Biomimicking Platelet-Monocyte Interactions as a Novel Targeting Strategy for Acute Myocardial Infarction
Development of effective cardioprotective treatment strategies continues to be a challenge as many potential cardioprotective drugs fail to translate from the bench into clinical results. One of the key issues is the optimization of targeting to the infarcted heart. Although several drug delivery systems claim to actively deliver encapsulated drugs to the infarct area, the functionalized surfaces on these delivery systems only allows them to be better retained at targeted sites or have a higher circulation half-life. Thus, an enhanced permeability and retention (EPR) effect is still required as a main route of delivery for these delivery systems. Herein, we present an alternative that allowed cardioprotective drugs to be actively delivered without relying on the EPR effect. To mimic platelet interaction with the circulating monocytes during post-myocardial infarction (MI), platelet-like proteoliposomes (PLPs) were fabricated using purified human platelet membrane proteins and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. In vitro data showed that PLPs displayed a strong affinity for monocytes and macrophages but not for endothelial cells. Intravital multiphoton imaging revealed PLPs had better targeting to the tissue injury site than the plain liposome control. When injected at 72 hours of reperfusion, which is when the monocyte recruitment is at its maximum, there were significantly more PLPs compared to the plain liposomes in the infarct area of the heart. Moreover, cobalt protoporphyrin (CoPP) encapsulated in PLPs (PLP-CoPP) was shown to improve the cardiac function in a murine model of MI while reducing the adverse effect of the encapsulated drug.
Dr. Bill Cheng graduated from the University of New South Wales (Sydney, Australia) with a PhD degree in Biomedical Engineering, under the supervision of Prof. John Whitelock in 2012. He then moved to France to start his postdoctoral research at Dr. Patricia Rousselle’s lab at Institute of Biology and Chemistry of Proteins (Lyon, France), investigating the roles of syndecan-4 in cell polarization. In 2013, Dr. Cheng moved back to Taiwan, and started his postdoctoral research at Prof. Patrick Hsieh’s lab at Academia Sinica, developing novel drug delivery systems for cardiovascular diseases. Dr. Cheng is a now faculty member with Graduate Institute of Biomedical Engineering, National Chung-Hsing University. His past achievement includes, 1st Place in Postdoc & Young Investigator Oral Presentation Award in 2016 TERMIS-AP Meeting, and Top 5 Winners at 2017 Global Young Scientist Summit. His primary research interest is to develop cell-mediated drug delivery systems for heart targeting.
Bead-Based Sensors for Rapid Biomedical Diagnoses
Early diagnosis is an ideal measure for effective treatments. However, dilute analytes form a high barrier in detection. To tackle the problem, micro/nano-beads are commonly functionalized with various ligands to capture target molecules. As a result, they can be employed as smart probes to alarm users the occurrences of diseases or pathogens in the early stage. In this presentation, two self-developed techniques in our laboratory involving with functional micro/nano-beads will be introduced to show how they were used in some specific biomedical applications. In the first case, an optoelectrokinetic technique, termed rapid electrokinetic patterning (REP), was developed to manipulate micro/nano-beads. A comprehensive repertoire of manipulation, including size sorting, translation, concentration, and single particle trapping were all explored. By properly concentrating dye-labelled beads, weak biosignal can be enhanced. Two biomarkers in tears, LCN1 and TNF-α, for diabetic retinopathy were successfully detected in a concentration level down to 100 pg/mL. In another case, Brownian motion, a thermal random noise, was employed here to harness functional micro/nano-beads. According to the Einstein relation, particle diameter is inversely proportional to diffusivity. Therefore, the concentration variation of any target in a sample medium can be simply estimated from its diffusivity change. Micro/nano-beads were coated with specific antibodies to induce immunoreactions with their corresponding targets. For microbial tests, bacteria, P. aeruginosa, S. aureus, and E. coli were respectively studied for their diffusivity responses. It was found that the diffusivity changes were in relation to the numbers of the bacteria. A limit of detection (LOD) as low as one bacterium per particle was achieved. The result was further used to improve the anti-microbial susceptibility testing (AST). The turnaround time for each AST measurement was then significantly reduced from days to a few hours (~3 h). Notably, the same platform but conjugated with additional gold nanoparticles was further attempted to challenge the detection limit of a trace amount of food toxin, botulinum. The LOD for such a configuration eventually achieved 10 pg/mL. Overall, the diffusion-enabled technique requires neither complicated fabrication nor sophisticated instruments. In conclusion, both proposed techniques provide great insight to saving lives with micro/nano-beads by enabling early diagnosis. An attempt to build a prototype to bring the diffusion-enabled technique from theory to practice is also under way.
Han-Sheng Chuang is currently an associate professor in the Department of Biomedical Engineering at National Cheng Kung University in Taiwan. Dr. Chuang received his bachelor and master degrees from the Department of Mechanical Engineering at National Cheng Kung University in 1998 and 2000, respectively. He joined Industrial Technology Research Institute (ITRI) as a R&D engineer in 2001. In 2005, he was awarded a competitive fellowship from Ministry of Education, Taiwan. After then, he worked with Professor Steve T. Wereley for advanced microfluidics and received his Ph.D. from the School of Mechanical Engineering at Purdue University in 2010. He and his research colleagues entered the finalists of the prestigious Burton D. Morgan Business Competition in 2008 and 2009, respectively. After graduation, he received an appointment as a postdoctoral researcher at University of Pennsylvania and worked with Professor Haim H. Bau on cell sorting and Caenorhabditis elegan. Since starting the current position, he has received the 2014 Young Researcher Career Grant from the Ministry of Science and Technology, the 2015 Young Scholar Award from the Taiwan Comprehensive University System, and the 2016 Excellent Teaching Award from NCKU. In addition, he is also a cofounder of a US-based start-up, Microfluidic Innovations, since 2009 and a board member of the Association for Chemical Sensors in Taiwan. Dr. Chuang has dedicated to the field of microfluidics for more than 10 years. His research interests are mainly focused on bio-micro/nano-fluidics, Bio-MEMS/NEMS, optical diagnostics, and C. elegans.
Han-Sheng Chuang, PhD
Associate Professor, Department of Biomedical Engineering
Division Director of Common Lab, Centers for Micro/Nano Science Technology
National Cheng Kung University
Tainan, Taiwan
Email: oswaldchuang[at]mail.ncku.edu.tw
Electrochemical DNA Biosensing for Methicillin-resistant Staphylococcus aureus Using Nanoparticle Modified Probe
Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen organism that causes infection disease in a hospital. To prevent epidemic, it’s needed that simple and quick detection system for pathogen organism. Herein, we present probe based electrochemical biosensing system for detection of MRSA without DNA amplification. Two complimentary probe for mecA region of MRSA genome were modified to gold nanoparticle (AuNP) and magnetic nanoparticle (MNP), respectively, and ferrocene was also modified to AuNP in order to obtain the electronic signal. DNA from MRSA was hybridized with the two types of nanoparticle. After B/F separation of hybridized products magnetically, including AuNP, MNP and target DNA, were detected by oxidation of ferrocene on the AuNPs surface.
DNA was extracted from MRSA cells and obtained as single-strand DNA by heat treatment. Single-strand DNA, probe/ferrocene modified AuNP and probe modified MNP was mixed and hybridized. Then hybridized products were obtained with magnetic separation. The hybridization products were measured on screenprinted electrode which is three electrode system using carbon working, Ag/AgCl reference and carbon counter electrode. Oxidation current could be obtained of ferrocene when a constant potential of +250 mV vs. Ag/AgCl was applied. The hybridization products were transferred to electrode and then D-glucose and glucose dehydrogenase was added to make oxidation current of ferrocene amplify.
The current response increased linearly with increasing concentration of genomic DNA from MRSA in the range of 0-166 pM and its detection limit was 10 pM without amplification of the target DNA. In selectivity, we measured DNA from Staphylococcus aureus (SA) and it was found that this system could distinguish SA from MRSA. We demonstrated a PCR-free electrochemical biosensing system for MRSA using nanoparticle modified probe.
Reference:
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K. Watanabe, N. Kuwata, H. Sakamoto, Y. Amano, T. Satomura, S. Suye, Biosens. and Bioelectron., 67, 419 (2015).
Hiroaki Sakamoto received his PhD from Kyushu Institute of Technology (2009). From 2009–2012, he was postdoctoral fellow at Ritsumeikan University. From 2012–2013 assistant professor at University of Fukui. From 2014-2018 he is Lecturer at Tenure-Track Program for Innovative Research, University of Fukui. Since 2018 he is Associate professor at department of frontier fiber technology and science, University of Fukui. His current research interests include bioelectronics, biosensing system and nanomaterials.
Hiroaki Sakamoto, PhD
Associate Professor
Department of Frontier Fiber Technology and Science
Graduate School of Engineering
University of Fukui
Fukui, Japan
Email: hi-saka[at]u-fukui.ac.jp
Thin-Film Transistor and Photonic Crystal Biosensors
This talk is aiming on presenting two novel detecting schemes that were developed in our lab. First of all, we demonstrate a highly sensitive IGZO TFT (thin film transistor) with dual gate electrodes that detects electric charges of the target biomaterial. The TFT also functions as a transducer for signal readout. An IGZO TFT-based biosensor integrated with a microfluidic channel can be further used to monitor biochemical reactions between proteins and small molecules. Using Biotin-Streptavidin reaction as an example, the specific diffusion time of biotin, streptavidin, or streptavidin-biotin complex across the fluidic channel are benchmarked. By intentionally delaying the application of one specie to another, we can study properties of biochemical reactions, such as excess and exhaust conditions of the reactants, and the formation of the products while diffusing in the channel. The proposed TFT sensor provides an excellent platform in understanding the biochemical functions for the advance of biological science
Second, the target biomaterial can also be detected by a compact 2-dimensional hexagonal photonic crystals (PhCs). A critical wavelength that satisfies the phase matching or becomes evanescent is employed to benchmark the refractive index of the target analyte applied on the sensor. Using glucose solution as the example, our sensor demonstrates very high sensitivity and low limit of detection. It shows the diffraction mechanism from the hexagonal photonic crystals can be used for sensors when the compact size is a concern.
The talk will spend a little bit of time comparing the sensitivity of both TFT and PhC sensors. Pros and cons of both approaches will be briefly mentioned.
JianJang Huang received the B.S. degree in Electrical Engineering (EE) and the M.S. degree in Graduate Institute of Photonics and Optoelectronics (GIPO) from National Taiwan University (NTU), Taipei, Taiwan, in 1994 and 1996, respectively, and the Ph.D. degree in Electrical Engineering from the University of Illinois, Urbana-Champaign, in 2002. Prof. Huang is a member of the Phi Tau Phi Scholastic Honor Society. He received “Wu Da-Yu” award in 2008, the most prestigious one for young researchers in Taiwan sponsored by National Science Council. And in the same year, he received the award for the most excellent young electrical engineer from the Chinese Institute of Electrical Engineering. He is the chairman of GIPO NTU and the director of Innovative Photonics Advanced Research Center, NTU. He was the chair of SPIE (San Diego, CA, USA), Optics & Photonics, International Conference on Solid State Lighting from 2011~2015, and the executive secretary of Taiwan Photonic Society from 2013 to 2016. He has been involved in several industrial and venture positions, including the board director of Unity Opto in Taiwan (2019~now), Global Communication Semiconductor, Inc. in CA, USA (2011~2019), Tacbright Optronics Corp. in Taiwan (2013~2016) and TMP co. in Taiwan (2012~2013). In academia, he currently serves as the Editor of IEEE, Transactions on Electron Devices, the Associate Editor of IEEE, Transactions on Nanotechnology, and the IEEE EDS committee member of Optoelectronics. He is a fellow of SPIE and OSA.
Jian-Jang Huang, PhD
Professor
Graduate Institute of Photonics and Optoelectronics &
Department of Electrical Engineering
National Taiwan University
Taipei, Taiwan
Email: jjhuang[at]ntu.edu.tw
Tape-Stripping Detection of Cancer Biomarkers by Scanning Electrochemical Microscopy
Scanning electrochemical microscopy (SECM) is a scanning probe technique that is composed of a micro- or nanoelectrode that can be scanned in close proximity to an interface. Faradaic current signals can be recorded due to the flux of redox active species between the sample and an amperometric SECM probe. SECM can be used to image the topography and reactivity of biological specimens for mapping localized biochemical activity. Although SECM has been applied to different biological systems, SECM studies of tissues are still under exploration. The reason is due to the shape and high roughness of such real samples and requires overcoming major drawbacks in conventional SECM instrumentation when scanning large, i.e. square centimeter sized, areas with irregular surface keeping a constant working distance.
My research aims to develop various reliable SECM bioimaging techniques for the study of the distribution of biomarkers and nanomaterials in tape-stripping cancer samples and human tumor samples. Particularly the last is of major importance, because melanoma is the most lethal form of skin cancer striking thousands of people around the world. The survival rate depends on the stage of the cancer when it is diagnosed. Therefore, reliable methodologies for early diagnosis and unequivocal identification of cancer stages are of high relevance. It is demonstrated that SECM could improve the diagnosis and understanding of different melanoma stages based on highly resolved maps of the tyrosinase distribution while being immune against optical interferences, e.g. from the presence of melanin in the skin samples [1].
The increasing levels of biomarker tyrosinase (TYR), were recorded in cells that were collected from the skin of melanoma mouse models representing three different stages of tumor growth [2]. Additionally, SECM results of tape-stripped different human melanoma cell lines were confirmed by previous studies based on traditionally fixed and permeabilized cells. The presented electrochemical TYR detection approach can potentially be integrated into point of-care testing (POCT) devices. SECM scanning parameters, such as microelectrode size and step size can be used to determine experimentally the best design before fabricating microelectrode array prototypes [3].
References:
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Lin TE, Bondarenko A, Lesch A, Pick H, Cortés-Salazar F, Girault H. Monitoring tyrosinase expression in non‐metastatic and metastatic melanoma tissues by scanning electrochemical microscopy Angew. Chem. Int. Ed. 2016;55:3813–3816.
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Lin TE, Lu YJ, Sun CL, Pick H, Chen JP, Lesch A, Girault H. Soft electrochemical probes for mapping the distribution of biomarkers and injected nanomaterials in animal and human tissues Angew. Chem. Int. Ed. 2017;56:16498–16502.
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Darvishi S, Pick H, Lin TE, Zhu Y, Li X, Ho PC, Girault HG, Lesch A. Tape-Stripping Electrochemical Detection of Melanoma Anal. Chem. 2019, 91, 12900–12908.
Dr. Tzu-En Lin is currently an assistant professor in National Chiao Tung University, Institute of Biomedical Engineering. She received her Ph.D. degree in Chemistry and Chemical Engineering at the École Polytechnique Fédérale de Lausanne, Switzerland (瑞士聯邦理工學院, EPFL) with a specialization in electrochemistry. Her supervisor is Prof. Hubert Girault, the expert of bio-analytical chemistry, battery technology and hydrogen cars development. In 2012, she obtained her master’s degree in National Taiwan University under the supervision of Prof. Huan-Tsung Chang (張煥宗) who is specialized in the applications of nanoparticles. Tzu-En Lin received her bachelor’s degree in National Taiwan University, department of biochemical science and technology. She was a postdoc researcher in EPFL, department of Chemistry and Chemical Engineering, and was a visiting scholar in Stanford University, department of chemical engineering. In 2018, she was a postdoc in National Taiwan University, department of material science.
Tzu-En Lin, PhD
Assistant Professor
Institute of Biomedical Engineering
National Chiao Tung University
Hsinchu, Taiwan
Email: telin[at]nctu.edu.tw
KEYNOTE SPEAKERS - Biomedical
Bill Cheng, PhD
Assistant Professor
Graduate Institute of Biomedical Engineering
National Chung Hsing University
Taichung, Taiwan
Email: bcheng[at]dragon.nchu.edu.tw
Beyond the Debye Length and Ideal Nernst Sensitivity for FET Sensors: From Fundamental Breakthrough to Real Applications for Mobile Diagnostic and Personal Healthcare
Field-effect transistors have been widely demonstrated for protein detection due to their extremely high sensitivity, label-free detection, tiny size and the potential to integrate with electronic devices for personal use. However, several problems, including the materials stability, severe charge-screening effect in high salt clinical samples, and electric isolation between fluids and metal interconnect for miniaturized FETs, need to be solved prior to the application for medical use. In order to allow people to monitor the risk of disease by themselves timely, the sensor and the readout device need to be small, portable, cheap, and easy to operate. The detection has to be quick and accurate. In this presentation, a new methodology that can overcome severe charge-screening effect for FET-biosensors will be presented. Furthermore, a new miniaturized FET-sensor package and a handheld device with readout circuits were demonstrated with clinical whole blood samples for acute myocardial infarction (AMI) and chronic heart failure (CHF), without any sample purification procedure. The test takes only 5 mins and only one drop of whole blood is needed. The same methodology is also used for heavy metal ion detection, which shows sensitivity higher than that of the ideal Nernst limit. Systematic investigation indicates the high sensitivity driven by the high field-modulated electrolyte-gated FET sensors. This sensor and the device are able to allow people to monitor their health and food at anytime and anywhere.
Dr. Yu-Lin Wang received his B.S. degree in chemistry from Tunghai University and M.S. degree from National Taiwan University, in 1993 and 1995, respectively. He had worked in semiconductor industry from 1997~2006. He received his Ph.D. in materials science and engineering from University of Florida, in 2009. He has been with the Institute of NanoEngineering and Microsystems, Department of Power Mechanical Engineering, at National Tsing Hua University, Hsinchu, Taiwan since 2010. His research interests are semiconductor-based sensors and the device for medical use and personal healthcare. His team has won several awards in recent years including the Top 10 pioneering technology worldwide by Google-X in 2016, Merit of Asia Pacific ICT Aliance in 2016, Gold Medal by Spintech Inc. in 2018, Silver Medal for Shark Tank by uTAS in 2018, and Silver Medal by EpiStar Inc. in 2019.
Yu-Lin Wang, PhD
Professor
Institute of Nanoengineering and Microsystems
Department of Power Mechanical Engineering
National Tsing Hua University
Hsinchu, Taiwan
Email: ylwang[at]mx.nthu.edu.tw
To M, or not to M cells
Oral vaccine has been become the most effective strategy in the fight against intestinal infections because of its ease, economy, and capability on inducing responses not only systemic but also local, especially in mucosal compartments. The major obstruction to potent vaccine development is antigen dispersion and tolerance. To overcome this hindrance, we aimed to target M cells, which are main sentinel gateway for taking up luminal antigens and initiating specific mucosal responses.
In this review, candidate peptides/protein derived from experimentally proven proteins were predicted using molecular docking and molecular dynamics simulations. To confirm the interaction between the peptides/protein and M cell receptor, the coding gene for peptides/protein were fused, cloned and expressed with Green Fluorescent Protein (GFP), then purified, evaluated in vitro and ex vivo. Next, the fusion protein were orally administrated into mice for immune response evaluation. As a result, via silicon nanowire field-effect transistors chip (SiNW FET), fusion protein showed interaction with its cognate receptor. In line with SiNW FET result, ex vivo assessment on murine M cells demonstrated that the fusion protein showed binding on murine M cells surface. Lastly, secretory IgA and serum IgG measurement showed M cell targeting peptides/protein fusion enhanced immune response in comparison to non-fused counterpart. These present results suggest that M cell targeting peptides/protein could be competent candidates for oral vaccine development.
Hieu Tran-Van completed his PhD from University of Würzburg, Germany. He is now the Vice Dean of Faculty of Biology and Biotechnology, University of Science, VNU-HCM, in charge for External Relations and Science-Technology. He has published 27 scientific papers and got funded for more than 10 projects.
His research interests mainly focus on i) recombinant proteins for therapeutic and cosmeceutical applications, ii) novel vaccine adjuvants and their controlled release, iii) functional foods, iv) oral vaccine, v) nano medicine, and vi) point-of-care testing.
Hieu Tran-Van, Dr.rer.nat
Associate Professor in Biotechnology
Faculty of Biology and Biotechnology
University of Science, VNU-HCM