Polystyrene beads as probes of the surface-enhanced Raman scattering response characteristics of silver nanorod arrays.

Polystyrene microspheres were used to interrogate the surface-enhanced Raman scattering (SERS) response of silver nanorod (AgNR) array substrates. It has been suggested that enhancement between nanorods is significantly greater than that at the top due to contributing electromagnetic fields from each nanostructure. To test this theory, two different sized fluorescent polystyrene microspheres were used. The SERS intensity of beads small enough to fit within the array was compared with that of larger beads confined to rest on top of the array. Location of the beads within the array was established using optical fluorescence and scanning electron microscopy. The findings presented herein suggest that evaporation of the sample produces a nonuniform distribution of scatterers across the AgNR array and that the enhancement found for beads located on top of the AgNRs was significantly greater than that for beads located within the array.

Limitations of surface enhanced Raman scattering in sensing DNA hybridization demonstrated by label-free DNA oligos as molecular rulers of distance-dependent enhancement.

This article presents a critical evaluation of silver nanorod arrays as substrates for assaying nucleic acid hybridization by surface enhanced Raman scattering (SERS). SERS spectra acquired on complementary oligos, alone or in combination, contain the known spectral signatures of the nucleotides that comprise the oligo; however, no signature bands characteristic of the hybrid were observed. Spectra acquired on an oligo with a 5'- or 3'-thiol were distinctly different from that acquired on the identical oligo without a thiol pendant group suggesting a degree of control over the orientation of the oligo on the nanorod surface. A set of oligos consisting of adenine tracts in a polycytosine chain served as molecular rulers to probe the distance dependence of the SERS enhancement. Using these, we have identified the point at which the characteristic bands for the nucleotides that comprise the oligo disappear from the spectrum. These findings suggest that the applicability of SERS for label-free detection of nucleic acid hybridization is limited to short oligos of less than nine nucleotides.

Myocardial tissue elastic properties determined by atomic force microscopy after stromal cell-derived factor 1α angiogenic therapy for acute myocardial infarction in a murine model.

OBJECTIVES: Ventricular remodeling after myocardial infarction begins with massive extracellular matrix deposition and resultant fibrosis. This loss of functional tissue and stiffening of myocardial elastic and contractile elements starts the vicious cycle of mechanical inefficiency, adverse remodeling, and eventual heart failure. We hypothesized that stromal cell-derived factor 1α (SDF-1α) therapy to microrevascularize ischemic myocardium would rescue salvageable peri-infarct tissue and subsequently improve myocardial elasticity. METHODS: Immediately after left anterior descending coronary artery ligation, mice were randomly assigned to receive peri-infarct injection of either saline solution or SDF-1α. After 6 weeks, animals were killed and samples were taken from the peri-infarct border zone and the infarct scar, as well as from the left ventricle of noninfarcted control mice. Determination of tissues' elastic moduli was carried out by mechanical testing in an atomic force microscope. RESULTS: SDF-1α-treated peri-infarct tissue most closely approximated the elasticity of normal ventricle and was significantly more elastic than saline-treated peri-infarct myocardium (109 ± 22.9 kPa vs 295 ± 42.3 kPa; P < .0001). Myocardial scar, the strength of which depends on matrix deposition from vasculature at the peri-infarct edge, was stiffer in SDF-1α-treated animals than in controls (804 ± 102.2 kPa vs 144 ± 27.5 kPa; P < .0001). CONCLUSIONS: Direct quantification of myocardial elastic properties demonstrates the ability of SDF-1α to re-engineer evolving myocardial infarct and peri-infarct tissues. By increasing elasticity of the ischemic and dysfunctional peri-infarct border zone and bolstering the weak, aneurysm-prone scar, SDF-1α therapy may confer a mechanical advantage to resist adverse remodeling after infarction.

Computational protein design to reengineer stromal cell-derived factor-1α generates an effective and translatable angiogenic polypeptide analog.

BACKGROUND: Experimentally, exogenous administration of recombinant stromal cell-derived factor-1α (SDF) enhances neovasculogenesis and cardiac function after myocardial infarction. Smaller analogs of SDF may provide translational advantages including enhanced stability and function, ease of synthesis, lower cost, and potential modulated delivery via engineered biomaterials. In this study, computational protein design was used to create a more efficient evolution of the native SDF protein. METHODS AND RESULTS: Protein structure modeling was used to engineer an SDF polypeptide analog (engineered SDF analog [ESA]) that splices the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a diproline segment designed to limit the conformational flexibility of the peptide backbone and retain the relative orientation of these segments observed in the native structure of SDF. Endothelial progenitor cells (EPCs) in ESA gradient, assayed by Boyden chamber, showed significantly increased migration compared with both SDF and control gradients. EPC receptor activation was evaluated by quantification of phosphorylated AKT, and cells treated with ESA yielded significantly greater phosphorylated AKT levels than SDF and control cells. Angiogenic growth factor assays revealed a distinct increase in angiopoietin-1 expression in the ESA- and SDF-treated hearts. In addition, CD-1 mice (n=30) underwent ligation of the left anterior descending coronary artery and peri-infarct intramyocardial injection of ESA, SDF-1α, or saline. At 2 weeks, echocardiography demonstrated a significant gain in ejection fraction, cardiac output, stroke volume, and fractional area change in mice treated with ESA compared with controls. CONCLUSIONS: Compared with native SDF, a novel engineered SDF polypeptide analog (ESA) more efficiently induces EPC migration and improves post-myocardial infarction cardiac function and thus offers a more clinically translatable neovasculogenic therapy.

Oxygen-dependent quenching of phosphorescence used to characterize improved myocardial oxygenation resulting from vasculogenic cytokine therapy.

This study evaluates a therapy for infarct modulation and acute myocardial rescue and utilizes a novel technique to measure local myocardial oxygenation in vivo. Bone marrow-derived endothelial progenitor cells (EPCs) were targeted to the heart with peri-infarct intramyocardial injection of the potent EPC chemokine stromal cell-derived factor 1α (SDF). Myocardial oxygen pressure was assessed using a noninvasive, real-time optical technique for measuring oxygen pressures within microvasculature based on the oxygen-dependent quenching of the phosphorescence of Oxyphor G3. Myocardial infarction was induced in male Wistar rats (n = 15) through left anterior descending coronary artery ligation. At the time of infarction, animals were randomized into two groups: saline control (n = 8) and treatment with SDF (n = 7). After 48 h, the animals underwent repeat thoracotomy and 20 µl of the phosphor Oxyphor G3 was injected into three areas (peri-infarct myocardium, myocardial scar, and remote left hindlimb muscle). Measurements of the oxygen distribution within the tissue were then made in vivo by applying the end of a light guide to the beating heart. Compared with controls, animals in the SDF group exhibited a significantly decreased percentage of hypoxic (defined as oxygen pressure ≤ 15.0 Torr) peri-infarct myocardium (9.7 ± 6.7% vs. 21.8 ± 11.9%, P = 0.017). The peak oxygen pressures in the peri-infarct region of the animals in the SDF group were significantly higher than the saline controls (39.5 ± 36.7 vs. 9.2 ± 8.6 Torr, P = 0.02). This strategy for targeting EPCs to vulnerable peri-infarct myocardium via the potent chemokine SDF-1α significantly decreased the degree of hypoxia in peri-infarct myocardium as measured in vivo by phosphorescence quenching. This effect could potentially mitigate the vicious cycle of myocyte death, myocardial fibrosis, progressive ventricular dilatation, and eventual heart failure seen after acute myocardial infarction.

Removal of surface contamination and self-assembled monolayers (SAMs) from silver (Ag) nanorod substrates by plasma cleaning with argon.

Surface contamination of surface-enhanced Raman (SERS)-active metallic substrates has been a limitation to the utility of SERS as an analytical technique, potentially affecting surface coverage, spectral reproducibility, and analytical limits of detection. We have developed a simple and versatile cleaning method for SERS-active Ag nanorod arrays that consists of a short (4 min) exposure of the substrate to an Ar(+) plasma in a low-pressure environment. The findings presented here demonstrate that this cleaning procedure essentially eliminates organic background contamination. This procedure works equally well for self-assembled monolayers of thiolates that strongly adsorb onto Au and Ag surfaces. For SERS-active surfaces composed of arrays of Ag nanorods prepared by oblique-angle vapor deposition, we investigated the (1) Raman band intensities, (2) nanorod morphology via scanning electron microscopy, and (3) surface hydrophobicity via static contact angle measurements, as a function of exposure time of the Ag nanorods to the Ar(+) plasma. Short (4 min) exposure to Ar(+) plasma eliminated background contamination but decreased the observed SERS intensity for re-adsorbed analytes by approximately a factor of 2 while leaving the nanorod morphology essentially unchanged. Prolonged exposure to Ar(+) plasma (>10 min) resulted in substantial morphological changes of the Ag nanorod lattice and led to a decrease in the observed SERS intensities by a factor of 10. The results presented here suggest that Ar(+) plasma cleaning is an efficient process for removing carbonaceous and organic contamination as well as thiolate monolayers from SERS-active Ag surfaces, as long as the plasma conditions and exposure times are carefully monitored.

Spliced stromal cell-derived factor-1α analog stimulates endothelial progenitor cell migration and improves cardiac function in a dose-dependent manner after myocardial infarction.

OBJECTIVES: Stromal cell-derived factor (SDF)-1α is a potent endogenous endothelial progenitor cell (EPC) chemokine and key angiogenic precursor. Recombinant SDF-1α has been demonstrated to improve neovasculogenesis and cardiac function after myocardial infarction (MI) but SDF-1α is a bulky protein with a short half-life. Small peptide analogs might provide translational advantages, including ease of synthesis, low manufacturing costs, and the potential to control delivery within tissues using engineered biomaterials. We hypothesized that a minimized peptide analog of SDF-1α, designed by splicing the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a truncated amino acid linker, would induce EPC migration and preserve ventricular function after MI. METHODS: EPC migration was first determined in vitro using a Boyden chamber assay. For in vivo analysis, male rats (n = 48) underwent left anterior descending coronary artery ligation. At infarction, the rats were randomized into 4 groups and received peri-infarct intramyocardial injections of saline, 3 µg/kg of SDF-1α, 3 µg/kg of spliced SDF analog, or 6 µg/kg spliced SDF analog. After 4 weeks, the rats underwent closed chest pressure volume conductance catheter analysis. RESULTS: EPCs showed significantly increased migration when placed in both a recombinant SDF-1α and spliced SDF analog gradient. The rats treated with spliced SDF analog at MI demonstrated a significant dose-dependent improvement in end-diastolic pressure, stroke volume, ejection fraction, cardiac output, and stroke work compared with the control rats. CONCLUSIONS: A spliced peptide analog of SDF-1α containing both the N- and C- termini of the native protein induced EPC migration, improved ventricular function after acute MI, and provided translational advantages compared with recombinant human SDF-1α.

Stromal cell-derived factor-1alpha activation of tissue-engineered endothelial progenitor cell matrix enhances ventricular function after myocardial infarction by inducing neovasculogenesis.

BACKGROUND: Myocardial ischemia causes cardiomyocyte death, adverse ventricular remodeling, and ventricular dysfunction. Endothelial progenitor cells (EPCs) have been shown to ameliorate this process, particularly when activated with stromal cell-derived factor-1α (SDF), known to be the most potent EPC chemokine. We hypothesized that implantation of a tissue-engineered extracellular matrix (ECM) scaffold seeded with EPCs primed with SDF could induce borderzone neovasculogenesis, prevent adverse geometric remodeling, and preserve ventricular function after myocardial infarction. METHODS AND RESULTS: Lewis rats (n=82) underwent left anterior descending artery ligation to induce myocardial infarction. EPCs were isolated, characterized, and cultured on a vitronectin/collagen scaffold and primed with SDF to generate the activated EPC matrix (EPCM). EPCM was sutured to the anterolateral left ventricular wall, which included the region of ischemia. Control animals received sutures but no EPCM. Additional groups underwent application of the ECM alone, ECM primed with SDF (ECM+SDF), and ECM seeded with EPCs but not primed with SDF (ECM+SDF). At 4 weeks, borderzone myocardial tissue demonstrated increased levels of vascular endothelial growth factor in the EPCM group. When compared to controls, Vessel density as assessed by immunohistochemical microscopy was significantly increased in the EPCM group (4.1 versus 6.2 vessels/high-powered field; P<0.001), and microvascular perfusion measured by lectin microangiography was enhanced 4-fold (0.7% versus 2.7% vessel volume/section volume; P=0.04). Comparisons to additional groups also showed a significantly improved vasculogenic response in the EPCM group. Ventricular geometry and scar fraction assessed by digital planimetric analysis of sectioned hearts exhibited significantly preserved left ventricular internal diameter (9.7 mm versus 8.6 mm; P=0.005) and decreased infarct scar formation expressed as percent of total section area (16% versus 7%; P=0.002) when compared with all other groups. In addition, EPCM animals showed a significant preservation of function as measured by echocardiography, pressure-volume conductance, and Doppler flow. CONCLUSIONS: Extracellular matrix seeded with EPCs primed with SDF induces borderzone neovasculogenesis, attenuates adverse ventricular remodeling, and preserves ventricular function after myocardial infarction.

Surface-enhanced Raman scattering of bacterial cell culture growth media.

The application of surface-enhanced Raman spectroscopy (SERS) to characterizing bacteria is an active area of investigation. Micro- and nano-structured SERS substrates have enabled detection of pathogens present in biofluids. Several publications have focused on determining the spectral bands characteristic of bacteria from different species and cell lines. In this report, the spectra of fifteen commonly used bacterial growth media are presented. In many instances, these spectra are similar to published spectra purportedly characteristic of specific bacterial species. The findings presented herein suggest that bacterial fingerprinting by SERS requires further examination.

Patterned silver nanorod array substrates for surface-enhanced Raman scattering.

A novel method for batch fabrication of substrates for surface-enhanced Raman scattering (SERS) has been developed. A modified platen that fits in a commercial electron beam evaporator enables the simultaneous deposition of Ag nanorod arrays onto six microscope slides by glancing angle deposition. Following removal of substrates from the evaporator, patterned wells are formed by contact printing of a polymer onto the surface. Well dimensions are defined by penetration of the polymer into the nanorod array and subsequent photochemical curing. Inherent advantages of this method include: (1) simultaneous production of several nanorod array substrates with high structural uniformity, (2) physical isolation of nanorod arrays from one another to minimize cross contamination during sample loading, (3) dimensional compatibility of the patterned array with existing SERS microscope, (4) large SERS enhancement afforded by the nanorod array format, (5) small fluid volumes, and (6) ease of use for manual delivery of fluids to each element in the patterned array. In this article, the well-to-well, slide-to-slide, and batch-to-batch variability in physical characteristics and SERS response of substrates prepared via this method is critically examined.