Multianalyte Detection of Breast Cancer by Fabrication of Hybridmicroarrays on Polymer-Based Microanalytical Devices

Breast cancer is one of the most common and fatal cancer diseases that affect women worldwide. As is true with most other cancer diseases, early detection of breast cancer is very crucial for proper medical treatment because treatment of advanced breast cancer will be much more difficult and inconsistent. Screening and testing of breast cancer biomarkers, either genetic or proteomic, are among techniques used for diagnosis of breast cancers. Nevertheless, none of the biomarkers is by itself sensitive and selective enough for diagnosis of breast cancer, and thus, multi-analyte assays towards detection of multiple breast cancer biomarkers from different classes are desired for accurate diagnosis of this disease. Described is a methodology with which both genetic and protein biomarkers of breast cancers are detected simultaneously on the same platform. This methodology consists of a novel hybrid biosensor system in a universal Zipcode DNA array format on the platform of polymer-based microfluidic devices. Detection of the genetic mutated material and the protein targeting material is through hybridization events between the arrayed universal Zipcode DNA sequences and the corresponding complementary Zipcode DNA sequences that are incorporated into both biomarkers during materials preparation. Signal generation and detection are through near-IR, laser-induced fluorescence imaging method. The hybrid biosensor system combines the strengths of microfluidic devices—high throughput, low sample consumption, and high kinetics—with that of the universal DNA array format, which uncouples detection from hybridization event, thereby increasing the sensitivity of detection. Near-IR laser-induced fluorescence detection method adds further sensitivity to this system. In this work, surface properties of the microfluidic device substrate, PMMA have been manipulated in surface functionalities, surface topography, and surface wettabilities. Biomolecules including both antibodies and DNA have been successfully immobilized onto the UV-modified PMMA surfaces. The targeting biomarker materials were prepared using distinct protocols: PCR/LDR combined assays were adopted to prepare the breast cancer gene marker BRCA1 mutated material, while the protein antigen CEA targeting complex was achieved by a semi-synthetic method. Monitoring and characterization of surface manipulation, bio-functionalization, and targeting materials preparation were accomplished by unique analytical tools

A modular multi electrode array system for electrogenic cell characterisation and cardiotoxicity applications

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities. [Continues.

Characterization of glutaraldehyde-immobilized chymotrypsin and an in-situ immobilized enzyme reactor using capillary electrophoresis-based peptide mapping

La digestion enzymatique des protéines est une méthode de base pour les études protéomiques ainsi que pour le séquençage en mode « bottom-up ». Les enzymes sont ajoutées soit en solution (phase homogène), soit directement sur le gel polyacrylamide selon la méthode déjà utilisée pour l’isolation de la protéine. Les enzymes protéolytiques immobilisées, c’est-à-dire insolubles, offrent plusieurs avantages tels que la réutilisation de l’enzyme, un rapport élevé d’enzyme-sur-substrat, et une intégration facile avec les systèmes fluidiques. Dans cette étude, la chymotrypsine (CT) a été immobilisée par réticulation avec le glutaraldehyde (GA), ce qui crée des particules insolubles. L’efficacité d’immobilisation, déterminée par spectrophotométrie d’absorbance, était de 96% de la masse totale de la CT ajouté. Plusieurs différentes conditions d’immobilisation (i.e., réticulation) tels que la composition/pH du tampon et la masse de CT durant la réticulation ainsi que les différentes conditions d’entreposage tels que la température, durée et humidité pour les particules GA-CT ont été évaluées par comparaison des cartes peptidiques en électrophorèse capillaire (CE) des protéines standards digérées par les particules. Les particules de GA-CT ont été utilisés pour digérer la BSA comme exemple d’une protéine repliée large qui requit une dénaturation préalable à la digestion, et pour digérer la caséine marquée avec de l’isothiocyanate de fluorescéine (FITC) comme exemple d’un substrat dérivé afin de vérifier l’activité enzymatique du GA-CT dans la présence des groupements fluorescents liés au substrat. La cartographie peptidique des digestions par les particules GA-CT a été réalisée par CE avec la détection par absorbance ultraviolet (UV) ou fluorescence induite par laser. La caséine-FITC a été, en effet, digérée par GA-CT au même degré que par la CT libre (i.e., soluble). Un microréacteur enzymatique (IMER) a été fabriqué par immobilisation de la CT dans un capillaire de silice fondu du diamètre interne de 250 µm prétraité avec du 3-aminopropyltriéthoxysilane afin de fonctionnaliser la paroi interne avec les groupements amines. Le GA a été réagit avec les groupements amine puis la CT a été immobilisée par réticulation avec le GA. Les IMERs à base de GA-CT étaient préparé à l’aide d’un système CE automatisé puis utilisé pour digérer la BSA, la myoglobine, un peptide ayant 9 résidus et un dipeptide comme exemples des substrats ayant taille large, moyenne et petite, respectivement. La comparaison des cartes peptidiques des digestats obtenues par CE-UV ou CE-spectrométrie de masse nous permettent d’étudier les conditions d’immobilisation en fonction de la composition et le pH du tampon et le temps de réaction de la réticulation. Une étude par microscopie de fluorescence, un outil utilisé pour examiner l’étendue et les endroits d’immobilisation GA-CT dans l’IMER, ont montré que l’immobilisation a eu lieu majoritairement sur la paroi et que la réticulation ne s’est étendue pas si loin au centre du capillaire qu’anticipée.Digesting proteins using proteolytic enzymes is a standard method in proteomic studies and bottom-up protein sequencing. Enzymes can be added in solution or gel phase depending on how the protein has been isolated. Immobilized, i.e., insoluble, proteolytic enzymes offer several advantages such as reusability of enzyme, high enzyme-to-substrate ratio, and integration with fluidic systems. In this study, we prepared glutaraldehyde-crosslinked chymotrypsin (GA-CT), which creates insoluble particles. The immobilization efficiency was determined by absorbance spectrophotometry and found to be 96% of the total amount of chymotrypsin added. Different immobilization (i.e., crosslinking) conditions such as buffer composition/pH and initial mass of CT during crosslinking as well as different storage conditions such as temperature, time and humidity for the GA-CT particles were evaluated by comparing capillary electrophoretic (CE) peptide maps of protein standards digested with the particles. The GA-CT particles were used to digest BSA as an example of a large folded protein that needs denaturation prior to digestion, and casein-fluorescein isothiocyanate (FITC) as an example of a small, labeled substrate to test enzyme activity in the presence of substrate-bound fluorescent groups. Peptide mapping of digests from GA-CT particles was achieved by CE with ultraviolet (UV) absorbance or laser induced fluorescence (LIF) detection. FITC-labeled casein was digested by GA-CT to the same extent as with free (i.e., soluble) CT. An immobilized enzyme microreactor (IMER) was fabricated by immobilizing CT inside a 250 µm i.d. fused-silica capillary tube pre-treated with 3-aminopropyltriethoxysilane to functionalize the inner walls with amine groups. Glutaraldehyde was reacted with the amine groups and then CT was immobilized by crosslinking to the GA. IMERs based on GA-CT were fabricated using an automated CE system and used to digest BSA, myoglobin, a 9-residue peptide and a dipeptide as examples of large, medium and small substrates. Digests were studied by comparing peptide maps obtained by CE coupled to either UV or mass spectrometric (MS) detection in order to evaluate immobilization conditions as a function of buffer composition/pH and reaction times. A separate study, which used fluorescence microscopy to investigate the extent and location of GA-CT immobilization in the IMER, showed that immobilization only takes place primarily near the capillary walls and that crosslinking does not extend as far into the center of the IMER as had been expected

Capillary-based Microreactor System Integrated with UHPLC/GC for High Throughput Screening of Catalysts for Organic Reactions

Catalyst discovery through high throughput screening can greatly take advantage of automated microreactor technology. Quantitative analysis of reaction outcomes is necessary for evaluating catalyst activities and GC and/or LC are common tools. Few microfluidic systems, however, have the capability of automatically screening catalysts in slow reactions with in situ chromatography analysis. Thus, a novel microreactor was developed that integrated sample loading, reaction and online analysis functions in a totally automated format. An autosampler and syringe pump load homogeneous catalysts and reagents. The chemicals are mixed and reacted in a long capillary followed by online analysis, either by GC or UHPLC. The approach of parallel reactions in a flow stream is good for either slow reactions or fast reactions depending on the operation mode. Some palladium and ligand complex catalysts for the Stille reaction were chosen and screened by a stop-flow approach with GC analysis for validation. The screening results were in good accordance with the literature.The first application of this microreactor was to discover peptidic catalysts for the direct aldol reaction. A major difficulty was the poor solubility of peptidic catalysts. This was solved by allowing catalysts to react with one of the aldol substrates to form soluble catalyst adduct, which was then loaded into the microreactor to complete the reaction. This two-step approach was used to screen a diverse set of amino acids and short peptidic catalysts, in which two groups of peptides containing r-Glu and b-Asp residues showed higher activities than other catalysts. The second application was to screen Bronsted or Lewis acid catalysts for an internal cyclization reaction. This reaction can be used to prepare large libraries of amide compounds for drug discovery. A continuous flow approach with online UHPLC analysis was applied to study this relatively fast reaction. The experimental throughput can reach 9 reactions/h. Results showed that strong acids are generally good for conversion and yield. Lanthanide triflate compounds, although having weak acidity, had better conversion and selectivity than other acids. Side reaction analysis by GC-MS and LC-MS indicated that strong and weak Bronsted acids can lead to the formation of different major byproducts

Micro/nanofluidic and lab-on-a-chip devices for biomedical applications

Micro/Nanofluidic and lab-on-a-chip devices have been increasingly used in biomedical research [1]. Because of their adaptability, feasibility, and cost-efficiency, these devices can revolutionize the future of preclinical technologies. Furthermore, they allow insights into the performance and toxic effects of responsive drug delivery nanocarriers to be obtained, which consequently allow the shortcomings of two/three-dimensional static cultures and animal testing to be overcome and help to reduce drug development costs and time [2–4]. With the constant advancements in biomedical technology, the development of enhanced microfluidic devices has accelerated, and numerous models have been reported. Given the multidisciplinary of this Special Issue (SI), papers on different subjects were published making a total of 14 contributions, 10 original research papers, and 4 review papers. The review paper of Ko et al. [1] provides a comprehensive overview of the significant advancements in engineered organ-on-a-chip research in a general way while in the review presented by Kanabekova and colleagues [2], a thorough analysis of microphysiological platforms used for modeling liver diseases can be found. To get a summary of the numerical models of microfluidic organ-on-a-chip devices developed in recent years, the review presented by Carvalho et al. [5] can be read. On the other hand, Maia et al. [6] report a systematic review of the diagnosis methods developed for COVID-19, providing an overview of the advancements made since the start of the pandemic. In the following, a brief summary of the research papers published in this SI will be presented, with organs-on-a-chip, microfluidic devices for detection, and device optimization having been identified as the main topics.info:eu-repo/semantics/publishedVersio

Von Plattformen zu miRNA-Biomarkern : Methoden zur miRNA-Molekulardiagnostik

An obvious way to improve human healthcare is to develop new and more effective drugs. Another opportunity is however to develop solutions that allow to utilize the available drugs better. This includes more accurate and early diagnosis of pathologies, improved therapy selection as well as digital and patient centric solutions in healthcare systems. Especially in molecular diagnostics new biomarkers have been developed and partially shown promising results in terms of improving patient care. In this work I describe the development of respective platform techniques, biomarkers and computational solutions during my PhD thesis. First, I briefly introduce the concept of a flexible microarray platform and assays, such as the MPEA assay, tailored for the fast and efficient quantification of miRNA signatures. Then, I describe how we made use of respective platforms along with computational solutions to improve the understanding of physiological and pathophysiological processes. Further, I present results on my efforts to develop new molecular diagnostic biomarkers based on circulating miRNAs. Here, my special focus was in cancer (most importantly lung cancer) and diseases affecting the Central Nervous System (most importantly Multiple Sclerosis, Alzheimer’s Disease and Parkinson’s Disease). Together with the supervisors of my thesis I was among the first researchers worldwide to recognize that small non-coding RNAs (most importantly microRNAs) measured from body fluids have a great potential as biomarkers. An obvious advantage to messenger RNAs is the small length of the molecules of only 17-22 nucleotides. This makes microRNAs stable in vivo but also in vitro. Finally, I will mention recent developments in patient care. The current trend is clearly the digitalization of central parts of healthcare. This affects all stakeholders in the healthcare system, most importantly medical doctors and patients. Especially patient empowerment and self-containment of medical data is becoming more important. Again, Multiple Sclerosis is used as an example. But also for physicians, computational tools have to be implemented to support them in making treatment decisions from highly complex data. In sum, my thesis describes the road from developing a molecular diagnostic platform over the research on biomarkers for detecting disease in time towards holistic computational solutions to improve patient care.Es ist offensichtlich, dass man Krankheiten besser behandeln kann, wenn man neue und effektivere Medikamente und Therapien entwickelt. Eine andere Möglichkeit ist es, Lösungen zu entwickeln, die es erlauben, vorhandene Medikamente besser einzusetzen. Das schließt die frühzeitige Diagnose von Erkrankungen, eine verbesserte Wahl der richtigen Therapie und die Entwicklung von patienten-zentrischen digitalisierten Lösungen mit ein. Insbesondere in der Molekulardiagnostik wurden neue vielversprechende Biomarker entwickelt. In dieser Arbeit führe ich meine Beiträge zur Entwicklung von Plattform Technologien zum Messen von Biomarkern aus, erläutere die Erforschung von Biomarkern selbst und beschreibe die Anwendung der dazugehörigen, computergestützten Methoden. Beginnen möchte ich mit einer Beschreibung der Entwicklung einer flexiblen Mikroarray Plattform und Assays, wie zum Beispiel des MPEA Assays, die maßgeschneidert für die schnelle und effiziente Quantifizierung von miRNA Biomarkern sind. Dann gehe ich darauf ein, wie wir Plattformen, Assays und computergestützte Lösungen eingesetzt haben, um physiologische und pathologische Prozesse besser zu verstehen. Außerdem präsentiere ich Resultate meiner Bemühung, neue molekulardiagnostische Biomarker basierend auf zirkulierenden miRNA Mustern zu entwickeln. Hierbei habe ich mich auf Krebs (vornehmlich Lungentumore) und Erkrankungen, die das Zentrale Nervensystem betreffen (Multiple Sklerose und die Alzheimer Erkrankung), konzentriert. Gemeinsam mit meinen Betreuern war ich unter den ersten Forschern weltweit, die das große Potenzial kleiner nicht-kodierender RNAs (am wichtigsten dabei microRNAs), die aus Blut gemessen werden können, erkannt haben. Ein offensichtlicher Vorteil gegenüber mRNA Biomarkern ist die kurze Länge von nur 17-22 Nukleotiden. Diese macht miRNAs sowohl in-vivo als auch in-vitro stabil. Letztlich gehe ich in meiner Arbeit auf momentane Entwicklungen in der Patientenversorgung ein. Ein klarer Trend ist die Digitalisierung zentraler Teile der Gesundheitsversorgung. Das betrifft alle Personen im Gesundheitswesen, allen voran Mediziner und Patienten. Selbstbestimmung des Patienten wird besonders wichtig werden. Hier dient mir wieder Multiple Sklerose als ein Beispiel. Auch für Ärzte müssen, angesichts der immer komplexeren Daten, computergestützte Lösungen entwickelt werden, die ihnen helfen, die richtige Therapieentscheidung zu treffen. Zusammenfassend halte ich fest, dass meine Arbeit den Weg von der Entwicklung einer molekulardiagnostischen Plattform über die Entwicklung von Biomarkern zur Frühdiagnose von Erkrankungen bis hin zu ganzheitlichen computergestützten Lösungen, die die Patientenversorgung verbessern, beschreibt

Selected Papers from the 1st International Electronic Conference on Biosensors (IECB 2020)

The scope of this Special Issue is to collect some of the contributions to the First International Electronic Conference on Biosensors, which was held to bring together well-known experts currently working in biosensor technologies from around the globe, and to provide an online forum for presenting and discussing new results. The world of biosensors is definitively a versatile and universally applicable one, as demonstrated by the wide range of topics which were addressed at the Conference, such as: bioengineered and biomimetic receptors; microfluidics for biosensing; biosensors for emergency situations; nanotechnologies and nanomaterials for biosensors; intra- and extracellular biosensing; and advanced applications in clinical, environmental, food safety, and cultural heritage fields

Factories of the Future

Engineering; Industrial engineering; Production engineerin