Characterization of cells from the cervical lymph nodes of C57BL/6 and C57BL/6.NOD-<i>Aec1Aec2</i> mice using microengraving.

<p><b>A.</b> Representative micrographs of live cells from C57BL/6 cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, C57BL/6 (B6) salivary glands proteins labeled with Alexa Fluor 594 and C57BL/6.NOD-<i>Aec1Aec2</i> (SjS) salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features (arrows) (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. <b>B.</b> Representative micrographs of live cells from C57BL/6.NOD-<i>Aec1Aec2</i> cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, B6 salivary glands proteins labeled with Alexa Fluor 594 and SjS salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features pointed by the arrows (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. All experiments were repeated at least twice for consistency.</p

Frequency of IgG1.

<p>Enumeration of IgG1-secretion cells from arrays of nanowells occupied by single cells from the spleens and cervical lymph nodes of C57BL/6 (n = 4) and C57BL/6.NOD-<i>Aec1Aec2</i> mice (n = 4). Data extracted from the image processing using Genepix software were used to identify the appropriate signals. The data were correlated with the nanowell image data in which nanowells contained a single cell positive for both Calcein (live cells) and CD19. The frequency was determined by using the ratio of positive IgG1 signal from wells with single cells and the total number of wells with single cells. *p<0.05 by unpaired t test. NS: not significant.</p

Illustration of microengraving.

<p>Arrays of nanowells with dimensions of 50 µm×50 µm×50 µm were used for microengraving. Spleen or cervical lymph nodes cells were loaded in the nanowells. Cells in the nanowells were imaged using an automated epifluorescence microscope. Micrograving is performed by hybridizing nanowells with capture slides containing anti-mouse Ig for 2 hrs at 37°C with 5% CO₂. After incubation, nanowells containing intact live cells and capture slides were separated. A mixture of antibodies containing IgG1-Alexa Fluor 488, B6 SG lysate-Alexa Fluor 594 and <i>Aec1Aec2</i> SG lysate-Alexa Fluor 555 were added to the capture slides. Micrographs of microarrays were generating by scanning using a Genepix 4200AL microarray scanner.</p

Driving Chemical Reactions in Plasmonic Nanogaps with Electrohydrodynamic Flow

Nanoparticles from colloidal solutionwith controlled composition, size, and shapeserve as excellent building blocks for plasmonic devices and metasurfaces. However, understanding hierarchical driving forces affecting the geometry of oligomers and interparticle gap spacings is still needed to fabricate high-density architectures over large areas. Here, electrohydrodynamic (EHD) flow is used as a long-range driving force to enable carbodiimide cross-linking between nanospheres and produces oligomers exhibiting sub-nanometer gap spacing over mm² areas. Anhydride linkers between nanospheres are observed <i>via</i> surface-enhanced Raman scattering (SERS) spectroscopy. The anhydride linkers are cleavable <i>via</i> nucleophilic substitution and enable placement of nucleophilic molecules in electromagnetic hotspots. Atomistic simulations elucidate that the transient attractive force provided by EHD flow is needed to provide a sufficient residence time for anhydride cross-linking to overcome slow reaction kinetics. This synergistic analysis shows assembly involves an interplay between long-range driving forces increasing nanoparticle–nanoparticle interactions and probability that ligands are in proximity to overcome activation energy barriers associated with short-range chemical reactions. Absorption spectroscopy and electromagnetic full-wave simulations show that variations in nanogap spacing have a greater influence on optical response than variations in close-packed oligomer geometry. The EHD flow–anhydride cross-linking assembly method enables close-packed oligomers with uniform gap spacings that produce uniform SERS enhancement factors. These results demonstrate the efficacy of colloidal driving forces to selectively enable chemical reactions leading to future assembly platforms for large-area nanodevices