Trehalose and its dihydrate: terahertz insights from solid to solution states

Carbohydrates are pivotal biomolecules in biochemistry; this study employs terahertz time-domain spectroscopy (THz-TDS) to investigate the spectral characteristics of trehalose and its hydrate across the 0.1 to 2.2 THz frequency range. Notable differences in spectra between the two compounds were observed. Density Functional Theory (DFT) simulations of the crystal structure were conducted to elucidate this phenomenon. The consistency between experimental results and simulations substantiates the reliability of the experimental findings. Additionally, the spectral characteristics of these carbohydrates in solution were examined using microfluidic chip technology. This approach facilitates a comprehensive comparison of their behaviors in both solid and solution states.


Introduction
Terahertz (THz) radiation, also often called far infrared radiation, occupies a special area in the electromagnetic spectrum.Its frequency range is between 0.1 and 10 THz, corresponding to the wavelength range of 3000 to 30 microns [1][2][3].In recent years, with the rapid development of THz detection technology, the application of THz spectroscopy technology in biochemical sample analysis has become a key tool for biochemical research.Among them, an important characteristic of THz radiation is that its photon energy in THz band is extremely low, only 0.4 meV, which is highly consistent with the rotation and vibration energy levels of many organic compounds and biological macromolecules.Therefore, THz spectroscopy can provide unique absorption spectra of these substances, which is helpful for nondestructive quantitative and qualitative analysis.It is worth mentioning that THz radiation can penetrate most nonmetallic materials and biological tissues without causing ionization, thus ensuring the safety of biological tissues.This characteristic makes it show great application potential in nondestructive testing and medical imaging.In addition, many molecules, especially macromolecules and biomolecules, exhibit unique absorption and scattering modes at THz frequency, which provides a new method for the detection and characterization of chemical and biological substances [4][5][6].
Trehalose, a naturally occurring carbohydrate primarily sourced from brown algae, possesses distinctive chemical attributes that set it apart from common monosaccharides like glucose and fructose [7,8].Trehalose exists in two primary forms: anhydrous trehalose and trehalose dihydrate, each distinguished by their structural and physical properties, and finding unique applications in diverse fields such as biology, medicine, and industry.Anhydrous trehalose represents the dehydrated variant of trehalose, and its chemical structure lacks the hydrogen bonds associated with water molecules in its crystal structure, imparting it with exceptional physical and chemical characteristics.For instance, anhydrous trehalose exhibits superior stability compared to other forms.In the realm of biology, anhydrous trehalose assumes a critical role as a stabilizer, contributing significantly to the crystallization and preservation of proteins.It effectively preserves the original protein structure, thereby enhancing sample stability.In the food industry, it serves as a natural sweetener and stabilizer due to its outstanding thermal stability and resistance to acidity [9][10][11].Trehalose dihydrate, on the other hand, incorporates two crystalline water molecules that form hydrogen bonds with trehalose, resulting in unique physical and chemical properties.In pharmaceutical preparations, it frequently functions as an adjuvant, particularly in freeze-dried biological products, to maintain the stability of active ingredients and optimize product quality and shelf life.We choose anhydrous trehalose and trehalose dihydrate as the research objects firstly because of their high application value.Secondly, taking trehalose as a sample, we aim to explore the correlation of THz spectra of different forms of the same carbohydrate.Given the central role of carbohydrate molecules in biological systems and their widespread practical applications, comprehending their fundamental properties, including spectral characteristics, is of paramount importance [12].Previous research, such as the work conducted by Upadhya and colleagues [13], involved measuring the high-resolution absorption spectrum of polycrystalline carbohydrates in the 0.1-3.0THz frequency range, unveiling the vibration characteristics of absorption peaks through simulation.Additionally, studies by Takeuchi and others [14] aimed to estimate the crystallinity of trehalose dihydrate microspheres by measuring THz spectra, Masae [15] measured and analyzed the low frequency vibration spectrum of α-trehalose dihydrate, and Huang [16] used THz technology to quantitatively detect the purity of trehalose.At present, most of the research focuses on the absorption characteristics of solid carbohydrates, while the research on carbohydrate solutions is relatively few.In natural biological systems, carbohydrate molecules mostly exist in liquid environment, especially water, a common solvent.Because of its special hydrogen bond structure, water molecules show remarkable absorption characteristics in THz frequency band, which is especially critical in the study of carbohydrate solutions.Considering the key role of carbohydrates in organisms and their ubiquitous existence in water environment, it is of great significance to study their absorption characteristics and characteristic fingerprints in liquid environment, especially the comparative analysis of THz absorption spectra and vibration modes of carbohydrates in solid and solution States, which may reveal the unique behavior and interaction mechanism of these molecules in different physical States and understand the complex molecular interaction and metabolic process in organisms.
The main challenge in this research involves the analysis of liquid biological samples using traditional technology, hindered by significant water absorption in the THz frequency band [17].To address this limitation, microfluidic chip technology has been employed.This innovative approach enables precise control and manipulation of fluids at a microscale, intersecting the fields of chemistry, fluid mechanics, microelectronics, and novel materials [18].Cycloolefin copolymer (COC) was selected as the substrate for the chip due to its high THz wave transmittance.Utilizing COC, microfluidic chips with exceptional transparency were fabricated, enhancing the efficient use of COC materials and reducing sample consumption.Furthermore, these uniquely designed chips demonstrated superior sealing performance during experimentation, and their cleanliness and reusability contributed to the convenience of the experimental setup.
In this research, anhydrous trehalose and its dihydrate were chosen as subjects, both significant in food and pharmaceutical industries.The study began with measuring the THz spectra in the solid state and conducting molecular dynamics simulations, where the experimental data closely matched the theoretical results.Subsequently, a new microfluidic chip was employed to ascertain the THz spectra of these carbohydrates in a solution environment.The research then delved into examining how various concentrations and temperatures affect the THz spectral intensity, including a comparative analysis of their absorption spectra in both solid and solution states.Ultimately, this study provides an analysis of THz spectral properties of anhydrous trehalose and its dihydrate, and clarifies the influence of environment on its molecular vibration mode.

Samples
Anhydrous trehalose, provided by Nanjing Quanlong Biotechnology Co., Ltd., was used without additional purification.Distilled water, with a resistivity of 18.2 Ω•cm, served as the solvent.We prepared five distinct concentrations of anhydrous trehalose solutions, ranging from 0 to 1000 mg/mL.Similarly, trehalose dihydrate, also sourced from Nanjing Quanlong Biotechnology Co., Ltd., underwent no further purification.Solutions of trehalose dihydrate were prepared in the same concentration spectrum, from 0 to 1000 mg/mL.The structural details of the samples are depicted in Fig. 1.
delved into examining how various concentrations and temperatures affect the THz spectral intensity, including a comparative analysis of their absorption spectra in both solid and solution states.Ultimately, this study provides an analysis of THz spectral properties of anhydrous trehalose and its dihydrate, and clarifies the influence of environment on its molecular vibration mode.

Samples
Anhydrous trehalose, provided by Nanjing Quanlong Biotechnology Co., Ltd., was used without additional purification.Distilled water, with a resistivity of 18.2 Ω•cm, served as the solvent.We prepared five distinct concentrations of anhydrous trehalose solutions, ranging from 0 to 1000 mg/mL.Similarly, trehalose dihydrate, also sourced from Nanjing Quanlong Biotechnology Co., Ltd., underwent no further purification.Solutions of trehalose dihydrate were prepared in the same concentration spectrum, from 0 to 1000 mg/mL.The structural details of the samples are depicted in Fig. 1.Polyethylene, a white powder procured from Sigma Aldrich Trading Co, Ltd., exhibits nearcomplete transparency to THz waves.Its THz spectrum is shown in the Fig. 2. For sample preparation, 100 mg each of anhydrous trehalose and trehalose dihydrate were finely ground and blended with polyethylene in a 1:5 ratio.Using a pressure of 5 tons, we fashioned samples with diameters of 1 cm, and thicknesses of 1.39 mm and 1.27 mm, respectively.Polyethylene, a white powder procured from Sigma Aldrich Trading Co, Ltd., exhibits nearcomplete transparency to THz waves.Its THz spectrum is shown in the Fig. 2. For sample preparation, 100 mg each of anhydrous trehalose and trehalose dihydrate were finely ground and blended with polyethylene in a 1:5 ratio.Using a pressure of 5 tons, we fashioned samples with diameters of 1 cm, and thicknesses of 1.39 mm and 1.27 mm, respectively.
delved into examining how various concentrations and temperatures affect the THz spectral intensity, including a comparative analysis of their absorption spectra in both solid and solution states.Ultimately, this study provides an analysis of THz spectral properties of anhydrous trehalose and its dihydrate, and clarifies the influence of environment on its molecular vibration mode.

Samples
Anhydrous trehalose, provided by Nanjing Quanlong Biotechnology Co., Ltd., was used without additional purification.Distilled water, with a resistivity of 18.2 Ω•cm, served as the solvent.We prepared five distinct concentrations of anhydrous trehalose solutions, ranging from 0 to 1000 mg/mL.Similarly, trehalose dihydrate, also sourced from Nanjing Quanlong Biotechnology Co., Ltd., underwent no further purification.Solutions of trehalose dihydrate were prepared in the same concentration spectrum, from 0 to 1000 mg/mL.The structural details of the samples are depicted in Fig. 1.Polyethylene, a white powder procured from Sigma Aldrich Trading Co, Ltd., exhibits nearcomplete transparency to THz waves.Its THz spectrum is shown in the Fig. 2. For sample preparation, 100 mg each of anhydrous trehalose and trehalose dihydrate were finely ground and blended with polyethylene in a 1:5 ratio.Using a pressure of 5 tons, we fashioned samples with diameters of 1 cm, and thicknesses of 1.39 mm and 1.27 mm, respectively.

THz-TDS system
In this study, the employed THz-TDS system is a transmission type, comprising a MaiTai femtosecond laser, a chopper, and a THz wave generation and detection apparatus, as depicted in Fig. 3.The MaiTai femtosecond laser, a titanium sapphire laser, features a wavelength range of 700-1200 nm, with a central wavelength of 800 nm, a repetition rate of 82 MHz, and an output power of 3.4 W. The laser pulses, modulated by the C-995 chopper for synchronization with the lock-in amplifier, are focused onto an InAs crystal using a lens and an electric translation stage.This crystal emits THz pulses induced by semiconductor surface radiation.These pulses, after being collimated and focused on the sample by a parabolic mirror, are then re-collimated by another parabolic mirror into a ZnTe crystal.Concurrently, a femtosecond probe beam reaches the ZnTe crystal, altering the polarization state of the probe beam through electro-optic effect modulation.The altered beam, after passing through a Wollaston prism, undergoes detection by a differential detector.The resulting weak current is fed into a lock-in amplifier for amplification, shaping, and phase adjustment, effectively enhancing the signal and suppressing incoherent noise.The signal-to-noise ratio thus achieved exceeds 800, within an effective spectral range of 0.2-2.2THz.The system employs a scanning step size of 1 µm, a scan length of 3 cm, resulting in a frequency domain resolution of 37 GHz and a time domain resolution of 66 fs.The relative optical path difference between the pump and probe beams is finely tuned using a linear translation stage, allowing for the acquisition of the time-domain waveform of the THz pulse through point-by-point scanning.

THz-TDS system
In this study, the employed THz-TDS system is a transmission type, comprising a MaiTai femtosecond laser, a chopper, and a THz wave generation and detection apparatus, as depicted in Fig. 3.The MaiTai femtosecond laser, a titanium sapphire laser, features a wavelength range of 700-1200 nm, with a central wavelength of 800 nm, a repetition rate of 82 MHz, and an output power of 3.4 W. The laser pulses, modulated by the C-995 chopper for synchronization with the lock-in amplifier, are focused onto an InAs crystal using a lens and an electric translation stage.This crystal emits THz pulses induced by semiconductor surface radiation.These pulses, after being collimated and focused on the sample by a parabolic mirror, are then re-collimated by another parabolic mirror into a ZnTe crystal.Concurrently, a femtosecond probe beam reaches the ZnTe crystal, altering the polarization state of the probe beam through electro-optic effect modulation.The altered beam, after passing through a Wollaston prism, undergoes detection by a differential detector.The resulting weak current is fed into a lock-in amplifier for amplification, shaping, and phase adjustment, effectively enhancing the signal and suppressing incoherent noise.The signal-to-noise ratio thus achieved exceeds 800, within an effective spectral range of 0.2-2.2THz.The system employs a scanning step size of 1 μm, a scan length of 3 cm, resulting in a frequency domain resolution of 37 GHz and a time domain resolution of 66 fs.The relative optical path difference between the pump and probe beams is finely tuned using a linear translation stage, allowing for the acquisition of the time-domain waveform of the THz pulse through point-by-point scanning.

Microfluidic Chip Device
This research utilized a microfluidic chip, as shown in Fig. 4, to mitigate the impact of liquid samples on THz absorption and ensure precision in results.COC chosen for its high average transmittance of 95% in the THz range and transparency to visible light, is particularly suited

Microfluidic chip device
This research utilized a microfluidic chip, as shown in Fig. 4, to mitigate the impact of liquid samples on THz absorption and ensure precision in results.COC chosen for its high average transmittance of 95% in the THz range and transparency to visible light, is particularly suited for microfluidic chip fabrication.It notably lacks absorption peaks within 2.2 THz.The chip comprises two COC sheets: one as the base and the other as the covering layer, with a 50-micrometer-thick double-sided adhesive tape serving as the spacer.This design not only reduces the THz absorption by water by managing the liquid layer's thickness but also minimizes Fabry-Perot interference by avoiding overly thin configurations.A channel, created by removing part of the adhesive tape, measures 2 cm in length and 1 cm in width.The base and cover are then bonded, with two 1 mm radius holes on the cover serving as inlet and outlet.This design effectively diminishes water's high absorption of THz waves in solvents, offering an economical, efficient, and eco-friendly approach.
effectively diminishes water's high absorption of THz waves in solvents, offering an economical, efficient, and eco-friendly approach.The temperature control system comprises the microfluidic chip and a temperature sensor.An annular heating plate made of alumina ceramics, with a 40 mm outer diameter and a 20 mm inner diameter, is mounted beside the chip, allowing unobstructed THz wave transmission through the liquid.The temperature sensor is connected on one side to the chip via heatconductive silica gel and on the other to a temperature controller (ST700 intelligent proportional integral temperature controller with a 220 V rating, a 0-200 ℃ range, and 0.1 ℃accuracy) via wires.In operation, the controller adjusts the temperature, modulating the rise and fall to maintain a constant value near the target temperature.The temperature sensor provides realtime feedback of the microfluidic chip's temperature, enabling indirect and qualitative assessment of the sample's temperature variations.

Calculation method
In this experiment, to mitigate the effects of Fabry-Perot oscillation, we adopted the flat-plate medium model grounded in the Fresnel formula, as proposed by Dorney, for data processing.The focus of our study was on employing a physical model for the THz optical parameters of the extracted substance.This model was instrumental in determining the sample's absorption coefficient [19,20].The model performs Fourier transform on the THz time-domain waveforms of the reference and transmitted samples to obtain the amplitude and phase information, and it calculates the refractive index of the sample with the known sample thickness d.
where  is the circular frequency, c is the speed of light in vacuum, and ( )   is the phase difference, which is calculated further to obtain the absorption coefficient.
where ( )   is the amplitude ratio of the sample and the reference.The temperature control system comprises the microfluidic chip and a temperature sensor.An annular heating plate made of alumina ceramics, with a 40 mm outer diameter and a 20 mm inner diameter, is mounted beside the chip, allowing unobstructed THz wave transmission through the liquid.The temperature sensor is connected on one side to the chip via heat-conductive silica gel and on the other to a temperature controller (ST700 intelligent proportional integral temperature controller with a 220 V rating, a 0-200 °C range, and 0.1 °Caccuracy) via wires.In operation, the controller adjusts the temperature, modulating the rise and fall to maintain a constant value near the target temperature.The temperature sensor provides real-time feedback of the microfluidic chip's temperature, enabling indirect and qualitative assessment of the sample's temperature variations.

Calculation method
In this experiment, to mitigate the effects of Fabry-Perot oscillation, we adopted the flat-plate medium model grounded in the Fresnel formula, as proposed by Dorney, for data processing.The focus of our study was on employing a physical model for the THz optical parameters of the extracted substance.This model was instrumental in determining the sample's absorption coefficient [19,20].The model performs Fourier transform on the THz time-domain waveforms of the reference and transmitted samples to obtain the amplitude and phase information, and it calculates the refractive index of the sample with the known sample thickness d.
where ω is the circular frequency, c is the speed of light in vacuum, and ϕ(ω) is the phase difference, which is calculated further to obtain the absorption coefficient.
where ρ(ω) is the amplitude ratio of the sample and the reference.

THz spectra of two solid carbohydrates
Fast Fourier transform analysis was utilized to produce frequency domain spectra, refractive index spectra, and absorption coefficient spectra.As depicted in Fig. 5

THz spectra of two solid carbohydrates
Fast Fourier transform analysis was utilized to produce frequency domain spectra, refractive index spectra, and absorption coefficient spectra.As depicted in Fig. 5

Simulation of two solid carbohydrates
To facilitate a more quantitative analysis of the data gathered, PXRD (Powder X-ray Diffraction) experiments were conducted on the samples, complemented by simulations of the crystal structure.Figure 6 presents both the experimentally measured and simulated PXRD spectra of the samples.The consistency between the peaks observed in the experimental results and the theoretical calculation values confirms the effectiveness of the crystal structure model.

Simulation of two solid carbohydrates
To facilitate a more quantitative analysis of the data gathered, PXRD (Powder X-ray Diffraction) experiments were conducted on the samples, complemented by simulations of the crystal structure.Figure 6 presents both the experimentally measured and simulated PXRD spectra of the samples.The consistency between the peaks observed in the experimental results and the theoretical calculation values confirms the effectiveness of the crystal structure model.
To explore deeper into the spectral characteristics of anhydrous trehalose and trehalose dihydrate and to elucidate the complex interplay between THz spectra and molecular vibrations, we utilized Density Functional Theory (DFT).This approach enabled us to optimize the geometric structures and calculate the vibrational modes of these molecules [21].The computational process entailed two primary stages: the geometric optimization followed by energy optimization, employing the To explore deeper into the spectral characteristics of anhydrous trehalose and trehalose dihydrate and to elucidate the complex interplay between THz spectra and molecular vibrations, we utilized Density Functional Theory (DFT).This approach enabled us to optimize the geometric structures and calculate the vibrational modes of these molecules [21].The computational process entailed two primary stages: the geometric optimization followed by energy optimization, employing the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation (GGA) [22].Figure 7(a) depicts the crystal structure of anhydrous trehalose.Our simulations revealed that the absorption peaks of solid anhydrous trehalose closely match the experimental data, occurring at frequencies of 0.66, 1.25, 1.70, and 1.97 THz, as shown in Fig. 7(b).Additionally, the calculations did not yield any negative frequencies, indicating successful attainment of the minimum energy structure in the geometric optimization.The vibrational modes and crystal structure of anhydrous trehalose are detailed in Fig. 7(c-f).According to our analyses, the absorption peaks primarily originate from the vibrations of -CHOH, -CH 2 OH, and -OH groups, along with collective molecular vibrations or intermolecular interactions.It is essential to note that intermolecular interactions, particularly hydrogen bonding, can significantly influence THz absorption peaks.This can lead to minor discrepancies between the simulated and experimental absorption peaks.For example, a slight deviation is observed between the simulated peak at 1.97 THz and its experimental counterpart at 1.96 THz, showing the intricacies of the relationship between molecular vibrations and THz absorption in anhydrous trehalose.Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation (GGA) [22].Figure 7(a) depicts the crystal structure of anhydrous trehalose.Our simulations revealed that the absorption peaks of solid anhydrous trehalose closely match the experimental data, occurring at frequencies of 0.66, 1.25, 1.70, and 1.97 THz, as shown in Fig. 7(b).Additionally, the calculations did not yield any negative frequencies, indicating successful attainment of the minimum energy structure in the geometric optimization.The vibrational modes and crystal structure of anhydrous trehalose are detailed in Fig. 7(c-f).According to our analyses, the absorption peaks primarily originate from the vibrations of -CHOH, -CH 2 OH, and -OH groups, along with collective molecular vibrations or intermolecular interactions.It is essential to note that intermolecular interactions, particularly hydrogen bonding, can significantly influence THz absorption peaks.This can lead Figure 8(a) illustrates the crystal structure of trehalose dihydrate.Through computational simulations, we ascertained the absorption peaks of solid trehalose dihydrate at frequencies of 1.12, 1.50, 1.75, and 2.03 THz, as shown in Fig. 8(b).These peaks show a strong correlation with the experimental findings, notably with the simulated peak at 1.50 THz corresponding closely to the experimentally observed peak at 1.42 THz.The other frequencies, 1.12, 1.75, and 2.03 THz, match exactly with their experimental counterparts, affirming their status as distinctive characteristics of trehalose dihydrate.The vibrational modes linked to these peaks are detailed in Fig. 8(c-f).On comparing trehalose dihydrate with anhydrous trehalose, it is  Several factors contribute to the differences between experimental and simulation results in this study.First, the computational models are based on an idealized crystal structure, which may not accurately reflect the complexities encountered in experimental settings.In practice, replicating an ideal crystal structure is often a challenging endeavor.Second, the absorption peaks of THz radiation are significantly affected by intermolecular interactions, especially hydrogen bonding.While these interactions are considered in simulations, they may not be completely or accurately represented, leading to minor differences in the absorption peak positions between simulated and experimental results.In summary, the variations between experimental and simulation outcomes arise from the idealization of the crystal structure and the intricate nature of intermolecular interactions.Table 1 provides a comprehensive summary of the vibration properties of anhydrous trehalose and trehalose dihydrate in the THz frequency range, so that the unique vibration modes of these carbohydrates in the THz spectrum can be more clearly understood.to minor discrepancies between the simulated and experimental absorption peaks.For example, a slight deviation is observed between the simulated peak at 1.97 THz and its experimental counterpart at 1.96 THz, showing the intricacies of the relationship between molecular vibrations and THz absorption in anhydrous trehalose.Figure 8(a) illustrates the crystal structure of trehalose dihydrate.Through computational simulations, we ascertained the absorption peaks of solid trehalose dihydrate at frequencies of 1.12, 1.50, 1.75, and 2.03 THz, as shown in Fig. 8(b).These peaks show a strong correlation with the experimental findings, notably with the simulated peak at 1.50 THz corresponding closely to the experimentally observed peak at 1.42 THz.The other frequencies, 1.12, 1.75, and 2.03 THz, match exactly with their experimental counterparts, affirming their status as distinctive characteristics of trehalose dihydrate.The vibrational modes linked to these peaks are detailed in Fig. 8(c-f).On comparing trehalose dihydrate with anhydrous trehalose, it is observed that the vibration mode at 2.03 THz in trehalose dihydrate is similar to the 1.97 THz mode in anhydrous trehalose.This additional peak arises predominantly due to hydration, which amplifies hydrogen bond vibrations and certain intermolecular interactions.This finding shows the significant impact of water molecules on the THz absorption spectrum, leading to unique peaks specific to trehalose dihydrate, thereby differentiating it from its anhydrous counterpart.
Several factors contribute to the differences between experimental and simulation results in this study.First, the computational models are based on an idealized crystal structure, which may not accurately reflect the complexities encountered in experimental settings.In practice, replicating an ideal crystal structure is often a challenging endeavor.Second, the absorption peaks of THz radiation are significantly affected by intermolecular interactions, especially hydrogen bonding.While these interactions are considered in simulations, they may not be completely or accurately represented, leading to minor differences in the absorption peak positions between simulated and experimental results.In summary, the variations between experimental and simulation outcomes arise from the idealization of the crystal structure and the intricate nature of intermolecular interactions.Table 1 provides a comprehensive summary of the vibration properties of anhydrous trehalose and trehalose dihydrate in the THz frequency range, so that the unique vibration modes of these carbohydrates in the THz spectrum can be more clearly understood.

THz spectra of two carbohydrate solutions
Given that carbohydrates predominantly function in biological processes as solutions, and considering water's strong absorption in the THz range, measuring spectra becomes notably

THz Spectra of Two Carbohydrate Solutions
Given that carbohydrates predominantly function in biological processes as solutions, and considering water's strong absorption in the THz range, measuring spectra becomes notably challenging.To address this, our study leverages microfluidic technology to precisely limit the liquid layer to a thickness of 50 μm, effectively reducing water's absorption.We initially investigated the relationship between concentration and THz spectral intensity.Solutions of varying concentrations of anhydrous trehalose and trehalose dihydrate were prepared and sequentially introduced into microfluidic chips for measurement at room temperature.The corresponding THz time-domain and frequency-domain spectra are presented in Fig. 9. Our observations reveal that as the concentration increases, both the time-domain and frequencydomain spectra of the solutions are relatively intensified, indicating a decrease in absorption and an increase in transmittance.This enhancement suggests that the THz absorption spectra of these carbohydrate solutions are influenced by the concentration of molecules present, showing the interplay between molecular density and THz spectral characteristics.Our research subsequently concentrated on the highest concentration levels of both carbohydrates.To obtain the liquid THz absorption spectra, we applied formula 2 for calculating the absorption coefficients of the sample (comprising both solvent and solute) and separately for the solvent (deionized water).The ratio of these absorption coefficients yielded the relative absorption coefficient spectrum, as depicted in Fig. 10.This data processing approach effectively accentuates the relative absorption peak of the sample while minimizing the solvent's with changes in intramolecular and intermolecular interactions causing slight variances in the absorption spectra between solid and solution states.It is also crucial to recognize that the THz absorption spectrum of the aqueous solution not only reduces the amplitude of the sample's absorption peaks due to the sample-water interaction and the strong absorption by hydrogen bonds but also introduces new absorption bands not present in the solid state.This phenomenon further reflects the important influence of water on THz absorption spectrum and the complex nature of carbohydrate behavior in solution environment.Furthermore, we selected two carbohydrate samples with a concentration of 1 g/ml and introduced them into a microfluidic chip equipped with a temperature control mechanism.The control system monitored and adjusted the temperature within the chip.At 10 °C intervals, the THz Time-Domain Spectroscopy (THz-TDS) system gathered data, producing both timedomain and frequency-domain THz spectra as depicted in Fig. 11.Notably, with an increase in temperature, the THz transmission intensity of both anhydrous trehalose and trehalose dihydrate progressively decreased.During the injection of samples, special attention was given to removing air from the liquid pool to maintain data integrity and prevent liquid leakage.However, in the temperature experiment, bubbles formed in the chip when the liquid pool was continuously heated above 80°C, thus setting the experiment's maximum temperature at 80 °C.This indicated that evaporation was altering the sample concentration, modifying the experimental conditions.The THz time-domain and frequency-domain spectra of trehalose at challenging.To address this, our study leverages microfluidic technology to precisely limit the liquid layer to a thickness of 50 µm, effectively reducing water's absorption.We initially investigated the relationship between concentration and THz spectral intensity.Solutions of varying concentrations of anhydrous trehalose and trehalose dihydrate were prepared and sequentially introduced into microfluidic chips for measurement at room temperature.The corresponding THz time-domain and frequency-domain spectra are presented in Fig. 9. Our observations reveal that as the concentration increases, both the time-domain and frequencydomain spectra of the solutions are relatively intensified, indicating a decrease in absorption and an increase in transmittance.This enhancement suggests that the THz absorption spectra of these carbohydrate solutions are influenced by the concentration of molecules present, showing the interplay between molecular density and THz spectral characteristics.
Our research subsequently concentrated on the highest concentration levels of both carbohydrates.To obtain the liquid THz absorption spectra, we applied formula 2 for calculating the absorption coefficients of the sample (comprising both solvent and solute) and separately for the solvent (deionized water).The ratio of these absorption coefficients yielded the relative absorption coefficient spectrum, as depicted in Fig. 10.This data processing approach effectively accentuates the relative absorption peak of the sample while minimizing the solvent's influence, suggesting its efficacy in extracting absorption peaks in liquid samples.Despite water's damping effect on the sample's absorption peaks, it was still possible to discern that the absorption peaks of anhydrous trehalose and trehalose dihydrate in both solid and solution states exhibit a significant degree of consistency.Notably, the absorption peak of solid anhydrous trehalose at 2.04 THz undergoes a slight shift in solution.In contrast, trehalose dihydrate interacts with increasing temperature.This phenomenon is largely attributed to non-covalent bonds, like hydrogen bonds and electrostatic forces, crucial in stabilizing the molecule.Classified as a polar liquid, trehalose solution exhibits significantly higher (10 to 100 times) THz absorption coefficient due to hydrogen bond interactions between dipoles and polar molecules, compared to nonpolar liquids [23].Therefore, the observed increase in THz absorption in both anhydrous trehalose and trehalose dihydrate solutions is primarily due to dipole-molecular interactions, particularly influenced by temperature changes.

Conclusion
In this study, we employed a THz-TDS system and density functional theory for detecting and analyzing the vibration absorption of anhydrous trehalose and trehalose dihydrate.By discussing the vibration absorption characteristics of these carbohydrates, distinct THz absorption peaks were identified.To validate the experimental data, optimized stable structures and vibration features were obtained through simulation calculations, aligning well with some experimental findings.Moreover, we measured the spectral characteristics of the two carbohydrates in solution using microfluidic chip technology and compared their spectra in solid and solution states.A change in their absorption bands was observed, indicating that intramolecular and intermolecular interactions in anhydrous trehalose and trehalose dihydrate molecules are attenuated by water molecules.Nonetheless, differences in their THz absorption spectra persist.Additionally, we compared spectra at various concentrations and temperatures, establishing a correlation between the THz absorption spectrum of the same substance in solution and its concentration and temperature.These results show the significant impact of the water at 2.10 THz, resulting in a new absorption peak.These findings imply that trehalose molecules are substantially affected by the presence of water in aqueous solutions, with changes in intramolecular and intermolecular interactions causing slight variances in the absorption spectra between solid and solution states.It is also crucial to recognize that the THz absorption spectrum of the aqueous solution not only reduces the amplitude of the sample's absorption peaks due to the sample-water interaction and the strong absorption by hydrogen bonds but also introduces new absorption bands not present in the solid state.This phenomenon further reflects the important influence of water on THz absorption spectrum and the complex nature of carbohydrate behavior in solution environment.Furthermore, we selected two carbohydrate samples with a concentration of 1 g/ml and introduced them into a microfluidic chip equipped with a temperature control mechanism.The control system monitored and adjusted the temperature within the chip.At 10 °C intervals, the THz Time-Domain Spectroscopy (THz-TDS) system gathered data, producing both time-domain and frequency-domain THz spectra as depicted in Fig. 11.Notably, with an increase in temperature, the THz transmission intensity of both anhydrous trehalose and trehalose dihydrate progressively decreased.During the injection of samples, special attention was given to removing air from the liquid pool to maintain data integrity and prevent liquid leakage.However, in the temperature experiment, bubbles formed in the chip when the liquid pool was continuously heated above 80°C, thus setting the experiment's maximum temperature at 80 °C.This indicated that evaporation was altering the sample concentration, modifying the experimental conditions.The THz time-domain and frequency-domain spectra of trehalose at varying temperatures showed a consistent pattern of decreasing THz transmission intensity with increasing temperature.This phenomenon is largely attributed to non-covalent bonds, like hydrogen bonds and electrostatic forces, crucial in stabilizing the molecule.Classified as a polar liquid, trehalose solution exhibits significantly higher (10 to 100 times) THz absorption coefficient due to hydrogen bond interactions between dipoles and polar molecules, compared to nonpolar liquids [23].Therefore, the observed increase in THz absorption in both anhydrous trehalose and trehalose dihydrate solutions is primarily due to dipole-molecular interactions, particularly influenced by temperature changes.

Conclusion
In this study, we employed a THz-TDS system and density functional theory for detecting and analyzing the vibration absorption of anhydrous trehalose and trehalose dihydrate.By discussing the vibration absorption characteristics of these carbohydrates, distinct THz absorption peaks were identified.To validate the experimental data, optimized stable structures and vibration features were obtained through simulation calculations, aligning well with some experimental findings.Moreover, we measured the spectral characteristics of the two carbohydrates in solution using microfluidic chip technology and compared their spectra in solid and solution states.A change in their absorption bands was observed, indicating that intramolecular and intermolecular interactions in anhydrous trehalose and trehalose dihydrate molecules are attenuated by water molecules.Nonetheless, differences in their THz absorption spectra persist.Additionally, we compared spectra at various concentrations and temperatures, establishing a correlation between the THz absorption spectrum of the same substance in solution and its concentration and temperature.These results show the significant impact of the surrounding environment on molecular structure and vibrational modes, laying a foundation for the application of THz in carbohydrate biology research.

Fig. 4 .
Fig. 4. Device diagram of temperature-controllable microfluidic chip and its THz transmission spectrum diagram.(a) Device schematic diagram.(b) THz transmission spectrum of microfluidic chip.

Fig. 4 .
Fig. 4. Device diagram of temperature-controllable microfluidic chip and its THz transmission spectrum diagram.(a) Device schematic diagram.(b) THz transmission spectrum of microfluidic chip.
(a) and 5(d), the spectra demonstrate a remarkable signal-to-noise ratio across the 0-2.2THz range, confirming the credibility of the acquired data.Notably, anhydrous trehalose displays five distinct absorption peaks within the 0.1-2.2THz band, aligning with frequencies at 0.66, 1.25, 1.61, 1.96, and 2.04 THz as seen in Fig.5(c).These peaks' varied positions and intensities reflect their different generative mechanisms.In comparison, trehalose dihydrate exhibits four absorption peaks at frequencies 1.12, 1.42, 1.75, and 2.03 THz, as illustrated in Fig.5(f).These data show the unique spectral characteristics of both anhydrous trehalose and trehalose dihydrate, revealing their molecular properties and interactions within the THz range.Regarding refractive indices, anhydrous trehalose ranges from 1.60 to 1.65, and trehalose dihydrate from 1.60 to 1.70, as shown in Fig.5(b) and Fig.5(e).The indices at the absorption peaks decrease with increasing frequency, indicating anomalous dispersion, aligning with the Kramers-Kronig relationship.
(a) and 5(d), the spectra demonstrate a remarkable signal-to-noise ratio across the 0-2.2THz range, confirming the credibility of the acquired data.Notably, anhydrous trehalose displays five distinct absorption peaks within the 0.1-2.2THz band, aligning with frequencies at 0.66, 1.25, 1.61, 1.96, and 2.04 THz as seen in Fig.5(c).These peaks' varied positions and intensities reflect their different generative mechanisms.In comparison, trehalose dihydrate exhibits four absorption peaks at frequencies 1.12, 1.42, 1.75, and 2.03 THz, as illustrated in Fig.5(f).These data show the unique spectral characteristics of both anhydrous trehalose and trehalose dihydrate, revealing their molecular properties and interactions within the THz range.Regarding refractive indices, anhydrous trehalose ranges from 1.60 to 1.65, and trehalose dihydrate from 1.60 to 1.70, as shown in Fig.5(b) and Fig.5(e).The indices at the absorption peaks decrease with increasing frequency, indicating anomalous dispersion, aligning with the Kramers-Kronig relationship.

Fig. 5 .
Fig. 5. THz spectra of solid trehalose.(a) Spectra of anhydrous trehalose and reference signal; (b) Refractive index spectrum and (c) absorption spectrum of anhydrous trehalose; (d) Spectra of trehalose dihydrate and reference signal; (e) Refractive index spectrum and (f) absorption spectrum of trehalose dihydrate.

Fig. 5 .
Fig. 5. THz spectra of solid trehalose.(a) Spectra of anhydrous trehalose and reference signal; (b) Refractive index spectrum and (c) absorption spectrum of anhydrous trehalose; (d) Spectra of trehalose dihydrate and reference signal; (e) Refractive index spectrum and (f) absorption spectrum of trehalose dihydrate.

Fig. 7 .
Fig. 7. Simulated results of the THz spectrum and the corresponding vibrational modes of anhydrous trehalose.(a)Crystal structure of anhydrous trehalose.(b)Simulation spectrogram of anhydrous trehalose.(c)Vibration mode of anhydrous trehalose at 0.66 THz.(d)Vibration mode of anhydrous trehalose at 1.25THz.(e)Vibration mode of anhydrous trehalose at 1.70 THz.(f)Vibration mode of anhydrous trehalose at 1.97 THz.

Fig. 7 .
Fig. 7. Simulated results of the THz spectrum and the corresponding vibrational modes of anhydrous trehalose.(a)Crystal structure of anhydrous trehalose.(b)Simulation spectrogram of anhydrous trehalose.(c)Vibration mode of anhydrous trehalose at 0.66 THz.(d)Vibration mode of anhydrous trehalose at 1.25THz.(e)Vibration mode of anhydrous trehalose at 1.70 THz.(f)Vibration mode of anhydrous trehalose at 1.97 THz.

Fig. 8 .
Fig.8.Simulated results of the THz spectrum and the corresponding vibrational modes of hydrate dihydrate.(a)Crystal structure of hydrate dihydrate.(b)Simulation spectrogram of hydrate dihydrate.(c)Vibration mode of hydrate dihydrate at 1.12 THz.(d)Vibration mode of hydrate dihydrate at 1.50THz.(e)Vibration mode of hydrate dihydrate at 1.75 THz.(f)Vibration mode of hydrate dihydrate at 2.03 THz.

Fig. 8 .
Fig. 8. Simulated results of the THz spectrum and the corresponding vibrational modes of hydrate dihydrate.(a)Crystal structure of hydrate dihydrate.(b)Simulation spectrogram of hydrate dihydrate.(c)Vibration mode of hydrate dihydrate at 1.12 THz.(d)Vibration mode of hydrate dihydrate at 1.50THz.(e)Vibration mode of hydrate dihydrate at 1.75 THz.(f)Vibration mode of hydrate dihydrate at 2.03 THz.

Fig. 9 .
Fig. 9. THz spectra at different concentrations.THz time-domain spectra (a) and frequency-domain spectra (b) of anhydrous trehalose at different concentrations.THz time-domain spectra (c) and frequency-domain spectra (d) of trehalose dihydrate at different concentrations.

Fig. 9 .
Fig. 9. THz spectra at different concentrations.THz time-domain spectra (a) and frequencydomain spectra (b) of anhydrous trehalose at different concentrations.THz time-domain spectra (c) and frequency-domain spectra (d) of trehalose dihydrate at different concentrations.

Fig. 11 .
Fig. 11.THz spectra of anhydrous trehalose and trehalose dihydrate at different temperatures.THz time-domain spectra (a) and THz frequency-domain spectra (c) of anhydrous trehalose at different temperatures.THz time-domain spectra (b) and THz frequency-domain spectra (d) of trehalose dihydrate at different temperatures.

Fig. 11 .
Fig. 11.THz spectra of anhydrous trehalose and trehalose dihydrate at different temperatures.THz time-domain spectra (a) and THz frequency-domain spectra (c) of anhydrous trehalose at different temperatures.THz time-domain spectra (b) and THz frequency-domain spectra (d) of trehalose dihydrate at different temperatures.

Funding.
Beijing Municipal Natural Science Foundation (4232066); National Natural Science Foundation of China (61575131); National Key Research and Development Program of China (2021YFB3200100).