System for detecting pka step and pulse temperature combined two-dimensional infrared spectroscopy and method thereof

Abstract

This invention discloses a two-dimensional infrared spectroscopy system and its detection method that combines acid-base step and pulsed heating. The system includes a femtosecond laser, an optical parametric amplifier and difference frequency generator, a frequency divider, an electronic time delay generator, a nanosecond laser, a detachable optical parametric oscillator providing 355nm light pulses, an optical parametric oscillator providing 2020nm light pulses, a monochromator and a mercury cadmium telluride array detector, an integrator and data acquisition card, a computer, a chopper, a time-resolved pulse generation chamber, and a sample chamber. This invention retains the original pulsed heating trigger mode by using a nanosecond light source with a detachable optical parametric oscillator providing 355nm light pulses. It achieves convenient switching between the acid-base step and pulsed heating modes through a dichroic mirror and a reversible reflector. Before and after switching, the propagation direction of the nanosecond pulses is completely consistent, and the relative time delay is corrected by the electronic time delay device.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of spectral detection, and relates to an acid-base step and pulse temperature-rising compound two-dimensional infrared spectrum system and a detection method thereof, in particular to a time-resolved infrared spectrum system based on the combination of femtosecond and nanosecond pulses. BACKGROUND

[0002] The binding and dissociation kinetics process between biological macromolecules (DNA, polypeptide and protein) and small molecule ligands is the basis for realizing the functions of signal transmission, metabolism and pathogen immunity of living bodies, and thus has important significance in the field of biological medicine. Research on the intermediate state structure of the kinetics process and discovery of specific groups with regulatory functions become the key to the development of new drugs and treatment methods, in which the structural characterization of biological macromolecules and the analysis of intermolecular weak interactions (hydrogen bond, hydrophobic interaction, pi-pi stacking and the like) are involved. Infrared spectrum technology is a mainstream means for characterizing the above-mentioned content, and in particular, two-dimensional infrared spectrum technology can solve the problem of overlapping of characteristic peaks in the frequency domain, utilize femtosecond infrared pulse pumping and detection of molecular vibration, and reveal the interaction relationship between molecules and between molecules, and has strong sensitivity to the structural change of biological macromolecules. However, the technology for realizing transient infrared spectrum detection is very limited, and the main problem is that it is difficult to simultaneously meet the dual requirements of time resolution and time window width. Common means include microfluidic chips (Int. J. Mol. Sci. 2011, 12, 3263) and pulse temperature rising (Rev. Sci. Instrum. 78, 063101). The former can obtain a time resolution of hundreds of microseconds through fast mixing technology, and cannot meet the demand of ultrafast kinetics research. The latter can improve the resolution to the nanosecond level through nanosecond pulse to rapidly heat the water solution, but due to the heat dissipation of the sample window sheet, the temperature of the excited area will quickly decrease back to the initial temperature, and only a time window of microsecond level can be provided, which cannot capture the complete kinetics process. In addition, the signal acquisition mode of transient two-dimensional infrared spectrum is usually step scan, that is, the time delay line moves once and the detector collects one spectrum. However, due to the high accuracy requirement of two-dimensional infrared spectrum for the position of the delay line (4 fs, corresponding delay line moving distance 0.06 μm), the delay line needs a certain time to balance at each target position to ensure the position accuracy, so this acquisition mode is easy to introduce the position error of the delay line, and the data acquisition efficiency is low. SUMMARY

[0003] The purpose of this invention is to provide a two-dimensional infrared spectroscopy system and its detection method that combines pH step and pulsed heating. This system, based on the existing pulsed heating two-dimensional infrared spectroscopy system (Rev. Sci. Instrum. 78, 063101), replaces the nanosecond light source with a nanosecond laser that can provide both 1064nm and 355nm wavelengths. This nanosecond laser has a detachable optical parametric oscillator that provides 355nm light pulses. Before and after detachment, the light pulse propagation direction is completely consistent, allowing for convenient switching between pulsed heating and pH step triggering modes (pH step refers to a change in the pH of the sample solution within a very short time; pulsed heating refers to rapidly increasing the temperature of the sample solution using a light pulse) via a dichroic mirror and a flip-flop mirror. The system employs a fast scan signal acquisition method, where the delay line moves from point A to point B at a fixed speed, and the detector simultaneously acquires the spectrum and provides the delay time based on the speed. This method minimizes the positional error of the delay line, reduces the time required for delay line balancing, and greatly improves signal acquisition efficiency.

[0004] The technical solution adopted in this invention is as follows:

[0005] A composite two-dimensional infrared spectroscopy system combining acid-base step and pulsed heating is disclosed. This system includes a femtosecond laser (50 fs, 800 nm, 1 kHz), an optical parametric amplifier (TOPAS) and a difference frequency generator (DFG), a frequency divider, an electronic time delay generator, a nanosecond laser (7 ns, 1064 nm, 1-50 Hz adjustable), a detachable optical parametric oscillator providing 355 nm light pulses, an optical parametric oscillator providing 2020 nm light pulses, a monochromator and a mercury cadmium telluride (MCT) array detector (128 channels, single row), an integrator and data acquisition card, a computer, a controller, a time-resolved pulse generation chamber, and a sample chamber. The femtosecond laser, optical parametric amplifier, and difference frequency generator work together to generate mid-infrared pulses of the desired wavelength, which enter the time-resolved pulse generation chamber. The frequency signal (1 kHz) output from the femtosecond laser is converted into a 1-50 Hz signal by the frequency divider, and the time difference between this signal and the femtosecond laser frequency signal is adjusted by the electronic time delay generator, then input to the nanosecond laser to generate 1064 nm wavelength light pulses.

[0006] pH step mode: A 1064nm wavelength light pulse enters a detachable optical parametric oscillator that provides a 355nm light pulse, is converted into a 355nm light pulse, and then the angle is adjusted by the dichroic mirror DM and the reflector M in sequence, illuminating the flip-flop reflector FM. The pulse energy is then adjusted by the attenuator A, and the direction is adjusted by the reflector M before entering the sample chamber. The chopper CH is placed on the 355nm light pulse path to control the switching state of the light pulse. Its controller is controlled by the electronic pulse signal provided by the frequency divider, and the working frequency is set to half the repetition frequency of the nanosecond laser.

[0007] Pulse heating mode: The detachable optical parametric oscillator that provides 355nm light pulses is disassembled. The 1064nm light pulse passes through the dichroic mirror DM and enters the optical parametric oscillator that provides 2020nm light pulses. The 2020nm light pulse is generated and the angle is adjusted by the reflector M to irradiate the back of the flip-up reflector FM. The pulse energy is then adjusted by the attenuator A and the direction is adjusted by the reflector M before entering the sample chamber.

[0008] In the sample chamber, the aforementioned 355nm or 2020nm light pulses are angled by the reflector M so that they spatially overlap with the femtosecond light generated by the femtosecond laser on the sample cell S.

[0009] The time-resolved pulse generation chamber generates the pump light and probe light required for two-dimensional infrared spectroscopy detection. The optical path difference between the two is adjusted using a time delay line DS2 to achieve time resolution. Mid-infrared pulses entering the time-resolved pulse generation chamber are split into three beams (k1, k2, and k3) by a wedge mirror W and a beam splitter BS (50:50 splitting ratio), with an energy ratio of approximately 47:47:6. Pulses k1 and k2 generate interference pulses on the second beam splitter BS (50:50 splitting ratio). Half of this interference pulse is used as pump light and enters the sample chamber via a half-wave plate WP and a polarizer P. The other half is used for signal correction and detected by a single-channel MCT detector. Pulse k3 enters the sample chamber via a dispersion compensation plate C and a polarizer P and is used as probe light.

[0010] In the sample chamber, a parabolic mirror PM focuses the parallel-incident pump beams k1 & k2 and probe beams k3 into the sample cell S. Another parabolic mirror PM then collects the probe beam, which is then converged via a mirror M, a lens L, and a polarizer P onto the monochromator and MCT array detector. Nanosecond pulses used to trigger pH steps or temperature increases enter the sample chamber, are focused by the lens L, and illuminate the sample cell through the mirror M, completely overlapping the light spots of the k1, k2, and k3 pulses.

[0011] The time-resolved pulse generation chamber and the sample chamber are covered by a box made of 5mm thick acrylic sheet. The box is connected to a dry air generator via an air valve and PTFE tubing to control the humidity inside the box to below 5%.

[0012] For both acid-base step and pulsed temperature rise triggering methods, this invention employs two sample cell configurations:

[0013] Samples used for acid-base step spectroscopy detection need to be doped with photo-induced acid / base generators (such as o-nitrobenzaldehyde), whose (de)protonation process is irreversible, requiring sample flow during spectral acquisition. Therefore, in acid-base step spectroscopy detection mode, the sample cell adopts a windowless design (patent application number 202223024867.X), mainly composed of two parallel tungsten filaments, avoiding the protein aggregation problem caused by calcium fluoride windows. The liquid sample is pushed to the nozzle by a peristaltic pump, forming a liquid film between the two parallel tungsten filaments; the peristaltic pump flow rate is determined according to the repetition frequency of the nanosecond pulse.

[0014] The sample cell in pulse-heated two-dimensional infrared spectroscopy detection mode has a "sandwich" structure similar to that of a conventional infrared sample cell. The liquid sample is sandwiched between two calcium fluoride windows coated with a thin film of perfluoroethylene (FEP), and the sample thickness is adjusted (typically 50 micrometers) by a polytetrafluoroethylene (PTFE) gasket. This "sandwich" structure is fixed in a copper module and temperature is controlled by a water bath circulation pump. To mitigate heat dissipation in the pulse-heated region and thus widen the time window, this invention coats the inner side of the calcium fluoride windows (the side in contact with the liquid sample) with a thin film approximately 12 micrometers thick, made of perfluoroethylene propylene copolymer (FEP). This method effectively widens the time window of pulse-heated spectroscopy experiments by 5 times (from the original 3 ms to 15 ms).

[0015] The integrator, triggered by the electronic pulse signal input from the chopper, integrates the signal input from the MCT detector (integration time is typically set to 2000 ns) and then divides it into odd and even sequences, sending them to the data acquisition card for recording. These two sequences represent whether the k2 pulse is blocked by the chopper's CH phase. The signal input from the single-channel MCT detector during the same time period is also recorded by the integrator and the signal acquisition card for signal correction.

[0016] A detection method based on a two-dimensional infrared spectroscopy system combining acid-base step and pulsed temperature rise includes the following steps:

[0017] 1) Set the division frequency n of the frequency divider (adjustable from 1 to 50). n should be a number divisible by 1000. That is, after the nanosecond and femtosecond lasers are synchronized, each nanosecond pulse corresponds to a set of femtosecond pulses with a quantity of 1000 / n and a femtosecond pulse interval of 1 millisecond.

[0018] 2) Set the time delay τ using an electronic time delay device. τ is the time difference between the nanosecond pulse and the first pulse in the corresponding femtosecond pulse group, and therefore should be a value less than 1 millisecond;

[0019] 3) The two-dimensional infrared spectrum includes frequency coordinate axes ω1 and ω3. The resolution of the ω3 coordinate axis is determined by the total number of channels of the MCT detector, while the resolution of the ω1 coordinate axis is determined by the step size dt of the relative time delays of pump light k1 and k2 (usually set to 4 fs). The moving speed v of the delay line is set according to the step size dt, and the calculation formula is v = dt * c * n / 2, with units of meters per second, where c is the speed of light, i.e., 3 * 10⁻⁶. 8 meters per second;

[0020] 4) Set the start and end positions ts and te for the delay line movement, i.e., the relative time delays of k1 and k2 (usually -300fs and 2500fs). Set the waiting time t for the delay line at the start and end positions, which should be an integer multiple of 1000 / n (milliseconds) and greater than 0.1 seconds;

[0021] 5) The delay line moves from the starting position to the ending position (forward movement). The signal acquisition card is triggered by the frequency signal of the femtosecond laser to record the corresponding spectral signal of each femtosecond pulse, which is stored as a p*q*m matrix M1. p is the number of pulses in a group of femtosecond pulses, 1000 / n, q is the total number of femtosecond pulse groups (te-ts) / dt, and m is the total number of channels of the MCT array detector. In this matrix, when p is odd, k2 is "on", that is, not blocked by the chopper; when p is even, k2 is "off", that is, blocked by the chopper.

[0022] 6) The delay line moves from the termination position to the starting position (reverse movement), and the signal acquisition method is the same as the previous step, resulting in a p*q*m matrix M2. At this point, k2 is "off" when p is odd, and "on" when p is even. The data in the matrix difference M1-M2 where p is even are negative to obtain the final chopped signal;

[0023] 7) Repeat steps 5 and 6 j times according to the set average number of times j. The computer records the average value of the chopper signal and generates a data file.

[0024] 8) In the data file, the q*m matrix corresponding to p=1 is the two-dimensional infrared spectrum corresponding to the time delay τ (the time difference between nanosecond and femtosecond pulses). When p=2,3,4…, the corresponding time delay is τ+(p-1), in milliseconds. The corresponding ω1 and ω3 frequency axes can be obtained using conventional two-dimensional infrared spectroscopy frequency calibration methods.

[0025] This invention provides a two-dimensional infrared spectroscopy system and its detection method that combines pH step and pulsed heating. It retains the original pulsed heating trigger mode by using a nanosecond light source with a detachable optical parametric oscillator providing 355nm light pulses. The system achieves convenient switching between the pH step and pulsed heating modes via a dichroic mirror (DM) and a foldable reflector (FM). Before and after switching, the propagation direction of the nanosecond pulses is completely consistent, and the relative time delay is corrected by an electronic time delay device. The two trigger modes employ a windowless sample cell and an FEP-coated sample cell, respectively. The former solves the protein aggregation problem caused by sample circulation and the window in the pH step mode, allowing for free adjustment of the time observation window width (20ms to 1s); the latter effectively widens the time window of the pulsed heating spectroscopy experiment by 5 times, reducing protein sample aggregation caused by the window's charge.

[0026] The beneficial effects of this invention are:

[0027] 1. Acidity / alkalinity step triggering mode:

[0028] (1) Accurately simulates the internal environment of living organisms: Taking the photoacid generator o-nitrobenzaldehyde as an example, in the pH range of 4-9, it can induce a decrease in the pH of the solution by up to 3 units after being excited by a 355nm pulse, and the local temperature change caused by the light pulse is very small (<1K). Currently, a large number of photoacid and photoalkali generators (ref) reported in the literature can be excited in the 355nm band, and they vary in (de)protonation rate, lifetime, and pKa range, which makes them more flexible for the research subjects.

[0029] (2) Adjustable pH step amplitude: The single pulse energy required to achieve a pH unit step is only 20 μJ, while the maximum energy of the 355 nm nanosecond light pulse used in this invention can reach 6 mJ.

[0030] (3) Adjustable time window width: The repetition frequency of the nanosecond laser used in this invention is adjustable in the range of 1 to 50 Hz. By adjusting the peristaltic pump to control the sample flow rate, a time window width of up to 1 s can be achieved.

[0031] 2. Pulse heating trigger mode:

[0032] (1) The present invention provides a 2020nm optical pulse optical parametric oscillator that is compatible with the conventional pulse heating triggering method, and achieves quick switching between the two modes through a detachable 355nm optical parametric oscillator module, a dichroic mirror and a flip-up reflector.

[0033] (2) Wider time window: Compared with the sample cell of conventional pulse-heated infrared systems, this invention uses FEP-coated calcium fluoride windows. This method solves the problem of protein aggregation caused by conventional sample windows and effectively prevents heat dissipation of the windows, increasing the time window width by 5 times.

[0034] 3. Signal acquisition for transient two-dimensional infrared spectroscopy:

[0035] This invention proposes a rapid scanning signal acquisition method. This method reduces the positional error of the delay line and the time required for delay line balancing in conventional step-scanning methods by moving the delay line at a fixed speed and simultaneously acquiring the spectrum with the detector, thus significantly improving signal accuracy and acquisition efficiency. Attached Figure Description

[0036] Figure 1 This is the optical path diagram of the present invention.

[0037] Figure 2 This is an optical path diagram of the time-resolved pulse generation chamber of the present invention.

[0038] In the diagram: 1-Femtosecond laser; 2-Optical parametric amplifier TOPAS and difference frequency generator DFG; 3-Frequency divider; 4-Electronic time delay generator; 5-Nanosecond laser; 6-Detachable optical parametric oscillator providing 355nm light pulses; 7-Optical parametric oscillator providing 2020nm light pulses; 8-Monochromator and mercury cadmium telluride MCT array detector; 9-Integrator and data acquisition card; 10-Computer; 11-Controller; 12-Controller; 13-Time-resolved pulse generation chamber; 14-Sample chamber; M-Mirror; DM-Dialectical mirror; L-Lens; PM-Parabolic mirror; FM-Flip-flop mirror; P-Polarizer; A-Attenuator; S-Sample cell; CH-Chopper; D-Beam collector; BS-Beam splitter; C-Compensator; W-Wedge mirror; WP-Half-wave plate; MCT-Single-channel MCT detector; DS-Time delay line. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0040] like Figure 1 As shown, the spectral system of the present invention includes: a femtosecond laser (50 fs, 800 nm, 1 kHz) 1, an optical parametric amplifier TOPAS and a difference frequency generator DFG 2, a frequency divider 3, an electronic time delay generator 4, a nanosecond laser (7 ns, 1064 nm, 1-50 Hz) 5, a detachable optical parametric oscillator OPO 6 providing 355 nm light pulses, an optical parametric oscillator OPO 7 providing 2020 nm light pulses, a monochromator and a mercury cadmium telluride MCT array detector 8, an integrator and a data acquisition card 9, a computer 10, a controller 11, a controller 12, a time-resolved pulse generation chamber 13, and a sample chamber 14.

[0041] This invention includes a nanosecond light source generated by a nanosecond laser and a femtosecond light source generated by a femtosecond laser. The nanosecond light source is used for triggering acid-base steps and pulsed heating modes, while the femtosecond light source is used for two-dimensional infrared spectroscopy detection. The specific implementation of these two parts is further described below with reference to the accompanying drawings.

[0042] Acid-base step and pulse heating mode triggering: The electronic frequency signal (1kHz) output by femtosecond laser 1 is converted into a 1-50Hz signal by frequency divider 3. A time delay (i.e., the relative time difference between nanosecond and femtosecond light pulses) is applied by electronic time delay generator 4, triggering nanosecond laser 5 to generate a 1064nm wavelength light pulse. In acid-base step mode, the 1064nm light pulse enters the detachable optical parametric oscillator 6, is converted into a 355nm light pulse, and its angle is adjusted by dichroic mirror DM and reflector M. It then passes through chopper CH2 and illuminates the flip-flop reflector FM. The controller 12 of chopper CH2 is controlled by the electronic pulse signal provided by frequency divider 3, and its rotation frequency is set to half the repetition frequency of the nanosecond light source. In pulse heating mode, optical parametric oscillator 6 is detached, and the 1064nm light pulse passes through dichroic mirror DM and enters optical parametric oscillator 7, generating a 2020nm light pulse which is then irradiated onto the back of FM by reflector M with an adjusted angle. The main purpose of the dichroic mirror DM and the reflector M in the 355nm and 2020nm optical paths is to spatially overlap the two beams at the flip-flop mirror FM, ensuring that the propagation directions of the nanosecond light pulses are completely consistent before and after the flip-flop mirror FM is flipped. The energy of these two light pulses is adjusted by the attenuator A, and their direction is adjusted by the reflector M before entering the sample chamber 14. In the sample chamber 14, the nanosecond light is focused by a lens, and its angle is adjusted by the reflector M to spatially overlap with the femtosecond light on the sample cell S. The spot diameter of the nanosecond pulse on the sample is approximately 200 micrometers, and 4 millimeters in front of the lens.

[0043] Two-dimensional infrared spectroscopy detection section: Femtosecond laser 1 generates light pulses with a wavelength of 800nm ​​and a repetition frequency of 1kHz. These pulses are adjusted in angle by two reflecting mirrors M and then incident on the optical parametric amplifier and difference frequency generator 2. The software integrated into the optical parametric amplifier TOPAS and the difference frequency generator DFG controls the corresponding parameters to generate mid-infrared pulses of the desired wavelength (typically set to 6 micrometers). The mid-infrared pulses then enter the time-resolved pulse generation chamber 13 (…). Figure TwoThe pump light, composed of interference from k1 and k2, and the probe light, k3, enter the sample chamber 14. A parabolic mirror PM in the sample chamber 14 focuses the parallel-incident pump light k1 and k2 and the probe light k3 into the sample cell S. Another parabolic mirror PM collects the probe light, which is then adjusted in angle by a mirror M, converged by a lens L, and polarized by a polarizer P before finally entering the slit of the monochromator 8. The polarization direction of the polarizer P should be completely consistent with the probe light to filter signal interference caused by sample scattering. The monochromator and the MCT array detector 8 are connected to the integrator 9 via a data cable. After the integrator 9 is triggered by an electronic pulse signal input from the controller 11, it integrates the signal input from the MCT array detector 8 (the integration time is typically set to 2000 ns), which is then recorded by the data acquisition card 9 and sent to the computer 10 to generate a data file. The signal input from the single-channel MCT detector during the same time period is also recorded by the integrator and the signal acquisition card 9 for signal correction.

[0044] In pulse heating mode, the sample cell S adopts a "sandwich" structure, in which the liquid sample is sandwiched between two FEP-coated calcium fluoride windows, and the sample thickness is adjusted by a PTFE gasket (typically 50 micrometers). The "sandwich" structure is fixed in a copper module and the temperature is controlled by a water bath circulation pump.

[0045] In the pH step mode, the sample cell S adopts a windowless configuration (patent application number 202223024867.X). The liquid sample is pushed to the nozzle by a peristaltic pump, forming a liquid film between two parallel tungsten filaments. The flow rate of the peristaltic pump needs to be determined according to the repetition frequency of the nanosecond pulse, and the reference formula is: flow rate = nanosecond spot diameter * film thickness * film width * nanosecond pulse repetition frequency.

[0046] In this embodiment, the triggering mode is a pH step, and the frequency division of the frequency divider is 20. Delay line DS1 ( Figure Two The scanning range is -300 fs to 2500 fs, with a step size of 4 fs. The time delay of nanosecond and femtosecond optical pulses is 10 microseconds. The signal averaging count is 100.

[0047] This embodiment presents a rapid scanning detection method based on a two-dimensional infrared spectroscopy system combining acid-base step and pulsed heating, comprising the following steps:

[0048] 1) Set the frequency division of frequency divider 3 to 20.

[0049] 2) Set the time delay to 10 microseconds using time delay unit 4.

[0050] 3) Set the moving speed of the delay line to v = dt * c * n / 2, which is 1.2 * 10 -5 meters per second.

[0051] 4) Set the start and end positions of the delay line movement to -300fs and 2500fs, respectively. Set the waiting time for the delay line at the start and end positions to 0.1 seconds.

[0052] 5) The delay line moves from the starting position to the ending position (moving in the forward direction) and is stored as a 50*700*128 matrix M1.

[0053] 6) Move the delay line from the termination position to the starting position (reverse movement) to obtain a 50*700*128 matrix M2. Negate the data in the matrix difference M1-M2 where p is an even number (p=2,4…50) to obtain the final chopper signal.

[0054] 7) Repeat steps 5 and 6 100 times, and the computer records the average value of the chop signal to generate a data file.

[0055] 8) In the data file, the 700*128 matrix corresponding to p=1 is the transient two-dimensional infrared spectrum with a time delay of 10 microseconds. The corresponding ω1 and ω3 frequency axes can be obtained by the frequency calibration method of conventional two-dimensional infrared spectroscopy.

Claims

1. A two-dimensional infrared spectroscopy system with pH step and temperature pulse, characterized in that, The system comprises a femtosecond laser (1), an optical parametric amplifier and a difference frequency generator (2), a frequency divider (3), an electronic time delay generator (4), a nanosecond laser (5), a detachable optical parametric oscillator (6) for providing 355 nm light pulses, an optical parametric oscillator (7) for providing 2020 nm light pulses, a monochromator and a mercury cadmium telluride array detector (8), an integrator and a data acquisition card (9), a computer (10), a controller, a time-resolved pulse generation chamber (13) and a sample chamber (14); the femtosecond laser (1) and the optical parametric amplifier and the difference frequency generator (2) cooperate to generate mid-infrared pulses of a desired wavelength, which enters the time-resolved pulse generation chamber (13); the frequency signal output by the femtosecond laser (1) is converted into a signal of 1-50 Hz through the frequency divider, and the time difference between the signal and the frequency signal of the femtosecond laser (1) is adjusted through the electronic time delay generator (4), and the signal is input to the nanosecond laser (5) to generate 1064 nm wavelength light pulses; The acid-base step mode: the 1064 nm wavelength light pulses enter the detachable optical parametric oscillator (6) for providing 355 nm light pulses, are converted into 355 nm light pulses, and are then adjusted in angle by a dichroic mirror (DM) and a mirror (M) in turn, are irradiated onto a foldable mirror (FM), are adjusted in pulse energy by an attenuator (A), are adjusted in direction by the mirror (M) to enter the sample chamber (14); a chopper (CH) is placed on the 355 nm light pulse path, and is used for regulating the on-off state of the light pulses, and a controller thereof is controlled by an electronic pulse signal provided by the frequency divider (3), and the working frequency is set as half of the repetition frequency of the nanosecond laser (5); The pulse heating mode: the detachable optical parametric oscillator (6) for providing 355 nm light pulses is detached, the 1064 nm wavelength light pulses pass through the dichroic mirror (DM) to enter the optical parametric oscillator (7) for providing 2020 nm light pulses, 2020 nm light pulses are generated, the angle of the light pulses is adjusted by the mirror (M) to irradiate onto the back of the foldable mirror (FM), the pulse energy is adjusted by the attenuator (A), and the direction is adjusted by the mirror (M) to enter the sample chamber (14); In the sample chamber (14), the above-mentioned 355 nm light pulses or 2020 nm light pulses are adjusted in angle by the mirror (M) so as to be spatially overlapped with femtosecond light generated by the femtosecond laser (1) on a sample cell (S).

2. The two-dimensional infrared spectroscopy system according to claim 1, wherein, The time-resolved pulse generation chamber (13) is used for generating pump light and probe light required by two-dimensional infrared spectrum detection, and the optical path difference of the two is adjusted by a time delay line (DS2) to realize time resolution; the mid-infrared pulse entering the time-resolved pulse generation chamber (13) is divided into three beams k1, k2 and k3 through a wedge mirror (W) and a beam splitter (BS), and the pulse energy ratio is 47:47:6; the light pulses k1 and k2 generate interference light pulses on the second beam splitter (BS), half of which is used as pump light and enters the sample chamber (14) through a half-wave plate (WP) and a polarizer (P), and the other half is used for signal correction and detected by a single-channel MCT detector; the light pulse k3 enters the sample chamber (14) through a dispersion compensation plate (C) and a polarizer (P) and is used as probe light.

3. The two-dimensional infrared spectroscopy system of claim 2, wherein the pH jump and temperature jump are combined. The parabolic mirror (PM) in the sample chamber (14) focuses the parallel incident pump light k1&k2 and probe light k3 into the sample cell (S), and then another parabolic mirror (PM) collects the probe light and converges it onto the monochromator and MCT array detector through a mirror (M), a lens (L) and a polarizer (P); the nanosecond pulse for triggering the pH step or pulse heating is focused by a lens (L) after entering the sample chamber, and is irradiated to the sample cell through a mirror (M), and the spatial spot of the three beams of pulses k1, k2 and k3 completely overlaps.

4. The two-dimensional infrared spectroscopy system of claim 3, wherein the pH jump and temperature jump are combined. The time-resolved pulse generation chamber (13) and the sample chamber (14) are covered by a box made of 5mm thick acrylic plates, and the box is connected to a dry air generator through a gas valve and a fluorotube to control the humidity in the box below 5%.

5. The system according to claim 4, wherein the system is characterized by, The sample cell (S) adopts a windowless design in the pH step spectrum detection mode, and the liquid sample is pushed to the nozzle by a peristaltic pump to form a liquid film between the two parallel tungsten filaments; The flow rate of the peristaltic pump needs to be determined according to the repetition frequency of the nanosecond pulse.

6. The two-dimensional infrared spectroscopy system of claim 5, wherein the pH jump and temperature jump are combined. In the pulse heating spectrum detection mode, the sample cell (S) adopts a "sandwich" structure, i.e. the liquid sample is sandwiched between two pieces of thin-film-coated calcium fluoride window sheets, and the sample thickness is adjusted by a polytetrafluoroethylene gasket; the "sandwich" structure is fixed in a copper module and temperature-controlled by a water bath circulating pump; and the inside of the calcium fluoride window sheet is coated with a 12-micron-thick film made of perfluoroethylene propylene copolymer.

7. The system according to claim 6, wherein the system is characterized by, The integrator is triggered by the electronic pulse signal input by the chopper, integrates the signal input by the MCT detector, and then sends the odd and even columns to the data acquisition card for recording; the two sets of columns represent whether the k2 pulse is blocked by the chopper (CH); the signal input by the single-channel MCT detector in the same period is also recorded by the integrator and the data acquisition card (9) for signal correction.

8. A detection method of the pH step and pulse heating combined two-dimensional infrared spectrum system of claim 7, comprising the following steps: 1) The frequency divider is set to have a frequency n adjustable within 1-50 Hz, and n should be an integer divisible by 1000, i.e. after synchronization with a nanosecond and femtosecond laser, each nanosecond pulse corresponds to a group of 1000 / n femtosecond pulses, and the interval between the femtosecond pulses is 1 millisecond; 2) Set the time delay τ by the electronic time delay generator, τ is the time difference between the nanosecond pulse and the first pulse in the corresponding femtosecond pulse group, so it should be a value less than 1 millisecond; 3) Two-dimensional infrared spectrum contains ω1 and ω3 frequency coordinate axes; the resolution of ω3 coordinate axis is determined by the total number of channels of MCT detector, and the resolution of ω1 coordinate axis is determined by the step size dt of relative time delay of pump light k1 and k2, which is set to 4 fs; the moving speed v of delay line is set according to the step size dt, and the calculation formula is v = dt * c * n / 2, with the unit of meter per second, wherein c is the speed of light, that is, 3 * 10 8 meter per second; 4) The two-dimensional infrared spectrum is obtained by scanning the delay line from 0 to 2 * n * dt, and the scanning step size is dt, and the scanning time is 2 * n * dt / v, wherein n is the total number of channels of MCT detector, and v is the moving speed of delay line. 4) Set the start and end positions ts and te of the delay line movement, i.e. k1 and k2 are-300 fs and 2500 fs respectively relative to the time delay; Set the waiting time t of the delay line at the start and end positions, which should be an integer multiple of 1000 / n milliseconds and greater than 0.1 seconds; 5) The delay line moves from the start position to the end position, moving forward, and the data acquisition card records the corresponding spectral signal of each femtosecond pulse triggered by the frequency signal of the femtosecond laser, stored as a p*q*m matrix M1; p is the number of pulses in a group of femtosecond pulses 1000 / n, q is the total number of femtosecond pulse groups (te-ts) / dt, and m is the total number of channels of the MCT array detector; In this matrix, k2 is "on" when p is odd, i.e. not blocked by the chopper; k2 is "off" when p is even, i.e. blocked by the chopper; 6) The delay line moves from the end position to the start position, moving backward, and the signal acquisition method is the same as the previous step, obtaining a p*q*m matrix M2; At this time, k2 is "off" when p is odd, and k2 is "on" when p is even; Take the negative of the data in the matrix difference M1-M2 when p is even to get the final chopping signal; 7) According to the set average number j, repeat steps 5 and 6 j times, and record the average value of the chopping signal by the computer to generate a data file; 8) In the data file, the q*m matrix corresponding to p=1 is the two-dimensional infrared spectrum corresponding to the time delay τ; When p=2, 3, 4…, the corresponding time delay is τ+(p-1) with the unit of millisecond; The corresponding ω1 and ω3 frequency coordinate axes can be obtained by the conventional frequency calibration method of two-dimensional infrared spectrum.

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