Label-Free Impedance Detection of Cancer Cells

Ovarian cancer cells, SKOV3, have been immobilized onto platinum microelectrodes using anti-EPCAM capture antibodies and detected with high sensitivity using electrochemical impedance. The change in impedance following cell capture is strongly dependent on the supporting electrolyte concentration. By controlling the concentration of Dulbecco’s phosphate buffered saline (DPBS) electrolyte, the double layer thickness can be manipulated so that the interfacial electric field interacts with the bound cells, rather than simply decaying across the antibody capture layer. Significantly, the impedance changes markedly upon cell capture over the frequency range from 3 Hz to 90 kHz. For example, using an alternating-current (ac) amplitude of 25 mV, a frequency of 81.3 kHz, and an open circuit potential (OCP) as the direct-current (dc) voltage, a detection limit of 4 captured cells was achieved. Assuming an average cell radius of 5 μm, the linear dynamic range is from 4 captured cells to 650 ± 2 captured cells, which is approximately equivalent to fractional coverages from 0.1% to 29%. An equivalent circuit that models the impedance response of the cell capture is discussed

RGD Labeled Ru(II) Polypyridyl Conjugates for Platelet Integrin α_IIbβ₃ Recognition and as Reporters of Integrin Conformation

The ability of two novel ruthenium­(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin α_IIbβ₃ in solution and in live cells was described. The compounds are [Ru­(bpy)₂PIC-RGD]²⁺, bpy-RGD, and [Ru­(dpp)₂PIC-RGD]²⁺, dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2′-bipyridine, and PIC is 2-(4-carboxyphenyl)­imidazo­[4,5-<i>f</i>]­[1,10]­phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified α_IIbβ₃ in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (<i>K</i>_d) in the micromolar range for each complex. The <i>K</i>_d for each conjugate/α_IIbβ₃ assembly were compared following treatment of the integrin with the activating agents, Mn²⁺ and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/α_IIbβ₃ <i>K</i>_d showed relatively little variation with integrin activation, presenting the following trend: denatured α_IIbβ₃ > resting α_IIbβ₃ = pretreated DTT = pretreated Mn²⁺. <i>K</i>_d for dpp-RGD/ α_IIbβ₃ showed greater variation with integrin activation and the following trend was followed: denatured α_IIbβ₃ > resting α_IIbβ₃ > pretreated Mn²⁺ = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 ± 0.03 μs and 2.58 ± 0.04 μs, respectively, when the complexes were bound to resting integrin. Addition of Mn²⁺ to bpy-RGD/α_IIbβ₃ or dpp-RGD/α_IIbβ₃ reduced the rotational correlation time of the ruthenium center to 1.29 ± 0.03 μs and to 1.72 ± 0.03 μs, respectively. Following treatment, the rotational correlation time decreased to 1.04 ± 0.01 μs and 1.29 ± 0.03 μs for bpy-RGD/α_IIbβ₃, and dpp-RGD/α_IIbβ₃, respectively. The large relative changes in rotational correlation times observed for Mn²⁺ or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of α_IIbβ₃. In each case the probe colocalized with α_IIb specific SZ22 antibody. Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells

Peptide-Bridged Dinuclear Ru(II) Complex for Mitochondrial Targeted Monitoring of Dynamic Changes to Oxygen Concentration and ROS Generation in Live Mammalian Cells

A novel mitochondrial localizing ruthenium­(II) peptide conjugate capable of monitoring dynamic changes in local O₂ concentrations within living cells is presented. The complex is comprised of luminescent dinuclear ruthenium­(II) polypyridyl complex bridged across a single mitochondrial penetrating peptide, FrFKFrFK-CONH₂ (r = d-arginine). The membrane permeability and selective uptake of the peptide conjugate at the mitochondria of mammalian cells was demonstrated using confocal microscopy. Dye co-localization studies confirmed very precise localization and preconcentration of the probe at the mitochondria. This precision permitted collection of luminescent lifetime images of the probe, without the need for co-localizing dye and permitted semiquantitative determination of oxygen concentration at the mitochondria using calibration curves collected at 37 °C for the peptide conjugate in PBS buffer. Using Antimycin A the ability of the probe to respond dynamically to changing O₂ concentrations within live HeLa cells was demonstrated. Furthermore, based on lifetime data it was evident that the probe also responds to elevated reactive oxygen species (ROS) levels within the mitochondria, where the greater quenching capacity of these species led to luminescent lifetimes of the probe at longer Antimycin A incubation times which lay outside of the O₂ concentration range. Although both the dinuclear complex and a mononuclear analogue conjugated to an octaarginine peptide sequence exhibited some cytotoxicity over 24 h, cells were tolerant of the probes over periods of 4 to 6 h which facilitated imaging. These metal-peptide conjugated probes offer a valuable opportunity for following dynamic changes to mitochondrial function which should be of use across domains in which the metabolic activity of live cells are of interest from molecular biology and drug discovery

Peptide-Mediated Platelet Capture at Gold Micropore Arrays

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine–glycine–aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin α_IIbβ₃), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, α_IIbβ₃. Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells

Enhanced electrochemiluminescence and charge transport through films of metallopolymer-gold nanoparticle composites

Water-soluble 4-(dimethylamino) pyridine (DMAP) stabilized gold nanoparticles (DMAP-AuNP) were synthesized by ligand exchange and phase transfer (toluene/water). The DMAP-AuNPs are positively charged with the core diameter of 4 +/- 1 nm. Metallopolymer-gold nanocomposites were prepared by mixing gold nanoparticles and [Ru(bpy)(2)PVP(10)](ClO(4))(2), in water at different mole ratios: bpy is 2,2'-bipyridyl and PVP is poly (4-vinylpyridine). The photoluminescence emission intensity of the metallopolymer decreases with increasing AuNP loading and approximately 57% of the emission intensity is quenched when the Au NP:Ru mole ratio is 14.8 x 10(-2). The rate or homogeneous charge transfer through thin layers of the nanocomposite deposited oil glassy carbon electrodes increases with increasing nanoparticle loading. The homogeneous charge transport diffusion coefficient, D(CT)., for the composite (AuNP:Ru mole ratio 13.2 x 10(-2)) is (2.8 +/- 0.8) X 10(-11) cm(-2) s(-1) and is approximately 3-fold higher than that found for the pure metallopolymer. Significantly, despite the ability of the metal nanoparticles to quench the ruthenium-based emission, the electrochemiluminescence of the nanocomposite with a AuNP:Ru mole ratio of 4.95 x 10(-2) is approximately three times more intense than the parent metallopolymer. This enhancement arises from the increased rate of charge transport that leads to it greater number of excited states per unit time while minimizing lie quenching effects. The implications of these findings for the design of electrochemiluminescent sensors are discussed

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S_N2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu²⁺ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis­(2,2′-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)­Os­(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10⁶ nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD₅₀ and Comet assay results

Electrochemiluminescent Array to Detect Oxidative Damage in ds-DNA Using [Os(bpy)₂(phen-benz-COOH)]²⁺/Nafion/Graphene Films

Reactive oxygen species (ROS) oxidize guanosines in DNA to form 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a biomarker for oxidative stress. Herein we describe a novel 64-microwell electrochemiluminescent (ECL) array enabling sensitive multiplexed detection of 8-oxodG in ds-DNA without hydrolysis. Films of Nafion and reduced graphene oxide containing ECL dye [Os­(bpy)₂(phen-benz-COOH)]²⁺ (OsNG, {bpy= 2,2′-bipyridine and phen-benz-COOH = (4-(1,10-phenanthrolin-6-yl)­benzoic acid)}) were assembled into microwells on a pyrolytic graphite wafer to detect 8-oxodG in oligonucleotides by electrochemiluminescence (ECL). DNA oxidation by Fenton’s reagent or by ROS formation during redox cycles involving NADPH, Cu^II, and model metabolites was monitored. UPLC-MS/MS of oxidized DNA samples were used for calibration. Detection limit for the fluidic arrays was one 8-oxodG per 670 intact nucleobases, or 0.15%. The method is sensitive enough to evaluate DNA oxidation from biologically relevant ROS-generating reactions of Cu^II, NADPH, and model metabolites

Galectin‑3 Binding to α₅β₁ Integrin in Pore Suspended Biomembranes

Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane’s admittance and fluidity, as well as integrin’s lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan–Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5β1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3−α5β1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid–lectin (GL–Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins