a digital processing method, and accurate quantitative analysis of ultra-low concentration molecules can be realized, and the detection sensitivity can be improved, and the sensitivity level reaches the single-molecule level. It has great application value in the fields of clinical medicine, life sciences, food testing, environmental protection, national defense and public security, and basic research. For example, in clinical medicine, it can realize the tracking detection of specific pathological molecules, in the field of life sciences, it can realize the detection of targeted DNA and RNA, and in food testing and environmental protection, it can realize ultra-sensitive quantitative detection of pesticide residue molecules. It can also be applied to single-molecule immunoassays to realize single-molecule immunoassays with ultra-low detection limits. It is expected to realize early and very early immune diagnosis, and more accurate real-time detection and pathological tracking can be realized for various diseases such as neurological disease detection, tumor detection, and infectious disease detection. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0209] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A single molecule sensor array module, characterized in that: The single-molecule sensor array module comprises: an optical waveguide, a multi-mode interferometer cascade array and a waveguide array connected in sequence; The multi-mode interferometer at each level in the multi-mode interferometer cascade array splits the excitation light transmitted from the optical waveguide; The multi-mode interferometers of the last level in the multi-mode interferometer cascade array are connected to the waveguides in the waveguide array in a one-to-one correspondence; The waveguide includes a waveguide layer with a high refractive index and an optical cladding layer with a low refractive index, wherein the optical cladding layer covers the waveguide layer and forms a surface evanescent wave on the surface of the waveguide layer; The waveguide is provided with a plurality of waveguide single-molecule sensing units along its length direction, and the minimum distance between the surface of the optical cladding for carrying the sample to be measured 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 sensor array module according to claim 1, characterized in that: The minimum distance between the surface of the optical cladding layer used to carry the sample to be measured and the surface of the waveguide layer in each of the waveguide single-molecule sensing units is less than 200 nm.

3. The single molecule sensor array module according to claim 2, characterized in that: The minimum distance between the surface of the optical cladding used to carry the sample to be measured and the surface of the waveguide layer in each of the waveguide single-molecule sensing units is 40 nm to 60 nm.

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

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

6. The single molecule sensor array module according to claim 1, characterized in that: Each of the waveguide single-molecule sensing units is formed by arranging a heterogeneous material layer in a circumferential direction around the optical cladding with a planar structure where the waveguide single-molecule sensing unit is located, and the heterogeneous material layer is used to prevent nonspecific binding of the detected molecules, and the minimum distance is the distance between the surface of the optical cladding with a planar structure and the surface of the waveguide layer.

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

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

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

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

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

13. The single molecule sensor array module according to claim 12, characterized in that: The coupling-in grating is a periodic grating, a fan-shaped grating or a sub-wavelength grating; the excitation light is coupled into the coupling-in grating through an optical fiber or the excitation light is coupled into the coupling-in grating through a spatial light mode.

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

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

16. A single molecule quantitative analysis chip, characterized in that: The chip comprises: a single molecule sensor 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 sensor array module; The microfluidic liquid loading module includes: a liquid inlet, a liquid inlet channel, a fluid containing cavity, a liquid outlet channel and a liquid outlet which are arranged in sequence and connected in a carrier plate; the fluid containing cavity is arranged above the waveguide array in the single-molecule sensor array module, and the fluid containing 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 set to a single-channel liquid inlet mode or a multi-layer cascade liquid inlet mode, wherein each layer of the liquid inlet channel in the multi-layer cascade liquid inlet mode divides a main line into two sub-lines; the liquid outlet channel is set to a single-channel liquid outlet mode or a multi-layer cascade liquid outlet mode, wherein each layer of the liquid outlet channel in the multi-layer cascade liquid outlet mode merges two sub-lines into one main line.

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 sensor array module.

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

20. A single molecule quantitative analysis method, characterized in that: The analysis method comprises: A single molecule quantitative analysis chip and an optical detection device as claimed in any one of claims 16 to 19 are provided, wherein the optical detection device comprises an optical signal collection system and a spectrum detection system, wherein the optical signal collection system is used to collect optical signals excited in the single molecule quantitative analysis chip; The liquid to be detected is introduced into the liquid inlet of the microfluidic liquid loading module, and the liquid to be detected flows into the fluid containing cavity through the liquid inlet channel and is dispersed in the waveguide single molecule sensing unit of the waveguide array and 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 spectrum detection system; The spectrum detection system performs digital processing on the collected optical signal, and the principle of the digital processing is: setting an optical signal threshold, when the detected optical signal is not less than the threshold, it is recorded as the waveguide single molecule sensing unit detecting a target single molecule, and the count is 1; when the detected optical signal is less than the threshold, it is recorded as the waveguide single molecule sensing unit not detecting the 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 the liquid to be detected is introduced into the liquid inlet of the microfluidic liquid loading module, the surface of the waveguide single-molecule sensing unit needs to be subjected to hydrophilic surface treatment, specific site modification, streptavidin modification and biotinylated antibody modification in sequence.

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 type signal or a spectral intensity signal.

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