Download or read book Microfluidic Devices with Integrated Sample Preparation for Improved Analysis of Protein Biomarkers written by Pamela Nsang Nge and published by . This book was released on 2012 with total page 113 pages. Available in PDF, EPUB and Kindle. Book excerpt: To fabricate polymeric microdevices, I prepared templates from silicon wafers by the process of photolithography. The design on the template was transferred to a polymer piece by hot embossing, and a complete device was formed by bonding the imprinted piece with a cover plate. I prepared affinity columns in these devices and used them for protein extraction. The affinity monolith was prepared from reactive monomers to faciliate immobilization of antibodies. Extraction and concentration of biomarkers on this column showed specificity to the target molecule. This shows that biomarkers could be extracted, purified and concentrated with the use of microfluidic affinity columns.
Download or read book Microfluidics Diagnostics written by Valérie Taly and published by Springer Nature. This book was released on with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Total Integrated Sample Preparation for Microfluidic Immunoassays in Complex Biological Matrices written by Akwasi Asare Apori and published by . This book was released on 2011 with total page 140 pages. Available in PDF, EPUB and Kindle. Book excerpt: A high-throughput protein analysis platform with integrated sample preparation is developed to address the identified technology gaps in biomarker validation, clinical and point-of-care diagnostics. The goals of the technology are to automate and integrate protein sample preparation with electrokinetic separations, implement immunoassays capable of processing raw biological fluids, and perform high-throughput protein assays targeted for disease diagnosis. Integration of multiple functions is a hallmark of microfluidic tools. Efforts to fully incorporate required sample preparation steps within electrokinetic assays would benefit from a surge in technology development. On-chip fluorescence labeling, analyte enrichment, buffer exchange, reagent-sample mixing and matrix protein depletion are essential to realize the "sample-to-answer" paradigm of lab-on-a-chip systems for targeted proteomics--which hold the key to rigorous validation of potentially high impact biomarkers relevant for drug discovery, clinical diagnostics, and patient therapy and monitoring. Integration of sample preparation techniques would also expand the protein analysis application space to point-of-care assays in low resource settings. Presently, introduction of in-situ fabrication methods has enabled advances in integration of multiple reaction zones into microfluidic geometries. Fabrication is performed at the microscale using chemically and physical functionalized polyacrylamide gels photo-patterned on microfluidic chips. These zones facilitate precise control of assay performance parameters including sample resolution, background noise, dynamic range, binding kinetics, and dispersion among others while taking advantage of microscale interactions to reduce assay time, cost, and reagent consumption while increasing sensitivity, and portability. In this dissertation several novel techniques for on-chip proteomics and immunoassays with integrated sample preparation are introduced for the analysis of serum or proximal fluids. These methods are applied to develop laboratory medicine and point-of-care assays for the following applications: 1) immunosubtraction polyacrylamide gel electrophoresis for detection of protein mobility and binding specificity, 2) on-chip fluorogenic labeling integrated with native electrophoresis for mass isoform detection, 3) low molecular weight compound sandwich fragment antibody assays for detecting drugs of abuse, 4) integrated enrichment and labeling of proteins facilitated with size-exclusion membranes, 5) cerebrospinal fluid rhinorrhea detection for emergency room screening of traumatic brain injury, and 6) rapid single-step electrophoretic and isotachophoretic immunoassays for serum Hepatitis C confirmatory diagnostics. The unifying theme of all techniques presented herein is the precise electrokinetic control and manipulation of sample and reagents on-chip to automate and expedite time consuming and manual labor intensive benchtop immunoassays based on diffusion driven interactions.
Download or read book Microfluidics and Biosensors in Cancer Research written by David Caballero and published by Springer Nature. This book was released on 2022-06-27 with total page 599 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book offers a comprehensive overview of the development and application of microfluidics and biosensors in cancer research, in particular, their applications in cancer modeling and theranostics. Over the last decades, considerable effort has been made to develop new technologies to improve the diagnosis and treatment of cancer. Microfluidics has proven to be a powerful tool for manipulating biological fluids with high precision and efficiency and has already been adopted by the pharmaceutical and biotechnology industries. With recent technological advances, particularly biosensors, microfluidic devices have increased their usefulness and importance in oncology and cancer research. The aim of this book is to bring together in a single volume all the knowledge and expertise required for the development and application of microfluidic systems and biosensors in cancer modeling and theranostics. It begins with a detailed introduction to the fundamental aspects of tumor biology, cancer biomarkers, biosensors and microfluidics. With this knowledge in mind, the following sections highlight important advances in developing and applying biosensors and microfluidic devices in cancer research at universities and in the industry. Strategies for identifying and evaluating potent disease biomarkers and developing biosensors and microfluidic devices for their detection are discussed in detail. Finally, the transfer of these technologies into the clinical environment for the diagnosis and treatment of cancer patients will be highlighted. By combining the recent advances made in the development and application of microfluidics and biosensors in cancer research in academia and clinics, this book will be useful literature for readers from a variety of backgrounds. It offers new visions of how this technology can influence daily life in hospitals and companies, improving research methodologies and the prognosis of cancer patients.
Download or read book Microfluidic Devices for In line Sample Preparation of Biological Mixture for Mass Spectrometry Based Downstream Analyses written by Tristan F. Kinde and published by . This book was released on 2013 with total page 213 pages. Available in PDF, EPUB and Kindle. Book excerpt: For a majority of analytical chemistry studies, effective sample preparation is tantamount to the ability to answer "What is it?" and "How much is there?". In academia and industry, methods of sample preparation traditionally aim to make a sample "cleaner" so as to minimize interference from other species in the mixture and to do so in a timely, cost-effective manner. Ultimately, a rapidly produced "clean" sample allows for lower limits of detection for the analyte and quicker sample turnover. The goal of our work was to expand the knowledge of experimental microfluidics (also known as Lab-on-a-chip), explore designs to better exploit microfluidic phenomena, and develop microfluidic technologies readily integrated to mass spectrometers, all while focusing towards improving sample preparation and assay procedures. Through our efforts we created three novel devices that accomplished these goals. First, we designed an H-shaped microfluidic channel (H-cell) laden with gold internal electrodes which induced a continuous electrophoretic separation transverse to the direction of bulk fluid flow. When combined with a surface coating to suppress electroosmotic flow, the H-cell with internal electrodes removed approximately 96% of a peptide from a synthetic mixture of nucleic acid and peptide in less than 10 seconds. Once integrated with on-chip electrospray ionization functionality, we explored the dynamics of H-cell separations and improved the limit of detection for the peptide while integrated on-line with a mass spectrometer. Second, the original H-cell was modified by replacing the internal electrodes with a series of channels. In the place of electrodes, sub 100 nanometer channels were studied as well as covalently bound cross-linking polyacrylamide gel membranes. With these modifications, we achieved more challenging separations within high conductivity solutions. More specifically, we achieved continuous-flow electrophoretic separations in solutions containing 6.25% v/v formic acid or 1% w/v sodium dodecyl sulfate. In addition, we used these modified H-cell devices to fractionate a sample with analyte molecules of the same charge; essentially, these microfluidic platforms can operate as a continuous flow low-pass/high-pass fluidic mass filter (membrane H-cell) or a band-pass fluidic mass filter (nanochannel H-cell). Finally, we designed a microfluidic device using the concept of concentration polarization at the interface of a microchannel/nanochannel junction to explore sample preconcentration phenomena with a dilute analyte. Integrating this device with a mass spectrometer, we reduced the limit of detection by over two orders of magnitude in less than two minutes compared to detecting the analyte without the microfluidic device.
Download or read book Microfluidic Technologies for Human Health written by Utkan Demirci and published by World Scientific. This book was released on 2012 with total page 495 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ch. 1. A microscale bioinspired cochlear-like sensor / Robert D. White, Robert Littrell, and Karl Grosh -- ch. 2. Systematic evaluation of the efficiencies of proteins and chemicals in pharmaceutical applications / Morgan Hamon and Jong Wook Hong -- ch. 3. Microfluidic glucose sensors / Jithesh V. Veetil [und weitere] -- ch. 4. Applications of microfabrication and microfluidic techniques in mesenchymal stem cell research / Abhijit Majumder [und weitere] -- ch. 5. Patient-specific modeling of low-density lipoprotein transport in coronary arteries / Ufuk Olgac -- ch. 6. Point-of-care microdevices for global health diagnostics of infectious diseases / Sau Yin Chin [und weitere] -- ch. 7. Integrated microfluidic sample preparation for chip-based molecular diagnostics / Jane Y. Zhang [und weitere] -- ch. 8. Microfluidic devices for cellular proteomic studies / Yihong Zhan and Chang Lu -- ch. 9. Microfluidics for neuroscience: novel tools and future implications / Vivian M. Hernandez and P. Hande Ozdinler -- ch. 10. Microfluidics: on-chip platforms as in vitro disease models / Shan Gao, Erkin Seker, and Martin L. Yarmush -- ch. 11. Application of microfluidics in stem cell and tissue engineering / Sasha H. Bakhru, Christopher Highley, and Stefan Zappe -- ch. 12. Microfluidic "on-the-fly" fabrication of microstructures for biomedical applications / Edward Kang, Sau Fung Wong, and Sang-Hoon Lee -- ch. 13. Microfluidics as a promising tool toward distributed viral detection / Elodie Sollier and Dino Di Carlo -- ch. 14. Electrophoresis and dielectrophoresis for lab-on-a-chip (LOC) analyses / Yagmur Demircan, Gurkan Yilmaz, and Haluk Kulah -- ch. 15. Ultrasonic embossing of carbon nanotubes for the fabrication of polymer microfluidic chips for DNA sample purification / Puttachat Khuntontong, Min Gong, and Zhiping Wang -- ch. 16. Ferrofluidics / A. Rezzan Kose and Hur Koser -- ch. 17. Antibody-based blood bioparticle capture and separation using microfluidics for global health / ZhengYuan Luo [und weitere] -- ch. 18. Applications of quantum dots for fluorescence imaging in biomedical research / ShuQi Wang [und weitere]
Download or read book Lab on a chip Devices for Advanced Biomedicines written by Arpana Parihar and published by Royal Society of Chemistry. This book was released on 2024-08-14 with total page 694 pages. Available in PDF, EPUB and Kindle. Book excerpt: The global miniature devices market is poised to surpass a valuation of $12–$15 billion USD by the year 2030. Lab-on-a-chip (LOC) devices are a vital component of this market. Comprising a network of microchannels, electrical circuits, sensors, and electrodes, LOC is a miniaturized integrated device platform used to streamline day-to-day laboratory functions, run cost-effective clinical analyses and curb the need for centralized instrumentation facilities in remote areas. Compact design, portability, ease of operation, low sample volume, short reaction time, and parallel investigation stand as the pivotal factors driving the widespread acceptance of LOC within the biomedical community. In this book, the Editors meticulously explore LOC through three key ‘Ts’: Theories (microfluidics, microarrays, instrumentation, software); Technologies (additive manufacturing, artificial intelligence, computational thinking, smart consumables, scale-up tactics, and biofouling); and Trends (biomedical analysis, point-of-care diagnostics, personalized healthcare, bioactive synthesis, disease diagnosis, and space applications) This comprehensive text not only provides readers with a thorough understanding of the current advancements in the LOC domain but also offers valuable insights to support the utilization of miniaturized devices for enhanced healthcare practices. Aimed at career researchers looking for instruction in the topic and newcomers to the area, the book is also useful for undergraduate and postgraduate students embarking on new studies or for those interested in reading about the LOC platform.
Download or read book Microfluidic Modules for Enabling Point of Care Biopsy based Cancer Diagnostics written by Debkishore Mitra and published by . This book was released on 2013 with total page 144 pages. Available in PDF, EPUB and Kindle. Book excerpt: The analyses of patient tumor biopsy samples and biopsy-based diagnostics have emerged as important tools for cancer diagnostics. However, the techniques employed currently are restricted to centralized laboratories as they are time-consuming, manual labor intensive and vary considerably in their effectiveness amongst institutions and countries. Point of Care testing (POCT) for cancer with the capacity for multiplexed detection of numerous biomarkers in biopsy samples in a rapid, precise and portable manner is emerging as an area with enormous potential to disseminate universal diagnostics to cancer patients. Additionally, POCT can be used as a screening tool, to discern malignant from benign tumors at the physician's office, and lead to reduction in the need for expensive and time-consuming laboratory tests, hence minimizing the cost and anxiety, for patients with benign tumors. A POCT platform for multiple biomarker analysis can not only improve the operational characteristics of assays but can also help ascertain drug efficacy, ushering in personalized medicine for the patients. The reduced volumes and diffusion distances, which enable multiplexed, portable and quick assays, in microfluidic devices makes such devices a promising platform to realize POCT systems. But current microfluidic devices for cancer diagnostics suffer from the lack of a generalized on-chip sample preparation module and a simplified fluid actuation technique. The overall goal of the reported dissertation research is to develop microfluidic modules that will enable the development of integrated microfluidic diagnostic platforms for the multiplexed detection of cancer biomarkers in tumor biopsy samples. The main focus of the thesis is on the development of novel microfluidic sample preparation modules. The purpose of the sample preparation component is to pre-concentrate cancerous cells, remove background proteins in the sample and to subsequently lyse the cells to release the proteins of interest. The pre-concentration of the adherent cells, including the cancerous cells, in the sample is reported by trapping them using a novel hydrodynamic cell trap. The sample washing methods, to remove extracellular proteins that could interfere with downstream assays, is also optimized. An electrochemical lysis technique is then integrated to the cell pre-concentration module, to effectively lyse the cells without having to add external reagents. Microfluidic modules for the separation of bacterial and mammalian cells from mixed samples are also reported. The immortalized cancer cell lines used in this research include the human breast cancer cell lines BT-474, known to over express the Her-2 protein, and T47D along with cervical cancer cell line HeLa. The development of a novel fluid actuation technique, termed Proximal Degas-driven Flow (PDF), is also reported in this thesis. PDF takes advantage of the high porosity and air solubility of PDMS to reduce the pressure inside the fluidic channels leading to fluid flow in the channel. This actuation technique enables bubble-free fluid flow, can be used to fill up dead-end chambers in contrast to traditional pressure (positive or negative) driven flows and does not require the priming of the channels. Unlike degas-driven flow, PDF alleviates the need for pre-degassed and sealed devices, enabling consistent and longer-lasting fluid flow. This portable technique also requires very simple and cheap hardware like a vacuum bulb or membrane pump (thumb pump). In conclusion, several microfluidic modules to enable Point of Care biopsy-based cancer diagnostics are introduced. The research presented in this dissertation is an effort to transform point-of-care cancer testing and provide universal diagnostics and personalized medicine to cancer patients.
Download or read book Microfluidic Systems for Cancer Diagnosis written by Jose L. Garcia-Cordero and published by Springer Nature. This book was released on 2023-06-10 with total page 327 pages. Available in PDF, EPUB and Kindle. Book excerpt: This detailed volume explores recent developments in microfluidics technologies for cancer diagnosis and monitoring. The book is divided into two sections that delve into techniques for liquid biopsy for cancer diagnosis and platforms for precision oncology or personalized medicine in order to create effective patient avatars for testing anti-cancer drugs. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Microfluidic Systems for Cancer Diagnosis serves as an ideal guide that will be helpful to either replicate the construction of microfluidic devices specifically developed for cancer diagnosis or to catalyze development of new and better cancer diagnostic devices.
Download or read book Microfluidic Lab on a Chip for Chemical and Biological Analysis and Discovery written by Paul C.H. Li and published by CRC Press. This book was released on 2005-11-01 with total page 529 pages. Available in PDF, EPUB and Kindle. Book excerpt: The microfluidic lab-on-a-chip allows scientists to conduct chemical and biochemical analysis in a miniaturized format so small that properties and effects are successfully enhanced, and processes seamlessly integrated. This microscale advantage translates into greater sensitivity, more accurate results, and better information. Microfluidic
Download or read book Encyclopedia of Microfluidics and Nanofluidics written by Dongqing Li and published by Springer Science & Business Media. This book was released on 2008-08-06 with total page 2242 pages. Available in PDF, EPUB and Kindle. Book excerpt: Covering all aspects of transport phenomena on the nano- and micro-scale, this encyclopedia features over 750 entries in three alphabetically-arranged volumes including the most up-to-date research, insights, and applied techniques across all areas. Coverage includes electrical double-layers, optofluidics, DNC lab-on-a-chip, nanosensors, and more.
Download or read book Algorithms for Sample Preparation with Microfluidic Lab on Chip written by Sukanta Bhattacharjee and published by CRC Press. This book was released on 2022-09-01 with total page 179 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent microfluidic technologies have brought a complete paradigm shift in automating biochemical processing on a tiny lab-on-chip (a.k.a. biochip) that replaces expensive and bulky instruments traditionally used in implementing bench-top laboratory protocols. Biochips have already made a profound impact on various application domains such as clinical diagnostics, DNA analysis, genetic engineering, and drug discovery, among others. They are capable of precisely manipulating micro-/pico-liter quantities of fluids, and provide integrated support for mixing, storage, transportation, and sensing, on-chip. In almost all bioprotocols, sample preparation plays an important role, which includes dilution and mixing of several fluids satisfying certain volumetric ratios. However, designing algorithms that minimize reactant-cost and sample-preparation time suited for microfluidic chips poses a great challenge from the perspective of protocol mapping, scheduling, and physical design. Algorithms for Sample Preparation with Microfluidic Lab-on-Chip attempts to bridge the widening gap between biologists and engineers by introducing, from the fundamentals, several state-of-the-art computer-aided-design (CAD) algorithms for sample preparation with digital and flow-based microfluidic biochips. Technical topics discussed in the book include: Basics of digital and flow-based microfluidic lab-on-chipComprehensive review of state-of-the-art sample preparation algorithmsSample-preparation algorithms for digital microfluidic lab-on-chipSample-preparation algorithms for flow-based microfluidic lab-on-chip
Download or read book Proteomic Profiling written by Anton Posch and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Electrokinetically Operated Integrated Microfluidic Devices for Preterm Birth Biomarker Analysis written by Mukul Sonker and published by . This book was released on 2017 with total page 130 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the future, these optimized microfluidic components can be integrated into a single platform for automated immunoaffinity extraction, preconcentration, fluorescent labeling, andseparation of PTB biomarkers. This integrated microfluidic platform could significantly improve human health by providing early diagnosis of PTBs.
Download or read book Sample Preparation with Nanomaterials written by Chaudhery Mustansar Hussain and published by John Wiley & Sons. This book was released on 2021-04-30 with total page 304 pages. Available in PDF, EPUB and Kindle. Book excerpt: Discover this timely, comprehensive, and up-to-date exploration of crucial aspects of the use of nanomaterials in analytical chemistry Sample Preparation with Nanomaterials: Next Generation Techniques for Sample Preparation delivers insightful and complete overview of recent progress in the use of nanomaterials in sample preparation. The book begins with an overview of special features of nanomaterials and their applications in analytical sciences. Important types of nanomaterials, like carbon nanotubes and magnetic particles, are reviewed and biological sample preparation and lab-on-a-chip systems are presented. The distinguished author places special emphasis on approaches that tend to green and reduce the cost of sample treatment processes. He also discusses the legal, economical, and toxicity aspects of nanomaterial samples. This book includes extensive reference material, like a complete list of manufacturers, that makes it invaluable for professionals in analytical chemistry. Sample Preparation with Nanomaterials offers considerations of the economic aspects of nanomaterials, as well as the assessment of their toxicity and risk. Readers will also benefit from the inclusion of: A thorough introduction to nanomaterials in the analytical sciences and special properties of nanomaterials for sample preparation An exploration of the mechanism of adsorption and desorption on nanomaterials, including carbon nanomaterials used as adsorbents Discussions of membrane applications of nanomaterials, surface enhanced raman spectroscopy, and the use of nanomaterials for biological sample preparation A treatment of magnetic nanomaterials, lab-on-a-chip nanomaterials, and toxicity and risk assessment of nanomaterials Perfect for analytical chemists, materials scientists, and process engineers, Sample Preparation with Nanomaterials: Next Generation Techniques for Sample Preparation will also earn a place in the libraries of analytical laboratories, universities, and companies who conduct research into nanomaterials and seek a one-stop resource for sample preparation.
Download or read book Molecular Diagnostics written by George P. Patrinos and published by Academic Press. This book was released on 2016-10-27 with total page 526 pages. Available in PDF, EPUB and Kindle. Book excerpt: Molecular Diagnostics, Third Edition, focuses on the technologies and applications that professionals need to work in, develop, and manage a clinical diagnostic laboratory. Each chapter contains an expert introduction to each subject that is next to technical details and many applications for molecular genetic testing that can be found in comprehensive reference lists at the end of each chapter. Contents are divided into three parts, technologies, application of those technologies, and related issues. The first part is dedicated to the battery of the most widely used molecular pathology techniques. New chapters have been added, including the various new technologies involved in next-generation sequencing (mutation detection, gene expression, etc.), mass spectrometry, and protein-specific methodologies. All revised chapters have been completely updated, to include not only technology innovations, but also novel diagnostic applications. As with previous editions, each of the chapters in this section includes a brief description of the technique followed by examples from the area of expertise from the selected contributor. The second part of the book attempts to integrate previously analyzed technologies into the different aspects of molecular diagnostics, such as identification of genetically modified organisms, stem cells, pharmacogenomics, modern forensic science, molecular microbiology, and genetic diagnosis. Part three focuses on various everyday issues in a diagnostic laboratory, from genetic counseling and related ethical and psychological issues, to safety and quality management. Presents a comprehensive account of all new technologies and applications used in clinical diagnostic laboratories Explores a wide range of molecular-based tests that are available to assess DNA variation and changes in gene expression Offers clear translational presentations by the top molecular pathologists, clinical chemists, and molecular geneticists in the field
Download or read book Bioanalytical Microfluidic Devices and Methods for Analysis of Cancer Gene Expression written by Nadia Del Bueno and published by . This book was released on 2011 with total page 160 pages. Available in PDF, EPUB and Kindle. Book excerpt: Transcriptional profiling is essential in fundamental studies of pathogenesis. In particular, multiplexed analysis of gene expression enables the identification of cancer-specific expression signatures that correlate with clinical disease and which can be used for the prediction of tumor presence and disease progression. Microfabricated capillary electrophoresis (CE) devices feature reduced sample and reagent requirements, faster analysis times, and increased automation. These advantages make microdevices ideal analytical platforms for the quantitative monitoring of biomarkers, and their potential as point-of-care devices for facilitating cancer detection in the clinical setting is explored in this thesis. First, an integrated microdevice capable of performing low-volume, rapid, and highly sensitive expression analysis was developed. To further the goal of quantitative measurements of transcript levels from a small amount of sample, an affinity capture gel approach was used to address the problem of inefficient CE sample injection which limits sensitivity. Photopolymerization protocols were developed to define a small plug of oligonucleotide-modified polyacrylamide gel inline with the CE channel in order to accomplish efficient capture and sample microinjection for quantitative analysis. This concentration and purification step also demonstrated increased detection sensitivity and improved separation resolution. To perform multiplexed analysis of cancer genes, an optimized protocol for transcript analysis with aid of affinity capture was used in conjunction with the integrated CE microdevice. The expression of genes implicated in prostate cancer was assayed directly from cells by solution hybridization with complementary fluorescently-labeled detection probes, followed by affinity bead capture of mRNA-probe complexes. Released detection probes were then injected on-chip and captured on a photopolymerized capture gel for concentration prior to CE separation and detection. The ability of the assay and microdevice system to evaluate gene expression was demonstrated by the measurement of absolute transcript levels of ten genes, enabling the successful identification of distinct expression signatures for the human prostate cancer cell lines LNCaP, VCaP, 22Rv1, and PC-3 with high sensitivity. Finally, a microdevice also including on-chip PCR amplification is presented for improving the sensitivity of detection to enable the analysis of clinical samples. The functionality of the proposed microsystem is further expanded by the integration of upstream sample processing steps on-chip. By performing several bioanalytical processes on a single microfabricated platform, high-sensitivity expression analysis from complex biological samples should be possible, all the while reducing cost and analysis time. Based on the technologies developed in this thesis, these fully integrated devices could be implemented as diagnostic tools and play a key role in the future of clinical detection.
