Terahertz Spectra Of Living Cells With The Construction Of Label-free Metamaterial-based Biosensor

Background:As the basic structural unit of life,the analysis of cellular function and structure plays a key role in the research area of life science.Living cell analysis method can be divided into labeled and label-free method.Based on reporter molecules such as fluorescein and nuclide,labeled living cell analysis method is the core method for the research of cell biology,which can reveal the pathophysiological characteristics of cells,tissues and organs at the molecular level.However,the labeled method may affect the structure and function of target biomolecules,and the operation process is complicated,making it difficult to obtain the characteristic information under the intrinsic state of living cells.The label-free method,analyzing the biophysical characteristics of living cells（mechanical,electrical and optical properties）,can reveal the basic life process such as cell proliferation,division,apoptosis and interaction,which has a great potential in the analysis of blood cells and circulating tumor cells（phenotypic analysis and chemosensitivity test）.At present,it is still necessary to expand the depth and breadth of research on the biophysical characteristics of living cells,improve and increase the systems for label-free detection of living cells,and provide multiple platforms for mature industrial application such as clinical laboratory diagnosis,drug screening,food engineering and environmental toxicity assessment.Terahertz(THz,1 THz=10¹² Hz)wave technology is a research hotspot in the interdisciplinary field of biophysics,with unique advantages different from traditional label-free method for living cells:（1）THz wave can obtain the low-frequency collective vibration pattern of biomolecules,especially for cellular genome methylation status whose characteristic vibrational mode locates in THz band（¹.6 THz）.（2）THz spectroscopy can obtain intracellular hydration dynamics parameters at the timescale of sub-picosecond to picosecond.（3）With a short measurement time,THz spectroscopy is suitable for medical imaging,especially for label-free in vivo detection.（4）As a non-ionizing method,THz spectroscopy will not damage the biologic activity of cells.At present,THz attenuated total reflection（THz-ATR）spectroscopy and metamaterial technique are mainstream methods for detecting living cells,which could solve the problem from strong absorption of culture medium and mismatch between THz wavelength and cell size.These methods have been used for label-free measurement of the intracellular hydration dynamics mode,saponin-induced cellular permeability change,anti-tumor drug induced cellular apoptosis and cellular oxidative stress state.However,the THz-ATR platform needs to culture cell monolayer at the interface of the ATR prism with the help of a fixed incubation chamber to maintain a suitable environment,resulting in a low detection flux（only one sample can be detected at a time）,long detection cycle（It will take 48 h for cell suspension to grow into cell monolayer）.With the advantages of high sensitivity and portability,THz metamaterial chips can reflect the relative complex dielectric changes of the nearby environment corresponding to the change of cell pathophysiological state.However,current researches are based on transmission mode and need to remove extracellular medium,which may affect the normal physiological state of living cells.Finally,the changes of metabolic state of malignant tumor cells often occur before the changes of cellular morphology.The pathway of glucose metabolism and intracellular glucose content are crucial characteristics of the metabolic state for tumor cells and normal cells.Unfortunately,due to the strong attenuation of water,THz wave cannot specifically distinguish glucose molecules in liquid phase.Aiming at the above problems,we firstly tried to build a THz-ATR platform combined with algorithm model to extract the complex dielectric constant of cells seeded onto the porous membrane,which could separate the cell culture process and detection process,and achieve label-free as well as rapid measurement.The high-sensitive and smart THz metamaterial chip was used to develop a cell sensor.Simulations and experiments were conducted to verify the feasibility of this self-referenced metamaterial in reflection mode,which proved to be a rapid and portable cell sensor.To improve the polarization-dependent property and reflection intensity of the above metamaterial cell sensor,a polarization-insensitive circular ring THz metamaterial array based on EOT（extraordinary optical transmission）effect was designed for detecting cell density variation,thrombin-induced cellular morphology change,cervical cells with different degrees of malignancy and docetaxel-induced cell apoptosis in a label-free manner.Finally,the strong absorption of water in THz band and the specific water content change of stimuli-responsive hydrogels were organically combined to improve the specificity of THz detection,and the hydrogel-functionalized THz metamaterial chip was also constructed.Through above research,we built a set of complementary and flexible THz platforms and methods for label-free sensing living cells,namely THz-ATR device for extracting absolute value of complex dielectric constant of living cells,high-sensitive and portable THz metamaterial cell sensor array,and hydrogel-functionalized THz metamaterial chip for specific detection of glucose,which could pave the way for further clinical applications of THz technology,such as detecting circulating tumor cells,analyzing target biomolecules in the body fluids,and intraoperative imaging.Methods:1.THz spectra of living cells and evaluation of cell migration by THz-ATR spectroscopy:The laser scanning confocal microscope was used to acquire the thickness of normal cervical epithelium cell line（HCerEpiC cell）,cervical precancerous lesion cell line（Ect1/E6E7 cell）,and cervical cancer cell line（Hela cell）.THz-ATR platform and matched algorithm model were built to extract the complex dielectric constants of cell monolayer adhered to the porous thin-film.Then the Hela cells with starvation treatment for 24 h were seeded into the Transwell chamber,and different volume fraction（1%,2.5%,5%,10%）of fetal bovine serum were used as the chemokines.The built THz-ATR platform was used to detect the density distribution of Hela cells on the lower surface of porous thin-film to assess cell migration ability,and compared with crystal violet stain method.2.The research of self-referenced THz metamaterial cell sensor:THz metamaterial chip was fabricated on a 550-μm-thick high-resistance Si substrate,and the resonator arrays of split square structures were defined with a central gap on each side.Finite difference time-domain（FDTD）simulation was conducted to investigate the response of living cells,the distribution of resonant electric field,the saturated sensing area,and the signal variation of water content change in living cells.We used the self-referenced THz metamaterial to detect the artificial density distribution of MDCK cells and saponin-induced cell permeability change,and analyze the relation between cell density and resonant peak amplitude.3.The research of polarization-insensitive circular ring THz metamaterial sensor array:The circular ring metamaterial based on EOT effect,was integrated with a self-designed holder,in order to assembled into a THz metamaterial array with 2×3 holes inside.FDTD simulation was conducted to analyze the response of living cells,the signal variation of water content change in living cells and sensitive sensing area.Then the metamaterial array was used to detect the density distribution of HUVEC cell,thrombin-induced morphology change of HUVEC cell,cervical cells with different degrees of malignancy and docetaxel-induced apoptosis of Hela cells.The CCK8 counting method was used as contrast method.4.THz spectra of glucose-sensitive hydrogels and the construction of hydrogel-functionalized THz metamaterials chip:Glucose-sensitive hydrogel was prepared by ultraviolet crosslinking method.The 3-（acrylamido）phenylboronic acid（AAPBA）content of hydrogel was optimized by weighing analysis.THz-ATR spectroscopy and weighing analysis were used to obtain THz absorption spectra and water content changes of glucose-sensitive hydrogels presented in various concentrations of glucose solution.The changes of THz absorption and water content of hydrogels were fitted and analyzed.The sensitivity,anti-interference ability and repeatability of the proposed method for measuring glucose solution were obtained.A 20-μm-thick hydrogel layer was crosslinked on the surface of THz metamaterial chip with the help of aluminium foil gasket.The response of resonance peak,sensitivity,anti-interference ability and repeatability of the hydrogel-functionalized metamaterial chip were also obtained.Results:1.We obtain the THz absorption coefficient,refractive index,real and imaginary part of complex dielectric constant of normal cervical epithelium cell line（HCerEpiC cell）,cervical precancerous lesion cell line（Ect1/E6E7 cell）,and cervical cancer cell line（Hela cell）.The main difference between culture medium and living cells were the imaginary part of complex dielectric constant.These absorption differences also existed between three cell lines within0.5-1.2 THz,showing an increasing trend of dielectric loss and absorption with the increasing degree of malignancy.For the migration experiment,the distribution density of Hela cells on the porous thin-film changed with the volume fraction of fetal bovine serum.The THz absorption coefficient decreased with the increase of cell distribution density.The trend of THz absorption variation was linearly correlated with crystal violet staining analysis results,and the R² was determined as 0.99107.2.The resonant peak of reflective self-referenced THz metamaterial chip in liquid phase was in good agreement with the simulation signal.The resonant peak located near 0.9 THz and the amplitude changes was about 12%.Compared with the high-resistance Si substrate,it can obtain about 10 times signal enhancement.The resonant peak of the sensor was not affected by the thickness variation of the solution layer（>100μm）.Within the common cell thickness range（5-20μm）,the change of dielectric loss caused by the change of cellular water content can be effectively detected.Without any need for reference measurement,this chip can monitor saponin-induced cell permeability change in a real-time as well as label-free manner.The plateau phase for saponin-membrane interaction and concentration-dependent resonance peak variation can be observed.The resonance peak amplitude was used to evaluate the cell density distribution on the surface of the metamaterial chip.There was a good linear correlation between the change of peak amplitude and the average number of cells adhered to the resonant structure,and the R² was determined as 0.9914.3.The resonant peak of the circular ring metamaterial array was in good agreement with the simulation signal.The resonant peak was near 0.5 THz,and the peak amplitude variation between cell and culture medium was about 21%.The simulation results showed that the resonant peak can effectively respond to the change of dielectric loss caused by cellular water content change,with a good linear correlation（R²=0.9995）.The enhanced electric field of the metamaterial chip mainly located inside the ring slit.More than 90%variations of the resonance peak corresponded to the 10-μm-wide simulated cell monolayer in the center of the ring slit.And the resonant peak amplitude changed exponentially with the width of simulated cell monolayer varying from 0μm to 10μm.Correspondingly,this metamaterial array could monitor the thrombin-induced morphology change of HUCVEC cells real-timely.Meanwhile,the resonance peak variation for HCerEpiC,Ect1/E6E7,and Hela cells correlated well with the change of their extinction coefficients.The signal for cell density change and docetaxel-induced cell apoptosis showed a good linear correlation with CCK8 method.4.The optimum mass fraction of AAPBA for the glucose-sensitive hydrogel was determined as 9%.The water content of the hydrogel（measured by weighing analysis）increased with the augment of the concentration of glucose solution,and therewith the increase of THz absorption coefficient.The water content change of glucose-sensitive hydrogel showed a good linear correlation with the change of absorption coefficient at 1 THz.The limit of detection（LOD）for this THz-ATR method was determined as 16.18 mg/dL.The interference of lactic acid,fructose and galactose can be eliminated obviously with a good repeatability.The resonance peak amplitude of hydrogel-functionalized metamaterial chip increased as the augment of the concentration of glucose solution.The LOD of this metamaterial was 3.92 mg/dL,with a good specificity and repeatability.Conclusion:1.The built THz-ATR platform can obtain the complex dielectric constants of living cells adhered to the porous film in a high-throughput manner.The imaginary part of the complex dielectric constants of living cells proved to be related to the degree of cell malignancy,namely the rise of hydration state and metabolic activity for tumor cells,which was useful for label-free analysis of circulating tumor cells.The cell distribution density of lower part of Transwell chamber can be measured within 1 minute in a label-free manner by THz-ATR platform.It is expected to develop a series of new methods for evaluating the ability of cellular invasion and migration in Transwell experiment.2.The self-referenced THz metamaterial cell sensor in reflection mode can effectively acquire the characteristic signals of living cells in liquid phase,and detect the changes of cellular dielectric properties and cell density distribution.This self-referenced method proved to be suitable for long-term monitoring without any requirements for reference measurement.It could be integrated into common cell culture plates to develop a label-free,in situ cell sensor.3.The THz circular ring metamaterial array was not sensitive to the polarization state of THz wave,which is convenient for practical measurements.It can also effectively acquire characteristic signals of living cells in liquid phase.The sensitive sensing area locating in the slit,was helpful to monitor subcellular structure change,and detect cell density distribution,cells with different degrees of malignancy and cell apoptosis in a label-free manner.Further research will focus on developing a label-free and convenient system for the tumor chemosensitivity analysis of circulating tumor cell.4.THz-ATR platform can effectively evaluate the degree of water content change during the specific swelling process of stimuli-responsive hydrogel presented in targeted biomolecule solution.With the help of stimuli-responsive hydrogel,the biomolecule solutions could be quantitatively determined with high specificity.With the LOD of 3.92 mg/dL for glucose sensing,the hydrogel-functionalized THz metamaterial chip could provide great potential to analyze cellular glucose metabolism,and develop a series of THz biosensors with high biocompatibility,specificity and sensitivity.