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    CHM4400 Advanced Analytical Chemistry: Microchip and Microarray

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    Koon Sing Chan
    The document discusses microarrays and describes their components and uses. Microarrays are small analytical chips composed of spots containing target molecules like DNA, RNA, proteins or small molecules. Each spot represents a collection of target molecules that bind specifically to labeled probe molecules, allowing detection of binding events. DNA microarrays allow large-scale gene analysis and are commonly used to study gene expression and find mutations. Protein and small-molecule microarrays have also been developed for various applications.

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    CHM4400 Advanced Analytical Chemistry: Microchip and Microarray

    Uploaded by

    Koon Sing Chan
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    The document discusses microarrays and describes their components and uses. Microarrays are small analytical chips composed of spots containing target molecules like DNA, RNA, proteins or small molecules. Each spot represents a collection of target molecules that bind specifically to labeled probe molecules, allowing detection of binding events. DNA microarrays allow large-scale gene analysis and are commonly used to study gene expression and find mutations. Protein and small-molecule microarrays have also been developed for various applications.

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    The document discusses microarrays and describes their components and uses. Microarrays are small analytical chips composed of spots containing target molecules like DNA, RNA, proteins or small molecules. Each spot represents a collection of target molecules that bind specifically to labeled probe molecules, allowing detection of binding events. DNA microarrays allow large-scale gene analysis and are commonly used to study gene expression and find mutations. Protein and small-molecule microarrays have also been developed for various applications.

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    31 views29 pages

    CHM4400 Advanced Analytical Chemistry: Microchip and Microarray

    Uploaded by

    Koon Sing Chan
    The document discusses microarrays and describes their components and uses. Microarrays are small analytical chips composed of spots containing target molecules like DNA, RNA, proteins or small molecules. Each spot represents a collection of target molecules that bind specifically to labeled probe molecules, allowing detection of binding events. DNA microarrays allow large-scale gene analysis and are commonly used to study gene expression and find mutations. Protein and small-molecule microarrays have also been developed for various applications.

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    CHM4400

    Advanced Analytical Chemistry

    Microchip and Microarray


    Bo Zheng
    bozheng@cuhk.edu.hk
    Science Center Rm. 165
    Microarray

    Microarray is a small analytical chip, which is used to


    analyze biological or chemical problems.

    Usually microarray is composed of a piece of glass with


    arrays of spots on it. The diameter of the spots is smaller
    than 1.0 mm, usually between 10 and 500 mm.

    Each spot in the microarray represents a collection of


    target molecules. The target molecules can be DNA, RNA,
    protein, or small molecules. F
    probe
    Target molecules bind specifically to probe molecules in
    solution. The probe molecules are usually labeled for
    target
    detection of the binding events.

    The most often used microarrays are DNA microarrays.


    Protein microarrays and small-molecule microarrays are
    also developed and used in many application.
    Structure of Nucleic Acids
    Bases in Nucleic Acid
    Chemical Structure of RNA and DNA
    Hydrogen-Bonding Patterns in the Base Pairs

    The sequence of the DNA determines the


    sequence of the corresponding protein.
    Why Is DNA Important
    Chromosome, Gene, and DNA

    Chromosome is a large DNA-


    protein complex. Each
    chromosome consists of a
    single, extremely long linear
    DNA molecule associated with
    protein that fold and pack the
    DNA into a compact structure.

    Gene is a segment of DNA


    coding for a single protein.

    The most important function of


    DNA is to carry genes, the
    information that specifies all the
    proteins that make up an
    organism, including information
    of when, in what types of cells,
    and in what quantity each
    protein is to be synthesized.
    Why Is DNA Important

    For a cell to make protein, the information from a gene is copied,


    base by base, from DNA into new strands of messenger RNA
    (mRNA). Then mRNA travels out of the nucleus into the cytoplasm,
    to cell organelles called ribosomes. There, mRNA directs the
    assembly of amino acids that fold into completed protein molecule.
    DNA Microarray

    Target DNA with different sequences fluorescent label

    Probe DNA that


    binds to one of
    the targets

    DNA microarray allows large scale gene exploration with high speed and
    precision.
    The usual applications include analysis of gene expression related to disease,
    ageing, drug action, diet, and etc. DNA microarrays is also used to find mutations
    in gene sequences for gene testing and diagnostics.
    DNA Microarray
    DNA Microarray Fabrication
    Liquid Dispenser for Preparation of DNA Microarray

    A piezoelectric microarrayer spots DNA on functionalized glass.


    During contact printing, sample is held in the pin by the combined forces of
    adhesion (pin-liquid and surface-liquid) and surface tension. On such small
    length scale, gravity is a minor downward force.
    DNA Fixation on Surface

    1) Surface pretreatment

    2) DNA fixation
    DNA Fixation on Surface

    1) DNA fixation by forming Schiff base

    2) Strengthen the fixation by


    reduction through NaBH4
    Oligonucleotide Synthesis by Phosphoramidite
    Chemistry

    (dimethoxytrityl)

    CPG: controlled pore glass


    Oligonucleotide Synthesis by Phosphoramidite
    Chemistry
    DNA Microarray Prepared by Photolithography

    (methylnitropiperonyloxy
    carbonyl)
    DNA Microarray by Photolithography

    A glass substrate modified with photoprotecting groups is aligned with a checkerboard


    photomask that contains a Cr coating (black) interspersed with transparent regions.
    If the mask is exposed to UV light, select regions of the chip become deprotected and
    active for chemical coupling. The activated chip is flooded with a reactive DNA base,
    allowing coupling to selective regions defined by the photomask.
    Scanner for Analyzing the Signal of DNA Microarray

    1”x3” glass slide


    Scanner for Analyzing the Signal of DNA Microarray
    Isolate mRNAs from both
    normal and cancerous
    hepatocells; each mRNA
    sample represents all the
    genes expressed in the cells
    at that stage.

    Detection of
    Differentially expressed
    genes in hepatocellular
    carcinoma using DNA
    microarray
    Detection Results of the DNA Microarray

    Normal hepatocell

    Cancerous hepatocell

    Y. Li, et al Yi Chuan Xue Bao, 2000, 27, 1042


    Protein Microarray
    Polypeptides:

    O O O O O
    A: +H3N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-O-
    R1 R2 R3 R4 R5

    B: +H3N-Asp-Glu-Pro-Gly-Ser-COO-

    Proteins are directly involved in all aspects of cell activities. Protein


    microarray allows researchers to study proteins on a much greater scale.

    Depending on the purpose, protein microarray can be classified into two


    groups: (1) Quantitative microarray. Similar to DNA microarrays, the
    purpose of quantitative protein microarrays is to measure levels of protein
    expression in a cell or tissue; (2) Functional microarray. The purpose is to
    quickly probe the activity of a given protein against many targets
    simultaneously.
    Protein Microarray Formats
    Target element Synthesis Applications Comments

    Synthetic peptides Solid phase Epitope analysis, folding Easy to manufacture and purify, amenable to
    (10-80 amino acids) chemical synthesis studies, diagnostics chemical modifications and mass spec
    analysis, and scalable to kg quantities

    Recombinant proteins Expression in Proteomic studies of Best way to study diversity of protein
    (100-4000 amino recombinant host protein-protein binding, functions though each protein possesses
    acids) cells or production protein-drug binding, and idiosyncratic biochemical properties,
    in vitro enzyme-substrate including differences in solubility and stability
    specificity
    Monoclonal Hybridoma Protein expression Excellent biochemical stability and binding
    antibodies technology in profiling and antigen specificity, can be produced in unlimited
    cultured cell lines detection for serum-based quantity but relatively expensive to prepare
    diagnostics and screen
    Polyclonal antibodies Immunization and Same as above Excellent biochemical stability and binding
    isolation from affinity but requires a relatively large amount
    injected animals of antigen, supply is limited to injected animal,
    and can exhibit cross-reactivity
    Protein Microarray

    Protein microarrays use the same fundamental concepts as DNA


    microarrays, i.e., five-step assay cycle (biological question, sample
    preparation, biochemical reaction, detection, and data analysis and
    modeling).

    A major difference between DNA and protein microarray pertains to the


    structure of the target and probe molecules. Denaturation of protein must
    be prevented.

    Most protein microarrays can only be synthesized using contact printing or


    other delivery techniques. Adding one amino acid residue per cycle to each
    element of a peptide array would require 20 cycles of wet chemistry, and
    many more photomasks will be needed. The cost of in situ synthesis makes
    this method not feasible..
    Protein Microarrays Fabrication

    1. aldehyde-coated glass

    2. Ni-NTA
    coated glass
    Protein Microarrays Fabrication

    3. self-assembled x=
    monolayer on gold biotin

    N-hydroxysuccinimide (NHS) esters

    epoxy

    succinimide
    Small-Molecule Microarrays

    (i) Incubate with labeled binding


    <10,000 compounds partner
    immobilized on solid support

    (ii) Fluorescence
    microscopy to detect
    binding partners
    Different small
    molecules attached to
    the surface

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