Abstract-High-sensitivity detection of metastatic breast cancer cells via terahertz chemical microscopy using aptamers

Eman M.Hassan, Ahmed Mohamed, Maria C.DeRosa, William G.Willmore, Yuki Hanaoka, Toshihiko Kiwa, Tsuneyuki Ozaki


https://www.sciencedirect.com/science/article/pii/S0925400519302229?via%3Dihub

We demonstrate high-sensitivity detection of metastatic breast cancer cells using terahertz (THz) chemical microscopy (TCM) with aptamers as ligands. For the aptamers, we use previously developed synthetic single stranded (ss) DNA aptamers mammaglobin B1 (MAMB1) and mammaglobin A2 (MAMA2) that bind to mammaglobin B and mammaglobin A proteins, respectively, which are overexpressed on the surface of MCF7 and MDA-MB-415 breast cancer cells. Each aptamer was immobilized on the surface of a sensing plate, and the amplitude of the THz signal was measured upon the binding of each aptamer to different number (10–106) of its target breast cancer cells. A change in the THz amplitude was observed when MAMB1 and MAMA2 bind to MCF7 and MDA-MB-415, respectively. We find that this change was linear as a function of the log number of breast cancer cells used. No change in the THz amplitude was observed when the same number of normal breast cells (MCF 10A) were used. Moreover, MAMB1 and MAMA2 did not show binding to the counter breast cancer cells, indicating high selectivity. We have demonstrated that the TCM using aptamers as ligands has a limit of detection as small as 1 breast cancer cell in 100 μL of sample. Results described here indicate that the TCM could be a powerful tool to detect metastatic breast cancer cells.

Okayama University Research: Technology to Rapidly Detect Cancer Markers for Cancer Diagnosis

Breast-cancer cells bound to aptamers have a different terahertz response than freestanding aptamers. This notion can be exploited to detect, with high sensitivity, breast-cancer cells.

https://finance.yahoo.com/news/okayama-university-research-technology-rapidly-053400536.html

OKAYAMA, Japan, April 29, 2019 /PRNewswire/ -- Researchers at Okayama University report in the journal Sensors and Actuators B: Chemical that terahertz radiation can be used to rapidly detect makers for breast-cancer cells. The scientists present a technique that makes use of the binding properties of aptamers, synthetic organic molecules acting as probes for cancer cells.

Breast cancer is the most common cancer in women. Detecting it in time is crucial for treatment to be successful. X-ray screening (mammography) is the standard detection technique but is not without risk as it involves exposure of a patient to ionizing radiation. Another approach for detecting breast cancer cells is based on terahertz (THz) radiation, which is sensitive to polar molecules like water — normal and cancer tissues do not have the same water content. Associate Professor Toshihiko Kiwa (Okayama University, Japan) and Professor Tsuneyuki Ozaki (INRS : Institut national de la recherche scientifique, Canada) and colleagues have now discovered a way to increase the sensitivity of THz radiation for the detection of makers of breast cancer cells, implying that 'THz chemical microscopy' could become a powerful alternative screening technique.

The key principle underlying the method of Associate Professor Kiwa and colleagues is that cancer and normal breast cells bind and don't bind, respectively, to certain molecules known as aptamers.  The aptamers consist of (single-stranded) DNA or RNA fragments; they have a high affinity for particular molecules — in the experiments of the team of Associate Professor Kiwa, these molecules were breast cancer cells.

The experimental setup of the researchers involved a sensing plate, consisting of a sapphire substrate, and silicon and silicon dioxide thin films. The aptamers were fixed to the top layer.  Irradiating the plate with a specific laser creates charge carriers, the motion of which generates electromagnetic radiation in the THz range. The precise THz response depends, however, on whether the aptamers are 'freestanding' (no cells attached) or not (breast-cancer cells attached).  The former corresponds to the situation where the sample on the sensing plate only consists of normal cells; for the latter, the sample contains breast-cancer cells.

The scientists noted that the detection method is highly sensitive: the so-called 'limit of detection' was found to be as low as 1 cancer cell in 0.1 milliliter of sample. Moreover, a qualitative assessment of a sample seems possible, as the change in THz signal can be associated with the number of cancer cells per aptamer. Although further investigation on the latter association is needed, Associate Professor Kiwa and colleagues conclude that " the results obtained from this study can be the spark of new evolution in the detection of breast cancer."

Background

Mammography

Mammography is the procedure in which low-energy X-rays are used to screen the human breast for cancer. Although it is the standard technique for breast cancer detection, the use of X-rays implies exposure to ionizing radiation. Because there have also been cases of false positives (apparent detection of breast cancer in a healthy patient) and false negatives (missed detections), alternative detection methods are desired. Associate Professor Toshihiko Kiwa and Professor Tsuneyuki Ozaki and colleagues have now reported a promising detection technique, in which a tissue sample is investigated by terahertz (THz) radiation.

Aptamers

Aptamers are short DNA or RNA molecules, or molecules built from peptides, that can bind to specific molecules. Associate Professor Kiwa and colleagues used the binding properties of two aptamers, mammaglobin B1 (MAMB1) and mammaglobin A2 (MAMA2). These aptamers bind to proteins (mammaglobin B and mammaglobin A, respectively) that are overexpressed in typical breast-cancer cells.  Attached to a special engineered sensing plate, freestanding and bound (to cancer cells) aptamers have a different response to terahertz radiation. This difference can be used to distinguish a tissue sample with breast-cancer cells from a sample with only normal cells.

Caption

Breast-cancer cells bound to aptamers have a different terahertz response than freestanding aptamers. This notion can be exploited to detect, with high sensitivity, breast-cancer cells.

Reference

Eman M. Hassan, Ahmed Mohamed, Maria C. DeRos, William G. Willmore, Yuki Hanaoka, Toshihiko Kiwa, Tsuneyuki Ozaki. High-sensitivity detection of metastatic breast cancer cells via terahertz chemical microscopy using aptamers. Sensors & Actuators: B. Chemical, 287 (2019), 595–601.

DOI : https://doi.org/10.1016/j.snb.2019.02.019

Abstract-Imaging of Chemical Reactions Using a Terahertz Chemical Microscope

Toshihiko Kiwa  , Tatsuki Kamiya , Taiga Morimoto , Kentaro Fujiwara , Yuki Maeno , Yuki Akiwa, Masahiro Iida, Taihei Kuroda, Kenji Sakai, Hidetoshi Nose, Masaki Kobayashi, Keiji Tsukada

https://www.mdpi.com/2304-6732/6/1/10

This study develops a terahertz (THz) chemical microscope (TCM) that visualizes the distribution of chemical reaction on a silicon-based sensing chip. This chip, called the sensing plate, was fabricated by depositing Si thin films on a sapphire substrate and thermally oxidizing the Si film surface. The Si thin film of the sensing plate was irradiated from the substrate side by a femtosecond laser, generating THz pulses that were radiated into free space through the surface field effect of the Si thin film. The surface field responds to chemical reactions on the surface of the sensing plate, changing the amplitude of the THz pulses. This paper first demonstrates the principle and experimental setup of the TCM and performs the imaging and measurement of chemical reactions, including the reactions of bio-related materials

Abstract-Evaluation of Bio-materials Using a Laser-excited Terahertz Wave

Toshihiko Kiwa, Tatsuki Kamiya, Masahiro Iida, Hirofumi Inoue, Kenji Sakai, Shinichi Toyooka, Keiji Tsukada,

https://www.jstage.jst.go.jp/article/jslsm/39/4/39_jslsm-39_0031/_article/-char/en

We have developed various types of terahertz sensing systems for evaluation of bio-related materials. Here, we describe a terahertz chemical microscopy that we have invented and demonstrate a label-free immune assay and evaluation of penetration speed of cosmetic liquid, each of which was realized using different way of use of the terahertz chemical microscopy.

Okayama University Research: Measuring ion Concentration in Solutions for Clinical and Environmental Research

Schematic of the sensing plate when it is illuminated by the femtosecond laser. Inset is the microphotograph of the microsolution wells. (PRNewsfoto/Okayama University)

Okayama University researchers describe in the journal Optics Express the use of Terahertz (THz) chemical microscopy to measure the pH of water-based solutions with a volume as small as 16 nL. The findings are important to be able to measure pH concentrations in small-volume solutions for clinical and environmental analyses.

https://www.prnewswire.com/news-releases/okayama-university-research-measuring-ion-concentration-in-solutions-for-clinical-and-environmental-research-686667031.html

For clinical and environmental research and monitoring it is important to be able to measure pH concentrations in small-volume solutions. However, conventional systems used to measure the concentration of ions require the use of reference electrodes that end up reducing the volume of the solution, setting a limit on the minimum volume that it is possible to analyze.

Now, Dr. Toshihiko Kiwa and colleagues at the Graduate School of Natural Science and Technology in Okayama University, Japan, demonstrated the use of Terahertz (THz) chemical microscopy to measure the pH of water-based solutions with a volume as small as 16 nL. The results are published in Optics Express. This type of microscope has a sensing plate with patterned micro wells hosting the solution; an ultrafast laser pulse directed on the sensing plate generates a photocurrent with ultrafast modulation that, in turn, emits THz radiation into free space. Because the amplitude of the THz radiation depends on the concentration of ions in the micro wells, this method opens up the possibility of imaging the concentration of ions without the need of using electrodes. This enables the measurement of volumes of solution that would be too small for conventional methods.

The THz chemical microscope, which was developed by this same group in 2007, features a semiconducting (silicon) thin film mounted on a sapphire substrate that acts as the sensing plate. A layer of oxide naturally forms on the silicon film, providing an insulating layer between the silicon surface and the solution. The researchers added a resin on top of the oxide layer and used conventional photolithographic techniques to pattern micro wells in it, obtaining wells with a volume of 16 nL. They also optimized the laser pulses to stabilize the signal, and integrating this method into the microscope is part of the next steps the researchers intend to take.

Thinking about the future directions the team is interested to explore, the author says that "we will attempt the integration for multi-ion sensing and reducing the laser spot size to improve the accuracy of THz chemical microscopy."

About Okayama University 

Okayama University is one of the largest comprehensive universities in Japan with roots going back to the Medical Training Place sponsored by the Lord of Okayama and established in 1870. Now with 1,300 faculty and 13,000 students, the University offers courses in specialties ranging from medicine and pharmacy to humanities and physical sciences.

Okayama University is located in the heart of Japan approximately 3 hours west of Tokyo by Shinkansen.

Website: http://www.okayama-u.ac.jp/index_e.html

Correspondence to 

Associate Professor Toshihiko Kiwa, Ph.D.

Advanced Electro Measurement Technology Laboratory,

Graduate School of Interdisciplinary Science and Engineering

in Health Systems, Okayama University,

3-1-1 Tsushimanaka, Kita-Ku, Okayama 700-8530, Japan

kiwa@okayama-u.ac.jp

http://www.ec.okayama-u.ac.jp/~sense/index.html

Further information
Okayama University
1-1-1 Tsushima-naka , Kita-ku , Okayama 700-8530, Japan
Public Relations and Information Strategy
E-mail: www-adm@adm.okayama-u.ac.jp

Website: http://www.okayama-u.ac.jp/index_e.html
Okayama Univ. e-Bulletin: http://www.okayama-u.ac.jp/user/kouhou/ebulletin/
About Okayama University (YouTube): https://www.youtube.com/watch?v=iDL1coqPRYI
Okayama University Image Movie (YouTube): https://www.youtube.com/watch?v=KU3hOIXS5kk

Reference  

Toshihiko Kiwa, Tatsuki Kamiya, Taiga Morimoto, Kenji Sakai, And Keiji Tsukada. pH measurements in 16-nL-volume solutions using terahertz chemical microscopy. Optics Express, 26(7), 8232-8238, 2018.

DOI: https://doi.org/10.1364/OE.26.008232

https://www.osapublishing.org/oe/fulltext.cfm?uri=oe-26-7-8232&id=383910

Reference (Okayama Univ. e-Bulletin): Associate Professor Kiwa's team 

e-Bulletin Vol.4：Unique terahertz chemical microscope for mapping chemical reactions

e-Bulletin Vol.10：Simple, compact, highly sensitive SQUID based magnetic field measurement sysytem to detection of a very small magnetic signals

e-Bulletin Vol.11：High-performance Terahertz Project kick-off symposium

e-Bulletin Vol.13：Terahertz chemical microscope: Innovative terahertz technology for high resolution mapping of chemical reactions, label free immunoassays, cosmetics research, and more.

SOURCE Okayama University

Abstract-pH measurements in 16-nL-volume solutions using terahertz chemical microscopy

Toshihiko Kiwa, Tatsuki Kamiya, Taiga Morimoto, Kenji Sakai, and Keiji Tsukada

https://www.osapublishing.org/oe/fulltext.cfm?uri=oe-26-7-8232

Terahertz chemical microscopy has been developed for measuring the pH of a solution using only a small volume. The microsolution wells were fabricated on the surface of the sensing plate using a conventional photolithograph technique. Because the pH value can be calculated from the amplitude of a terahertz wave directly radiated from a sensing plate by a femtosecond laser irradiation, this method does not require any reference electrode in the solution. Thus, pH measurement can be achieved with a volume as small as 16 nL.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Abstract-Stabilization method for signal drifts in terahertz chemical microscopy

Toshihiko Kiwa, Kenji Sakai, and Keiji Tsukada 
 »View Author Affiliations
http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-22-2-1330

A stabilization method for signal drifts in terahertz chemical microscopy (TCM) due to unexpected chemical potential changes in sample solutions was proposed and developed. The sensing plate was separated into two areas: a detection area and a control area. The detection area radiated a THz pulse whose amplitude was related to both the chemical reactions in the sample solutions and unexpected potential changes. The THz pulse from the control area was related only to unexpected potential changes. In the proposed system, the THz pulse from each area was interfered and detected. By adjusting the timing of the positive peak of the THz pulse from the detection area and the negative peak of the THz pulse from the control area, we detected the difference in both peaks as the interference signal. Thus, the signal deviation of 390 when the environmental condition changes in the temperature range of 38 °C and the pH range of 8.33 was stabilized to be the signal deviation of 31. As the result, the TCM with stabilization method could detect the signal shift of 121 when the 275-nmol/L immunoglobulin G was immobilized on the sensing plate.

© 2014 Optical Society of America

Imaging chemical reactions with terahertz

Imaging chemical reactions http://spie.org/x105177.xml?highlight=x2406&ArticleID=x105177 Toshihiko Kiwa, Kenji Sakai and Keiji Tsukada A novel laser-terahertz emission microscope is used to visualize changes in chemical and electrical potential. 31 December 2013, SPIE Newsroom. DOI: 10.1117/2.1201312.005281 In the technique of terahertz (THz) time-domain spectroscopy, the amplitude and phase of short-pulsed incident THz (i.e., 1012Hz) radiation are sensitive to a sample's material properties. Imaging systems that use THz time-domain spectroscopy are attractive for novel nondestructive testing (NDT) applications.1 The spatial resolution of such systems, however, has so far been limited by the wavelength of the THz radiation, which is about 300μm at 1THz. A laser-THz emission microscope (LTME) has been proposed and developed for NDT of semiconductor devices, such as large-scale integrated circuits and solar panels.2–4 The spatial resolution of the laser-THz emission technique is not limited by the THz radiation wavelength, but by the wavelength of the femtosecond (i.e., 10−15s) laser that is used to generate the THz radiation. This means that higher spatial resolutions can be achieved using an LTME than with a conventional THz imaging system. We have developed a THz chemical microscope (TCM) that can be used to visualize chemical and/or electrical potential shifts caused by chemical reactions.5, 6 Our TCM has a sensing plate that is made of thin silicon dioxide (SiO2) and silicon (Si) films on a sapphire substrate, as shown in Figure 1(a). When a femtosecond laser pulse travels through the substrate and hits the thin Si film, a THz pulse is generated and radiates into free space. As a chemical reaction progresses on the sensing plate, the chemical and/or electrical potential at the surface of the plate shifts. This causes a change in the magnitude of the local field and results in variation of the generated THz pulse's amplitude. The THz pulse contains information about the chemical reaction at precisely the point it is illuminated by the laser pulse. We are therefore able to create a map of the chemical reaction by scanning the laser across the surface of the sensing plate. The spatial resolution of our TCM is currently about 50μm, but this can be improved by optimizing the aperture of the objective lens. A photograph of our prototype TCM is shown in Figure 1(b).   Figure 1. (a) Schematic diagram of the terahertz (THz) chemical microscope (TCM) sensing plate. A femtosecond laser is focused on the thin silicon (Si) film from the substrate side of the plate, and a THz pulse radiates into the free space. (b) Photograph of the prototype TCM system. SiO2: Silicon dioxide. An image of an immune reaction that we obtained with our TCM is shown in Figure 2. During this reaction, mouse immunoglobulin G (IgG), at a concentration of about 275nmol/L, was initially immobilized by being covalently bound to the sensing plate. Anti-mouse-IgG (also with a concentration of 275nmol/L) was then reacted with the IgG on the sensing plate. We obtained a TCM image by measuring the shift in the THz pulse amplitude before and after the reaction. The image in Figure 2 shows that the THz pulse was enhanced at locations where IgG–anti-IgG bindings formed. Unlike conventional immunoassays (biochemical tests), the sensitivity of our TCM is independent of the sample material's molecular weight. We were able to detect small molecules (e.g., biotin) that are generally difficult to measure using conventional methods. Our results indicate that the TCM system can be used to perform label-free immunoassays.7   Figure 2. TCM image of the reaction between mouse immunoglobulin G (IgG) and anti-mouse-IgG. Our TCM can also be used to detect ions (e.g., potassium, K+, and sodium, Na+) in water solutions: see Figure 3(a). A difference in electrical potential arises between membrane surfaces when the sensing membrane (on the sensing plate) comes into contact with the water solution containing the specific ions. This causes a change in the amplitude of the THz pulse. Figure 3(b) shows the TCM image we obtained when the concentration of sodium ions in a water solution was changed from 10−4mol/L to 10−1mol/L. The amplitude of the THz pulse increased at the points where the sensing membranes for Na+ ions were immobilized.   Figure 3. (a) Photograph of the TCM sensing plate, with immobilized sensing polymer membranes for sodium (Na+) and potassium (K+) ions. (b) TCM image obtained when the concentration of Na ions in the water solution on the sensing plate was changed from 10−4mol/L to 10−1mol/L. We have designed and developed a TCM instrument that can detect several types of chemical reactions. Many different materials can be detected with a single scan of a femtosecond laser on a 10mm2 sensing chip. We are currently working to integrate biomaterials on a single chip. The TCM technique will be a useful screening tool, with applications in medical diagnosis and materials research. We are also developing fuel cells that will be mounted on the sensing chip so that catalytic reactions of their electrodes can be measured. This work was partly supported by an Industry-Academia Collaborative R&D grant from the Japan Science and Technology Agency. Toshihiko Kiwa, Kenji Sakai, Keiji Tsukada Okayama University Okayama, Japan Toshihiko Kiwa is an associate professor. He received his PhD from Osaka University in 2003. He subsequently worked as a JSPS fellow at Osaka University's Research Center for Superconductor Photonics. His research interests include THz-sensing systems. Kenji Sakai is an assistant professor and is involved in the research of magnetic sensors and superconducting quantum interference devices (SQUIDs) and their applications, nondestructive evaluation systems, and gas sensors. He was a research fellow of the Japan Society for the Promotion of Science between 2009 and 2010. Keiji Tsukada is a professor whose research deals with hydrogen gas, ion, and magnetic sensors, as well as SQUIDs and their applications. From 1982 to 2003 he worked at the central research laboratory of Hitachi Ltd., where he was a project leader of the SQUID application research group. References: 1. M. Tonouchi, Cutting-edge terahertz technology, Nat. Photon. 1, p. 97-105, 2007. 2. T. Kiwa, M. Tonouchi, M. Yamashita, K. Kawase, Laser terahertz-emission microscope for inspecting electrical faults in integrated circuits, Opt. Lett. 28, p. 2058-2060, 2003. 3. M. Yamashita, K. Kawase, C. Otani, T. Kiwa, M. Tonouchi, Imaging of large-scale integrated circuits using laser terahertz emission microscopy, Opt. Express 13, p. 115-120, 2005. 4. H. Nakanishi, K. A. Salek, S. Fujiwara, K. Takayama, I. Kawayama, H. Murakami, M. Tonouchi, Development of solar cell inspection system based on a laser terahertz emission microscope, 3rd EOS Topical Mtg. Terahertz Sci. Technol., p. 5291, 2012. 5. T. Kiwa, Y. Kondo, Y. Minami, I. Kawayama, M. Tonouchi, K. Tsukada, Terahertz chemical microscope for label-free detection of protein complex, Appl. Phys. Lett. 96, p. 211114, 2010. 6. T. Kiwa, T. Hagiwara, M. Shinomiya, K. Sakai, K. Tsukada, Work function shifts of catalytic metals under hydrogen gas visualized by terahertz chemical microscopy, Opt. Express 20, p. 11637-11642, 2012. 7. T. Kiwa, A. Tenma, S. Takahashi, K. Sakai, K. Tsukada, Label free immune assay using terahertz chemical microscope, Sens. Actuators B: Chem. 187, p. 8-11, 2013.