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Sk microfluidics and lab on-a-chip-ch3 Sk microfluidics and lab on-a-chip-ch3 Presentation Transcript

  • Microfluidics and Lab-on-a-Chip for biomedical applications Chapter 3 : Molecular biology and selected analytical tools. By Stanislas CNRS Université de Lyon, FRANCE Stansan International Group
  • CONTEN T Chapter 1: Introduction. Chapter 2 : Basic principles of Microfluidics. Chapter 3 : Basis of molecular biology and analytical tools. Chapter 4 : Micromanufacturing. Chapter 5 : Lab-on-a-Chip & applications. Chapter 6 : Cancer diagnostics and monitoring.
  • There is a wide range of applications of Lab-on-aChip in the field of chemistry, biochemistry, environmental control, bio defense…. Our objective today is to speak about applications in the field of medical diagnostics and monitoring. We will choose the field of the cancer. Thus, a little bit of (molecular) biology….
  • Biological cell The cell is the basic unit of life. It was discovered by Robert Hooke (1665) and is the functional unit of all known living organisms. It is classified as a living thing, and is often called the building block of life. There are two types of cells: Prokaryotic cells are usually independent, while Eukaryotic cells are often found in multicellular organisms. Some organisms, such as most bacteria consist of a single cell. Other organisms, such as humans, are multicellular. Humans have about 100 trillion or 1014 cells ; a typical cell size is 10 µm; a typical cell mass is 1 nanogram. The largest cells are about 135 µm, the smallest, can be some 4 µm. The largest known cells are unfertilised ostrich egg cells which weigh 3.3 pounds. The word cell comes from the Latin cellula, meaning, a small room. The human body contains many different organs, such as the heart, lung, and kidney... Cells also have a set of "little organs," called organelles.
  • Typical Eukaryotic Cell 1) Nucleolus 2) Nucleus 3) Ribosome 4) Vesicle 5) Rough endoplasmic reticulum (ER) 6) Golgi apparatus 7) Microtubule 8) Smooth ER 9) Mitochondria 10) Vacuole 11) Cytoplasm 12) Lysosome 13) Centrioles
  • DNA
  • DNA
  • BASES
  • Few Numbers
  • DN A the genetic instructions used DNA is a nucleic acid that contains in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the longterm storage of information. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore antiparallel. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins.
  • DN A
  • Chromosome A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Scheme of a Chromosome : (1) Chromatid. One of the two identical parts of the chromosome after S phase. (2) Centromere. The point where the two chromatids touch, and where the microtubules attach. (3) Short arm (4) Long arm. In accordance with the display rules in Cytogenetics, the short arm is on top.
  • A gene is a unit of heredity in a living organism. It is normally a stretch of DNA that codes for a type of protein or for an RNA chain that has a function in the organism. All proteins and functional RNA chains are specified by genes. All living things depend on genes. Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring. A modern working definition of a gene is "a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions, and or other functional sequence regions ". GENE S
  • Telomere A telomere is a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. If cells divided without telomeres, they would lose the ends of their chromosomes, and the necessary information they contain. The telomeres protect the chromosomes and are consumed during cell division due to an enzyme, the telomerase reverse transcriptase. The telomere shortening mechanism normally limits cells to a fixed number of divisions, and this is responsible for aging on the cellular level. Most cancers are "immortal" cells which have ways of evading this programmed destruction. Activation of the Alternative Lengthening of Telomeres (ALT) pathway which involves p53 and pRb may lead to the arrest of cell proliferation. Elizabeth Blackburn, Carol Greider, and Jack Szostak were awarded the 2009 Nobel Prize in Physiology or Medicine for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.
  • Telomere Three-dimensional molecular structure of a telomere. Human chromosomes (grey) capped by telomeres (white). Telomeric repeat (human) : TTAGGG.....
  • DNA Analysis
  • Genome Projects The Human Genome sequence is complete approximately 3.2 billion base pairs
  • Genomics Technologies • Next-Generation DNA sequencing • Automated annotation of sequences • DNA microarrays – gene expression (measure RNA levels) – single nucleotide polymorphisms (SNPs) – ChIP-chip, genomic tiling, etc • Proteomics (mass-spec) • Protein chips • Protein-protein interactions
  • Single Molecular DNA manipulation At Humboldt University of Berlin the new approach was developed for DNA manipulation of single macromolecules on solid substrate. No one other known approach allows that.
  • The picture demonstrates ability of assembling of the word "SCIENCE" on atomically flat substrate from DNA molecules
  • Cycle cellulaire Phase M : mitose, séparation d’une cellule mère en 2 cellules filles. Phase G1: phase de croissance et contrôle avant réplication. Phase S : synthèse de l’ADN, réplication des chromosomes. Phase G2 : Contrôle de la qualité de réplication avant division.
  • Transcription & Translation
  • Proteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. Proteins The amino acids in a polymer are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids Proteins are a primary constituent of living things and one of the chief classes of molecules studied in biochemistry. 3D structure of myoglobin showing coloured alpha helices. This protein was the first to have its structure solved by X-ray crystallography.
  • Measuring Gene Expression
  • Types of Microarrys (BioChips)
  • Oligonucleotide Array: a "DNA Chip" A collection of microscopic DNA spots attached to a solid surface forming an array; used to measure the expression levels of large numbers of genes simultaneously. An oligonucleotide array consists of a series of short (typically 20~30 bases) single-stranded DNA sequences (oligonucleotides, or "oligos") attached to a glass chip about the size of a microscope cover slip. In the arrangement shown here, each adjacent oligo differs from its neighbor only at the last base. In the example, the first four oligos in block 1 begin with GAGCCAAGCTG and end with A, G, C, or T, respectively.
  • Fluorescence Detection DNA Chip
  • Exemple of a DNA Chip Called the "GreeneChip," this device consists of a glass slide onto which are attached nearly 30,000 pieces of genetic material taken from thousands of different viruses, bacteria, fungi and parasites. When human fluid and tissue samples are applied to the chip, these probes will stick to any closely related genetic material in the samples. This allows the rapid and specific identification of any pathogens therein-even those related to but genetically distinct from the ones represented on the chip.
  • Example of a System for Biochips Fabrication SonoPlot provides innovative fluid dispensers and chemical surface treatments for microelectronics and the life sciences. The Microplotter line of dispensers can print microcircuitry on a desktop for research and development or deposit high-density grids of biomolecules such as DNA or proteins for fabrication of microarrays. A wide variety of materials can be treated with the SonoCoat surface modification process, producing ideal surfaces for adhering molecules in a microarray.
  • DNA Chip Reader A DNA chip reader is used to analyze colossal amounts of genetic information. On the DNA chip, hybridization is performed on the DNA labeled by a fluorescent dye. The DNA chip is then scanned by laser beam and by measuring the fluorescent intensity of the hybridized DNA spot, the genetic information is acquired from among the targeted DNA. (Hybridization is process to link 2 chains of DNA each having a complementary base.)
  • In the 1990s, DNA arrays provided the means to analyze patterns of gene expression in a living cell. DNA microarrays often consist of glass slides with spots of attached DNA fragments. The DNA fragments act as probes for specific sequences in a sample.
  • In the early 1990s, Stephen Fodor and his team developed a technique to produce miniature arrays of biological molecules. Their work led to the first DNA chip, and became the basis of techniques for largescale genomic studies. The company Affymetrix was spun off in the early 1990s to focus on DNA GeneChips®.
  • Examples of DNA Microarrys (BioChips)
  • Application of BioChips DNA chips have been extensively used for research applications in academia and in industrial laboratories. A big progress in bioinformatics is still needed in order to be able to explore in a reliable way the DNA data in the field of medicine. It is expected that many DNA chip for medical diagnostics and monitoring will be developed in the next few years. One of challenges for the Biochip industry is also the integration of microarrays with microfluidics, in order to achieve Microsystems, which include the extraction of the genetic material (e.g. DNA or RNA from white blood cells or from rare circulating tumor cells), purification and amplification of the extracted material and, finally, the analysis of this material with DNA chips.
  • Protein biochip and other microarray technologies Microarrays are not limited to DNA analysis; protein microarrays, antibody microarray, chemical compound microarray can also be produced using biochips. Randox Laboratories Ltd. launched Evidence, the first protein Biochip Array Technology analyzer in 2003. In protein Biochip Array Technology, the biochip replaces the ELISA plate or cuvette as the reaction platform. The biochip is used to simultaneously analyze a panel of related tests in a single sample, producing a patient profile. The patient profile can be used in disease screening, diagnosis, monitoring disease progression or monitoring treatment.
  • Antibody Microarrays for Biomarker Discovery Antibodies have the potential to identify biomarkers that are novel, unusually spliced or modified or are present in a differential concentration in cancer serum samples with respect to normal samples. They combine the advantages of an unbiased discovery approach (as is the case for Mass Spectrometry techniques) with the sensitivity of an immunoassay for detecting low abundance serum proteins (such as ELISA). As an added advantage, the antibodies can be used for discovery, purification, identification and characterization of the novel biomarker molecules.
  • Schematic description of protein immobilization
  • Antibody microarray An antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens. Antibody microarray is often used for detecting protein expressions from cell lysates in general research and special biomarkers from serum or urine for diagnostic applications.
  • Antibody microarray
  • ELISA - immunoassay Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In ELISA, an antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
  • ELISA - immunoassay
  • ELISA - immunoassay i) Test serum is incubated with antigen immobilised on a 96-well plate or microscope slide ii) Secondary antibodies labelled with an enzyme are added Iii) After washing, any bound secondary antibodies can be detected using the marker. The label used is an enzyme which induces a colour change when the substrate is added.
  • BioChip Applications
  • Electrophoretic Flow (EF)
  • Electro-Osmotic Flow (EOF)
  • Working Principle of CE
  • Capillary Electrophoresis (CE) Schematic representation of the arrangement of the main components of a typical Capillary Electrophoresis (CE) instrument.
  • Capillary Electrophoresis
  • Capillary Electrophoresis (CE)
  • Capilary Electrophoresis - Design Consideration
  • Some Issues about CE
  • Gel Electrophoresis
  • Gel Electrophoresis Gel Electrophoresis is a technique used to separate macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used technique in Molecular Biology. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. Proteins, which can have either a net positive or net negative charge Nucleic acids have a consistent negative charge and migrate toward the anode. Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab.. The gel is immersed within an electrophoresis buffer. The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks.
  • Agarose Concentration Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.
  • Gel Electrophoresis Apparatus (e.g. forensic investigations)
  • Gel Electrophoresis In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix.
  • First commercial system : Agilent 2100 Bioanalyzer Agilent Technologies, Waldbronn, Germany Caliper Technologies, Mountain View, CA
  • Lab-on-a-Chip Products  Cutting edge Lab-on-a-Chip Products Agilent Technologies is the leader in commercial microfluidic Lab-on-a-Chip technology. This technology utilizes a network of channels and wells that are etched onto glass or polymer chips to build mini-labs. Pressure or electrokinetic forces move pico-liter volumes in finely controlled manner through the channels. Lab-on-a-Chip enables sample handling, mixing, dilution, electrophoresis and chromatographic separation, staining and detection on single integrated systems. The main advantages of Lab-on-a-Chip are ease-of-use, speed of analysis, low sample and reagent consumption and high reproducibility due to standardization and automation.
  • Applications of Electrophoresis
  • Chromatography Family of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a “stationary phase”, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column).
  • Schematic Diagram of Liquid Chromatography
  • echniques by physical state of mobile phase Affinity chromatography It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained. Size exclusion chromatography Size exclusion chromatography separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to enter the pores of the media and, therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster.
  • Schematic of pore vs. analyte size
  • Chromatogram of Orange Juice Compounds
  • High Performance Liquid Chromatography
  • Electrophoresis vs. Chromatography
  • Polymerase Chain Reaction (PCR)
  • How PCR is done ?
  • How PCR is done ? Liquid-Phase PCR Reactors : Three different phases: denaturing, hybridization, extension (1)Denaturing : A heating temperature above 90°- 95°C breaks adouble-stranded DNA molecule into two complementary singlestranded DNA molecules. (2) Hybridization : The single-stranded DNA molecules is cooled at a lower temperature 50°C - 60°C and seek their complementary strands to create double-stranded DNA molecules. (3) Extension : The incomplete double-stranded DNA molecules are extended with the help of an enzyme called DNA polymerase. It occurs at a temperature of 70° - 75°C.
  • PCR Thermal Steps
  • PCR Thermal Steps
  • Polymerase Chain Reaction Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA.
  • PCR Thermocycler Instruments
  • PCR - Miniaturization
  • Integrated PCR and detection microsystem
  • Channel size: 40 μm×90 μm×2.2 m ⇒ The sample is forced to through three temperature zones with a constant velocity. ⇒ The cycle time is proportional to the capillary length. ⇒ The process only depends on the speed of the fluid flow and not on the thermal constant of the system. ⇒ Relatively fast cycling. Continuous Flow - PCR Miniaturisation
  • Continuous Flow PCR - Miniaturisation
  • PCR + CE Chip
  • Cell Sorter
  • Schematic of a modern conventional FACS
  • icrofabricated fluorescence-activate cell sorter (µFACS)
  • Principle of confocal Microscopy
  • Fluorescence microscope
  • THANK YOU FOR YOUR ATTENTION Any question ?