| The damage of animal-derived food caused by antibiotics residues induces serious economic losses directly and indirectly on a global scale. Many antibiotics can cause serious harm to human health and even death in people through antibiotics-contaminated food, especially tetracyclines residues which are the most serious. Traditional detection methods such as microbiological methods, chromatography, capillary electrophoresis, and immunoassays are limited in detection time, operation, method establishment, and molecular structure information provision though they are sensitive. Detection methods based on spectroscopy have the advantages of high speed, simple operation, and reflecting the molecular structure information, which are perhaps one of the most powerful and potential approach available. Wherein, terahertz (THz) spectroscopy as one kind of emerging spectroscopy technologies, has received much attention in the past decade due to its unique optical properties.In this study, four kinds of tetracyclines hydrochloride (TCsH) in animal-derived food, including tetracycline hydrochloride (TCH), oxytetracycline hydrochloride (OTCH), doxycycline hydrochloride (DTCH), and chlortetracycline hydrochloride (CTCH) were selected as detection target of interest. Using THz time-domain spectroscopy (THz-TDS) as a main line, detection methods and quantitative detection models for the detection of TCsH antibiotics have been established. It is the first time to provide the spectral features of TCsH pellets, and explore the feasibility and methods of using THz spectroscopy to detect TCsH antibiotics, providing a new platform for the detection of antibiotics residues in animal-derived food.The main contents and results are summarized as follows:(1) The THz spectra of TCsH pellets were studied initially. TCsH samples were prepared as pellets. The analysis for absorbance spectra of TCsH pellets in the 0.3-1.8 THz showed that TCH had three absorption peaks at 0.79,1.40, and 1.60 THz. DTCH exhibited three strong absorption peaks at 0.76,1.20, and 1.57 THz, and a very weak absorption peak at 0.54 THz. CTCH showed four absorption peaks at 0.76,1.00,1.34, and 1.62 THz. In contrast, OTCH demonstrated a broad absorption peak centered at-1.3 THz, and very weak absorption peaks at 1.48,1.58, and 1.70 THz. The densiy functional theory (DFT) calculations of TCsH isolated-molecules indicated that most of the absorption peaks of TCsH located in the 0.3-1.8 THz were mainly from the intermolecular vibrations or phonon modes, but a few of them were due to the rocking (in plane) and wagging (out of plane) from the atomic group which belonged to the intramolecular vibration. It could be concluded that TCsH had their distinctive THz fingerprint features. DFT predictions showed fairly well agreement with the experimental data and the assignment of absorption peaks of TCsH was achieved.(2) A method based on THz spectroscopy combined with liquid cell was established for the detection of TCsH in solutions. A demountable liquid cell was used as a sample holder. The transmittances of PE and SiO2 in the 0.2-1.0 THz ranged from 0.67-0.90 and 0.41-0.85, respectively, while their corresponding absorption coefficients ranged from 0.28-1.28 and 0.54-2.97 cm-1, respectively. All things considered, SiO2 was more suitable for window material in the THz region for the measurement of liquid sample. The results from the absorption coefficient of pure water with different thickness showed that the liquid cell with an optical path of 0.2 mm could be used as a sample holder to obtain the optical parameters of pur water in the 0.2-1.0 THz with a high accuracy and reliability, while too long or too short optical paths affected the accuracy of optical parameters. The analysis for absorption spectra of TCsH solutions in the 0.2-1.0 THz showed that the absorption spectra of four kinds of TCsH aqueous solutions at 5 mg/mL overlapped together, thus qualitative detection of TCsH in pure water could not be realized by visual observation of their THz absorption spectra. The absorption spectra of TCsH milk solutions at 5 mg/mL and pure milk overlapped together, thus the presence of TCsH in pure milk could not be detected by visual observation of their THz absorption spectra. Took TCH as an example, the absorption spectra of TCH aqueous solutions at concentrations of 0,2.5,5,10, and 20 mg/mL could be easily distinguished from each other and the absorption coefficient decreased with increasing TCH concentration in the 0.45-0.55 THz. Thus the minimum detectable TCH concentration was 2.5 mg/mL by visual observation of their THz absorption spectra. The SLR model of the average absorption coefficient in this region and the TCH concentration showed that the absorption coefficient lineally decreased as the TCH concentration increased, with a determination coefficient (R2) of 0.98, a root mean square error (RMSE) of 1.15 mg/mL, and a limit of detection (LOD) of 0.73 mg/mL. It could be concluded that TCH in pure water can be quantitatively analyzed using THz spectroscopy based on liquid cell method.(3) A method based on THz spectroscopy combined with attenuated total reflection (ATR) technology was established for the detection of TCsH in solutions. The testing results of pure water demonstrated that this method could be used to measure the complex refractive index of pure water in the 0.3-2.0 THz with a high accuracy and reliability. The analysis for complex refractive index spectra of TCsH solutions in the 0.3-1.0 THz showed that the complex refractive index of four kinds of TCsH solutions at 5 mg/mL overlapped together, both in the case of pure water and pure milk used as a solvent, however, the complex refractive index of these TCsH solutions was smaller than that of their corresponding solvents (pure water or pure milk) at the same frequency. Therefore, qualitative detection of TCsH in pure water or in pure milk could not be realized, while the presence of TCsH in pure water or in pure milk could be detected by visual observation of their THz complex refractive index spectra. Took TCH as an example, the shapes of complex refractive index spectra for TCH solutions were similar, and their complex refractive indexes decreased with increasing TCH concentration at the same frequency. The SLR model of the complex refractive index of TCH solutions at 0.5 THz and the TCH concentration showed that the complex refractive index lineally decreased as the TCH concentration increased, and the rate of decline in the extinction coefficient was faster than that in the refractive index. All the SLR models performed well in predicting TCH concentration in solutions, with R2 ranging from 0.95 to 0.98, RMSE ranging from 0.61 to 0.99 mg/mL, and LOD ranging from 0.45 to 1.29 mg/mL. It could be concluded that TCH in pure water and in pure milk can be quantitatively analyzed using ATR-THz spectroscopy.(4) A method based on THz spectroscopy combined with metallic holes array (MHA) was established for the detection of TCsH. The measured transmission spectra of MHA showed a transmission peak at 0.43 THz, which agreed well with the simulation data. The simulation result showed that the transmission peak frequency experienced red-shift as the thickness of photoresist layer increased, with a sensitivity of 12.5 GHz/μm. In using pure water as a solvent, the analysis for transmission spectra of TCsH films on the MHA showed that the transmission spectra of four kinds of TCsH films at 1000 mg/L overlapped together, however, both transmission peak amplitude value and the transmission peak frequency of these TCsH films were smaller than those of empty MHA (pure water on the MHA after drying process). Therefore, qualitative detection of TCsH in pure water could not be realized, while the presence of TCsH in pure water could be detected by visual observation of their THz transmission spectra. In using pure milk as a solvent, the transmission spectra of TCsH milk films at 1000 mg/L and pure milk film overlapped together, thus the presence of TCsH in pure milk could not be detected by visual observation of their THz transmission spectra. Took TCH aqueous solutions as an example, the analysis for transmission spectra of TCsH films at different concentrations on the MHA showed that when the TCH concentration was below 0.1 mg/L, no difference was observed in their transmission spectra; when the TCH concentration ranged from 0.1-10 mg/L, the transmission peak amplitude increased as the TCH concentration increased; when the TCH concentration was greater than 10 mg/L, both red-shift in the transmission peak frequency and attenuation in the transmission peak amplitude could be observed. PCA analysis for the transmission spectra of TCH films showed that the minimum detectable TCH concentration on the MHA was 0.1 mg/L, which was about 105 times enhancement compared to the measurement of TCH on a high-resistivity silicon, and was comparable to the maximum limit residue of tetracycline antibiotics in food matrix set by law. It could be concluded that TCH in pure water can be quantitatively analyzed with high sensitivity using THz spectroscopy based on MHA. |
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