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Noninvasive ovarian cancer biomarker detection via an optical nanosensor implant

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Science Advances  18 Apr 2018:
Vol. 4, no. 4, eaaq1090
DOI: 10.1126/sciadv.aaq1090
  • Fig. 1 Design and in vitro characterization of optical nanosensor for HE4.

    (A) Scheme of Ab-DNA-SWCNT complex synthesis and proposed nanosensor function. a.u., arbitrary units. (B) Correlograms from a dynamic light scattering instrument showing correlation coefficient of pre-Ab– and post-Ab–conjugated ssDNA-SWCNT samples. n = 3 for each complex. (C) Electrophoretic light scattering of ssDNA-SWCNT before and after anti-HE4 antibody conjugation. n = 3, mean ± SD; **P < 0.01, t test. (D) Representative absorbance spectra of the hybridized ssDNA-SWCNT before and after conjugation of the anti-HE4 antibody. Inset: Representative PL excitation/emission plot of the Ab-DNA-SWCNT sensor. (E) Dose-response curve of the Ab-DNA-SWCNT sensor emission [of the (9,4) nanotube species] as a function of HE4 concentration in 10% fetal bovine serum (FBS). Each point is the mean of three experiments ± SD. (F) Response of the Ab-DNA-SWCNT complex to interferent proteins. n = 3, mean ± SD; control and HE4, P = 1.0 × 10−4; control and bovine serum albumin (BSA), P = 0.998; control and CA-125, P = 0.163; control and urokinase plasminogen activator (uPA), P = 1.0 × 10−3; control and FBS, P = 0.64 [two-sided one-way analysis of variance (ANOVA) with Dunnett’s post hoc analysis]. NS, not significant. (G) Representative kinetic response of nanotube emission upon introducing recombinant HE4.

  • Fig. 2 Single-sensor HE4 measurements in patient biofluids.

    (A) NIR image of Ab-DNA-SWCNT complexes adsorbed to a glass surface. Scale bar, 5 μm. (B) Representative spectra of a single complex, denoted by the green circle in (A), before and 10 min after introducing recombinant HE4. RFU, relative fluorescence units. (C) Shift in sensor emission wavelength 10 min after addition of recombinant HE4 or 10% FBS. Pre- to post-FBS, *P = 0.03; pre-HE4 to post-HE4, *P = 0.04 (two-sided t test). n = 82 single nanotubes before and 98 after FBS, 100 before HE4, and 97 after HE4. Data shown are means ± SEM. (D) Sensor response to serum. n = 3 HGSC patients or patients with benign conditions. HE4 concentrations, measured independently via enzyme-linked immunosorbent assay (ELISA), are specified for each sample. *P = 0.015, two-sided t test. (E) Sensor response to HGSC patient ascites or peritoneal fluid from patients with benign conditions. n = 3. HE4 concentrations, measured independently via ELISA, are specified for each sample, with one exception due to sample volume limitation. *P = 0.03, two-sided t test.

  • Fig. 3 Implantable nanosensor device.

    (A) Semipermeable 500-kDa MWCO membrane capillary incorporating Ab-DNA-SWCNT complexes. Spectrum of the nanosensor acquired through the capillary wall. (B) Emission wavelength from the sensor after introducing recombinant HE4. n = 3, mean ± SD; ***P = 7.2 × 10−6, two-sided t test. (C) NIR image of nanosensor emission from the implanted device, overlaid onto a reflected light image of the mouse. (D) Photograph of typical data acquisition from the probe-based system used to excite/acquire NIR emission from the implanted sensor in mice. (E) Representative NIR fluorescence spectra of sensors implanted into healthy mice and measured 1 hour and 38 days after implantation.

  • Fig. 4 In vivo measurement of HE4 in orthotopic ovarian cancer models.

    (A) Change in emission center wavelength in mice following intraperitoneal injection of 10 pmol of BSA or HE4, compared to emission in uninjected mice. Bold lines represent mean ± SD. n = 3. Lighter lines represent measurements from each mouse. (B) Change in emission center wavelength at 60 min after injection of BSA or HE4 compared to uninjected mice. n = 3, mean ± SD; *P = 0.016, two-sided t test. (C) Representative bioluminescence images denoting tumor burden in the peritoneal cavity of nude mice inoculated with luciferase-expressing cell lines. (D) Schematic method of HE4 measurement in live tumor-bearing mice. (E) Change in sensor emission center wavelength following implantation into mice bearing four different orthotopic intraperitoneal tumor models. Bold lines represent mean ± SD. n = 4. Lighter lines represent traces from the emission within each mouse. (F) Sensor response from all mice at 60 min after implantation. n = 4, mean ± SD. Sensor wavelength difference between models and statistical analysis: SK-OV-3 and OVCAR-3 (0.79 nm; P = 4.4 × 10−3), SK-OV-3 and OVCAR-5 (1.07 nm; P = 3.9 × 10−4), OVCAR-8 and OVCAR-3 (1.07 nm; P = 3.9 × 10−4), OVCAR-8 and OVCAR-5 (1.34 nm; P = 4.5 × 10−5), SK-OV-3 and OVCAR-8 (0.27 nm; P = 0.46), and OVCAR-3 and OVCAR-5 (0.27 nm; P = 0.48) by two-sided one-way ANOVA with Tukey’s post hoc analysis.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaaq1090/DC1

    Supplementary Text

    fig. S1. Characterization of sensor function in vitro.

    fig. S2. Single-sensor HE4 response characterization.

    fig. S3. Images taken during the procedure to surgically implant the sensor devices.

    fig. S4. Baseline subtraction procedure of a spectrum obtained from the implanted sensor device in vivo (E3 = one thousand units).

    fig. S5. Raw and fitted sensor data from all mice in the exogenous protein injection experiment.

    fig. S6. H&E stain of tumor nodules in tissue resected from each murine model of ovarian cancer.

    fig. S7. Sensor implant emission data from all murine orthotopic ovarian cancer models.

    fig. S8. In vivo sensor stability.

    table S1. Change in the nanotube emission wavelength of the DNA-SWCNT following conjugation of the anti-HE4 antibody to the DNA.

    table S2. Concentration of HE4 in conditioned cell culture media and ascites from representative mouse ascites samples as determined by ELISA of SK-OV-3, OVCAR-8, OVCAR-3, and OVCAR-5 models in vitro and in vivo.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • fig. S1. Characterization of sensor function in vitro.
    • fig. S2. Single-sensor HE4 response characterization.
    • fig. S3. Images taken during the procedure to surgically implant the sensor devices.
    • fig. S4. Baseline subtraction procedure of a spectrum obtained from the implanted sensor device in vivo (E3 = one thousand units).
    • fig. S5. Raw and fitted sensor data from all mice in the exogenous protein injection experiment.
    • fig. S6. H&E stain of tumor nodules in tissue resected from each murine model of ovarian cancer.
    • fig. S7. Sensor implant emission data from all murine orthotopic ovarian cancer models.
    • fig. S8. In vivo sensor stability.
    • table S1. Change in the nanotube emission wavelength of the DNA-SWCNT following conjugation of the anti-HE4 antibody to the DNA.
    • table S2. Concentration of HE4 in conditioned cell culture media and ascites from representative mouse ascites samples as determined by ELISA of SK-OV-3, OVCAR-8, OVCAR-3, and OVCAR-5 models in vitro and in vivo.

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