Open Access
Add to BibSonomy
Abstract
A high-sensitivity and label-free method for identifying a transgenic plant genome is highly desirable in plant biotechnology. Here, we present a terahertz (THz) metamaterial (MM)-based biosensor that comprises a planar array of gold split-ring (SR) resonators capable of sensing a considerable shift in the resonance frequency due to the change of the dielectric environment on the MM chip. The meta-sensor is employed to detect transgenic tomato genome DNAs by THz time-domain spectroscopy. The experimental results were confirmed by finite-element modeling through varying the thickness and dielectric constant of the DNA overlayer. Consequently, high-efficiency and label-free discrimination between the wild-type and transgenic genome DNA was achieved.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Plant biotechnology has opened a novel avenue in the development of plants for the yield, resistance, and important metabolism of desired plants, more recently, new types of plants as bio-reactors (pharmaceuticals, vaccines, nutraceuticals, etc.) [1]. One of the prominent challenges in plant biotechnology is the development of a rapid and flexible system for genetic analysis. An early and essential step among the analyses for the transgenic material is to determine which plants contain the transgene and in how many copies. This is because the integrated genes with multiple copies in transgenic plants have been shown to be more silent and single-copy transformation events are preferred due to their stability over several generations of subsequent breeding [2]. Furthermore, for the uncertainty of transgenic safety and the right of consumers to know, nearly 40 countries and areas have issued qualitative or quantitative labeling regulations in terms of the copy of specific insert sequence for the transgenic product [3]. In the past decades, advancements have been achieved in identifying transgenic plants and quantifying transgene number, including Southern blot analysis, real-time PCR, and so on [2,3]. Unfortunately, these methods are quite costly in terms of reagents, labor, and time, and deplete a relatively large amount of plant material to start with, limiting their utilization on a large scale for the genetic determination. In addition, applications of these methods have been also hindered in part due to their low efficiency, accuracy, and scalability. Thus, it is highly desired to develop innovative detection that is more sensitive, rapid, high-throughput and especially in a label-free way.
The presence of interhelical excitations highlight the feasibility of terahertz time-domain spectroscopy (THz-TDS) as a label-free detection of the binding state of DNA molecule and the observation of a transmission change has experimentally verified its unique potential in the efficient detection on double-strand DNA (hybridized/denatured), oligo- and poly-nucleotides [4–8]. However, as to the transgenic plants, the minute distinction only exists in the insert sequence, not in the phonon or vibration modes. In many cases, the insert is only a gene fragment with a small number of nucleotides, so the sensitivity of THz-TDS in transgene detection requires enhancement, and furthermore, reducing the amount of DNA. Recently, metamaterial (MM)-based terahertz biosensor has overcome the limit in sensitivity with typical THz-TDS systems. This sensing modality is based on changes in the dielectric environment and minute quantities of biomaterials above the MM chips. Up to now, a variety of methods have been attempted to reach a greater frequency shift or a higher Q-factor to improve the sensitivity of MMs’ sensing [9,10]. One efficient strategy to achieve an extremely sharp resonance is to introduce an asymmetric split resonator by means of the excitation of “trapped mode” [11,12]. Moreover, the sensitivity of the THz biosensor can be significantly enhanced by fabricating THz MMs on some thin, low-permittivity substrates to reduce the transmission loss and the induced capacitance. An order of magnitude improvement of frequency shift in LC resonator was experimentally observed of planar THz MMs fabricated on thin membranes like silicon nitride (400 nm thick) and polymers in comparison to counterparts fabricated on bulk Si substrates [13–18]. Here, we exploit these two distinct technologies to realize an easily accessible and high-sensitivity identification of single-copy transgenic genome.
In this article, we demonstrate two circular MM split-ring (SR) resonators with single or double splits to distinguish wild-type native genome and transgenic genome with single-copy foreign DNA fragment. We also compare the performance of two SR sensors fabricated on an ultrathin polyethylene terephthalate (PET) film with a low intrinsic loss, where the double-split ring (DSR) sensor with an asymmetric structure exhibits a strong and sensitive Fano resonance and shows superior performance for minute detection of a single-copy transgenic genome. In addition, the sensing of analyte was demonstrated as a function of content, thickness, and dielectric constant by detailed measurement and simulation. Based on the data fitting, the function was constructed between the sensitivity and the variants in order to achieve a quantitative identification.
2. Experimental methods
2.1 DNA sample
Genome DNA is taken as the target for identification because of its stability and high detectability. Wild-type, near-isogenic lines of tomatoes (Solanum lycopersicum L. cv Micro-Tom) were used in the experiment. Genomic DNA was isolated from the 1 g two-week tomato leaves by CTAB DNA extraction method. The foreign DNA fragment of a precursor sequence of microRNA160 is about 650 bp in length, amplified by PCR and recovered from agarose gel. The concentration of the native genome (without any foreign DNA fragment) and fragment DNA is 0.944 μg·μL−1 and 2.3 ng·μL−1, respectively, determined by Spectrophotometer.
To obtain standard genome DNA with a single-copy transgene, in the study, based on the molecular numbers of an exogenous gene and plant genome, the two types of DNA were mixed at a certain concentration ratio as a mimic for the transgenic genome, while the wild-type genome was considered as the non-transgenic control. By calculation, the fragment DNA solution was diluted to 1000 times and 6 μL of the diluted solution was added to 10 μL native genome DNA solution, then the copy of genome and gene equals in the mixed solution.
2.2 Design and preparation of THz MM chips
Two circular SR resonators comprised of single or double splits were designed to realize the high-sensitivity THz chips due to their excellent sensing characteristics of the resonance shift loaded with a dielectric material [19,20]. A single-split ring (SSR) resonator, as illustrated in Fig. 1(a), has a cell size of 90 × 90 μm2 with an inner radius, outer radius, width, and gap of 24, 30, 6, and 2 μm, respectively. For comparison, another DSR resonator, as schematically shown in Fig. 1(b), was also prepared by adding a second gap to the SSR and breaking the symmetry. Two structures were fabricated by conventional photolithography and metallization processing. A 200-nm-thick Au film was deposited on a PET sheet with a thickness of 25 microns. Finally, a lift-off process was performed to form the designed metamaterials.
Fig. 1 Designed SSR (a) and DSR (b) cell in a square lattice with dimensions of g, R1, R2, and w. All dimensions are in μm. (c) Schematic diagram of the metamaterial-based biosensor chip.
The structure enables resonances that are highly sensitive to the change of dielectric environment. In order to verify the resonance behavior and the possible applicability as a high-sensitivity chip for diagnosing of a transgenic genome, the thin sample layers were formed by depositing 3 μL aqueous DNA solution onto an area of 5 × 5 mm2, as shown in Fig. 1(c). The water in the solution was evaporated in an oven set at a low temperature 42°C in order not to denature the DNA. Multilayers were made by repeating the deposition procedure anew several times.
2.3 THz spectroscopy measurement
A setup based on photoconductive antennas is used to characterize the THz transmission spectra of the samples [21,22]. The transmission measurements were performed with linearly polarized THz waves at normal incidence and the electric field is parallel to the gap, as illustrated in Fig. 1(c). A blank PET sheet identical to the array substrate alone was used to obtain the reference THz pulse. The THz MM chips with or without the DNA film were used as the sample THz pulse. Then the sample and reference amplitude spectra were obtained from the time-domain data by Fourier transform and denoted as As(ω) and Ar(ω), respectively. For all of our measurements, the amplitude transmission spectra were obtained by As(ω)/ Ar(ω).
To evaluate the potential of our method on the DNA samples, we conducted the following three sets of experiments. In the first set of experiments, the blank PET films coated with native genome as control or transgenic genome solutions at a volume of 15 μL were detected to verify the necessity of the THz MM. In the second set of experiments, the SSR or DSR resonators coated with transgenic genome DNA solution at a volume of 5 μL were used to compare the performance of the two resonators. In the third set of experiments, the relative change of resonance frequencies of the DSR resonator with different contents of the native or transgenic genome was measured by THz-TDS.
2.4 Finite-element simulations
To compare and give an insight into the frequency shift in response to a deposited overlayer, the THz biosensing sensitivity was investigated numerically by the finite-element simulations using CST Microwave Studio. For that comparison, all geometric parameters of the SSR and DSR cells are identical to the designed structures, as illustrated in Fig. 1. The complex permittivity of the PET film is ε = 3.1 + i 0.28, which was experimentally determined and extracted by applying the multiple-reflection approach [23,24].
3. Experimental results and discussion
3.1 Transmission response of the blank PET
We first describe the THz transmission experiments for the native and transgenic genome with 15 μL that were dripped to a blank PET substrate without THz MM patterns, as shown in the inset of Fig. 2(b). The THz time-domain waveforms and the corresponding frequency-domain transmission spectra of the two samples are obtained and plotted in Figs. 2(a) and 2(b) respectively. The native and transgenic genome DNAs do not have spectral fingerprints in the THz frequency range, thus it is basically transparent to the THz waves. No noticeable change in transmission (amplitude and phase change) was found following the deposition of the DNAs and these two types of genomes cannot be distinguished. To be detectable and reliable, a large amount of DNA sample is required, thus the first set of experiment is not practical in the genetic analysis.
Fig. 2 Time-domain waveforms (a) and the corresponding frequency-domain transmission spectra (b) transmitted through a blank PET substrate without and with coverage of native and transgenic DNA layer.
3.2 Biosensing characteristics of SSR and DSR MMs
A representative result on the highly sensitive detection by using the THz MM chips is demonstrated in the following. For comparison, the measured transmission spectra of SSR and DSR without and with DNA thin films are shown in Fig. 3. Unlike the results on the transmission through the blank substrate (Fig. 2), the presence of the 5 μL DNA layer induces a distinct transmission change and a significant red-shift in the resonance frequency for obvious sensing due to modification of the dielectric constant of the medium surrounding the MMs. Two distinct resonances are observed as transmission dips in the uncoated THz MM sensors. The low-frequency resonance mode (Dip 1) originates from inductor-capacitor (LC) oscillation, whereas the high-frequency resonance mode (Dip 2) is attributed to the electric dipole oscillator [25–28]. In addition, the presence of the overlayer causes both LC and dipole resonances to shift to lower frequencies. In the case of SSR, the LC and dipole resonances for bare SSR chip locate at 0.571 and 1.838 THz, while the corresponding LC and dipole modes at 0.535 and 1.769 THz for SSR chip with DNA, respectively. For DSR, the dips 1 and 2 are at the sites of 0.974 and 1.736 THz before coating, while after coating, at 0.897 and 1.641THz, respectively. The redshifts normalized to the resonance frequencies of low- and high-frequency resonators for SSR and DSR are 6.5% as well as 3.8%, and 7.9% as well as 5.5%, respectively, as indicated in Table 1. Our results thus indicate that the detection sensitivity can be improved by increasing the split numbers and this enhanced effect on the LC mode is more obvious than that on the dipole mode.
Fig. 3 Resonance frequency shift upon addition of 5μL overlayer of transgenic samples onto the SSR (a) and DSR (b).
Table 1. Frequency shift of the LC and dipole resonances for SSR and DSR
To give an insight into the underlying transmitted responses, Figs. 4(a) and 4(c) show the simulated frequency-dependent transmission of two kinds of SR resonators with and without a DNA overlayer under different thicknesses. The relationship between the normalized frequency shift and the thickness of overlayer film for both SSR and DSR patterns is shown in Figs. 4(b) and 4(d). Similar to the experimental observation, DSR shows a greater resonance shift than SSR, confirming that the SR resonators with more splits have a higher sensing capability.
Fig. 4 Simulated transmission spectra and resonance frequencies as a function of overlayer thickness. (a) Transmission spectra of SSR for various overlayer thicknesses; (b) Normalized frequency shift (Δf /f0) of the LC and dipole resonances for SSR; (c) Transmission spectra of DSR for various overlayer thicknesses; (d) Normalized frequency shift (Δf /f0) of dip 1 and 2 for DSR.
By utilizing the LC circuit model, the resonance frequency of the LC resonator (Dip 1) is defined as , where C and L are the capacitance and inductance of the SR resonator, respectively. Resonance frequency shifts induced by the dielectric overlayer are mainly due to alterations in the SR capacitance because the inductance in a single SR or the mutual inductance between SRs is inertia to the environmental change. According to O’Hara’s suggestion [29], the total capacitance (C = C1 + C2 + C3 + C4 + C5) in our case can be divided into five parts: the capacitance due to flux within the substrate (C1), fringing flux between substrate and SRs (C2), flux within the gap of SRs (C3), flux within the overlayer (C4), and fringing flux between SRs and overlayer (C5). Here, with coating DNA overlayer onto SRs, the main capacitance change is C3, C4, and C5, which induces the frequency redshift of the Dip 1. More splits will induce larger gap capacitance (C3), which eventually causes the greater redshift and the higher sensitivity. Moreover, a low-permittivity and thin PET substrate will have a minor capacitive contribution (C1 and C2) in comparison with high-permittivity and thick substrates. This substantially improves the change in capacitance and increases the amount of resonance shift resulted from the overlayer, which also leads to a higher sensitivity. Unlike the LC mode, the dipole mode (Dip 2) arises from the dipole resonance characteristic of , which is mainly related to the micro-environment dielectric change, resulting in much larger redshift [30].
3.3 Quantitative analysis of the DSR biosensor chip
To further evaluate its potential as a thin-film sensor, the dose-dependent manner was investigated through the DSR chip. The concentration of native and transgenic samples for detection was fixed, but in different volume for detection. The two kinds of the prepared overlayer were coated with DNA solution ranging from 0 to 9 μL. The measured transmission spectra of dip 1 with native and transgenic sample overlayer are shown in Fig. 5. It is remarkable to see that the two types of DNA behave in a very similar way and present that the transgenic DNA can be distinguished from native DNA due to its higher frequency shift. The circular DSR features a LC resonator at 0.970, 0.908, 0.878 and 0.864 THz for 0, 3, 6 and 9 μL native sample coverage, respectively [Fig. 5(a)]. This is slightly more than the values observed from the transgenic sample, whose corresponding resonant frequencies are 0.970, 0.905, 0.872 and 0.850 THz [Fig. 5(b)].
Fig. 5 THz transmission spectra of the DSR metamaterial after deposition of native (a) and transgenic (b) DNA at various doses: 0 (black), 3 (red), 6 (green), and 9 μL (blue). The error bars represent fluctuation of the amplitude only while the positions of the resonance frequencies of the dips remain unchanged.
They also show a similarly nonlinear sensitivity for the redshift of the central frequency, as shown in Fig. 6(a). Quite obviously, a greater red-shift of the resonance frequency is observed for the DSR biosensor with transgenic DNA compared to the case of native DNA, so that the transgenic DNA can be distinguished with unprecedented sensitivity. Also, the nonlinear resonance shifts are in quite adequate agreement with the simulated results [Fig. 4(d)]. Here, we assume that the thickness of the deposited overlayers is proportional to the volume of the DNA samples. In addition, we observe from Fig. 6(a) that the experimental data matched reasonably well with the polynomial function, which would be the basis for quantitative analysis of the content and copy number in transgenic genome samples. The fitting functions are described by Δf = 0.92 + 23.05x-1.32x2 and Δf = 1.09 + 24.17x-1.22x2 for native and transgenic DNAs, respectively.
Fig. 6 (a) Measured frequency shift as a function of contents for the native and transgenic DNA, and (b) Simulated frequency shift as a function of thickness and dielectric constant for the native and transgenic DNA with εeff = 2.8 and 3.1, respectively.
As mentioned previously, the LC resonance frequency of the SR resonator is strongly dependent on the capacitance of the dielectric overlayer and a shift of the resonance frequency occurs when the dielectric permittivity changes. The different shift between native and transgenic DNA is due to their different dielectric constants. According to the experimental results presented by Brucherseifer et al. [31], a significantly higher refractive index and absorption was observed for hybridized DNA in comparison with pure DNA, so did in our case, where the native and transgenic DNAs are corresponding to the pure and hybridized DNA, respectively. To understand the experimental observations, Fig. 6(b) shows the simulated frequency shifts with the dielectric constants of the native and transgenic DNAs set to εeff = 2.8 and 3.1, respectively. The resonance frequency shift increases nonlinearly with the increasing overlayer thickness (proportional to the content), which is apparently larger for the higher dielectric constants. Results of these simulations successfully reproduce our experimental findings, which enable the label-free identification of the transgenic DNA.
Based on the previous results that the redshift of the transgenic genome is greater than the native genome, we suppose that an increase of the copy number of foreign DNA fragment will lead to a higher refractive index (or effective permittivity) of the transgenic genome. As a consequence, the resonance frequency shift (Δf) increases in the same way. We plotted the simulated frequency shift against the refractive index of the DNA overlayer for fixed analyte thicknesses of 2 and 5 μm while varying the refractive indices from n = 1.0 to 1.8 in incremental steps of 0.1 in Fig. 7. More importantly, the frequency shifts fitted linearly with the changes of the refractive indices. The sensitivity of the DSR sensor, which equaled to the slope of lines in Fig. 7, reached 90 GHz/RIU and 122 GHz/RIU when the thickness of the analyte was 2 and 5 μm, respectively. Thus, we are hopeful to realize qualitative and quantitative sensing of the content (or thickness) and copy number (refractive index) simultaneously with the THz metamaterial-based biosensor.
4. Conclusions
Two circular SR arrays with single or double splits were presented to distinguish the wild-type plant genome DNA and transgenic genome with a single-copy foreign gene. A clear frequency shift, due to the change in the effective dielectric constant and thickness of the DNA overlayers, was experimentally and theoretically studied by THz-TDS and finite-element simulation. In addition, the simulated results are in good agreement with the experimental data and give a good insight into sensing response to the deposited DNA film. The study demonstrates the capability of the THz MM biosensors to identify the transgenic plants in an unprecedentedly sensitive, high-throughput, label-free way. Further refinement of the approach is required for the large-scale application, for example, real-time monitoring with microfluidic devices and experimentally finding a special correlation between the copy number as well as the refractive index.
Funding
National Key R&D Program of China (2017YFB0405400); National Natural Science Foundation of China (11574408, 61627814, 61675238); Hebei Economic and Business University Fund Project (2015KYZ04); Hebei Province Natural Science Foundation (D2015207013); Young-talent Plan of State Affairs Commission (2016-3-02); Technology Foundation for Selected Overseas Chinese Scholar.
Acknowledgments
The authors would like to acknowledge Dr. Zhaoxin Geng from Minzu University of China for the fabrication of chips, and Prof. Ramanjulu Sunkar from Oklahoma State University for the experimental support. Yuping Yang and Dongqian Xu also thank the sponsorship of the China Scholarship Council.
The authors declare that there are no conflicts of interest related to this article.
References
1. A. K. Sharma and M. K. Sharma, “Plants as bioreactors: Recent developments and emerging opportunities,” Biotechnol. Adv. 27(6), 811–832 (2009). [CrossRef] [PubMed]
2. B. Bubner and I. T. Baldwin, “Use of real-time PCR for determining copy number and zygosity in transgenic plants,” Plant Cell Rep. 23(5), 263–271 (2004). [CrossRef] [PubMed]
3. L. Yang, S. Xu, A. Pan, C. Yin, K. Zhang, Z. Wang, Z. Zhou, and D. Zhang, “Event Specific Qualitative and Quantitative Polymerase Chain Reaction Detection of Genetically Modified MON863 Maize Based on the 5′-Transgene Integration Sequence,” J. Agric. Food Chem. 53(24), 9312–9318 (2005). [CrossRef] [PubMed]
4. X. Wu, Y. E. X. Xu, and L. Wang, “Label-free monitoring of interaction between DNA and oxaliplatin in aqueous solution by terahertz spectroscopy,” Appl. Phys. Lett. 101(3), 033704 (2012). [CrossRef]
5. M. Nagel, P. Haring Bolivar, M. Brucherseifer, H. Kurz, A. Bosserhoff, and R. Büttner, “Integrated THz technology for label-free genetic diagnostics,” Appl. Phys. Lett. 80(1), 154–156 (2002). [CrossRef]
6. M. Nagel, P. Haring Bolivar, M. Brucherseifer, H. Kurz, A. Bosserhoff, and R. Büttner, “Integrated planar terahertz resonators for femtomolar sensitivity label-free detection of DNA hybridization,” Appl. Opt. 41(10), 2074–2078 (2002). [CrossRef] [PubMed]
7. M. Nagel, F. Richter, P. Haring-Bolívar, and H. Kurz, “A functionalized THz sensor for marker-free DNA analysis,” Phys. Med. Biol. 48(22), 3625–3636 (2003). [CrossRef] [PubMed]
8. P. Haring Bolívar, M. Nagel, F. Richter, M. Brucherseifer, H. Kurz, A. Bosserhoff, and R. Büttner, “Label-free THz sensing of genetic sequences: towards THz biochips,” Philos Trans A Math Phys Eng Sci 362(1815), 323–335 (2004). [CrossRef] [PubMed]
9. J. F. O’Hara, W. Withayachumnankul, and I. Al-Naib, “A review on thin-film sensing with Terahertz waves,” J Infrared Milli Terahz Wave 33(3), 245–291 (2012). [CrossRef]
10. W. Xu, L. Xie, and Y. Ying, “Mechanisms and applications of terahertz metamaterial sensing: a review,” Nanoscale 9(37), 13864–13878 (2017). [CrossRef] [PubMed]
11. R. Singh, I. A. Al-Naib, M. Koch, and W. Zhang, “Asymmetric planar terahertz metamaterials,” Opt. Express 18(12), 13044–13050 (2010). [CrossRef] [PubMed]
12. I. Al-Naib, C. Jansen, and M. Koch, “Thin-film sensing with planar asymmetric metamaterial resonators,” Appl. Phys. Lett. 93(8), 083507 (2008). [CrossRef]
13. H. Tao, A. C. Strikwerda, M. Liu, J. P. Mondia, E. Ekmekci, K. Fan, D. L. Kaplan, W. J. Padilla, X. Zhang, R. D. Averitt, and F. G. Omenetto, “Performance enhancement of terahertz metamaterials on ultrathin substrates for sensing applications,” Appl. Phys. Lett. 97(26), 261909 (2010). [CrossRef]
14. C. Zhang, L. Liang, L. Ding, B. Jin, Y. Hou, C. Li, L. Jiang, W. Liu, W. Hu, Y. Lu, L. Kang, W. Xu, J. Chen, and P. Wu, “Label-free measurements on cell apoptosis using a terahertz metamaterial-based biosensor,” Appl. Phys. Lett. 108(24), 241105 (2016). [CrossRef]
15. H. Tao, L. R. Chieffo, M. A. Brenckle, S. M. Siebert, M. Liu, A. C. Strikwerda, K. Fan, D. L. Kaplan, X. Zhang, R. D. Averitt, and F. G. Omenetto, “Metamaterials on paper as a sensing platform,” Adv. Mater. 23(28), 3197–3201 (2011). [CrossRef] [PubMed]
16. L. Cong, S. Tan, R. Yahiaoui, F. Yan, W. Zhang, and R. Singh, “Experimental demonstration of ultrasensitive sensing with terahertz metamaterial absorbers: A comparison with the metasurfaces,” Appl. Phys. Lett. 106(3), 031107 (2015). [CrossRef]
17. R. Yahiaoui, S. Tan, L. Cong, R. Singh, F. Yan, and W. Zhang, “Multispectral terahertz sensing with highly flexible ultrathin metamaterial absorber,” J. Appl. Phys. 118(8), 083103 (2015). [CrossRef]
18. M. Maasch, M. Mueh, and C. Damm, “Sensor array on structured PET substrates for detection of thin dielectric layers at Teraherz frequencies,” in 2017 IEEE MTT-S International Microwave Symposium (IMS) (2017), pp. 1030–1033. [CrossRef]
19. C. Jansen, I. A. I. Al-Naib, N. Born, and M. Koch, “Terahertz metasurfaces with high Q-factors,” Appl. Phys. Lett. 98(5), 051109 (2011). [CrossRef]
20. Z. Geng, X. Zhang, Z. Fan, X. Lv, and H. Chen, “A route to terahertz metamaterial biosensor integrated with microfluidics for liver cancer biomarker testing in early stage,” Sci. Rep. 7(1), 16378 (2017). [CrossRef] [PubMed]
21. M. Chen, L. Singh, N. Xu, R. Singh, W. Zhang, and L. Xie, “Terahertz sensing of highly absorptive water-methanol mixtures with multiple resonances in metamaterials,” Opt. Express 25(13), 14089–14097 (2017). [CrossRef] [PubMed]
22. H. Li, H. M. Ye, and Y. Yang, “Characterizing polymorphism and Crystal Transformation of Polylactide by Terahertz Time-domain Spectroscopy,” Polym. Test. 57, 52–57 (2017). [CrossRef]
23. T. I. Jeon, D. Grischkowsky, A. K. Mukherjee, and R. Menon, “Electrical and optical characterization of conducting poly-3-methylthiophene film by THz Time-domain spectroscopy,” Appl. Phys. Lett. 79(25), 4142–4144 (2001). [CrossRef]
24. T. D. Dorney, R. G. Baraniuk, and D. M. Mittleman, “Material parameter estimation with terahertz time-domain spectroscopy,” J. Opt. Soc. Am. A 18(7), 1562–1571 (2001). [CrossRef] [PubMed]
25. X. Wu, B. Quan, X. Pan, X. Xu, X. Lu, C. Gu, and L. Wang, “Alkanethiol-functionalized terahertz metamaterial as label-free, highly-sensitive and specific biosensor,” Biosens. Bioelectron. 42(1), 626–631 (2013). [CrossRef] [PubMed]
26. S. J. Park, J. T. Hong, S. J. Choi, H. S. Kim, W. K. Park, S. T. Han, J. Y. Park, S. Lee, D. S. Kim, and Y. H. Ahn, “Detection of microorganisms using terahertz metamaterials,” Sci. Rep. 4(1), 4988 (2015). [CrossRef] [PubMed]
27. Z. Zhang, H. Ding, X. Yan, L. Liang, D. Wei, M. Wang, Q. Yang, and J. Yao, “Sensitive detection of cancer cell apoptosis based on the non-bianisotropic metamaterials biosensors in terahertz frequency,” Opt. Mater. Express 8(3), 659–667 (2018). [CrossRef]
28. S. J. Park, S. H. Cha, G. A. Shin, and Y. H. Ahn, “Sensing viruses using terahertz nano-gap metamaterials,” Biomed. Opt. Express 8(8), 3551–3558 (2017). [CrossRef] [PubMed]
29. J. F. O’Hara, R. Singh, I. Brener, E. Smirnova, J. Han, A. J. Taylor, and W. Zhang, “Thin-film sensing with planar terahertz metamaterials: sensitivity and limitations,” Opt. Express 16(3), 1786–1795 (2008). [CrossRef] [PubMed]
30. S. J. Park, B. H. Son, S. J. Choi, H. S. Kim, and Y. H. Ahn, “Sensitive detection of yeast using terahertz slot antennas,” Opt. Express 22(25), 30467–30472 (2014). [CrossRef] [PubMed]
31. M. Brucherseifer, M. Nagel, P. Haring Bolivar, H. Kurz, A. Bosserhoff, and R. Büttner, “Label-free probing of the binding state of DNA by time-domain terahertz sensing,” Appl. Phys. Lett. 77(24), 4049–4051 (2000). [CrossRef]
