Impact of Redox-Active Molecules on the Fluorescence of Polymer-Wrapped Carbon Nanotubes

The near-infrared (nIR) fluorescence of polymer-wrapped single-walled carbon nanotubes (SWCNTs) is very sensitive to the local chemical environment. It has been shown that certain small reducing molecules can increase the fluorescence of SWCNTs. However, so far the role of the polymer around the SWCNT as well as the mechanism is not understood. Here, we investigated how reducing and oxidizing small molecules affect the nIR fluorescence of polymer-wrapped SWCNTs. Our results show that the polymer plays an essential role. Reducing molecules such as ascorbic acid, epinephrine, and trolox increased the nIR fluorescence up to 250% but only if SWCNTs were suspended in negatively charged polymers such as DNA or poly­(acrylic acid) (PAA). In comparison, phospholipid–poly­(ethylene glycol) wrapped SWCNTs did not respond at all while positively charged polyallylamine-wrapped SWCNTs were quenched. Oxidized equivalents such as dehydroascorbic acid did not show a clear tendency to quench or increase fluorescence. Only riboflavin with an intermediate oxidation potential and light absorption in the visible range quenched all polymer-wrapped SWCNTs. In general, polymer-wrapped SWCNTs that responded to reducing molecules (e.g., +141%, ascorbic acid) also responded to oxidizing molecules (e.g., −81%, riboflavin). Nevertheless, several reducing molecules showed only a small fluorescence increase (NADH, +21%) or even a decrease (glutathione, −14%), which highlights that the redox potential alone cannot explain fluorescence changes. Furthermore, we show that neither changes of absorption cross sections, scavenging of reactive oxygen species (ROS), nor free surface areas on SWCNTs explain the observed patterns. However, results are in agreement either with a redox reaction of the polymer or conformational changes of the polymer that change fluorescence decay routes. In summary, we show that the polymer around SWCNTs governs how redox-active molecules change nIR fluorescence (quantum yield) of SWCNTs. Molecules with a low redox potential (<−0.4 V) are more likely to increase SWCNT fluorescence, but a low redox-potential alone is not sufficient

Control of Integrin Affinity by Confining RGD Peptides on Fluorescent Carbon Nanotubes

Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via noncovalent adsorption of single-stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near-infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing. The basic idea of this approach is that the DNA adsorbed on the SWCNT surface determines the conformational freedom of the RGD motif and affects binding affinities. The RGD motif was conjugated to different ssDNA sequences in both linear ssDNA–RGD and bridged ssDNA–RGD–ssDNA geometries. Molecular dynamics (MD) simulations show that the RGD motif in all the synthesized systems is mostly exposed to solvent and thus available for binding, but its flexibility depends on the exact geometry. The affinity for the human platelet integrin α_IIbβ₃ could be modulated up to 15-fold by changing the ssDNA sequence. IC₅₀ values varied from 309 nM for (C)₂₀–RGD/SWCNT hybrids to 29 nM for (GT)₁₅–RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)₁₅–RGD/SWCNTs. (GT)₁₅–RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, α_IIbβ₃ integrins on human platelets were labeled in the nIR by incubating them with these ssDNA–peptide/SWCNT hybrids. In summary, we show that ssDNA–peptide hybrid structures noncovalently adsorb onto SWCNTs and serve as recognition units for cell surface receptors such as integrins. The DNA sequence affects the overall RGD affinity, which is a versatile and straightforward approach to tune binding affinities. In combination with the nIR fluorescence properties of SWCNTs, these new hybrid materials promise many applications in integrin targeting and bioimaging

Chemotaxis of <em>Dictyostelium discoideum:</em> Collective Oscillation of Cellular Contacts

<div><p>Chemotactic responses of <em>Dictyostelium discoideum</em> cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single <em>Dictyostelium discoideum</em> cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of <em>Dictyostelium discoideum</em> cells.</p> </div

Impedance signal of <i>D. discoideum</i>.

<p>Magnitude of detrended impedance signal at 4 kHz |<i>Z</i>_Detrend|_{4 kHz} of <i>D. discoideum</i> (3,750 cells mm⁻² added in Sorenseńs buffer) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054172#pone-0054172-g001" target="_blank">Figure 1</a> C. Cells were seeded at <i>t</i> = 0 min on a circular gold electrode ( = 250 µm). Boxes highlight magnification of the impedance signal. Data were smoothed by subtracting a moving average algorithm (box size: 800 points) to remove long-term trends.</p

D-QCM of <i>D. discoideum</i> chemotaxis.

<p>D-QCM measurement of starved <i>D. discoideum</i> amebas. Shift in resonance frequency (red) and damping (black) of an oscillating quartz crystal as a function of time. <i>D. discoideum</i> cells (10,000 cells mm⁻²) were seeded at <i>t</i> = 0 on a gold-electrode. The black box highlights the time period during which collective oscillations occur.</p

Changes in circularity of <i>D. discoideum</i>.

<p>A) Optical micrographs of the ameba-covered electrode taken at two different times as marked by the dashed line in (B). Cells’ perimeters were manually surrounded by a green line to compute circularity. For sake of clarity, the electrode is eradicated from the bottom part of the images. B) Circularity <<i>C</i>> of <i>D. discoideum</i> cells determined as a function of time (red dots). Additionally, the corresponding time series of the detrended impedance values |<i>Z</i>_Detrend|_{4 kHz} from the same area is shown in blue.</p

Origin of impedance oscillation

<p>. The scheme illustrates how circularity , number of isolated amebas no., light intensity of subtracted bright field BF, and fluorescence intensity from TIRF mages (TIRF) correspond temporally to the measured impedance spikes |<i>Z</i>|.</p

TIRF analysis of <i>D. discoideum</i> chemotaxis.

<p>A) Bright field and B) TIRF microscopy image sections of cells (8,000 cells mm^-²) starved for 5 h on a glass substrate recorded at three distinct time points (I, II, III). The bright field images are recorded 10 second earlier than the TIRF images presented beneath. The arrow exemplarily highlights one particular cell during the stages I-III. C) Plot of the measured total fluorescence intensity of the whole TIRF image as a function of time shows an oscillation, which is cropped and magnified in the second plot. The first image (I) in B corresponds to a peak minimum of the fluorescence intensity and the third image (III) to a peak maximum.</p

Cross correlation coefficients.

<p>Cross-correlation coefficient <i>ρ</i> of the corresponding descriptors, cell number <i>N</i>, mean covered area per cell <i>A</i>, mean circularity <<i>C</i>>, TIRF intensity (time axis multiplied with 1.94 to match periodicity of impedance spikes), and number of isolated cells <i>N</i>_s with impedance time traces. Amebas were displaying oscillations in impedance recorded at 4 kHz generator frequency.</p

Periodic clustering of <i>D. discoideum</i>.

<p>A) Optical micrographs (bright field images) of the cell-covered electrode at <i>t_I</i> = 387.2 min and <i>t</i>_II = 390.2 min after seeding of <i>D. discoideum</i> cells (3750 cells mm⁻²). The cells marked in green are isolated amebas, while blue color indicates cells belonging to a 2-D aggregate or cluster. B) Time series of the number of isolated cells derived from image analysis (red curve). Time points labeled with gray lines correspond to the images shown in (A). Additionally, the corresponding time series of the simultaneously acquired detrended impedance values |<i>Z</i>_Detrend| are shown as a blue line.</p