A dual channel differential total internal reflection infrared spectroscopy detection system

Abstract

The application discloses a kind of dual-channel differential total internal reflection infrared spectrum detection systems.It will be adjustable mid-infrared laser beam by beam splitter into two routes, power is substantially equal, respectively into the dual-channel of total internal reflection ATR crystal, the evanescent wave of two light beams respectively and the target substance and standard reference substance in sample channel and reference channel interact;First photodetector and second photodetector receive two routes of emergent light respectively, data processing module carries out differential demodulation to two signals, effectively suppresses laser relative intensity noise and common-mode noise, extracts the absorption spectrum of target substance.The system also integrates temperature control subsystem to precisely control crystal temperature, SEIRA nano antenna array enhances evanescent field interaction, microfluidic microchannel cover plate realizes fixed flow and fixed pressure transmission, and detachable ATR sensing subunit and integrated RFID / NFC label realize automatic optical path correction.

Description

Technical Field

[0001] This invention relates to the field of infrared spectroscopy detection technology, specifically to a dual-channel differential total internal reflection infrared spectroscopy detection system, which can be used for high-sensitivity, high signal-to-noise ratio qualitative and quantitative analysis of trace molecules of target substances in liquid or solid samples using infrared absorption spectroscopy. Background Technology

[0002] Attenuated total reflectance infrared spectroscopy (ATR-IR) is an important technique for analyzing the molecular structure of liquid and solid samples. This technique utilizes evanescent waves to detect samples on the surface of a total internal reflectance crystal, offering advantages such as no complex sample preparation required and high detection speed. It is widely used in biochemical analysis, food testing, medical diagnostics, and environmental monitoring. Traditional ATR-IR systems typically use Fourier transform infrared spectroscopy (FTIR) as the core light source. However, due to the low brightness (similar to the difference between candlelight and laser light) and wide bandwidth characteristics of thermal light sources, they cannot achieve total internal reflection of the ATR crystal beyond the third order, nor can they achieve beam splitting for differential spectral detection of weak signals. This results in insufficient detection sensitivity, making it difficult to effectively detect trace molecules.

[0003] Therefore, there is an urgent need for a high-precision ATR infrared spectroscopy detection system that can achieve dual-channel spatial synchronous differential, has multiple total internal reflection cumulative enhancement effects, and integrates a multi-dimensional noise suppression structure, in order to overcome the bottlenecks of existing technologies in terms of sensitivity, stability, and differential noise suppression. At the same time, it is necessary to utilize a dual-channel differential mechanism to eliminate common-mode noise and integrate temperature control compensation and surface enhancement methods to achieve a high-sensitivity ATR infrared spectroscopy detection technology solution, so as to solve the problems of insufficient signal-to-noise ratio, severe baseline drift, and limited trace detection capability in the existing technologies. Summary of the Invention

[0004] To achieve a high-precision ATR infrared spectroscopy detection system with dual-channel spatial synchronous differential detection, multiple total internal reflection accumulation enhancement effects, and integrated multi-dimensional noise suppression structure, and to overcome the shortcomings of existing ATR infrared spectroscopy detection systems such as low single-channel detection signal-to-noise ratio, insufficient common-mode noise suppression capability, and severe baseline thermal drift, this invention provides a dual-channel differential total internal reflection infrared spectroscopy detection system. By splitting the laser beam into two paths with essentially equal power, these paths interact with the target material and the standard reference material respectively on the same ATR sensing interface using evanescent waves, and then undergo differential demodulation processing, thereby significantly improving the system's detection signal-to-noise ratio and stability.

[0005] The technical solution adopted in this invention is as follows: A dual-channel differential total internal reflection infrared spectroscopy detection system includes: The light source modulation module (e.g., a light source) is configured to generate a mid- to far-infrared laser beam with continuously tunable wavelength; the light source module includes a single-longitudinal-mode quantum cascade laser (QCL) or an interband cascade laser (ICL), which outputs a collimated and shaped laser beam with a tuning range covering the characteristic fingerprint spectral region of the target substance. A beam splitting coupling module (e.g., a beam splitter) is configured to split the laser beam into a first beam and a second beam with substantially equal power. A total internal reflection (ATR) sensing system includes one or more infrared transmittance sensors, each having an incident end face, an exit end face, and an upper surface serving as a sensing interface. The sensor internally generates one or more total internal reflections, with two beams output from the exit end face in spatially symmetrical and non-interfering optical paths. The upper surface is configured with a sample microfluidic channel and a reference microfluidic channel, which are independent and non-contacting channel spaces, respectively covering the evanescent wave regions generated by the first and second beams at the sensing interface. The evanescent waves interact with the target material and the standard reference material within their respective channels. The detection module (e.g., a detector) includes a first photodetector and a second photodetector, which respectively receive the first beam and the second beam after the interaction of evanescent waves, and respectively generate a first detection signal and a second detection signal. A data processing module (e.g., a data processor) is connected to the detection module and configured to receive the first detection signal and the second detection signal and perform differential demodulation processing to extract the spectral signal of the target substance.

[0006] In some embodiments, the one or more infrared transmittance sensors include a single, integrally formed ATR crystal that simultaneously guides the first beam and the second beam; or they include multiple discrete infrared transmittance ATR crystals, each embedded in a metal or polymer holder, with adjacent ATR crystals achieving continuous beam transmission through optical coupling or spatial alignment, thereby creating multiple cumulative total internal reflections along the total sensing path.

[0007] In some embodiments, the ATR crystal is made of diamond (C), zinc selenide (ZnSe), zinc sulfide (ZnS), germanium (Ge), or silicon (Si), and both the incident and exit surfaces are coated with a broadband infrared antireflection coating (AR Coating) to minimize Fresnel reflection loss and suppress parasitic interference fringes; an ultra-hard protective film may be provided on the upper surface of the ATR crystal, made of sapphire, silicon nitride, diamond-like carbon (DLC), or diamond, with a thickness between a few nanometers and several hundred nanometers.

[0008] In some embodiments, the upper surface of the ATR crystal includes a surface-enhanced infrared absorption (SEIRA) structure; the SEIRA structure includes a nano-metal antenna array or metasurface fabricated at the evanescent wave interaction site, the size and design of the nano-metal antenna resonating with the incident laser wavelength and the vibrational frequency of the target molecule, thereby increasing the interaction cross section between infrared light and the target material through the local field enhancement effect.

[0009] In some embodiments, the ATR sensing system further includes a temperature control subsystem, which includes a thermoelectric cooler (TEC), a temperature sensor, and a closed-loop feedback control circuit, configured to maintain the overall temperature of the sensor and the microfluidic channel cover at a preset constant value, thereby suppressing baseline thermal drift noise caused by spectral distortion, refractive index drift, and thermal expansion due to ambient temperature fluctuations.

[0010] In some embodiments, the system further includes a microfluidic channel cover plate inverted on the upper surface of the ATR sensor, the microfluidic channel cover plate being engraved with at least two physically isolated microfluidic channels as the sample microfluidic channel and the reference microfluidic channel; the system further includes a fluid system containing a sample injection pump and a precision flow rate control module, configured to ensure that the flow rate and pressure of the fluid in the two channels are consistent in real time; an online degasser or bubble trapping device is integrated upstream of the fluid system, including at least one of a vacuum membrane degasser, a geometric expansion bubble trap, a hydrophobic venting membrane, or a porous diffusion barrier, to prevent bubbles from precipitating at the sensing interface and interfering with detection.

[0011] In some embodiments, the total internal reflection ATR sensing system is packaged as a detachable, replaceable independent module (Cartridge) that integrates an RFID or NFC electronic tag chip to store the crystal's factory calibration parameters for external systems to read and automatically perform optical path normalization correction.

[0012] In some embodiments, the light source module is configured to perform amplitude modulation (AM) or phase modulation (PM) on the light beam; the data processing module includes a lock-in amplifier that demodulates the detector output signal using a reference signal synchronized with the light source modulation frequency to suppress noise and improve the signal-to-noise ratio; the data processing module can also dynamically adjust the gain parameters of the two spectra to achieve dynamic gain equalization of the differential demodulation system to compensate for spectral baseline drift caused by crystal surface contamination, system aging, or environmental changes; and can monitor the characteristic peak position of the infrared inert internal standard mixed in the target liquid to provide real-time feedback and adjust the laser wavelength to compensate for wavelength drift.

[0013] In some embodiments, a capture probe that specifically recognizes biomolecules, including antibodies, antigens, nucleic acid aptamers, or molecularly imprinted polymers, is prepared in the evanescent field region to specifically capture and enrich trace amounts of target molecules from flowing liquid or solid samples to the crystal surface, thereby achieving highly sensitive immunoinfrared spectroscopy detection.

[0014] This invention discloses a dual-channel differential total internal reflection infrared spectroscopy detection system. In some embodiments, the technical problems to be solved include the insufficient signal-to-noise ratio, severe baseline drift, and limited trace detection capability of existing single-channel ATR-IR systems due to relative intensity noise of the light source, crystal refractive index drift, and common-mode noise.

[0015] The technical solution employed is as follows: a tunable mid-to-far-infrared laser beam is split into two paths of approximately equal power by a beam splitter, and these paths are respectively introduced into the dual channels of a total internal reflection ATR crystal. The evanescent waves of the two beams interact with the target material and standard reference material in the sample channel and reference channel, respectively. A first photodetector and a second photodetector receive the two outgoing beams, respectively. The data processing module performs differential demodulation on the two signals, effectively suppressing relative intensity noise and common-mode noise of the laser, and extracting the absorption spectrum of the target material. The system also integrates a temperature control subsystem for precise control of the crystal temperature, a SEIRA nano-antenna array to enhance evanescent field interaction, a microfluidic microchannel cover plate to achieve constant current and constant pressure transmission, and a detachable ATR crystal with integrated RFID / NFC tags for automatic optical path correction. It is mainly used for trace qualitative and quantitative analysis of target substances in fields such as biochemical samples, medical diagnostics, drug detection, and environmental monitoring.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. By adopting a dual-channel differential detection structure, the inherent relative intensity noise (RIN) of the laser source is used as common-mode noise to cancel it out. Compared with a single-channel ATR system, the signal-to-noise ratio can be improved by more than an order of magnitude, effectively reducing the detection limit and achieving high-sensitivity detection of trace target molecules.

[0017] 2. By integrating a temperature control subsystem to precisely control the temperature of the ATR crystal and the microfluidic channel cover, the refractive index drift of the crystal and the optical path change caused by thermal expansion due to ambient temperature fluctuations are effectively suppressed, thereby minimizing the baseline thermal drift noise of the differential spectrum and improving the long-term stability of the detection.

[0018] 3. By fabricating SEIRA nano-metal antenna arrays or metasurfaces on the ATR crystal sensing surface, the interaction cross section between evanescent waves and target materials can be significantly improved by utilizing the local electromagnetic field enhancement effect, thereby further enhancing the detection sensitivity to the level of a single molecular layer or even a sub-single molecular layer.

[0019] 4. By immobilizing specific capture probes at the sensing interface, selective enrichment of target biomolecules in complex matrices can be achieved. Combined with differential ATR-IR spectroscopy detection, the system is endowed with label-free, real-time online immunosensing capabilities.

[0020] 5. By designing the ATR sensing system as a detachable cartridge and integrating RFID / NFC electronic tags, the automatic reading of factory calibration parameters and optical path normalization correction are realized, which simplifies the user operation process and ensures the comparability and traceability of measurement results between different batches of crystals.

[0021] 6. Through precise flow rate control and online degassing / bubble capture mechanism, the fluid conditions of the sample channel and the reference channel are highly consistent, eliminating the interference of crystal micro-deformation caused by flow rate and pressure difference on differential spectroscopy, and significantly improving the measurement repeatability under liquid flow conditions. Attached Figure Description

[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It will be apparent that the drawings described below are merely some embodiments of this disclosure, and that those skilled in the art can derive other drawings from these drawings without inventive effort. The drawings are not intended to be drawn to scale. In the drawings, each identical or substantially identical component shown in the various figures may be represented by the same reference numerals. For clarity, not every component is labeled in every figure.

[0023] Figure 1 This is an overall system block diagram of a dual-channel differential total internal reflection infrared spectroscopy detection system according to an embodiment of the present invention.

[0024] Figure 2 This is a partially enlarged planar schematic diagram of an embodiment of the dual-channel ATR crystal module of the present invention performing a few-time total internal reflections.

[0025] Figure 3 This is a partially enlarged planar schematic diagram of an embodiment of the dual-channel ATR crystal assembly of the present invention performing multiple total internal reflections.

[0026] Figure 4 This is a side view of a stacked multi-ATR crystal assembly according to an embodiment of the present invention.

[0027] Figure 5 This is a three-dimensional structural diagram of an ATR sensor module with a microfluidic channel cover plate according to an embodiment of the present invention.

[0028] Figure 6 This is a three-dimensional structural diagram of an ATR sensor module with a microfluidic channel cover plate according to an embodiment of the present invention.

[0029] Figure 7 This is a cross-sectional schematic diagram of the SEIRA nano-antenna array region according to an embodiment of the present invention.

[0030] Figure 8 This is a bottom view schematic diagram of a microfluidic channel cover plate assembly according to an embodiment of the present invention.

[0031] Figure 9 This is a block diagram of a temperature control subsystem according to an embodiment of the present invention.

[0032] Figure 10 This is a block diagram of a fluid control subsystem according to an embodiment of the present invention.

[0033] Figure 11 This is a flowchart of an embodiment of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the described embodiments are only for explaining the invention and are not intended to limit the scope of the invention.

[0035] like Figure 1 As shown, this embodiment of the invention provides a dual-channel differential total internal reflection infrared spectroscopy detection system 10. In some embodiments, the dual-channel differential total internal reflection infrared spectroscopy detection system 10 mainly includes a light source modulation module 100, a beam splitting and coupling module 110, a total internal reflection sensing system 200, a detection module 300, and a data processing module 350. Specifically, the light source modulation module 100 is configured to generate a continuously tunable mid-to-far infrared laser beam and apply high-frequency modulation to the beam. Its output beam is split into two paths by the beam splitting and coupling module 110 and then introduced into the total internal reflection sensing system 200. After the evanescent wave in the total internal reflection sensing system 200 interacts with the substances in the sample channel and the reference channel, respectively, the two output beams are received by the detection module 300 and converted into electrical signals, which are then differentially demodulated and spectrally analyzed by the data processing module 350.

[0036] The light source modulation module 100 includes a drive modulation circuit 101 and a QCL laser 102. The drive modulation circuit 101 provides drive current to the QCL laser 102 and simultaneously outputs a high-frequency modulation signal to modulate the intensity of the laser emitted from the QCL laser 102 by amplitude modulation (AM) or phase modulation (PM). The QCL laser 102 has a built-in single-longitudinal-mode quantum cascade laser core, and its wavelength tuning range covers the infrared fingerprint region of the target material (typically 500 nm). 1800cm -¹). In some embodiments, the QCL laser 102 can also be replaced with an interband cascaded laser (ICL) to cover a longer mid-infrared band, which can be selected according to the detection target. Optionally, the output beam of the QCL laser 102 is incident on the polarization controller 104 through the lens 103. The polarization controller 104 optimizes the polarization state of the output beam of the QCL laser 102 to improve the uniformity and consistency of the power distribution of the two beams after subsequent 50 / 50 beam splitting, thereby reducing the differential residual noise introduced by the unequal power of the two beams. The modulation reference signal output by the light source modulation module 100 is synchronously transmitted to the data processing module 350 for correlation demodulation by the lock-in amplifier in the data processing module 350.

[0037] In some embodiments, the frequency of the QCL laser 102 is set between 100 kHz and 1 MHz, and in conjunction with a lock-in amplifier, the noise level can be reduced to the microabsorbance (μAU) level.

[0038] In some embodiments, the light source modulation module 100 may be configured to: 1) adjust the polarization state of the first beam and the second beam to make them exhibit p-polarization state (or TM mode) so that the electric field vector of the evanescent wave is perpendicular to the sensing interface, thereby maximizing the excitation of the local electromagnetic field of the surface-enhanced infrared absorption (SEIRA) structure; 2) collimate and shape the first beam and the second beam so that their wavelength tuning range covers the characteristic fingerprint spectral region of the target material or target biomass molecules.

[0039] The beam splitting coupling module 110 includes a 50 / 50 beam splitter 111 and corresponding coupling optical elements. The 50 / 50 beam splitter 111 splits the incident laser beam into a first beam and a second beam with substantially equal power. In some embodiments, the 50 / 50 beam splitter 111 may be a composite beam splitting film made of CaF2 or ZnSe, or may be implemented using a diffraction method. It is not limited to a specific beam splitting structure and can be applied to any beam splitting form suitable in the art. The first beam and the second beam are respectively coupled into the interior of the total internal reflection sensing system 200 and correspond to one or more incident end faces of the dual optical paths within the total internal reflection sensing system 200.

[0040] The total internal reflection sensing system 200 includes a multichannel multiple ATR crystal assembly. Figure 2-4Several embodiments of the total internal reflection (TIR) ​​sensing system 200 are shown. Multiple beams of light are emitted from the TIR sensing system 200. In some embodiments, the first beam and the second beam are incident at an angle greater than the critical angle for total internal reflection between the crystal and the target or reference material above it, thereby generating single or multiple TIR reflections internally. The first beam emitted from the TIR sensing system 200 is received by the sample detector 303 through a first lens 301, and the second beam emitted from the TIR sensing system 200 is received by the reference detector 304 through a second lens 302. The first lens 301 and the second lens 302 are optional components.

[0041] The detection module 300 includes a sample detector 303 and a reference detector 304. The sample detector 303 and the reference detector 304 respectively receive the emitted light from the total internal reflection sensing system 200. Both the sample detector 303 and the reference detector 304 are high-sensitivity infrared photon detectors. The sample detector 303 receives the first emitted light beam and outputs a first detection signal containing infrared absorption information of the sample channel. The reference detector 304 receives the second emitted light beam and outputs a second detection signal containing infrared absorption information of the reference channel. In some embodiments, both the sample detector 303 and the reference detector 304 can be high-sensitivity infrared photon detectors such as mercury cadmium telluride (HgCdTe, i.e., MCT) or indium antimonide (InSb). The specific selection can be determined according to the target spectral range and sensitivity requirements, and this invention does not limit this. Specifically, the sample detector 303 and the reference detector 304 should use devices of the same model and batch to ensure the consistency of the dual-channel response and reduce the differential residual introduced by detector differences.

[0042] The data processing module 350 includes a differential processor 310 and a data analysis terminal 320. The differential processor 310 receives the first and second detection signals, and synchronously demodulates them using the modulation reference signal output from the light source modulation module 100. It then performs differential operations on the two demodulated signals to extract the differential absorption spectrum belonging only to the target substance after removing common-mode noise, and inputs this spectrum to the data analysis terminal 320. At this point, a lock-in amplifier should be installed within the differential processor 310 to enable demodulation using the modulation reference signal. Furthermore, a lock-in amplifier can also be built into the data analysis terminal 320. In this case, correlation demodulation can be performed using the modulation reference signal output from the drive modulation circuit 101 through the data analysis terminal 320, effectively extracting the detection signal from broadband noise and significantly improving the signal-to-noise ratio. Figure 1 The figure shows an embodiment in which a lock-in amplifier is installed in the data analysis terminal 320, namely Figure 1 The figure shows an embodiment of demodulation using a modulation reference signal via a data analysis terminal 320.

[0043] In some embodiments, the data analysis terminal 320 can also be configured to dynamically adjust the gain parameters of the two signals to achieve dynamic gain equalization of the differential demodulation system, thereby compensating for spectral baseline drift caused by crystal surface contamination, system aging, or environmental changes, and further improving long-term measurement stability. In some embodiments, during operation, the driving modulation circuit 101 applies periodic high-frequency modulation to the output light intensity of the QCL laser 102, and the modulation frequency can be much higher than the 1 / f noise cutoff frequency, so that the dual-channel differential total internal reflection infrared spectroscopy detection system 10 of the present invention operates in the frequency range with the lowest noise. The first beam and the second beam are reflected by the first mirror 112 and the second mirror 113, respectively, and are simultaneously coupled into the corresponding incident end face within the total internal reflection sensing system 200. They interact with the target material and the reference material in their respective evanescent field regions. The first beam, which absorbs the target spectral information, and the second beam, which contains only background absorption information, flow through the sample channel and reach the corresponding detectors. After being differentially processed by the differential processor 310, the common-mode noise (including the relative intensity noise RIN of the laser source, global temperature fluctuations, laser interference fringes, etc.) is effectively suppressed, thereby significantly improving the detection limit of the sample absorption characteristics.

[0044] In some embodiments, the total internal reflection (TIR) ​​sensing system 200 of the dual-channel differential total internal reflection infrared spectroscopy detection system 10 is designed as a detachable and replaceable independent cartridge module, which can be quickly coupled and separated from the fixed optical support of the light source modulation module 100 and the beam splitting coupling module 110. The designed cartridge module can integrate an RFID or NFC electronic tag chip to store the factory calibration parameters of the TIR sensing system 200 (including precise optical path length, lens parameters, and background transmittance). After being read by a reader, the data analysis terminal 320 automatically performs optical path normalization correction on the measurement results and records the usage status of the independent cartridge module. This design can extend the maintenance and replacement cycle of the TIR sensing system 200 and ensure the comparability and traceability of measurement results between different batches of TIR sensing systems 200.

[0045] In some embodiments, the dual-channel differential total internal reflection infrared spectroscopy detection system 10 achieves real-time wavelength compensation for the mid- and far-infrared laser source through an internal standard feedback tuning mechanism. Specifically, a known concentration of infrared inert internal standard is mixed into the target substance to be tested. This internal standard does not broaden or interfere with the characteristic absorption peaks of the target substance within the detection spectral range. For the internal standard (such as a highly fluorinated organic solvent), its absorption peak position is constant and does not change with the chemical environment of the target substance. By monitoring the shift of this internal standard peak in real time, the current or temperature controller of the QCL laser 102 can be driven in a reverse closed loop to achieve real-time automatic calibration of the wavelength axis. The internal standard can be selected from standard substances with low infrared activity and stable chemical properties; this invention is not limited to these options.

[0046] During each spectral scan, the data processing module 350 monitors the spectral position of the internal standard characteristic absorption peak in real time and compares it with the factory-simulated peak position or the initial reference. When a systematic shift in the internal standard peak position is detected, this shift indicates a deviation between the actual output wavelength of the laser source and the commanded wavelength. The data processing module 350 feeds this deviation signal back to the drive modulation circuit 101, which fine-tunes the drive current or temperature of the QCL laser 102 in real time, locking the actual output wavelength of the QCL laser 102 in the target fingerprint spectral region. This compensates for spectral wavelength drift caused by changes in the viscosity of the target material, flow rate fluctuations, or crystal surface contamination, ensuring high-precision alignment between the laser spectrum and the target fingerprint spectral region throughout the entire scan.

[0047] In other embodiments, the real-time infrared absorbance reading of the internal standard can also serve as an internal sensitivity reference standard. By comparing it with the initial standard, it can sensitively reveal the changes in spectral intensity caused by light intensity drift or crystal surface contamination, providing additional real-time reference information for differential spectral baseline correction.

[0048] like Figure 2 As shown, the single ATR crystal assembly in this embodiment of the invention demonstrates a dual-channel, low-order total internal reflection optical path layout. The left side of the single ATR crystal is the incident end face 202a, the right side is the exit end face 203a, and the upper surface 201a serves as the sensing interface. Figure 2As shown, the first beam 206a is reflected by mirror 112a from the upper left corner of the incident end face 202a at a defined incident angle, and the second beam 207a is reflected by mirror 113a from the lower left corner of the incident end face 202a at a symmetrical incident angle. Both beams undergo two total internal reflections within the crystal, ultimately exiting from the exit end face 203a with symmetrical optical paths. The first and second beams undergo total internal reflection at two contact points 250a and 250b on the upper surface 201a, respectively, generating evanescent waves above the upper surface 201a. Microfluidic channels are configured above the upper surface 201a, and the evanescent fields extend into the corresponding sample and reference channels, interacting with the target and reference materials in their respective channels. The first emitted beam 208a and the second emitted beam 209a at the exit end face are reflected by mirrors 204a and 205a, respectively, and enter the corresponding sample detector 303 and reference detector 304. In some embodiments, a few-order total internal reflection design (such as...) Figure 2 (As shown) It is suitable for applications that require high evanescent field penetration depth, such as the analysis of water-soluble samples containing a strong water absorption background.

[0049] Figure 2 To visually distinguish the first beam 206a and the second beam 207a, the two beams appear not to overlap in a side view. In some embodiments, the first beam and the second beam form a dual-channel parallel transmission side-by-side. That is, the two beams... Figure 2 In the side view, they are completely overlapping, but they are offset by a certain distance (e.g., 10mm apart) in the direction perpendicular to the paper (Z-axis), and "run side by side" below the left and right channels respectively.

[0050] like Figure 3As shown, this embodiment of the invention provides a dual-channel ATR crystal assembly layout with multiple total internal reflections. Specifically, the upper surface 201b of a single ATR crystal serves as the sensing interface, the left side is the incident end face 202b, and the right side is the exit end face 203b. The first beam 206b and the second beam 207b are reflected by mirrors 112b and 113b, respectively, and enter from the upper and lower parts of the incident end face 202b. Multiple total internal reflections occur inside the crystal, and finally, the first exit beam 208b and the second exit beam 209b are emitted from the exit end face 203b in a symmetrical optical path. They are then reflected by mirrors 204b and 205b, respectively, and reach the sample detector 303 and the reference detector 304. In the design with multiple total internal reflection points, the first beam 206b and the second beam 207b generate multiple evanescent field reflection points at multiple contact points with the upper surface 201b, such as evanescent field reflection points 250b, 251b, 252b, and 253b. Each evanescent field reflection point contributes to the sample absorption signal, and the absorption signals of all evanescent field reflection points are accumulated in a single transmission, thereby significantly enhancing the absorption intensity of the target substance compared to a single reflection and reducing the detection limit. In some embodiments, the two beams may undergo 2, 4, 10, or more total internal reflections within the crystal, respectively. The specific number of reflections can be selected according to the crystal geometry, sample concentration, and detection sensitivity requirements, and is not limited in this invention.

[0051] Figure 3 To visually distinguish the first beam 206b and the second beam 207b, the two beams appear not to overlap in a side view. In some embodiments, the first beam and the second beam form a dual-channel parallel transmission side-by-side. That is, the two beams... Figure 3 In the side view, they are completely overlapping, but they are offset by a certain distance (e.g., 10mm apart) in the direction perpendicular to the paper (Z-axis), and "run side by side" below the left and right channels respectively.

[0052] like Figure 4 As shown, this embodiment of the invention further provides a stacked multi-block ATR crystal configuration. This configuration can be used to significantly increase the total internal reflection count without changing the geometry of a single ATR crystal, further enhancing the cumulative absorption sensitivity of the evanescent field to the target material. Figure 4As shown, the overall optical path consists of three ATR crystals arranged in series along the horizontal direction. From left to right, they are the first crystal (incident surface 202c, exit surface 203c), the second crystal (incident surface 262c, exit surface 263c), and the third crystal (incident surface 272c, exit surface 273c). The upper surfaces of the three ATR crystals are all marked with 201c, forming a continuous sensing interface that can cover a larger area of ​​evanescent field and increase the number of interactions between the evanescent wave and the target material.

[0053] Specifically, the first beam 206c and the second beam 207c, after being reflected by mirrors 112c and 113c respectively, enter the first ATR crystal at symmetrical incident angles from above and below its incident end face 202c. After undergoing multiple total internal reflections within the first crystal, they exit from the exit end face 203c, are reflected by mirror 210c, and then couple into the incident end face 262c of the second ATR crystal. The two beams continue to undergo multiple total internal reflections within the second crystal before exiting from the exit end face 263c. They are then reflected by mirror 210c in the same manner, coupled into, and pass through the third ATR crystal, finally exiting from the exit end face 273c of the third ATR crystal as the first exit beam 204c and the second exit beam 205c respectively. All three ATR crystals are embedded and fixedly supported by a bottom holder made of metal or polymer material to ensure optical alignment accuracy and mechanical stability between adjacent ATR crystal end faces. Each ATR crystal has a broadband infrared antireflection coating on both its incident and exit surfaces to minimize Fresnel reflection loss at the interfaces between ATR crystals and suppress parasitic interference fringes. In some embodiments, the number of ATR crystal blocks is not limited to three and can be further increased according to the detection sensitivity requirements; this invention does not impose such a limitation.

[0054] Figure 4 To visually distinguish the first beam 206c and the second beam 207c, the two beams appear not to overlap in a side view. In some embodiments, the first beam and the second beam form a dual-channel parallel transmission side-by-side. That is, the two beams... Figure 4 In the side view, they are completely overlapping, but they are offset by a certain distance (e.g., 10mm apart) in the direction perpendicular to the paper (Z-axis), and "run side by side" below the left and right channels respectively.

[0055] In some embodiments, the incident end faces 202a, 202b, 202c, 262c, and 272c, and the exit end faces 203a, 203b, 203c, 263c, and 273c are all coated with a broadband infrared antireflection coating (AR Coating) to minimize Fresnel reflection loss when the laser enters and exits the ATR crystal and suppress parasitic interference fringes caused by end face reflection. The surface of the ATR crystal is precision polished to meet the surface smoothness requirements of total internal reflection. In some embodiments, the upper surface (sensing surface) of the ATR crystal may be further provided with an ultra-hard, ultra-smooth infrared protective film 221, which is made of sapphire, silicon nitride, diamond-like carbon (DLC), or diamond, with a thickness between a few nanometers and several hundred nanometers, to protect the crystal from chemical erosion and mechanical wear without affecting the evanescent wave penetration depth.

[0056] In some embodiments, each ATR crystal monomer can be made of the same or different materials. For example, the first ATR crystal monomer can be ZnSe, while the next can be Ge. This combination can simultaneously improve sensitivity and spectral range.

[0057] In some embodiments, both the incident and exit faces of the ATR crystal are coated with a broadband infrared antireflection film to ensure optical alignment accuracy between adjacent ATR crystal faces, thereby reducing Fresnel reflection loss at the interface between ATR crystals. In other embodiments, multiple ATR crystals may be made of the same material, or a combination of different ATR crystal materials may be used to optimize the durability and ease of cleaning of the microfluidic channel. The holder can be sealed to the microfluidic channel cover plate 214 to form a well-isolated sample microfluidic channel and a reference microfluidic channel.

[0058] In some embodiments, the ATR crystal is selected from diamond (C), zinc selenide (ZnSe), zinc sulfide (ZnS), germanium (Ge), or silicon (Si), all of which have a higher refractive index than common sample media, thereby ensuring that the total internal reflection condition is met. Specifically, different materials have significant differences in infrared transmission window, refractive index, and chemical resistance, and can be selected according to the absorption spectrum region of the target substance and the chemical properties of the sample. This invention does not limit the selection. Diamond material has the widest infrared transmission range (approximately 500 cm⁻¹). - ¹ to tens of thousands of cm - ¹), with a refractive index of approximately 2.4, moderate evanescent wave penetration depth, and extremely high hardness and chemical inertness, allows it to directly contact strongly acidic, strongly alkaline, or highly abrasive samples, making it suitable for long-term stable detection of corrosive or highly abrasive biological or chemical samples, but its processing cost is relatively high. Germanium (Ge) material has a high refractive index (approximately 4.0) and a shallow evanescent wave penetration depth (approximately 0.65 μm @ 1000 cm). -¹), suitable for aqueous sample detection, the shallow penetration depth effectively suppresses interference from the strong infrared absorption background of water on the target absorption peak. Zinc selenide (ZnSe) material has a penetration depth of 600 to 20000 cm⁻¹. - ¹ It exhibits good light transmittance and a moderate refractive index (approximately 2.4) within the specified range, along with good chemical stability, making it suitable for broad-spectrum detection of general organic solutions and biological liquid samples. Zinc sulfide (ZnS) has similar properties to ZnSe, but slightly higher mechanical hardness. Silicon (Si) offers low cost and high processing precision, but its light transmittance is limited to 1500 cm⁻¹. - ¹The following methods are limited by phonon absorption and are suitable for specific applications with restricted mid-infrared windows. In other embodiments, multiple ATR crystals connected in series in the same sensing path can be a combination of different materials to optimize the evanescent field penetration depth distribution while maintaining broad spectral coverage and durability. Regardless of the material chosen, the refractive index of the ATR crystal should be higher than that of the sample medium to meet the critical angle condition for total internal reflection, thereby ensuring effective generation of evanescent waves at the sensing interface.

[0059] In some embodiments, both the incident and exit faces of the ATR crystal are coated with a broadband infrared antireflection coating (AR Coating). This AR Coating can employ a multilayer dielectric thin-film structure, such as alternating ZnS / ZnSe or Ge / YbF3 layers. Through precise design of the film thickness and refractive index, the target laser wavelength (typically 500–1800 cm⁻¹) can be optimized. - ¹) reduces Fresnel reflection loss to below 0.5%. The role of AR Coating is not only to reduce power loss when the laser enters and exits the crystal, but more importantly, to suppress parasitic interference fringing caused by residual reflection at the end face.

[0060] It should be noted that the parasitic interference fringes superimpose periodic baseline ripples on the scanning spectrum, significantly affecting the signal-to-noise ratio and baseline flatness of the differential spectrum. The coating process can employ electron beam evaporation, ion-assisted deposition (IAD), or sputtering deposition, and the choice can be made based on coating uniformity, adhesion requirements, and production conditions; this invention is not limited to these methods. In some embodiments, the end face can also be slightly wedge-shaped (e.g., wedge angle 0.5°–2°) to further disrupt the coherence conditions of end face reflection, working in conjunction with AR Coating to suppress interference effects. In a stacked multi-block ATR crystal structure, the interface between the exit end face of an adjacent crystal and the incident end face of the next ATR crystal is small. Interface reflection loss can be further reduced through refractive index matching gel or precision optical coupling, maintaining high transmission efficiency throughout the multiple total internal reflection optical path.

[0061] like Figure 5As shown, this embodiment of the invention provides an overall three-dimensional structure (including a microfluidic channel cover plate 214) of a total internal reflection sensing system 200. The thermally conductive base 211 serves as the bottom support structure of the module, responsible for the installation, positioning, and temperature transfer of the total internal reflection sensing system 200. A thermally conductive layer 212 is vertically embedded above the thermally conductive base 211. The thermally conductive layer 212 is enclosed on both sides, housing the thermoelectric cooler (TEC), temperature sensor, and crystal holding assembly. The incident end 202 of one or more ATR crystals 213 is located on the left side of the base 211, and the exit end 203 of one or more ATR crystals 213 is located on the right side of the thermally conductive base 211, corresponding to the incident and exit ports of the two laser beam paths, respectively. A light guide element (not separately labeled) is located on the inner surface of the incident end 202, used to precisely couple the two beams into the incident end face of the crystal. The microfluidic channel cover 214 is inverted on the upper surface of the module, and its lower surface without engraved microfluidic channels is sealed and fitted to the upper surface of the sensing interface.

[0062] The microfluidic channel cover 214 has multiple sets of microfluidic channel inlets and outlets, such as the sample microfluidic channel inlet 216 and sample microfluidic channel outlet 217, as well as the reference microfluidic channel inlet 215 and reference microfluidic channel outlet 218. Liquid can flow in from the microfluidic channel inlet and then flow out from the microfluidic channel outlet. The microfluidic channel cover 214 is used to seal and secure the cover plate to the module base. The material of the microfluidic channel cover 214 can be selected according to the application scenario, such as polydimethylsiloxane (PDMS) or ethylene fluoride (PTFE), which have good mid-infrared light transmittance and chemical resistance. The sample inlet and outlet ports 217 on the top surface of the cover plate are connected to the internally engraved microfluidic channels, and the width, depth, and shape of the channels can be customized according to application requirements.

[0063] In some embodiments, the lower surface of the microfluidic channel cover 214 is engraved with at least two completely isolated microfluidic channels, corresponding to the sample microfluidic channel and the reference microfluidic channel, respectively, with the channel centerline aligned with the position where the evanescent wave is formed on the upper surface of the sensing interface. After the lower surface of the microfluidic channel cover 214 is sealed and bonded to the sensing interface, the two channels respectively form sealed microfluidic spaces with the lower surface of the ATR crystal as the upper surface, namely the sample microfluidic channel and the reference microfluidic channel. The width and shape of the two microfluidic channels can generally be configured to cover the evanescent field regions generated by the first beam and the second beam on the sensing interface, respectively. The microfluidic channel cover 214 is also loaded with a temperature sensor (not separately labeled) for real-time monitoring of the cover's own temperature, used in conjunction with the closed-loop feedback control circuit of the temperature control subsystem.

[0064] like Figure 6 As shown, this embodiment of the invention provides an overall three-dimensional structure (including microfluidic channel cover plate 214) of a total internal reflection sensing system 200. Figure 6 Can be Figure 5A cross-sectional view. The structure includes a single or multiple ATR crystals 213, a microfluidic channel cover 214, a thermally conductive base 211, a thermally conductive layer 212, and an incident end 202, etc. Figure 5 Consistent. The attached figures clearly show the arrangement of one or more ATR crystals 213 inside the ATR sensor module, as well as the cross-section 219 of the sample microfluidic channel and the cross-section 220 of the reference microfluidic channel. The upper surfaces (i.e., sensing interfaces) of some of the single or multiple ATR crystals 213 respectively constitute the lower surfaces of the sample microfluidic channel and the reference microfluidic channel, facilitating the installation of the microfluidic channel cover plate 214 and the functionalization of the sensing interface.

[0065] In practical implementation, the temperature control subsystem components (thermoelectric cooler TEC, temperature sensor) can be directly attached to the inner side of the holder or the outer layer of the thermally conductive base 211 to achieve precise temperature control of the entire ATR crystal. In some embodiments, a heat sink or fan can be connected below the thermally conductive base 211 to work with the thermoelectric cooler TEC to control heat dissipation at the bottom of the module. The thermoelectric cooler TEC is not only used for temperature control but also to compensate for the local photothermal expansion caused by the high-power laser emitted from the QCL laser 102 irradiating the ATR system interface. At the same time, the fluid control subsystem 500 ensures that the hydrostatic pressure of the sample and the reference channel is completely consistent to eliminate the interference of strain-induced birefringence on the differential signal caused by pressure difference. An RFID / NFC electronic tag chip can also be integrated into the module and installed inside the thermally conductive layer 212 to store the crystal's factory calibration parameters and record the cartridge's usage history.

[0066] In some embodiments, the total internal reflection sensing system 200 (such as...) Figure 5 and Figure 6 The crystal (as shown) is designed as a detachable and replaceable independent cartridge structure. Compared to traditional fixed ATR designs, the detachable cartridge design allows users to quickly replace the crystal without moving the light source module and detector. Positioning guides and positioning surfaces are provided around the cartridge to ensure micron-level alignment accuracy during each installation, eliminating the need for recalibration. In other embodiments, the cartridge's outer wall may employ a leak-proof design to isolate the internal optical path and temperature control space from external moisture, further reducing the impact of ambient temperature fluctuations.

[0067] like Figure 7As shown in the figure, this embodiment of the invention illustrates a cross-sectional schematic diagram of the SEIRA nano-antenna array region. A thermally conductive base 211 and a thermally conductive layer 212 constitute the module base, with a microfluidic channel cover 214 inverted on top. Inside the module, the upper surface of one or more ATR crystals 213 forms the lower surface of the sample microfluidic channel and, after laser introduction, forms an evanescent field region. This portion of the upper surface (i.e., within the sample microfluidic channel) has a nano-antenna array region (e.g., in cross-section 219 of the sample microfluidic channel). The upper surface of another portion of one or more ATR crystals 213 forms the lower surface of the reference microfluidic channel and, after laser introduction, forms an evanescent field region. This portion of the upper surface (i.e., within the reference microfluidic channel) has a nano-antenna array region (e.g., in cross-section 220 of the reference microfluidic channel).

[0068] In some embodiments, each nanoantenna array region comprises periodically arranged nanoantennas 222, which cover the underlying ATR crystal region. The size and geometry of each nanoantenna 222 are configured to achieve localized surface plasmon resonance with the wavelength of the incident laser and the specific vibrational frequency of the target molecules, significantly enhancing the interaction cross-section between infrared light and target molecules adsorbed on the antenna surface through localized electromagnetic field enhancement. The nanoantenna 222 array covers the region with the greatest evanescent field penetration depth and spatially corresponds to the microfluidic channel. The combined effect of localized electromagnetic field enhancement and evanescent wave transmission enables even a small number of target molecules adsorbed on the antenna surface to generate a sufficiently strong detectable infrared absorption signal, further elevating the detection limit to the monolayer or even sub-monolayer level.

[0069] In some embodiments, the nanoantenna 222 may be in the form of nanopillars, nanoplates, nanodisks, or nanoslot arrays made of noble metals such as gold, platinum, or lead; the length, width, and spacing of the antenna can be optimized through digital simulation to maximize the local electric field enhancement factor for the absorption peak of the target substance. In other embodiments, the nanoantenna array may also be replaced by a metasurface, i.e., a dynamically tunable metasurface composed of two-dimensional periodic nanostructures, thereby achieving simultaneous enhancement of vibrational modes of multiple target molecules. In other embodiments, the nanoantenna array regions corresponding to the sample channel and the reference channel (e.g., in cross section 219 of the sample microfluidic channel) and (e.g., in cross section 220 of the reference microfluidic channel) both employ the same design parameters to ensure consistent enhancement characteristics in the differential schemes of the two channels and reduce systematic errors introduced by differences in antenna manufacturing.

[0070] In some embodiments, the evanescent field region may be further prepared with capture probes (not separately labeled) that specifically recognize biomolecules. The capture probes may be antibodies, antigens, nucleic acid aptamers, or molecularly imprinted polymers, etc., which are then bonded via covalent chemical bonds (such as thiol-based self-assembled monolayers of SAM to Au on noble metal surfaces). S-bonds are fixed to the SEIRA antenna surface or crystal surface, enabling targeted chemical functional modification of target molecules. When the sample liquid flows through the sample channel, the target molecules are specifically enriched on the crystal surface by the capture probe. Combined with the SEIRA enhancement effect of the nanoantenna, highly sensitive label-free infrared spectroscopy detection of trace target molecules can be achieved. In some embodiments, the enhancement factor of the SEIRA antenna depends on the aspect ratio of the nanoantenna and its resonance relationship with the incident laser wavelength. For 1650 cm⁻¹... - ¹(amide I band) detection shows that the length of the gold nanorods in the SEIRA antenna can be between 1.5 and 2.5 μm.

[0071] like Figure 8 As shown in the figure, this embodiment of the invention illustrates a bottom view of the microfluidic channel cover 214. The microfluidic channel cover 214 mainly consists of two physically isolated microfluidic channels 223 and 224. For example, microfluidic channel 223 has a reference microfluidic channel inlet 215 and a reference microfluidic channel outlet 218. Since the upper surface of the ATR crystal can serve as its lower surface, the microfluidic channel (223) in this figure is a groove structure etched on the lower surface of the cover. Microfluidic channel 224 also has a similar structure to microfluidic channel 223. Thus, microfluidic channel 223 and microfluidic channel 224 correspond to the sample channel and the reference channel, respectively. After the microfluidic channel cover 214 is installed, it is inverted and placed on the upper surface of the ATR crystal assembly. The lower surface of the cover is sealed and fitted to the upper surface of the sensing interface, forming a closed microfluidic fluid channel.

[0072] In some embodiments, the microfluidic channel cover 214 may be made of biocompatible and chemically resistant materials, such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or photocurable resin. The material selection can be determined based on the sample liquid properties, sealing requirements, and processing technology, and is not limited by this invention. In other embodiments, a hydrophobic or hydrophilic layer may be further prepared on the inner surface of the channel to control liquid fluid behavior, reduce non-specific adsorption, and promote the migration of target molecules to the sensing interface. The channel cross-sectional dimensions and length can be customized according to sample volume, flow rate, and residence time requirements. The microfluidic channel cover may be designed as a disposable or limited-use replaceable component, allowing for routine updates and replacements of the microfluidic channel without replacing the ATR crystal.

[0073] In some embodiments, the upper surface (sensing surface) of one or more ATR crystals 213 may be further provided with an ultra-hard, ultra-smooth infrared protective film 221. The infrared protective film 221 is made of sapphire, silicon nitride (Si3N4), diamond-like carbon (DLC), or diamond, and its thickness is between a few nanometers and several hundred nanometers. It is used to protect the crystal from chemical erosion and mechanical wear without affecting the evanescent wave penetration depth. In some embodiments, the infrared protective film 221 may be prepared using chemical vapor deposition (CVD) or atomic layer deposition (ALD) processes to ensure that the film matches the crystal surface profile and has a uniform thickness distribution.

[0074] In some embodiments, the thickness of the infrared protective film 221 should be chosen to balance the protective effect with the impact on the evanescent wave penetration depth. Ultrathin (a few nanometers) protective films have negligible impact on the evanescent wave penetration depth, making them particularly suitable for quantitative analysis applications requiring precise penetration depth. Thicker films, on the order of several hundred nanometers, provide stronger mechanical protection and chemical isolation, suitable for applications where severe wear or chemical corrosion of the crystal surface is expected. Especially for ZnSe and ZnS crystals, which have weak acid and alkali resistance, applying diamond-like carbon (DLC) or silicon nitride (Si3N4) protective films can effectively extend the crystal's lifespan in strongly acidic or alkaline environments. Diamond protective films, being themselves effective infrared transmitting materials, have minimal impact on mid-infrared light transmittance, making them suitable for applications with the most stringent requirements for infrared transmission of the protective film. In some embodiments, the infrared protective film 221 layer can also undergo surface functionalization treatments, such as depositing a hydrophobic or hydrophilic layer, to control the wetting behavior of the sample on the protective film surface and reduce non-specific adsorption. In quantitative analysis applications, if the thickness of the protective film needs to be included in the evanescent field model, the thickness can be used as a correction parameter to calculate the evanescent field intensity distribution, which can further improve the quantitative accuracy of the detection.

[0075] like Figure 9 As shown, this embodiment of the invention provides an overall architecture block diagram of a temperature control subsystem. The temperature control subsystem 400 mainly includes a heat dissipation device 401, a temperature controller 402, a temperature regulator 403, and a temperature sensor 404. The temperature controller 402 is the core controller of the temperature control subsystem, connected to the heat dissipation device 401, with the temperature sensor 404 connected to its left side and the temperature regulator 403 connected to its right side. The temperature sensor 404 is attached to one or more infrared light-transmitting sensors, microfluidic channel covers, or thermally conductive bases, collecting the current temperature in real time and feeding the data back to the temperature controller 402. The temperature regulator 403 can employ a thermoelectric cooler (TEC) or a heating-cooling combination, configured to actively heat or cool the crystal assembly and the microfluidic channel cover. The heat dissipation device 401 is responsible for rapidly dissipating the heat generated by the TEC cooling into the environment to maintain the cooling capacity.

[0076] The temperature controller 402 incorporates a closed-loop feedback control algorithm, comparing the real-time measurement value of the temperature sensor 404 with a preset target temperature. Based on the deviation, it adjusts the operating power of the temperature regulator 403, thereby maintaining the overall temperature of the sensor and the microfluidic channel cover at a preset constant value. In some embodiments, the temperature control algorithm may employ PID control, fuzzy control, or adaptive control, etc. The specific control strategy can be selected according to the measurement dynamics and environmental conditions; this invention is not limited thereto. Precise control of the overall temperature of the sensor and the microfluidic channel cover ensures that common-mode temperature drift in the evanescent differential signals corresponding to the two optical paths is suppressed to the maximum extent. The temperature control subsystem 400 is configured to maintain the overall temperature of the sensor and the microfluidic channel cover at a preset constant value, thereby suppressing spectral distortion caused by sample temperature changes due to ambient temperature fluctuations, refractive index drift of the sensor, and effective optical path changes caused by crystal thermal expansion, thus minimizing baseline thermal drift noise in the differential spectrum.

[0077] like Figure 10 As shown, this embodiment of the invention provides an overall block diagram of a fluid control subsystem. The fluid control subsystem 500 includes a fluid controller and two precision liquid inlet devices serving the sample channel and reference channel, respectively. The sample channel liquid inlet device 501 includes a precision syringe pump 503 and an online degasser, configured to deliver a liquid target substance to the sample channel at a precision flow rate; the reference channel liquid inlet device 502 includes a precision syringe pump and an online degasser 504, configured to deliver a reference liquid to the reference channel at a precision flow rate. The fluid controller 500 is responsible for synchronously controlling the operating status of the precision syringe pumps in both devices, ensuring that the flow rate and pressure of the fluid in the sample channel and reference channel are consistent in real time.

[0078] Furthermore, this invention not only utilizes a temperature control system to suppress refractive index drift, but also extends it to a thermodynamic analysis tool for macromolecules. When analyzing macromolecules such as serum proteins, antibody drugs, or peptides, a linearly programmed temperature rise (e.g., from 20°C to 95°C at a rate of 1°C / min) is performed on one or more infrared transmittance sensors via a temperature control subsystem. During this thermal denaturation process, the unfolding of the macromolecular secondary structure leads to a mid-infrared fingerprint region (particularly 1600-1700 cm⁻¹). - The spectral characteristics of the amide I band (¹) show a significant red shift and morphological changes. By capturing this temperature-spectral two-dimensional data array in real time, the data analysis terminal can accurately determine key physicochemical parameters such as the thermal melting temperature T_m and aggregation initiation temperature T_agg of macromolecules, meeting the high-level needs of biopharmaceutical target screening and formulation stability assessment.

[0079] The online degasser 504 in the sample channel liquid inlet device 501 and the online degasser 504 in the reference channel liquid inlet device 502 are configured to actively remove gases (such as N2, O2, CO2) dissolved in the liquid before the fluid enters the sensing interface microchannel, preventing them from precipitating and forming bubbles on the crystal surface due to temperature or pressure changes. In some embodiments, the two online degassers ensure that the probability of bubble precipitation in both microchannels is minimized simultaneously through synchronous pressure feedback, and that the environments on both sides are consistent.

[0080] In some embodiments, the online degasser can be a vacuum membrane degasser, employing a degassing chamber encapsulated by a highly permeable and hydrophobic microporous membrane (preferably polytetrafluoroethylene PTFE or an amorphous fluoropolymer membrane). Fluid flows through one side of the membrane, while the other side is maintained under negative pressure by a vacuum pump, actively removing dissolved gases from the liquid using the partial pressure difference. In other embodiments, a geometric expansion bubble trap can be integrated into the fluid channel, utilizing the kinetic energy loss caused by a sudden drop in flow velocity and the buoyancy of the bubbles themselves to intercept and accumulate discrete bubbles in the channel within a dead zone at the top of the trapping chamber. Furthermore, a hydrophobic venting membrane or a porous diffusion barrier can be integrated into the fluid channel for automatic online venting of bubbles and mechanical breaking up of larger bubbles, respectively, and, in conjunction with hydrophilic surface treatment, to guide tiny bubbles away from the evanescent wave detection area.

[0081] The fluid control subsystem 500 is also configured to monitor the flow rate and pressure in both channels in real time, and dynamically adjust the pump rate of the two units through the fluid controller 500 to ensure that the flow rate and pressure in the sample channel and the reference channel are consistent in real time. The high consistency of fluid conditions in the two channels can eliminate the interference of crystal micro-deformation caused by flow rate and pressure differences on differential spectroscopy, significantly improving the measurement repeatability under liquid flow conditions. In some embodiments, the fluid control subsystem 500 may also integrate an external pressure sensor or flow sensor to provide more accurate fluid state information for closed-loop feedback control. In other embodiments, an online analysis module may also be integrated into the fluid channel, working in parallel with the spectral sensing module to achieve real-time analysis of physicochemical parameters such as sample temperature, pH, and conductivity, further enriching the system's multi-dimensional measurement capabilities.

[0082] Figure 11This is a flowchart according to an embodiment of the present invention. In some embodiments, the dual-channel differential total internal reflection infrared spectroscopy detection system 10 performs a real-time differential signal processing method 1100. Method 1100 includes step S1101, splitting the laser beam into a first beam and a second beam; step S1102, conveying the target sample and the standard reference material to the upper surface of one or more infrared transmittance sensors through the sample channel and the reference channel, respectively; step S1103, receiving the first beam and the second beam after the evanescent wave interacts with the corresponding material, and outputting the first signal and the second signal, respectively; step S1104, performing synchronous demodulation and differential operation on the first signal and the second signal to extract the absorption spectrum of the target material. In some embodiments, the dual-channel differential total internal reflection infrared spectroscopy detection system 10 firstly drives the modulation circuit 101 to apply high-frequency amplitude modulation (AM) or phase modulation (PM) to the output light intensity of the QCL laser 102, and synchronously outputs a modulation reference signal to the data processing module 350 for subsequent synchronous demodulation. Secondly, the beam splitting coupling module 110 splits the laser beam into a first beam and a second beam with substantially equal power, which are respectively input into the two incident end faces of the total internal reflection sensing system 200.

[0083] The fluid control subsystem 500 delivers the target sample and standard reference material to the upper surface of the sensor through the sample channel and reference channel, respectively, with the flow rates and pressures of the two fluids matched in real time. The first and second beams interact with the corresponding materials in the sample and reference channels within the evanescent field region, respectively, through evanescent wave interactions. After exiting through the exit face, the sample detector 303 and reference detector 304 receive the first and second beams and output the first and second detection signals, respectively. The differential processor 310 in the data processing module 350 uses the modulation reference signal output by the driving modulation circuit 101 to synchronously demodulate the first and second signals, then performs differential operations on the demodulated signals to extract the differential absorption spectrum belonging only to the sample after removing common-mode noise (including laser source relative intensity noise RIN, ambient temperature fluctuations, optical path interference fringes, and other common-mode interference), and inputs it to the data analysis terminal 320.

[0084] Finally, based on the real-time operating status, the system automatically invokes one or more compensation mechanisms from the temperature control subsystem 400, differential gain dynamic equalization, and internal standard feedback tuning to correct the differential spectral baseline in real time, ensuring that the final output differential absorption spectrum has the optimal signal-to-noise ratio, minimal baseline drift, and highest measurement repeatability. In some embodiments, the real-time differential signal processing structure can be implemented by software, hardware, or a combination thereof. The specific implementation method can be selected by those skilled in the art as needed, and this invention is not limited thereto.

[0085] Example 1: Online detection of trace organic pollutants in water. In this embodiment, the dual-channel differential total internal reflection infrared spectroscopy detection system described in this invention is applied to the online continuous monitoring of trace pesticides (such as glyphosate and diuron) and endocrine disruptors (such as bisphenol A) in surface water or industrial wastewater. The ATR crystal is a single piece of germanium (Ge) crystal (5-times total internal reflection), which has a short evanescent field penetration depth (approximately 0.65 μm) against the strong infrared absorption background of water, effectively reducing the interference of the water background on the target absorption peak. Blank ultrapure water filtered through a membrane is injected into the reference channel, and the water sample to be tested is injected into the sample channel. The flow rates of the two liquids are synchronously controlled at 2.0 μL / min by the fluid control subsystem to ensure that the fluid conditions of the two channels are completely consistent. The driving modulation circuit applies 100 kHz amplitude modulation to the QCL laser 102, and the lock-in amplifier in the data processing module demodulates synchronously at this frequency, suppressing the broadband background noise to the Johnson noise level. After differential processing, the water background absorption, acting as a common-mode signal, is canceled out, leaving only the characteristic infrared absorption differential spectrum of the target pollutant. Laboratory evaluation of glyphosate indicates that at 1020 cm⁻¹... - ¹(P O At the characteristic peak of C-stretching vibration, the system's lowest detection limit (LOD) can reach below 50 ppb, which is about an order of magnitude better than the traditional single-channel FTIR-ATR system.

[0086] Example 2: Label-free quantitative detection of glucose concentration in serum. In this embodiment, the system described in this invention is used for real-time label-free quantitative analysis of glucose concentration in human serum, with the target application scenario being point-of-care testing (POCT). The ATR crystal is a single-piece multi-internal reflection crystal (10 times total internal reflection) made of diamond material. Its chemical inertness and high hardness allow it to directly contact serum without a protective film and withstand repeated cleaning. The serum sample to be tested (after centrifugation to remove cellular components) flows into the sample channel, and isotonic phosphate buffer (PBS) flows into the reference channel as a baseline reference. The flow rates of both channels are synchronously set to 1.0 μL / min. The temperature control subsystem maintains the overall module temperature stably at 37.0 ± 0.05 °C to eliminate the spectral distortion of protein and lipid components in serum caused by temperature fluctuations, which affects the glucose characteristic peak (1030). 1080 cm - ¹, C O The interference of C-stretching vibration. After extracting the differential absorption spectrum, the data processing module uses a multivariate correction model established by partial least squares (PLS) to quantitatively regress glucose concentration. In 2.0 Within the clinical concentration range of 30.0 mmol / L, the root mean square error of prediction (RMSEP) is better than 0.4 mmol / L, meeting the accuracy requirements of ISO 15197 for blood glucose monitoring systems. At the same time, the absorption differences of coexisting components in serum such as albumin and triglycerides are significantly suppressed through the differential mechanism, improving quantitative selectivity.

[0087] Example 3: protein Real-time kinetic characterization of small molecule interactions. In this embodiment, the system described in this invention is used to monitor the binding and dissociation processes between target proteins and candidate drug molecules, enabling label-free real-time kinetic (kon, koff) and affinity constant (KD) measurements, suitable for early screening in the drug discovery stage. The ATR crystal is made of ZnSe, and the evanescent field region on the crystal surface is pre-modified with a self-assembled monolayer (SAM) to covalently and directionally immobilize the target protein (such as a kinase or G protein-coupled receptor extracellular domain fragment) at the sensing interface. The reference channel corresponds to a crystal region immobilized with a control protein (BSA) to eliminate non-specific adsorption background. The system is first filled with blank running buffer (PBS, pH 7.4) to establish a stable differential baseline between the two channels; then, candidate small molecule solutions of different concentration gradients (e.g., 1 nM to 10 μM) are injected into the sample channel, while the reference channel continues to receive running buffer. In the differential absorption signal curve (sensing map) after phase-locked demodulation, the increase in the intensity of the binding phase signal corresponds to the local refractive index and infrared absorption cross-section changes caused by the enrichment of small molecules at the sensing interface and their binding to the target protein; the signal decay trajectory of the dissociation phase (injected pure buffer) is used to extract the dissociation rate constant koff. By globally fitting the multi-concentration sensing maps (Langmuir 1:1 kinetic model), both kon and KD values ​​can be obtained simultaneously. After combining the SEIRA nanoantenna array enhancement structure, the system also has sufficient sensitivity to weakly interacting systems with KD down to the nanomolar level, providing a complementary alternative to traditional surface plasmon resonance (SPR) technology that also provides molecular structure fingerprint information.

Claims

1. A dual-channel differential total internal reflection infrared spectroscopy detection system, characterized in that, The detection system includes: The light source modulation module is configured to generate a mid- to far-infrared laser beam with continuously tunable wavelength; The beam splitting coupling module is configured to split the mid- and far-infrared laser beam into two beams with substantially equal power to form a first beam and a second beam. A total internal reflection sensing system, the total internal reflection sensing system including one or more infrared transmittance sensors, the one or more infrared transmittance sensors having an incident end face, an exit end face and an upper surface as a sensing interface; The one or more infrared light-transmitting sensors receive the incident first beam and second beam, generate one or more total internal reflections inside the one or more infrared light-transmitting sensors, and output from the output end face in a spatially symmetrical and non-interfering light path. The upper surface of the one or more infrared transmittance sensors is provided with a sample channel and a reference channel. The sample channel and the reference channel are independent and non-contacting channel spaces, and respectively cover the total internal reflection area generated by the first beam and the second beam at the sensing interface. The evanescent waves generated by the first and second beams at the sensing interface interact with the target material and the standard reference material in the corresponding channels, respectively. The detection module includes a first photodetector and a second photodetector, which are respectively used to receive a first light beam emitted from the output end face that has interacted with the target material through evanescent waves and a second light beam that has interacted with a standard reference material, and respectively generate a first detection signal and a second detection signal. A data processing module, connected to the detection module, is configured to receive and process the first and second detection signals to extract the spectral signal of the target substance.

2. The system according to claim 1, characterized in that, The one or more infrared transmittance sensors include a single, integrally formed ATR crystal that simultaneously guides the first beam and the second beam. Alternatively, multiple discrete infrared-transmitting ATR crystals may be used, each corresponding to the transmission path of the first beam and the second beam, respectively.

3. The system according to claim 2, characterized in that, The one or more infrared transmittance sensors include a holder made of metal or polymer, and a plurality of discrete infrared transmittance ATR crystals embedded in the holder. Continuous transmission of light beams is achieved between adjacent ATR crystals through optical coupling or spatial alignment, thereby creating multiple cumulative total internal reflections along the total length of the sensing path.

4. The system according to claim 2, characterized in that, The upper surface of the ATR crystal includes a surface-enhanced infrared absorption structure; The surface-enhanced infrared absorption structure includes a nano-metal antenna array or metasurface fabricated at specific locations on the upper surface where it interacts with the evanescent wave. The design of the nano-metal antenna resonates with the wavelength of the incident laser and the vibrational frequency of the target molecules, thereby increasing the interaction cross-section between infrared light and the target material through the local electromagnetic field enhancement effect.

5. The system according to claim 1, characterized in that, The light source module includes: Single-mode, collimated output quantum cascade laser or interband cascade laser; The light source modulation module is configured as follows: Adjust the polarization states of the first and second beams to make them exhibit p-polarization or TM mode, so that the electric field vector of the evanescent wave is perpendicular to the sensing interface. The first and second beams are collimated and shaped so that their wavelength tuning range covers the characteristic fingerprint spectral region of the target substance or target biomass molecules.

6. The system according to claim 1, characterized in that: Both the incident and exit surfaces are coated with broadband infrared antireflection films, configured to minimize Fresnel reflection loss when the laser enters and exits the total internal reflection sensing system, and suppress parasitic interference fringes caused by end-face reflection.

7. The system according to claim 1, characterized in that, The one or more infrared transmittance sensors include one or more ATR crystals: The ATR crystal is made of diamond, zinc selenide, zinc sulfide, germanium or silicon, and its surface is polished with high precision to meet the surface smoothness requirements of total internal reflection.

8. The system according to claim 7, characterized in that, The sensing surface of the ATR crystal includes an ultra-hard, ultra-smooth infrared protective film. The infrared protective film is made of sapphire, silicon nitride, diamond-like carbon, or diamond, and its thickness ranges from a few nanometers to several hundred nanometers.

9. The system according to claim 1, characterized in that: The region in which the evanescent wave interacts with the target material or standard reference material in the corresponding channel is the evanescent field region. The system includes: a capture probe specifically designed to recognize biomolecules, prepared within the evanescent field region; The capture probe includes antibodies, antigens, nucleic acid aptamers, or molecularly imprinted polymers; The capture probe is used to specifically capture and enrich trace amounts of target molecules from flowing liquid or solid samples to the crystal surface to achieve highly sensitive immunoinfrared spectroscopy detection.

10. The system according to claim 1, characterized in that, The total internal reflection sensing system also includes a temperature control subsystem: The temperature control subsystem includes a thermoelectric cooler, a temperature sensor attached to one or more infrared light-transmitting sensors, a microfluidic channel cover or a thermally conductive base, and a closed-loop feedback control circuit. The temperature control subsystem is configured to maintain the overall temperature of the total internal reflection sensing system and the microfluidic channel cover plate at a preset constant value. The system controls the temperature of the total internal reflection sensing system and the microfluidic channel cover to suppress spectral distortion caused by sample temperature changes due to ambient temperature fluctuations, refractive index drift of the one or more infrared transmittance sensors, and effective optical path changes caused by crystal thermal expansion, thereby minimizing baseline thermal drift noise in the differential spectrum.

11. The system according to claim 10, characterized in that: The temperature control subsystem is also configured to perform programmable temperature ramp-up or temperature ramp-down scans; the data processing module is configured to continuously acquire the differential infrared spectrum of the sample during the temperature scan, and monitor the changes in the secondary structure of macromolecules in real time by tracking the peak position shift, peak width change or intensity attenuation of specific infrared characteristic peaks, thereby calculating the thermodynamic property parameters of the sample, including the thermal melting temperature T_m and the aggregation initiation temperature T_agg.

12. The system according to claim 1, characterized in that, Also includes: A microfluidic channel cover plate is inverted on the upper surface of one or more infrared light-transmitting sensors. The lower surface of the microfluidic channel cover plate without engraved microfluidic channels is sealed and fitted to the upper surface of the sensing interface. The engraved microfluidic channels form at least two physically isolated microfluidic channels as the sample channel and the reference channel. The position of the microfluidic channels overlaps with the position of the evanescent wave formed on the upper surface of the sensing interface. A fluid system comprising a sample pump and a precision flow rate control module, configured to ensure that the fluid flow rate and pressure in the sample channel and the reference channel remain consistent in real time, so as to eliminate the interference of crystal micro-deformation caused by flow rate and pressure difference on the two differential spectra.

13. The system of claim 12, further comprising at least one of the following online degassing machines or bubble traps integrated upstream of the fluid passage of the fluid system: When an online degasser is integrated, the online degasser includes a vacuum membrane degasser, wherein, The vacuum degasser employs a degassing chamber encapsulated with a microporous membrane that is highly permeable and hydrophobic. Fluid flows through one side of the membrane, while the other side of the membrane is maintained under negative pressure by a vacuum pump. The gas dissolved in the liquid is actively removed by utilizing the partial pressure difference, preventing it from precipitating on the crystal surface and forming bubbles due to temperature or pressure changes. When integrated with a bubble trapping device, the bubble trapping device includes one or more of a geometric expansion bubble trap, a hydrophobic venting membrane, or a porous diffusion barrier, wherein... When the bubble trapping device uses a geometric expansion bubble trap: before entering the microchannel on the upper surface of the ATR crystal, a trapping cavity with a suddenly expanded cross-sectional area is provided in the geometric expansion bubble trap; by utilizing the kinetic energy loss caused by the sudden drop in flow velocity and the buoyancy characteristics of the bubble itself, the discrete bubbles in the flow channel are intercepted and gathered in the dead zone at the top of the trapping cavity; When the bubble trapping device employs a hydrophobic venting membrane, a T-shaped or Y-shaped branch is integrated at the top or corner of the fluid channel. The open end of this branch is covered with a hydrophobic venting membrane that allows only gas to pass through while preventing liquid penetration, thus achieving online automatic bubble discharge; and, When the bubble trapping device uses a porous diffusion barrier: a set of micron-sized column arrays or porous sintered metal blocks are set at the inlet of the flow channel to form a porous diffusion barrier. The porous diffusion barrier is used to mechanically break up larger bubbles, and a hydrophilic surface treatment is used to guide tiny bubbles away from the evanescent wave detection area.

14. The system according to claim 1, characterized in that, The light source modulation module is configured to perform amplitude modulation or phase modulation on the first beam and the second beam. The data processing module includes a lock-in amplifier configured to demodulate the output signals of the first photodetector and the second photodetector using a reference signal synchronized with the modulation frequency of the light source, so as to suppress noise and improve the signal-to-noise ratio.

15. The system according to claim 14, characterized in that: The detection module is configured to continuously record the signal channel spectrum and reference channel spectrum of the first photodetector and the second photodetector after demodulation by the lock-in amplifier during the simultaneous injection of background buffer into the sample channel and reference channel on the lower surface of the microfluidic channel cover plate. The data processing module is configured to dynamically adjust the gain parameter of either of the two spectra to minimize the difference between the spectral signals of the two channels, thereby achieving dynamic gain balance of the differential demodulation system and compensating for spectral baseline drift caused by factors such as crystal surface contamination, system aging, or environmental changes.

16. The system according to claim 1, characterized in that, The ATR sensing system is packaged as a detachable and replaceable independent module; The independent module integrates an RFID or NFC electronic tag chip, and the tag stores the factory calibration parameters of the specific crystal. The electronic tag is configured to allow external systems to read the tag data, automatically perform optical path normalization correction on the measurement results, and record the usage status of the independent module.

17. The system according to claim 1, characterized in that, The target substance includes the target liquid and an infrared inert internal standard liquid of known concentration mixed in; The data processing module is configured to monitor the position of the characteristic peak of the infrared inert internal standard substance in the spectral scanning window, provide real-time feedback and adjust the wavelength of the mid- and far-infrared laser beam, and compensate for spectral wavelength drift caused by changes in the viscosity of the target substance, flow rate fluctuations or crystal surface contamination.

18. The system according to any one of claims 1 to 17, configured as follows: The laser beam is split into the first beam and the second beam; The target sample and the standard reference material are respectively delivered to the upper surface of the one or more infrared transmittance sensors through the sample channel and the reference channel; The system receives the first beam and the second beam after the evanescent wave interacts with the corresponding matter, and outputs the first signal and the second signal, respectively. The first and second signals are simultaneously demodulated and differentially processed to extract the absorption spectrum of the target substance.

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