A Versatile Terahertz Chemical Microscope and Its Application for the Detection of Histamine

Terahertz waves have gained increasingly more attention because of their unique character-istics and great potential in a variety of ﬁelds. In this study, we introduced the recent progress of our versatile terahertz chemical microscope (TCM) in the detection of small biomolecules, ions, cancer cells, and antibody–antigen immunoassaying. We highlight the advantages of our TCM for chemical sensing and biosensing, such as label-free, high-sensitivity, rapid response, non-pretreatment, and minute amount sample consumption, compared with conventional methods. Furthermore, we demonstrated its new application in detection of allergic-related histamine at low concentration in buffer solutions.


Introduction
Terahertz (THz) waves are a type of electromagnetic wave that located in the region between radio waves and light waves. Although THz as an electromagnetic wave that has been studied for a long time, which was mainly used for spectroscopic analysis, the milestone of the rapid development of THz waves in recent years is established by DH Auston et al. [1,2]. It is considered that THz waves are generated, and time-domain waveforms are acquired, using a photo-conducting switch. Since then, a THz time-domain spectroscopy (THz-TDS) has been proposed and developed [3,4]. At present, not only the above-mentioned generation detection method but also various THz wave generation/detection methods have been proposed, and the generation of THz waves is becoming closer to practical use in frequency bands and intensities for quantum cascade lasers and resonant tunnel diodes. Moreover, by using ultra-short laser pulses, THz-TDS, measuring the optical properties of materials, has become a promising technique in the academic field and industrial application [5,6]. It is possible to identify the material from the absorption spectrum peculiar to the molecule of the material existing in the THz wave region [7][8][9], and its application for airport security inspection and non-destructive material inspection have been proposed and put into practical use. Moreover, a biosensor using a metal mesh structure designed for steep absorption peaks in the THz wave region was demonstrated [10]. The specific binding of the antigen to the antibody immobilized on the sensor changes the permittivity of the sensor; as a result, the frequency of the absorption peak of the sensor changes. This technique uses a THz spectroscopy system to measure the shift of absorption peaks. Extremely sensitive measurement is realized with high peak Q value.
As a technology different from THz spectroscopy, a highly stable femtosecond laserexcited THz emission microscopy (LTEM) has also been developed to evaluate the dynamics of carriers or electric dipoles in materials [11][12][13][14][15]. Specifically, a semiconductor integrated circuit chip is irradiated with a femtosecond laser, and THz waves are emitted from the integrated circuit chip is irradiated with a femtosecond laser, and THz waves are emitted from the integrated circuit chip itself. Since the generated THz waves contain information about the electric field inside the integrated circuit chip, as well as the dynamic behavior of carriers inside the semiconductor, the distribution of THz wave radiation can be obtained by scanning the femtosecond laser on the integrated circuit chip [15]. Furthermore, this information enables failure inspection and the analysis of integrated circuit chips. In general, the spatial resolution of THz imaging has a spatial resolution of about the length of a THz wave, which is 300 μm at 1 THz, whereas the spatial resolution of LTEM is determined by the center wavelength of the femtosecond laser used for excitation (around 790 nm in the case of Ti:Sapphire laser). Recently, a spatial resolution of 20 nm is obtained by integrating LTEM with scanning near-field optical microscopy [16,17].
Our group has developed an advanced version of LTEM, named the THz chemical microscope (TCM) [12,14,[18][19][20][21][22][23], which could be used for visualization of chemical reaction and bio-reaction, including small molecules and ions visualization [18,[24][25][26][27][28][29][30], cancer cell detection [31,32], antibody-antigen immunoassaying [33], enzyme kinetics analysis [34], and cosmetic and lithium ion battery evaluation [35][36][37][38][39], by measuring the electrochemical potential distribution on the sensing plate. Based on reviewing the recent progress of TCM, we believe that this versatile TCM is promising in academic research and industrial applications ( Figure 1).   Figure 2 illustrate the schematic of the optical setup of the TCM. The femtosecond laser pulse is focused by the objective lens on the back surface of the sensing plate, at an incident angle of 45 degrees. A mode-locked Ti: sapphire laser was used as the femtosecond laser light source. The pulse width was about 100 fs, and the central wavelength was 790 nm. The THz wave emitted by the femtosecond laser irradiation is guided to the detector by an off-axis parabolic mirror pair. A low temperature growth GaAs photoconductive antenna is used for the detector. The THz wave detection optical system is almost the same as THz-TDS; however, the antenna arrival time of the trigger pulse is fixed at the point where the maximum amplitude intensity of the THz wave was obtained. The sensing plate is installed in a stepping motor-driven x-y automatic stage and can be replaced. By driving the x-y automatic stage, the THz wave radiation intensity was measured while the laser focusing position on the back surface of the sensing plate was relatively changed. This makes it possible to obtain a distribution image related to the potential of the sensing plate. The spatial resolution of the obtained TCM image is about 5 µm, and a resolution of 1 µm or less can be achieved by improving the focusing optical system [22]. Figure 2 illustrate the schematic of the optical setup of the TCM. The femtosecond laser pulse is focused by the objective lens on the back surface of the sensing plate, at an incident angle of 45 degrees. A mode-locked Ti: sapphire laser was used as the femtosecond laser light source. The pulse width was about 100 fs, and the central wavelength was 790 nm. The THz wave emitted by the femtosecond laser irradiation is guided to the detector by an off-axis parabolic mirror pair. A low temperature growth GaAs photoconductive antenna is used for the detector. The THz wave detection optical system is almost the same as THz-TDS; however, the antenna arrival time of the trigger pulse is fixed at the point where the maximum amplitude intensity of the THz wave was obtained. The sensing plate is installed in a stepping motor-driven x-y automatic stage and can be replaced. By driving the x-y automatic stage, the THz wave radiation intensity was measured while the laser focusing position on the back surface of the sensing plate was relatively changed. This makes it possible to obtain a distribution image related to the potential of the sensing plate. The spatial resolution of the obtained TCM image is about 5 μm, and a resolution of 1 μm or less can be achieved by improving the focusing optical system [22].

Terahertz Chemical Microscope
. In TCM, the semiconductor device named sensing plate, shown in Figure 3a, is used to measure the electrochemical potential distribution. The sensing plate was made by forming a silicon thin film (Si film) on a sapphire substrate and then forming a silicon thermal oxide film (SiO2 film). The film thicknesses of the Si film and the SiO2 film are 150 nm and several nm, respectively. The size of the sensing plate is 15 mm square for ease of handling, but it can be expanded to the wafer size, according to the area to be measured. In the sensing plate, there are defects near the boundary between the Si and SiO2 films, so the energy band bends toward the boundary surface and a depletion layer electric field is generated. The bending direction depends on the doping type of the Si film. When this sensing plate is irradiated with a femtosecond laser pulse having photon energy equal to or higher than the Si band gap from the substrate side, the carriers inside the Si film are excited and accelerated by the depletion layer electric field. The movement of this photoexcited carrier can be regarded as a high-speed current modulation. According to classical electromagnetism, an electromagnetic wave is generated by a change in current; however, since the change is on the order of femtoseconds to picoseconds, the frequency of the generated electromagnetic wave is also on the order of THz. In addition, the amplitude inten- In TCM, the semiconductor device named sensing plate, shown in Figure 3a, is used to measure the electrochemical potential distribution. The sensing plate was made by forming a silicon thin film (Si film) on a sapphire substrate and then forming a silicon thermal oxide film (SiO 2 film). The film thicknesses of the Si film and the SiO 2 film are 150 nm and several nm, respectively. The size of the sensing plate is 15 mm square for ease of handling, but it can be expanded to the wafer size, according to the area to be measured. In the sensing plate, there are defects near the boundary between the Si and SiO 2 films, so the energy band bends toward the boundary surface and a depletion layer electric field is generated. The bending direction depends on the doping type of the Si film. When this sensing plate is irradiated with a femtosecond laser pulse having photon energy equal to or higher than the Si band gap from the substrate side, the carriers inside the Si film are excited and accelerated by the depletion layer electric field. The movement of this photoexcited carrier can be regarded as a high-speed current modulation. According to classical electromagnetism, an electromagnetic wave is generated by a change in current; however, since the change is on the order of femtoseconds to picoseconds, the frequency of the generated electromagnetic wave is also on the order of THz. In addition, the amplitude intensity of the generated electromagnetic wave is proportional to the electric field. In the sensing plate, when the electrochemical potential of the SiO 2 film changes, the bending of the energy band changes accordingly. As a result, the depletion layer electric field changes, and the intensity of the radiated electromagnetic waves also changes. The femtosecond laser is focused and the THz wave intensity at each position is measured while scanning [22]. sity of the generated electromagnetic wave is proportional to the electric field. In the sensing plate, when the electrochemical potential of the SiO2 film changes, the bending of the energy band changes accordingly. As a result, the depletion layer electric field changes, and the intensity of the radiated electromagnetic waves also changes. The femtosecond laser is focused and the THz wave intensity at each position is measured while scanning [22]. In the sensing plate, there are defects near the boundary between the Si and SiO2 films, so the energy band bends toward the boundary surface and a depletion layer electric field is generated. The electrical or chemical reaction on the sensing plate surface could shift the electric potential, it simultaneously changes the magnitude of the depletion field. (b) Chemical modification and bio-modification on the sensing plate are applied for chemical or bio-related substances detection and evaluation. Created with BioRender.com.
The amplitude of the radiated terahertz wave is expressed by the equation (1): where ETHz(t) is the electric field of the terahertz wave, J(t) is the instantaneous current density, e is the elementary charge, n(t) is the carrier density, and v(t) is the velocity of the carriers accelerated in the Si layer. Because the carrier acceleration∂v/∂t is proportional to El,, it indicates that ETHz(t) is proportional to the square root of electric potential. By doing so, it is possible to obtain a THz wave intensity distribution that reflects the electrochemical potential distribution on the surface of the SiO2 film. Figure 3b shows the chemical or bio-modification methods on the sensing plate, for interest of substances measurements.
Our TCM is different from the conventional terahertz imaging and terahertz spectroscopy, regarding to the principle used. The terahertz wave intensity is changed at the boundary of Si layer when the surface potential changes, due to the chemical reaction on the semiconductor sensing plate. Furthermore, the spatial resolution of TCM (~5 μm) is independent of the wavelength of the generated terahertz, determined by the wavelength of the femtosecond laser (~790 nm), and can be improved by using better condensing optics. In the sensing plate, there are defects near the boundary between the Si and SiO 2 films, so the energy band bends toward the boundary surface and a depletion layer electric field is generated. The electrical or chemical reaction on the sensing plate surface could shift the electric potential, it simultaneously changes the magnitude of the depletion field. (b) Chemical modification and bio-modification on the sensing plate are applied for chemical or bio-related substances detection and evaluation. Created with BioRender.com.

A versatile TCM for Biological Substances Detection
The amplitude of the radiated terahertz wave is expressed by the Equation (1): where E THz (t) is the electric field of the terahertz wave, J(t) is the instantaneous current density, e is the elementary charge, n(t) is the carrier density, and v(t) is the velocity of the carriers accelerated in the Si layer. Because the carrier acceleration∂v/∂t is proportional to E l , it indicates that E THz (t) is proportional to the square root of electric potential. By doing so, it is possible to obtain a THz wave intensity distribution that reflects the electrochemical potential distribution on the surface of the SiO 2 film. Figure 3b shows the chemical or bio-modification methods on the sensing plate, for interest of substances measurements.
Our TCM is different from the conventional terahertz imaging and terahertz spectroscopy, regarding to the principle used. The terahertz wave intensity is changed at the boundary of Si layer when the surface potential changes, due to the chemical reaction on the semiconductor sensing plate. Furthermore, the spatial resolution of TCM (~5 µm) is independent of the wavelength of the generated terahertz, determined by the wavelength of the femtosecond laser (~790 nm), and can be improved by using better condensing optics.

A versatile TCM for Biological Substances Detection
TCM has shown great potential in the detection of biological substances, based on recent progress, which is summarized in Figure 4. Specifically, the detection of those biological substances, including ions, small biomolecules, large antibodies, and cancer cells, by TCM is elucidated. Furthermore, its new application in detection of histamine was demonstrated. TCM has shown great potential in the detection of biological substances, based on recent progress, which is summarized in Figure 4. Specifically, the detection of those biological substances, including ions, small biomolecules, large antibodies, and cancer cells,  [28] and ions distribution and THz visualization results through array-based microwells [18]. Reprinted with permission from [18,28]. Copyright 2018 Optical Society of America and SPIE. (b) Cross-sectional schematic of the sensing plate with specific antibody immobilized. (c) THz amplitude change before and after the reaction of mannose and THz amplitude change versus three different concentrations of mannose [24]. Reprinted with permission from [24]. Copyright 2016 The Japan Society of Applied Physics. (d) THz images of three different concentrations of anti-IgG on four regions of the sensing plate and a plot of THz amplitude corresponding to different anti-IgG concentrations [33]. Reprinted with permission from [33]. Copyright 2012 Elsevier B.V. (e) THz amplitude mapping of with/without adding avidin and real-time THz signal of forming the biotin-avidin protein complex [26]. Reprinted with permission from [26]. Copyright 2010 American Institute of Physics. (f) The differential THz amplitude distribution between before and after reaction on the sensing plate and THz amplitude changes as a function of PC9 concentration [40]. Copyright 2021 MDPI (Basel, Switzerland).

pH and Ion Measurement
Measuring pH and ion concentrations is an effective way to evaluate health condition in medical applications or environmental analysis. Electrochemical sensors or ion-sensitive field effect sensors (ISFETs) are often utilized for specific ions measurement. Basically, pH sensitive materials such as IrO2 [41], ZnO [42], and popular polymer polyaniline (PAN1) [43,44] are developed for pH sensing to be cost-effective and highly sensitive. These materials could accumulate the H + and OHions and are coated to form the electrode. Moreover, nanostructures have been designed for better pH measurements, due to  [28] and ions distribution and THz visualization results through array-based microwells [18]. Reprinted with permission from [18,28]. Copyright 2018 Optical Society of America and SPIE. (b) Cross-sectional schematic of the sensing plate with specific antibody immobilized. (c) THz amplitude change before and after the reaction of mannose and THz amplitude change versus three different concentrations of mannose [24]. Reprinted with permission from [24]. Copyright 2016 The Japan Society of Applied Physics. (d) THz images of three different concentrations of anti-IgG on four regions of the sensing plate and a plot of THz amplitude corresponding to different anti-IgG concentrations [33]. Reprinted with permission from [33]. Copyright 2012 Elsevier B.V. (e) THz amplitude mapping of with/without adding avidin and real-time THz signal of forming the biotin-avidin protein complex [26]. Reprinted with permission from [26]. Copyright 2010 American Institute of Physics. (f) The differential THz amplitude distribution between before and after reaction on the sensing plate and THz amplitude changes as a function of PC9 concentration [40]. Copyright 2021 MDPI (Basel, Switzerland).

pH and Ion Measurement
Measuring pH and ion concentrations is an effective way to evaluate health condition in medical applications or environmental analysis. Electrochemical sensors or ion-sensitive field effect sensors (ISFETs) are often utilized for specific ions measurement. Basically, pH sensitive materials such as IrO 2 [41], ZnO [42], and popular polymer polyaniline (PAN1) [43,44] are developed for pH sensing to be cost-effective and highly sensitive. These materials could accumulate the H + and OHions and are coated to form the electrode. Moreover, nanostructures have been designed for better pH measurements, due to higher surface-to-volume ratio. Different from electrochemical or FET sensors [45,46], in TCM system, the Si-OH groups titrate with the protons in the solution and exist as either uncharged Si-OH or negatively charged SiO − (Equations (2) and (3)) [22,28]. An electric Photonics 2022, 9, 26 6 of 13 double layer is, thus, formed at the SiO 2 surface. The electric potential ψ at the surface is determined by the Nernst Equation, which depends on the proton concentration.
Based on this detection mechanism, as shown in Figure 4a, an extremely small volume of 16 nL buffer solutions were successfully measured through array-based microwells [28].
Ion measurements play a very important role in evaluating the biological activity. In ion measurement by TCM, a sensitive membrane, on which the electrical potential changes depending on a specific ion concentration, is immobilized on the surface of the sensing plate. In the presence of ions with a certain concentration C 0 , the chemical potential (µ 0 ) on the surface of the sensitive membrane is expressed as follows: Here, Gi 0 represents the standard generated Gibbs free energy, and zi, F, R, and T represent the valence, Faraday constant, gas constant, and temperature, respectively. Also, Ψ 0 is an electrical potential. This equation indicates that the surface electrical potential of the membrane changes as the ion concentration in the liquid changes, so it can be measured by TCM.
For Na + ions, ETH2120 as an ionophore was used. Dioctyl adipate (DOA) was used as the plasticizer and sodium tetraphenylboron (NaTPB) was used as an additive to stabilize the potential. For K + ions, valinomycin as an ionophore was used, and as an additive, potassium tetraphenylboron (KTPB) was used. They are mixed with resins chloride (PVC), which is the base material of the membrane, and dissolved in tetrahydrofuran (THF), respectively. Then the liquid membrane solution was dropped onto the sensing plate overnight to volatilize THF at room temperature [22,25]. Figure 4a showed the mapping terahertz images obtained with sodium (Na + ) and potassium (K + ) sensitive membranes, immobilized on the sensing plate. It visualized the distribution of changes in THz wave intensity, when the Na + ion concentration changes from 10 −4 mol/L to 10 −1 mol/L [18,25]. In the future, we are considering developing a plate with laminated multi-sensitive membranes and applying it to multi-ion screening.

Lectin-Sugar/Sugar Chain Interactions and Antibody-Antigen Immunoassay
Small molecules, with or without charge, could also be measured by TCM. Figure 4b shows a cross-sectional schematic of the sensing plate with antibodies on the surface of the sensing plate and a photograph of the four solution wells formed through an engineering plastic on the sensing plate. Each well was 3 × 3 mm 2 in area and 3 mm deep. The antibodies were immobilized on the surface of the sensing plate by a covalent binding method. Different concentrations of the small antigen molecules (about 30 µL in each well) were introduced, and the surface potential was changed because of the charged molecules capture. Meanwhile, the change in THz amplitudes were monitored.
Lectin-sugar/sugar chain interactions take an important part in various bioactivities, such as cell recognition, adhesion, blood typing, and ligand-receptor recognition. Accurate and efficient techniques for the screening of lectin-sugar/sugar chain interactions are important to understand and elucidate the mechanisms of complicated bio-reactions and improve to discovery novel drugs. Figure 4c showed the amplitude was changed in the THz pulses before and after adding the D-(+)-Mannose (MW: 180), reacting with Con A immobilized on the sensing plate and plotted the amplitude changes against three different mannose concentrations. As shown, the amplitude change increased by increasing the mannose concentration in the dashed line region. The sensitivity calculated as the slope of the linear fitting was 3.3 mV/dec. The sensitivity could be enhanced by increasing the Photonics 2022, 9, 26 7 of 13 signal-to-noise of the THz detectors. The limit of detection (LOD) was calculated as 0.3 mM at the background THz amplitude 0.3 mV [24].
Antigen-antibody immunoassay plays an important role in a wide range of biotechnology fields, such as pathological examination, drug discovery, and life science research. The enzyme-linked immuno-sorbent assay (ELISA) method as gold standard is widely used for immunoassays. There are several methods, such as the direct, indirect, sandwich, and competitive methods, for optimized measurement, depending on the antigen-antibody combination. In which, it requires an enzyme labeled antigen or antibody or secondary antibody to trigger the coloration reaction. Procedures including labeling secondary antibody and washing of the unbound label are required, and it often takes several hours or more. Several label-free techniques have been developed for high sensitivity and high performances of immunoassays. Surface plasmon resonance (SPR) sensor is a promising approach to detect interaction especially between large biomolecules without labeling in real-time measurement [47][48][49][50]. SPR measures the changes of refractive index occurred at the gold or silver surface, on which antibody is usually immobilized. When the target substances bond to the sensor surface, the resonance angle shifted, which is proportional to the biomolecule concentration near the surface. However, it is still difficult to detect antigens with a small mass (1000 Da or less). FET is another promising label-free approach for immunoassays [51][52][53]. Basically, the gate electrode of FET was functionalized with specific antibody to react with the antigen. The change in the distribution of electrons during the reaction can be measured as a change in the threshold voltage, which could provide higher sensitivity. Dealing with Debye length issue is still a great challenge for high-throughput measurement.
On the other hand, in the measurement using the TCM system, the potential change on the surface of the sensing plate, due to the binding of the protein itself or the adsorption reaction, is measured [33]. Therefore, unlabeled antigen-antibody reaction measurement can be realized without depending on the mass of the measurement sample. Here, an example of measuring the binding reaction of mouse IgG (Ig: immunoglobulin, antibody) was shown. Mouse IgG was immobilized on the sensing plate by covalent bonding. Then, sheep anti-mouse IgG was applied to the surface of the sensing plate to which IgG was bound. Figure 4d showed the difference between the TCM images measured before and after binding the sheep anti-mouse IgG, that was the change in the terahertz wave intensity distribution. In this way, TCM can capture the antigen-antibody reaction distribution as an image. In the future, we aim to measure the distribution of several proteins with the aim of increasing sensitivity and resolution. Moreover, visualization of a biotin-avidin protein complex was demonstrated by using TCM as shown in Figure 4e. A half area of the sensing plate was immobilized with avidin through amine-coupling. Real-time recording of the THz amplitude changes during biotin introduced in a flow channel was obtained. A low concentration 10 −12 mol/L of biotin was detected as an initial demonstration [26]. By analyzing the results, we believe that our TCM could provide rapid, real-time, high sensitivity, and label-free immunoassays.

Detection of Cancer Cells
THz technology has showed great potential in biomedical diagnosis due to its noninvasive and label-free property. Significant progress has been made to accelerate the THz imaging in this field. Son et al. have developed THz-TDS imaging coupled with magnetic resonance (MR) imaging for cancer cells by modifying cancer cells with superparamagnetic iron oxide nanoparticles both in vivo and in vitro [54]. Seo et al. developed a mouse brain tissue THz imaging using large-area array-based terahertz metamaterials with real-time historical analysis. Ultrasensitive imaging of real bio-samples was realized [55]. Serita et al. developed a terahertz near-field microscopy for label-free observation of human breast cancer cell density [56][57][58][59]. Their results may further explore the application of terahertz imaging for cancer tissue biopsy. Conventional evaluation the ratio of cancer cells includes several steps: the specimen tissue should be first fixed to make formalin-fixed paraffin-embedded (FFPE) by replacing water with formalin degreased with alcohol, followed by paraffin embedding; tissue sliced and stained, then visually observed using an optical microscope by pathologists. The sophisticated progress required more than two days and skilled pathologist [60][61][62].
Different from the THz imaging system mentioned above, the TCM exhibits unique detection advantage. In recent investigation, Ozaki et al. demonstrated the high-sensitivity detection of metastatic breast cancer cells using TCM. In their study, single stranded (ss) DNA aptamer named mammaglobin B1 (MAMB1) and mammaglobin A2 (MAMA2) were immobilized on the sensing plate. These aptamers could specific bound to mammaglobin B and mammaglobin A proteins, which were overexpressed on the surface of MCF7 and MDA-MB-415 breast cancer cells. By measuring the THz amplitude change, one breast cancer cell in a 100 µL of sample was detected [31]. Furthermore, biotin-labeled cytokeratin conjugated with avidin immobilized on a sensing plate surface was developed for human lung adenocarcinoma cells (PC9) detection. Figure 4f showed the THz amplitude for different concentrations of lung cancer cells and the response curve. After each measurement, the THz amplitude was normalized by pH measurement, which aims to compensate for the variability of the sensitivity of the sensing plate [40]. There results indicated that the TCM could be a novel tool to detect cancer cells rapidly, label-free, and with high sensitivity.

Detection of Histamine Released from Allergic Response
A histamine is a vital biomarker during allergic march which it is released from the cells after allergens contact ( Figure 5). It could cause edema, bronchial asthma, and lead to different diseases. Current in-vivo and in-vitro inspections, including prick test, oral food challenge, and specific IgE tests, as well as a histamine release test (HRT), are recognized as the most reliable methods and, thus, widely used [63][64][65][66]. However, these kinds of inspections most require injection of allergen into the body at a risk of causing anaphylactic shock, delicate supervision by a physician, or large amount of blood sample consumption and difficult to discriminate specific allergen among many types of candidates. For novel diagnosis of allergic march at the early stage, the TCM was utilized to detect the histamine level in buffer solution for the fast screening of allergens. Serita et al. developed a terahertz near-field microscopy for label-free observation of human breast cancer cell density [56][57][58][59]. Their results may further explore the application of terahertz imaging for cancer tissue biopsy.
Conventional evaluation the ratio of cancer cells includes several steps: the specimen tissue should be first fixed to make formalin-fixed paraffin-embedded (FFPE) by replacing water with formalin degreased with alcohol, followed by paraffin embedding; tissue sliced and stained, then visually observed using an optical microscope by pathologists. The sophisticated progress required more than two days and skilled pathologist [60][61][62].
Different from the THz imaging system mentioned above, the TCM exhibits unique detection advantage. In recent investigation, Ozaki et al. demonstrated the high-sensitivity detection of metastatic breast cancer cells using TCM. In their study, single stranded (ss) DNA aptamer named mammaglobin B1 (MAMB1) and mammaglobin A2 (MAMA2) were immobilized on the sensing plate. These aptamers could specific bound to mammaglobin B and mammaglobin A proteins, which were overexpressed on the surface of MCF7 and MDA-MB-415 breast cancer cells. By measuring the THz amplitude change, one breast cancer cell in a 100 μL of sample was detected [31]. Furthermore, biotin-labeled cytokeratin conjugated with avidin immobilized on a sensing plate surface was developed for human lung adenocarcinoma cells (PC9) detection. Figure 4f showed the THz amplitude for different concentrations of lung cancer cells and the response curve. After each measurement, the THz amplitude was normalized by pH measurement, which aims to compensate for the variability of the sensitivity of the sensing plate [40]. There results indicated that the TCM could be a novel tool to detect cancer cells rapidly, label-free, and with high sensitivity.

Detection of Histamine Released from Allergic Response
A histamine is a vital biomarker during allergic march which it is released from the cells after allergens contact ( Figure 5). It could cause edema, bronchial asthma, and lead to different diseases. Current in-vivo and in-vitro inspections, including prick test, oral food challenge, and specific IgE tests, as well as a histamine release test (HRT), are recognized as the most reliable methods and, thus, widely used [63][64][65][66]. However, these kinds of inspections most require injection of allergen into the body at a risk of causing anaphylactic shock, delicate supervision by a physician, or large amount of blood sample consumption and difficult to discriminate specific allergen among many types of candidates. For novel diagnosis of allergic march at the early stage, the TCM was utilized to detect the histamine level in buffer solution for the fast screening of allergens.   for 1 h. The ester group was produced. By immersing the sensing plate in 35% HCl (35-37%, Fujifilm Wako, Osaka, Japan) at room temperature for 24 h, the carboxylation reaction was realized. After that, 3 mM N-hydroxysuccinimide (NHS) (98.0~102.0%, Fujifilm Wako, Osaka, Japan) and 1 mM 1-ethyl-3-(3-dimethrlaminopropyl)-carbodimide hydrochloride (EDC) (Over 98.0%, Fujifilm Wako, Osaka, Japan) in phosphate-buffered saline (PBS) (pH 7.4, Thermo Fisher Scientific, Waltham, MA, USA) were prepared, and the carboxyl group was activated by immersing the sensing plate in an NHS & EDC solution at pH 7.4 for 30 min at room temperature. The sensing plate was glued to a measuring substrate with four wells, and avidin (affinity purified, Vector Laboratories, Burlingame, CA, USA), diluted to 0.147 µM with PBS and 30 µL, was pipetted into each well for immobilization at 4 • C for 24 h. Then, the surface was blocked by 1 mM 2-Aminoethanol (Over 99.0%, Tokyo Chemical Industry, Tokyo, Japan) at room temperature for 15 min. Finally, biotin-labeled anti-histamine (Monoclonal Mouse Histamine Antibody, Protein A, Protein G affinity chromatography, LifeSpan BioSciences, Inc., Seattle, WA, USA) antibody was diluted with PBS to a final concentration 0.33 µM, incubated at room temperature shaking at 45 rpm for 30 min. Atomic Force Microscope (AFM) (Hitachi High-Tech Science Corporation.) was performed for surface morphology observation during the modification procedure. As shown in Figure 6b, the surface modification was confirmed by surface morphology and height profile observation. The average height was 1.80 nm, 1.97 nm, 2.69 nm and 10.4 nm for before avidin immobilization, after avidin immobilization, after surface blocking, after biotin-labeled anti-histamine antibody immobilization, respectively.
Photonics 2022, 9, x FOR PEER REVIEW 9 of 13 Figure 6a shows the procedure of surface modification on the sensing plate. First, the sensing plate was ultrasonically cleaned with acetone and ethanol. Second, the surface was soaked in a 0.5 mM 2-carbomethoxy ethyltrichlorosilane (CMETS) (Fujifilm Wako, Osaka, Japan) solution of CMETS in toluene (99.5%, Fujifilm Wako, Osaka, Japan) at −15 °C for 1 h. The ester group was produced. By immersing the sensing plate in 35% HCl (35-37%, Fujifilm Wako, Osaka, Japan) at room temperature for 24 h, the carboxylation reaction was realized. After that, 3 mM N-hydroxysuccinimide (NHS) (98.0 ~ 102.0%, Fujifilm Wako, Osaka, Japan) and 1 mM 1-ethyl-3-(3-dimethrlaminopropyl)-carbodimide hydrochloride (EDC) (Over 98.0%, Fujifilm Wako, Osaka, Japan) in phosphate-buffered saline (PBS) (pH 7.4, Thermo Fisher Scientific, Massachusetts, USA) were prepared, and the carboxyl group was activated by immersing the sensing plate in an NHS & EDC solution at pH 7.4 for 30 min at room temperature. The sensing plate was glued to a measuring substrate with four wells, and avidin (affinity purified, Vector Laboratories, Burlingame, USA), diluted to 0.147 μM with PBS and 30 μL, was pipetted into each well for immobilization at 4 °C for 24 h. Then, the surface was blocked by 1 mM 2-Aminoethanol (Over 99.0%, Tokyo Chemical Industry, Tokyo, Japan) at room temperature for 15 min. Finally, biotin-labeled anti-histamine (Monoclonal Mouse Histamine Antibody, Protein A, Protein G affinity chromatography, LifeSpan BioSciences, Inc., Seattle, WA, USA) antibody was diluted with PBS to a final concentration 0.33 μM, incubated at room temperature shaking at 45 rpm for 30 min. Atomic Force Microscope (AFM) (Hitachi High-Tech Science Corporation.) was performed for surface morphology observation during the modification procedure. As shown in Figure 6b, the surface modification was confirmed by surface morphology and height profile observation. The average height was 1.80 nm, 1.97 nm, 2.69 nm and 10.4 nm for before avidin immobilization, after avidin immobilization, after surface blocking, after biotin-labeled anti-histamine antibody immobilization, respectively. The measurement procedure was described as follows. First, reference solution and three different concentrations of histamine 3 nM, 30 nM, and 300 nM were pipetted into four wells to interact with biotin-labeled anti-histamine antibody modified sensing plate shaking at 45 rpm for 1 h. After reaction, the wells were washed with PBS buffer for 10 times to remove the unbound histamine. Figure 7a showed the THz amplitude changes before and after histamine reaction with antibody. The terahertz amplitude was automatedly calculated by lab-developed program with MATLAB software (R2017a, The MathWorks, Inc., Japan) in the 1.5 square mm area excluding the singularities, which was marked with black line in Figure 7a. Figure 7b showed that a linear relationship was observed by plotting the THz amplitude against the histamine concentrations in logarithmic scale, in which the THz amplitude was offset by 3 nM. Large variation in the THz amplitude was obtained at concentration of 30 nM because of the variation using different sensing plates. However, the variation among the sensing plates can be compensated by pH measurement for more accurate detection [40]. By using TCM, trace level down to nM concentration of histamine could be detected, and high correlation coefficient (R 2 = 0.995) was obtained. The total measurement time was about 20 min, which was significantly shorter than current wildly used methods. The results demonstrated that TCM could be a novel approach for many small biomolecules monitoring.
The measurement procedure was described as follows. First, reference solution and three different concentrations of histamine 3 nM, 30 nM, and 300 nM were pipetted into four wells to interact with biotin-labeled anti-histamine antibody modified sensing plate shaking at 45 rpm for 1 h. After reaction, the wells were washed with PBS buffer for 10 times to remove the unbound histamine. Figure 7a showed the THz amplitude changes before and after histamine reaction with antibody. The terahertz amplitude was automatedly calculated by lab-developed program with MATLAB software (R2017a, The Math-Works, Inc., Japan) in the 1.5 square mm area excluding the singularities, which was marked with black line in Figure7a. Figure 7b showed that a linear relationship was observed by plotting the THz amplitude against the histamine concentrations in logarithmic scale, in which the THz amplitude was offset by 3 nM. Large variation in the THz amplitude was obtained at concentration of 30 nM because of the variation using different sensing plates. However, the variation among the sensing plates can be compensated by pH measurement for more accurate detection [40]. By using TCM, trace level down to nM concentration of histamine could be detected, and high correlation coefficient (R 2 = 0.995) was obtained. The total measurement time was about 20 min, which was significantly shorter than current wildly used methods. The results demonstrated that TCM could be a novel approach for many small biomolecules monitoring.

Conclusions
TCM has been proposed and developed not only for small molecular weight molecules such as ions, proton, chemical substances with/without charge, but also large molecular weight biomolecules including cancer marker, proteins, antibodies, enzymes. Various types of surface functionalization method can be achieved on the sensing plate for interest of substances. The new application in detection of histamine that released from allergic response for fast screening of allergen was also explored. Very low concentration of histamine (nM level) could be measured. By using TCM, label-free, rapid, and highly sensitivity, accurate measurements could be achieved. These features demonstrate that TCM has a great potential in future chemical sensing and biosensing. More impressive progress is that TCM is now being developed and applied to detect SARS-CoV-2, liquid

Conclusions
TCM has been proposed and developed not only for small molecular weight molecules such as ions, proton, chemical substances with/without charge, but also large molecular weight biomolecules including cancer marker, proteins, antibodies, enzymes. Various types of surface functionalization method can be achieved on the sensing plate for interest of substances. The new application in detection of histamine that released from allergic response for fast screening of allergen was also explored. Very low concentration of histamine (nM level) could be measured. By using TCM, label-free, rapid, and highly sensitivity, accurate measurements could be achieved. These features demonstrate that TCM has a great potential in future chemical sensing and biosensing. More impressive progress is that TCM is now being developed and applied to detect SARS-CoV-2, liquid biopsy, and neurotransmitters, as well as other biological substances, which aims to provide an effective and accurate method to fight against diseases and environmental threats around us. Data Availability Statement: Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.