Detailed Study on the Failure of the Wedge Calibration Method at Nanonewton Setpoints for Friction Force Microscopy

The wedge calibration method is the most popular calibration technique in friction force microscopy for converting raw lateral laser deflection signals (in volt) into forces (in newton). Recent trends in nanotribology demand the use of the method at nanonewton (nN) force ranges; however, this method fails at these small forces. The objective of the present work is to identify the reason why the conventional wedge calibration method fails at nN force ranges. We found that the equation used in the model in this method amplifies experimental errors by orders of magnitude only at small setpoints purely due to its mathematical expression. This low tolerance against experimental errors in nanonewton force ranges is the reason for the failure. We identified that the condition, under which the method operates accurately, is adhesion ≪ setpoint. Discovery of this operation range (adhesion ≪ setpoint) is important because performing the calibration under other conditions can wrongly calibrate the system by orders of magnitude

Directed Self-Assembly of Lipid Nanotubes from Inverted Hexagonal Structures

Conventional lipid-tube formation is based on either a tube phase of certain lipids or the shape transformation of lamellar structures by applying a point load. In the present study, lipid blocks in inverted hexagonal phase made of 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) were shown to protrude lipid nanotubes upon a fluid-dynamic flow on polyelectrolyte-functionalized surfaces in physiological buffer solution. The outer diameter of the tubes is 19.1 ± 4.5 nm and their lengths are up to several hundred micrometers. The method described enables the alignment and patterning of lipid nanotubes into various (including curvy) shapes with a microfluidic system

Directed Self-Assembly of Lipid Nanotubes from Inverted Hexagonal Structures

Conventional lipid-tube formation is based on either a tube phase of certain lipids or the shape transformation of lamellar structures by applying a point load. In the present study, lipid blocks in inverted hexagonal phase made of 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) were shown to protrude lipid nanotubes upon a fluid-dynamic flow on polyelectrolyte-functionalized surfaces in physiological buffer solution. The outer diameter of the tubes is 19.1 ± 4.5 nm and their lengths are up to several hundred micrometers. The method described enables the alignment and patterning of lipid nanotubes into various (including curvy) shapes with a microfluidic system

A possible role of LR161-170 motif derived from other organisms for exerting of EGCG’s activities.

<p><b>A</b>) LR161-170 motif of other organisms. <b>B</b>) The neutralizing activity of LR161-170 derived from other organisms for the cell-surface binding of EGCG. After incubation of EGCG with each peptide at a molar ratio of 1∶1 in PBS, interactions between these EGCG-peptide mixtures and the 67LR-overexpressed HepG2 cells were measured by a SPR assay. Sensorgrams of the net binding of EGCG, which is the value of the subtracted peptide-binding signal from the total mixture-binding signal, are shown. The results are represented as EGCG alone (blue line) and EGCG plus deletion mutant of LR161-170 (red line). <b>C</b>) The neutralizing activity of LR161-170 derived from other organisms on the EGCG-induced inhibition of cancer cell growth. After incubation of EGCG with each peptide, HepG2 cells were treated with the mixtures for 5 days and the cell number was assessed. The results, EGCG plus peptide (closed bar), are shown as the relative cell number to the EGCG-nontreated control (open bar), and the data presented are the means ± S.D. (n = 3) (Student’s <i>t</i>-test, *, <i>p</i><0.05).</p

The nature of the EGCG-LR peptide interactions.

<p>Electrospray ionization mass spectrum of peptide (<b>A</b>) or peptide-EGCG mixture (<b>B, C</b>). The peptide solution was prepared by incubating each peptide (5 µM) with or without EGCG (5 µM) in water at room temperature for 15 min. Solutions of the mixture were analyzed in the positive ion mode. The arrows in the figure show the observed ion peaks. <b>A</b>) The Cys residue-containing peptide human LR161-170 and (<b>B</b>) its mixture with EGCG. <b>B</b>) An inset is the figure zooming in the relevant peak to see the difference between non-covalent (<i>m/z</i> 749.7 [LR161-170+EGCG+2H]²⁺) and covalent binding (<i>m/z</i> 748.7 [(LR161-170+EGCG-2H)+2H]²⁺). <b>C</b>) Mass spectrum of the mixture of the Cys residue-lacking peptide soy LR168-177 with EGCG. [LR161-170+2H]²⁺ or [Soy LR168-177+2H]²⁺ represents the doubly protonated form of each peptide. [LR161-170+EGCG+2H]²⁺ or [Soy LR168-177+EGCG+2H]²⁺ represents the peptide ion with added EGCG (EGCG-peptide complexes).</p

The neutralization of the inhibitory effect of EGCG on cancer cell growth by peptides deduced from the extracellular domain of 67LR.

<p>After pre-incubation of EGCG (1 µM) with each peptide (1 µM): (A) 20-amino acid segment peptides, (B) 10-amino acid segment peptides, or (C) 9-amino acid segment peptides (single amino acid deletion form of the N- or C-terminus of the peptide LR 161-170), the 67LR-overexpressed HepG2 cells were treated with these mixtures for 5 days and the cell number was assessed. The results, EGCG plus peptide (closed bar), are shown as the relative cell number to the EGCG-nontreated control (open bar), and the data presented are the means ± S.D. (n = 3) (Student’s <i>t</i>-test, *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001). D) SDS-PAGE of recombinant LR (r-hLR_102-295 and the mutant r-hLR_102-295Δ161-171 expressed in <i>E. coli</i>). The lanes of Wild and Δ161-171 are the wild-type r-hLR_102-295 and the mutant r-hLR_102-295Δ161-171, respectively. Molecular mass markers (in kDa) are indicated at the left. E) Western blot analysis of the recombinant LR was performed by using the anti-LR antibody F18. F) The effect of r-hLR_102-295 and the mutant r-hLR_102-295Δ161-170 on the cancer cell growth inhibition by EGCG. After incubation of each r-hLR protein or LR161-170 peptide with or without EGCG, HepG2 cells were treated with these mixtures for 5 days and the cell number was assessed. The results are shown as the relative cell number of EGCG-, EGCG plus LR peptide-, or EGCG plus LR protein-treated cells (closed bar) to the EGCG-nontreated control cells (open bar) under each mixture condition (none, LR peptide, or LR protein), and the data presented are the means ± S.D. (n = 3) (Student’s <i>t</i>-test, ***, <i>p</i><0.001).</p

The relationship between the responsiveness of EGCG to the HepG2 cells and 67LR expression.

<p><b>A</b>) Chemical structure of green tea polyphenol EGCG. <b>B</b>) Western blot analysis of whole cell lysate from HepG2 cells using anti-LR antiserum (I) and anti-LR antibody F-18 (II). <b>C</b>) To examine the expression of 67LR on cell membrane in HepG2 cells, both cytosolic and membrane fractions were prepared, and the 67LR were detected by western blot analysis using anti-LR antiserum. This test was performed under reducing (2-Me (+)) or non-reducing (2-Me (−)) conditions. 2-Me indicates 2-mercaptoethanol. The lower panel displays protein levels from the same filter blotted again with the anti-β-actin antibody used as a quantitative loading control. <b>D</b>) The cells transfected with either the empty vector (−) or the 67LR gene expression vector (+) were lysed and total cellular protein was subjected to western blot analysis using the cell-surface LR-specific antibody MLuC5. The lower panel displays protein levels from the same filter blotted again with the anti-β-actin antibody used as a quantitative loading control. <b>E</b>) Both transfected cells were fixed on the sensor chip. The cell-surface binding of EGCG to immobilized 67LR-overexpressed or control HepG2 cells were measured using a surface plasmon resonance (SPR) biosensor. EGCG was injected at a concentration of 10 µM for the time indicated interval in the figure. <b>F</b>) Both types of cells were treated with 1 µM EGCG for 5 days. The results are shown as the relative cell number to untreated control and the data presented are means ± S.D. (n = 3) (Student’s <i>t</i>-test, **, <i>p</i><0.01).</p

Importance of basic amino acid resides in the EGCG sensing motif for EGCG’s activity.

<p><b>A</b>) Basic amino acid replacements of LR161-170. Replaced peptide sequences are shown in the list. <b>B</b>) The neutralizing activity of several basic amino acid-replaced LR161-170 segments for the cell-surface binding of EGCG. After incubation of EGCG with each peptide at a molar ratio of 1∶1 in PBS, interactions between these EGCG-peptide mixtures and the cells were measured by a SPR assay. Sensorgrams of net binding of EGCG, which is the value of the subtracted peptide-binding signal from the total mixture-binding signal, are shown. The results are represented as EGCG alone (blue line) and EGCG plus deletion mutant of LR161-170 (red line). <b>C</b>) The neutralizing activity of several basic amino acid-replaced LR161-170 segments on the EGCG-induced inhibition of cancer cell growth. After incubation of EGCG with each peptide, the 67LR-overexpressed HepG2 cells were treated with the mixtures for 5 days and the cell number was assessed. The results, EGCG plus peptide (closed bar), are shown as the relative cell number to the EGCG-nontreated control (open bar), and the data presented are the means ± S.D. (n = 3) (Student’s <i>t</i>-test, *, <i>p</i><0.05, **, <i>p</i><0.01).</p

Dual Nanofriction Force Microscopy/Fluorescence Microscopy Imaging Reveals the Enhanced Force Sensitivity of Polydiacetylene by pH and NaCl

Polydiacetylene (PDA) is a popular mechanochromic material often used in biosensing. The effect of its headgroup–headgroup interactions on thermochromism such as pH or salt concentration dependency has been extensively studied before; however, their effect on mechanochromism at the nanoscale is left unstudied. In this work, nanofriction force microscopy and fluorescence microscopy were combined to study the effect of pH and ionic strength on the polydiacetylene (PDA) force sensitivity at the nanoscale. We found that the increase in pH from 5.7 to 8.2 caused an 8-fold enhancement in force sensitivity. The elevation of NaCl concentration from 10 to 200 mM also made the PDA 5 times more force-sensitive. These results suggest that the PDA force sensitivity at the nanoscale can be conveniently enhanced by “pre-stimulation” with pH or ionic strength

Switching Transport through Nanopores with pH-Responsive Polymer Brushes for Controlled Ion Permeability

Several nanoporous platforms were functionalized with pH-responsive poly­(methacrylic acid) (PMAA) brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). The growth of the PMAA brush and its pH-responsive behavior from the nanoporous platforms were confirmed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The swelling behavior of the pH-responsive PMAA brushes grafted only from the nanopore walls was investigated by AFM in aqueous liquid environment with pH values of 4 and 8. AFM images displayed open nanopores at pH 4 and closed ones at pH 8, which rationalizes their use as gating platforms. Ion conductivity across the nanopores was investigated with current–voltage measurements at various pH values. Enhanced higher resistance across the nanopores was observed in a neutral polymer brush state (lower pH values) and lower resistance when the brush was charged (higher pH values). By adding a fluorescent dye in an environment of pH 4 or pH 8 at one side of the PMAA-brush functionalized nanopore array chips, diffusion across the nanopores was followed. These experiments displayed faster diffusion rates of the fluorescent molecules at pH 4 (PMAA neutral state, open pores) and slower diffusion at pH 8 (PMAA charged state, closed pores) showing the potential of this technology toward nanoscale valve applications