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, which can achieve high-throughput parallel excitation and sensing of single molecules, and the detection sensitivity limit can reach the single-molecule level; a standard curve can be formed for the optical signals of target molecules at ultra-low concentrations, and the problems of uneven and non-repetitive optical signal intensities can be solved by using digital processing methods, enabling accurate quantitative analysis of ultra-low concentration molecules, improving the detection sensitivity, and the sensitivity level reaching the single-molecule level. It has great application value in the fields of clinical medicine, life sciences, food detection, environmental protection, national defense and public safety, and basic research. It can also be applied to single-molecule immunoassay to achieve single-molecule immunoassay with ultra-low detection limits.

Description

Technical Field

[0001] The present invention relates to the technical field of single-molecule detection, and particularly to a single-molecule sensing 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, which is a long-term goal pursued by people. It has important application values in many fields such as "rediscovery of traditional biomarkers", "development of new biomarkers", and "new drug research and development". Currently, single-molecule detection mainly uses atomic force microscopy methods and laser-induced fluorescence techniques. Among them, the laser-induced fluorescence method is the most effective method for single-molecule detection, especially suitable for the study of single molecules in solution. However, the traditional laser-induced fluorescence technology for single-molecule detection often has a low throughput, and it is difficult to improve the detection efficiency and accuracy. High throughput means that the quantity of data or information processed within a certain time reaches a relatively high level. It can greatly accelerate the generation and processing of data, thereby enabling a deeper understanding of many scientific problems and promoting the progress of human science and technology. In addition, in an unknown complex background, it is very difficult to achieve accurate quantitative analysis of the single-molecule sensitivity for ultra-low concentration target molecules, and the single-molecule signal intensity is often uneven and non-repetitive. Summary of the Invention

[0003] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a single-molecule sensing 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 laser-induced fluorescence technology for single-molecule detection has a low throughput, it is difficult to improve the detection efficiency and accuracy, and it is very difficult to achieve accurate quantitative analysis of the single-molecule sensitivity, especially for ultra-low concentration target molecules.

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

[0005] Each layer of multimode interferometer in the cascaded array of multimode interferometers splits the excitation light transmitted from the optical waveguide;

[0006] The last layer of multimode interferometer in the cascaded array of multimode interferometers is connected to the waveguides in the waveguide array one by one;

[0007] The waveguide includes a waveguide layer with a high refractive index and an optical cladding layer with a low refractive index. The optical cladding layer wraps the waveguide layer inside it and forms a surface evanescent wave on the surface of the waveguide layer;

[0008] A plurality of waveguide single-molecule sensing units are arranged along the length direction of the waveguide. In each waveguide single-molecule sensing unit, the minimum distance between the surface of the optical cladding layer for carrying the sample to be measured and the surface of the waveguide layer is controlled within the wave field range of the surface evanescent wave.

[0009] Optionally, in each waveguide single-molecule sensing unit, the minimum distance between the surface of the optical cladding layer for carrying the sample to be measured and the surface of the waveguide layer is less than 200 nm.

[0010] Further, in each waveguide single-molecule sensing unit, the minimum distance between the surface of the optical cladding layer for carrying the sample to be measured and the surface of the waveguide layer is 40 nm to 60 nm.

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

[0012] Further, a heterogeneous material layer is provided 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 molecules to be detected; or the heterogeneous material layer is provided on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall of the micropores.

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

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

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

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

[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 layer is silicon oxide.

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

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

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

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

[0022] Optionally, the waveguide array is a single-mode waveguide array; each multi-mode interferometer in each layer of the multi-mode interferometer cascaded array splits the excitation light in a 1:1 ratio; the single-molecule sensing array module further includes a plurality of output gratings, and the output gratings are connected to the waveguides in a one-to-one correspondence.

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

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

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

[0026] Optionally, the liquid inlet channel is set to a single-channel liquid inlet mode or a multi-level cascaded liquid inlet mode, wherein in the multi-level cascaded liquid inlet mode, each layer of the liquid inlet channel divides a main pipeline into two sub-pipelines; the liquid outlet channel is set to a single-channel liquid outlet mode or a multi-level cascaded liquid outlet mode, wherein in the multi-level cascaded liquid outlet mode, each layer of the liquid outlet channel combines two sub-pipelines into a main pipeline.

[0027] Optionally, the liquid flow direction of the microfluidic liquid loading module is perpendicular to the extending 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 closely attached through a rubber ring and are hermetically fixed by a mechanical structure externally; or the single-molecule sensing array module and the microfluidic liquid loading module are hermetically fixed by an adhesive, and the adhesive satisfies biocompatibility.

[0029] The present invention also provides a single-molecule quantitative analysis method, which includes:

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

[0031] A 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 accommodation cavity through the liquid inlet channel and is dispersed in the waveguide single-molecule sensing units of the waveguide array and is excited by the surface evanescent wave generated by the waveguide array to generate the optical signals;

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

[0033] The spectral detection system performs digital processing on the collected optical signals, 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 that a target single molecule is detected by the waveguide single-molecule sensing unit, and the count is 1; when the detected optical signal is less than the threshold, it is recorded that no target single molecule is detected by the waveguide single-molecule sensing unit, 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 liquid inlet of the microfluidic liquid loading module, it is necessary to first perform a hydrophilic surface treatment, a specific site modification, a streptavidin modification, and a biotinylated antibody modification on the surface of the waveguide single-molecule sensing unit in sequence.

[0036] Optionally, the optical signals are fluorescence spectral signals or Raman spectral signals.

[0037] Furthermore, the optical signals are spectral pattern signals or spectral intensity signals.

[0038] As described above, the single-molecule sensing array module, quantitative analysis chip, and quantitative analysis method of the present invention can achieve high-throughput parallel excitation and sensing of single molecules, and the detection sensitivity limit can reach the single-molecule level. A standard curve can be formed for the optical signals of target molecules at ultra-low concentrations. The problem of uneven and non-repetitive optical signal intensities can be solved using digital processing methods, enabling accurate quantitative analysis of ultra-low concentration molecules, improving detection sensitivity, and reaching the single-molecule level of sensitivity. It has great application value in the fields of clinical medicine, life sciences, food detection, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it can achieve the tracking detection of specific pathological molecules; in the field of life sciences, it can achieve the detection of targeted DNA and RNA; in food detection and environmental protection, it can achieve ultra-sensitive quantitative detection of pesticide residues, etc. 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 extremely early immunodiagnosis, and more accurate real-time detection and pathological tracking can be achieved for various diseases such as the detection of neurological diseases, tumor detection, and infectious disease detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0041] Figure 3 It shows 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 It shows schematic cross-sectional structure diagrams 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 It shows a top view of the single-molecule quantitative analysis chip of the present invention.

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

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

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

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

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

[0049] Figure 18 It shows Figure 17 Partial micropore fluorescence time-sequential signal diagram of p-Tau single-molecule immunoassay.

[0050] Figure 19 It shows Figure 17 Standard curve of the "1" occupancy ratio of digital processing statistics of p-Tau single-molecule immunoassay.

[0051] Figures 20 to 25 It shows the schematic diagram of the preparation of surface chemical modification 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] Figure 26 It shows the schematic diagram of single-molecule immunomagnetic bead detection based on the single-molecule quantitative analysis chip in Experimental Example 2.

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

[0054] Figure 32 It shows Figure 26 Partial micropore fluorescence time-sequential signal diagram of single-molecule immunomagnetic bead capturing protein.

[0055] Figure 33 It shows Figure 26 Standard curve of the "1" occupancy ratio of digital processing statistics of single-molecule immunomagnetic bead capturing protein detection.

[0056] Figure 34 It shows the cross-sectional structure schematic diagram of the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip based on the surface plasmon enhanced mode, where there are 2 gold balls accommodated in the micropores of the waveguide single-molecule sensing unit.

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

[0058] Figure 36 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 enhanced mode, where there is 1 gold ball accommodated in the micropores of the waveguide single-molecule sensing unit.

[0059] Figure 37 Shown is a schematic cross-sectional structure diagram of a waveguide single-molecule sensing unit of a single-molecule quantitative analysis chip based on surface plasmon enhancement, where a gold material layer is formed on the surface of the waveguide single-molecule sensing unit and 2 gold balls are accommodated in the micropores.

[0060] Figure 38 Shown as Figure 37 The electric field simulation diagram of the waveguide single-molecule sensing unit.

[0061] Figure 39 Shown is the electric field simulation diagram of a waveguide single-molecule sensing unit of a single-molecule quantitative analysis chip based on surface plasmon enhancement, where a gold material layer is formed on the surface of the waveguide single-molecule sensing unit and 1 gold ball is accommodated in the micropores.

[0062] Figure 40 Shown is a schematic cross-sectional structure diagram of a waveguide single-molecule sensing unit of a single-molecule quantitative analysis chip based on surface plasmon enhancement, where 3 stacked gold balls are accommodated in the micropores of the waveguide single-molecule sensing unit.

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

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

[0065] Figure 51 Shown are the standard Raman curve and partial micropore Raman signal diagrams for detecting glucose solution by a single-molecule quantitative analysis chip based on surface plasmon enhancement in Experimental Example 3.

[0066] Figure 52 Shown is the standard curve of the ratio of "1" statistically obtained by digital processing for detecting glucose solution by a single-molecule quantitative analysis chip based on surface plasmon enhancement in Experimental Example 3.

[0067] Figure 53 Shown are partial micropore fluorescence time-sequential signal diagrams for detecting Cy3 by a single-molecule quantitative analysis chip based on surface plasmon enhancement in Experimental Example 4.

[0068] Figure 54 Shown is the standard curve of the ratio of "1" statistically obtained by digital processing for detecting Cy3 by a single-molecule quantitative analysis chip based on surface plasmon enhancement in Experimental Example 4.

[0069] Figure 55Schematic cross-sectional structure diagram of the waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip shown in Experimental Example 5 based on the surface plasmon enhancement method.

[0070] Figure 56 Schematic diagram of the principle of fluorescence-free and label-free single-molecule detection of the single-molecule quantitative analysis chip shown in Experimental Example 5 based on the surface plasmon enhancement method.

[0071] Figure 57 Graph showing the shift of the surface plasmon resonance peak in a waveguide single-molecule sensing unit of the single-molecule quantitative analysis chip shown in Experimental Example 5 based on the surface plasmon enhancement method.

[0072] Figure 58 Standard curve of the ratio of "1" in the digital processing statistics of fluorescence-free and label-free single-molecule detection of the single-molecule quantitative analysis chip shown in Experimental Example 5 based on the surface plasmon enhancement method.

[0073] Description of component labels

[0074] 1 Single-molecule quantitative analysis chip

[0075] 10 Single-molecule sensing array module

[0076] 11 Coupling 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 wave

[0084] 134 Micropore

[0085] 135 Waveguide single-molecule sensing unit

[0086] 136 Heterogeneous material layer

[0087] 137 Gold material layer

[0088] 14 Coupling-out grating

[0089] 15 Optical waveguide

[0090] 16 Optical fiber

[0091] 20 Microfluidic liquid loading module

[0092] 21 Liquid inlet

[0093] 22 Liquid inlet channel

[0094] 220 Main pipeline

[0095] 221 Sub - pipeline

[0096] 23 Fluid accommodation cavity

[0097] 24 Liquid outlet channel

[0098] 240 Sub - pipeline

[0099] 241 Main pipeline

[0100] 25 Liquid outlet

[0101] 26 Connecting pipeline

[0102] 27 Carrier plate

[0103] 30 Optical detection device

[0104] 31 Optical signal collection system

[0105] 310 Objective lens

[0106] 311 Reflecting mirror

[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 Optical signal

[0114] 34 Immunomagnetic beads

[0115] 35 Gold ball

[0116] 36 Single molecule Detailed implementation manners

[0117] The following illustrates the implementation manners of the present invention through specific specific examples. 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 implementation manners, 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.

[0118] Please refer to Figures 1 to 58 . It should be noted that the illustrations provided in this embodiment only schematically illustrate the basic concept of the present invention. Therefore, only the components related to the present invention are shown in the illustrations, rather than being drawn according to the number, shape, and size of the components in actual implementation. The types, quantities, and proportions of the components in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

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

[0120] Each layer of multimode interferometer 120 in the multimode interferometer cascade array 12 splits the excitation light transmitted from the optical waveguide 15;

[0121] The multimode interferometer 120 of the last layer in the multimode interferometer cascade array 12 is connected to the waveguide 130 in the waveguide array 13 one by one;

[0122] As Figure 1 and Figure 2 shown, the waveguide 130 includes a waveguide layer 131 with a high refractive index and an optical cladding layer 132 with a low refractive index. The optical cladding layer 132 wraps the waveguide layer 131 inside it, and a surface evanescent wave 133 is formed on the surface of the waveguide layer 131;

[0123] As Figure 1 and Figure 2 shown, a plurality of waveguide single-molecule sensing units 135 are arranged along the length direction of the waveguide 130. In each waveguide single-molecule sensing unit 135, the minimum distance D between the surface of the optical cladding layer 132 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.

[0124] It should be noted here that throughout the entire optical path of the single-molecule sensing array module 10, the optical path connection between all component structures is realized through the optical waveguide.

[0125] The core technical principle of signal detection of the single-molecule sensing array module 10 in this embodiment is as Figure 1 and Figure 2As shown, 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 an optical wave propagating in the waveguide layer with a high refractive index. When total internal reflection occurs at the interface between the waveguide layer and the optical cladding with a low refractive index, the surface evanescent wave 133 will be formed on the surface of the waveguide layer, and the field strength of the surface evanescent wave will decay exponentially with the outward radiation distance (as Figure 3 shown). On the other hand, an appropriate amount of the single-molecule sample to be measured is dispersed on the surface of the waveguide array 13. By controlling the concentration or incubation time, only a few single molecules (such as one or two single molecules) are present on the surface of the optical cladding 132 of each waveguide single-molecule sensing unit 135 in the waveguide array 13 for carrying the sample to be measured, as Figure 2 shown. In this way, several 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 realized by judging the optical signal information. The core technical principle of the high-throughput detection of the single-molecule sensing array module 10 in this embodiment is as Figure 1 shown. By setting the optical waveguide 15, the multi-mode interferometer cascade array 12 and the waveguide array 13, and connecting these structures in sequence, the optical waveguide 15 transmits the excitation light into the single-molecule sensing array module 10. The multi-mode interferometer cascade array 12 splits the excitation light. After passing through multiple cascaded multi-mode interferometers 120, the initial beam of excitation light is split into hundreds or even thousands of required beams and enters each waveguide 130 of the waveguide array 13 respectively. And several (such as hundreds or even thousands) of waveguide single-molecule sensing units 135 are arranged along the length direction of each waveguide 130, so that a large number of waveguide single-molecule sensing units 135 are formed in the waveguide array 13. Each waveguide single-molecule sensing unit 135 is similar to a pixel for optical signal detection, that is, countless optical signal detection pixels are formed in the waveguide array 13, so that high-throughput single-molecule detection can be realized.

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

[0127] AsFigure 2 and Figure 4 As shown in Figure 2 , the waveguide single-molecule sensing unit 135 can be set to two structures. For the first structure, a plurality of microholes 134 are provided on the optical cladding 132 of the waveguide 130. For example, in Figure 2 , one microhole 134 is provided on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. 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. For the second structure, as shown in Figure 4 , the surface of the optical cladding 132 of the waveguide 130 is set to a planar structure. However, in order to define a single waveguide single-molecule sensing unit 135, a hetero material layer 136 needs to be circumferentially provided around the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135. For example, in Figure 8 , the hetero material layer 136 is provided on the surface of the optical cladding 132 circumferentially around the optical cladding 132 of the waveguide single-molecule sensing unit 135 where it is located. For example, in Figure 9 , the hetero material layer 136 is provided inside the optical cladding 132 circumferentially around the optical cladding 132 of the waveguide single-molecule sensing unit 135 where it is located. Specifically, it is embedded inward from the surface of the optical cladding 132. The hetero 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 hetero material layer 136. At this time, the minimum distance D refers to the distance between the surface of the optical cladding 132 with 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 providing microholes 134 on the optical cladding 132, at least part of the surface of the optical cladding 132 of the waveguide single-molecule sensing unit 135 can also be provided with the hetero material layer 136 to control the binding position and binding method of the single molecule to be detected in the microhole 134. For example, in Figure 5 , the hetero material layer 136 is formed on the optical cladding 132 outside the microhole 134, so the single molecule to be detected can only bind in the microhole 134. For example, in Figure 6 , the hetero material layer 136 is formed on the optical cladding 132 outside the microhole 134 and on the inner sidewall of the microhole 134, so the single molecule to be detected can only bind to the bottom wall of the microhole 134.

[0129] As an example, the material of the hetero material layer 136 can be selected from alumina, hafnium oxide, titanium nitride, tantalum oxide, or other materials suitable for preventing the binding of biomolecules.

[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 layer 132 is silicon oxide. At this time, the single-molecule sensing array module can be implemented based on silicon photonics technology, forming an optical cladding layer 132 of silicon oxide material on a silicon-based material and forming a waveguide layer 131 in the optical cladding layer 132 of silicon oxide material. Silicon photonics technology is a low-cost and high-speed optical communication technology based on silicon photonics. It combines the characteristics of ultra-large scale and ultra-high-precision manufacturing of integrated circuit technology and the advantages of ultra-high speed and ultra-low power consumption of photonics technology, and is a disruptive technology to cope with the failure of Moore's Law. With the help of this silicon-based single-molecule sensing array module, high-throughput analysis can be achieved, and detection arrays of millions or even tens of millions can be easily realized, which is a detection throughput that is almost impossible to achieve by traditional detection technologies, and has great application value in the fields of clinical medicine, life science, food detection, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, the tracking detection of specific pathological molecules can be realized, and in food detection and environmental protection, the ultrasensitive quantitative detection of pesticide residues can be realized.

[0131] As an example, the excitation light can be directly coupled into the optical waveguide 15 by means of end-face coupling, and then the excitation light is transmitted to the multimode interferometer cascade array 12 through the optical waveguide 15. As Figure 1 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 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 fan-shaped grating, or a sub-wavelength grating; the light coupling method can be selected as: the first, as Figure 12 shown, the excitation light is coupled into the coupling grating 11 through the optical fiber 16; the second, the excitation light is coupled into the coupling grating 11 by means of spatial light. In addition, the excitation light can be coupled into the coupling grating 11 at a preset required angle.

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

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

[0134] As Figure 1As shown, the single-molecule sensing array module 10 further includes a plurality of output gratings 14, and the output gratings 14 are connected to the waveguides 130 in a one-to-one correspondence. The output gratings 14 can effectively prevent the formation of interface reflection of the excitation light, generate a standing-wave interference effect in the waveguides 130, and export the excitation light in each waveguide 130 through the output gratings 14 to improve 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. Additionally, 64, 128, 256, 512, 1024, 2048 or even more waveguide single-molecule sensing units 135 can be identically arranged 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 arranging micropores 134 on the optical cladding 132, a gold material layer 137 can be arranged 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 can be arranged 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 arranged as Figure 7 shown on the upper surface of each waveguide single-molecule sensing unit 135, the inner sidewall and the inner bottom wall of the micropore 134. The surface plasmon effect is a phenomenon in which an electromagnetic wave (such as the surface evanescent wave in this embodiment) excites free charges in a metal and causes collective periodic oscillations. The surface plasmon effect can confine light to the surface of the metal or micro-nano structure, forming a highly enhanced electric field, thereby effectively enhancing the optical signal of surface-adsorbed molecules. As Figures 34 to 36 shown, the gold sphere 35 is in the micropore 134 of the waveguide single-molecule sensing unit 135, and a surface plasmon-enhanced local electric field is excited on the surface of the gold sphere 35, and the local electric field is confined to the surface of the micro-nano structure (such as the gap between two gold spheres and the gap where the gold sphere contacts the bottom wall of the micropore), as Figures 37 to 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 waveguide single-molecule sensing unit 135 and the inner side wall 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 using the gold ball 35 for single-molecule detection, there may be multiple gold balls in the micropore 134. The multiple gold balls may be stacked, and there are differences in size. Their diameter distribution generally follows a Poisson distribution. Multiple enhanced local electric fields will appear between the gold balls 35. As long as the molecule to be detected diffuses into the enhanced local electric field of the gold ball, an enhanced optical signal will be generated, such as Figures 41 to 46 As shown, there may be 2, 3, 4, 5, 6, 7... gold balls 35 in the micropore 134. 2, 3, 4, 5, 6, 7... enhanced local electric fields will appear between the gold balls 35. As long as the molecule to be detected diffuses into the range of the enhanced local electric field of the gold ball 35, an enhanced optical 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 triangular, pentagonal, square, and hexagonal respectively, but they can also be other shapes, which are specifically set according to the actual situation.

[0137] Such as Figures 10 to 12 As shown, 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; for the specific structure of the single-molecule sensing array module 10, please refer to the above description and will not be elaborated below;

[0138] The microfluidic liquid loading module 20 is hermetically fixed above the single-molecule sensing array module 10;

[0139] The microfluidic liquid loading module 20 includes: a liquid inlet 21, a liquid inlet channel 22, a fluid accommodation cavity 23, a liquid outlet channel 24, and a liquid outlet 25 that are sequentially connected in the carrier plate 27; the fluid accommodation cavity 23 is arranged above the waveguide array 13 in the single-molecule sensing array module 10, and the fluid accommodation cavity 23 is a blind cavity extending upward from the bottom, that is, the opening of the fluid accommodation cavity 23 faces the waveguide array 13 and does not penetrate the carrier plate 27.

[0140] The liquid is injected through the liquid inlet 21 of the microfluidic liquid loading module 20, enters the fluid accommodating cavity 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 accommodating cavity 23, and is excited by the surface evanescent wave 133 generated by the waveguide array 13 to generate an optical signal, realizing the detection of single-molecule signals. The microfluidic liquid loading module 20 realizes the circulation control of the liquid, and the single-molecule sensing array module 10 realizes the excitation and sensing of high-throughput parallel optical signals. Sealing and fixing the two together can achieve precise control of the fluid and realize the excitation and sensing of high-throughput parallel single molecules in the detection area. The detection sensitivity limit can reach the single-molecule level, and it has great application value in the fields of clinical medicine, life science, food detection, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it can realize the tracking detection of specific pathological molecules, in the field of life science, it can realize the detection of targeted DNA and RNA, and in food detection and environmental protection, it can realize the ultrasensitive quantitative detection of pesticide residues, etc.; it can also be applied to single-molecule immunoassay to realize single-molecule immunoassay with an ultra-low detection limit. It is expected to realize early and very early immunodiagnosis, and more accurate real-time detection and pathological tracking can be achieved for various diseases such as the detection of neurological diseases, tumor detection, and infectious disease detection.

[0141] As Figure 10 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-level cascaded liquid inlet mode. In the multi-level cascaded liquid inlet mode, each layer of the liquid inlet channel divides a main pipeline 220 into two sub-pipelines 221. Through the multi-level cascaded mode, the initially one-way liquid can be evenly divided into several paths and enter the fluid accommodating cavity 23; the liquid outlet channel 24 can also be set to a single-channel liquid outlet mode or a multi-level cascaded liquid outlet mode. In the multi-level cascaded liquid outlet mode, each layer of the liquid outlet channel combines two sub-pipelines 240 into one main pipeline 241. Through the multi-level cascaded mode, the liquid in the fluid accommodating cavity 23 can be combined through several sub-pipelines 240 and flow out through the final main pipeline 241. The setting of the liquid inlet channel 22 in this multi-level cascaded mode can evenly circulate the liquid entering from the liquid inlet 21 to the fluid accommodating cavity 23, thereby effectively improving the evenness of the liquid spreading in the detection area, improving the detection accuracy, and combining the setting of the liquid outlet channel 24 in the multi-level cascaded mode can evenly discharge the liquid in the fluid accommodating cavity 23, further ensuring the evenness of the liquid spreading in the detection area and improving the detection accuracy.

[0142] As an example, the liquid can be equally dispersed into 1, 2, 4, 8, 16, 32, 64, 128, 256 or even more sub-pipe lines 221, and finally be evenly dispersed into the fluid accommodation cavity 23; in addition, the liquid can equally pass through 1, 2, 4, 8, 16, 32, 64, 128, 256 or even more sub-pipe lines 240 and finally converge into a main pipe line 241 and flow out.

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

[0144] As Figure 11 shown, as an example, the liquid flow direction of the microfluidic liquid loading module 20 can be set perpendicular to the extending direction of the waveguide array 13 in the single molecule sensing array module 10, so as to further improve the evenness of the liquid spreading 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 in a separable and sealed fixing manner. For example, they can be closely attached by a rubber ring, sealed and fixed by an external mechanical structure or sealed and fixed by using a biocompatible adhesive. It is preferably sealed and fixed by using a glue that meets the ISO-10993 biocompatibility certification, such as a cured epoxy adhesive.

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

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

[0148] S2. Introduce the liquid to be detected into the liquid inlet 21 of the microfluidic liquid loading module 20. The liquid to be detected flows through the liquid inlet channel 22 into the fluid accommodation cavity 23 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 signals.

[0149] S3. The optical signal collection system 31 collects the optical signals and transmits them to the spectral detection system 32.

[0150] S4. The spectral detection system 32 performs digital processing on the collected optical signals. The principle of the digital processing is as follows: Set an optical signal threshold. When the detected optical signal is not less than this threshold, it is recorded that a target single molecule is detected by the waveguide single-molecule sensing unit 135, and the count is 1; when the detected optical signal is less than this threshold, it is recorded that the waveguide single-molecule sensing unit 135 does not detect a target single molecule, and the count is 0.

[0151] S5. Statistically analyze the number of target single molecules that appear in the single-molecule quantitative analysis chip 1 through the digital processing.

[0152] As Figures 14 to 16 shown, when processing the optical signals, the single-molecule quantitative analysis method of this embodiment adopts a digital processing method. Set an optical signal threshold. When the detected optical signal is greater than or equal to this threshold, it is determined that a target molecule is detected by the waveguide single-molecule sensing unit 135, and the system count is 1 (as Figure 15 shown), when the detected optical signal is less than this threshold, it is determined that the waveguide single-molecule sensing unit 135 does not detect a target molecule, and the system count is 0 (as Figure 16 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 in this single-molecule counting method, a single-molecule detection array diagram as Figure 14 can be obtained. Each 1 or 0 represents the single-molecule detection result of the corresponding waveguide single-molecule sensing unit 135, so as to obtain the number of target molecules, and realize the quantitative analysis and detection of target molecules. The single-molecule quantitative analysis method of this embodiment can form a standard curve for the optical signals of ultra-low concentration target molecules. Using the digital processing method can solve the problems of uneven and non-repetitive optical signal intensity, and can realize accurate quantitative analysis of ultra-low concentration molecules, improve the detection sensitivity, and the sensitivity level reaches the single-molecule level.

[0153] As an example, as Figure 13 shown, 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 that are optically connected. 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, and is specifically determined by the detection method selected according to the characteristics of the target molecule to be detected actually. In addition, the detection method of the optical signal 33 is often selected as a spectrum pattern signal or a spectrum intensity signal.

[0155] As an example, according to the characteristics of different target molecules, before detection, 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. The optical signal 33 obtained after processing the single-molecule quantitative analysis chip 1 is a fluorescence spectrum signal. The hydrophilic surface treatment can be realized by means of plasma treatment, for example. In addition, according to the characteristics of different target molecules, before detection, 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, and subsequent processes such as specific site modification are not required. Specific selection is made according to actual needs.

[0156] The single-molecule quantitative analysis method of the present invention will be described in detail below in conjunction with specific experimental examples. Obviously, the described experimental examples are only a part of the experimental examples of the present invention, rather than all the experimental examples. Based on the experimental examples of the present invention, all other experimental examples obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0157] Experimental Example 1

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

[0159] Figure 17 The specific principle of this experimental example is shown, where the tau protein is an important characteristic protein of Alzheimer's disease. By performing surface chemical modification on the waveguide single-molecule sensing unit 135 on the single-molecule quantitative analysis chip 1 and connecting the p-Tau protein ( Figure 17 the antigen in Figure 17 ), finally, the specific antibody ( the antibody-Dye in

[0160] Figures 20 to 25

[0161] The surface chemical modification preparation process of the single-molecule quantitative analysis chip 1:

[0162] 1. Provide a single-molecule quantitative analysis chip 1. The waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is as follows Figure 20 shown. Its structure is to set the surface of the optical cladding 132 of the waveguide 130 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, ultrasonically clean the single-molecule quantitative analysis chip 1 in absolute ethanol for 3 min, and then put it into a vacuum plasma device for O2 plasma for 2 min;

[0163] 2. As Figure 21 shown, immerse the plasma-treated single-molecule quantitative analysis chip 1 in a 1:100 Silane-PEG / Silane-PEG-Biotin solution (1% water / ethanol), incubate at room temperature for 4 h, and after incubation, wash the surface of the single-molecule quantitative analysis chip 1 with a large amount of deionized water;

[0164] 3. As Figure 22 shown, spread a PBST solution containing 2 mg / ml streptavidin protein (such as Streptavidin in Figure 17 ) on the surface of the modified single-molecule quantitative analysis chip 1, incubate at room temperature for 30 min, and after incubation, wash the surface of the single-molecule quantitative analysis chip 1 with the PBST solution multiple times;

[0165] 4. As Figure 23 shown, spread a PBST solution containing biotinylated p-Tau181 antibody (such as antibody-biotin in Figure 17 ) on the surface of the single-molecule quantitative analysis chip 1 incubated with streptavidin protein, incubate at room temperature for 2 h. After the antibody is fully bound to the surface of the single-molecule quantitative analysis chip 1, wash the surface of the single-molecule quantitative analysis chip 1 with the PBST solution multiple times to remove non-specifically adsorbed antibodies;

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

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

[0168] 2. Wash the surface of the blocked single-molecule quantitative analysis chip 1 with the PBST solution multiple times;

[0169] 3. As Figure 24 shown, the p-Tau181 protein (such as Figure 17The antigen in [[]] was diluted to a protein solution with a concentration gradient of 1 pg / ml - 100 pg / ml, and slowly flowed into the liquid inlet channel from the liquid inlet, and incubated at room temperature for 1 h to achieve the immune reaction between the antigen and the antibody protein;

[0170] 4. After the reaction was completed, the surface of the single-molecule quantitative analysis chip 1 was washed with PBST solution to remove the remaining unreacted samples;

[0171] 5. As Figure 25 shown, the Tau protein capture antibody conjugated with fluorescent dye molecules (such as the antibody-Dye in Figure 17 [[]]) was diluted with the blocking buffer and incubated with the chip surface at room temperature for 1 h to achieve the binding of the fluorescently labeled antibody to the protein to be detected;

[0172] 6. The surface of the single-molecule quantitative analysis chip 1 was washed multiple times with PBST solution;

[0173] 7. Based on the optical detection device, the real-time fluorescence signals 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) were 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.

[0174] Figure 18 and Figure 19 showed some nanopore fluorescence time-series signals and standard curves of p-Tau single-molecule immunoassay. As Figure 19 shown, the R 2 reached 0.98 under the results of 3 repeated tests, showing high reproducibility. In practical applications, the high reproducibility between the analyte concentration and the single-molecule events is sufficient to convert the measured proportion of "1" into the actual concentration.

[0175] Experimental Example 2

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

[0177] Figure 26 illustrated the specific principle of this experimental example, in which the waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 was as Figure 26 shown, and its structure was to set a micropore 134 on the optical cladding 132 corresponding to each waveguide single-molecule sensing unit 135, and this micropore 134 was used to accommodate the single molecules to be detected. The surface of the immunomagnetic bead 34 was modified with a specific ligation antibody (such as the antibody-biotin in Figure 26 [[]]) to capture the protein to be detected (such as the antigen in Figure 26 [[]]), and finally the specific antibody conjugated with fluorescent dye molecules (such as Figure 26The antibody-Dye binds to the protein to be detected. The surface evanescent wave 133 of the waveguide single-molecule sensing unit 135 is used to excite the fluorescent dye molecules Dye, and the fluorescence of the fluorescent dye molecules is digitally recorded.

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

[0179] 1. As Figure 27 shown, modify Silane-PEG-Biotin on the surface of the immunomagnetic beads 34. As Figure 28 shown, incubate the PBST solution containing 2 mg / mL streptavidin protein (such as Streptavidin in Figure 26 ) with the immunomagnetic beads 34 at room temperature for 30 min, transfer to a magnetic stand, and wash multiple times with the PBST solution to remove the unbound streptavidin protein.

[0180] 2. As Figure 29 shown, incubate the capture antibody with a biotin tag at one end (such as antibody-biotin in Figure 26 ) with the immunomagnetic beads 34 at room temperature for 30 min. Subsequently, transfer to a magnetic stand and resuspend and wash the immunomagnetic beads 5 times with 5 column volumes (CV) of 1×PBST washing solution, with each washing time being 10 min, to remove non-specifically adsorbed antibodies.

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

[0182] 4. As Figure 30 shown, add the test samples with a concentration gradient of 1 pg / ml - 100 pg / ml, and incubate at room temperature for 2 h to enable specific antigen-antibody binding between the target protein (such as the antigen in Figure 26 ) and the capture antibody. After the incubation, transfer to a magnetic stand, and resuspend and wash the immunomagnetic beads 34 multiple times with the PBST solution to remove non-specifically adsorbed impurities.

[0183] 5. As Figure 31 shown, add the labeled antibody with a fluorescent molecule (such as antibody-Dye in Figure 26 ), incubate at room temperature for 1 h to achieve specific binding between the fluorescently labeled antibody and the target protein. Subsequently, transfer to a magnetic stand, and resuspend and wash the immunomagnetic beads 34 multiple times with the PBST solution to remove non-specifically bound chromogenic antibodies.

[0184] 6. As Figure 26As shown, the resuspended immunomagnetic bead 34 sample is added to the surface of the single-molecule quantitative analysis chip 1 and freely diffuses in the solution. When the immunomagnetic bead 34 conjugated with the target protein and the labeled antibody falls to 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.

[0185] Figure 32 and Figure 33 show the partial nanopore fluorescence time-series signals of the captured protein and the standard curve. As Figure 33 shown, the R value 2 reached 0.98 under the results of 3 repeated tests.

[0186] Experimental Example 3

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

[0188] Among them, the waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is as Figure 34 shown. Its structure is to set a micropore 134 on the corresponding optical cladding 132 of each waveguide single-molecule sensing unit 135. This micropore 134 is used to accommodate the single molecules to be measured. The diameter of the micropore 134 is 300 nm, the pore depth is 200 nm, the spacing between two adjacent micropores 134 is 10 μm, the excitation light wavelength is 638 nm, the excitation light 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:

[0189] 1. Configure a series of glucose solutions with concentrations ranging from 10 -14 -10 -10 M. After mixing with gold nanoparticles with a concentration of 1 nM at a ratio of 1:10, ultrasonically disperse for 5 min.

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

[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 signals of each waveguide single-molecule sensing unit 135 are obtained by scanning, and digital processing is performed. The occupancy ratio of the "1" signal is obtained through the digital result, and the standard curve is fitted.

[0192] Figure 51 show the standard Raman curve of the glucose solution and the partial nanopore Raman signals. Figure 52 shows the occupancy ratio of "1" statistically digitized. The R value 2 reached 0.98 under the results of 3 repeated tests, showing high repeatability.

[0193] Experimental Example 4

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

[0195] Among them, the waveguide single-molecule sensing unit 135 of the single-molecule quantitative analysis chip 1 is as Figure 37 shown. Its structure is to set 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 molecules 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 sensing unit 135; the diameter of the micropore 134 is 300 nm, the pore depth is 200 nm; the distance between two adjacent micropores 134 is 10 μm; the excitation light wavelength is 638 nm, the excitation light 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:

[0196] 1. Configure a series of Cy3 solutions with concentrations ranging from 10 -15 -10 -12 M. After mixing with gold nanoparticles with a concentration of 1 nM at a ratio of 1:10, ultrasonically disperse for 5 min.

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

[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 signals of each waveguide single-molecule sensing unit 135 are obtained by scanning, and digital processing is performed. The ratio of the "1" signal in the digital result is obtained, and a standard curve is fitted.

[0199] Figure 53 Shows the partial nanopore fluorescence time-series signals of Cy3. Figure 54 Then shows the standard curve of the "1" ratio in the digital statistics. Under the results of 3 repeated tests, R 2 reached 0.99, showing high repeatability.

[0200] Experimental Example 5

[0201] This experimental example is for label-free single-molecule detection without fluorescence based on the waveguide single-molecule sensing unit 135 enhanced by surface plasmons.

[0202] Since the surface plasmons of metals exhibit dielectric-sensitive characteristics, the refractive index of the surrounding environment will affect the position of the plasmon resonance peak. Figure 56It shows the basic principle that 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] Among them, the waveguide single molecule sensing unit 135 of the single molecule quantitative analysis chip 1 is as Figure 55 shown. Its structure is to set a microhole 134 on the optical cladding 132 corresponding to each waveguide single molecule sensing unit 135. This microhole 134 is used to accommodate the single molecule to be measured; the diameter of the microhole 134 is 300 nm, the hole depth is 200 nm, and the distance between two adjacent microholes 134 is 10 μm; the excitation light wavelength is 550 nm - 700 nm, the excitation light 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:

[0204] 1. Prepare a series of solutions with concentrations ranging from 10 -11 -10 -7 M. After mixing with gold nanoparticles with a concentration of 1 nM at a ratio of 1:10, ultrasonically disperse for 5 min.

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

[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. By scanning, the scattered spectral signal of each waveguide single molecule sensing unit 135 is obtained. When the resonance peak position shows a 3 nm shift, it is recorded as the number "1". Finally, the occupancy ratio of the "1" signal is statistically analyzed, and a standard curve is fitted.

[0207] Figure 57 and Figure 58 show the shift of the plasmon resonance peak of one of the microholes and the occupancy ratio of the digitized "1". Under the results of 3 repeated tests, R 2 reached 0.97, showing a high degree of repeatability.

[0208] In summary, the present invention provides a single-molecule sensing array module, a quantitative analysis chip and a quantitative analysis method, which can achieve high-throughput parallel excitation and sensing of single molecules, and the detection sensitivity limit can reach the single-molecule level; a standard curve can be formed for the optical signals of target molecules at ultra-low concentrations, and the problems of uneven and non-repetitive optical signal intensities can be solved by using digital processing methods, enabling accurate quantitative analysis of ultra-low concentration molecules, improving the detection sensitivity, and the sensitivity level reaching the single-molecule level. It has great application value in the fields of clinical medicine, life sciences, food detection, environmental protection, national defense and public safety, and basic research. For example, in clinical medicine, it can achieve the tracking detection of specific pathological molecules; in the field of life sciences, it can achieve the detection of targeted DNA and RNA; in food detection and environmental protection, it can achieve ultra-sensitive quantitative detection of pesticide residues, etc. It can also be applied to single-molecule immunoassay to achieve single-molecule immunoassay with ultra-low detection limits. It is expected to achieve early and very early immunodiagnosis, and more accurate real-time detection and pathological tracking can be achieved for various diseases such as the detection of neurological diseases, tumor detection, and infectious disease detection. Therefore, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0209] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by 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 each layer of the cascaded array of multimode interferometers, the multimode interferometer splits the excitation light transmitted from the optical waveguide; The multimode interferometer in the last layer of the cascaded array of multimode interferometers is connected to the waveguides in the waveguide array one by one; The waveguide includes a waveguide layer with a high refractive index and an optical cladding with a low refractive index. The optical cladding wraps the waveguide layer inside it and forms a surface evanescent wave on the surface of the waveguide layer; Along the length direction of the waveguide, a number of waveguide single-molecule sensing units are provided. In each waveguide single-molecule sensing unit, the minimum distance between the surface of the optical cladding for carrying the sample to be measured and the surface of the waveguide layer 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 waveguide single-molecule sensing unit, the minimum distance between the surface of the optical cladding for carrying the sample to be measured 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: In each waveguide single-molecule sensing unit, the minimum distance between the surface of the optical cladding for carrying the sample to be measured and the surface of the waveguide layer is 40 nm to 60 nm.

4. The single-molecule sensing array module according to claim 1, wherein: Each waveguide single-molecule sensing unit is formed by setting micropores on the optical cladding, and the minimum distance is the distance between the bottom of the micropores 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 provided 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 a heterogeneous material layer is provided on the upper surface of each waveguide single-molecule sensing unit and the inner sidewall of the micropores.

6. The single-molecule sensing array module according to claim 1, characterized in that: Each waveguide single-molecule sensing unit is formed by circumferentially providing 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, and 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, wherein: The heterogeneous material layer is provided 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 alumina or hafnium oxide or titanium nitride or tantalum oxide.

9. The single-molecule sensing array module according to claim 4, wherein: A gold material layer is provided on the upper surface and the inner sidewall of the micropores of each waveguide single-molecule sensing unit; or a gold material layer is provided on the upper surface, the inner sidewall, and the inner bottom wall of the micropores of each waveguide single-molecule sensing unit.

10. The single-molecule sensing array module according to claim 1, wherein: 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.

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

12. The single-molecule sensing array module according to claim 1, wherein: The single-molecule sensing array module further includes a coupling grating connected to the optical waveguide. The coupling grating is 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 or a fan-shaped grating or a sub-wavelength grating; the excitation light is coupled into the coupling grating through an optical fiber or the excitation light is coupled into the coupling grating by 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; each multimode interferometer in each layer of the multimode interferometer cascade array splits the excitation light in a 1:1 ratio; the single-molecule sensing array module further includes a plurality of output gratings, and the output gratings are connected to the waveguides in one-to-one correspondence.

16. A single molecule quantitative analysis chip, characterized in that, The chip includes: the single-molecule sensing array module as described in any one of claims 1 to 15 and a microfluidic liquid loading module; The microfluidic liquid loading module is hermetically fixed above the single-molecule sensing array module; The microfluidic liquid loading module includes: a liquid inlet, a liquid inlet channel, a fluid accommodating cavity, a liquid outlet channel, and a liquid outlet that are sequentially connected in a carrier plate; the fluid accommodating cavity is arranged above the waveguide array in the single-molecule sensing array module, and the fluid accommodating cavity is a blind cavity extending upward 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-level cascade liquid inlet mode, wherein in the multi-level cascade liquid inlet mode, each layer of the liquid inlet channel divides a main pipeline into two sub-pipelines; the liquid outlet channel is set to a single-channel liquid outlet mode or a multi-level cascade liquid outlet mode, wherein in the multi-level cascade liquid outlet mode, each layer of the liquid outlet channel combines two sub-pipelines into a main pipeline.

18. The single-molecule quantitative analysis chip according to claim 16, wherein: 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, wherein: The single-molecule sensing array module and the microfluidic liquid loading module are closely attached through a rubber ring and hermetically fixed by a mechanical structure externally; or the single-molecule sensing array module and the microfluidic liquid loading module are hermetically fixed by an adhesive, and the adhesive meets biocompatibility.

20. A single-molecule quantitative analysis method, characterized in that, The analysis method includes: Providing the single-molecule quantitative analysis chip as described in any one of claims 16 to 19 and an optical detection device, the optical detection device includes an optical signal collection system and a spectral detection system, and the optical signal collection system is used to collect the optical signals excited in the single-molecule quantitative analysis chip; Injecting the liquid to be detected into the liquid inlet of the microfluidic liquid loading module, and the liquid to be detected flows into the fluid accommodating cavity through the liquid inlet channel and is dispersed in the waveguide single-molecule sensing units of the waveguide array and is excited by the surface evanescent wave generated by the waveguide array to generate the optical signals; The optical signal collection system collects the optical signals and transmits them to the spectral detection system; The spectral detection system performs digital processing on the collected optical signals, 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 that a target single molecule is detected by the waveguide single-molecule sensing unit, 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 does not detect the target single molecule, and the count is 0; Counting the number of target single molecules appearing in the single-molecule quantitative analysis chip through the digital processing.

21. The single-molecule quantitative analysis method according to claim 20, characterized in that: Before injecting the liquid to be detected into the liquid inlet of the microfluidic liquid loading module, it is necessary to perform hydrophilic surface treatment, specific site modification, streptavidin modification, and biotinylated antibody modification on the surface of the waveguide single-molecule sensing unit in sequence.

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

23. The single-molecule quantitative analysis method according to claim 22, wherein: The detection method of the optical signal is a spectral pattern signal or a spectral intensity signal.

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