A single molecule force spectroscopy-infrared spectroscopy combined method, system and device

Abstract

The present invention discloses a method, system and device for combining single-molecule force spectroscopy and infrared spectroscopy, and relates to the field of single-molecule force spectroscopy. An optical path system module obtains and sends a deflection signal of a microcantilever; a phase-locked amplifier obtains a resonance frequency; a processor module assigns the resonance frequency to a signal generator as a pulse repetition frequency; an electrical signal of the same frequency is generated; a trigger signal is transmitted to an infrared laser; a reference signal is transmitted to the phase-locked amplifier; a photothermal detector converts the laser intensity into laser power; the phase-locked amplifier demodulates the deflection signal of the microcantilever to obtain the amplitude of the microcantilever; an amplitude-wavenumber curve is obtained by the processor module; the raw data of the single-molecule force spectroscopy and infrared spectroscopy of the sample under test are obtained; the raw data of the single-molecule force spectroscopy and infrared spectroscopy of the sample under test are divided by the laser power, and the photoacoustic noise is removed to obtain a true detection result. The present invention improves the accuracy and signal-to-noise ratio of the AFM infrared spectrometer, and realizes the data acquisition of the single-molecule force spectroscopy and infrared spectroscopy.

Description

Technical Field

[0001] The present invention relates to the technical field of single molecule force spectroscopy, and in particular to a single molecule force spectroscopy-infrared spectroscopy combined method, system and equipment. Background Art

[0002] Single-molecule force spectroscopy based on atomic force microscopy (AFM) can use the AFM tip to stretch a single polymer. By recording the deformation of the stretched polymer molecule and the mechanical force it is subjected to, rich physical and chemical information can be obtained. This technology can be used to accurately study polymer mechanochemistry, mechano-melting of semi-crystalline polymers, supramolecular assembly, protein-protein / protein-DNA and other biomacromolecule interactions at the single-molecule level.

[0003] The AFM probe consists of a microcantilever and a needle tip, which is used to stretch polymer molecules and detect their vibrations. FFT transform: FFT is an efficient algorithm of DFT, called fast Fourier transform. Fourier transform converts time domain signals into frequency domain signals. Mechanochemical reaction: A chemical reaction corresponding to the breaking of old chemical bonds and the formation of new chemical bonds caused by mechanical force. β folding: A state in which polypeptide molecular chains exist in proteins. Its characteristics are that several peptide segments are arranged in parallel, and adjacent peptide segments are bonded by hydrogen bonds to form a folded structure, which can be unfolded under the action of external force and become a linear structure.

[0004] Combining single-molecule force spectroscopy with fluorescence microscopy or fluorescence spectroscopy can achieve direct detection of single-molecule fluorescence while mechanically manipulating a single molecule. Professor Hermann E. Gaub of Germany, Professor Julio M. Fernández of the United States, and Professor Martin Vacha of Japan have successively established related technologies and have realized single-molecule shear and paste technology, energy measurement of the evanescent field at the total internal reflection interface, and detection of single-molecule fluorescence behavior of conjugated polymer condensed states. The development of technology for in-situ analysis of target molecules of single-molecule force spectroscopy by fluorescence microscopy or fluorescence spectroscopy has not stopped in the past 20 years.

[0005] AFM infrared spectroscopy technology has continued to develop in recent years. This technology irradiates a beam of modulated infrared light onto the surface of the AFM sample and uses the AFM probe as a spectral detector to increase the resolution of infrared imaging from micrometers to nanometers. This technology was originally proposed by Professor Alexandre Dazzi and has been fully applied in the field of polymer structure analysis.

[0006] The combination of AFM and spectroscopy has made many leaps and bounds, which has strongly promoted scientific progress in various fields, but the technology has not paid enough attention to the study of force-induced structural transformation. At present, most of the AFM and infrared spectroscopy technologies only sense the vibration caused by light excitation by pressing down the AFM tip. It is difficult to establish a quantitative relationship between external force and mechanochemical reaction dynamics, etc., and cannot meet the needs of in-depth discussion of microscopic mechanochemistry. The single-molecule force spectroscopy method can manipulate a single polymer molecule in situ. Since the repeating units containing mechanochromophores are connected to each other in series, the uniformity of the force on these mechanochromophores is guaranteed. However, during the experiment, only the length change and mechanical force from a single molecule can be obtained, and the chemical changes or supramolecular structural changes occurring inside the molecule cannot be directly identified. The single-molecule force spectroscopy-fluorescence microscopy / fluorescence spectroscopy combined technology can only monitor the structural changes of light-responsive molecules, and cannot directly provide information on changes in chemical bonds.

[0007] In terms of instruments and equipment, commercial AFM infrared spectrometers are mainly designed for infrared imaging experiments and control systems. The equipment uses an open-loop scanner as the motion control module of the AFM probe, and the repositioning accuracy of the AFM probe is insufficient; its infrared laser beam quality is poor, and the laser energy near the AFM probe tip is insufficient to excite single-molecule vibrations; its control system cannot operate the AFM probe to perform complex movements, and cannot perform complex single-molecule force spectroscopy experiments. The above limitations make commercial AFM infrared spectrometers unable to meet the testing needs of single-molecule force spectroscopy.

[0008] In July 2017, Professor Mikhail A Belkin and others from the University of Texas at Austin reported a method for acquiring underwater AFM-IR signals, demonstrating the ability to collect weak AFM-IR signals. However, the research team used a germanium prism to construct a total internal reflection optical path and could not use visible light as a guide laser, making it difficult to achieve precise alignment between the infrared spot and the AFM tip.

[0009] In November 2022, Xiaoji G. Xu (Lehigh University) and others published a research paper that showed that AFM-based Fourier transform infrared spectroscopy technology can be realized by upward incidence (Nano Lett. 2022, 22, 9174-9180). The AFM used in this technology uses a closed-loop scanner as the motion control module of the AFM tip, but it is impossible to achieve multi-level force clamps through the force spectrum programming function.

[0010] In May 2022, Xia Xinghua and others from Nanjing University submitted a patent for "A method for nanoscale subsurface infrared imaging". This technology uses surface enhancement to achieve subsurface nano-infrared imaging. However, the instrument design of this invention adopts the upper incident infrared laser introduction method. This technical means will bring extremely strong photoacoustic noise and cannot meet the high precision and high signal-to-noise ratio required for SMFS-IR data acquisition.

[0011] In May 2023, Wang Hailong and others from Xiamen University submitted a patent for "Infrared, electrical and mechanical imaging device and method for obtaining nanomaterials", which can perform infrared imaging while testing the surface modulus and electrochemical properties of the sample. However, the infrared optical path used in this invention is different from that of the present invention, which results in the electric vector mode of the infrared laser formed by this invention being perpendicular to the detection direction of the AFM probe, and cannot directly induce the vibration of the target molecule, making it difficult to collect SMFS-IR signals. Summary of the invention

[0012] The purpose of the embodiments of the present invention is to provide a single-molecule force spectroscopy-infrared spectroscopy combined method, system and equipment, which uses mathematical methods to remove photoacoustic signals, programs the force spectrum to realize multi-level force clamp function, and improves the accuracy and signal-to-noise ratio of the AFM infrared spectrometer.

[0013] To achieve the above purpose, the embodiment of the present invention provides the following solution:

[0014] A single molecule force spectroscopy-infrared spectroscopy system, comprising:

[0015] An optical system module is used to use the micro-cantilever to sense and amplify the force between the sharp probe on the cantilever and the polymer molecule to be tested, and obtain and send a deflection signal of the micro-cantilever;

[0016] A lock-in amplifier, connected to the optical path system module, for calculating the resonant frequency by FFT transformation of the deflection signal;

[0017] A processor module, connected to the lock-in amplifier, for assigning the resonance frequency as a pulse repetition frequency to a signal generator;

[0018] A signal generator, connected to the processor module, is used to:

[0019] generating an electrical signal of the same frequency according to the pulse repetition frequency;

[0020] Transmitting the electrical signal of the same frequency as a trigger signal to the infrared laser in the optical path system module;

[0021] Transmitting the electrical signal of the same frequency as a reference signal to the lock-in amplifier;

[0022] The photothermal detector is connected to the infrared laser and the lock-in amplifier in the optical path system module, respectively, and is used for:

[0023] When collecting the infrared laser background data, converting the received laser intensity into laser power;

[0024] and transmitting the laser power to the lock-in amplifier; the lock-in amplifier transmits the laser power to the processor module;

[0025] The lock-in amplifier is also used to demodulate the deflection signal of the micro-cantilever according to the reference signal to obtain the micro-cantilever amplitude, and transmit the micro-cantilever amplitude to the processor module; the micro-cantilever amplitude represents the original spectral data;

[0026] The processor module obtains an amplitude-wavenumber curve according to the micro-cantilever amplitude and the laser power; divides the infrared laser background data by the amplitude-wavenumber curve, performs gain adjustment according to the radiation intensity of each wavenumber segment of the infrared laser, and obtains single-molecule force spectrum-infrared spectrum data of the tested sample.

[0027] Optionally, the optical path system module specifically includes:

[0028] Infrared laser, used for emitting infrared laser;

[0029] A guiding laser emitter, used for emitting a guiding laser;

[0030] A plane reflector unit, arranged on the infrared laser optical path, for controlling the spatial position of the infrared laser;

[0031] A beam combiner, disposed on a common optical path of the infrared laser and the guide laser, for adjusting the guide laser and the infrared laser to be collinear to obtain collinear laser;

[0032] A reflective beam expander is arranged on the collinear laser optical path and is used to expand the collinear laser to obtain an N-fold expanded laser.

[0033] An off-axis parabolic mirror movable in three-dimensional space is arranged on the beam expansion laser optical path, and is used to focus the N-fold beam expansion laser onto the surface of an infrared transparent prism loaded with a sample to be tested; the infrared transparent prism at least includes: infrared transparent glass of zinc selenide, infrared transparent glass of zinc sulfide, infrared transparent glass of calcium fluoride, infrared transparent glass of magnesium fluoride or infrared transparent glass of potassium bromide.

[0034] Optionally, the optical path system module further includes:

[0035] The flip mirror is arranged on the collinear laser light path and is used for reflecting the collinear laser light directly to the photothermal detector.

[0036] Optionally,

[0037] The spectral raw data is collected under the condition that the sharp probe is connected to the target molecule of the sample under test; after the collection is completed, the target molecule is pulled apart, and the sharp probe is controlled to move to the position where the spectral signal is collected to re-collect the signal, thereby obtaining pure photoacoustic noise;

[0038] Afterwards, the original spectrum data and the simple photoacoustic noise are normalized and subtracted to obtain single-molecule force spectroscopy-infrared spectrum data with the photoacoustic noise removed.

[0039] Optionally,

[0040] After the sharp probe forms a connection with the target molecule of the sample to be tested, a first clamping force is preset; when the force applied to the sharp probe reaches the first clamping force, the process of the sharp probe moving away is stopped and the sharp probe is stopped;

[0041] The pointed probe performs infrared laser intervention and infrared spectrum data collection during the dwelling stage;

[0042] After completing the infrared spectrum data acquisition, the pointed probe continues to move away from the target molecule of the sample under test until the target molecule is broken.

[0043] Optionally,

[0044] For a target molecule with a mechanochemical response, a M-level clamp force is preset after the sharp probe forms a connection with the target molecule of the sample to be tested; specifically, it includes:

[0045] When the force applied to the pointed probe reaches the first clamping force and stops once, the operation steps of secondary stretching and secondary stopping are continued; if a force chemical reaction is induced in the secondary stretching stage, the second clamping force of the secondary stopping is set to the same magnitude as the first clamping force of the primary stopping;

[0046] The pointed probe performs infrared laser intervention and collects infrared spectrum data during the first dwelling stage and the second dwelling stage to obtain in-situ mechanochemical identification results.

[0047] To achieve the above purpose, the embodiment of the present invention also provides the following solution:

[0048] A single molecule force spectroscopy-infrared spectroscopy combined method, comprising:

[0049] A micro-cantilever is used to sense and amplify the force between the sharp probe on the cantilever and the polymer molecule to be tested, and a deflection signal of the micro-cantilever is obtained and sent;

[0050] Calculate the resonant frequency of the deflection signal through FFT transformation; assign the resonant frequency to the signal generator as the pulse repetition frequency;

[0051] The signal generator generates an electrical signal of the same frequency according to the pulse repetition frequency; transmits the electrical signal of the same frequency as a trigger signal to the infrared laser in the optical path system module; and transmits the electrical signal of the same frequency as a reference signal to the lock-in amplifier;

[0052] When collecting the infrared laser background data, converting the received laser intensity into laser power;

[0053] Demodulating the deflection signal of the micro-cantilever according to the reference signal to obtain the micro-cantilever amplitude, and transmitting the micro-cantilever amplitude to the processor module; the micro-cantilever amplitude represents the original spectral data;

[0054] An amplitude-wavenumber curve is obtained according to the micro-cantilever amplitude and the laser power; the amplitude-wavenumber curve is divided by the infrared laser background data, and gain adjustment is performed according to the radiation intensity of each wavenumber segment of the infrared laser to obtain single-molecule force spectrum-infrared spectrum data of the tested sample.

[0055] An electronic device comprises a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor implements the single-molecule force spectroscopy-infrared spectroscopy combined method when executing the computer program.

[0056] A non-transitory computer-readable storage medium stores a computer program, which implements the single-molecule force spectroscopy-infrared spectroscopy combined method when executed.

[0057] In the embodiment of the present invention, the AFM device of the closed-loop scanner is first used in combination with an infrared laser, and a high-precision, high-signal-to-noise ratio AFM infrared spectrometer is built through laser beam expansion and large numerical aperture focusing light path. Subsequently, a new single-molecule force spectroscopy test method is established. While obtaining single-molecule force spectroscopy data, an infrared laser is used to excite the vibration of the target molecule and synchronously collect single-molecule infrared signals. The chemical sensitivity of the infrared spectrum to the sample is used to solve the technical defect that the single-molecule force spectroscopy technology cannot directly obtain the chemical information of the sample to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0059] Figure 1 A schematic diagram of the structure of a single-molecule force spectroscopy-infrared spectroscopy system provided in an embodiment of the present invention;

[0060] Figure 2 A schematic diagram of the process of the single molecule force spectroscopy-infrared spectroscopy combined method provided in an embodiment of the present invention;

[0061] Figure 3 A schematic diagram of the structure of a single-molecule force spectroscopy-infrared spectroscopy signal collection optical path system provided in an embodiment of the present invention;

[0062] Figure 4 A schematic diagram of a laser background collection optical path system provided in an embodiment of the present invention;

[0063] Figure 5 A schematic diagram of a data processing flow provided by an embodiment of the present invention;

[0064] Figure 6 A schematic diagram of a linked target molecule and an unlinked molecule provided by an embodiment of the present invention;

[0065] Figure 7 A schematic diagram of establishing a programmed force spectrum function to realize a force clamp working mode provided by an embodiment of the present invention;

[0066] Figure 8 A schematic diagram of a multi-stage force clamp working mode provided by an embodiment of the present invention;

[0067] Fig. 9 A single molecule force spectrum-infrared spectrum diagram of a protein provided in an embodiment of the present invention;

[0068] Fig.10 This is a single molecule force spectrum-infrared spectrum diagram of the dichlorocyclopropane polymer provided in an embodiment of the present invention.

[0069] Explanation of symbols:

[0070] Optical path system module-1, phase-locked amplifier-2, processor module-3, signal generator-4, photothermal detector-6. DETAILED DESCRIPTION

[0071] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] The purpose of the present invention is to provide a single-molecule force spectroscopy-infrared spectroscopy combined method, system and equipment to solve the problem that the chemical information of the sample cannot be obtained when performing the existing single-molecule force spectroscopy experiment.

[0073] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0074] Figure 1 An exemplary structure of the above-mentioned single-molecule force spectroscopy-infrared spectroscopy system is shown. The following is a detailed introduction to each module.

[0075] The optical path system module 1 is used to use the micro-cantilever to sense and amplify the force between the sharp probe on the cantilever and the polymer molecule to be tested, and obtain and send the deflection signal of the micro-cantilever;

[0076] See also Figure 3 and Figure 4 , the optical path system module 1 specifically includes:

[0077] Infrared laser, used for emitting infrared laser;

[0078] A guiding laser emitter, used for emitting a guiding laser;

[0079] A plane reflector unit, arranged on the infrared laser optical path, for controlling the spatial position of the infrared laser;

[0080] A beam combiner, disposed on a common optical path of the infrared laser and the guide laser, for adjusting the guide laser and the infrared laser to be collinear to obtain collinear laser;

[0081] A reflective beam expander is arranged on the collinear laser optical path and is used to expand the collinear laser to obtain an N-fold expanded laser.

[0082] An off-axis parabolic mirror movable in three-dimensional space is arranged on the beam expansion laser optical path, and is used to focus the N-fold beam expansion laser onto the infrared transparent prism surface loaded with the tested polymer molecules; the infrared transparent prism at least includes: zinc selenide infrared transparent glass, zinc sulfide infrared transparent glass, calcium fluoride infrared transparent glass, magnesium fluoride infrared transparent glass or potassium bromide infrared transparent glass.

[0083] The flip mirror is arranged on the collinear laser light path and is used for reflecting the collinear laser light directly to the photothermal detector.

[0084] In one example, A plane mirrors are used to control the spatial position of the laser, a beam combiner is used to adjust the guide laser (visible light) and the infrared laser (invisible to the naked eye) to be collinear, a reflective beam expander is used to achieve 6-fold beam expansion, and finally a three-dimensional movable off-axis parabolic mirror is used to focus the laser beam onto the surface of an infrared transparent prism loaded with a sample. In the system, the reflective beam expander can be replaced by a pair of off-axis parabolic mirrors with overlapping focal points, and the infrared transparent prism can be selected from zinc selenide, zinc sulfide, calcium fluoride, magnesium fluoride, potassium bromide or other infrared transparent glass. Since the infrared laser has an emission background, a flip mirror can be used to reflect the infrared laser directly to the photothermal detector, thereby detecting the emission background signal of the laser. Exemplarily, the value of A can be 2, 3, 4, etc., which will not be repeated here.

[0085] The lock-in amplifier 2 is connected to the optical path system module 1, and is used to calculate the resonant frequency by FFT transformation of the deflection signal;

[0086] The processor module 3 is connected to the lock-in amplifier 2, and the processor module 3 is used to assign the resonance frequency as the pulse repetition frequency to the signal generator;

[0087] The signal generator 4 is connected to the processor module 3, and the signal generator 4 is used for:

[0088] generating an electrical signal of the same frequency according to the pulse repetition frequency;

[0089] Transmitting the electrical signal of the same frequency as a trigger signal to the infrared laser in the optical path system module 1;

[0090] Transmitting the electrical signal of the same frequency as a reference signal to the lock-in amplifier 2;

[0091] The photothermal detector 6 is connected to the infrared laser in the optical path system module 1 and the lock-in amplifier 2 respectively, and the photothermal detector 6 is used for:

[0092] When collecting the infrared laser background data, converting the received laser intensity into laser power;

[0093] and transmits the laser power to the lock-in amplifier 2; the lock-in amplifier 2 transmits the laser power to the processor module 3;

[0094] The lock-in amplifier 2 is also used to demodulate the deflection signal of the micro-cantilever according to the reference signal to obtain the micro-cantilever amplitude, and transmit the micro-cantilever amplitude to the processor module 3; the micro-cantilever amplitude represents the original spectral data;

[0095] The spectral raw data is collected under the condition that the sharp probe is connected to the target molecule of the sample under test; after the collection is completed, the target molecule is pulled apart, and the sharp probe is controlled to move to the position where the spectral signal is collected to re-collect the signal, thereby obtaining pure photoacoustic noise;

[0096] Afterwards, the original spectrum data and the simple photoacoustic noise are normalized and subtracted to obtain single-molecule force spectroscopy-infrared spectrum data with the photoacoustic noise removed.

[0097] After the sharp probe forms a connection with the target molecule of the sample to be tested, a first clamping force is preset; when the force applied to the sharp probe reaches the first clamping force, the process of the sharp probe moving away is stopped and the sharp probe is stopped;

[0098] The pointed probe performs infrared laser intervention and infrared spectrum data collection during the dwelling stage;

[0099] After completing the infrared spectrum data acquisition, the pointed probe continues to move away from the target molecule of the sample under test until the target molecule is broken.

[0100] For a target molecule with a mechanochemical response, a M-level clamp force is preset after the sharp probe forms a connection with the target molecule of the sample to be tested; specifically, it includes:

[0101] When the force applied to the pointed probe reaches the first clamping force and stops once, the operation steps of secondary stretching and secondary stopping are continued; if a force chemical reaction is induced in the secondary stretching stage, the second clamping force of the secondary stopping is set to the same magnitude as the first clamping force of the primary stopping;

[0102] The pointed probe performs infrared laser intervention and collects infrared spectrum data during the first dwelling stage and the second dwelling stage to obtain in-situ mechanochemical identification results.

[0103] The processor module 3 obtains an amplitude-wavenumber curve based on the micro-cantilever amplitude and the laser power; divides the infrared laser background data by the amplitude-wavenumber curve, and performs gain adjustment according to the radiation intensity of each wavenumber segment of the infrared laser to obtain the single-molecule force spectrum-infrared spectrum data of the tested sample; after the tested polymer molecule is broken, the micro-cantilever is repositioned to the spatial position in the single-molecule force spectrum-infrared spectrum data collection process to collect the photoacoustic noise signal; the original spectrum data and the simple photoacoustic noise are normalized and subtracted to obtain the single-molecule force spectrum-infrared spectrum data with the photoacoustic noise removed.

[0104] See also Figure 5, the AFM system transmits the deflection signal of the micro-cantilever to the phase-locked amplifier, which calculates the resonant frequency of the system through FFT transformation and transmits it to the processor module 3 (host). The host assigns the resonant frequency of the system to the signal generator 4 as the pulse repetition frequency. The signal generator 4 generates an electrical signal of the same frequency according to the written data, which is transmitted to the infrared laser and the phase-locked amplifier 2 as a trigger signal and a reference signal respectively. The phase-locked amplifier 2 demodulates the micro-cantilever deflection signal according to the reference signal to obtain the micro-cantilever amplitude, and transmits it to the host as the spectral raw data. When collecting the laser background, the photothermal detector 6 converts the received light intensity into an electrical signal (laser power), and transmits it to the phase-locked amplifier 2, which is finally transmitted to the host by the phase-locked amplifier 2 as a laser intensity signal.

[0105] The data processing flow is as follows Figure 5 The laser background is collected by the photothermal detector and recorded in the software. The microcantilever deflection signal collected by the AFM is demodulated by the reference signal provided by the signal generator 4 through the phase-locked amplifier 2 to obtain the amplitude of the microcantilever. The obtained amplitude-wavenumber curve is divided by the laser background data, and the gain is adjusted according to the radiation intensity of each wavenumber band of the laser to obtain the single-molecule force spectrum-infrared spectrum data of the sample.

[0106] The photoacoustic noise removal process is as follows Figure 6 During the detection process, there is a photoacoustic noise signal that interferes with the identification of single-molecule infrared signals. The principle is that the silver nanoparticles near the target molecule have a scattering effect on the infrared laser. The scattered infrared light heats the air below the probe, producing a periodically changing air layer. The air layer is transmitted to the microcantilever to stimulate the cantilever to vibrate, and its amplitude is superimposed with the amplitude of the target molecule vibration, resulting in a more complex signal. In order to solve the photoacoustic signal, the collected original spectral data is processed using mathematical subtraction. That is, the original infrared spectral signal is collected under the condition that the AFM probe is connected to the target molecule. After the collection is completed, the target molecule is pulled off, and the probe is controlled to move to the position where the spectral signal is collected (the same XYZ spatial coordinates), and the signal is re-collected at this position to obtain pure photoacoustic noise. After the collection is completed, the two parts of the signal are normalized, and then the two are subtracted to achieve mathematical methods to remove the photoacoustic noise.

[0107] Conventional single-molecule force spectroscopy uses a constant-speed stretching method to collect data, and the AFM probe approaches the sample at a constant speed. After the tip and the target molecule form a connection, the AFM probe moves away from the sample surface at a constant speed to apply mechanical force to the target molecule. However, this working method will constantly change the resonance frequency of the series system formed by the microcantilever and the target molecule, and thus cannot complete the above-mentioned resonance frequency acquisition and pulse repetition frequency assignment process.

[0108] See also Figure 7, establish a programmed force spectrum function to realize the force clamp working mode. When the AFM probe and the target molecule form a connection, set a certain "first clamp force". When the force on the AFM probe reaches the first clamp force, stop the further distance process and start to stay. In the stay stage, the infrared laser is intervened and the infrared spectrum is collected. After the spectrum collection is completed, the probe continues to move away from the sample until the target molecule is broken.

[0109] See also Figure 8 For target molecules with mechanochemical response, an M-level force clamp operation mode was established. After the clamp force was reached for the first time and a dwell was performed, the operation steps of secondary stretching and secondary dwell were added to induce a mechanochemical reaction in the secondary stretching stage, and the clamp force of the secondary dwell was set to the same magnitude as the clamp force of the primary dwell. Infrared absorption during the primary and secondary dwell stages was collected for in-situ mechanochemical identification.

[0110] The embodiment of the present invention firstly builds a high-precision, high signal-to-noise ratio AFM infrared spectrometer, then establishes a new single-molecule force spectrum acquisition method, uses the instrument and data acquisition method to obtain the original data of the single-molecule infrared spectrum, and finally, establishes a background noise removal method to achieve the synchronous acquisition of the single-molecule force spectrum and the single-molecule infrared spectrum.

[0111] In summary, due to the limitations of instrumentation methods, single-molecule force spectroscopy technology cannot directly obtain the chemical information of the target molecule. When the stretched molecule changes in length, it is impossible to know the chemical bonds or supramolecular interactions corresponding to the length change. The present invention introduces infrared spectroscopy technology into the single-molecule force spectroscopy test process, and uses the infrared spectrum's ability to resolve chemical bonds to achieve the goal of in-situ identification of changes in the chemical information of the target molecule before and after the mechanochemical reaction occurs. This characterization method significantly improves the credibility of single-molecule force spectroscopy experiments, so as to directly display the mechanical characteristics of single-chain molecules and the spectroscopic characteristics of single molecules synchronously. This scientific progress will help solve the confusion that there is a large difference between the calculation results of theoretical chemistry "isolated molecules" and the spectral experimental results of "aggregated molecules" in actual systems. It is of great significance to promote the development of related disciplines.

[0112] At present, the AFM technology combined with spectroscopy is developing very rapidly, and there is a huge demand for instruments in this research field. The instrument development of the present invention combines technological innovation with specific applications, and through the guidance of new technologies at key points, similar instruments and equipment with independent intellectual property rights are created.

[0113] To achieve the above purpose, the embodiment of the present invention also provides the following solution:

[0114] A single-molecule force spectroscopy-infrared spectroscopy method, see Figure 2 ,include:

[0115] Step S1: using a microcantilever to sense and amplify the force between the sharp probe on the cantilever and the polymer molecular chain to be measured, and obtaining and sending a deflection signal of the microcantilever;

[0116] Step S2: Calculate the resonant frequency of the deflection signal through FFT transformation; assign the resonant frequency to the signal generator as the pulse repetition frequency;

[0117] Step S3: the signal generator generates an electrical signal of the same frequency according to the pulse repetition frequency; the electrical signal of the same frequency is transmitted as a trigger signal to the infrared laser in the optical path system module; the electrical signal of the same frequency is transmitted as a reference signal to the lock-in amplifier;

[0118] Step S4: when collecting the infrared laser background data, converting the received laser intensity into laser power;

[0119] Step S5: demodulating the deflection signal of the micro-cantilever according to the reference signal to obtain the micro-cantilever amplitude, and transmitting the micro-cantilever amplitude to the processor module; the micro-cantilever amplitude represents the original spectral data;

[0120] Step S6: obtaining an amplitude-wavenumber curve according to the micro-cantilever amplitude and the laser power; dividing the infrared laser background data by the amplitude-wavenumber curve, and performing gain adjustment according to the radiation intensity of each wavenumber band of the infrared laser to obtain the single-molecule force spectrum-infrared spectrum data of the tested sample; after breaking the tested polymer molecule, repositioning the micro-cantilever to the spatial position in the single-molecule force spectrum-infrared spectrum data collection process to collect the photoacoustic noise signal; normalizing and subtracting the original spectrum data from the simple photoacoustic noise to obtain the single-molecule force spectrum-infrared spectrum data with the photoacoustic noise removed.

[0121] Example 1: Single molecule force spectroscopy-infrared spectroscopy experiment of protein.

[0122] See also Fig. 9 The protein molecules were connected to the surface of the zinc selenide prism using an interface connection method, and the AFM probe and protein were covalently connected to collect single-molecule force spectroscopy-infrared spectroscopy signals under different clamping forces. -1 A higher mechanical force (1100 pN) resulted in a lower absorption of ~1630 cm -1 The absorption peak corresponds to the opening of the protein β-folded structure under the action of mechanical force, which leads to a decrease in signal intensity.

[0123] Example 2: Single molecule force spectroscopy-infrared spectroscopy experiment of dichlorocyclopropane polymer.

[0124] See also Fig.10 The horizontal axis is the height of the microcantilever during the force spectrum experiment (Height), and the vertical axis is the force (Force) on the microcantilever during the force spectrum experiment. The multi-level force spectrum function is used to collect single-molecule force spectroscopy-infrared spectrum data for dichlorocyclopropane polymer. Since the mechanical force threshold for the ring-opening rearrangement of the dichlorocyclopropane structure under the action of mechanical force is 2nN, a clamp force of 800pN is used for infrared spectrum collection, and the maximum mechanical force for the secondary stretching of the polymer is set to 2.3nN. By comparing the infrared spectra collected before and after the mechanochemical reaction, it can be observed that after the mechanochemical reaction occurs, 1690cm -1 A new absorption peak appears at , corresponding to the C=C double bond generated after the ring-opening reaction.

[0125] Furthermore, the present invention also provides an electronic device, which may include: a processor, a communication interface, a memory and a communication bus. The processor, the communication interface and the memory communicate with each other through the communication bus. The processor may call a computer program in the memory to execute the single molecule force spectroscopy-infrared spectroscopy method when the processor executes the computer program.

[0126] In addition, when the computer program in the above-mentioned memory is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art or the part of the technical solution, can be embodied in the form of a software product, which is stored in a storage medium and includes several instructions for a computer device (which can be a personal computer, server or network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes: various media that can store program codes, such as a USB flash drive, a mobile hard disk, a read-only memory, a random access memory, a magnetic disk or an optical disk.

[0127] Furthermore, the present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, and the computer program, when executed, implements the single-molecule force spectroscopy-infrared spectroscopy combined method.

[0128] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the system disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0129] This article uses specific examples to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only used to help understand the methods and core ideas of the embodiments of the present invention. At the same time, for those skilled in the art, according to the ideas of the embodiments of the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the embodiments of the present invention.

Claims

1. A single molecule force spectroscopy-infrared spectroscopy system, characterized in that: include: An optical system module is used to use the micro-cantilever to sense and amplify the force between the sharp probe on the cantilever and the polymer molecule to be tested, and obtain and send a deflection signal of the micro-cantilever; A lock-in amplifier, connected to the optical path system module, for calculating the resonant frequency by FFT transformation of the deflection signal; A processor module, connected to the lock-in amplifier, for assigning the resonance frequency as a pulse repetition frequency to a signal generator; A signal generator, connected to the processor module, is used to: generating an electrical signal of the same frequency according to the pulse repetition frequency; Transmitting the electrical signal of the same frequency as a trigger signal to the infrared laser in the optical path system module; Transmitting the electrical signal of the same frequency as a reference signal to the lock-in amplifier; The photothermal detector is connected to the infrared laser in the optical path system module and the lock-in amplifier respectively, and is used for: When collecting the infrared laser background data, converting the received laser intensity into laser power; and transmitting the laser power to the lock-in amplifier; the lock-in amplifier transmits the laser power to the processor module; The lock-in amplifier is also used to demodulate the deflection signal of the micro-cantilever according to the reference signal to obtain the micro-cantilever amplitude, and transmit the micro-cantilever amplitude to the processor module; the micro-cantilever amplitude represents the original spectral data; The processor module obtains an amplitude-wavenumber curve according to the micro-cantilever amplitude and the laser power; divides the infrared laser background data by the amplitude-wavenumber curve, performs gain adjustment according to the radiation intensity of each wavenumber segment of the infrared laser, and obtains single molecule force spectrum-infrared spectrum data of the tested polymer molecule; The spectral raw data is collected under the condition that the sharp probe is connected to the tested polymer molecule; after the collection is completed, the tested polymer molecule is pulled apart, and the sharp probe is controlled to move to the position where the spectral signal is collected to re-collect the signal, thereby obtaining pure photoacoustic noise; Then, the original spectrum data and the pure photoacoustic noise are normalized and subtracted to obtain single-molecule force spectroscopy-infrared spectrum data with the photoacoustic noise removed; Presetting a first clamping force after the sharp probe forms a connection with the polymer molecule to be tested; When the force applied to the sharp probe reaches the first clamp force, the process of moving the sharp probe away is stopped and the sharp probe is stopped; The pointed probe performs infrared laser intervention and infrared spectrum data collection during the dwelling stage; After completing the infrared spectrum data collection, the pointed probe continues to move away from the tested polymer molecule until the tested polymer molecule is broken; For the tested polymer molecule with mechanochemical response, after the sharp probe forms a connection with the tested polymer molecule, a M-level clamping force is preset; specifically, it includes: When the force applied to the pointed probe reaches the first clamping force and stops once, the steps of secondary stretching and secondary stopping are continued; If a mechanochemical reaction is induced in the secondary stretching stage, the second clamp force of the secondary dwell is set to the same magnitude as the first clamp force of the primary dwell; The pointed probe performs infrared laser intervention and collects infrared spectrum data during the first dwelling stage and the second dwelling stage to obtain in-situ mechanochemical identification results.

2. The single-molecule force spectroscopy-infrared spectroscopy system according to claim 1, characterized in that: The optical path system module specifically includes: Infrared laser, used for emitting infrared laser; A guiding laser emitter, used for emitting a guiding laser; A plane reflector unit, arranged on the infrared laser optical path, for controlling the spatial position of the infrared laser; A beam combiner, disposed on a common optical path of the infrared laser and the guide laser, for adjusting the guide laser and the infrared laser to be collinear to obtain collinear laser; A reflective beam expander is arranged on the collinear laser optical path and is used to expand the collinear laser to obtain an N-fold expanded laser. An off-axis parabolic mirror movable in three-dimensional space is arranged on the beam expansion laser optical path, and is used to focus the N-fold beam expansion laser onto the infrared transparent prism surface loaded with the tested polymer molecules; the infrared transparent prism at least includes: zinc selenide infrared transparent glass, zinc sulfide infrared transparent glass, calcium fluoride infrared transparent glass, magnesium fluoride infrared transparent glass or potassium bromide infrared transparent glass.

3. The single-molecule force spectroscopy-infrared spectroscopy system according to claim 2, characterized in that: The optical path system module also includes: The flip mirror is arranged on the collinear laser light path and is used for reflecting the collinear laser light directly to the photothermal detector.

4. A single molecule force spectroscopy-infrared spectroscopy method, characterized in that: The single-molecule force spectroscopy-infrared spectroscopy method uses the single-molecule force spectroscopy-infrared spectroscopy system according to any one of claims 1 to 3, and the single-molecule force spectroscopy-infrared spectroscopy method comprises: A micro-cantilever is used to sense and amplify the force between the sharp probe on the cantilever and the polymer molecule to be tested, and a deflection signal of the micro-cantilever is obtained and sent; Calculate the resonant frequency of the deflection signal through FFT transformation; assign the resonant frequency to the signal generator as the pulse repetition frequency; The signal generator generates an electrical signal of the same frequency according to the pulse repetition frequency; transmits the electrical signal of the same frequency as a trigger signal to the infrared laser in the optical path system module; and transmits the electrical signal of the same frequency as a reference signal to the lock-in amplifier; Demodulating the deflection signal of the micro-cantilever according to the reference signal to obtain the micro-cantilever amplitude, and transmitting the micro-cantilever amplitude to the processor module; the micro-cantilever amplitude represents the original spectral data; An amplitude-wavenumber curve is obtained according to the micro-cantilever amplitude and the laser power; the amplitude-wavenumber curve is divided by the infrared laser background data, and gain adjustment is performed according to the radiation intensity of each wavenumber band of the infrared laser to obtain single molecule force spectrum-infrared spectrum data of the tested polymer molecule.

5. An electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the single-molecule force spectroscopy-infrared spectroscopy method as described in claim 4 is implemented.

6. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed, the single-molecule force spectroscopy-infrared spectroscopy method as claimed in claim 4 is implemented.

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