(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
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`(43) International Publication Date
`17 October 2013 (17.10.2013)
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`WIPOI PCT
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`\9
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`(10) International Publication Number
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`WO 2013/154770 A1
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`(51)
`
`International Patent Classification:
`G01N 21/65 (2006.01)
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`(21)
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`International Application Number:
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`PCT/U82013/032347
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`(22)
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`International Filing Date:
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`15 March 2013 (15.03.2013)
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`(25)
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`(26)
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`(30)
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`(71)
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`(72)
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`(74)
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`(81)
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`Filing Language:
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`Publication Language:
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`English
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`English
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`Priority Data:
`61/622,226
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`10 April 2012 (10.04.2012)
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`US
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`Applicant: THE TRUSTEES OF PRINCETON UNI-
`VERSITY [US/US]; 701 Carnegie Center, Suite 438, Prin-
`ceton, NJ 08540 (US).
`
`Inventors: CHOU, Stephen Y.; 7 Foulet Drive, Princeton,
`New Jersey 08540 (US). ZHOU, Liang-Cheng; 465
`Meadow Road, Apt. 8307, Princeton, New Jersey 08540
`(US).
`
`Agent: KEDDIE, James S.; Bozicevic, Field & Francis
`LLP, 1900 University Avenue, Suite 200, East Palo Alto,
`California 94303 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`
`A0, AT, AU, Az, BA, BB, BG, BH, BN, BR, Bw, BY,
`BZ, CA, CII, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`IIN, IIR, IIU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, Mw, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, R0, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ,
`TM, TN, TR, TT, Tz, UA, UG, US, UZ, VC, VN, ZA,
`ZM, zw.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`Published:
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`with international search report (Art. 21(3))
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`with sequence listing part ofdescription (Rule 5.2(a))
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`(54) Title: ULTRA-SENSITIVE SENSOR
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`
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`(57) Abstract: This disclosure provides, among other things, a nanosensor comprising a substrate and one or a plurality of pillars ex-
`tending from a surface of the substrate, where the pillars comprise a metallic dot structure, a metal disc, and a metallic back plane.
`The nanosensor comprises a molecular adhesion layer that covers at least a part of the metallic dot structure, the metal disc, and/or
`the metallic back plane and a capture agent bound to the molecular adhesion layer. The nanosensor amplifies a light signal from an
`analyte, when the analyte is specifically bound to the capture agent.
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`WO 2013/154770
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`PCT/USZOl3/032347
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`ULTRA-SENSITIVE SENSOR
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`CROSS-REFERENCING
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`This application claims the benefit of U.S. provisional application serial no.
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`61/622,226 filed on April 10, 2012, which application is incorporated by reference
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`herein for all purposes.
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`STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
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`This invention was made with United States government support under Grant
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`No. FA9550-08—1-0222 awarded by the Defense Advanced Research Project
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`Agency (DARPA) The United States government has certain rights in this invention.
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`BACKGROUND
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`There is a great need to enhance a luminescence signal (e.g. a fluorescence
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`signal) and detection sensitivity of biological and chemical assays. The application
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`is related to the micro/nanostructures and molecular layers and methods for
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`achieving an enhancement (namely amplification of luminescence and improvement
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`of detection sensitivity), their fabrication and applications.
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`SUMMARY
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`This disclosure provides, among other things, a nanosensor comprising a
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`substrate and one or a plurality of pillars extending from a surface of the substrate,
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`with a metallic dot structure on pillar’s sidewall, a metal disc on top of the pillar, and
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`a metallic back plane covering a significant area near the foot of the pillar. The
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`nanosensor further comprises a molecular adhesion layer that covers at least a part
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`of the metallic dot structure, and/or the metal disc, and/or the metallic back plane
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`and that binds a capture agent. The nanosensor is coated with capture agent that
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`specifically captures targeted analytes (e.g. molecules, which can be proteins or
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`nucleic acids). The analytes can be optically labeled directly or indirectly.
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`In indirect
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`labeling, a secondary capture agent with an optical label (Le. a labeled detection
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`agent) is used to bind and hence identify the presence of the captured analyte. The
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`nanosensor amplifies a light signal from a the analyte, when the analyte is bound to
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`the capture agent.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`The skilled artisan will understand that the drawings, described below, are for
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`illustration purposes only. The drawings are not intended to limit the scope of the
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`present teachings in any way. Some of the drawings are not in scale.
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`Fig. 1 panels A and B schematically illustrate some features of embodiment
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`of a subject nanodevice. Panel C schematically illustrates one way in which a
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`subject nanodevice can be manufactured.
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`Fig. 2 schematically illustrates an exemplary system.
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`Fig. 3 schematically illustrates an exemplary self-assembled monolayer.
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`Fig. 4 schematically illustrates an exemplary antibody detection assay.
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`Fig. 5 schematically illustrates an exemplary nucleic acid detection assay.
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`Fig. 6 schematically illustrates another embodiment nucleic acid detection
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`assay.
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`Fig. 7 Disk-coupled dots-on-pillar antenna array (D2PA) plate and
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`immunoassay.
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`(a) Schematic (overview and cross—section) of D2PA plate without
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`an immunoassay. D2PA has an array of dense three—dimensional (3D) resonant
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`cavity nanoantennas (formed by the gold disks on top of periodic nonmetallic pillars
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`and the gold backplane on the pillar foot) with dense plasmonic nanodots inside,
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`and couples the metallic components through nanogaps. (b) Schematic of the
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`immunoassay on the D2PA, consisting of a self-assembled monolayer (SAM) of
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`adhesion layer, Protein-A (as capture layer) and human-lgG pre-labeled with lRDye-
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`800cw (as pre-labeled biomarker). (c) Scanning electron micrograph (SEM) of D2PA
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`with 200 nm period (overview and cross-section). The gold nanodots rested on the
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`silica nano—pillar sidewalls are clearly observed.
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`Fig. 8 Measured absorbance spectrum of D2PA with (blue line) and without
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`(red line) the immunoassay being deposited. The peak absorbance is 98% and
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`97%, and the resonance peak width is 165 nm and 145 nm, respectively, with and
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`without the immunoassay. Deposition of the immunoassay slightly blue-shifted the
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`absorption peak from 795 nm to 788 nm and widened the absorption wavelength
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`range
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`Fig. 9 Measured area-average fluorescence intensity spectrum of the human-
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`lgG labeled with lRDye800CW captured by the assay on the D2PA (red line) and
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`the glass plate (blue line, which is amplified 1000 times to be visible at given
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`scales), respectively. Compared with the assay on the glass plate, the average
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`fluorescence enhancement (dashed line) is 7,440 fold at the peak wavelength of
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`fluorescence (800 nm) and 7,220 fold when average over the FWHM fluorescence.
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`The plasmonic fluorescence enhancement factor (EF) spectrum has much broader
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`FWHM than the fluorescence spectrum, which is consistent with the observed D2PA
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`plasmonic resonance spectrum (Fig. 5).
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`Fig. 10 Measured uniformity of fluorescence enhancement over large area.
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`(a) Measured immunoassay fluorescence enhancement (factor) map over a total 5
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`mm x 5 mm area of the D2PA. The map has total 2,500 tiles (50 X 50), measured by
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`using each tile area (i.e. laser probe area) of 100 um x 100 um and a step—and—
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`repeat distance of 100 um. (b) The corresponding histogram of the measured
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`enhancement factor gives a Gaussian distribution variation of 19%.
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`Fig. 11 A model direct assay of protein A and lgG. Fluorescence intensity vs.
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`lgG concentration on D2PA (squares) and glass plate reference (circles). The
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`squares and circles are measured data, and the curves were the fittings using five-
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`parameter logistic regression model to allow an extrapolation of the data points
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`between the measured ones. The limit of detection (LoD) of D2PA and glass plate
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`was found to be 0.3 fM and 0.9 nM, respectively, giving an enhancement of LoD of
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`3,000,000 fold. Schematic of the immunoassay on the D2PA, consisting of a self-
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`assembled monolayer (SAM) of adhesion layer, Protein-A (as capture layer) and
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`human—lgG pre—labeled with lRDye—800cw (as pre—labeled biomarker).
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`Fig. 12 Single molecule fluorescence of lRDye800CW labeled lgG on D2PA
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`plate. (a) 2D fluorescence image of 50 um x 50 um area of a Protein A/lgG
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`immunoassay on D2PA plate with an lgG concentration of 10'10 M. Distinct “bright
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`spots” are visible. And (b) Fluorescence vs. time of a single bright spot. The binary
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`stepwise behavior indicates that the fluorescence is from a single dye molecule
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`placed at a hot spot (large electric field location) of D2PA. Compared with the
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`immunoassay on the glass reference, the single molecule fluorescence at a hot spot
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`is enhanced by 4 x 106 fold.
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`Fig. 13. PSA immunoassay on D2PA plates. The experiment data was fitted
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`using 5-parameter logstic model
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`(solid curve) in order to calculate the LoD. An LoD
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`~ 10 aM was achieved on D2PA. Compared to glass plates, whose LoD was 0.9
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`pM, the sensitivity of D2PA is 90,000 folds better. (Chou Group, to be published)
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`Fig. 14 CEA immunoassay on D2PA plates. Similar configuration is used as
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`the PSA immunoassay. For the tentative trial so far, we managed to achieve an
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`LoD~ 28aM. Better sensitivity (lower LoD) is expected once we manage to raise the
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`signal to noise ratio. (Chou Group, to be published)
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`Fig. 15 CA15.3 immunoassay on D2PA plates. A similar configuration is used
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`as the PSA immunoassay. For the tentative trial so far, we managed to achieve an
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`LoD~ 0.01 U/mL. Better sensitivity (lower LoD) is expected once we manage to
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`raise the signal to noise ratio. (Chou Group, to be published)
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`Fig. 16 is two graphs showing the correlation between spiked concentration
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`and observed concentration.
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`Fig. 17 is two graphs showing the crossreactivity between two antibodies.
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`Fig. 18 is two graphs showing reproducibility of results.
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`Fig. 19 shows the results of a DNA hybridization assay, and a schematic
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`illustration of the same.
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`Fig. 20 shows a series of scanning electron micrographs.
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`Fig. 21 schematically illustrates an alternative embodiment.
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`Corresponding reference numerals indicate corresponding parts throughout
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`the several figures of the drawings. It is to be understood that the drawings are for
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`illustrating the concepts set forth in the present disclosure and are not to scale.
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`Before any embodiments of the invention are explained in detail, it is to be
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`understood that the invention is not limited in its application to the details of
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`construction and the arrangement of components set forth in the following
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`description or illustrated in the drawings.
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`DEFINITIONS
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`Before describing exemplary embodiments in greater detail, the following
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`definitions are set forth to illustrate and define the meaning and scope of the terms
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`used in the description.
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`The term “molecular adhesion layer” refers to a layer or multilayer of
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`molecules of defined thickness that comprises an inner surface that is attached to
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`the nanodevice and an outer (exterior) surface can be bound to capture agents.
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`The term “capture agent-reactive group” refers to a moiety of chemical
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`function in a molecule that is reactive with capture agents, i.e., can react with a
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`moiety (e.g., a hydroxyl, sulfhydryl, carboxy or amine group) in a capture agent to
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`produce a stable strong, e.g., covalent bond.
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`The term “capture agent” as used herein refers to an agent that binds to a
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`target analyte through an interaction that is sufficient to permit the agent to bind and
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`concentrate the target molecule from a heterogeneous mixture of different
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`molecules. The binding interaction is typically mediated by an affinity region of the
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`capture agent. Typical capture agents include any moiety that can specifically bind
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`to a target analyte. Certain capture agents specifically bind a target molecule with a
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`dissociation constant (KB) of less than about 10"5 M (e.g., less than about 10'7 M,
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`less than about 10'8 M, less than about 10'9 M, less than about 10'10 M, less than
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`about 10'11 M, less than about 10'12 M, to as low as 10'16 M) without significantly
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`binding to other molecules. Exemplary capture agents include proteins (e.g.,
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`antibodies), and nucleic acids (e.g., oligonucleotides, DNA, RNA including
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`aptamers).
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`The terms “specific binding” and “selective binding” refer to the ability of a
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`capture agent to preferentially bind to a particular target molecule that is present in a
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`heterogeneous mixture of different target molecule. A specific or selective binding
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`interaction will discriminate between desirable (e.g., active) and undesirable (e.g.,
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`inactive) target molecules in a sample, typically more than about 10 to 100-fold or
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`more (e.g., more than about 1000- or 10,000-fold).
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`The term "protein" refers to a polymeric form of amino acids of any length, i.e.
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`greater than 2 amino acids, greater than about 5 amino acids, greater than about 10
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`amino acids, greater than about 20 amino acids, greater than about 50 amino acids,
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`greater than about 100 amino acids, greater than about 200 amino acids, greater
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`than about 500 amino acids, greater than about 1000 amino acids, greater than
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`about 2000 amino acids, usually not greater than about 10,000 amino acids, which
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`can include coded and non—coded amino acids, chemically or biochemically
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`modified or derivatized amino acids, and polypeptides having modified peptide
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`backbones. The term includes fusion proteins, including, but not limited to, fusion
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`proteins with a heterologous amino acid sequence, fusions with heterologous and
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`homologous leader sequences, with or without N-terminal methionine residues;
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`immunologically tagged proteins; fusion proteins with detectable fusion partners,
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`e.g., fusion proteins including as a fusion partner a fluorescent protein, [3-
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`galactosidase, luciferase, etc.; and the like. Also included by these terms are
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`polypeptides that are post—translationally modified in a cell, e.g., glycosylated,
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`cleaved, secreted, prenylated, carboxylated, phosphorylated, etc, and polypeptides
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`with secondary or tertiary structure, and polypeptides that are strongly bound, e.g.,
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`covalently or non-covalently, to other moieties, e.g., other polypeptides, atoms,
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`cofactors, etc.
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`The term “antibody” is intended to refer to an immunoglobulin or any
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`fragment thereof, including single chain antibodies that are capable of antigen
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`binding and phage display antibodies).
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`The term “nucleic acid” and “polynucleotide” are used interchangeably herein
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`to describe a polymer of any length composed of nucleotides, e.g.,
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`deoxyribonucleotides or ribonucleotides, or compounds produced synthetically (e.g.,
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`PNA as described in US. Patent No. 5,948,902 and the references cited therein)
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`which can hybridize with naturally occurring nucleic acids in a sequence specific
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`manner analogous to that of two naturally occurring nucleic acids, e.g., can
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`participate in Watson-Crick base pairing interactions.
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`The term “complementary” as used herein refers to a nucleotide sequence
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`that base-pairs by hydrogen bonds to a target nucleic acid of interest. In the
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`canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine
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`(T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by
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`uracil (U). As such, A is complementary to T and G is complementary to C.
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`Typically, “complementary” refers to a nucleotide sequence that is fully
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`complementary to a target of interest such that every nucleotide in the sequence is
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`complementary to every nucleotide in the target nucleic acid in the corresponding
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`positions. When a nucleotide sequence is not fully complementary (100%
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`complementary) to a non-target sequence but still may base pair to the non-target
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`sequence due to complementarity of certain stretches of nucleotide sequence to the
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`non-target sequence, percent complementarily may be calculated to assess the
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`possibility of a non-specific (off-target) binding. In general, a complementary of 50%
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`or less does not lead to non-specific binding. In addition, a complementary of 70%
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`or less may not lead to non—specific binding under stringent hybridization conditions.
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`The terms “ribonucleic acid” and “RNA” as used herein mean a polymer
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`composed of ribonucleotides.
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`The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer
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`composed of deoxyribonucleotides.
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`The term “oligonucleotide” as used herein denotes single stranded nucleotide
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`multimers of from about 10 to 200 nucleotides and up to 300 nucleotides in length,
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`or longer, e.g., up to 500 nt in length or longer. Oligonucleotides may be synthetic
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`and, in certain embodiments, are less than 300 nucleotides in length.
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`The term “attaching” as used herein refers to the strong, e.g, covalent or non—
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`covalent, bond joining of one molecule to another.
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`The term “surface attached” as used herein refers to a molecule that is
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`strongly attached to a surface.
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`The term “sample” as used herein relates to a material or mixture of materials
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`containing one or more analytes of interest. In particular embodiments, the sample
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`may be obtained from a biological sample such as cells, tissues, bodily fluids, and
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`stool. Bodily fluids of interest include but are not limited to, amniotic fluid, aqueous
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`humour, vitreous humour, blood (e.g., whole blood, fractionated blood, plasma,
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`serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime,
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`endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including
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`nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus,
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`rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit,
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`urine and exhaled condensate. In particular embodiments, a sample may be
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`obtained from a subject, e.g., a human, and it may be processed prior to use in the
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`subject assay. For example, prior to analysis, the protein/nucleic acid may be
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`extracted from a tissue sample prior to use, methods for which are known. In
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`particular embodiments, the sample may be a clinical sample, e.g., a sample
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`collected from a patient.
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`The term “analyte” refers to a molecule (e.g., a protein, nucleic acid, or other
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`molecule) that can bound by a capture agent and detected.
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`The term “assaying” refers to testing a sample to detect the presence and/or
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`abundance of an analyte.
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`As used herein, the terms “determining, measuring,” and “assessing,” and
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`“assaying” are used interchangeably and include both quantitative and qualitative
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`determinations.
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`As used herein, the term “light-emitting label” refers to a label that can emit
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`light when under an external excitation. This can be luminescence. Fluorescent
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`labels (which include dye molecules or quantum dots), and luminescent labels (e.g.,
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`electro- or chemi-luminescent labels) are types of light-emitting label. The external
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`excitation is light (photons) for fluorescence, electrical current for
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`electroluminescence and chemical reaction for chemi-luminscence. An external
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`excitation can be a combination of the above.
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`The phrase “labeled analyte” refers to an analyte that is detectably labeled
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`with a light emitting label such that the analyte can be detected by assessing the
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`presence of the label. A labeled analyte may be labeled directly (i.e., the analyte
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`itself may be directly conjugated to a label, e.g., via a strong bond, e.g., a covalent
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`or non—covalent bond), or a labeled analyte may be labeled indirectly (i.e., the
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`analyte is bound by a secondary capture agent that is directly labeled).
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`The term “hybridization” refers to the specific binding of a nucleic acid to a
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`complementary nucleic acid via Watson-Crick base pairing. Accordingly, the term
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`“in situ hybridization” refers to specific binding of a nucleic acid to a metaphase or
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`interphase chromosome.
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`The terms “hybridizing” and “binding”, with respect to nucleic acids, are used
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`interchangeably.
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`The term "capture agent/analyte complex" is a complex that results from the
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`specific binding of a capture agent with an analyte. A capture agent and an analyte
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`for the capture agent will usually specifically bind to each other under “specific
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`binding conditions” or “conditions suitable for specific binding”, where such
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`conditions are those conditions (in terms of salt concentration, pH, detergent,
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`protein concentration, temperature, etc.) which allow for binding to occur between
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`capture agents and analytes to bind in solution. Such conditions, particularly with
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`respect to antibodies and their antigens and nucleic acid hybridization are well
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`known in the art (see, e.g., Harlow and Lane (Antibodies: A Laboratory Manual Cold
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`Spring Harbor Laboratory, Cold Spring Harbor, NY. (1989) and Ausubel, et al, Short
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`Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002).
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`The term “specific binding conditions” as used herein refers to conditions that
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`produce nucleic acid duplexes or protein/protein (e.g., antibody/antigen) complexes
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`that contain pairs of molecules that specifically bind to one another, while, at the
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`same time, disfavor to the formation of complexes between molecules that do not
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`specifically bind to one another. Specific binding conditions are the summation or
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`combination (totality) of both hybridization and wash conditions, and may include a
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`wash and blocking steps, if necessary.
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`For nucleic acid hybridization, specific binding conditions can be achieved by
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`incubation at 4290 in a solution: 50 % formamide, 5 x SSC (150 mM NaCl, 15 mM
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`trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt's solution, 10%
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`dextran sulfate, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by
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`washing the filters in 0.1 x SSC at about 659C.
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`For binding of an antibody to an antigen, specific binding conditions can be
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`achieved by blocking a substrate containing antibodies in blocking solution (e.g.,
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`PBS with 3% BSA or non-fat milk), followed by incubation with a sample containing
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`analytes in diluted blocking buffer. After this incubation, the substrate is washed in
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`washing solution (e.g. PBS+TWEEN 20) and incubated with a secondary capture
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`antibody (detection antibody, which recognizes a second site in the antigen). The
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`secondary capture antibody may conjugated with an optical detectable label, e.g., a
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`fluorophore such as lRDye8OOCW, Alexa 790, Dylight 800. After another wash, the
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`presence of the bound secondary capture antibody may be detected. One of skill in
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`the art would be knowledgeable as to the parameters that can be modified to
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`increase the signal detected and to reduce the background noise.
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`The term “a secondary capture agent” which can also be referred to as a
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`“detection agent" refers a group of biomolecules or chemical compounds that have
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`highly specific affinity to the antigen. The secondary capture agent can be strongly
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`linked to an optical detectable label, e.g., enzyme, fluorescence label, or can itself
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`be detected by another detection agent that is linked to an optical detectable label
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`through bioconjugatio (Hermanson, “Bioconjugate Techniques” Academic Press,
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`2nd Ed., 2008).
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`The term “biotin moiety” refers to an affinity agent that includes biotin or a
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`biotin analogue such as desthiobiotin, oxybiotin, 2’-iminobiotin, diaminobiotin, biotin
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`sulfoxide, biocytin, etc. Biotin moieties bind to streptavidin with an affinity of at least
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`10-8M. A biotin affinity agent may also include a linker, e.g., —LC-biotin, —LC-LC-
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`Biotin, —SLC-Biotin or —PEGn-Biotin where n is 3-12.
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`The term “streptavidin” refers to both streptavidin and avidin, as well as any
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`variants thereof that bind to biotin with high affinity.
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`The term “marker” refers to an analyte whose presence or abundance in a
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`biological sample is correlated with a disease or condition.
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`The term “bond” includes covalent and non-covalent bonds, including
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`hydrogen bonds, ionic bonds and bonds produced by van der Waal forces.
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`The term “amplify” refers to an increase in the magnitude of a signal, e.g., at
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`least a 10-fold increase, at least a 100-fold increase at least a 1,000-fold increase,
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`at least a 10,000-fold increase, or at least a 100,000-fold increase in a signal.
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`Other specific binding conditions are known in the art and may also be
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`It must be noted that as used herein and in the appended claims, the singular
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`forms “a”, “an”, and “the” include plural referents unless the context clearly dictates
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`othen/vise, e.g., when the word “single” is used. For example, reference to “an
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`analyte” includes a single analyte and multiple analytes, reference to “a capture
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`agent” includes a single capture agent and multiple capture agents, and reference to
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`“a detection agent” includes a single detection agent and multiple detection agents.
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`DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
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`The following detailed description illustrates some embodiments of the
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`invention by way of example and not by way of limitation.
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`With reference to Fig. 1A and 1B, disclosed herein is nanodevice 100
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`comprising: (a) substrate 110; and (b) one or a plurality of pillars 115 extending
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`from a surface of the substrate, wherein at least one of the pillars comprises a
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`pillar body 120, metallic disc 130 on top of the pillar, metallic back plane 150 at
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`the foot of the pillar, the metallic back plane covering a substantial portion of the
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`substrate surface near the foot of the pillar; metallic dot structure 130 disposed
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`on sidewall of the pillar and molecular adhesion layer 160 that covers at least a
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`part of the metallic dot structure, and/or the metal disc, and/or the metallic back
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`plane. The underlying structure in this device has been referred as “disk-coupled
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`dots-on-pillar antenna array, (D2PA)” and examples are them have been
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`described (see, e.g., Li et al Optics Express 2011 19, 3925-3936 and
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`WO2012/O24006, which are incorporated by reference).
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`The exterior surface of molecular adhesion layer 160 comprises a
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`capture—agent—reactive group, Le, a reactive group that can chemically react with
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`capture agents, e.g., an amine-reactive group, a thiol-reactive group, a hydroxyl-
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`reactive group, an imidazolyl-reactive group and a guanidinyl-reactive group. For
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`illustrative purposes, the molecular adhesion layer 160 covers all of the exposed
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`surface of metallic dot structure 160, metal disc 130, and metallic back plane
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`150. However, for practical purposes, adhesion layer 160 need only part of the
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`exposed surface of metallic dot structure 160, metal disc 130, or metallic back
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`plane 150. As shown, in certain cases, substrate 110 may be made of a
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`dielectric (e.g., SiOz) although other materials may be used, e.g., silicon, GaAs,
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`polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA). Likewise, the
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`metal may be gold, silver, platinum, palladium, lead, iron, titanium, nickel,
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`copper, aluminum, alloy thereof, or combinations thereof, although other
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`materials may be used, as long as the materials’ plasma frequency is higher
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`than that of the light signal and the light that is used to generate the light signal.
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`Nanodevice 100 is characterized in that it amplifies a light signal that is
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`proximal to the exterior surface of the adhesion layer.
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`In some embodiments, the dimensions of one or more of the parts of the
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`pillars or a distance between two components may be that is less than the
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`wavelength of the amplified light. For example, the lateral dimension of the pillar
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`body 120, the height of pillar body 120, the dimensions of metal disc 130, the
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`distances between any gaps between metallic dot structures 140, the distances
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`between metallic dot structure 140 and metallic disc 130 may be smaller than the
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`wavelength of the amplified light. As illustrated in Fig. 1A, the pillars may be
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`arranged on the substrate in the form of an array. In particular cases, the nearest
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`pillars of the array may be spaced by a distance that is less than the wavelength of
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`the light. The pillar array can be periodic and aperiodic.
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`The nanodevice may be disposed within a container, e.g., a well of a
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`multi—well plate. The nanodevice also can be the bottom or the wall of a well of a
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`multi—well plate. The nanodevices may be disposed inside a microfluidic channel
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`(channel width of 1 to 1000 micrometers) or nanofluidic channel (channel width
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`less 1 micrometer) or a part of inside wall of such channels.
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`As will be described in greater detail below (and as illustrated in Fig. 1C), a
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`subject nanodevice 100 may be fabricated by coating a so-called “disc-coupled
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`dots-an-pillar antenna array" 200 (Le, a “D2PA”, which is essentially composed of
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`substrate 110 and a plurality of pillars that comprise pillar body 120, metallic disc
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`130, metallic back plane 150 and metallic dot structures 140 with a molecular
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`adhesion layer 160. A detailed description an exemplary D2PA that can be
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`employed in a subject nanodevice are provided in WO2012/024006, which is
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`incorporated by reference herein for disclosure for all purposes.
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`The first part of the description that follows below describes certain features
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`(i.e., the substrate, the pillar body, the metallic disc, the metallic back plane and the
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`metallic dot structures) of the underlying D2PA structure. The second part of the
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`description that follows below describes the molecular adhesion layer, the capture
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`Disc-coupled dots-on-pillar antenna arrays (D2PA)
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`A disc-coupled dots-on-pillar antenna array has a 3D plasmon cavity antenna
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`with a floating metallic disc or nanodisc that is coupled to nanoscale metallic dots on
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`a pillar body. Specifically,
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`in some embodiments the D2PA has a substrate, a pillar
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`array on the substrate, a metallic disc or nanodisc on top of each of the pillars,
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`nanoscale metallic dots on the pillar sidewall, with gaps between the disc and some
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`of the dots, gaps between the neighboring dots, and a metallic back-plane which
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`covers the most of the substrate areas that are not occupied by the pillars.
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`In one embodiment, the pillar array is fabricated from SiOz with a 200 nm
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`pitch, 130 nm height, and 70 nm diameter on the substrate, formed from silicon. The
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`metallic back-plane may be formed from a 40 nm thick layer of gold, deposited on
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`the pillar array structures and substrate using e-beam evaporation along the normal
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`direction. The deposition process forms the metallic discs in gold on top of each
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`SiOz pillar while simultaneously forming the gold nanohole metallic back plane on
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`the surface of the silicon substrate. Each disc has a thickness of 40 nm and
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`diameter about 110 nm. During the evaporation process, with a deposition rate of
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`about 0.4 A/s, the gold atoms diffuse onto the sidewalls of the SiOg pillars and
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`congregate into random particles with granule sizes between 10 nm and 30 nm,
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`forming the nanoscale metallic dots.
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`A substrate with the gold nanodiscs, random gold nanoparticle metallic dots,
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`and bottom gold nanohole plate (back-plane) is formed by the evaporation process.
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`The gold nanoparticles scattered on the sidewall of the SiOz pillars, forming the
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`nanoscale metallic dots, have narrow gaps of about 0.5 nm — 20 nm between them,
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`which can induce highly enhanced electrical fields. As used herein, the term “gap” is
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`defined as the m