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[Pracha] Seminar Ph.D.
 

[Pracha] Seminar Ph.D.

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"Metallic Barcodes for Multiplexed Bioassays" Seminar in 2010

"Metallic Barcodes for Multiplexed Bioassays" Seminar in 2010

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  • Thank you,chairman for kind introductionAndGd afternoon, teachers and all audiencesToday, I will give the seminar in the topic ofMetallic barcodes for multiplexed bioassays,
  • For this seminar, I will talk aboutWhat’s the Metallic Barcodes,How they can be synthesized and readout,Surface functionalizationandapplications for Multiplexed Bioassays will be presentedand finally, conclusion
  • Why must be barcode?I think everyone, has ever seen the barcode. Barcode is a symbol that represents somedata in the parallel line pattern or square pattern.The data in barcode can be easily and rapidly identified By using optical scanner.
  • In the field of science, the barcode pattern can be made from some metalsSuch as gold, silver, nickel, copper, palladium, platinum or cobalt.So they can be called “Metallic barcodes”Metallic barcodes are cylindrical particles with stripes of metals along the length.They are several micrometersin length, and thirty-to-three hundred nanometer in diameter, The right image shows appearanceof Au-Ag-Au metallicbarcodefrom Scanning ElectronMicroscope.
  • Metallic barcodes can be synthesized by using templated sequential electrodepositionMetal ions of interestwere reduced and deposited into the pores of alumina or polycarbonate as membrane template.The membrane template is coated on metal film served as working electrode for reduction of metal ions.For example, if we want to prepare the metallic barcode pattern Such a Au-Ag-Au pattern like this.The template will be immersed in the gold solution and amount of current will be applied into the solution.Gold ion will be reduced to gold zero And deposited into the pores of template. After that, the solution will be changed into silver solution and gold solution in order and also apply the current for silver deposition and gold deposition, respectively
  • Then, the metal film and membrane template will be dissolvedby usingNitric acid and sodium hydroxide.Finally, the metallic barcode such Au-Ag-Au pattern will be obtained.
  • The structure of metallic barcode can be controlled by three factors.The first is membrane pore size, To control the width of metallic barcodeThe second is sequence of metal ions introduced into solution, To defines the number and pattern of metallic barcode; and the last is Amount of current applied in each step, To control the length of metallic barcode
  • The readout of stripepattern on metallic barcodedepend on different reflectivity properties of metal.The left figure shows the wavelength-dependent reflectance for the bulk metal.You can see the different reflectivity properties of various metals, As could be seen, at 430 nm, reflectivity of silver is higher than gold about 2.5 timesWhereas, at 600 nmthey are equivalent.From this property,the stripe pattern on metallic barcode can be visualized by optical reflectance microscopy.The right figure shows reflectance image of Au-Ag-Au metallic barcode at two wavelengths.At 430 nm, we can distinguish the stripe pattern on metallic barcode, the brighter sections corresponds to silver and the darker section corresponds to gold.Whereas, the same particle at 600 nm, The stripe pattern on metallic barcode cannot be distinguished.
  • From previous slide, The optical reflectivity between Au and Ag metal is very different (especially at ~430 nm) and easy to differentiate. So, Au and Ag have been often used to be composition in metallic barcodes.Normally, researcher often specified number one represent Ag stripe and number zero represent Au strip. So, Large numbers of stripe pattern of metallic barcode can be synthesized such as 01010, 000001, 01100100
  • Some advantages of metallic barcode are shown in this slide such asEase of preparationLarge numbers of metallic barcode patterns can be synthesized.Stripping pattern can be visualized by differential optical reflectivity.Require no specialized instrumentationImportantly,The ability to be functionalized with biological probe molecules is possible for detection of specific analytes
  • Therefore, the metallic barcode have beenused as an substrate to be functionalized with biological probe molecules for bioassay such as antibody, molecular beacon and oligonucleotide.Each probe can bind with specific targetsFor example, antibody probe – it can be used to detect antigen and protein targets.molecular beacon probe or even oligonucleotide probe - They can be used to detect DNA targets.So if we have various patterns of metallic barcode that functionalizes with various biological probe molecule.Multiplexed detection for bioassays will be possible.
  • Multiplexed detection in biological sample or multiplexed bioassayis a procedure that simultaneously measures multiple analyte(such as proteins, nucleic acids) in a single assay within a biological sample.Metallic barcode in multiplexed bioassay Show some advantages over the conventional available assaysSuch asReduce assay complexity Reducethe risk of contamination More rapid assay timesCost-efficiencyUse less sample and reagent
  • In this seminar, I will give two applications of Metallic barcode for multiplexed bioassay The first is multiplexed immunoassay for cancer detection And the latter, is multiplexed nucleic acid detection for viral DNA detection
  • The first applicationis multiplexed detection of protein cancer markerson metallic barcode using fluorescent-conjugated polymers as optical probe.
  • For sure,I think all of you must know…………….CANCER.Cancer is a class of diseases that caused from abnormalities in the genetic of the cells.It can occur in various organs or tissue in the body.As could be seen in this figure.Such as lung cancer, prostate cancer or skin cancer.
  • Possible way to detect Cancer that is the use of…….cancer markers,Cancer marker is a protein that produced from cancer cell.It can be used to state kinds of cancers.For examples, PSA-prostate specific antigen, can be found in prostate cancer. CEA can be found in colorectal cancerbHCGcan be found in testicular cancer, respectively.
  • Normally,ELISA and DNA microarray have been used for cancer detection,However, there are still some limitations from these techniquesFor examples,ELISA cannot be used for the detection of many targets in a single sample and DNA microarray still requirethe acquisition of dedicatedinstrumentation
  • In this application, three metallic barcode patterns such as 000100, 01010, and 011110were individually functionalized with antibody probes, that are specific for each cancer marker proteinFor examples, pattern 000100 for PSA-specific probeThe latter pattern for CEA-specific probeAnd the last pattern for bHCG-specific probe
  • A schematic of overall detection strategy of protein cancer detection on metallic barcode is shown in this slide.Assume that, only PSA-specific metallic barcode was incubated in sample solution that containing non-specific protein and CEA target protein,As could be seen, only CEA target protein were recognized onto the antibody.After that, 2nd antibody-(dsDNA)n bound fluorescent-conjugated polymer was added for binding with the recognized target protein and will give the fluorescence at 505 nm by excitation at 423 nm
  • Optical readout of PSA-specific metallic barcode can be visualize by optical reflectance and fluorescence microscopy. Figure 7a and 7c showreflectance images of PSA-specific metallic barcode in the nonspecific protein solution and target protein PSA solution, the position and pattern of metallic barcodes were clearly identified in both solutions. In figure 7b, You can see no fluorescence in nonspecific protein solution. Because nonspecific protein cannot recognize onto the PSA-specific metallic barcode. Whereas, in figure 7d, fluorescence on metallic barcode can be observed in the target protein PSA solution.
  • Effect of cancer marker concentration was studied. This figure show fluorescence images of PSA-specific metallic barcode incubated with PSA at various concentration ranging from zero to ten thousand ng/ml.As could be seen, the Fluorescence intensity significantly increase when the concentration of PSA increases.The fluorescence intensity was plotted against the concentration of PSAfrom 0.1 to 1,000 ng/mL.The red point is the control sample or without PSA.The detection limit can be calculated by defined as 3 times of standard deviation of the background signal.
  • This slide show reflectanceand corresponding fluorescence images in various cases of sample solution.In all reflectance images, you can see that metallic barcode patternwere clearly observed and each cancan be identified as red, yellow and green circle.For fluorescence image,In the case of no cancer marker protein,only weak background wasobserved from the metallic barcodes.In the case of solution containing βhCG only,only βhCG-specific metallic barcode or green circle showed strongfluorescence,whereas the othersshowed near background signal. Similarly, In the case of solution containing CEA and βhCG,only CEA and βhCG-specific metallic barcode or yellow and green circle, showed strong fluorescence,whereas the another one showed near background signal. Also, In the case of solution containing all three cancer marker proteins, all three types of metallic barcodes showed strong fluorescence.
  • This figure show a quantitative plot between the fluorescent intensity from each type of the metallic barcodes in different as shown in this slide.x-axis represent cancer marker proteins present in the sample solutionand y-axisrepresent thefluorescence intensityAs could be seen,The detection of cancer marker proteins was very clear and simultaneous,
  • In summation for the first application, that is The metallic barcode particles allowed simultaneous detection of three cancer marker with good specificity.
  • The latter application is multiplexed nucleic acid detection by using metallic barcode functionalized with molecular beacon as biological probe molecule
  • A virus is a small infectious agent or a piece of nucleic acid (DNA or RNA) that can infect all of organisms.In this application, The researcher want to apply the metallic barcode functionalized with molecular beacons to simultaneously detect three DNA viruses that are HCV, HIV and SARS DNA viruses.
  • In the same way as previous applications, Three different patterns of metallic barcode were functionalized with different molecular beacon probes, That are specific for each viral DNAFor examples, pattern 00001 for SARS virus detectionThe latter pattern for HIV virus detectionAnd the last pattern for HCV virus detection
  • To revise mechanism of molecular beacon As Pornwilard gave us in the first seminar..Molecular beacons are oligonucleotide probes moleculecontainingfluorophore and quencher attached at the end of molecule as shown. Molecular beacons can provide nucleic acid detection by emitting fluorescence signal when the loop of molecular beacon was hybridized by target DNA.
  • Multiplexed detection was performed as shown in Scheme 2.For example,Three different patterns of metallic barcodes functionalized with different molecular beacon probesAll metallic barcodes were mixed and added into sample with two targets That are SARS- and HIV-target DNA.Produce as a result,Fluorescence can only occurs on the SARS- and HIV-specific metallic barcode,Whereas no fluorescence occur on another metallic barcode.
  • This figure show reflectance and corresponding fluorescence images.In reflectance images, all three patterns of metallic barcode were observed,In this assay, only HIV and SARS targets were added.So Only HIV-and SARS- specific metallic barcodes can be visible in the fluorescence image as shown.
  • This figure show quantification for the assay by plotting between fluorescence intensity and combination of targets in sample.As you can see, There was good discrimination in triplexed samples and also one or two targets can be observed.
  • Metallic barcode functionalized with molecular beacon could be early prepared and stored in citrate buffer before use.This figure show preservation of assay by plotting between fluorescence intensities and the day of storage in citrate buffer.As could be seen,More than 3 months, metallic barcode remained possible to determine all target DNA and also, assay performance was not lost.
  • In summation for this application, the metallic barcoded particles functionalized with molecular beacon - provides multiplexed detection of target DNA sequencessuch as viral DNA detection- Retain their selectivity and sensitivity in a triplexedassay after storage for over 3 months
  • In conclusions,Metallic barcodeare cylindrical particles with stripes of Au, Ag and/or other metals along their length and can be synthesized via electrodeposition method.The stripe pattern of metallic barcode can be visualized by optical-reflectance microscopy. A variety of bioassay formats for the detection of proteins or nucleic acid target can be multiplexed by using different barcode patterns with different biological probe molecule such as antibody or even molecular beacon
  • These are some references for this seminar.
  • And finally, Thank you for your attention.
  • Nanoparticles can be used in a variety of bioanalytical formats.Nanoparticles as quantitation tagssuch as the optical detection of quantum dots and the electrochemical detection of metallic nanoparticles.(A hybridization assay in which a gold nanoparticle is used as a tag in a DNA sandwich assay.) (b) Nanoparticles that leverage signal transductionfor example in colloidal gold-based aggregation assays.(An assay in which gold nanoparticles undergo aggregation due to cDNA sequences bringing the nanoparticles into close proximity.)(c) Functional nanoparticlesthat use specific physical or chemical properties of nanoparticles to carry out novel functions, such as the catalysis of a biological reaction.(The upper cartoon depicts localized heating of a single DNA sequence tagged with a gold nanoparticle, leading to selective dehybridization.)(The lower cartoon depicts a cellular assay in which only gold nanoparticles of a particular size can enter a cell.)(d) Encoded nanoparticles as substrates for multiplexed bioassayssuch as striped metallic nanoparticles.(A multiplexed assay, in which only one sequence is complementary to the sample and hence fluorescent.)
  • How the metallic barcode differentiate.
  • A barcode is an optical machine-readable representation of data
  • Illustration of examples of surface-based bioassays
  • The binding curve show that the detection limit of the multiplexed nanowire-bound molecular beacon assay is 100 pM when using molecular beacon probe to capture HCV virus DNA target.
  • The binding curve show that the detection limit of the multiplexed nanowire-bound molecular beacon assay is 100 pM when using molecular beacon probe to capture HCV virus DNA target.
  • The binding curve show that the detection limit of the multiplexed nanowire-bound molecular beacon assay is 100 pM when using molecular beacon probe to capture HCV virus DNA target.
  • The binding curve show that the detection limit of the multiplexed nanowire-bound molecular beacon assay is 100 pM when using molecular beacon probe to capture HCV virus DNA target.

[Pracha] Seminar Ph.D. [Pracha] Seminar Ph.D. Presentation Transcript

  • SCCH 656 Current Topics in Analytical Chemistry I
    Doctor of Philosophy Program in Analytical Chemistry
    Metallic Barcodes
    for Multiplexed Bioassays
    Presented by
    PrachaCheajesadagul
    Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand
  • Outline
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Introduction to Metallic Barcodes
    Why Barcodes?
    EAN barcode
    QR Code
    UPC barcode
    Representation /
    Identification of data
    Figure 1. Barcode with Parallel line pattern
    http://en.wikipedia.org
    http://www.nytimes.com/2009/06/26/technology/26barcode.html?_r=1
    SCCH 656 Current Topics in Analytical Chemistry I
    3
  • Introduction to Metallic Barcodes
    Metallic Barcodes
    Metallic barcodes are cylindrical particles with stripes of Au, Ag and/or other metals along their length.
    Ni
    Ag
    Examples of metals
    for striping pattern
    Pd
    Au
    Figure 3. SEM image of the Au-Ag-Au metallic barcode
    Pt
    Co
    Cu
    Figure 2. Schematic image demonstrating range of
    possible dimensions and one possible striping pattern
    Science 294, 137, 2001
    Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2010
    SCCH 656 Current Topics in Analytical Chemistry I
    4
  • Synthesis of Metallic Barcodes
    Templated sequential electrodeposition
    Au – Ag - Au
    Membrane template
    (Alumina or polycarbonate)
    Pore
    3. Au+ + e- Au0 3x Coulomb
    2. Ag+ + e- Ag0 2x Coulomb
    1. Au+ + e- Au0 x Coulomb
    Metal film
    served as the working electrode for reduction of metal ions from solution
    SCCH 656 Current Topics in Analytical Chemistry I
    5
  • Synthesis of Metallic Barcodes
    Templated sequential electrodeposition
    HNO3
    NaOH
    1. Metal film dissolution with HNO3
    2. Template dissolution with NaOH
    Metallic barcodes
    SCCH 656 Current Topics in Analytical Chemistry I
    6
  • Synthesis of Metallic Barcodes
    The structure of metallic barcode particles is controlled by
    SCCH 656 Current Topics in Analytical Chemistry I
    7
  • Optical Readout of Metallic Barcode
    The readout of stripe pattern on metallic barcode
    depend on different reflectivity properties of metal.
    Ag
    600 nm
    Au
    430 nm
    Ag
    Ag
    Au
    Ag
    Figure 5. Optical image of Ag-Au-Ag barcode particle
    at two wavelengths.
    Figure 4. The wavelength-dependent
    reflectance for the bulk metal
    Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141
    SCCH 656 Current Topics in Analytical Chemistry I
    8
  • Stripe Patterning
    The optical reflectivity between Au and Ag metal
    is very different !! (especially at ~430 nm)
    Ag stripe = 1
    Au stripe = 0
    SCCH 656 Current Topics in Analytical Chemistry I
    9
  • Advantages of Metallic Barcode
    The ability to be functionalized with biological probe molecules
    is possible for detection of specific analytes
    SCCH 656 Current Topics in Analytical Chemistry I
    10
  • Surface Functionalization
    Targets
    DNA targets
    Protein target
    Molecular
    beacon
    Antibody
    Oligonucleotide
    Biological-probe molecules
    s
    Metallic barcode particle as an substrate
    SCCH 656 Current Topics in Analytical Chemistry I
    11
  • Multiplexed Bioassays
    A procedure that simultaneously measures multiple analyte
    (such as proteins, nucleic acids) in a single assay within a biological sample.
    SCCH 656 Current Topics in Analytical Chemistry I
    12
  • Multiplexed Bioassays
    SCCH 656 Current Topics in Analytical Chemistry I
    13
  • Application I
    Multiplexed Immunoassay
    SCCH 656 Current Topics in Analytical Chemistry I
    14
  • Application I
    Cancer by Types
    Figure 6. Cancer by types
    http://www.metrothinkpink.co.uk/think/thinkhealth_guide.html
    SCCH 656 Current Topics in Analytical Chemistry I
    15
  • Application I
    Protein Cancer Markers
    Prostate cancer marker
    Colorectal cancer marker
    Testicular cancer marker
    http://www.alternative-cancer.net/Cell_photos.htm
    SCCH 656 Current Topics in Analytical Chemistry I
    16
  • Application I
    Cancer Detection

    Well accepted for clinical work

    Not used for the detection
    of many targets in a single sample

    Provide genome-level multiplexing

    Require the acquisition of dedicated
    instrumentation
    SCCH 656 Current Topics in Analytical Chemistry I
    17
  • Application I
    Immobilization
    functionalized with
    Antibody Probes
    CEA-specific probes
    PSA-specific probes
    βhCG-specific probes
    Metallic barcode pattern
    (000100)
    Metallic barcode pattern
    (01010)
    Metallic barcode pattern
    (011110)
    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
    SCCH 656 Current Topics in Analytical Chemistry I
    18
  • Application I
    Detection of Protein Cancer Markers
    PSA-specific metallic barcode
    505 nm
    423 nm
    Non-specific
    protein
    PSA target
    protein
    2nd antibody-(dsDNA)n bound
    fluorescent-conjugated polymer
    Scheme 1. A schematic of overall detection strategy of protein cancer detection on metallic barcode.
    SCCH 656 Current Topics in Analytical Chemistry I
    19
  • Application I
    Protein Detection: PSA
    Reflectance
    Fluorescence
    Nonspecific
    protein
    PSA-specific
    metallic barcode
    Target
    protein PSA
    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
    SCCH 656 Current Topics in Analytical Chemistry I
    20
  • Application I
    Protein Detection: PSA
    Fluorescence
    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
    SCCH 656 Current Topics in Analytical Chemistry I
    21
  • Application I
    Multiplexed
    Reflectance
    Fluorescence
    No cancer
    marker proteins
    βhCG only
    CEA and βhCG
    All three cancer
    marker proteins
    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).
    SCCH 656 Current Topics in Analytical Chemistry I
    22
  • Application I
    Multiplexed Detection
    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
    SCCH 656 Current Topics in Analytical Chemistry I
    23
  • Application I
    Summary: Multiplexed Immunoassay
    SCCH 656 Current Topics in Analytical Chemistry I
    24
  • Application II
    Multiplexed Nucleic Acid Detection
    SCCH 656 Current Topics in Analytical Chemistry I
    25
  • Application II
    Viral Infections
    SARS
    (Severe Acute
    Respiratory Syndrome)
    HCV
    (Hepatitis C Virus)
    HIV
    (Human Immunodeficiency Virus)
    Figure 11.
    Viral infections
    http://en.wikipedia.org
    SCCH 656 Current Topics in Analytical Chemistry I
    26
  • Application II
    Immobilization
    functionalized with
    Molecular Beacon Probes
    s
    s
    s
    s
    s
    s
    s
    s
    s
    HIV-specific probes
    SARS-specific probes
    HCV-specific probes
    Metallic barcode pattern
    (00001)
    Metallic barcode pattern
    (00100)
    Metallic barcode pattern
    (00010)
    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
    SCCH 656 Current Topics in Analytical Chemistry I
    27
  • Application II
    Molecular Beacon
    • Oligonucleotide probes molecule 
    • Can provide nucleic acid detection
    • Simplifies assay performance and greatly reduces contamination risk 
    Fluorescence signal (real-time)
    Figure 13. Molecular beacon
    J. AM. CHEM. SOC. 2006, 128, 16892-16903
    http://www.cellscience.com/reviews6/Molecular_beacons.html
    SCCH 656 Current Topics in Analytical Chemistry I
    28
  • Application II
    Detection of Nucleic Acid Target
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    s
    SARS-target DNA
    Fluorescence !!
    Without HCV-target DNA
    HIV-target DNA
    No changing
    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
    SCCH 656 Current Topics in Analytical Chemistry I
    29
  • Application II
    Multiplexd Detection of Viral DNA
    Reflectance
    Fluorescence
    SARS-specific probes
    (00001)
    (00100)
    HCV-specific probes
    (00010)
    (00010)
    HIV-specific probes
    (00100)
    (00001)
    Figure 14. The reflectance image (left) show the barcode pattern and the fluorescence image (right) show which pathogenic oligonucleotide sequences are present
    SCCH 656 Current Topics in Analytical Chemistry I
    30
  • Application II
    Multiplexd Detection of Viral DNA
    Figure 15. Multiplexed detection of viral DNA using molecular beacons on metallic barcodes
    SCCH 656 Current Topics in Analytical Chemistry I
    31
  • Application II
    Preservation of Assays
    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.
    SCCH 656 Current Topics in Analytical Chemistry I
    32
  • Summary: Multiplexed Nucleic Acid Detection
    SCCH 656 Current Topics in Analytical Chemistry I
    33
  • Conclusions
    SCCH 656 Current Topics in Analytical Chemistry I
    34
  • References
    SCCH 656 Current Topics in Analytical Chemistry I
    35
  • Thank you
    for your attention
  • Nanoparticles for Bioassays
    (d) Encoded substrates
    (a) Quantitation tags
    (b) Signal transducers
    (c) Functional tags
    Fig. 1  Four formats in which nanoparticles can be divided, in the field of bioanalysis.
    Current Opinion in Chemical Biology 2003, 7:609–615
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Optical Properties of Cylindrical Striped Particles
    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).
    Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Optical Properties of Cylindrical Striped Particles
    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.
    Wavelength dependence of reflectivity for bulk metals
    Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141
    Anal. Chem., 2002, 74 (10), pp 2240–2247
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Optical Reflectivity and Patterned Fluorescence
    Figure… Fluorescence Patterning on Metallic Barcodes
    J. Phys. Chem. B, 2003, 107 (30), pp 7360–7367
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Multiplexed Detection
    Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Strategies for Particle Encoding
    Solution-phase synthesis
    Templated synthesis
    Lithographic particle
    fabrication
    Encoded anisotropic particles for multiplexed bioanalysis.
    Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2010
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Synthesis of Barcoded Particles
    Nature Biotechnology 19, 1122 - 1123 (2001)
    Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Nanorod / Nanowire
    Nanorods:  Each of their dimensions range from 1–100 nm.
    Synthesized from metals or semiconducting materials.
    Standard aspect ratios (length divided by width) are 3-5.
    Nanowires  Exhibit aspect ratios (length-to-width ratio) of 1000 or more.
    Often referred to as 1-Dimensional materials.
    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.
    Many techniques have been developed to synthesize these structures and can be grouped into four categories:
    1) Spontaneous growth
    2) Template synthesis
    3) Electrospinning
    4) Lithography
    http://www.nanogallery.eu/nanotopics/10-nanorod-nanowire.html
    http://www.engr.colostate.edu/ECE581/fall07/Nanowires%20and%20Nanorods.pdf
    SCCH 656 Current Topics in Analytical Chemistry I
    2
  • Electrochemistry
    Metallic elements, including several non-noble metals, sorted by their chemical "nobility" (noble metals bolded):
    SCCH 656 Current Topics in Analytical Chemistry I
    2
    http://en.wikipedia.org
  • Template based Synthesis
    - Involves an electrolysis process
    that results in the deposition of solid material in an electrode
    The most commonly used - Anodized alumina membranes
    - Radiation track etched polymer membranes/mica
    - Nanochannel array glass
    - Mosoporous materials
    Cross section of anodized Al
    Al membrane
    450 nm pores in glass
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Electrochemical Deposition
    (also knows as electrodeposition , electroplating, cathode deposition)
    - Involves an electrolysis process that results in the deposition of solid material in an electrode
    E0 = the electrode potential
    ai = the activity of the ions
    Rg = the gas constant
    F = the Faraday’s constant
    T = the temperature
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Electrochemical Deposition
    A typical electrolytic process composes
    of a series of steps:
    a- Mass transfer through the solution
    from one electrode to another
    b- Chemical reactions at the interfaces between electrode-solution
    c- Electrons transfer at the electrode surfaces and through the external circuit
    d- Other surface reactions such as adsorption, desorption or recrystallization
    An experimental setup for electrochemical deposition
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Optical Readout of Metallic Barcode
    430 nm
    520 nm
    600 nm
    Ratio of reflected intensities for various metals versus Au
    Reflectance optical microscopy images
    Line profiles of
    each particle
    SCCH 656 Current Topics in Analytical Chemistry I
    Science 5 October 2001: Vol. 294. no. 5540, pp. 137 - 141
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  • Decoding the Metal Striping Pattern
    SCCH 656 Current Topics in Analytical Chemistry I
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  • No. of Striping Patterns
    Anal. Chem., 2002, 74 (10), pp 2240–2247
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Barcoded Segment Measurements
    Anal. Chem., 2002, 74 (10), pp 2240–2247
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Florophore (Fluorescent Dye)
    Fluorescein
    isothiocyanate (FITC)
    Texas Red
    http://info.med.yale.edu/genetics/ward/tavi/FISHdyes2.html
    http://en.wikipedia.org
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Cationic Fluorescent Conjugated Polymer
    Polythiophene derivative
    {poly(1H-imidazolium-1-methyl-3-{2-[(4-methyl-3-thienyl)oxy]ethyl}bromide)}
    J. Am. Chem. Soc., 2009, 131 (10), pp 3432–3433
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Applications I : Protein Detection
    Fig. 1  A schematic illustration of protein detection on Au/Ag-barcodednanorods using fluorescent-conjugated polymers. 
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Cationic Fluorescent Conjugated Polymer
    Figure…Conjugated π-orbitals of a coplanar and a twisted substituted polythiophene.
    http://en.wikipedia.org
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Applications I : Polymer Concentration
    Without
    PSA
    1 μg/mL
    of PSA
    25 μM
    50 μM
    10 μM
    Concentration of conjugated polymers
    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
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Applications I : Simultaneous Detection
    Reflectance
    Fluorescence
    incubated in a solution containing PSA, CEA, and βhCG of 10, 100, and 1,000 ng/mL
    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
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Applications I : Nanowires in Different Solutions
    in bovine serum
    in PBS buffer
    in bovine serum
    containing 10 ng/mL PSA
    in bovine serum
    containing 1 ng/mL PSA
    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
    SCCH 656 Current Topics in Analytical Chemistry I
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  • DNA Microarray
    http://www.medscape.com
    http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/work.htm
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Bioassays
     A method for the quantitative estimation of the effects that result in a biological system after its exposure to a substance
    DNA microarray
    relying on a two-color fluorescent dye system for hybridization elucidation
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Enzyme-linked Immunosorbent Assay
    http://www.51protocol.com/
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Surface-Based Bioassays
    DNA microarray
    relying on a two-color fluorescent dye system for hybridization elucidation
    Planar array with multicolored
    nanoparticle tags (i.e. QDs)
    For hybridization determination instead of organic dye
    Suspension array
    where the encoding element,
    is nanoparticle shape
    http://wires.wiley.com/WileyCDA/WiresArticle/wisId-WNAN96.html
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Multiplexed Assays
    A type of laboratory procedure that simultaneously measures multiple analytes in a single assay
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Nucleotides
    Oligonucleotide (from Greek prefix oligo-, "having few, having little")
    a short nucleic acid polymer, typically with twenty or fewer bases. 
    http://en.wikipedia.org
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Probe Sequences
    Table 1. Probe Sequences Used in This Work
    * The italic portions of the sequences indicate complementary stem regions
    * TAMRA = florophore (tetramethylrhodamine)
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Multiplexd Detection of Viral DNA
    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.
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Multiplexd Detection of Viral DNA
    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.
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Multiplexd Detection of Viral DNA
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Antibodies Immobilization
    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
    Anal. Chem., 2001, 73 (3), pp 471–480
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Adsorption Isotherm
    To obtain more useful data, it was necessary to measure the adsorption isotherms.
    Adsorption experiments data may be fitted to Langmuir, Sips, and Toth isotherms.
    J. Chem. Eng. Data, 2006, 51 (2), pp 451–456
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Binding Isotherm
    Fractional coverage
    estimated on the basis of the fluorescence intensity as compared to the maximum intensity
    Limit of detection
    calculated by taking the average fluorescence
    intensity for the control (background signal) and adding 2 times its standard deviation
    - mean fluorescence units translates a concentration of <100 pM
    - with dynamic range of 3-4 orders of magnitude in concentration
    Figure… Binding isotherm for molecular beacon probes on barcodednanowires
    SCCH 656 Current Topics in Analytical Chemistry I
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  • Sips Isotherm
     - Used to assess the heterogeneity in the Target−Probe binding in solution
    - Antigen−Antibody probe
    - DNA−Molecular beacon probe
    • Relates the fractional coverage of antibody binding sites to the concentration of antigen in solution.
    f = the fractional coverage of target binding sites on the probes bound on the substrate
    K= the average equilibrium constant for adsorption
    C = the concentration of target in solution
    a = the heterogeneity index
    If a = 1 the antigen-antibody binding affinity is described by a single value and the antibodies are homogeneous
    If a < 1 indicate a wider affinity distribution and thus increasing heterogeneity
    Anal. Chem., 2001, 73 (3), pp 471–480
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