[Pracha] Seminar in Ph.D.

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[Pracha] Seminar in Ph.D.

  1. 1. SCCH 656 Current Topics in Analytical Chemistry I<br />Doctor of Philosophy Program in Analytical Chemistry<br />Metallic Barcodes <br />for Multiplexed Bioassays<br />Presented by <br />PrachaCheajesadagul<br />Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand<br />
  2. 2. Outline<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  3. 3. Introduction to Metallic Barcodes<br />Why Barcodes?<br />EAN barcode<br />QR Code<br />UPC barcode<br />Representation / <br />Identification of data<br />Figure 1. Barcode with Parallel line pattern <br />http://en.wikipedia.org<br />http://www.nytimes.com/2009/06/26/technology/26barcode.html?_r=1<br />SCCH 656 Current Topics in Analytical Chemistry I<br />3<br />
  4. 4. Introduction to Metallic Barcodes<br />Metallic Barcodes<br />Metallic barcodes are cylindrical particles with stripes of Au, Ag and/or other metals along their length.<br />Ni<br />Ag<br />Examples of metals <br />for striping pattern<br />Pd<br />Au<br />Figure 3. SEM image of the Au-Ag-Au metallic barcode<br />Pt<br />Co<br />Cu<br />Figure 2. Schematic image demonstrating range of <br />possible dimensions and one possible striping pattern <br />Science 294, 137, 2001<br />Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2010<br />SCCH 656 Current Topics in Analytical Chemistry I<br />4<br />
  5. 5. Synthesis of Metallic Barcodes<br />Templated sequential electrodeposition<br />Au – Ag - Au<br />Membrane template<br />(Alumina or polycarbonate)<br />Pore<br />3. Au+ + e- Au0 3x Coulomb <br />2. Ag+ + e- Ag0 2x Coulomb <br />1. Au+ + e- Au0 x Coulomb <br />Metal film <br />served as the working electrode for reduction of metal ions from solution<br />SCCH 656 Current Topics in Analytical Chemistry I<br />5<br />
  6. 6. Synthesis of Metallic Barcodes<br />Templated sequential electrodeposition<br />HNO3<br />NaOH<br />1. Metal film dissolution with HNO3<br />2. Template dissolution with NaOH<br />Metallic barcodes <br />SCCH 656 Current Topics in Analytical Chemistry I<br />6<br />
  7. 7. Synthesis of Metallic Barcodes<br />The structure of metallic barcode particles is controlled by<br />SCCH 656 Current Topics in Analytical Chemistry I<br />7<br />
  8. 8. Optical Readout of Metallic Barcode <br />The readout of stripe pattern on metallic barcode<br />depend on different reflectivity properties of metal.<br />Ag<br />600 nm<br />Au<br />430 nm<br />Ag<br />Ag<br />Au<br />Ag<br />Figure 5. Optical image of Ag-Au-Ag barcode particle <br />at two wavelengths.<br />Figure 4. The wavelength-dependent <br />reflectance for the bulk metal<br />Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141<br />SCCH 656 Current Topics in Analytical Chemistry I<br />8<br />
  9. 9. Stripe Patterning<br />The optical reflectivity between Au and Ag metal <br />is very different !! (especially at ~430 nm)<br />Ag stripe = 1<br />Au stripe = 0<br />SCCH 656 Current Topics in Analytical Chemistry I<br />9<br />
  10. 10. Advantages of Metallic Barcode<br />The ability to be functionalized with biological probe molecules <br />is possible for detection of specific analytes<br />SCCH 656 Current Topics in Analytical Chemistry I<br />10<br />
  11. 11. Surface Functionalization<br />Targets<br />DNA targets<br />Protein target<br />Molecular <br />beacon<br />Antibody<br />Oligonucleotide<br />Biological-probe molecules<br />s<br />Metallic barcode particle as an substrate<br />SCCH 656 Current Topics in Analytical Chemistry I<br />11<br />
  12. 12. Multiplexed Bioassays<br />A procedure that simultaneously measures multiple analyte<br />(such as proteins, nucleic acids) in a single assay within a biological sample.<br />SCCH 656 Current Topics in Analytical Chemistry I<br />12<br />
  13. 13. Multiplexed Bioassays<br />SCCH 656 Current Topics in Analytical Chemistry I<br />13<br />
  14. 14. Application I<br />Multiplexed Immunoassay<br />SCCH 656 Current Topics in Analytical Chemistry I<br />14<br />
  15. 15. Application I<br />Cancer by Types<br />Figure 6. Cancer by types<br />http://www.metrothinkpink.co.uk/think/thinkhealth_guide.html<br />SCCH 656 Current Topics in Analytical Chemistry I<br />15<br />
  16. 16. Application I<br />Protein Cancer Markers<br />Prostate cancer marker<br />Colorectal cancer marker<br />Testicular cancer marker<br />http://www.alternative-cancer.net/Cell_photos.htm<br />SCCH 656 Current Topics in Analytical Chemistry I<br />16<br />
  17. 17. Application I<br />Cancer Detection<br /><br />Well accepted for clinical work<br /><br />Not used for the detection <br />of many targets in a single sample <br /><br />Provide genome-level multiplexing<br /><br />Require the acquisition of dedicated<br /> instrumentation<br />SCCH 656 Current Topics in Analytical Chemistry I<br />17<br />
  18. 18. Application I<br />Immobilization <br />functionalized with <br />Antibody Probes<br />CEA-specific probes<br />PSA-specific probes<br />βhCG-specific probes<br />Metallic barcode pattern<br />(000100)<br />Metallic barcode pattern<br />(01010)<br />Metallic barcode pattern<br />(011110)<br />Figure 7. Illustration of metallic barcodes functionalized with antibody probes, patterned 000100 (left), 01010 (middle), and 011110 (right) are coated with antibody probes for PSA, CEA and βhCG, respectively<br />SCCH 656 Current Topics in Analytical Chemistry I<br />18<br />
  19. 19. Application I<br />Detection of Protein Cancer Markers<br />PSA-specific metallic barcode<br />505 nm<br />423 nm<br />Non-specific <br />protein<br />PSA target<br />protein<br />2nd antibody-(dsDNA)n bound <br />fluorescent-conjugated polymer <br />Scheme 1. A schematic of overall detection strategy of protein cancer detection on metallic barcode.<br />SCCH 656 Current Topics in Analytical Chemistry I<br />19<br />
  20. 20. Application I<br />Protein Detection: PSA<br />Reflectance<br />Fluorescence<br />Nonspecific <br />protein <br />PSA-specific <br />metallic barcode<br />Target <br />protein PSA <br />Figure 7.  Reflectance (a, c) and fluorescence images (b, d) of PSA-specific metallic barcode at the presence of target protein PSA (c, d) and nonspecific protein BSA (a, b). Scale bars in the images = 5 μm<br />SCCH 656 Current Topics in Analytical Chemistry I<br />20<br />
  21. 21. Application I<br />Protein Detection: PSA<br />Fluorescence<br />Figure 8.  a Fluorescence images of PSA-specific metallic barcode incubated with PSA cancer marker at various concentration ranging from 0 to 10,000 ng/mL(a–g; scale bar = 5 μm). b The quantitative plot of fluorescence signal intensity against the concentration of PSA. The straight line is a linear fitting of the data collected between 0.1 and 1,000 ng/mL. The control sample where PSA was absent was plotted in red<br />SCCH 656 Current Topics in Analytical Chemistry I<br />21<br />
  22. 22. Application I<br />Multiplexed<br />Reflectance<br />Fluorescence<br />No cancer <br />marker proteins<br />βhCG only<br />CEA and βhCG<br />All three cancer <br />marker proteins<br />Figure 9. Corresponding reflectance (a–d) and fluorescence (e–h) images of the mixture of three antibody-bound metallic barcodes. The concentration of each target protein was kept constant at 100 ng/mLin all assays (scale bars = 5 μm). <br />SCCH 656 Current Topics in Analytical Chemistry I<br />22<br />
  23. 23. Application I<br />Multiplexed Detection<br />Figure 10. A quantitative plot between the fluorescent intensity from each type of the metallic barcodes in different sample solutions. Cancer marker proteins present in the sample solution were labeled inx-axis, and the corresponding fluorescence readouts were recorded in y-axis. The color/pattern of the columns corresponded to the capture antibodies immobilized on different metallic barcodes were specified in the legend<br />SCCH 656 Current Topics in Analytical Chemistry I<br />23<br />
  24. 24. Application I<br />Summary: Multiplexed Immunoassay<br />SCCH 656 Current Topics in Analytical Chemistry I<br />24<br />
  25. 25. Application II<br />Multiplexed Nucleic Acid Detection<br />SCCH 656 Current Topics in Analytical Chemistry I<br />25<br />
  26. 26. Application II<br />Viral Infections<br />SARS<br />(Severe Acute <br />Respiratory Syndrome)<br />HCV <br />(Hepatitis C Virus)<br />HIV<br />(Human Immunodeficiency Virus)<br />Figure 11. <br />Viral infections<br />http://en.wikipedia.org<br />SCCH 656 Current Topics in Analytical Chemistry I<br />26<br />
  27. 27. Application II<br />Immobilization<br />functionalized with <br />Molecular Beacon Probes<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />HIV-specific probes<br />SARS-specific probes<br />HCV-specific probes<br />Metallic barcode pattern<br />(00001)<br />Metallic barcode pattern<br />(00100)<br />Metallic barcode pattern<br />(00010)<br />Figure 12. Illustration of different patterns of metallic barcode functionalized with molecular beacon probes, patterned 00001 (left), 00100 (middle), and 00010 (right) are coated with MB probes SARS, HIV, and HCV, respectively<br />SCCH 656 Current Topics in Analytical Chemistry I<br />27<br />
  28. 28. Application II<br />Molecular Beacon<br /><ul><li>Oligonucleotide probes molecule 
  29. 29. Can provide nucleic acid detection
  30. 30. Simplifies assay performance and greatly reduces contamination risk </li></ul>Fluorescence signal (real-time)<br />Figure 13. Molecular beacon<br />J. AM. CHEM. SOC. 2006, 128, 16892-16903<br />http://www.cellscience.com/reviews6/Molecular_beacons.html<br />SCCH 656 Current Topics in Analytical Chemistry I<br />28<br />
  31. 31. Application II<br />Detection of Nucleic Acid Target<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />s<br />SARS-target DNA<br />Fluorescence !!<br />Without HCV-target DNA<br />HIV-target DNA<br />No changing<br />Scheme 2. Illustration of multiplexed detection of nucleic acid targets by metallic barcodes functionalized with molecular beacon probes and complementary target sequences have been added for SARS and HIV only<br />SCCH 656 Current Topics in Analytical Chemistry I<br />29<br />
  32. 32. Application II<br />Multiplexd Detection of Viral DNA<br />Reflectance<br />Fluorescence<br />SARS-specific probes<br />(00001)<br />(00100)<br />HCV-specific probes<br />(00010)<br />(00010)<br />HIV-specific probes<br />(00100)<br />(00001)<br />Figure 14. The reflectance image (left) show the barcode pattern and the fluorescence image (right) show which pathogenic oligonucleotide sequences are present<br />SCCH 656 Current Topics in Analytical Chemistry I<br />30<br />
  33. 33. Application II<br />Multiplexd Detection of Viral DNA<br />Figure 15. Multiplexed detection of viral DNA using molecular beacons on metallic barcodes<br />SCCH 656 Current Topics in Analytical Chemistry I<br />31<br />
  34. 34. Application II<br />Preservation of Assays<br />Figure 16. Triplex beacon assay using wires pre-coated in beacons and stored in citrate buffer for various numbers of days. It shows target versus no target data for days of storage up to 110 days. Intensities for all three probes on each day have been normalized to the HIV intensity at day 0.<br />SCCH 656 Current Topics in Analytical Chemistry I<br />32<br />
  35. 35. Summary: Multiplexed Nucleic Acid Detection <br />SCCH 656 Current Topics in Analytical Chemistry I<br />33<br />
  36. 36. Conclusions<br />SCCH 656 Current Topics in Analytical Chemistry I<br />34<br />
  37. 37. References<br />SCCH 656 Current Topics in Analytical Chemistry I<br />35<br />
  38. 38. Thank you <br />for your attention<br />
  39. 39. Nanoparticles for Bioassays<br />(d) Encoded substrates<br />(a) Quantitation tags<br />(b) Signal transducers<br />(c) Functional tags<br />Fig. 1  Four formats in which nanoparticles can be divided, in the field of bioanalysis.<br />Current Opinion in Chemical Biology 2003, 7:609–615<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  40. 40. Optical Properties of Cylindrical Striped Particles<br />Optical properties of cylindrical striped particles. (A) FE-SEM image (left) and optical microscope image (right) of the same Au-Ag-Au particle. Contrast in the FE-SEM image results from differences in backscattered electron intensity from the two metals. [A reflective Au background is used in (A) but not in the other optical images here.] (B) Upper panel: Wavelength dependence of reflectivity for bulk metals [this graph was generated from values reported in (17)]. Lower panel: Ratio of reflected intensities for various metals versus Au at 430 nm for bulk materials (open triangles) and for striped particles. For the latter, values were experimentally determined using reflectance optical microscopy. (C) Reflectance optical microscopy image of an Ag-Au-Ag barcode rod (length ~10 µm). Top: High contrast was observed between Ag (brighter sections) and Au (dark middle section) with 430-nm illumination. Bottom: No contrast using 600-nm excitation. (D) Reflectance optical microscopy images and line profiles for a particle of composition Au-Ag-Ni-Pd-Pt with illumination at 430 nm, 520 nm, and 600 nm, respectively. Samples were mounted on glass slides and imaged with a Nikon TE-300 inverted microscope equipped with a bright-field reflectance filter set (containing a 50/50 beam splitter) using a 100× oil immersion lens (NA = 1.3). A 100-W Hg lamp with a 430-, 520-, or 600-nm (±10 nm) bandpass filter was used for excitation, as indicated. All particles shown here and in subsequent figures were prepared by electrodeposition in the pores of 0.2-µm (11) Anodisc (Whatman) alumina membranes (8-10).<br />Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  41. 41. Optical Properties of Cylindrical Striped Particles<br />Nanobarcodes particles (NBC). (A) Schematic image demonstrating range of possible dimensions and one possible striping pattern. (B) Optical microscope image of a single particle. The particle contains alternating sections of Ag and Au and is 6.3 μm in length. Ag is the brighter material at this wavelength (405 nm). The apparent difference in the thickness of the Ag and Au stripes is due to the difference in brightness, not to a true difference in particle diameter.<br />Wavelength dependence of reflectivity for bulk metals<br />Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141<br />Anal. Chem., 2002, 74 (10), pp 2240–2247<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  42. 42. Optical Reflectivity and Patterned Fluorescence<br />Figure… Fluorescence Patterning on Metallic Barcodes<br />J. Phys. Chem. B, 2003, 107 (30), pp 7360–7367<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  43. 43. Multiplexed Detection<br />Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  44. 44. Strategies for Particle Encoding<br />Solution-phase synthesis<br />Templated synthesis<br />Lithographic particle <br />fabrication<br />Encoded anisotropic particles for multiplexed bioanalysis. <br />Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2010<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  45. 45. Synthesis of Barcoded Particles<br />Nature Biotechnology 19, 1122 - 1123 (2001)<br />Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  46. 46. Nanorod / Nanowire<br />Nanorods:  Each of their dimensions range from 1–100 nm. <br /> Synthesized from metals or semiconducting materials. <br /> Standard aspect ratios (length divided by width) are 3-5.<br />Nanowires  Exhibit aspect ratios (length-to-width ratio) of 1000 or more. <br /> Often referred to as 1-Dimensional materials. <br />Nanowires have many interesting properties that are not seen in bulk or 3-D materials. This is because electrons in nanowires are quantum confined laterally and thus occupy energy levels that are different from the traditional continuum of energy levels or bands found in bulk materials.<br />Many techniques have been developed to synthesize these structures and can be grouped into four categories:<br /> 1) Spontaneous growth<br /> 2) Template synthesis<br /> 3) Electrospinning<br /> 4) Lithography<br />http://www.nanogallery.eu/nanotopics/10-nanorod-nanowire.html<br />http://www.engr.colostate.edu/ECE581/fall07/Nanowires%20and%20Nanorods.pdf<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  47. 47. Electrochemistry<br />Metallic elements, including several non-noble metals, sorted by their chemical "nobility" (noble metals bolded):<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />http://en.wikipedia.org<br />
  48. 48. Template based Synthesis<br />- Involves an electrolysis process <br /> that results in the deposition of solid material in an electrode<br />The most commonly used - Anodized alumina membranes<br /> - Radiation track etched polymer membranes/mica <br /> - Nanochannel array glass<br /> - Mosoporous materials<br />Cross section of anodized Al<br />Al membrane<br />450 nm pores in glass<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  49. 49. Electrochemical Deposition<br />(also knows as electrodeposition , electroplating, cathode deposition)<br />- Involves an electrolysis process that results in the deposition of solid material in an electrode<br />E0 = the electrode potential <br />ai = the activity of the ions <br />Rg = the gas constant <br />F = the Faraday’s constant <br />T = the temperature<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  50. 50. Electrochemical Deposition<br />A typical electrolytic process composes <br />of a series of steps:<br />a- Mass transfer through the solution <br />from one electrode to another<br />b- Chemical reactions at the interfaces between electrode-solution<br />c- Electrons transfer at the electrode surfaces and through the external circuit<br />d- Other surface reactions such as adsorption, desorption or recrystallization<br />An experimental setup for electrochemical deposition<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  51. 51. Optical Readout of Metallic Barcode <br />430 nm<br />520 nm<br />600 nm<br />Ratio of reflected intensities for various metals versus Au <br />Reflectance optical microscopy images<br />Line profiles of <br />each particle<br />SCCH 656 Current Topics in Analytical Chemistry I<br />Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141<br />2<br />
  52. 52. Decoding the Metal Striping Pattern<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  53. 53. No. of Striping Patterns<br />Anal. Chem., 2002, 74 (10), pp 2240–2247<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  54. 54. Barcoded Segment Measurements<br />Anal. Chem., 2002, 74 (10), pp 2240–2247<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  55. 55. Florophore (Fluorescent Dye)<br />Fluorescein<br />isothiocyanate (FITC)<br />Texas Red<br />http://info.med.yale.edu/genetics/ward/tavi/FISHdyes2.html<br />http://en.wikipedia.org<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  56. 56. Cationic Fluorescent Conjugated Polymer<br />Polythiophene derivative<br />{poly(1H-imidazolium-1-methyl-3-{2-[(4-methyl-3-thienyl)oxy]ethyl}bromide)}<br />J. Am. Chem. Soc., 2009, 131 (10), pp 3432–3433<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  57. 57. Applications I : Protein Detection <br />Fig. 1  A schematic illustration of protein detection on Au/Ag-barcodednanorods using fluorescent-conjugated polymers. <br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  58. 58. Cationic Fluorescent Conjugated Polymer<br />Figure…Conjugated π-orbitals of a coplanar and a twisted substituted polythiophene.<br />http://en.wikipedia.org<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  59. 59. Applications I : Polymer Concentration<br />Without<br />PSA<br />1 μg/mL<br />of PSA<br />25 μM<br />50 μM<br />10 μM <br />Concentration of conjugated polymers<br />Fig. 2 (Top) Fluorescence images of anti-PSA-bound nanorods incubated with different concentrations of conjugated polymers in the absence (a–c) and the presence (d–f) of 1 μg/mL of PSA. Scale bar=5 μm. (Bottom) A quantitative plot of fluorescence intensities (counts/pixel) as a function of polymer concentration. The image labels were placed atop the corresponding columns<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  60. 60. Applications I : Simultaneous Detection <br />Reflectance<br />Fluorescence<br />incubated in a solution containing PSA, CEA, and βhCG of 10, 100, and 1,000 ng/mL<br />Fig. 5 (Top) Simultaneous detection of three cancer markers at different concentrations. A mixture of three antibody-bound nanorods was incubated in a solution containing PSA, CEA, and βhCG of 10, 100, and 1,000 ng/mL, respectively. particle patterns were labeled in the legend of panel B. (Bottom) Corresponding fluorescence intensities calculated from each type of particles corresponding to different cancer markers<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  61. 61. Applications I : Nanowires in Different Solutions<br />in bovine serum <br />in PBS buffer<br />in bovine serum <br />containing 10 ng/mL PSA<br />in bovine serum <br />containing 1 ng/mL PSA<br />Fig. 6 (Left) Fluorescence images of anti-PSA-coated nanowires incubated in different solutions. Scale bars = 5 μm. (Right) Corresponding fluorescence readouts from the nanowires in different solutions, as specified in the top panel<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  62. 62. DNA Microarray<br />http://www.medscape.com<br />http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/work.htm<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  63. 63. Bioassays<br /> A method for the quantitative estimation of the effects that result in a biological system after its exposure to a substance<br />DNA microarray <br />relying on a two-color fluorescent dye system for hybridization elucidation <br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  64. 64. Enzyme-linked Immunosorbent Assay<br />http://www.51protocol.com/<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  65. 65. Surface-Based Bioassays<br />DNA microarray <br />relying on a two-color fluorescent dye system for hybridization elucidation <br />Planar array with multicolored<br />nanoparticle tags (i.e. QDs)<br />For hybridization determination instead of organic dye<br />Suspension array<br />where the encoding element, <br />is nanoparticle shape <br />http://wires.wiley.com/WileyCDA/WiresArticle/wisId-WNAN96.html<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  66. 66. Multiplexed Assays<br />A type of laboratory procedure that simultaneously measures multiple analytes in a single assay<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  67. 67. Nucleotides<br />Oligonucleotide (from Greek prefix oligo-, "having few, having little") <br />a short nucleic acid polymer, typically with twenty or fewer bases. <br />http://en.wikipedia.org<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  68. 68. Probe Sequences<br />Table 1. Probe Sequences Used in This Work<br />* The italic portions of the sequences indicate complementary stem regions<br />* TAMRA = florophore (tetramethylrhodamine)<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  69. 69. Multiplexd Detection of Viral DNA<br />Figure 2. Effect of loop length on fluorescence intensity for molecular beacon probes bound to Ag/Au striped nanowires in the presence and absence of complementary target strands. Stem length was held constant at 5 base pairs. Error bars are the 95% confidence interval.<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  70. 70. Multiplexd Detection of Viral DNA<br />Figure 3. (A) Effect of stem length on fluorescence intensity for molecular beacon probes bound to Ag/Au striped nanowires in the presence and absence of complementary target strands. Hybridization was performed at 25 °C in 500 mMNaCl CAC buffer. Loop length was held constant at 21 bases. Error bars are 95% confidence intervals. (B) Effect of hybridization temperature on quenching efficiency for four stem lengths. Lines connecting the points are present only to guide the eye.<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  71. 71. Multiplexd Detection of Viral DNA<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  72. 72. Antibodies Immobilization<br />Figure 2. Monoclonal antibodies immobilized on silica fiber-optic probes via five different strategies. Antibodies were randomly oriented on the surface via their primary amine groups<br />Anal. Chem., 2001, 73 (3), pp 471–480<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  73. 73. Adsorption Isotherm<br />To obtain more useful data, it was necessary to measure the adsorption isotherms. <br />Adsorption experiments data may be fitted to Langmuir, Sips, and Toth isotherms.<br />J. Chem. Eng. Data, 2006, 51 (2), pp 451–456<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  74. 74. Binding Isotherm<br />Fractional coverage<br />estimated on the basis of the fluorescence intensity as compared to the maximum intensity<br />Limit of detection<br />calculated by taking the average fluorescence<br />intensity for the control (background signal) and adding 2 times its standard deviation<br />- mean fluorescence units translates a concentration of <100 pM<br />- with dynamic range of 3-4 orders of magnitude in concentration<br />Figure… Binding isotherm for molecular beacon probes on barcodednanowires<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />
  75. 75. Sips Isotherm<br /> - Used to assess the heterogeneity in the Target−Probe binding in solution<br /> - Antigen−Antibody probe<br /> - DNA−Molecular beacon probe<br /><ul><li> Relates the fractional coverage of antibody binding sites to the concentration of antigen in solution.</li></ul>f = the fractional coverage of target binding sites on the probes bound on the substrate <br />K= the average equilibrium constant for adsorption <br />C = the concentration of target in solution<br />a = the heterogeneity index<br /> If a = 1 the antigen-antibody binding affinity is described by a single value and the antibodies are homogeneous<br /> If a < 1 indicate a wider affinity distribution and thus increasing heterogeneity<br />Anal. Chem., 2001, 73 (3), pp 471–480<br />SCCH 656 Current Topics in Analytical Chemistry I<br />2<br />

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