Impedance Sensing of Cancer Cells Directly on Sensory Bioscaffolds of Bioceramics Nanofibers

Cancer cell research has been growing for decades. In the field of cancer pathology, there is an increasing and long-unmet need to develop a new technology for low-cost, rapid, sensitive, selective, label-free (i.e. direct), simple and reliable screening, diagnosis, and monitoring of live cancer and normal cells in same shape and size from the same anatomic region. For the first time on using an impedance signal, the breast cancer and normal cells have been thus screened, diagnosed and monitored on a smart bioscaffold of entangled nanowires of bioceramics titanate grown directly on the surface of implantable Ti-metal and characterized by SEM, XRD, etc. following a technology patented by Tian-lab. In experiment in the aqueous solution of phosphate buffer saline (PBS), human breast benign (MCF7) and aggressive (MDA-MB231) cancer cells, normal (MCF10A) cells, and colon cancer cells (HCT116) showed characteristic impedance spectrum highly different than that of the blank sensor (i.e. no cells on the bioscaffold surface). For two sets of mixtures each containing the normal and cancer cells over a wide range of mixing ratios, the shift of impedance signals has been linearly correlated with the mixing ratios which supports the biosensor’s selectivity and reliability. After being treated with pure glucose and chemotherapeutic drug (i.e. doxorubicin of DOX) and with one after the other, the breast cancer cells showed different impedance signals corresponding to their difference in glucose metabolisms (i.e. Warburg Effect) and resistances to the Dox, thus-fingerprinting the cells easily. Based on the nanostructure chemistry, impedance equivalent circuitry and cancer cell biology, it’s the different cells surface binding on the nanowires, and different cancer cells metabolic wastes from the different treatments on the nanowires that changed the charge density on the scaffolding nanowire surface and in turn changed the impedance signals. This new method is believed expandable to quantifying and characterizing live cells and even biological tissues of different types in general

MEMS-based Lab-on-chip platform with integrated 3D and planar microelectrodes for organotypic and cell cultures

La presente tesis doctoral se centra en el desarrollo y la validación de plataformas lab on chip (LOC) para su aplicación en el campo de la Biología, la Medicina y la Biomedicina, particularmente relacionados con el cultivo de células y tejidos, así como su tratamiento mediante electroestimulación y su actividad eléctrica. Actualmente, las investigaciones centradas en el desarrollo de LOCs han experimentado un crecimiento considerable, gracias, en gran medida, a la versatilidad que ofrecen. Dicha versatilidad se traduce en numerosas aplicaciones, de las cuales, aquellas relacionadas con la Biología y la Medicina, están alcanzando especial relevancia. La integración de sensores, actuadores, circuitos microfluídicos y circuitos electrónicos en la misma plataforma, permite fabricar sistemas con múltiples aplicaciones. Esta tesis se centra fundamentalmente en el desarrollo de plataformas para el cultivo in vitro de tejidos y células, así como para la monitorización y la interacción con dicho cultivo. Los cultivos in vitro resultan de vital importancia para realizar estudios en células o tejidos, experimentar con medicamentos o estudiar su proliferación y morfología. De esta manera, se cubriría la creciente necesidad de encontrar una alternativa para replicar modelos humanos de enfermedades in vitro para poder desarrollar nuevos fármacos y avanzar en la medicina personalizada. Por tanto, la posibilidad de realizar cultivos de media o larga duración en plataformas que no precisen de un equipamiento costoso como las incubadoras de CO2 y que puedan ser monitorizadas mediante aplicaciones ópticas, supone un salto cualitativo en el desarrollo de los cultivos in vitro. En este contexto, se presenta el trabajo relacionado con esta tesis que ha sido desarrollada dentro del grupo de Microsistemas de la Escuela Superior de Ingeniería de la Universidad de Sevilla. La tesis está estructurada de manera que a lo largo de este escrito se da respuesta a los distintos aspectos anteriormente descritos. En primer lugar, se hace una breve introducción a la tecnología MEMS y a los principios básicos de la microfluídica. Dado que este trabajo se ha desarrollado en un ambiente multidisciplinar, esta sección resulta necesaria para dar una visión general a aquellos no familiarizados con esta disciplina. Tras esa introducción se realiza una descripción del estado del arte en el que se encuadra este trabajo, incluyendo los sistemas LoCs, y sus principales aplicaciones en el campo de la Biología, Medicina y Biomedicina, prestando especial atención a las aplicaciones de los LoCs relacionadas con cultivos organotípicos y de células. Tras la introducción y el estado del arte en el que se enmarca la tesis, se explican los resultados obtenidos durante este trabajo. Durante la primera parte, se describe el desarrollo, fabricación y caracterización de un sistema autónomo para el cultivo y electroestimulación de tejidos que integra un lab on PCB (LOP) formado por un array de microelectrodos en 3D (MEA) formado por hilos de oro de 25 µm en sustrato transparente, sensores y actuadores, junto con una plataforma microfluídica fabricada en metacrilato (PMMA) y polidimetilsiloxano (PDMS). El LOP permite mantener las condiciones de temperatura idóneas para llevar a cabo cultivos de media-larga duración sin necesidad de usar incubadoras deCO2 , así como su seguimiento de forma continua a través de un microscopio, gracias al uso de materiales transparentes. Este sistema también incluye una electrónica suplementaria y un programa que permite la monitorización del sistema y la electrostimulación de la muestra biológica. Tras explicar detalladamente el diseño y el novedoso proceso de fabricación del LOP, se presentan los resultados experimentales. En primer lugar, se ha demostrado que es posible desarrollar cultivos organotípicos de retinas de ratón durante más de 7 días, obteniendo resultados muy similares a los conseguidos para las mismas muestras biológicas, pero mediante métodos de cultivo tradicionales. Además, se ha logrado la neuro-protección mediante la electroestimulación de retinas de ratón con la enfermedad de la retinosis pigmentaria, logrando de esta manera ralentizar la degeneración de la muestra debido a la enfermedad. Otra de las aplicaciones que se quiere conseguir con el desarrollo del LOP anteriormente descrito se centra en la adquisición de señales eléctricas procedentes de las muestras biológicas cultivadas en el dispositivo, así como extrapolar su uso a cultivos celulares. Para la adquisición de señales procedentes del cultivo, la impedancia de los electrodos fabricados con hilos de oro de 25 µm ha resultado ser demasiado alta como para discernir entre el ruido base y la actividad eléctrica del cultivo. Por ello, la segunda parte de esta tesis doctoral se centra en la mejora de la MEA para la adquisición de actividad eléctrica. Dado el objetivo marcado en esta segunda parte, durante esta tesis se ha realizado una estancia en la Universidad de Bath. En dicha estancia, se ha caracterizado la actividad eléctrica de células del cáncer de próstata (PC-3), que fueron cultivadas en chips de silicio con electrodos de oro. La experiencia obtenida durante la estancia ha permitido avanzar en el desarrollo y la fabricación de nuevas MEAs para la adquisción de señales eléctricas de cultivos celulares. La primera aproximación para mejorar la MEA se ha realizado sobre PCB. Se trata de un dispositivo compuesto por pilares de oro en 3D fabricados mediante la técnica de Resumen XXV electroplating. Estos electrodos tienen 100 µm de diámetro y una altura de 25 µm que aseguran el contacto en el caso de cultivos de tejidos. Se ha demostrado una mejora significativa, traducida tanto en una impedancia más baja, como en una línea base de ruido menor con respecto a la MEA con hilos de oro. Asimismo, se han obtenido patrones de actividad eléctrica en las células PC-3 muy similares a los obtenidos con el chip de silicio y oro empleado en la estancia. Como mejora de la MEA 3D se ha cambiado el sustrato por otro transparente, como vidrio o PMMA, para permitir su uso en aplicaciones ópticas. Dichas MEAs integran electrodos planares fabricados mediante la técnica de sputtering de oro sobre su superficie. Estas MEAs están en una fase preliminar de desarrollo, y se está probando en primer lugar su biocompatibilidad y viabilidad para el desarrollo de cultivos celulares. Para finalizar, se exponen las conclusiones de esta tesis doctoral, entre las que destacan: el proceso de fabricación del LOP con electrodos de oro y la aplicación del sistema completo para desarrollar cultivos organotípicos, monitorizarlos y aplicar electroestimulación, logrando la neuro-protección de retinas de ratón con la retinosis pigmentaria; la transición hacia el desarrollo de una plataforma para cultivos celulares mejorando la MEA y su fabricación usando diferentes sustratos; la caracterización de la actividad eléctrica de las células PC-3. También se incluyen las líneas de investigación abiertas para continuar lo que se ha empezado en esta tesis doctoral. Para facilitar la comprensión del lector, se adjuntan los apéndices complementarios a esta tesis doctoral.The presented thesis is focused on the development and validation of lab on chip (LOC) platforms for their application on Biology, Medicine and Biomedicine, particularly those related with cells and tissues cultures, as well as their treatment through electrostimulation and their electrical behavior. Nowadays, research works focused on the development of LOCs have significantly increased, mostly thanks to its high versatility, which involves countless applications. Among all this applications, those related with Biology and Medicine are becoming more and more important. The integration of sensors, actuators, microfluidic circuits and electronic circuits in the same platform allows the fabrication of systems with lots of applications. This thesis is focused on the development of platforms for in vitro cultures of cells and tissues, to monitor their behavior and interact with the biological samples. The importance of in vitro cultures lies on the study of cells and tissues proliferation and morphology or performing drug delivery experiments. In this respect, through LOC technologies, it would be possible to model human diseases in vitro, in order to improve the development of new drugs and advance personalized medicine. Thus, the possibility of carrying out medium-long term cultures on platforms without the need of any expensive equipment, such as CO2 incubators, with software and monitoring, implies a qualitative step forward in the development of in vitro cultures. Within this framework, the work related to this thesis is presented. This PhD has been undertaken in the Microsystem group of the High School Engineering of the University of Seville. The structure of this thesis is organized in such a way that, all along the text, the different aspects previously described are explained in detail. Firstly, a brief introduction about MEMS technology and the basic principles of Microfluidics is presented. Due to this work has been developed in a multidisciplinary environment, this section becomes necessary in order to give a wide view to those non XXVII XXVIII Abstract directly familiarized with these fields. Subsequently, a description of the state of the art is presented, including LOC systems and their applications in Biology, Medicine and Biomedicine, taking special attention to those applications related to organotypic and cell cultures. After the introduction and the state of the art of the framework of this thesis, the results obtained are presented. In the first part of this PhD, the development, fabrication and characterization of the autonomous system for culture and electrostimulation of tissues is described. This system includes a lab on PCB (LOP) composed of a 3D microelectrode array (MEA), with gold wires of 25 µm on transparent substrate, sensors and actuators, together with a microfluidic platform made of PMMA and PDMS. This LOP allows to maintain the appropriate temperature conditions to carry out medium-long term cultures without using a CO2 incubator, as well as its continuous monitoring through an inverted microscope, thanks to the transparent materials used for its fabrication. This system is connected to an external electronic circuit and a software to control the whole system, including the electrostimulation of the biological sample. After explaining the design and the innovative fabrication process of the LOP, the experimental results are presented. Firstly, it has been demonstrated the suitability of this system to perform organotypic cultures of mice retinas for longer than 7 days, obtaining similar results to the same samples, but cultured through traditional methods. In addition, it has been provided neuroprotection to mice retinal explants with the retinitis pigmentosa (RP) disease through the electrostimulation of the samples, being able to slowdown the degeneration of the retinas caused by RP

Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions. Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL-1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface. Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl -D-mannopyranoside (Man-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1→4)-D-Glc1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A to mannoside and soybean agglutinin to galactose in these SAMs were found to be different on NPG than on flat polycrystalline gold, and was also sensitive to the chemical composition of the modified surfaces

Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

Applications of Graphene Quantum Dots in Biomedical Sensors

Due to the proliferative cancer rates, cardiovascular diseases, neurodegenerative disorders, autoimmune diseases and a plethora of infections across the globe, it is essential to introduce strategies that can rapidly and specifically detect the ultralow concentrations of relevant biomarkers, pathogens, toxins and pharmaceuticals in biological matrices. Considering these pathophysiologies, various research works have become necessary to fabricate biosensors for their early diagnosis and treatment, using nanomaterials like quantum dots (QDs). These nanomaterials effectively ameliorate the sensor performance with respect to their reproducibility, selectivity as well as sensitivity. In particular, graphene quantum dots (GQDs), which are ideally graphene fragments of nanometer size, constitute discrete features such as acting as attractive fluorophores and excellent electro-catalysts owing to their photo-stability, water-solubility, biocompatibility, non-toxicity and lucrativeness that make them favorable candidates for a wide range of novel biomedical applications. Herein, we reviewed about 300 biomedical studies reported over the last five years which entail the state of art as well as some pioneering ideas with respect to the prominent role of GQDs, especially in the development of optical, electrochemical and photoelectrochemical biosensors. Additionally, we outline the ideal properties of GQDs, their eclectic methods of synthesis, and the general principle behind several biosensing techniques.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz

Principios básicos y uso en medicina de la espectroscopia de impedancia

Introducción: La impedancia de los tejidos biológicos o bioimpedancia es la relación entre la diferencia de potencial de 2 puntos en el tejido evaluado. Esta medida, depende de los componentes de tipo resistivo (resistencia al flujo iónico), capacitivo e inductivo (permitividad de la membrana). Los tejidos no presentan un comportamiento puramente resistivo, por lo cual su impedancia depende de la frecuencia y su caracterización, por lo tanto, necesita del uso de corrientes a diferentes frecuencias. Esta forma de medición se denomina espectroscopia de impedancia eléctrica. Se realizó una revisión bibliográfica, en los últimos 5 años, en las bases de datos PubMed, Medline, Scielo, LILACS, EBSCO y Excerpta Medica. Objetivo: Describir los principios básicos de la espectroscopia de impedancia y profundizar en sus aplicaciones médicas. Desarrollo: Se encontraron aplicaciones de la espectroscopia de impedancia para el diagnóstico en: 1) Oncología, 2) Vías digestivas, 3) Enfermedades pulmonares y 4) Enfermedades neurológicas. Sobre la aplicación diagnóstica, en la literatura consultada se obtuvieron resultados que corroboran la utilidad de la espectroscopia de impedancia como un método diagnóstico. Conclusiones: Mediante la espectroscopia de impedancia es posible conocer la respuesta eléctrica de tejidos biológicos en un amplio rango de frecuencias. Esto permite la caracterización de los tejidos biológicos; así como la evaluación de su transformación de sanos a patológicos

Synthesis, characterization, and evaluation of metal complexes with cancer selective anti-proliferative effects and hydrogen evolution catalytic properties.

Bis-thiosemicarbazones (BTSC) and their metal chelates have properties that are useful in several different scientific fields. These systems have already received attention in major fields of biology and engineering. Hydrogen evolution reaction (HER) catalysts need to be cheap and operate under minimal overpotentials with a long lifetime. The treatment of cancer requires, novel agents that have potent cytotoxic activity against cancer cells while displaying minimal side effects. In this dissertation the modular synthesis of these bis-thiosemicarbazone systems is utilized to regulate the redox chemistry for employment in the desired sector of chemistry. The ligand and metal chelates synthesized were characterized via cyclic voltammetry, NMR, UV-visible spectroscopy, FT-IR, EPR, and single crystal X-ray crystallography. The first generation replaced the pendent amine functionality of BTSCs with an alkoxy group. This replacement allowed for the structure to retain its physical characteristics that make these systems of interest in biological settings. However the change in functional group allows for tuning of the reduction potential, which is crucial for their activity. The copper diacetyl-bis(4-methylthiosemicarbazonato) (CuATSM) analog lays 250 mV more anodic for its CuII/I couple. Immobilization of NiATSM (2), NiATSDM (28), and NiATSM-F6 (29) on a glassy carbon electrode surface revealed that after reductive cycling, 200-300 cycles, improved the overpotentials of 2 and 28 by 250 mV from 700 mV to 450 mV. The lack of a dramatic effect for 29, is due to solid state interactions between molecules and electrode surface. Raman spectroscopy and SEM reveal that 29, does not remain on the electrode surface, whereas 2 and 28 undergo dynamic rearrangement to improve overall performance. A combination of both the pendant amine and alkoxy functionalities gave rise to the a new BTSC analog which chelated Ni, Cu and Zn. The electrochemical characterization revealed the copper chelates to have reduction potentials around 0.950 V CuII/I . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed high selectivity and potency with 50% growth inhibitory (GI50) concentrations of 0.09 and 2.0 μM for carcinogenic and healthy cell lines respectively. Further the National Cancer Institute (NCI) 60 profile demonstrates that the copper chelate is effective against a wide variety of cancer types