Correction: Ion concentration polarization (ICP) of proteins at silicon micropillar nanogaps.

[This corrects the article DOI: 10.1371/journal.pone.0223732.].

Ion concentration polarization (ICP) of proteins at silicon micropillar nanogaps.

Fast detection of low-abundance protein remains a challenge because detection speed is limited by analyte transport to the detection site of a biosensor. In this paper, we demonstrate a scalable fabrication process for producing vertical nanogaps between micropillars which enable ion concentration polarization (ICP) enrichment for fast analyte detection. Compared to horizontal nanochannels, massively paralleled vertical nanogaps not only provide comparable electrokinetics, but also significantly reduce fluid resistance, enabling microbead-based assays. The channels on the device are straightforward to fabricate and scalable using conventional lithography tools. The device is capable of enriching protein molecules by >1000 fold in 10 min. We demonstrate fast detection of IL6 down to 7.4 pg/ml with only a 10 min enrichment period followed by a 5 min incubation. This is a 162-fold enhancement in sensitivity compared to that without enrichment. Our results demonstrate the possibility of using silicon/silica based vertical nanogaps to mimic the function of polymer membranes for the purpose of protein enrichment.

Germanium as a scalable sacrificial layer for nanoscale protein patterning.

We demonstrate the use of germanium (Ge) films as water-soluble features that allow the patterning of proteins onto surfaces with commonly used organic solvents. This technique is scalable for manufacturing and is compatible with nano- and microfabrication processes, including standard lithography. We use Ge as a sacrificial layer to mask and protect areas of the substrate during surface functionalization. Since Ge dissolves in 0.35% hydrogen peroxide (H2O2) in water but not in organic solvents, Ge can be removed after patterning without significantly affecting protein activities. In this paper, we present examples of protein patterning with two different techniques. We show that 50 nm thick Ge layers can be completely removed in 10 min without residues and, importantly, nanoscale resolution and misalignment can be achieved with conventional photolithography equipment. Both biotin and streptavidin maintain ~80% and >50% activity after 10 min and 360 min incubation in 0.35% H2O2, respectively. Lastly, the process can be used to functionalize sidewalls with proteins, a capability of recent interest for cell-cell adhesion studies.

Modic type 1 change is an autoimmune response that requires a proinflammatory milieu provided by the 'Modic disc'.

BACKGROUND CONTEXT: Modic changes (MCs) are magnetic resonance imaging (MRI) evidence of inflammatory and fibrotic vertebral bone marrow lesions that associate with adjacent disc degeneration and end plate damage. Although MC etiology is uncertain, historical data suggest a linkage to an autoimmune response of bone marrow triggered by the nucleus pulposus (NP). PURPOSE: The aim of this study was to test whether bone marrow has an autoimmune response to NP cells that is amplified by an inflammatory milieu and ultimately leads to MC development in vivo. We hypothesized that an inflammatory co-stimulus is required for bone marrow/NP crosstalk to stimulate MC. STUDY DESIGN: This is an in-vitro cell co-culture study plus in-vivo experiments in rat caudal vertebrae. METHODS: In in-vitro study, bone marrow mononuclear cells (BMNCs) and NP cells (NPCs) from rats were co-cultured with and without interleukin (IL)-1α stimulation. Cell viability (n=3) of BMNCs and NPCs and gene expression (n=7) were analyzed. In in-vivo study, proinflammatory lipopolysaccharide (LPS) and control disc nucleus surrogates (NP micromass pellets) were generated in vitro from rat NPCs and implanted into rat tail vertebrae, and the response was compared with sham surgery (n=12 each). Tissue changes were investigated with T1w and T2w MRI (7T), histology, and immunohistochemistry (tumor necrosis factor, CD3) 1 (n=6) and 2 weeks (n=6) after implantation. RESULTS: BMNC/NPC co-culture significantly increased lymphocyte viability (42%-69%, p<.05) and reduced NPC viability (96%-88%, p<.001), indicating immunogenicity of NPC. However, IL-1α was required to cause significant transcriptional upregulation of IL-1, IL-6, IL-10, and tropomyosin receptor kinase A. Therefore, an inflammatory activation is required to amplify the immune response. Immunogenicity of the NP was corroborated in vivo by CD3 cell accumulation around LPS and control disc surrogates at Day 7. However, only the LPS disc surrogate group demonstrated infiltration of CD3 cells at Day 14. Furthermore, end plate defects (p<.05, LPS: n=4/6, Ctrl: n=0/6, sham: n=0/6) and MC1-like MRI changes (T2w hyperintensity, p<.05) were only seen with LPS disc surrogates. CONCLUSIONS: NPCs are immunogenic but cannot trigger MC without an additional proinflammatory stimulus. Our data suggest that MC requires end plate defects that allow marrow/NPC co-mingling plus an adjacent inflammatory "MC disc" that can amplify the immune response.