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Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain

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Science Advances  04 Sep 2015:
Vol. 1, no. 8, e1500487
DOI: 10.1126/sciadv.1500487
  • Fig. 1 Scheme of the whole process from peptide extraction to Raman detection.

    (A) A mixture of peptides is extracted from the BRCT domain derived from the BRCA1 protein. (B) The mixture is collected in aqueous solution at a concentration of 100 pM. (C to E) A drop is deposited and dried on a matrix array of SSCs (each chain is a pixel). (F) The whole content of peptides is spread over the matrix, and some (three different types per pixel, on average) are deposited in the smallest gap. These are the only ensembles per pixel that contribute to the Raman signal. (G) Representative Raman signal from pixel i,j of the matrix array composed of, on average, three different types of peptides.

  • Fig. 2 Fabrication process of silver SSC.

    (A) After electron beam lithography and surface treatment with 2 M HF, the sample is immersed in HF/AgNO3 aqueous solution, where Ag+ is reduced to silver metal through a redox reaction chain. (B) In nanowells (reduction surface), silver growth follows a spherical symmetry and generates three spheres of appropriate diameter and interdistance. (C) Redox reactions inside a nanowell starting from the silicon surface. (D) SSC architecture and 2D map of electric field. Evidence of external laser polarization along the chain axis. The electric hotspot is localized in the smallest gap. (E to H) SEM images of silver SSCs and possible combinations in monomer, dimer, trimer, and tetramer. Scale bars, 50 nm. PMMA, polymethyl methacrylate.

  • Fig. 3 Effects of disorder and sphere positioning on hotspot localization and enhancement.

    (A) Three-dimensional field enhancement and localization of (top) ideal SSC with zero sphere roughness and (bottom) fabricated SSC with 2-nm surface roughness. (B) Two-dimensional field map with zero roughness (top) and average surface disorder of 2 nm (bottom). (C) Comparison of enhancement between ideal SSC and fabricated SSC. A systematic electric field enhancement of a factor of 2 (as a function of wavelength) is expected of a 2-nm disorder. E0 is the maximal field value used as normalization factor in the 3D plot. In all simulations, laser polarization occurred along the SSC axis. au, arbitrary units; dis., disorder; ord., order.

  • Fig. 4 Linear combination fitting procedure of peptide spectra.

    Raman spectra were collected in a representative pixel of the array. The fit gives a weighted composition of peptides 1, 8, and 12.

  • Fig. 5 Matrix array and data acquisition.

    (Top left) SEM image of a 10 × 10 SSC array. Scale bar, 2 μm. Each SSC represents a pixel element i,j of the matrix. (Top right) In each pixel, color code is associated with a specific peptide. (Bottom left) A submatrix of 3 × 3 pixels is evidenced. Scale bar, 2 μm. (Bottom right) A detailed SEM image of the SSC representing the pixel. Scale bar, 50 nm. Empty pixels represent specific positions in the array where no peptides are detected.

  • Fig. 6 Raman spectra of pure wild-type and mutated peptides.

    (A and B) Raman spectra (B) show a net difference between two peptides differentiated by only the exchange of one amino acid (A; a methionine with an arginine). These spectra constitute the base set for the fitting procedure. Their net difference allows identification of mutated peptides in the mixture. (C and D) The results of PCA are also shown: a 2D map of the PC2 coefficients of two pixels (C), one pixel for each peptide, where color code is proportional to the significance of the PC2 parameter over the map; the PC2 parameter load curve (D) takes into account spectral differences between the two peptides. The combination of PC2 mapping and PC2 load curve allows identification of pixels dominated by wild-type or mutated species.

  • Table 1 Fractional peptide content of M1775 (wild type) and M1775R (mutated).

    The point mutation (arginine replaces methionine in peptide 1) is underlined.

    Number of
    peptides
    Fractional M1775 (wild type)
    sequence peptides
    Composition (%)
    (error, 8%)
    Fractional M1775R (mutated)
    sequence peptides
    Composition (%)
    (error, 8%)
    1ICCYGPFTNMPTDQLE11.18ICCYGPFTNRPTDQLE6.36
    2APVVTREWVLD7.85SDPSEDRAPE9.92
    3ADALYTNPAQARE2.56TSYLPRQDLE5.91
    4TAANLHAPVILAGTPGTFT HAGTE14.25SARVGNIPSSTSALKVPQLK VAE10.25
    5NLVQRVPKDVFMGVDE7.30ASHLPFAQNISRVKE7.72
    6GAILVVAATDGPMPQTRE11.76VYILSKDE8.76
    7GGDALIPMLKE9.26TFNVGSFASGKE5.76
    8KFMKIISLAPE11.34RYLGAKFPGAKRFSLE7.75
    9VIAHLVNWE10.22AAKAKGAMALFGEKYDE6.98
    10RINKALDFIAE8.25GRQGGTLQLFRTE4.22
    11LRAKNQITLPVILKNE5.03KFTALTAELTAE7.44
    12GGRTVGAGVVAKVLS6.38
    13RFQADTLARFE6.15
    14FLKAGGVFTDE6.57

Supplementary Materials

  • Supplementary materials for this article are available at http://advances.sciencemag.org/cgi/content/full/1/8/1500487/DC1

    Crystallographic model of human BRCA1-BRCT protein

    Fig. S1. Three-dimensional crystal structure of the BRCA1-BRCT protein.

    Design and fabrication of device

    Fig. S2. Top-down and bottom-up process fabrication.

    Fig. S3. SEM images of different SSCs.

    Theoretical comparison of ordered and disordered SSC device

    Fig. S4. FDTD analysis of the role of disorder.

    Complete peptide content of all the solutions resolved using the nanolens SERS device

    Fig. S5. Nanolens matrix position of measurement points for the synthetic peptide solution.

    Fig. S6. Nanolens matrix position of measurement points for the wild-type peptide solution.

    Fig. S7. Nanolens matrix position of measurement points for the mutated peptide solution.

    Table S1. Peptide fraction calculated for each measurement point in the grid of nanolens devices and χ2 and r2 statistics for the synthetic peptide solution.

    Table S2. Relative content of each peptide in the synthetic peptide solution.

    Table S3. Peptide fraction calculated for each measurement point in the grid of nanolens devices and χ2 and r2 statistics for the wild-type peptide solution.

    Table S4. Relative content of each peptide in the wild-type peptide solution.

    Table S5. Peptide fraction calculated for each measurement point in the grid of nanolens devices and χ2 and r2 statistics for the mutated peptide solution.

    Table S6. Relative content of each peptide in the mutated peptide solution.

    List of synthetic peptides utilized in the work and corresponding IDs

    Extraction of wild-type and mutated BRCA1-BRCT protein domain

  • Supplementary Materials

    This PDF file includes:

    • Crystallographic model of human BRCA1-BRCT protein
    • Fig. S1. Three-dimensional crystal structure of the BRCA1-BRCT protein.
    • Design and fabrication of device
    • Fig. S2. Top-down and bottom-up process fabrication.
    • Fig. S3. SEM images of different SSCs.
    • Theoretical comparison of ordered and disordered SSC device
    • Fig. S4. FDTD analysis of the role of disorder.
    • Complete peptide content of all the solutions resolved using the nanolens SERS device
    • Fig. S5. Nanolens matrix position of measurement points for the synthetic peptide solution.
    • Fig. S6. Nanolens matrix position of measurement points for the wild-type peptide solution.
    • Fig. S7. Nanolens matrix position of measurement points for the mutated peptide solution.
    • Table S1. Peptide fraction calculated for each measurement point in the grid of nanolens devices and x2 and r2 statistics for the synthetic peptide solution.
    • Table S2. Relative content of each peptide in the synthetic peptide solution.
    • Table S3. Peptide fraction calculated for each measurement point in the grid of nanolens devices and x2 and r2 statistics for the wild-type peptide solution.
    • Table S4. Relative content of each peptide in the wild-type peptide solution.
    • Table S5. Peptide fraction calculated for each measurement point in the grid of nanolens devices and x2 and r2 statistics for the mutated peptide solution.
    • Table S6. Relative content of each peptide in the mutated peptide solution.
    • List of synthetic peptides utilized in the work and corresponding IDs
    • Extraction of wild-type and mutated BRCA1-BRCT protein domain

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