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Terahertz range occupies an intermediate position between the microwave and optical parts of the electromagnetic spectrum. In this range are line rotational transitions of molecules, as well as vibrational and vibrational-rotational transitions of large molecules, including organic, which opens up opportunities for their research and for selective effect on them.
Теги: nanoparticles nano-sized imaging contrast agents spectroscopy terahertz radiation наноконтрастные агенты наночастицы спектроскопия терагерцевое излучение
Due to low-energy quanta, THz radiation is relatively safe for living organisms, and can be used to identify pathologies and foreign matter by methods of THz tomography. For example, THz time domain spectroscopy is based on the use of broadband coherent pulses and allows you to identify the "fingerprints" of different molecules, which is important for detection and identification of hazardous chemicals, drugs, express-analysis of the composition of the exhaled gases to diagnose diseases, and quality control of food and agricultural products.
Many processes in liquids occur in the THz range and can be investigated by THz waves. THz spectroscopy allows to obtain information about the structure and dynamics of proteins. Many amino acids can be distinguished on the basis of their THz spectrum, especially in crystalline form. THz spectra of individual pairs of DNA allow us to understand the dynamics of bioprocesses. Label-free measurements of protein-protein interaction for the study of cellular activity is possible. Differences in humidity of tissues, structure and chemical composition effectively determined using THz waves, since the latter are strongly absorbed by water, which can be used for early diagnosis of diseases.
Table 1 presents the basic characteristics of THz radiation, and table 2 presents emitters of THz waves.
THz radiation in bioanalysis
Researches [1] of application of THZ for detection of protein structural states, monitoring of receptor relationships, markerless DNA sequencing, imaging and cataloguing of acquisitions and contrast mechanism in tissues, studies of radiation effects in bioprocesses and samples are known [1]. Since the energy of the photons is small (1-12 meV), the damage to the cells or tissue due to thermal effects is negligible (strong resonant absorption is unlikely). At the same time, these energies are consistent with the molecular vibration, torsion and librational modes in liquids and solids, what is important for spectroscopy. With the passage of THZ through the tissue dominates the scattering mechanism of Tyndall, not Rayleigh, which is typical for IR and visible ranges, because the size of the cells is much less than the wavelength. Thus, the most important properties of materials – electric susceptibility and bulk conductivity. In the interaction of THZ with biomaterials there is a strong absorption due to dielectric polarization, which follows from the relaxation Debye model in polar liquids up to 1 THz (the exponential dependence of passing power Pout/Pin=e-αx, the absorption coefficient α of more than 500 cm-1 to 3 THz or more than 2000 dB/cm). Fig.1 and 2 shows absorption spectra and refractive index of deionized water, skin, fat and muscle tissues [2].
Blood has a low frequency resistance up to 140 Ohm∙cm and, therefore, is characterized by loss even without taking into account the absorption of water. Typical tissue – fat, cerebral cortex, liver, muscles have a higher resistivity (>1000 Ohm∙cm), at least up to megahertz frequencies. Because real materials consist of conductive and non-conductive particles in suspension or layers, high-frequency parameters will be different. However, these differences are small in the overall high level of absorption (according to [3], at 120 GHz, the absorption rate was 75, 71, 79, 83 cm-1 for blood, serum, saline and nutrient medium).
Moving from absorption to reflection, it is possible to observe a much more informative picture. For example, the refractive index of distilled water (1.33 in the visible range) varies from 80 (1 GHz) up to 2 (1 THz), as well as of blood and tissue. Thus, to identify the types of tissues more actual is the measurement of frequency spectrum of reflection than absorption. At the same time, high absorption coefficient, which restricts the penetration of THz radiation through tissue, allows to obtain a high contrast between tissues with different water content. This is important, for example, when examining degree of burns on necrotic skin samples and morphology of tumors.
THz spectroscopy is highly relevant, for example, when comparing signatures of reflection or absorption of the samples exposed to chemical or physical changes, such as differences of conformal states, the changes in the density or polarization, dehydration, temperature.
Prospective are measurements of Avidin-Biotin interaction [4], DNA hybridization [5]. Avidin-Biotin interaction is used in biotechnology for fixing of the produced proteins to surfaces in selective chromatography, transportation of drugs, fluorescent labeling. The binding leads to a change in refractive index of a surface film that changes the reflection of the THz beam. A similar change of the refraction occurs when the DNA hybrid in the solution. More difficult to quantify conformal changes of bundles or bend of molecular chains.
The study of rhodopsin is particularly interesting, allowing you to perform a real-time monitoring of changes after the selection of the appropriate frequency [6]. In addition to molecular signatures (based on THz spectroscopy) has already made a image catalogs (THz vision) of normal and damaged tissue in the range of 500-1500 GHz [3].
THz radiation in medicine and pharmacy
One of the most important applications of THz radiation in medicine is the early detection and diagnosis of diseases. Successful examples are the identification of caries [7], the assessment of the degree of skin burns [8], the control of wound healing and scarring [9], the detection of subdermal carcinoma [9]. The transparency of materials in the THz range allows to examine the wound without removal of plaster or bandage. IR and microwave thermography is used in neurology, oncology, rheumatology, ophthalmology, cardiology, dermatology and surgery. For forming of a thermal image the resolution less than 0.1 K is required. Typical temperature gradients in the skin (from inner to outer surface) are from 0.2 to 0.5 К/mm [10]. Modern uncooled thermal infrared microbolometer camera have a resolution of less than 0.04 K at frame rate of 30 Hz. In the THz range such permission has yet not been achieved. However, the advantage of THz camera is a penetrating power – the obtaining of 3D images (in particular, the ability to detect subcutaneous "hot areas") is possible. In addition, THz vision enables identification of diseases in the respiratory, digestive and vascular systems by endoscopy or catheters. Due to the difference of the reflective signatures of tissue, the results of the application of THz systems for identifying areas of atherosclerosis, plaque formation, fat layers, scars, and other endothelial abnormalities already received [2].
Since the invention of the first submillimeter generators in the mid 1960's explores possible harmful effects of THz on the health. The study examined not only thermal effects (tissue heating), but also informational effects [11], including permeability and adhesion of the cell membrane, ATP synthesis, immune response, metabolism rate, stimulation of receptors in the Central nervous system, electric effect on the cerebral cortex, and many other biological functions, as well as the positive therapeutic effect of submillimeter radiation. Were found the effects of "memory" when the state changes of water or moisture-containing tissue was maintained for 10 minutes after irradiation. In 1968 Frohlich one of the first predicted THz cell response [12]. Extensive research in this field has been conducted in Russia [13].
Since in proteins and oligonucleotides resonance mode was detected, the effects of THz radiation at the cellular and subcellular processes is possible. Under THz irradiation at the resonant frequencies are possible changes in the molecular or crystalline structure of the substance, therefore, it is possible to create materials with new properties. For example, the function of biomolecular proteins vary depending on their form. Such proteins include prions, the infectious agents that cause so-called mad cow disease (bovine spongiform encephalopathy) – a neurodegenerative disease of cattle. Normal prion molecules consist of 4 spiral structures (α-helices), and in the abnormal prions 2α-helix are unwounded in β-sheets. Currently an innovative method to transform the abnormal prions in normal molecules under the influence of THz irradiation is developed [14].
The rapid development of nanotechnology stimulates the expansion of applications of THz radiation both from the point of view of development of new sources, detectors, waveguides, and of creation of nano-contact agents for THz vision [15]. Nano-contact agents are used to increase image contrast of healthy and pathological areas of tissue or molecules. As such agents can be applied spherical particles, carbon nanotubes, fullerenes, quantum dots, nanorods, nanoshells, nanocages, nanowires, various metal and oxide nanoparticles. Nanoparticles capable of creating surface plasmons (plasmonic nanoparticles), are especially interesting for therapy and vision [15]. The most demanded metal for bioprinting is gold due to its biocompatibility, strong scattering near the resonant frequencies of the local surface plasmons (LSP), the ability to accept bioconjugation processes [16].
Nano-contact agents allow you to use the effect of hyperthermia [17] due to surface-plasmon polaritons (SPP) under irradiation of nanoparticles by an IR laser. As a result of this effect, the temperature of the water in the cancer cells is increased, and, since the THz signal is sensitive to changes in water temperature, the cancer cells can be detected and visualized (fig.3) [17].
Studies of nanocomposites from hydroxyapatite/gold and gold nanorods (GNRs) have shown, that contrast agents can enhance the sensitivity of THz signals and to be restricted by cancer cells, and thus can be targeted to cancer tumor [18]. Also as contrast agents for THz vision were investigated nanoparticles of gadolinium oxide (Gd2O3) [19]. The results showed, that these particles absorb THz waves by three orders of magnitude higher than water, and allow medical THz vision. Since nanoparticles of gadolinium oxide has been used for magnetic resonance vision (MRI), they give the opportunity to combine the methods. The simultaneous use of nanoparticles for THz vision, and as hyperthermal therapeutic agents will implement a diagnosis in the early stages of cancer and therapy. Moreover, the technique of THz vision can be used to monitor drug delivery and the use of IR laser with THz technique opens the possibilities of practical THz endoscopy [20].
Pulsed THz spectroscopy is used for research and non-destructive testing of multicomponent drugs in pill form [21]. The experimental installation is shown in fig.4. In particular, we have obtained THz image of the distribution of polymorphic forms of famotidine ties with D-mannitol, and determined the vibrational modes of the peaks in the spectra of famotidine polymorphic forms. The measurements were carried out at temperatures from 77 to 298 K. Fig.5 [21] shows the THz transmission spectra of different forms of famotidine at different temperatures. Fig.6 [21] shows THz spectroscopic image of compressed tablets of 10 mm diameter and 2 mm thick, containing famotidine polymorphic forms A and B, and D-mannitol, measured at a temperature of 220 K and 298 K.
Very actual application of pulsed THz spectroscopy and vision is a diagnostics of the brain gliomas [22]. Fig.7 shows the diagram of THz spectroscopy in the time domain. In particular, in the frequency range of 0.2–2 THz were investigated normal and diseased brain cells, placed in paraffin capsules. Fig.8 [22] shows the spectra of refraction and absorption in normal and diseased brain cells.
Technical perspectives
Further progress in the development of THz systems for biomedical research depends primarily on the development of light sources and detectors of THz radiation with improved characteristics (power, sensitivity, spectral range, operating temperature, power consumption, weight and size parameters). Among the most promising directions it should be noted quantumcascade and graphene lasers, and a photoconductive antenna with a plasmon nanoelectrodes. All these devices do not require cryogenic cooling systems and have the ability to adjust the operating frequency and bandwidth, and most importantly can operate in mode emitters, and in the mode receivers (detectors). ■
Many processes in liquids occur in the THz range and can be investigated by THz waves. THz spectroscopy allows to obtain information about the structure and dynamics of proteins. Many amino acids can be distinguished on the basis of their THz spectrum, especially in crystalline form. THz spectra of individual pairs of DNA allow us to understand the dynamics of bioprocesses. Label-free measurements of protein-protein interaction for the study of cellular activity is possible. Differences in humidity of tissues, structure and chemical composition effectively determined using THz waves, since the latter are strongly absorbed by water, which can be used for early diagnosis of diseases.
Table 1 presents the basic characteristics of THz radiation, and table 2 presents emitters of THz waves.
THz radiation in bioanalysis
Researches [1] of application of THZ for detection of protein structural states, monitoring of receptor relationships, markerless DNA sequencing, imaging and cataloguing of acquisitions and contrast mechanism in tissues, studies of radiation effects in bioprocesses and samples are known [1]. Since the energy of the photons is small (1-12 meV), the damage to the cells or tissue due to thermal effects is negligible (strong resonant absorption is unlikely). At the same time, these energies are consistent with the molecular vibration, torsion and librational modes in liquids and solids, what is important for spectroscopy. With the passage of THZ through the tissue dominates the scattering mechanism of Tyndall, not Rayleigh, which is typical for IR and visible ranges, because the size of the cells is much less than the wavelength. Thus, the most important properties of materials – electric susceptibility and bulk conductivity. In the interaction of THZ with biomaterials there is a strong absorption due to dielectric polarization, which follows from the relaxation Debye model in polar liquids up to 1 THz (the exponential dependence of passing power Pout/Pin=e-αx, the absorption coefficient α of more than 500 cm-1 to 3 THz or more than 2000 dB/cm). Fig.1 and 2 shows absorption spectra and refractive index of deionized water, skin, fat and muscle tissues [2].
Blood has a low frequency resistance up to 140 Ohm∙cm and, therefore, is characterized by loss even without taking into account the absorption of water. Typical tissue – fat, cerebral cortex, liver, muscles have a higher resistivity (>1000 Ohm∙cm), at least up to megahertz frequencies. Because real materials consist of conductive and non-conductive particles in suspension or layers, high-frequency parameters will be different. However, these differences are small in the overall high level of absorption (according to [3], at 120 GHz, the absorption rate was 75, 71, 79, 83 cm-1 for blood, serum, saline and nutrient medium).
Moving from absorption to reflection, it is possible to observe a much more informative picture. For example, the refractive index of distilled water (1.33 in the visible range) varies from 80 (1 GHz) up to 2 (1 THz), as well as of blood and tissue. Thus, to identify the types of tissues more actual is the measurement of frequency spectrum of reflection than absorption. At the same time, high absorption coefficient, which restricts the penetration of THz radiation through tissue, allows to obtain a high contrast between tissues with different water content. This is important, for example, when examining degree of burns on necrotic skin samples and morphology of tumors.
THz spectroscopy is highly relevant, for example, when comparing signatures of reflection or absorption of the samples exposed to chemical or physical changes, such as differences of conformal states, the changes in the density or polarization, dehydration, temperature.
Prospective are measurements of Avidin-Biotin interaction [4], DNA hybridization [5]. Avidin-Biotin interaction is used in biotechnology for fixing of the produced proteins to surfaces in selective chromatography, transportation of drugs, fluorescent labeling. The binding leads to a change in refractive index of a surface film that changes the reflection of the THz beam. A similar change of the refraction occurs when the DNA hybrid in the solution. More difficult to quantify conformal changes of bundles or bend of molecular chains.
The study of rhodopsin is particularly interesting, allowing you to perform a real-time monitoring of changes after the selection of the appropriate frequency [6]. In addition to molecular signatures (based on THz spectroscopy) has already made a image catalogs (THz vision) of normal and damaged tissue in the range of 500-1500 GHz [3].
THz radiation in medicine and pharmacy
One of the most important applications of THz radiation in medicine is the early detection and diagnosis of diseases. Successful examples are the identification of caries [7], the assessment of the degree of skin burns [8], the control of wound healing and scarring [9], the detection of subdermal carcinoma [9]. The transparency of materials in the THz range allows to examine the wound without removal of plaster or bandage. IR and microwave thermography is used in neurology, oncology, rheumatology, ophthalmology, cardiology, dermatology and surgery. For forming of a thermal image the resolution less than 0.1 K is required. Typical temperature gradients in the skin (from inner to outer surface) are from 0.2 to 0.5 К/mm [10]. Modern uncooled thermal infrared microbolometer camera have a resolution of less than 0.04 K at frame rate of 30 Hz. In the THz range such permission has yet not been achieved. However, the advantage of THz camera is a penetrating power – the obtaining of 3D images (in particular, the ability to detect subcutaneous "hot areas") is possible. In addition, THz vision enables identification of diseases in the respiratory, digestive and vascular systems by endoscopy or catheters. Due to the difference of the reflective signatures of tissue, the results of the application of THz systems for identifying areas of atherosclerosis, plaque formation, fat layers, scars, and other endothelial abnormalities already received [2].
Since the invention of the first submillimeter generators in the mid 1960's explores possible harmful effects of THz on the health. The study examined not only thermal effects (tissue heating), but also informational effects [11], including permeability and adhesion of the cell membrane, ATP synthesis, immune response, metabolism rate, stimulation of receptors in the Central nervous system, electric effect on the cerebral cortex, and many other biological functions, as well as the positive therapeutic effect of submillimeter radiation. Were found the effects of "memory" when the state changes of water or moisture-containing tissue was maintained for 10 minutes after irradiation. In 1968 Frohlich one of the first predicted THz cell response [12]. Extensive research in this field has been conducted in Russia [13].
Since in proteins and oligonucleotides resonance mode was detected, the effects of THz radiation at the cellular and subcellular processes is possible. Under THz irradiation at the resonant frequencies are possible changes in the molecular or crystalline structure of the substance, therefore, it is possible to create materials with new properties. For example, the function of biomolecular proteins vary depending on their form. Such proteins include prions, the infectious agents that cause so-called mad cow disease (bovine spongiform encephalopathy) – a neurodegenerative disease of cattle. Normal prion molecules consist of 4 spiral structures (α-helices), and in the abnormal prions 2α-helix are unwounded in β-sheets. Currently an innovative method to transform the abnormal prions in normal molecules under the influence of THz irradiation is developed [14].
The rapid development of nanotechnology stimulates the expansion of applications of THz radiation both from the point of view of development of new sources, detectors, waveguides, and of creation of nano-contact agents for THz vision [15]. Nano-contact agents are used to increase image contrast of healthy and pathological areas of tissue or molecules. As such agents can be applied spherical particles, carbon nanotubes, fullerenes, quantum dots, nanorods, nanoshells, nanocages, nanowires, various metal and oxide nanoparticles. Nanoparticles capable of creating surface plasmons (plasmonic nanoparticles), are especially interesting for therapy and vision [15]. The most demanded metal for bioprinting is gold due to its biocompatibility, strong scattering near the resonant frequencies of the local surface plasmons (LSP), the ability to accept bioconjugation processes [16].
Nano-contact agents allow you to use the effect of hyperthermia [17] due to surface-plasmon polaritons (SPP) under irradiation of nanoparticles by an IR laser. As a result of this effect, the temperature of the water in the cancer cells is increased, and, since the THz signal is sensitive to changes in water temperature, the cancer cells can be detected and visualized (fig.3) [17].
Studies of nanocomposites from hydroxyapatite/gold and gold nanorods (GNRs) have shown, that contrast agents can enhance the sensitivity of THz signals and to be restricted by cancer cells, and thus can be targeted to cancer tumor [18]. Also as contrast agents for THz vision were investigated nanoparticles of gadolinium oxide (Gd2O3) [19]. The results showed, that these particles absorb THz waves by three orders of magnitude higher than water, and allow medical THz vision. Since nanoparticles of gadolinium oxide has been used for magnetic resonance vision (MRI), they give the opportunity to combine the methods. The simultaneous use of nanoparticles for THz vision, and as hyperthermal therapeutic agents will implement a diagnosis in the early stages of cancer and therapy. Moreover, the technique of THz vision can be used to monitor drug delivery and the use of IR laser with THz technique opens the possibilities of practical THz endoscopy [20].
Pulsed THz spectroscopy is used for research and non-destructive testing of multicomponent drugs in pill form [21]. The experimental installation is shown in fig.4. In particular, we have obtained THz image of the distribution of polymorphic forms of famotidine ties with D-mannitol, and determined the vibrational modes of the peaks in the spectra of famotidine polymorphic forms. The measurements were carried out at temperatures from 77 to 298 K. Fig.5 [21] shows the THz transmission spectra of different forms of famotidine at different temperatures. Fig.6 [21] shows THz spectroscopic image of compressed tablets of 10 mm diameter and 2 mm thick, containing famotidine polymorphic forms A and B, and D-mannitol, measured at a temperature of 220 K and 298 K.
Very actual application of pulsed THz spectroscopy and vision is a diagnostics of the brain gliomas [22]. Fig.7 shows the diagram of THz spectroscopy in the time domain. In particular, in the frequency range of 0.2–2 THz were investigated normal and diseased brain cells, placed in paraffin capsules. Fig.8 [22] shows the spectra of refraction and absorption in normal and diseased brain cells.
Technical perspectives
Further progress in the development of THz systems for biomedical research depends primarily on the development of light sources and detectors of THz radiation with improved characteristics (power, sensitivity, spectral range, operating temperature, power consumption, weight and size parameters). Among the most promising directions it should be noted quantumcascade and graphene lasers, and a photoconductive antenna with a plasmon nanoelectrodes. All these devices do not require cryogenic cooling systems and have the ability to adjust the operating frequency and bandwidth, and most importantly can operate in mode emitters, and in the mode receivers (detectors). ■
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