THz Sensing Chip For Detection Of Breast Cancer Cells And MicroRNA Based On Optical Clearing Agent And Metamaterials

Background:Breast cancer is by far one of the most frequent tumors among women worldwide.Although the diagnosis and treatment techniques have been developed in the past few decades,it is still difficult to effectively reduce its late mortality rate.In recent years,the detection of various circulating markers with tumor molecular characteristics,including circulating tumor cells（CTCs）,circulating tumor DNA（ctDNA）,circulating microRNAs,and exosomes,is becoming a hotspot in the field of early diagnosis of tumors.Among them,tumor cells and microRNAs respectively represented the physiological and pathological characteristics of tumors at the cellular and molecular levels.They are expected to become new biomarkers for early diagnosis and prognosis monitoring.However,the existing detection methods for tumor cells and microRNA struggled to meet the demand of clinical application.Therefore,a new biomedical detection method is urgently needed.Terahertz（THz）technology is a rapid,label-free,and noninvasive optical detection method,which may provide new ideas for the detection of tumor cells and microRNA.However,there are still two significant bottlenecks before its applications in the detection of tumor cells and microRNA molecules:（1）Strong water absorption:Because THz radiation exhibits excellent sensitivity to water molecules,medium surrounding the living cells inevitably causes strong THz energy attenuation.Besides,the THz responses of the cells can be largely masked by the background signal interference of the medium,markedly restricting the detection of living cells.Considering that most biological samples from the human body are rich in water or must be cultured in a liquid phase environment,THz detection of tumor cells must overcome the interference of strong water absorption.（2）Low sensitivity:Without any signal amplification,the sensitivity of nucleic acid molecules detection using THz spectroscopy is only up to the nanomolar level,which struggles to meet the demand for detection of microRNA in clinical samples.Since the number of breast cancer-specific microRNA molecules is only about 0.01%of the total RNA in blood samples,to apply THz spectroscopy in the microRNA detection,it is necessary to find a method for sensitivity improvement.Optical clearing agent（OCA）is a class of organic solvent with excellent biocompatibility and low light scattering coefficient.It has been reported that OCA can enter the biological tissue and replace the water molecules in tissue,thus reducing THz signal attenuation and improving the penetration depth.This may provide great inspiration for eliminating the interference of strong water absorption,but the application of OCA in cell detection has not been reported.Therefore,screening a new OCA,which can eliminate the interference of strong water absorption during cell detection,become one of the leading research directions in this paper.Metamaterials are artificially engineered electromagnetic materials consisting of sub-wavelength metallic arrays.This material can adjust the propagation of electromagnetic waves,including THz radiation.As an attractive tool for improving the detection sensitivity of THz technology,metamaterials can induce localized electric field enhancement and thus probe tiny amounts of substances deposited over their surface.Besides,as the frequency shift of metamaterials increases with the increase of the refractive index of targets,samples combined with high refractive index particles（such as gold nanoparticles）can easily obtain more robust THz response on metamaterials.However,the mechanism of using nanoparticles to enhance THz metamaterial sensing has not been fully clarified,and the effects of different nanoparticles on the sensitivity of THz metamaterial biosensors have not been systematically investigated.Therefore,the combination of metamaterial technology and nanoparticle and its application to improve the sensitivity of THz spectroscopy is another direction of this paper.In this study,we carried out the theoretical basis,method construction,and practical application researches on eliminating the strong water absorption using OCA and improving the sensitivity of THz measurement using metamaterial.A THz sensing chip based on OCA and metamaterial was developed for the detection of breast cancer cells and microRNA.In the breast cancer cell detection,fluorinated oil was screened as OCA to solve the strong water absorption issue.It could replace the medium surrounding breast cancer MDA-MB-231 cells.Through the construction of a two-dimensional THz absorption model of cell samples in the fluorinated oil environment,the mechanism of THz signal difference between cells in water and fluorinated oil environment is revealed.This work proved the theoretical support for OCA technology in THz measurement of breast cancer cells.Subsequently,we combined OCA and microfluidic technology to construct a THz microfluidic chip.Its feasibility of quantifying cell viability was verified by detecting breast cancer cells treated with two anti-tumor drugs.Thus,a rapid,label-free,and noninvasive method for cell viability assays was developed.We also realized the uniform dispersion of cell suspension in OCA using the droplet-based microfluidics technology and constructed a new THz detection mode through measuring"cell droplet"consisting of tumor cell suspension.In the breast cancer-specific microRNA detection,we combined the metamaterial,nanoparticles,and strand displacement amplification（SDA）for the detection of microRNA-21,which improved the sensitivity of traditional THz measurement mode.Through the research on the combination of nanoparticles and SDA products,as well as the simulation and experimental investigation of the dependence of the metamaterial sensitivity on the nanoparticle diameter and metal type,we have screened out a nanoparticle with the best signal response on metamaterial.Besides,we constructed and optimized a THz metamaterial nanochip that integrates metamaterial,nanomaterial,and SDA.A series of key methodological parameters,such as sensitivity,specificity,and repeatability for microRNA-21 detection,were obtained and the clinical sample detection was carried out.Through the above work,this study summarized the methods of eliminating the strong water absorption and improving the sensitivity of THz measurement for the detection of breast cancer cells and microRNA molecules.The THz microfluidic chip can be used for the quantitative detection of breast cancer cell viability.THz metamaterial nanochip can be used for high sensitivity detection of breast cancer-specific microRNA molecules.All these findings provided a practical technical approach for the investigation of breast cancer circulating markers.Methods:1.OCA screening for breast cancer cells detection using THz spectroscopy:Nine common organic solvents were selected and CCK-8 method was used to measure the cell survival rate after interaction with breast cancer MDA-MB-231 cells to evaluate their cytotoxicity.The absorption coefficient and refractive index of candidate OCAs were obtained and compared with a DMEM medium to evaluate their THz absorption.The best OCA was selected based on the results of cytotoxicity and THz absorption.2.Replacing the water surrounding breast cancer cells using OCA:breast cancer MDA-MB-231 cells are cultured on PE sheets to make cell climbing sheet samples,which are loaded into a liquid sample detection pool and then replaced with the extracellular water by injecting OCA and flushing the PE sheets with a syringe.Breast cancer cells in the culture medium environment and OCA environment were measured using THz spectroscopy,respectively.3.Construction and application of OCA-based THz microfluidic sensing chip:The OCA-based THz microfluidic sensing chip is designed and fabricated using COP materials.The closed space inside the chip is used to realize accurate control of sample thickness and complete replacement of extracellular water.The THz microfluidic chip was applied in the detection of breast cancer cells subjected to two anti-tumor drugs.The feasibility of quantitative detection of cell viability is verified by comparing the results with traditional flow cytometry.4.Construction and application of THz droplet-based microfluidic chip:A droplet-based microfluidic chip is designed and fabricated using PDMS material.By controlling the flow rate ratio of OCA and tumor cell suspension entering the chip,uniform 50μm diameter OCA-coated tumor cell suspension droplets are stably generated.Theses droplets were measured using THz spectroscopy for the detection of suspended tumor cells.5.Construction of THz metamaterial sensing system for microRNA detection:We designed a single-stranded DNA probe with a specific recognition site and constructed the SDA reaction system for microRNA-21 detection.PAGE analysis was carried out to validate the performance of SDA.We also fabricated a metamaterial subwavelength resonant unit for detecting SDA products of microRNA-21.Ultraviolet-visible absorption spectrum,transmission electron microscope and Zeta potential measurement were carried out to validate the SDA products.6.Signal enhancement effect of nanoparticles on THz metamaterial:We carried out the researches on the combination of nanoparticles and SDA products,as well as the simulation and experimental investigations of dependence of the metamaterial sensitivity on the nanoparticle diameter and metal type.Based on the signal response of different nanoparticle on metamaterial,the optimal nanoparticle with the best signal enhancement effect was screened.7.Construction and performance characterization of THz metamaterial nano-sensing chip:Based on the previous studies on the THz metamaterial sensing system,we optimized various detection conditions and completed the construction of a THz metamaterial nano-sensing chip.A series of key methodological parameters,such as sensitivity,specificity,and repeatability for microRNA-21 detection,were characterized.8.Clinical value evaluation of THz metamaterial nano-sensor chip:The recovery rate experiment of THz metamaterial nanochip for detecting microRNA-21 molecules in clinical serum samples was carried out,which verified the effect of its application in practical sample detection.Results:1.Fluorinated oil retained cell viability of more than 85%over 60 min of treatment,which is better than any other candidate OCA.The absorption coefficient of fluorinated oil at1 THz at room temperature is about 10 cm^-1,which is only about 1/25 of that of DMEM.According to the cytotoxicity and THz absorption,fluorinated oil is selected as the best OCA for THz detection of living cells.After replacing the water molecules around breast cancer cells with fluorinated oil,the THz signal of cells,which is undetectable in the DMEM environment,becomes detectable in the fluorinated oil environment.This result confirmed the feasibility of fluorinated oil to reduce THz signal attenuation and eliminate strong water absorption.Through the construction of a two-dimensional THz absorption model of cell samples in the fluorinated oil environment,the mechanism of THz signal difference between cells in water and fluorinated oil environment is also revealed.2.After the precise control of the sample thickness and the complete replacement of the extracellular water using the closed space inside the THz microfluidic chip,the THz time-domain waveforms of cells are different from that of the pure fluorinated oil.By conducting an independent t-test,statistically significant di erences were observed between the fluorinated oil with and without cells（P<0.05 at 0.5,1.0,and 1.5 THz）,whereas no statistically significant di erences were observed between DMEM with and without cells.（P>0.05 at 0.5,1.0 and 1.5 THz）.3.After the treatment of cisplatin and paclitaxel with different concentrations,the THz normalized amplitude of human breast cancer MDA-MB-231 cells samples gradually decreases with the increasing drug concentration.The relative changes in the normalized THz transmission amplitude at 1 THz displayed obvious linear relationship with the cell survival rate measured by flow cytometry（cisplatin:y=0.99x-1.21,R²=0.98;Paclitaxel:y=0.96x-0.13,R²=0.96）.4.When the flow rate ratio of the two phases is 0.6 and the diameter of the microchannel is 50μm,the droplet with a diameter of 50μm and a volume of 250 pl can be stably generated to encapsulate the tumor cell suspension.The number of cells in the droplet continuously increased with the increasing concentration of the cell suspension,resulting in the increased power value at 1 THz.5.PAGE analysis of the SDA reaction system demonstrated that the SDA product presented the same position as that of synthesized product DNA,proving the performance of the designed SDA system for microRNA-21 amplification.TEM results show that SDA products and nanoparticles can be effectively captured by MBs.Zeta potential of captured MBs（-32.00±0.36 mV）is significantly lower than that of blank MBs（-28.97±0.17 mV）.The research on the effect of nanoparticles with different diameters and materials on THz metamaterial shows that AuNPs can generate larger metamaterial frequency shifts than AgNPs under the same diameter.With the same metal types,nanoparticles with a diameter of50 nm can be produced larger metamaterial frequency shifts than other diameters.The above results demonstrated the effectiveness of constructing the THz metamaterial sensing system for microRNA molecular detection.6.We constructed a THz metamaterial nanochip and verified its effectiveness in detecting microRNA-21.The resonant peak of the blank metamaterial is about 0.75 THz.When the sample is deposited on the metamaterial,the resonant peak starts to move toward the low frequency.The resonance peak frequency shift of the"SDA products of microRNA-AuNPs"complex on metamaterials is twice as much as that of the SDA products of microRNA molecules alone.The optimized sensing conditions of the constructed THz metamaterial nanochip are as follows:Klenow fragment polymerase concentration is 200U/mL,Nt.BsmAI nick endonuclease concentration is 200 U/mL,SDA reaction time is 2h,nanoprobe concentration is 50μg/mL,biotin probe concentration is 100μM,hybridization time of nanoprobe,trigger DNA and biotin probe is 60 min.7.linear relationship was found between microRNA-21 molecular concentrations and their THz response on THz metamaterial nanochip.Linear regression equation was?=3.04+13.37（=0.9985）.The detection limit（LOD）is 14.54 aM,and the linear range is 1 fM-10 pM.The?of microRNA-21 sample is significantly higher than that of microRNA-25,microRNA-139a,and Mix A（a mixture of microRNA-25 and microRNA-139a）（P<0.05）.The DI values of two interfering microRNA and Mix A are all lower than 10%（6.17%for miRNA-25,3.46%for miRNA-139a,and 5.16%for Mix A）.The RSD of THz metamaterial nanochip for detecting micro RNA-21 molecules with a concentration of 1 pM is 2.91%.The above results show that THz metamaterial nanochip has good sensitivity,specificity,and repeatability in detecting microRNA molecules.8.The THz metamaterial nanochip is used to detect microRNA-21 spiked clinical serum samples with concentrations of 10 pM,1 pM,100fM,and 10fM.The RSD of the prepared serum samples is between 2.31%and 4.77%,and the recovery rate is between 90.92%and107.01%,indicating that the designed THz metamaterial nanochip has good performance and can effectively detect target microRNA in practical samples.Conclusion:1.The fluorinated oil with low THz absorption and low cytotoxicity can reduce the attenuation of THz detection signals when passing through cell samples,improve the intensity of output THz signals,and effectively eliminate the strong water absorption during THz measurement.The two-dimensional THz absorption model of cell samples in the fluorinated oil environment clarifies the THz signal difference between cells in water and fluorinated-oil environment and provides theoretical support for OCA technology in THz detection of breast cancer cells.The proposed OCA method for replacing the extracellular water can eliminate the background signal interference and simultaneously maintain the cell activity and morphological characteristics,which is favorable for obtaining real information of cells under the physiological state.2.The constructed THz microfluidic sensor chip based on OCA can effectively control the thickness of the breast cancer cell sample and completely replace the extracellular water.A novel method for cell viability assay was developed based on this THz microfluidic sensor chip,which can quantify the viability of cells after the treatment of anti-tumor drugs.The number of cells required for an effective detection is as low as about 3600 cells.Compared with the traditional cell viability assay method,THz microfluidic sensor chip enables the rapid,label-free and non-destructive measurement,which is expected to provide a new research tool for clinical drug screening.The uniform dispersion of cell suspension in OCA was realized using droplet micro-fluid technology.A new THz detection mode through measuring"cell droplet"consisting of tumor cell suspension is constructed.3.THz spectroscopy coupling with the metamaterials,nanomaterial and SDA was developed for the highly sensitive detection of microRNA molecules.We have screened out a nanoparticle with the best signal response on metamaterial and clarified the principle of the signal response of nano particles on the metamaterials,which provides a theoretical basis for the use of nano materials to enhance the signal of THz metamaterials.Based on this work,we developed a microRNA-21 sensing system integrating metamaterial,nanomaterial,and SDA.This improved the THz detection sensitivity and provides a useful technical approach for the detection of microRNA molecules.4.A THz metamaterial nanochip integrating metamaterial,nanomaterial and SDA is constructed.A series of critical methodological parameters,such as sensitivity,specificity,and repeatability for microRNA-21 detection was obtained.THz metamaterial nanochip was also used to detect microRNA-21 spiked clinical serum samples,which verified the performance of detecting target microRNA molecules in practical samples.Compared with the existing electrochemical and optical sensors,the constructed THz metamaterial nanochip offered the same level of detection performance but the advantages of easy acquisition,simple implementation,low cost,which is expected to provide a promising analysis platform for nucleic acid analysis and tumor diagnosis research.