ALA-induced fluorescence detection with photoresist-based microfluidic cell sorter for bladder cancer diagnosis☆
Introduction
Early detection of cancer or precancerous lesions can contribute to timely clinical treatment and, when followed by appropriate treatment, will improve the prognosis of cancer patients. In general, there still exists a huge gap between current detection capabilities and requirements for early-stage cancer diagnosis. Improvements in existing diagnostic techniques or more efficient diagnostic approaches need to be explored by applying engineering technologies to the medical field. For example, detecting cancer cells and sorting them from a mixture of biological cells is an unmet need for early-stage cancer diagnostics and clinical management [1], [2]. To address this issue, microfluidic technologies, used extensively in academic research of cell sorting, have transitioned from a research tool primarily used in laboratories to powerful diagnostic tools for several diseases [2]. This is because microfluidic technology has several advantages in biomedical applications, including reduced sample volumes and analysis time, high sensitivity, and low device cost [3], [4], [5], [6], [7], [8], [9], [10]. Various microfluidic-based cancer cell sorting techniques exist, which have been reviewed elsewhere [1], [5], [11], [12], [13], [14], [15], foremost being fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), counter-flow centrifugal elutriation (CCE), and dielectrophoresis (DEP).
In the case of urogenital cancers such as bladder and prostatic cancer [16], the trend in detection assays is now shifting from urine cytology to fluorescence-based urinary cytology adopting the photodynamic diagnosis (PDD) concept [17], [18], [19], and an emerging approach is the combination of microfluidic-based cell sorter and fluorescence-based urinary cytology. PDD is an optical technique that uses aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence to enhance visual demarcation between normal and cancer cells. Studies suggest that cancer cells readily incorporate exogenous ALA intracellularly and metabolize ALA to PpIX; PpIX preferentially accumulates within cancer cells. When illuminated with near-UV light between 375 nm and 440 nm, PpIX returns to a lower energy level partly by emitting red fluorescence (635 nm). Since ALA is part of a regular diet, ALA-induced PpIX fluorescence has the potential to avoid the risk of prolonged phototoxicity associated with the conventional photosensitizer [20], [21]. This advantage is very attractive for polymerase chain reaction (PCR) analysis to obtain detailed genetic information, including the malignancy of sorted cancer cells. Furthermore, ALA-induced PpIX fluorescence can detect low-grade and low-stage bladder cancer cells at a higher rate than conventional urine cytology [22]. Since the accuracy of existing cytological diagnosis depends on the experience of the pathologist, there is an emergent need for an objective and easy cytological diagnosis adopting microfluidic technologies; however, microfluidic-based cell sorters tailored for PDD have not yet been reported. The authors therefore noted a new diagnostic approach for cancer cells in urine sediments: a combination of ALA-based photodynamic diagnostic tests using a microfluidic fluorescence-activated cell sorter (μFACS) system [23] and subsequent PCR analysis for early-stage cancer diagnosis.
Many challenges must be overcome before μFACS systems will see widespread use in fluorescence-based urine cytological testing. A basic problem is determining the material to use for the microfluidic device. The time and cost-effective advantages of polymer materials and their fabrication technologies have been widely pronounced, because the simplicity and low cost of polymer fabrication technologies have allowed designs to be produced on disposable devices [24], [25]. Most microfluidic devices in laboratory research and prototyping are made out of poly-dimethylsiloxane (PDMS) because of its ease of fabrication [26]. However, the poor mechanical properties of PDMS and its porosity (leading to cross-contamination or air-bubbles in microfluidic channels) can hinder the use of PDMS in clinical applications [27], [28]. Meanwhile, negative photoresist (SU-8, Microchem Co. [29], [30] or TMMR/F, Tokyo Ohka Kogyo Co., Ltd. [31], [32]) has attracted attention for microfluidic biomedical applications not simply because of its many physical attributes, including structural rigidity, chemical inertness, biocompatibility, mechanical robustness, electrical insulation, and optical transparency [30], [33], but because this material enables direct fabrication of three-dimensional (3D) microstructures at a nano/micrometer level using photolithographic technologies and straightforward integration of multiple functions on a single chip [25], [34]. Unfortunately, autofluorescence of negative photoresist is a significant drawback when used as a structural material for fluorescence-based detection devices [35], [36], [37], [38], [39]. In this case, high background noise during cancer cell sorting deteriorates the signal-to-noise ratio (S/N ratio), and a possible solution to avoid this noise is needed. Another major challenge is reducing the gap between proof-of-concept demonstrations in the laboratory and real use in the clinic with urine samples; unlike fluorescent beads and cell lines, urine samples from patients can contain mixtures of various biological cells [40]. However, even if the demonstration works well in laboratories, the results can be difficult to reproduce, i.e., no assay is clinically available. Hence, there are strong demands for (1) sensitive detection of cancer cells in a photoresist-based microchannel utilizing PDD concepts, (2) an automated and qualitative means to separate cancer cells and enrich them for subsequent gene alteration analysis, and (3) clinical tests demonstrating real potential of the method for effective screening and detailed diagnosis of bladder cancers.
We first present the development of a simple photoresist-based μFACS system for bladder cancer based on PDD concepts and assess its potential for clinical application. In the proposed μFACS, bladder cancer cells in urine sediment are detected using ALA-induced PpIX fluorescence, and then sorted at the Y-junction of the microchannel for gene alteration analysis. In this paper, the diagnosis protocol using the μFACS system and system structures are first introduced. Next, to achieve sensitive fluorescence detection in the photoresist-based microchannel, autofluorescence of photoresist materials and ways to improve detection sensitivity of cancer cells are investigated. Finally, we discuss the performance of the μFACS and its potential for diagnosis of bladder cancers.
Section snippets
Diagnosis protocol
The protocol for sorting cancer cells has two primary screening steps, which expedites diagnosis. The first screening is to determine whether the urine sample includes cancer cells, by means of a fluorescence spectrophotometer [22]. Then, given the presence of cancer cells, the second screening sorts these cells using the μFACS. This protocol facilitates the determination of appropriate countermeasures for an individual bladder cancer patient. To perform a detailed diagnosis, screening using
Biological materials and cell culture
The UMUC3 cell line (advanced high-grade human bladder cancer cells) and PC-3 cell line (advanced high-grade human prostate cancer cells) were used to prove the device concept. For a clinical test, urine samples from patients with confirmed bladder cancer were collected during routine clinical practice at Nara Medical University Hospital, and processed as soon as possible (within 2–4 h) after collection. Cell lines and urine samples were incubated in 50 μM h-ALA (Photocure Inc.) at 37 °C for 4 h in
Quantification of autofluorescence
Fluorescence intensity behavior under continuous laser illumination for 120 min is shown in Fig. 5. The autofluorescence of Si and glass, used as reference materials, remained essentially unaffected by illumination time. In contrast, the initial fluorescence intensity of TMMR S2000 was significantly higher than that of glass and Si. TMMR S2000 resin exhibited less initial fluorescence than TMMR S2000; however, PAG acted as sources of fluorescence in the photoresist. Additionally, the
Conclusions
We presented a μFACS system for bladder cancer diagnosis based on photodynamic analysis of urine and assessed its potential for clinical application. The developed μFACS detects cancer cells reliably using ALA-induced PpIX fluorescence and sorts them for subsequent gene alteration analysis. Fabrication of the microfluidic device was based on author-developed 3D lithography, which enables sensitive fluorescence detection in the photoresist-based microchannel. The autofluorescence in the
Acknowledgements
The authors thank the staff at Micronix Inc., Japan and Tokyo Ohka Kogyo Co., Ltd., Japan for support and useful discussions, S. Onishi at Nara Medical University for biological sample preparations, and S. Kinoshita at Kyoto University for capturing the SEM images. This work was supported in part by the subsidies for projects that promote the enhancement of manufacturing technology for small and medium enterprises (SMEs), administered by the Ministry of Economy, Trade and Industry, Japan. A
Yoshikazu Hirai received the M.S. degrees from Ritsumeikan University, Japan, in 2004, and the Ph.D. degree in the Department of Mechanical Engineering from Kyoto University, Japan, in 2007. He joined the Graduate School of Engineering at Kyoto University as a Postdoctoral Researcher in 2007. He was a Program-Specific Assistant Professor of the Advanced Biomedical Engineering Research Unit at Kyoto University from 2009 to 2013, and is currently an Assistant Professor with the Department of
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Yoshikazu Hirai received the M.S. degrees from Ritsumeikan University, Japan, in 2004, and the Ph.D. degree in the Department of Mechanical Engineering from Kyoto University, Japan, in 2007. He joined the Graduate School of Engineering at Kyoto University as a Postdoctoral Researcher in 2007. He was a Program-Specific Assistant Professor of the Advanced Biomedical Engineering Research Unit at Kyoto University from 2009 to 2013, and is currently an Assistant Professor with the Department of Micro Engineering, Kyoto University. From 2014, he is an Adjunct Assistant Professor with the Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University. His current research interests include three-dimensional microstructuring techniques for MEMS, atomic MEMS devices and microfluidic devices for biomedical applications.
Daisuke Takagi received B.E. and M.E. degrees in engineering science from Kyoto University, Japan, in 2012 and 2014. In 2014, he joined Hitachi Construction Machinery Co., Ltd, Ibaraki, Japan.
Satoshi Anai graduated from Toyama Medical and Pharmaceutical University, Japan, in 2000 and received the Ph.D. degree from Nara Medical University, Japan, in 2008. He is currently working as an assistant professor in the Department of Urology, Nara Medical University. His interests focuses on urological oncology, especially developing the new diagnostic method for bladder and prostate cancer.
Yoshitomo Chihara graduated from Nara Medical University, Japan, and passed Japanese national examinations for medical practitioners in 1999. He received a Ph.D. degree from the Graduate School Faculty of Medicine, Nara Medical University in 2005. He is currently working as a Chief of the Department of Urology, Saiseikai Tyuwa hospital in Japan. His clinical and research interests lie mainly urologic oncology.
Toshiyuki Tsuchiya received the M.S. degree from the University of Tokyo, Japan, in 1993, and the Ph.D. degree from Nagoya University, Japan, in 2002. He was with Toyota Central Research and Development Laboratories from 1993 to 2004. In 2004, he joined Kyoto University, Japan, as an Associate Professor, and he is currently with the Department of Micro Engineering of Kyoto University. He is currently engaged in the research of silicon surface micromachining, its application in MEMS, the mechanical property evaluation of micromaterials, and the reliability of MEMS devices.
Kiyohide Fujimoto graduated from Nara Medical University, Japan, and passed Japanese national examinations for medical practitioners in 1987. He received a Ph.D. degree from the Graduate School Faculty of Medicine, Nara Medical University in 1992. He is currently working as a Professor and Chair of the Department of Urology, Nara Medical University. His clinical and research interests lie mainly urologic oncology.
Yoshihiko Hirao received the B.M. from Nara Medical University, and medical qualification of Japan in 1972. He joined the Department of Urology of Nara Medical University as an Associate, Lecturer and Associate Professor since 1972 to 1996. He joined the Department of Pathology, Medical School of Northwestern University, U.S.A. as a Research Fellow between 1977 and 1979. He received Doctoral degree of Nara Medical University in 1984. He was a Professor and Chairman of Department of Urology, Nara Medical University from 1996 to 2012. He is Emeritus and Research Professor of Nara Medical University, and Honorary Director of Osaka Gyoumeikan Hospital. His clinical and research interests are mainly urologic oncology, including photodynamic diagnosis of bladder cancer, and urodynamics using Portable Uroflowdiary device for voiding dysfunction.
Osamu Tabata received the M.S. and Ph.D. degrees from Nagoya Institute of Technology, Japan, in 1981 and 1993, respectively. In 1981, he joined the Toyota Central Research and Development Laboratories, Inc., Japan. In 1996, he joined the Department of Mechanical Engineering, Ritsumeikan University, Japan. From September to December 2000, he was a Guest Professor at the Institute of Microsystem Technology, University of Freiburg, Germany, and from January to March 2001, he was a guest Professor at Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland. In 2003, he joined the Department of Mechanical Engineering, Kyoto University, Japan. Since April 2005, he has been a Professor in the Department of Micro Engineering, Kyoto University. He is currently engaged in research on micro/nano processes, MEMS, and micro/nano system synthetic engineering. He has begun research to realize a unique and novel nanosystem by assembling the various functional components such as a microchip, a particle, a microcapsule, a cell, etc., with sizes ranging from nanometer to micrometer scale on a few-millimeter-square MEMS substrate. This technology is termed synthetic engineering for nanosystems (SENS), and experimental and theoretical research on the establishment of SENS is pursued.
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Selected papers presented at EUROSENSORS 2014, the XXVIII edition of the conference series, Brescia, Italy, September 7–10, 2014.