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Abstract
The efficient sensing of amino acids, especially the distinction of their chiral enantiomers, is important for biological, chemical, and pharmaceutical research. In this work, a THz phase shift sensing method was performed for amino acid detection based on a polarization-dependent electromagnetically induced transparency (EIT) metasurface. More importantly, a method for binding the specific amino acids to the functional proteins modified on the metasurface was developed based on the isoelectric point theory so that the specific recognition for Arginine (Arg) was achieved among the four different amino acids. The results show that via high-Q phase shift, the detection precision for L-Arg is 2.5 × 10−5 g /ml, much higher than traditional sensing parameters. Due to the specific electrostatic adsorption by the functionalized metasurface to L-Arg, its detection sensitivity and precision are 22 times higher than the other amino acids. Furthermore, by comparing nonfunctionalized and functionalized metasurfaces, the D- and L-chiral enantiomers of Arg were distinguished due to their different binding abilities to the functionalized metasurface. Therefore, this EIT metasurface sensor and its specific binding method improve both detection precision and specificity in THz sensing for amino acids, and it will promote the development of THz highly sensitive detection of chiral enantiomers.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Terahertz (THz, 1 THz = 1012 Hz) radiation lies between the microwave and far-infrared region of the electromagnetic spectrum with a frequency range of 0.1–10 THz. It has been proven that many biological molecules’ vibrational modes can be observed in the THz range [1], and THz waves are non-ionizing, which is highly compatible with biological molecules and does not damage biological tissues [2,3]. These advantages make THz sensing become a promising technology for biological applications, detecting small biomolecules like amino acids [4], nucleic acids [5], and glucose [6], as well as biological macromolecules such as DNA [7,8], RNA [9], proteins [10], virus [11], antibody and antigen [12], cells [13], and biological tissues [14].
As the fundamental units that constitute proteins, amino acids are closely associated with life and various forms of life activities. For example, arginine (Arg) is an essential amino acid in the human body, which has a great influence on human cell division, brain chemistry, immune response, vascular dilation, and nerve transmission [15]. However, Arg has a pair of enantiomers, called D-Arg and L-Arg, respectively. Their properties of them are quite different: L-Arg is known to be a precursor of nitric oxide, a natural form found in proteins, and is now commonly used in clinical studies [16]. D-Arg is a synthetic amino acid, which is prepared from L-Arg by racemic reaction, it has important pharmacological functions in mammals. Therefore, it is of great significance to selectively identify L-Arg and D-Arg. The traditional methods for detecting amino acids mainly include liquid or gas chromatography, capillary electrophoresis, and infrared absorption spectroscopy, but their detection process is complicated, time-consuming, and high-cost. Recently, the surface-enhanced Raman scattering (SERS) for the identification of enantiomers of amino acid based on chiral plasmonic metals was reported with a low detection limit of 0.1 µmol/L [17], but the construction of SERS base and the synthesis of chiral selectors were very complicated. THz technology is promising to apply in the sensing of amino acids [18]. For example, Wang et al. [19] presented THz absorption spectroscopy of homocysteine solution, and the detection limit can reach 10 µmol/L, but this work is difficult to distinguish the differences between two chiral enantiomers of amino acid, and the detection for different amino acids also has no specificity. Recently, Choi et al. [20] reported THz chiral spectra of several kinds of amino acids and peptides, and this result shows the fingerprint characteristics of amino acid enantiomers in the THz band, but most of their circular dichroism angle is less than 5°, so the detection precision still needs to be improved. Therefore, it’s still a challenge to achieve specificity and high detection precision in THz sensing for amino acids, especially the identification of enantiomers.
One way to improve detection sensitivity is to use microstructured THz sensor chips, such as metasurfaces [21–23]. Metasurfaces can generate several kinds of highly localized resonances in designed THz frequency bands, such as multipole toroidal resonances [24], asymmetric Fano resonances [25], anapole-induced resonance [26], and electromagnetically induced transparency (EIT) [27–29]. EIT responses are mainly caused by interference between light and dark modes on the metasurface with a difference in Q-factor, resulting in an extremely narrowband transparency window over a wide absorption spectrum. For example, Zhang et al. [29] proposed a THz EIT metasurface to effectively distinguish the mutant and wild-type glioma cells without antibody introduction. Yao et al. [30] proposed a novel THz EIT biosensor, of which the detection limit for whey protein reaches 6.25 µg/L. Therefore, the EIT metasurfaces provide a potential way to achieve sensitive sensors in the THz region. In addition, the slow light effect can be excited in this transparency narrowband, which leads to strong interactions with the biochemical target. However, this strong group delay effect is mainly reflected in the phase information, but the current research ignores this important effect, which limits the detection precision of EIT sensors. Moreover, the EIT sensor itself remains unspecific to the biochemical target, so the functionalized metasurface modified by specific markers is still needed to binding target biomolecules on the metasurface.
In this work, we developed a polarization-dependent EIT metasurface sensor, and its high-Q birefringence phase shift due to the EIT effect was proposed as the sensing parameter to realize concentration sensing of amino acid solution with high detection precision. More importantly, based on the electrostatic adsorption of isoelectric point theory and peptide bond binding, we further proposed a method for binding the specific amino acids to the functional proteins modified on the surface of the metasurface sensor. And the specific THz detection experiments for L- Arg were performed: not only the recognition of L-Arg among the four different amino acid solutions but also the distinction between L- and D-Arg chiral enantiomers. Therefore, this work improves both detection precision and specificity in THz sensing for amino acids.
2. Methods
2.1 Device fabrication and experimental setup
The metallic EIT metasurface structure with double-ring array patterns was fabricated by conventional photolithography and lift-off. The substrate is the JGS1 quartz glass with 300 µm thickness, and the gold structure with a height of 200 nm is periodically attached to the surface. Figure 1(a) shows the structure parameters of the fabricated metasurface, where r = 60 µm and R = 90 µm denote the radius of the inner ring and outer ring, respectively, and the line width of each ring is 10 µm. Two opening angles of the outer ring are 6 °, and the period P of the unit cell is 200 µm.
Fig. 1. (a) Schematic of the all-dielectric metasurface consists of double-ring arrays. (b) Schematic diagram of THz-TDS system in the experiment.
The THz sensing experiments in this work were performed by a transmissive THz time-domain spectroscopy (THz-TDS) system shown in Fig. 1(b). And the numerical simulations of the metasurface are performed by using the time-domain solver of the commercial software CST Microwave Studio. Details of experimental and numerical methods can be found in Sec. S1 of Supplement 1.
2.2 Metasurface functionalization based on the isoelectric point theory
In this work, L-Arg is the biological target. L-Arg (99% purity, MW 174.2), D-Arg (98% purity, MW 174.2), L-Proline (Pro, 98% purity, MW: 115.13), L-Cysteine (Cys, 98% purity, MW: 121.16), and L-Alanine (Ala, 98% purity, MW: 89.09) used in our experiment are purchased from Macklin Biochemical Co., Ltd (Shanghai, China), and all samples were dissolved in phosphate buffer solution (PBS). The experiments of the specific detection of L-Arg require functionalization of the metasurface based on the isoelectric point theory of amino acids [31,32]. In the isoelectric point theory, if the pH value of the solution is less than the isoelectric point of amino acids, amino acids are positively charged, and when the pH value is greater than its isoelectric point, amino acids are negatively charged. As shown in Fig. 2(d), the isoelectric point of Arg is 10.7, which is positively charged in PBS buffer with its pH = 7.4. Three other amino acids Pro, Cys, and Ala, of which isoelectric points are 6.3, 5.05, and 6.0, respectively, will be negatively charged in PBS.
Fig. 2. Metasurface functionalization steps and microscope photographs. The microscope photographs of the metasurface (a) after soaking in PDDA solution, (b) after soaking in BSA solution, and (c) after soaking in Arg solution. (d) The relationship between the electrical properties of amino acids and the pH values.
Figures 2(a)–2(c) show the schematic workflow of the functionalization process: 1) Firstly, the metasurface chip is cleaned with deionized water at least three times, then the chip is blown dry with a small airflow, and the whole chip is immersed in 1% Poly dimethyl diallyl ammonium chloride (PDDA, (C8H16NCl)n) solution for 10 minutes. As a strong positive electrolyte, the PDDA solution can make the immersed gold structures of metasurface positively charged. 2) After soaking in PDDA solution, the metasurface chip was put in BSA solution at a concentration of 10 mg/ml for 5 minutes. A low BSA concentration weakens electrostatic interaction, while a high concentration reduces the binding efficiency of amino acids and BSA, so the concentration of BSA we used in the experiment is the appropriate concentration determined by many tests. The BSA is negatively charged in PBS buffer at pH = 7.4, so it can be adsorbed onto the chip by electrostatic adsorption. After the BSA binds to the surface of the metasurface chip, the functionalization of the metasurface is completed. Note that our functionalized metasurface refers to the metasurface modified by the BSA, not PDDA, and the nonfunctionalized metasurface means the bare metasurface without the chemical treatment. 3) Finally, the metasurface chip is soaked in the amino acid solutions, and waited for at least 5 minutes for the chip to fully contact the sample solution. Then, the chips are removed from the solution and dried for testing. All the samples tested in our experiment follow the above process.
According to the above isoelectric point theory and functionalization method, only Arg can bind to the BSA on the metasurface due to the electrostatic adsorption, while the other three amino acids Pro, Cys, and Ala cannot bind. In addition to the electrostatic adsorption, L-Arg can also bind to the BSA through the intermolecular interaction of peptide bonds. However, almost all D- amino acids obtained by artificial synthetic, including D-Arg, cannot bind to BSA through peptide bonds [33,34], or the binding efficiency is particularly low. Therefore, the functionalized metasurface sensor modified by the BSA is more capable of binding L-Arg, which is the methodological basis for the enantiomer recognition between L- and D-Arg.
3. Results and discussion
3.1 EIT response of the metasurface sensor
Firstly, we investigated the THz EIT responses of the metasurface sensor itself without an amino acid sample. Figures 3(a)–3(b) show the experimental transmission spectra, phase shift spectra, and time-domain signals of the metasurface, respectively. The group delay of time-domain signals in x and y-directions is significantly different, indicating a difference in polarization spectral response and strong phase shift. For y-polarization, there are two resonances at 0.53 THz and 0.71 THz, marked as fy1 and fy2 respectively, and between two resonance dips, a transparent window forms at 0.63 THz. For x-polarization, there is a resonance at 0.55 THz marked as fx. Fig. 3(b) shows that there is a phase shift peak at 0.54 THz marked as fp due to the different delay times in the x and y directions. Further simulation results can be found in Sec. S2 of Supplement 1. Both experiments and simulations confirm that this metasurface can excite a polarization-dependent EIT effect with a high-Q phase shift. In the following sensing experiments, we will use the frequency shifts of these four resonances fx, fy1, fy2, and fp as different characterization parameters to analyze and compare the sensing sensitivity and detection precision.
Fig. 3. Experimental EIT response of THz metasurface without sample. (a) transmission spectra for x- and y- polarizations. (b) Phase shift spectra between x- and y- polarizations and their time-domain signals.
3.2 Concentration sensing of L-arginine
In this section, we show the quantitative sensing results of L-Arg solutions with different concentrations on the EIT metasurface. The concentration of L-Arg solution ranges from 5 mg/ml to 40 mg/ml, and Fig. 4 shows the experimental THz intensity spectra and phase shift spectra for different concentrations of L-Arg. With increasing concentration of L-Arg, all the resonances occur red-shift: For the y-polarized case, the resonance fy1 varies from 0.53 THz to 0.45 THz with the growing concentration, fy2 varies from 0.71 THz to 0.61 THz, the maximum frequency shift is 100 GHz; in the case of x-polarization, fx varies from 0.55 THz to 0.468 THz, the frequency shift is 82 GHz. The corresponding simulation results can be found in Fig. S3 of Supplement 1, which fits well with the results shown in Figs. 4(a) and 4(b). For the phase shift between two orthogonal polarization directions shown in Fig. 4(c), a phase peak with a high-Q factor can be observed at resonance fp, with the change of concentration, it varies from 0.54 THz to 0.47 THz.
Fig. 4. Quantitative sensing experiments of L-Arg solutions with different concentrations on EIT metasurface: transmission spectra of the metasurface for (a) y-polarization and (b) x-polarization. (c) Phase shift spectra between y- and x-polarizations. (d) Frequency shifts v.s. different concentrations of L-Arg solutions at fy1, fy2, fx, and fp, respectively.
In addition, we compare the frequency shift value of four THz resonances (fy1, fy2, fx, and fp) shown in Fig. 4(d). All the frequency shifts have a good linear relationship with the concentrations, although Δfy2 has the highest value, all the frequency shifts with the same concentration are close, and only relying on these frequency shift values cannot fully reflect the sensing characteristics. To better evaluate the sensing performances for different characterization parameters at different resonances, we analyzed and calculated the Q-factor, sensitivity, and detection precision for these four characterization parameters. The Q-factor is calculated by $Q = {\omega _0}/FWHM,$ where FWHM represents full width at half maxima of the resonance, ${\omega _0}$ is the center frequency of the resonance dip or peak. The sensitivity is determined ${S_f} = \Delta f/\Delta c,$ where Δf is the frequency shift of the resonance peak, and Δc is the corresponding changes in sample concentration. To the minimum concentration change of amino acids that can be resolved in this sensing system, we also define the detection precision, which is dependent comprehensively on the performance of the sensor and measurement system, selection of sensing parameters, and samples. The detection precision is determined by the frequency resolution of the system (Δ=1.56 GHz in this work, some data points are skipped during plotting in the figures), the sensitivity Sf, and the Q-factor of the sensing parameter, which can be calculated as ${M_c} = \Delta /({{S_f} \cdot Q} ).$ The physical meaning of the detection accuracy we difined is the minimum change in sample concentration that can theoretically be detected by the sensor and experimental system we use. The detailed results are summarized in Table 1.
Table 1. Sensitivity and detection precision of different resonancea
According to Fig. 4 and Table 1, the resonance fy2 has the maximum sensitivity, which can reach 2.5 GHz·ml/mg, while the sensitivity is 2.1 GHz·ml/mg for x-polarization, indicating that the designed metasurface is more sensitive to the EIT response. All resonances basically satisfy linear change with the growing sample concentration, so we can conclude that the linear detection range of the sensor is from 5 mg/ml to 40 mg/ml, the limit of detection (LOD) is at least 5 mg/ml. It is important to note that due to the limitation of the Q-factor, the resonance with the highest sensitivity does not show the highest detection precision. Although the total frequency shift on the phase shift spectrum is not the largest, the resonance fp has the highest detection precision Mc =2.5 × 10−5 g/ml because its Q-factor is the highest among these resonances. Based on the high-Q phase shift generated by the polarization-dependent EIT effect, higher detection precision has been obtained in this phase shift sensing of the EIT metasurface sensor than that of the traditional resonance sensing.
3.3 Specific sensing of L-arginine
Next, we specifically identified L-Arg in four different kinds of L-amino acids by functionalized metasurface. The process of functionalized metasurface can be found in Section 2.2, where Arg is positively charged and the other three amino acids Pro, Cys, and Ala are negatively charged in PBS buffer. Therefore, L-Arg can be specifically recognized by the isoelectric point theory of amino acid on the EIT metasurface.
As shown in Fig. 5, Arg always has a more significant frequency shift than other amino acids at both high and low concentrations. When the amino acid concentration is 10 mg/ml, the functionalized metasurface has no response to Cys and Ala. This is because the BSA modified on the surface of the metasurface has the same charge as these three amino acids, and they repel each other and are difficult to bind together. We also observed that the metasurface had a slight response to Pro. This is because the isoelectric point of Pro was 6.3, close to 7.4, the electrostatic repulsion is weak, and a small number of Pro molecules are attached to the metasurface sensor. When the concentration of amino acids is 40 mg/ml, Cys and Ala also have a slight frequency shift, which indicates at a higher concentration, a small number of molecules will adhere to the chip surface and excite the response of the sensor. In Fig. 5 h, the frequency shifts of Pro for the different resonances are between 10 GHz and 20 GHz, for both Cys and Ala are below 10 GHz, while all for Arg are above 40 GHz, so we can effectively distinguish L-Arg from other amino acids. Additional, Since the metasurface modified by bovine serum protein, the resonant peak position will be different even if the amino acid concentration is the same, thus the resonant frequencies of L-Arg at concentrations of 10 mg/ml and 40 mg/ml in Fig. 5 are different from Fig. 4.
Fig. 5. Experimental results for four L-amino acids with the concentration of 10 mg/ml: (a) Transmission spectra for y-polarization and (b) x-polarization; (c) Phase shift spectra. 40 mg/ml: (d) Transmission spectra for y-polarization and (e) x-polarization; (f) Phase shift spectra. Frequency shifts for the four kinds of L-amino acids for the different resonances used as sensing parameters. The concentrations of the samples are (g) 10 mg/ml and (h) 40 mg/ml, respectively.
Since the phase shift has a higher Q-factor and detection precision, we focus on the comparison with the frequency shift of fp of the four amino acids for details. For 40 mg/ml in Fig. 5 h, the redshift of fp is 18.75 GHz for Pro, only 6.25 GHz for both Cys and Ala, but 46.875 GHz for Arg; and for 10 mg/ml, the redshift of fp is less than 1 GHz for both Cys and Ala, 8 GHz for Pro, while 22 GHz for Arg. This means that at the same concentration of 10 mg/ml, the detection sensitivity and precision of Arg are 2.75 times that of Pro and at least 22 times that of Cys and Ala, among these three amino acids, the isoelectric point of Pro is the closest to 7.4, it means that its molecular has the smallest negative charge in PBS solution and the electrostatic repulsion with BSA is the weakest, then the Pro binds to the protein much more than Cys and Ala, result in higher sensitivity. Thus, we can conclude that Arg can be specifically detected from the four kinds of amino acid samples. Moreover, this method is not only applicable to Arg but also effective for most amino acids with different isoelectric points. Other amino acids can be specifically detected by selecting an appropriate pH value based on the isoelectric point theory.
3.4 Distinguishing chiral enantiomers D-Arg and L-Arg
Finally, we prove that our functionalized metasurface can distinguish the two chiral enantiomers of Arg by the different abilities of BSA to bind them, compared with the nonfunctionalized metasurface. We measured two enantiomeric samples with the same concentrations at 40 mg/ml in Fig. 6. The results of other concentrations show the same conclusion, which can be found in Fig. S4 of Supplement 1. From Figs. 6(a)–6(c), it can be found that the spectral lines of D- and L-Arg are nearly overlapped with each other and have a similar frequency shift relative to the spectral lines of the nonfunctionalized metasurface. Therefore, it is impossible to distinguish the D- and L-amino acid enantiomers by the nonfunctionalized metasurface.
Fig. 6. (a)-(f) Enantiomer identification results for L-Arg and D-Arg at the concentration of 40 mg/ml. On nonfunctionalized metasurface: (a) Transmission spectra for y-polarization and (b) x-polarization; (c) phase shift spectra. On functionalized metasurface: (d) Transmission spectra for y-polarization and (e) x-polarization; (f) phase shift spectra. The comparisons for the frequency shifts of D-Arg and L-Arg with the sample of (g) 40 mg/ml and (h) 10 mg/ml.
Figures 6(d)–6(f) show the experimental results for D-Arg and L-Arg on functionalized metasurface, and we can see that there are significant differences in spectra between D-Arg and L-Arg. All the resonances of L-Arg have a larger frequency shift than that of D-Arg. As we mentioned at the end of Section 2.2, L-Arg can bind to BSA through both electrostatic adsorption and peptide bonding. However, D-Arg cannot easily bind to BSA through intermolecular interaction. Therefore, more L-Arg molecules are adsorbed by BSA on the sensing chip, and thus the functionalized metasurface modified by BSA is more sensitive to L-Arg solution.
Furthermore, when the concentration is 10 mg/ml shown in Fig. 6 h, the redshift of fp is 3.1 GHz for D-Arg while 22 GHz for L-Arg, the difference of frequency shift $\Delta {f_{10mg/ml}}$=18.75 GHz, so detection sensitivity and precision of L-Arg are 7.1 times that of D-Arg. As shown in Fig. 6 g, $\Delta {f_{40mg/ml}}$> 20 GHz for all four resonances, and the maximum value can reach 31.25 GHz. As the concentration increased, the difference between the frequency shifts of D-Arg and L-Arg also increases. Therefore, we conclude that this functionalized metasurface can effectively distinguish the chiral enantiomers L- and D-Arg. Furthermore, since the difference in binding capacity between the L- and D- enantiomers on the BSA peptide is the main mechanism in the enantiomer recognition, this method can not only be applied to distinguish the enantiomers of Arg, but also can be feasible to distinguish chiral enantiomers of other amino acids. Note that the matching of amino acid isoelectric points and the proper pH value is still important in this method of enantiomer recognition, which can maximize the adsorption of both enantiomers on the sensor and obtain higher detection sensitivity.
4. Conclusion
In conclusion, we performed specific sensing for the amino acid detection based on a THz EIT metasurface sensor and the specific electrostatic adsorption method. Three important results have been obtained: 1) Via high-Q phase shift generated by the EIT metasurface, a high precision quantitative concentration sensing of L-Arg is realized, of which the highest detection precision Mc =2.5 × 10-5 g/ml, much higher than that of other intensity sensing parameters. 2) L-Arg has been specifically identified in different kinds of amino acids by functionalized EIT metasurface. This specificity originates from the specific electrostatic adsorption based on the isoelectric point theory. Our results show that the detection sensitivity of Arg is 2.75 times that of Pro and at least 22 times that of Cys and Ala. 3) Our functionalized metasurface can distinguish the two chiral enantiomers of Arg by the different abilities of BSA to bind them, the detection sensitivity of L-Arg is 7.1 times that of D-Arg. This kind of molecular adsorption method has been proven to be effective in THz biochemical sensors for the first time. And the use of the phase-shift sensing method has further improved the detection precision of this specific sensing. We believe our sensing strategy will promote the development of THz highly sensitive biochemical sensors and specific sensing methods, especially the detection of chiral enantiomers.
Funding
Key Program of the Natural Science Foundation of Tianjin (19JCZDJC32700); National Key Research and Development Program of China (2017YFA0701000); National Natural Science Foundation of China (61831012, 61971242).
Acknowledgements
The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (61971242, 61831012); the National Key Research and Development Program of China (2017YFA0701000); the Key Program of the Natural Science Foundation of Tianjin (19JCZDJC32700).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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