Graphene Oxide Wrapped SERS Tags: Multifunctional Platforms toward Optical Labeling, Photothermal Ablation of Bacteria, and the Monitoring of Killing Effect

As novel optical nanoprobes, surface-enhanced Raman scattering (SERS) tags have drawn growing interests in the application of biomedical imaging and phototherapies. Herein, we demonstrated a novel in situ synthesis strategy for GO wrapped gold nanocluster SERS tags by using a tris­(2,2′-bipyridyl)­ruthenium­(II) chloride (Rubpy)/​GO nanohybrid as a complex Raman reporter, inspired by the role of GO as an artificial receptor for various dyes. The introduction of GO in the synthesis procedure provided systematic solutions for controlling several key parameters of SERS tags, including reproducibility, sensitivity, and colloidal and signal stability. An additional interesting thermal-sensitive SERS property (SERS intensity decreased upon increasing the temperature) was also achieved due to the heat-induced release/redistribution of reporter molecules adsorbed on GO. Combining the synergic effect of these features, we further fabricated multifunctional, aldehyde group conjugated Au@​Rubpy/​GO SERS tags for optical labeling and photothermal ablation of bacteria. Sensitive Raman imaging of gram-positive (<i>Staphylococcus aureus</i>) and gram-negative (<i>Escherichia coli</i>) bacteria could be realized, and satisfactory photothermal killing efficacy for both bacteria was achieved. Our results also demonstrated the correlation among the SERS intensity decrease ratio, bacteria survival rate, and the terminal temperature of the tag–bacteria suspension, showing the possibility to use SERS assay to measure antibacterial response during the photothermal process using this tag

Modelfree analysis.

<p>(A) Order parameters S² of the I214V mutant of RaPrP^C(121–228). Regular secondary structure elements are indicated on the top. The program Fastmodelfree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013273#pone.0013273-Cole1" target="_blank">[63]</a> was used to perform the calculation. Unavailable S² values for a few residues are due to the absence of data or failure in the data fitting. (B) Differences in S² values between the wild-type and the mutant. The difference is calculated according to the equation: ΔS² = S²_mutant−S²_wild-type. The absence of ΔS² values for residues result from unavailable S² values for either the wild-type or the mutant. (C) ΔS² values are mapped onto the tertiary structures of the I214V mutant: blue for ΔS²≥0, red for ΔS²<0, and grey for ΔS² unavailable. This ribbon diagram is generated by PyMol (kindly provided by Prof. DeLano WL).</p

Characterization of monomer and oligomer properties for human and rabbit prion proteins.

<p>Oligomerization of human prion protein (A) and rabbit prion protein (B) in the buffer (20 mM NaOAc, 150 mM NaCl, pH 4.0) was monitored by gel filtration chromatography. The prion protein oligomers were prepared with a buffer (20 mM NaOAc, 150 mM NaCl, pH 4.0) at 47°C. Particle sizes of both human prion protein (C) and rabbit prion protein (D) were analyzed by DLS spectroscopy. Secondary structures of human prion protein (E) and rabbit prion protein (F) were detected by Far-UV CD spectroscopy. Both DLS and CD experiments were performed at 25°C. The buffer used for prion monomers contained 20 mM NaOAc, pH 5.5.</p

Relaxation rates R₁, R₂ and {¹H}-¹⁵N heteronuclear NOEs of the I214V mutant of RaPrP^C(121–228).

<p>Regular secondary structure elements are indicated on the top. The relaxation constants and the experimental errors were extracted by a single exponential curve fitting of the peak heights using Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco).</p

Comparison of recHuPrP^O-induced and recRaPrP^O-induced toxicities on human glioblastoma cell lines U87.

<p>Cells were incubated with oligomeric PrP^O proteins at different concentrations for 48 h (37°C). Cytotoxicity was quanitified as a function of cell viability by the MTS assay (n = 3, mean±SD; *, <i>p</i><0.01; ***, <i>p</i><0.001; by Multiple Comparison Test).</p

Urea-induced transitions of the I214V mutant of RaPrP^C(121–228) characterized by CD spectroscopy.

<p>(A) The folded protein in the absence of urea (solid line) compared with the unfolded protein in the presence of 9 M urea (dashed line). (B) Mean residue ellipticity measured at 222 nm (θ₂₂₂) in the presence of urea of different concentration. θ₂₂₂ in denaturation is indicated by solid squares (▪), while in renaturation by open circles (○). The solid line presents the fitting curve on the basis of a two-state mechanism.</p

Thermal-induced unfolding transitions of recRaPrP^C and recHuPrP^C proteins analyzed by Far-UV CD spectroscopy.

<p>The buffer contained 20 mM NaOAc, 0–200 mM NaCl, pH 5.5. The unfolded fraction calculated from Δε at 222 nm is plotted as a function of temperature.</p

Apparent thermodynamic parameters associated with urea-induced unfolding transitions of recHuPrP^C and recRaPrP^C at 25°C.

<p>The buffer contained 20 mM NaOAc, pH 5.5. ΔG^H2O_N→U is designated as the apparent free energy of unfolding extrapolated to zero concentration of denaturant, m_N→U is the cooperativity of the unfolding transition, and C_m is the concentration of urea required to denature 50% of the protein. The CD spectrum was an average of three consecutive scans. Each experiment was repeated in triplicate for each sample.</p

NaCl concentration-dependent oligomerization of recHuPrP^C and recRaPrP^C monitored by gel filtration chromatography.

<p>The oligomerization experiments of prion proteins were conducted in the buffer (20 mM NaOAc, 50–200 mM NaCl, pH 4.0) at 57°C (n = 3; Error bars, S.D.).</p

Apparent thermodynamic parameters associated with thermal-induced unfolding transitions of recHuPrP^C and recRaPrP^C.

<p>The buffer contained 20 mM NaOAc, 0–200 mM NaCl, pH 5.5. ΔG^0°C_N→U is designated as the apparent free energy of unfolding extrapolated to 0°C, m_N→U is the cooperativity of the unfolding transition, and T_m is the temperature at the midpoint of unfolding. The CD spectrum was an average of three consecutive scans. One experiment was conducted for each sample.</p