Name: in situ characterization of shewanella oneidensis mr1 biofilms by salvi and tof-sims
Uploaded: 2017-08-18T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS at JoVE.com

We are grateful to the Pacific Northwest National Laboratory (PNNL) Earth and Biological Sciences (EBD) mission seed Laboratory Directed Research and Development (LDRD) fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) General User Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Dr. Yuanzhao Ding for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

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The authors have nothing to disclose.

After inoculating at log-phase, it is important to test the number of days and temperature at which the biofilm should grow before it is healthy and thick enough for imaging, as described in step 3.1. This procedure specifically covers culturing a S. oneidensis MR1 biofilm at room temperature; however different room temperatures can influence the rate of growth. Therefore, it is critical to use optical imaging to understand whether the biofilm is ready before proceeding to ToF-SIMS analysis. Similarly, different strains ...

Bacterial biofilms are surface-associated communities which have evolved over time as a defense for bacteria to survive varying adverse physical and mechanical stimuli, wherein cells are able to attach and survive in many possible environments.1,2 Biofilms are vastly investigated and have applications in many fields such as biomedicine, biomedical engineering, agriculture, and industrial research and development.1,2 Understanding the chemical mapping of these complex microbial communities, including their self-produced extracellular polymeric substances (EPS) and their local water-cluster environment, is essential to gaining an accurate and detailed depiction of their biological activities.2
Biofilms exist and grow within a highly hydrated state. This presents a great challenge in using vacuum-based surface analysis techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) due to the difficulty in studying volatile liquids in vacuum. As a result, vacuum-based surface analysis techniques have been limited almost exclusively to studying biofilm samples at only their dried state. However, studying a biofilm in its dried state inhibits the accurate investigation of its true biological microenvironment. It often causes drastic changes to the EPS integrity and biofilm morphology, which has been demonstrated after comparing dry biofilm mass spectral results to in situ liquid studies.3,4 This article presents a solution for studying biofilms within their natural hydrated state by employing the use of our System for Analysis at the Liquid Vacuum Interface (SALVI),5,6 a microfluidic reactor that contains liquid under its thin silicon nitride (SiN) membrane in a microchannel made of polydimethylsiloxane (PMDS), thus providing direct access to the secondary ion probe beam while still maintaining the structural integrity of the liquid matrix within a vacuum chamber.7,8
S. oneidensis MR-1 mutated to express green fluorescence protein (GFP) was chosen as a model organism for this biofilm procedure illustration due to its metabolic versatility and common use in environmental and applied microbiology, which was based heavily on its unique capability for metal reduction and extracellular electron transfer.9,10,11 Additionally, the presence of GFP allowed for easy continuous biofilm-thickness monitoring through fluorescence microscopy, using a fluorescein isothiocyanate (FITC) filter. Our previous studies have shown evidence of this bacteria favoring attachment to the SiN window using in operando fluorescence imaging for biofilm growth to a thickness of up to one hundred micrometers.4,12 While this paper will only discuss the confirmation of biofilm&#39;s presence through fluorescence microscopy, the SALVI is compatible with other optical imaging methods such as super-resolution fluorescence imaging (i.e., structured illumination microscopy (SIM)9) and confocal laser scanning microscopy (CLSM) imaging4). Optical imaging can serve to measure the biofilm thickness, and obtain a 3D image of, the shape of the biofilm as it appears, confirming its thickness and its attachment to the SiN window.9 While GFP was used in the SIMS analysis, S. oneidensis without GFP was used for the growth curve, as this only required measurement of optical density and did not require any fluorescent imaging. Generally, the difference between the GFP tagged and untagged species in the growth curve is insignificant. Additionally, while this protocol uses S. oneidensis MR-1 GFP as a model organism to describe the procedure, this procedure is designed for any bacterial strain that may be needed for cultivation within SALVI. Although, given knowledge of the bacterial strain needed, some growth conditions such as time, temperature, and oxygen environment may need to be modified to accommodate the strain of bacteria to be used. For growth medium, this procedure uses &#34;nanowires&#34; medium, tryptic soy broth (TSB) without dextrose, and tryptic soy agar (TSA) without dextrose for culturing. The composition of &#34;nanowires&#34; medium has been specially formulated for the growth and for monitoring of extensions of the membrane and periplasm of S. oneidensis that appear to take the shape of small wires, and the medium composition has been established within previous research.13,14
Our previous protocol on in situ liquid ToF-SIMS has illustrated the benefit that SALVI has to offer for protein immobilization and attachment to the SiN, as well as a detailed protocol on ToF-SIMS analysis and data reduction.12 Rather than reiterate data reduction steps, this paper will serve to instead focus on the unique approach of setting up and cultivating biofilms within our SALVI microchannel, as well as the imaging steps to detect biofilm presence and thickness prior to ToF-SIMS analysis. While biofilms have been previously limited to only dried samples within the chamber of vacuum-based surface analytical techniques, detailed EPS and biofilm chemical mapping of live biofilms can now be obtained in situ because of this new capability.

<table><tbody><tr><td>ToF-SIMS</td><td>IONTOF</td><td>TOF.SIMS 5</td><td>Resolution:&gt;10,000 m/&amp;Delta;m for mass resolution;&gt;4,000 m/&amp;Delta;m for high spatial resolution</td></tr><tr><td>System for Analysis at the Liquid Vacuum Interface (SALVI)</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td></tr><tr><td>-80&amp;deg;C Freezer</td><td>New Brunswick Scientific</td><td>N/A</td><td>U410 Premium Energy Efficient Ultra-Low Temperature Freezer</td></tr><tr><td>4&amp;deg;C Refrigerator</td><td>BioCold Scientific</td><td>N/A</td><td>COLDBOX1</td></tr><tr><td>Orbital Shaker</td><td>New Brunswick Scientific</td><td>N/A</td><td>Innova 4900 Multi-Tier Environmental Shaker, set at 30 degrees Celsius for serum bottle and flask culturing, set at 150rpm.</td></tr><tr><td>Syringe Pump</td><td>Cole-Parmer</td><td>EW-74905-02</td><td>Cole-Parmer Syringe Pump, Infusion Only, Touchscreen Control 74905-02, used for injecting liquid into the tubing system and SALVI at a constant flowrate.</td></tr><tr><td>Incubator</td><td>Barnstead International</td><td>LT1465X3</td><td>Lab-Line incubator, set at 30 degrees Celsius for plate culturing.</td></tr><tr><td>Autoclave</td><td>Getinge</td><td>533LS</td><td>Used to sterilize PEEK fittings, tubing systems, serum vials, and medium. Model 533LS Vacuum Steam Sterilizer</td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>4001-000</td><td>GENESYS 20 spectrophotometer for OD600 readings of cuvettes for growth curves.</td></tr><tr><td>Biological Safety Cabinet</td><td>Thermo Fisher Scientific</td><td>1385</td><td>1300 Series AZ Biological Safety Cabinet</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>N/A</td><td>Nikon OPTIPHOT-2 fluorescence microscope with camera and super high pressure mercury lamp power supply.</td></tr><tr><td>pH Meter</td><td>Mettler Toledo</td><td>51302803</td><td>Used to test the pH of the &amp;ldquo;nanowires&amp;rdquo; medium after finished and before autoclaving.</td></tr><tr><td>PEEK Union</td><td>Valco</td><td>ZU1TPK</td><td>For connecting the inlet and outlet of SALVI, the syringe to the tubing system, and the inlet of the SALVI to the drip chamber of the tubing system.</td></tr><tr><td>5 Axes Sample Stage</td><td>IONTOF</td><td>N/A</td><td>Stage is self-made for mounting SALVI in ToF-SIMS.</td></tr><tr><td>Barnstead Nanopure Water Purification System</td><td>Thermo Fisher Scientific</td><td>D11921</td><td>ROpure LP Reverse Osmosis filtration module (D2716)</td></tr><tr><td>Pipette</td><td>Thermo Fisher Scientific</td><td>21-377-821</td><td>Range: 100 to 1,000 &amp;micro;L.</td></tr><tr><td>Pipette Tip</td><td>Neptune</td><td>2112.96.BS</td><td>1,000 &amp;micro;L pipette tips</td></tr><tr><td>Razor Blade Handle</td><td>Stanley</td><td>N/A</td><td>Stanley Bostitch Razor Blade Scraper with 5 Single-Edge Blades, used for cutting PTFE tubing</td></tr><tr><td>Syringe</td><td>BD</td><td>309659</td><td>1 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>309657</td><td>3 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>309646</td><td>5 mL; Used for making the drip chamber</td></tr><tr><td>Syringe</td><td>BD</td><td>309604</td><td>10 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>302830</td><td>20 mL</td></tr><tr><td>Disposable Pipette</td><td>Thermo Fisher Scientific</td><td>13-678-11</td><td>25 mL Fisherbrand&amp;trade; Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe, for filling serum bottles.</td></tr><tr><td>Electric Pipette Filler</td><td>Pipet-aid</td><td>P-57260</td><td>Vacuum pressure electric serological pipette filler</td></tr><tr><td>Serum Bottle</td><td>Sigma</td><td>33109-U</td><td>Holds approximately 69 mL of liquid for culture growth, optimum for use of 20mL culture per bottle.</td></tr><tr><td>Anaerobic Culture Tube</td><td>VWR</td><td>89167-178</td><td>Anaerobic Tubes, 18 x 150 mm, Supplied with 20 mm Blue Butyl Rubber Stopper and Aluminum Seal.</td></tr><tr><td>Rubber Stopper</td><td>Sigma</td><td>27235-U</td><td>Silicone stopper, used for sealing serum bottles and for creating the tubing system/drip chamber.</td></tr><tr><td>Aluminum Crimp Seal (without septum)</td><td>Sigma</td><td>27227-U</td><td>Aluminum seal for top of serum bottle for use with serum bottle crimper.</td></tr><tr><td>Serum Bottle Aluminum Seal Crimper</td><td>Wheaton</td><td>224307</td><td>30 mm crimper with standard seal.</td></tr><tr><td>PTFE Tubing</td><td>Supelco</td><td>58697-U</td><td>1.58 mm OD x 0.5 mm ID 50 ft. PTFE Teflon tubing, used for creating the tubing system.</td></tr><tr><td>Disposable Cuvettes</td><td>GMBH</td><td>759085D</td><td>1.5 Ml for use with spectrophotometer.</td></tr><tr><td>Needle</td><td>BD</td><td>303015</td><td>22G; used for serum bottle injection.</td></tr><tr><td>Needle</td><td>BD</td><td>305120</td><td>23G; used for punching-through rubber stopper to create drip tubing system.</td></tr><tr><td>Shewanella oneidensis MR-1 with GFP</td><td>N/A</td><td>N/A</td><td>Matthysse AG, Stretton S, Dandie C, McClure NC, &amp;amp; Goodman AE (1996) Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145(1):87-94.&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;S25310A</td><td>95% Denatured</td></tr><tr><td>TSA</td><td>BD</td><td>212305</td><td>Tryptic soy agar for culturing the model organism (S. oneidensis) used in this protocol</td></tr><tr><td>PIPES Buffer</td><td>Sigma</td><td>P-1851</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S-5881</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Ammonium Chloride</td><td>Sigma</td><td>A-5666</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P-4504</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma</td><td>S-9638</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>S271-3</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium, and used to make mineral solution used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium lactate</td><td>Sigma</td><td>L-1375</td><td>60%(w/w) syrup @ 98% pure, d=1.3 g/mL, 7M, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S-5761</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nitrilotriacetic Acid Trisodium Salt</td><td>Sigma</td><td>N-0253</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Iron (III) Chloride</td><td>Sigma</td><td>451649</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma</td><td>208094</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Manganese (II) Sulfate Monohydrate</td><td>Sigma</td><td>M-7634</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Iron(II) Sulfate Heptahydrate</td><td>Sigma</td><td>215422</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma</td><td>223506</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Cobalt(II) Chloride</td><td>Sigma</td><td>60818</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Zinc Chloride</td><td>Sigma</td><td>229997</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Copper(II) Sulfate Pentahydrate</td><td>Sigma</td><td>C-8027</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Aluminum Potassium Sulfate Dodecahydrate</td><td>Sigma</td><td>237086</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Boric Acid</td><td>Sigma</td><td>B-6768</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Molybdate Dihydrate</td><td>Sigma</td><td>331058</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nickel(II) Chloride</td><td>Sigma</td><td>339350</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Tungstate Dihydrate</td><td>Sigma</td><td>14304</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>D-Biotin</td><td>Sigma</td><td>47868</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Folic Acid</td><td>Sigma</td><td>F-7876</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Pyridoxine Hydrochloride</td><td>Sigma</td><td>P-9755</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Riboflavin (B2)</td><td>Sigma</td><td>47861</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Thiamine Hydrochloride</td><td>Sigma</td><td>T-4625</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nicotinic Acid</td><td>Sigma</td><td>N4126</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>D-Pantothenic Acid Hemicalcium Salt</td><td>Sigma</td><td>21210</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Vitamin B12</td><td>Sigma</td><td>V-2876</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>4-Aminobenzoic Acid</td><td>Sigma</td><td>A-9878</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Thioctic Acid</td><td>Sigma</td><td>T-1395</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr></tbody></table>

in situ characterization of shewanella oneidensis mr1 biofilms by salvi and tof-sims

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

These representative results serve to show how the chemical profile of the attached biofilm can be identified and interpreted, as obtained through ToF-SIMS. After plotting mass spectra from ToF-SIMS data acquisition, highlighted briefly in the procedures section, peak identification should be conducted in order to assign identities to each respective m/z value. This can be done through extensive literature review on mass spectrometry on bacteria and specific chemical fragments that are ex...

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In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

in-situ-characterization-shewanella-oneidensis-mr1-biofilms-salvi-tof

Research

JoVE Journal

Chemistry

8.9K Views.  Pacific Northwest National Laboratory. Dit artikel presenteert een methode voor het kweken van een biofilm voor in situ secundaire ion time-of-flight massaspectrometrie voor toewijzing van de chemische in haar gehydrateerd staat, ingeschakeld door een microfluidic-reactor, systeem voor analyse op de vloeibare vacuüm-Interface. De heer Shewanella oneidensis -1 met groene fluorescentie eiwit werd gebruikt als een model.

Video: In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Cancer Research

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Biochemistry

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

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