The apoptosis of cancer cells was experimentally measured by terahertz (THz) biosensors based on the metamaterials (MMs). The non-bianisotropic resonance with an electric field of up to 106 V/m was exhibited at 0.85 THz, where the magnetic dipoles were cancelled in the unit cell. The simulate results show the dependence of the frequency shift on the occupying ratio and refractive index of analytes. The theoretical sensitivity was calculated to 182 GHz/RIU. The experimental results imply that the resonant frequency would red shift with the increase of the concentration of cancer cells. Furthermore, the apoptosis of cancer cells HSC3 under the effect of drug concentration from 1 to 15 μM and drug action time from 24 to 72 hours were also studied by the biosensors, respectively. It shows that the trend agrees with the results measured by the biological CCK-8 kits method. Our proposed MMs-based biosensors may supply a novel viewpoint on cell apoptosis from a physical perspective and be a valuable complementary reference for biological study.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Nowadays, terahertz (THz) science and technology is growing rapidly as a promising research field owing to the fast development in THz radiation sources and THz time domain spectroscopy (THz-TDS) [1–5]. The characteristics of THz waves in free space including transparent for most dielectric materials, non-destruction and broad bandwidth compared with millimeter enable THz technology to be widely used in variety of applications such as communications, medical sciences and security [6–8]. Further, the low average power and non-ionization of THz waves make it be promising candidate for the in situ diagnosis and non-destructive examination in bio- medical and chemical field. Recently, a portable THz spectroscopic tools facilitating on-site detection and identification of materials with high signal to noise ratio were reported [9,10]. Although this miniaturized THz spectroscopic technology has been conducted, this method is not suitable for trace amount analysis owing to the limitations of sample size and thickness. Alternatively, THz metamaterial sensing is not only signal-enhancing but also easy to operate, which attracts significant attentions from researchers, and many groups make great efforts to optimize the structure to obtain an improving sensitivity [11–15].
Metamaterials (MMs), a class of artificial arranged subwavelength structures, have been attracting enormous research concerns in community due to the extraordinary electromagnetic response that would not be possible with natural materials [16–20]. In addition, the strong localized electric and magnetic resonances generated by collective excitation of surface electrons of metal split ring resonators (SRRs) also lead to MMs showing significant application in biological sensing. Therefore, the biosensors combined with MMs owning typical resonance provide an excellent platform for ultrahigh sensitivity in THz devices, allowing the detection of extremely small amounts of biomolecules and/or cells. More importantly, in comparison to the culture-based detection methods used to sense even quantify cells, the MMs-based biosensors cost relatively low and require no complex fluorescence labelling procedure. Such advantages of the MMs-based biosensors open up a door for the cells detection into label-free, low cost and fast process, being promising method in future biological sensing and disease diagnosis.
Cancer is one of the leading causes of death worldwide, and its diagnosis is critical to initiate therapies . The apoptosis of cancer cells plays a pivotal role in shaping of organs in tandem with cell proliferation, regulation, and the removal of defective as well as excessive cells in immune system [22–24]. Hence, it is imperative to develop sensitive detection technologies that cannot only identify cancer cells at early stages, but also the cell apoptosis under the effect of anti-cancer drug action. In previous works, Liang et al. designed five concentric subwavelength gold ring resonant structures in order to sense the apoptosis of oral cancer cells under the influence of cisplatin. However, the induced magnetic dipoles resulting from circular rings produced resonant peaks with wide bandwidth. Besides, the dependence of cell apoptosis was investigated from only one perspective, namely, the effect of drug concentration . Here, we reported a THz MMs-based biosensor that consists of periodic metal SRRs array on dielectric layer. By well designing structural parameters, the MMs exhibits non-bianisotropic resonant response at 0.85 THz, where the magnetic dipoles were cancelled due to clockwise and counterclockwise components in adjacent regions of the unit cell, and the pure electric response produces a resonance undisturbed from magnetic overlying in THz range. In addition, the strong localized electric field up to 106 V/m make the MMs potentially be applied in sensitive biosensors. The simulate results show that the increasing of occupying ratio and refractive index of analytes supported on MMs would both lead to resonant frequency red shift, and the theoretical sensitivity defined by the slope of frequency shift function depending on refractive index can approach 182 GHz/RIU. In experiments, the proposed MMs-based biosensors present according red-shift with the rise of cells concentration from 1 × 105cell/ml to 3 × 105cell/ml. The apoptosis of cancer cells HSC3 under the effect of drug concentration from 1 to 15 μM and drug action time from 24 to 72 hours was also investigated by MMs-based biosensors. It is implied that the cancer cells HSC3 can be effectively killed under the improving of drug concentration and the prolong of drug action time. Such trend agrees with the results measured by biological CCK-8 kits method. It demonstrates the feasibility of our proposed MMs-based biosensors to sense the cancer cell apoptosis, and may supply a novel viewpoint on cells apoptosis process from physical perspective and be valuable complementary reference for biological study.
2. Simulation and experiment methods
In our works, the SRRs structures with double splits were designed on 10 μm-thick polyimide (PI) dielectric layer, as schematically shown in Fig. 1(a) and (b). The period of unit cell p is 50 μm and the metal thickness is 200 nm. The geometrical parameters of structure are: a = 36 μm, l = 4 μm, d = 3.5 μm and s = 4 μm. The structural size of MMs unit cells was optimized. The most suitable dimensions were adopted not only to acquire the strongest electric field but also to design the resonance frequency around 1 THz to be convenient for THz-TDS measurement. The full-wave numerical simulations are performed by commercial finite integration package CST Microwave Studio. In our simulation, the permittivity of PI layer is ε = 3.1 and loss tangent is 0.05. The magnetic and electric boundary conditions along x and y directions was adopted respectively to simulate y-polarized plane wave, which incident through the MMs from bottom dielectric to top metal layer. To verify the property of proposed biosensors, the MMs samples were fabricated in experiment. Firstly, 10 μm-thick PI layer was spin-coated on silicon wafer, followed by patterning of periodic SRRs structure by standard photolithography. Then the Ti and Au layer with the thickness of 20 nm and 200 nm were deposited on PI layer, respectively. Finally, the bottom silicon wafer was peeled off using HF solution. The microscope image of structure was presented in Fig. 1(c). To research the ability of cell detection of MMs-based biosensor, we select single layer epithelium-derived oral cancer cell (HSC3) as analytes. When cells of multicellular organisms are cultured in vitro, contact inhibition occurs when adherently growing cells touched each other, i.e., cells stop moving and migration in a particular direction, which leading to the conformation of single-layer structure of cancer cells to keep the same thickness of analytes layers. Based on this, we studied the adherent cells HSC3. Both cells were grown in Dulbecco's Modified Eagle's medium (DMEM) (HyClone, GE Healthcare, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO2 or 95% humidified atmosphere incubator. After the adherent cells are collected by 0.25% trypsin-EDTA (HyClone, GE Healthcare, USA), they are washed again with phosphate buffered saline (PBS) and then prepared into a single cell suspension. For seeding cells, THz MMs were sterilized with 75% ethanol and attached to the bottom of a 24-well culture plate. Subsequently, a single cell suspension of a certain concentration was seeded on the surface of the THz MMs as a monolayer and cultured at 37°C in a 5% CO2 or 95% humidified atmosphere incubator. The corresponding micrograph of MMs-based biosensor coated with cells was shown in Fig. 1(d), it needs to be mentioned that since the picture was taken by inverted microscope, the front seen in Fig. 1(d) is the bottom of MMs in fact. Before performing the THz-TDS measurement, the MMs with cells cultured on were taken out from 24-well culture plate, then immersed in water and dried in the nitrogen atmosphere for several seconds to eliminate the influence of other substances including residual drugs and water. To obtain the transmission spectrum, THz-TDS was performed to measure the MMs under the normal incidence.
3. Results and discussion
Due to the electromagnetic resonance resulted from the effect of surface plasmon resonance, the proposed MMs structure would exhibit typical resonant dips and/or peaks in certain frequency range under the THz waves radiation. At the same time, the strong localized electric field and the certain direction of surface current flow emerged on the designed MMs surface. The simulate and experimental transmission spectrum of our designed structure without cancer cells were shown in Fig. 2(a). It is observed that two curves reached a relatively well agreement except for a little discrepancy on the transmission amplitude, which can be attributed to the measuring atmosphere and fabrication deviation. Moreover, it can be found from experimental result that the structure exhibits a strong resonance at 0.85 THz with the transmission decreasing to 7.9% and a weak resonance at 3.6 THz with the near zero transmission, respectively. We first consider the lower frequency resonant response (0.85 THz) presented in MMs structure. This resonance originates from circulating currents in the ring components of SRR and results in a pure electric response as a result of magnetic dipoles eliminating due to the counter-circulating currents in each loop, more details will be discussed later. In addition to the lower frequency resonant response, there is a second transmission minimum at higher frequency (3.6 THz), which originates from the excitation of electric dipoles. For this higher resonance, the strong loss gives rise to a weak signal and bad Q factor in experimental measurement, thus in the remainder of this paper we will focus on the lower frequency resonant response to evaluate the performance of MMs-based biosensors.
More specifically, Fig. 2(b) and (c) show the surface current density and electric field at two resonant responses, respectively. It can be seen from Fig. 2(b) that the incident THz waves drive a circulating surface currents in four loops of SRR which results in the charge accumulation at capacitive split gap. Therefore, the electric field would be localized and enhanced strongly, as the electric field simulation reveals that the red regions at gap indicate a strong local field enhancement up to 106 V/m. On the other hand, the arrow orientation representing the flow of surface current enables to visualize the absence of magnetic response: from Fig. 2(b), one can see clearly that the magnetic dipole driven by circulating surface currents would be cancelled due to clockwise and counterclockwise components in adjacent regions of the unit cell. Thus, any resonant response must necessarily be of electrical origin since there is no net circulation of current in each unit cell. That is, these structures are not bianisotropic [26,27]. In comparison to the lower frequency resonance, the electric dipole resonant mode at higher frequency was presented, seen from Fig. 2(c), with a slight decline in local field amplitude.
As a result of the strong electric field localized on the proposed MMs structure, a small variation in dielectric environment would lead to the original resonance of MMs shift. Such relationship can be described according to perturbation theory, the relative change of the resonant angular frequency Δω/ω0 is :Eq. (1) were ignored. The advantage of such resonant feature is that it allows us to discuss the dependence of MMs resonant response on one factor without considering the perturbation of induced magnetic resonance along z direction. Moreover, according to Eq. (1), such structural design can avoid the sensitivity reduction as a result of the decline of electric field due to that the introduction of magnetic dipoles resulted from circulating current may transform the electric energy into magnetic ones. Besides, the coupling between electric and magnetic resonance can also be avoided, thus making the influence of biological analytes on MMs resonance more explicit. To have an insight to the dependence of biological analyte on resonance of MMs, we created and placed a dielectric disk with thickness of 4 μm and ε = 1.96 upon SRRs structure in simulation to model the average contact region of cells on MMs-based biosensors in practice.
Thus, we can define the parameter of occupying ratio (OR) that equals the ratio of analyte area to unit cell area to illustrate the influence of the biological cells number on resonant response of MMs, i.e., the larger OR value is, the more cells were supported on MMs. Figure 3(a) shows that the resonance dip in transmission of MMs emerged a red-shift relative to that of bare MMs with the increase of OR value, implying the potential ability of MMs structure to detect analytes supported on MMs as biosensors. To clarify this change more explicitly, the effect of OR value on resonance frequency shift Δf is extracted and shown in Fig. 3(b). The enhanced Δf with the increase of OR demonstrates that the incremental amounts of analytes can make the resonant response of biosensor shift away from original resonance position for bare condition more distance. On the other hand, we also investigated the effect of different refractive index analytes on the proposed MM-based biosensor through simulations. At the case of 4 μm-thick analyte layer, a series of THz spectrum of MMs under different refractive index n are shown in Fig. 3(c) and (d). The results reveal that the MMs structures were sensitive to the change of dielectric property of analyte, the according sensitivity S that was defined as derivative of Δf with respect to Δn can be obtained from linear relationship in Fig. 3(d), and the result S was calculated as 182 GHz/RIU.The experimental results exhibited a similar trend with simulations as well. In experiment, we cultured three different concentrations of cancer cells HSC3 on MMs as biosensor analytes. Figure 4(a) and (b) present corresponding transmission spectrum and their Δf at three concentrations, respectively. In order to make better comparison, the transmission spectrum of bare biosensor was also given. It can be clearly seen that when the cells concentration arrived at 1 × 105 cell/ml, the resonant frequency shift Δf is as small as 7.5 GHz. By reducing the cells concentration, the Δf cannot be presented any more (the result was not shown here) due to the restriction of resolution of THz-TDS instrument. Therefore, the detection limit of designed MMs biosensors in our works was approximately estimated to 1 × 105 cell/ml. Additionally, the increase of cell concentration will cause an increase of Δf, and the maximum shift reached 23 GHz when the cell concentration was at 3 × 105cell/ml. This dependence of the cell number on Δf is similar with the previous simulation results, which qualitatively shows the enhanced Δf under the increase of cells number.
Due to the sensitivity to the cell concentration of designed biosensors, the detection of apoptosis of cancer cells under the effect of cisplatin, a kind of chemotherapy medication, turns to be possible. In reference , the induced magnetic dipoles resulting from circular rings produced resonant transmission peaks with wide bandwidth. Besides, the dependence of cell apoptosis was investigated from only one perspective, namely, the effect of drug concentration. These disadvantages limit the comprehensive investigation on the cells apoptosis. To have an insight of such process, in our works, we not only obtained a resonant dip with narrow bandwidth, but also studied the apoptosis of cancer cells from drug concentration and drug action time, respectively. From biological perspective, such methodology is more reasonable for the research of cells apoptosis. For drug concentration effect, we first separated the natural growth cells with concentration of 2 × 106cell/ml into three groups, then treated with 1 μM, 8 μM and 15 μM of cisplatin for 72 hours to induce cell apoptosis, respectively. As for time effect, the same natural growth sample were also separated into three groups. The 15 μM cisplatin was added into samples for 24 hours, 48 hours and 72 hours, respectively. The transmission spectrum under the two conditions were given in Fig. 5. It can be seen from Fig. 5(a) and (b) that the resonant frequency of drug-induced samples emerged a shift compared with the bare one, illustrating that with the introduction of drug, there emerged an alteration in cell concentration on biosensors. Further, the fact that the extent of the shift in resonance is different with the change of drug concentration and action time was verified. To clearly observe the trend of this change, the dependence of frequency shift Δf on drug concentration and time duration were exhibited in Fig. 5(c) and (d), respectively. It can be found that the Δf declined from 75 to 45 GHz when the drug concentration increased from 1 to 15 μM. On the other hand, when the time duration of drug action expanded from 24 to 72 hours, the Δf declined from 76 to 46 GHz. Especially, no matter either the drug concentration or drug action time, the Δf has reached the minimum at the maximum of drug concentration and drug action time. Since the Δf is related to the cells number as previous discussed, it implies that with the improvement of drug concentration and the prolong of drug action time, the number of cancer cells would emerge a decrease, which demonstrates the effective kill of cisplatin to HSC3 cells and the apoptosis of cancer cells in progress.
Additionally, to verify the accuracy of MMs-based biosensors in the detection to the apoptosis of cancer cells, the CCK-8 method, a kind of biological method in terms of kits for diagnosis of cancer cells were used for comparison. As a result of the augment in apoptosis under the effect of anti-cancer drug, the measured data were dealed as the complement of apoptosis in percentage, i.e., the survival rate in nature. The results were also shown as the dashed line in Fig. 5(c) and (d). It can be clearly seen that with the increase of drug concentration and drug action time, the survival rate decreased from 86% to 31% and 66% to 31%, respectively. Such trend agrees with the measured Δf change, demonstrating the feasibility of our proposed MMs-based biosensors to sense the cancer cell apoptosis.
The apoptosis of cancer cells was investigated by MMs-based THz biosensors. The MMs structures were designed to be illuminated by THz beams from bottom dielectric layer to upper metal structures. The simulate results demonstrate that the structures show two resonant dips at 0.85 and 3.6 THz, respectively. The corresponding surface currents and electric field distribution at 0.85 THz reveal the non-bianisotropic resonant response. At the same time, the strong localized electric field up to 106 V/m make the MMs have ability to sense the change of dielectric environment. By changing the occupying ratio and permittivity of analytes on MMs, the resonant frequency exhibits a shift compared with that of bare MMs, illustrating the proposed MMs have potential to be sensors to detect different amounts and dielectric property of analytes. The theoretical sensitivity of such MMs-based sensors was calculated up to 182 GHz/RIU. In experiments, we cultured different concentrations of cancer cells HSC3 on the biosensors. The results verify that the frequency shift Δf would increase with the growing cancer cells concentration. Based on the Δf, the apoptosis of cancer cells HSC3 under the effect of drug concentration from 1 to 15 μM and drug action time from 24 to 72 hours was also investigated. It can be concluded that with the improving of drug concentration and prolonging of the drug action time, the Δf gradually decreases, illustrating that the cancer cells were effectively killed and apoptosis is in progress. The results measured by biological CCK-8 kits method agree with the trend of Δf measured by proposed MMs-based biosensors. It demonstrates the feasibility of our proposed MMs-based biosensors to sense the cancer cell apoptosis, and may supply a picture on cells apoptosis from physical perspective and be valuable complementary reference for biological study.
National Natural Science Foundation of China (61701434, 61735010); the Open Fund of Key laboratory of Opto-electronic Information Technology, Ministry of Education (Tianjin University); Key Laboratory of Optoelectronic information functional materials and micro-nano devices in Zaozhuang; the China Postdoctoral Science Foundation (2015M571263); the National Natural Science Foundation of Shandong Province (ZR2017MF005); A Project of Shandong Province Higher Education Science and Technology Program (J15LN36, J17KA087); the Program of Independent and Achievement Transformation plan for Zaozhuang (2016GH19, 2016GH31); Zaozhuang Engineering Research Center of Terahertz and the Doctoral Foundation (2015M571263).
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