Laser-Induced Cell Detachment, Patterning, and Regrowth on Gold Nanoparticle Functionalized Surfaces

We report on the selective cell detachment from nanoengineered gold nanoparticle (AuNP) surfaces triggered by laser irradiation, which occurs in a nonthermal manner. The gold nanoparticle-based surfaces reveal good adhesion of NIH3T3 fibroblast cells. Patterning is achieved by lithographic microcontact printing, selective gold nanoparticle deposition, and by laser beam profiling. It is shown that the effectiveness of fibroblast cell detachment depends on the cell age, laser power, and AuNP patterning profile. Heat distribution and temperature rise around gold nanoparticle functionalized surfaces is modeled, revealing low heating of nanoparticles by laser illumination. The nonthermal photochemical mechanism of cell detachment due to production of reactive oxygen species under illumination of gold nanoparticles by green laser light is studied. We also demonstrate that cells migrate from unirradiated areas leading to their reattachment and surface recovery which is important for controlled spatial organization of cells in wound healing and tissue engineering. Research presented in this work is targeted at designing biointerfaces for cell cultures

Protein A Functionalized Polyelectrolyte Microcapsules as a Universal Platform for Enhanced Targeting of Cell Surface Receptors

Targeted delivery systems recognizing specific receptors are a key element in personalized medicine. Such systems allow the delivery of therapeutics to desired sites of the body, increasing their local concentration and thus reducing the side effects. In this study, we fabricate chemically cross-linked (PAH/PAA)₂ microcapsules coated with specific cell-targeting antibodies in random (via direct covalent coupling to the surface) or optimized (via supporting layer of protein A) orientation. We use these antibody-functionalized capsules to target major histocompatibility complex (MHC) class I receptors in living cells and quantify the efficiency of targeting by flow cytometry. We show for the first time the selective binding of polyelectrolyte microcapsules to MHC class I receptors, and confirm that targeting is allotype-specific. Remarkably, protein A assisted immobilization of antibodies enhances targeting efficiency by 40–50% over capsules with randomly attached antibodies. Moreover, biofunctionalized capsules reveal low levels of cytotoxicity and nonspecific binding, excluding the need of additional modification with poly­(ethylene glycol). Thus, protein A coated (PAH/PAA)₂ microcapsules represent a unique example of universal targeting tools providing high potential for selective binding to a broad range of cell surface receptors

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids

<div><p>To detect and study diseases, research and clinical laboratories must quantify specific biomarkers in the plasma and urine of patients with precision, sensitivity, and cost-effectiveness. Newly developed techniques, such as particle-based immunoassays, must be validated in these terms against standard methods such as enzyme-linked immunosorbent assays (ELISAs). Here, we compare the performance of assays that use hollow polyelectrolyte microcapsules with assays based on solid plastic beads, and with standard microplate immunoassays. The polyelectrolyte microcapsules detect the disease biomarker beta-2 microglobulin with a fifty-fold increase in sensitivity than polystyrene (PS) beads. For sequence-specific nucleic acid detection, the oligonucleotide-coated microcapsules exhibit a two-fold lower increase in sensitivity over PS beads. The microcapsules also detect the presence of a monoclonal antibody in hybridoma supernatant at a fifty-six-fold increase in sensitivity compared to a microplate assay. Overall, polyelectrolyte microcapsule-based assays are more sensitive for the detection of protein and nucleic acid analytes than PS beads and microplate assays, and they are viable alternatives as a platform for the rapid quantitative detection of analytes at very low concentrations.</p></div

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids - Fig 3

<p><b>Comparison of microcapsules and PS beads for the detection of nucleic acids:</b> (A) Schematics. The anchor, Biotin-Oligo2, is attached to streptavidin-coated microcapsules or PS beads andhybridizes specifically to its complementary sequence on the analyte and on Oligo3, which in turn hybridizes to its complementary sequence on the detector, Oligo4-FITC. (B) Analyte dose-response of the assay for streptavidin-coated microcapsules and PS beads measured by flow cytometry. MFI values were normalized by the maximum value. The error bars are SD (n = 3). Invisible error bars are smaller than the size of the marker. (C) Background controls for the assay in (B). MFI values were normalized to the maximum value. The error bars indicate the SD (n = 3). (D) Hybridization scheme of the three oligonucleotides used in the current assay. Biotin-Oligo2 hybridizes to the 17 complementary nucleotide bases (brown) of analyte Oligo3, and the remaining free 17 bases of the analyte hybridize to the detector Oligo4-FITC (green).</p

Analytical performance of hβ₂m and oligonucleotide detection using microcapsule and PS beads: Best fit values were obtained with a four-parameter fit equation.

<p>Limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) were determined as described in the materials and methods.</p

Analytical performance of BBM.1 detection using microcapsule and microplate for BBM.1 hybridoma: Best fit values were obtained with a four-parameter fit equation.

<p>LoB, LoD, and LoQ were determined as described in the materials and methods.</p

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids - Fig 2

<p><b>Comparison of 6 μm microcapsules and 2.35 μm PS beads for binding of antibody and for the detection of a protein analyte:</b> (A+B) Detection of the capture antibody, BBM.1, on microcapsules and beads with fluorescently labeled goat anti-mouse antibody (GαM-AF488) by flow cytometry. (A) shows a representative experiment, (B) the average of two experiments with standard deviations (SD). (C) Schematics for the detection of human beta-2 microglobulin (hβ₂m). The capture antibody, BBM.1, is immobilized on the protein A-coated microcapsules/PS beads. BBM.1 antibody binds specifically to hβ₂m, which is sandwiched by the polyclonal rabbit anti-hβ₂M (Rαhβ₂M) antibody. The sandwich is then detected by adding AF488 labeled goat anti-rabbit (GαR-AF488) antibody. (D) Detection of hβ₂m in PBS. Dose-response curves for the assay performed as in (C) with microcapsules or PS beads. MFI values are normalized to the maximum values. Error bars are SD (n = 3). Invisible error bars are smaller than the size of the marker. (E) Control samples of hβ₂m plotted as histograms. Experiments were performed as in (C). Samples with analyte (10⁵ pg mL^-1 for microcapsules and 10⁶ pg mL^-1 for PS beads) were used as positive control and for normalization, which was done individually for microcapsules and PS beads. Error bars are SD (n = 3).</p