Label-free detection of living cervical cells based on microfluidic device with terahertz spectroscopy

Early diagnosis of cervical cancer is essential for a good prognosis. Terahertz wave detection technology is a nondestructive and label-free physical detection technology, which can detect and monitor the cancer cells in real time, especially for patients with deep or inaccessible tumors. In this study, a single-cell-layer microfluidic device was developed. After replacing the optical clearing agent, the characteristics of H8, HeLa and SiHa cell lines in adherent and suspended states were detected. Additionally, the absorption increased with increasing cell density. For the mixed suspension cell samples, principal component analysis–support vector machine method was used to identify benign and malignant cell component. After living cells formaldehyde, changes in cell membrane permeability were evaluated to identify the cell survival status (i.e., dead or living) based on terahertz spectroscopy amplitude differences. Therefore, extending the terahertz spectrum detection to the molecular level can characterize the life essence of cells and tissues.     

Detection of gene mutation responsible for Huntington's disease by terahertz attenuated total reflection microfluidic spectroscopy

Terahertz absorption spectroscopy based on attenuated total reflection (ATR) from a microfluidic sample cell was designed and implemented to detect gene mutations leading to Huntington's disease (HD). The self-developed compact ATR microfluidic system was employed to detect two groups of base-repeated DNA molecules combined with a terahertz time-domain spectrometer in a marker-free manner. The first group featured different repetition patterns of oligonucleotide fragments, and the second group included the HD gene. For the oligonucleotides of different repetition patterns, there were significant differences among the three oligonucleotides with three repeats of the double bases, which could be unambiguously classified and identified; For the HD gene, it was found that the magnitude of the terahertz absorption coefficients of the four oligonucleotide solutions was, in ascending order, CAG-4 < CAG-16 < CAG-32 < CAG-40 (the numbers are the repeat times of the CAG base segment, with 40 repeats belonging to the HD gene), when the concentration of oligonucleotide was 1 mg/mL. Principal component analysis result indicated that the spectral differences of the four oligonucleotide solutions with different CAG repeat times were statistically significant and clearly distinguishable. These results demonstrate the potential of terahertz spectroscopy as a noninvasive, unmarked, fast and low-cost assay for gene diagnosis and clinical disease detection.     

太赫兹辐射及其生物效应研究进展

随着太赫兹源和探测技术的不断进步，太赫兹技术迅速发展并在众多领域有着广泛的应用前景. 特别是在生物医学领域，太赫兹技术有望成为一种新型治疗手段. 本文首先介绍了太赫兹的电磁波特点及3种太赫兹波产生方式. 其次介绍了太赫兹辐射在生物上的两大效应：热效应和非热效应. 最后从细胞和生物体两大层面上，详细介绍了太赫兹辐射对不同细胞的生物效应和一些相关分子通路改变，以及太赫兹辐射在不同生物体上的作用效果，为太赫兹生物相关研究人员提供参考.     

THz-Spectroscopy of Biological Molecules

The terahertz frequency absorption spectraof DNA molecules reflect low-frequencyinternal helical vibrations involvingrigidly bound subgroups that are connectedby the weakest bonds, including thehydrogen bonds of the DNA base pairs,and/or non-bonded interactions. Althoughnumerous difficulties make the directidentification of terahertz phonon modes inbiological materials very challenging, ourresearch has shown that such measurementsare both possible and fruitful. Spectra ofdifferent DNA samples reveal a large numberof modes and a reasonable level ofsequence-specific uniqueness. In an attemptto show that the long wavelength absorptionfeatures are intrinsic properties ofbiological materials determined by phononmodes, a normal mode analysis has been usedto predict the absorption spectra ofpolynucleotide RNA Poly[G]-Poly[C]. Directcomparison demonstrated a correlationbetween calculated and experimentallyobserved spectra of the RNA polymers, thusconfirming that the fundamental physicalnature of the observed resonance structureis caused by the internal vibration modesin the macromolecules.In this work we demonstrate results fromFourier-Transform Infrared (FTIR)spectroscopy of DNA macromolecules andrelated biological materials in theterahertz frequency range. Carefulattention was paid to the possibility ofinterference or etalon effects in thesamples, and phenomena were clearlydifferentiated from the actual phononmodes. In addition, we studied thedependence of transmission spectra ofaligned DNA and polynucleotide film sampleson molecule orientation relative to theelectromagnetic field, showing the expectedchange in mode strength as a function ofsample orientation. Further, the absorptioncharacteristics were extracted from thetransmission data using the interferencespectroscopy technique, and a stronganisotropy of terahertz characteristics wasdemonstrated.     

Optimization for continuous-wave terahertz reflection imaging for biological tissues

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuous-wave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application.     

Rapid and label‐free detection and assessment of bacteria by terahertz time‐domain spectroscopy

Here we demonstrated the potential and applicability of terahertz (THz) spectroscopy to detect four commonly found bacteria in the infectious diseases. Besides the different spectral characteristics between bacterial species, THz absorption differences for living bacteria, dead bacteria and bacterial powder of the same species were also investigated. Our results revealed that small differences in water contents between bacterial cells account for distinct discrepancies of the absorption coefficients, which can be used for bacterial species identification. Furthermore, living and dead bacteria showed different absorption coefficients as a result of their different hydration levels, suggesting that THz spectroscopy can be used to rapidly assess the living state of bacteria under test. Our results clearly demonstrated the ability of THz spectroscopy for time‐saving and label‐free detection of bacteria with minimal sample preparation, potentially to be utilized for point‐of‐care tests in the near future.

Schematic representation of bacterial detection by THz spectroscopy. Different bacteria have distinctive absorption coefficients as a result of their different water contents.     

褪黑素的太赫兹时域光谱

太赫兹时域光谱技术是一种新兴的无损探测技术。利用太赫兹波的低能性以及大部分生物分子的振动跃迁和旋转在该频段表现出的强色散和吸收作用等特点,可以对生物分子及生命体的活动进行无损探测和研究。本文分别采用透射式和反射式太赫兹时域光谱系统,对不同质量比的褪黑素压片进行测试,分析它在太赫兹波段的光学性质,发现它在0.29、0.50、0.70、0.91、1.20、2.17和2.55 THz处存在特征吸收峰;频域谱的强度随样品浓度的变化呈线性关系。利用Gaussian 09及Gaussian VIEW软件进行模拟分析,得到褪黑素在0.46、0.91、1.15、2.01、2.23和2.61 THz处存在特征吸收峰,为实验结果提供了有力地支持。这些工作为褪黑素等生化样品的检测和鉴定提供了依据和参考。     

Studying the non‐thermal effects of terahertz radiation on E. coli/pKatG‐GFP biosensor cells

Studies of the impact of terahertz radiation on living objects present a significant interest since its use for security systems is currently considered promising. We studied the non‐thermal impact of terahertz radiation on E. coli/pKatG‐gfp biosensor cells. The Novosibirsk free electron laser (NovoFEL), which currently has the world's highest average and peak power, was used as the source of terahertz radiation. We demonstrated that exposure to terahertz radiation at the wavelengths of 130, 150, and 200 µm and a power of 1.4 W/cm² induces changes in green fluorescent protein (GFP) fluorescence values and thus induces the expression of GFP in E. coli/pKatG‐gfp biosensor cells. Possible mechanisms of the E. coli response to non‐thermal exposure to terahertz radiation are discussed. Bioelectromagnetics 34:15–21, 2013. © 2012 Wiley Periodicals, Inc.     

Visualization photofragmentation‐induced rhodamine B release from gold nanorod delivery system by combination two‐photon luminescence imaging with correlation spectroscopy

Plasmon‐enhanced gold nanorod (AuNR) with high photothermal conversion efficiency is a promising light‐controllable nanodrug delivery system for cancer therapy. Understanding the mechanism for the light‐controllable drug release of AuNR delivery systems is important for the development of nanomedicine. In this study, the rhodamine B (RB) released from AuNR‐RB nanodelivery system was quantitated and visualized by using two‐photon luminescence (TPL) imaging combined with correlation spectroscopy. The photofragmentation of AuNR induced by femtosecond pulsed laser was revealed by TPL correlation spectroscopy when the laser energy was above the thermal damage threshold of AuNR, and the RB released from this nanodrug delivery system was visualized by TPL imaging. Furthermore, the photofragmentation‐induced release of RB from AuNR‐RB nanodelivery system was visualized in living MCF‐7 breast cancer cells by TPL imaging combined with correlation spectroscopy. These results provided a novel optical approach to quantify the release of drugs from gold nanocarriers in complex biological media.     

Measurement of THz Spot Sizes with a λ/200 Diameter in the Near-Field of a Metal Tip

We report on a method to obtain asub-wavelength resolution in terahertz time-domain imaging. In our method,a sharp copper tip is used to locallydistort and concentrate the THz electricfield. The distorted electric field, presentmainly in the near field of the tip, iselectro-optically measured in an (100)oriented GaP crystal. By raster scanning the tipalong the surface of the crystal we find asmallest THz spot size of 10 m forfrequencies from 0.1 to 2.5 THz. For ourpeak frequency of 0.15 THz this corresponds to aresolution of /200. Our setup has thepotential to reach a resolution down to afew m, and is a promising candidate tostudy single, living cells in the THzfrequency range.     

In vivo terahertz reflection imaging of human scars during and after the healing process

We use terahertz imaging to measure four human skin scars in vivo. Clear contrast between the refractive index of the scar and surrounding tissue was observed for all of the scars, despite some being difficult to see with the naked eye. Additionally, we monitored the healing process of a hypertrophic scar. We found that the contrast in the absorption coefficient became less prominent after a few months post‐injury, but that the contrast in the refractive index was still significant even months post‐injury. Our results demonstrate the capability of terahertz imaging to quantitatively measure subtle changes in skin properties and this may be useful for improving scar treatment and management.

     

Light-induced Difference Terahertz Spectroscopy

Visible/near-infraredlaser-induced difference spectroscopy basedon a time-domain terahertz system has beendeveloped, and used to study copperpathancyonine. We find that the absorptionpeak of this molecule at 1.08 THz changessignificantly under 790 nm laserexcitation, suggesting that we haveobserved the first evidence of vibrationalmode changes in the THz range induced byvisible/near-infrared light.     

Low-power compact continuous-wave stimulated emission depletion microscopy

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.     

Terahertz Pulsed Imaging of Skin Cancer in the Time and Frequency Domain

Terahertz Pulsed Imaging(TPI) is a new medical imaging modality forthe detection of epithelial cancers. Overthe last two years this technique has beenapplied to the study of in vitrobasal cell carcinoma (BCC). Usingtime-domain analysis the contrast betweendiseased and normal tissue has been shownto be statistically significant, andregions of increased terahertz (THz)absorption correlated well with thelocation of the tumour sites in histology.Understanding the source of this contrastthrough frequency-domain analysis mayfacilitate the diagnosis of skin cancer andrelated skin conditions using TPI. Wepresent the first frequency-domain analysisof basal cell carcinoma in vitro,with the raw power spectrum giving aninsight into the surface features of theskin. Further data manipulation is requiredto determine whether spectral informationcan be extrapolated at depth. These resultshighlight the complexity of working inreflection geometry.     

In vivo sensing of rabbit cornea by terahertz technology

A Novel scalable approach using Terahertz (THz) waves together with the electromagnetic field simulation was applied to investigate four rabbits of eight rabbit corneas in vivo. One eye of each rabbits’ corneas was edema induced; the other eye of the corneas served as the control. The simulation revealed the propagation of THz waves at a certain distance along the sub-surface of the cornea. THz spectra have been collected close to the corneal surface by deviating the direct reflection of the THz beam for the edema cornea, the reflected wave intensity for edema corneas is generally larger compared with the control cornea. Upon edema becomes severe at the end of the observation, the reflected wave intensities obtained by detector corresponding to the corneal deep stroma layer approach to the same value for all observed corneas. Good correlation is observed between central corneal thickness measurements and THz wave reflection signal intensities. Our results demonstrated that THz spectroscopy technique could obtain the information from different corneal sublayers.     

Studying the genotoxic effects of high intensity terahertz radiation on fibroblasts and CNS tumor cells

The data is obtained on the effect of high-intensity pulses of terahertz (THz) radiation with a broad spectrum (0.2–3 THz) on cell cultures. We have evaluated the threshold exposure parameters of THz radiation causing genotoxic effects in fibroblasts. Phosphorylation of histone H2AX at Ser 139 (γH2AX) was chosen as a marker for genotoxicity and a quantitative estimation of γH2AX foci number in fibroblasts was performed after cell irradiation with THz pulses for 30 min. No genotoxic effects of THz radiation were observed in fibroblasts unless peak intensity and electric field strength exceeded 21 GW cm⁻² and 2.8 MV cm⁻¹, respectively. In tumor cell lines (neuroblastoma (SK-N-BE (2)) and glioblastoma (U87)), exposure to THz pulses with peak intensity of 21 GW cm⁻² for 30 min caused no morphological changes as well as no statistically significant increase in histone phosphorylation foci number.     

Front cover

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuouswave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application. Further details can be found in the article by Limin Wu, Yuye Wang, Haibin Li, Zelong Wang, Meilan Ge, Degang Xu, and Jianquan Yao ( e202100245 ).

     

Dendric nanostructures for SARS-CoV-2 proteins detection based on surface enhanced infrared absorption spectroscopy

Infrared spectroscopy is a non-destructive and rapid characterization tool that can distinguish different viral proteins by spectral details. However, traditional infrared spectroscopy has insufficient absorption signal intensity contrast when measuring low-concentration samples. In this work, surface enhanced infrared absorption (SEIRA) spectroscopy is proposed by deploying a novel nanostructure array as SEIRA substrates. An array of gold dendric nanostructures are designed and fabricated with a precision resonance control to achieve surface enhancement covering a broadband molecular “finger-print” region. The spectral positions of the multiple resonances accurately correspond to the characteristic absorption peaks of the SARS-CoV-2 proteins. An approach for SARS-CoV-2 protein detection based on SEIRA spectroscopy is then proposed. A low concentration detection of 40 μg/ml diluted SARS-CoV-2 nucleocapsid protein is experimentally demonstrated and the enhancement factor (EF) achieved is in good agreement with simulation results. The SEIRA methodology based on broadband resonance nanostructure design provides a systematic approach for sensitive, non-destructive and rapid protein molecular detection, which could be extended to various kind of molecular characterization and biomedical diagnostics.     

Cytological and spectroscopic characteristics of c‐KIT N822K mutation in core binding factor acute myeloid leukemia cells

The frequency of N822K mutation is high in the A‐loop region of c‐KIT which is highly associated with poor prognosis of core binding factor acute myeloid leukemia. The current work used common assays including cell cycle, apoptosis, clone formation and western blot to perform cytological detection for HL60 (wild type), NB4 (carrying t[15;17] chromosome translocation) and Kasumi‐1 (with c‐KIT N822K mutation); and meanwhile, the laser tweezers Raman spectroscopy (LTRS) was also used to perform label‐free detection of single living cells. The results demonstrated that Kasumi‐1 cell line bearing c‐KIT N822K mutation has a stable cell cycle, while there was a significant difference between early and late apoptosis within 48 hours. The LTRS detection initially reflected the spectral differences induced by genetic abnormalities and highlighted progressive patterns of DNA and amino acids band contents which were appropriately consistent with that of cell clone ratio and the c‐KIT phosphorylation level. It is concluded that methodology of LTRS‐based single living cell characterization could be potential and effective to reveal gene mutation‐induced cell differentiation.     

Generation of terahertz radiation from a low-density plasma slab irradiated by a laser pulse

The generation of terahertz electromagnetic radiation when a laser pulse propagates through a low-density plasma slab is considered. It is shown that terahertz waves are excited because of the growth of a weakly damped, antisymmetric leaking mode of the plasma slab. The spectral, angular, and energy parameters of the terahertz radiation are investigated, as well as the spatiotemporal structure of the emitted waves. It is demonstrated that terahertz electromagnetic wave fields are generated most efficiently when the pulse length is comparable to the slab thickness.