QCM-D sensors with gold coating (QSX301; Biolin Scientific, Västra Frölunda, Sweden) were used as received or after recoating with an additional 100 nm gold film. QCM-D sensors with a His-tag-capturing coating (QSX340; Biolin Scientific) were used as received or after regeneration with solutions of imidazole in ultrapure water (25 min at 500 mM) and, subsequently, CuSO4 in working buffer (15 min at 5 mM). Gold-coated AFM cantilevers with nominal spring constants of 30 or 6 pN/nm (Biolevers) and 60 pN/nm (NPG-10) were purchased from Bruker AFM Probes (Santa Barbara, CA).
Npg 10
Manufactured by Bruker
Sourced in United States
The NPG-10 is a compact and versatile lab equipment designed for precision gas flow control. It offers accurate and stable gas flow regulation across a wide range of flow rates, making it suitable for various laboratory applications. The core function of the NPG-10 is to provide precise and reliable gas flow management in a streamlined package.
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Lab products found in correlation
Afm multimode mmafm 2, by Bruker (2 mentions)
Qsx340, by Biolin Scientific (1 mentions)
Qsx301, by Biolin Scientific (1 mentions)
1 hexanethiol, by Merck Group (1 mentions)
6 mercaptohexanoic acid, by Merck Group (1 mentions)
Nickel 2 chloride, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
11 mercapto 1 undecanol, by Merck Group (1 mentions)
6 mercapto 1 hexanol, by Merck Group (1 mentions)
Peak force tuna application module, by Bruker (1 mentions)
Mfp 3d bio atomic force microscope, by Oxford Instruments (1 mentions)
1 octadecanethiol, by Merck Group (1 mentions)
7 protocols using npg 10
1
QCM-D and AFM Sensor Preparation
2
I-V Measurements of Au/Mica SAMs
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I–V measurements were performed on a Bruker AFM Multimode MMAFM-2 equipped with a Peak Force TUNA Application Module. The Au on mica substrates were removed from the flowbox immediately prior to measurement, which occurred under ambient conditions by contacting the SAM with a Au-coated SI3N4 tip with a nominal radius of 30 nm (NPG-10, Bruker; resonant frequency: 65 kHz, spring constant: 0.35 N m–1). The AFM tip was grounded and the samples were biased from –1.0 V → 1.0 V → –1.0 V on AuMica. 11 trace/re-trace cycles per junction were performed and the top electrode was removed from SAMs between junctions.
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I–V measurements were performed on a Bruker AFM Multimode MMAFM-2 equipped with a Peak Force TUNA Application Module. The Au on mica substrates were removed from the flowbox immediately prior to measurement, which occurred under ambient conditions by contacting the SAM with a Au-coated SI3N4 tip with a nominal radius of 30 nm (NPG-10, Bruker; resonant frequency: 65 kHz, spring constant: 0.35 N m–1). The AFM tip was grounded and the samples were biased from –1.0 V → 1.0 V → –1.0 V on AuMica. 11 trace/re-trace cycles per junction were performed and the top electrode was removed from SAMs between junctions.
3
Functionalization of Gold-Coated Tips and Wafers
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Gold coated silicon nitride tips (NPG-10, Bruker, Santa Barbara, CA, USA) and small parts (~ 3 × 3 mm2) of a previously broken gold-coated silicon wafer (AUSW-51, Ø 2″, ~ 50 nm gold layer, NanoAndMore, Wetzlar, Germany) were cleaned for 20 min in an UV/Ozone-cleaner (UV/OZON ProCleaner, Bioforce Nanosciences, Salt Lake City, UT, USA) to remove organic contaminations. Directly after cleaning, tips and wafer were placed in a thiol solution overnight. For COOH-tips, the material was placed in a 1 mM solution of 11-mercaptoundecanoic acid in ethanol. For OH-tips, the material was placed in a 1 mM solution of 11-mercapto-1-undecanol in ethanol. After functionalization, the tips and wafers were removed from the functionalization solution and dipped in ethanol and water to remove excess functionalization chemicals. The success of the thiol functionalization was verified with contact angle measurements in preliminary tests on the thiol coated gold wafer.
4
Characterization of Fibronectin Binding
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Full-length human plasma fibronectin (fc010) and recombinant human fibronectin fragment 3 protein, CF (FN-050; Carrier free histidine-tagged FNIII8–14 fragment) were obtained from Millipore Limited (Hertfordshire, UK) and R&D systems (Minneapolis, Minnesota, USA) respectively, and the trisNTA-EG3-C16-SH linker was synthesized as described previously37 (link)–39 (link). Gold-coated QCM crystals were supplied by openQCM (Novaetech S.r.l., Napoli, Italy) and gold-coated AFM chips (NPG-10) were purchased from Bruker (UK) Limited (Coventry, UK). The chemicals/reagents, ethanol (> 99.5% for HPLC), hydrogen peroxide solution (30% w/w in water), concentrated sulfuric acid (99.999%), 1-hexanethiol (HS–(CH2)5–CH3), 6-mercaptohexanoic acid (HS–(CH2)5–COOH), 6-amino-1-hexanethiol (HS–(CH2)6–NH2), 6-mercapto-1-hexanol (HS–(CH2)6–OH), 11-mercapto-1-undecanol (HS–(CH2)11–OH), nickel (II) chloride and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich (Dorset, UK), whereas tris(2-carboxyethyl)phosphine (TCEP), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate buffered saline (PBS) tablets and sodium chloride were acquired from Fisher Scientific UK Ltd. (Loughborough, UK). All solutions were prepared using deionized water of HPLC grade (Sigma Aldrich, Dorset, UK).
5
Conductive Probe AFM Characterization
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I–V measurements were performed
on a Bruker AFM Multimode MMAFM-2 equipped
with a Peak Force TUNA Application Module (Bruker). The SAMs were
contacted with an Au-coated silicon nitride tip with a nominal radius
of 30 nm (NPG-10, Bruker; tip A, resonant frequency = 65 kHz, spring
constant = 0.35 N/m; tip B, resonant frequency = 23 kHz, spring constant
= 0.12 N/m; tip D, resonant frequency = 18 kHz, spring constant =
0.06 N/m; tip A was chosen in this work) in TUNA mode. The AFM tip
was grounded and for all loading forces, T4C4 on AuTS were
biased from −1.0 to +1.0 V and from +1.0 to −1.0 V while
C10 on were biased from −1.5 to +1.5 V and from +1.5 to −1.5
V on AuTS to record the I–V curves: a max of 10 trace/retrace cycles per junction
were performed and the top electrode was removed from SAMs between
junctions. Between different samples, a new tip was used. The total
number of I–V traces recorded
by CP-AFM is summarized in theSupporting Information , Table S3. It is difficult to determine Vtrans for an individual I–V trace
due to the inherent noise in the raw data. The peaks of Gaussian fits
of histograms of I for each value of V at different loading forces obtained by CP-AFM were plotted and
transformed into axes of ln(I/V2) versus 1/V. The position of the Vtrans was determined manually by the center
of the dips in the plots.
on a Bruker AFM Multimode MMAFM-2 equipped
with a Peak Force TUNA Application Module (Bruker). The SAMs were
contacted with an Au-coated silicon nitride tip with a nominal radius
of 30 nm (NPG-10, Bruker; tip A, resonant frequency = 65 kHz, spring
constant = 0.35 N/m; tip B, resonant frequency = 23 kHz, spring constant
= 0.12 N/m; tip D, resonant frequency = 18 kHz, spring constant =
0.06 N/m; tip A was chosen in this work) in TUNA mode. The AFM tip
was grounded and for all loading forces, T4C4 on AuTS were
biased from −1.0 to +1.0 V and from +1.0 to −1.0 V while
C10 on were biased from −1.5 to +1.5 V and from +1.5 to −1.5
V on AuTS to record the I–V curves: a max of 10 trace/retrace cycles per junction
were performed and the top electrode was removed from SAMs between
junctions. Between different samples, a new tip was used. The total
number of I–V traces recorded
by CP-AFM is summarized in the
due to the inherent noise in the raw data. The peaks of Gaussian fits
of histograms of I for each value of V at different loading forces obtained by CP-AFM were plotted and
transformed into axes of ln(I/V2) versus 1/V. The position of the Vtrans was determined manually by the center
of the dips in the plots.
6
Quantifying LGG Cell Wall Adhesion Forces
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To study the interaction of the LGG cell wall with -CH3, -OH, and -NH2 functional groups, adhesion forces between functionalized AFM probes and the LGG cell wall were carried out. The experiments were performed at 21±1°C in liquid medium using a MFP-3D-BIO atomic force microscope (Oxford Instruments, Santa Barbara, CA, USA) equipped with a cell that was filled with 2.5 mL phosphate buffered saline (pH 6.8). Gold-coated AFM probes (NPG-10, Bruker) were functionalized with alkanethiols HS(CH2)11X (X=CH3, OH, or NH2) by exposure to 1 mM ethanolic solution of the corresponding alkanethiol. LGG cells at exponential growth phase suspended in phosphate buffered saline were immobilized (14 hours, 4C) through electrostatic interactions with -NH3 + terminal groups of SAM of 11-Amino-1-undecanethiol adsorbed onto Au/Cr/glass (Sputter Q150T, Quorum technologies). 31 The interactions between the functionalized probes and the LGG cell wall were quantified by scanning the surface and measuring the deflection of the cantilever as a function of the vertical displacement of the piezoelectric scanner. A total number of 1024 force-distance curves were recorded on the sample spot with the size of 100 µm 2 . These curves were analyzed using a custom program in MATLAB software for calculation of the frequency of appearance of an interaction force as a function of the probe-sample distance.
7
Functionalization of AFM Probes for Cell Adhesion
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Gold-coated AFM probes were functionalized with hydrophobic selfassembled monolayers (SAMs) using alkanethiols. Forming a SAMs assembly on gold surfaces is a well-controlled process having many practical advantages [12] . The SH-groups of alkanethiols has a high affinity on gold substrates [13] via chemical binding and it can remain stable during days to weeks after its formation [12] . Goldcoated cantilevers, with the nominal spring constant of 0.12 N/m and the nominal resonance frequency of 23 kHz, were purchased from Bruker (NPG-10, Santa Barbara, CA, USA). The self-assembled monolayers were formed on the gold-coated cantilevers according to a previous procedure [14] . Gold-coated cantilevers were immersed in 1 mM solutions of alkanethiols terminated with CH 3 (1octadecanethiol, Sigma-Aldrich) during 18 hours. Firstly, a teflon substrate (Sigma Aldrich) was scanned to verify the AFM tip functionalization. Secondly, the adhesion forces between the functionalized tips and cells were measured using contact imaging mode. AFM enables to probe the forces between the tip and sample by monitoring the deflection of the cantilever when it approaches,
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