Name: ptr-tof-ms coupled with an automated sampling system and tailored data analysis for food studies: bioprocess monitoring, screening and nose-space analysis
Uploaded: 2017-05-11T12:30:00
Description: Watch this Scientific Journal Video about PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis at

This work is supported by the European Commission's 7th Framework Programme under Grant Agreement Number 287382. SY is a beneficiary of a European Commission's 7th Framework Programme Grant Agreement Number 287382. IK is a beneficiary of a FIRST doctoral school grant from the Fondazione Edmund Mach. For his work at University of Foggia, VC is supported by the Apulian Region in the framework of 'Future In Research' program (practice code 9OJ4W81).

The protocol follows the guidelines of our institutional committee on human research ethics.
1. Sample Preparation and Autosampler Conditions
<ol>
	<li>On-line bioprocess monitoring: detecting VOCs released during lactic acid fermentation of yogurt 
	NOTE: This section of the protocol represents part of the procedure reported by Benozzi et al.32
	<ol>
		<li>Add 5 mL of pasteurized milk to each vial (20 mL glass vials equipped with PTFE/silicone septa). Note ...

Proton transfer reaction-mass spectrometry (PTR-MS) coupled to time of flight (ToF) mass analyzers represent a valid compromise between the need for identification and quantification of volatile organic compounds and the necessity for rapid analytical profiling. The high mass resolution that characterizes the ToF mass analyzer gives/provides relevant sensitivity and mass spectra with considerable informational content. Furthermore, the application of PTR-ToF-MS coupled with an auto-sampler and tailored data analysis tool...

Direct-Injection Mass Spectrometric (DIMS) technologies represent a class of analytical instrumental approaches that offer considerable mass and time resolution with high sensitivity and robustness, allowing the quick detection and quantification of volatile organic compounds (VOCs)1. These instrumental approaches include, among others, MS-e-noses, Atmospheric-Pressure Chemical Ionization Mass Spectrometry (APCI-MS), Proton-Transfer-Reaction Mass Spectrometry (PTR-MS), and Selected Ion-Flow-Tube Mass Spectrometry (SIFT-MS)1. The pros and the cons of each approach depend on: the kind of sample injection, the source and control of precursor ions, the control of the ionization process, and the mass analyzer1,2.
Proton-transfer-reaction mass-spectrometry (PTR-MS) was developed more than twenty years ago to monitor in real-time and with low detection limits (usually a few ppbv, part per billion by volume) most volatile organic compounds (VOCs) in air3,4. Current uses of PTR-MS range from medical applications, to food control, to environmental research5,6. The main features of this technique are: the possibility of rapid and continuous measurement, the intense and pure source of precursor ions, and the possibility to control ionization conditions (pressure, temperature and drift voltage). These features permit combining versatile uses with a high degree of standardization1,4. In fact, the method is based on reactions of hydronium ions (H3O+), which induce non-dissociative proton transfer in most volatile compounds (particularly in those characterized by a proton affinity higher than water), protonating neutral compounds (M) according to the reaction: H3O+ + M &#8594; H2O + MH+. In contrast to other techniques, e.g., APCI-MS, precursor ion generation and sample ionization are divided in two different instrumental compartments (a schematic representation of the PTR-MS instrument is given in Figure 1). An electrical discharge by water vapor in the hollow cathode ion source generates a beam of hydronium ions. After this phase, ions cross the drift tube, where the ionization of VOCs takes place7. Ions then enter a pulse extraction section and are accelerated into the TOF section. Through flight times, it is possible to determine the mass-to-charge ratios of the ions8. Each extraction pulse leads to a complete mass spectrum8 of the selected m/z range. Ion spectra are recorded by a fast data acquisition system7. A complete spectrum is typically acquired in one second although higher time resolution can be achieved according to the signal to noise level and a quantitative estimation of the VOC headspace concentration can be provided even without calibration9,10.
<img alt="Figure 1" src="/files/ftp_upload/54075/54075fig1.jpg" /> 
Figure 1: Schematic illustration of a PTR-MS. Schematic representation of the PTR-MS instrument. HC: external ion source with hollow cathode; SD: source drift; VI, venturi-type inlet; EM, electron multiplier; FC1-2, flow controllers. Reprinted with permission from Boschetti et al.7. <a href="http://cloudflare.jove.com/files/ftp_upload/54075/54075fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In general, the PTR technique assures fast analysis time, high detection sensitivity and a relatively compact instrument size, requires neither sample preparation nor sample destruction and thus allows real time investigations11. PTR is of considerable interest in the environmental, atmospheric, food, technological, medical and biological sciences12.
VOCs associated with food matrices are of outstanding interest in food science and technology because of their important role in the molecular basis of biological phenomena linked to odor and flavor perception and, thus, in food acceptance. Hence, our interest in real time and non-invasive detection of VOCs mainly deals with sensory qualities of food. In addition, if we consider the possibility to detect spoilage and pathogenic microorganisms by means of released VOCs13 and/or to monitor volatile organic compounds as markers following technological processes (e.g. Maillard by-products during thermal treatments)14, it becomes clear how VOC identification and quantification are fields of interest in food quality management6. Several recent uses of PTR-MS technologies for the rapid monitoring and quantification of VOCs in food matrices testify to the wide range of application of these analytical approaches (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Food matrix</td>
			<td>Kind of application</td>
			<td>Brief description</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Butter</td>
			<td>Screening/characterization</td>
			<td>Geographical origin of European butters</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Yogurt</td>
			<td>Bioprocess monitoring</td>
			<td>Evolution during lactic acid fermentation</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Cereal bars</td>
			<td>In vivo measurement</td>
			<td>Nosespace during consumption of cereal bars with varying sugar composition</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Liquid model systems</td>
			<td>Simulated oral conditions</td>
			<td>Evaluation of tongue pressure and oral conditions in a model mouth</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Apple</td>
			<td>In vivo measurement</td>
			<td>Nosespace during consumption apple with different genetic, textural, and physicochemical parameters</td>
			<td>19</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>Screening/characterization</td>
			<td>Differentiation of specialty coffees</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Grape must</td>
			<td>Screening/characterization</td>
			<td>Effect of cooking process</td>
			<td>21</td>
		</tr>
		<tr>
			<td>Flavored candies&#160;</td>
			<td>In vivo measurement</td>
			<td>Determination on panelists using different direct mass spectrometry methods</td>
			<td>22</td>
		</tr>
		<tr>
			<td>Ham&#160;</td>
			<td>Screening/characterization</td>
			<td>Effect of the pig rearing system</td>
			<td>23</td>
		</tr>
		<tr>
			<td>Bread</td>
			<td>Simulated oral conditions</td>
			<td>Simulating bread aroma during mastication</td>
			<td>24</td>
		</tr>
		<tr>
			<td>Milk</td>
			<td>Screening/characterization</td>
			<td>Monitoring photooxidation-induced dynamic changes in milk</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>Screening/characterization</td>
			<td>Diversity in roasted coffees from different geographic origins</td>
			<td>26</td>
		</tr>
		<tr>
			<td>Bread</td>
			<td>Bioprocess monitoring</td>
			<td>Effect of different yeast starters during alcoholic fermentation</td>
			<td>27</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>In vivo measurement</td>
			<td>Nosespace during consumption of different roasted coffee preparations</td>
			<td>28</td>
		</tr>
		<tr>
			<td>Tomatoes</td>
			<td>Screening/characterization</td>
			<td>Impact of Production Location, Production System, and Variety&#160;</td>
			<td>29</td>
		</tr>
		<tr>
			<td>Bread</td>
			<td>Bioprocess monitoring</td>
			<td>Effect of flour, yeast and their interaction during alcoholic fermentation</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Mushrooms&#160;</td>
			<td>Screening/characterization</td>
			<td>Shelf life of dried porcini mushrooms&#160;</td>
			<td>31</td>
		</tr>
		<tr>
			<td>Yogurt</td>
			<td>Bioprocess monitoring</td>
			<td>Effect of different starter cultures during lactic fermentation</td>
			<td>32</td>
		</tr>
		<tr>
			<td>Apple</td>
			<td>Screening/characterization</td>
			<td>Diversity in an apple germplasm collection</td>
			<td>33</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>Screening/characterization</td>
			<td>Tracing coffee origin&#160;</td>
			<td>34</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>In vivo measurement</td>
			<td>Combination of a dynamic sensory method and in-vivo nosespace analysis to understand coffee perception</td>
			<td>35</td>
		</tr>
	</tbody>
</table>
Table 1: List of scientific studies using PTR-ToF-MS in the food sector. Non-exhaustive list of scientific studies using PTR-based approaches to monitor VOC content in food-related experiments.
In recent studies, we reported on the application of PTR-ToF-MS coupled with an automated sampling system and tailored data analysis tools to increase sampling automation and reliability and, consequently, to enhance the potential of this technique7,10,13. This allowed us to screen, in terms of VOC content, large sample sets (e.g. food of different origins with many replicates, entire germoplasms), to analyze the influence of several experimental modes on VOC release (e.g. different concentrations of a given ingredient, diverse intensities of a specific technological parameter), and to monitor VOCs associated with a given bioprocess (e.g. enzymatic oxidation, alcoholic fermentation). Here, in order to exemplify the potential of PTR-ToF-MS in the agri-food sector, we present three paradigmatic applications: the detection of VOCs released during lactic acid fermentation of yogurt induced by different microbial starter cultures (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening) and the in vivo study of retronasal VOC release while drinking coffee (nosespace analysis).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PTR-TOF 8000 High-Resolution PTR-TOF-MS</td>
			<td>Ionicon Analytik Ges.m.b.H.</td>
			<td>PTR-TOF 8000</td>
			<td>An detector for volatile organic compounds (VOCs) that allows for continuous VOC quantification with a very high mass resolution</td>
		</tr>
		<tr>
			<td>GERSTEL&#160;MPS 2XL</td>
			<td>Gerstel</td>
			<td></td>
			<td>A multifunctional autosampler&#160;</td>
		</tr>
		<tr>
			<td>Gas Calibration Unit</td>
			<td>Ionicon Analytik Ges.m.b.H.</td>
			<td>GCU-s / GCU-a</td>
			<td>A dynamic gas dilution system that provides variable but known quantities of different standard compounds in a carrier gas stream</td>
		</tr>
		<tr>
			<td>TofDaq</td>
			<td>Tofwerk AG</td>
			<td>free available at http://soft.tofwerk.com/&#160;&#160;&#160;</td>
			<td>A data acquisition software&#160;(for spectra&#160; acquisition)</td>
		</tr>
		<tr>
			<td>MATLAB&#160;</td>
			<td>MathWorks</td>
			<td>http://it.mathworks.com/products/matlab/</td>
			<td>A technical computing language and interactive environment for algorithm development, data visualization, and data analysis</td>
		</tr>
		<tr>
			<td>R</td>
			<td>The R Foundation</td>
			<td>free available at https://cran.r-project.org/mirrors.html&#160;&#160;</td>
			<td>A language and environment for statistical computing and graphics</td>
		</tr>
	</tbody>
</table>

ptr-tof-ms coupled with an automated sampling system and tailored data analysis for food studies: bioprocess monitoring, screening and nose-space analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

The volatile profile of samples resulted in a complete mass spectrum for the desired mass range acquired each second. In Figure 2, an example of the acquired average spectra during the yogurt on-line bioprocess is given32. In every spectrum, more than 300 mass peaks in the m/z range up to 250 Th can be identified32.
<img alt="Figure 2" src="/files/ftp_upload/54075/...

Watch this Scientific Journal Video about PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis at 

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

The overall goal of this paper is to demonstrate the potential of PTR-ToF-MS for direct volatile organic compound analysis in food science and technology and in particular the importance of automated sampling and tailored data analysis. Volatile organic compound play a key role in food science and technology both because they control flavor and odor perception and because they provide a tool for the monitoring of food quality. The high sensitivity and the rapid and direct injection make PTRMS one of the best choices for the monitoring of volatile organic compound release for bio process monitoring, for the screening of large data sets and for nose space analysis.We also develop proper data and the analysis tools which are necessary for proper exploitation of large and complex data sets such as there was used by PTRMS in a very short time. Future demonstration of our methodology can inspire searches from different statistical and scientific fields such as SENSOMICS, metabolomics, path analysis, and phonemics. To begin the first experiment, add five milliliters of pasteurized milk to 20 milliliter glass vials equipped with PTFE silicone septa.Then transfer the samples to a multi functional GC auto sampler equipped with a temperature control tray. Use the robotic arm of the auto sampler to inoculate the vials with the microbial starter cultures, set the incubation time according to the typology of the desired process and the specifications reported by the starter culture manufacturer. Then set the auto sampler to analyzer one sample after the other obtaining an online volatile organic compound monitoring of lactic acid fermentation during yogurt preparation.The second experiment select at least five apples at the desired phase of ripening without any visible damage for each variety. Store the apples for the desired period at four degrees celsius. Following this, collect five cylindrical disks from each apple with a flesh blade sampler including part of the cortex tissue and avoiding the core portion with seeds.After freezing the samples and liquid nitrogen homogenize them. Place three replicates of 2.5 gram apple samples from each variety into 20 milliliter glass vials. Mix each sample with an antioxidant solution obtained by dissolving one gram of sodium chloride, 12.5 milligrams of ascorbic acid and 12.5 milligrams of citric acid in 2.5 milliliters of deionized water.Incubate the samples at 40 degree Celsius. Then set the auto sampler to automatically analyze the volatile organic compounds. For the last experiment, prepare brewed coffee from ground coffee samples with a six cup stove top coffee maker using 450 milliliters of water and 30 grams of coffee powder.Transfer the brewed coffee to a bottle and place the bottle in a 60 degree celsius thermostatic water bath. Report the water powder ratio, the type of mineral water used the type of coffee maker and the procedure adopted to obtain the coffee beverage. For each coffee brew, transfer 7.5 milliliter aliquots to a 40 milliliter polystyrene cup with a plastic cap.Have each panelist taste the beverage according to the protocol. While PTR-ToF-MS continuously samples the Nose space concentration via a single use ergonomic silicon rubber nose piece at the nose of each panelist. This nose piece is connected to the PTR-ToF-MS by a PIC tube with the part in contact with panelist at room temperature and the remaining tube heated at 110 degree Celsius and an inlet hose that connects the sampling interface with the instrument.To perform head space measurements of the samples directly inject air into the PTRMS drift tube head space by inserting the end of the PTRMS inlet into the sample head space. Before and during measurements, set and constantly verify the following ionization conditions in the drift tube to lead to an E/N ratio of 140 Townsend. If an auto sampler is used, set the minimum inlet flow to 40 standard cubic centimeters.Set the sampling time per channel of ToF acquisition to a master charge ratio range. Use the self developed software to correct the count losses due to the ion detector dead time and perform internal calibration, compound annotation noise reduction, and peak extraction. Mine the data by performing principle compound analysis analysis of variance Tukey's post-hoc test and other statistical analysis adapting existing packages developed using Matlab.The three experiments provide examples of applying our methodology bio process monitoring, screening of sample sets, and in vivo nose space analysis. The online volatile organic compound profile of samples gives a complete mass spectrum every second and more than 300 mass peaks can be identified in the acquired average spectra during the yogurt fermentation. Fermentation kinetics of nine selected mass peaks during yogurt fermentation using four different starter cultures are shown here.Most of these volatiles displayed classical microbial like kinetics with an initial lag phase followed by a growth phase and a post-log phase. Interestingly the online analysis highlighted particular depletion kinetics for four sulfur containing compounds which can have a sensory effect even at a very low concentration. The dendrogram based on volatile organic compounds associated with a large apple collection represented by a 198 sessions highlights the presence of six main clusters mainly determined by esters and alcohols.The cumulative profiles for five coffee testers represented by means of radial plots are shown here. The results demonstrate the presence of reproducible and relevant differences between panelists as evident for panelist P one and P two. The difference of samples, the most tried out methodology can provide reliable data on volatile organic compound release, which when compared with our methods can be obtained in a shorter time.The possibility of measuring many samples the key feature of our approach. Because it allows controlling and tracing viability of biological samples. After this development, this methodology paid well for the researchers in different fields related to volatile organic compound monitoring to explore the link between their release and genetics and the screening of populations.After watching this video, you should have a good idea of how to explore the potential of PTR-ToF-MS in food and technology analysis to analyze volatility by fruits. Please remember that samples and measuring line are exposed to air during your experiment. So avoid to introduce some sources of volatile organic compounds.Another advantage of PTRMS is that it doesn't involve the use of solvents or hazardous dyes and it is a green chemistry technique. However don't forget that your samples can be extremely hazardous.

ptr-tof-ms-coupled-with-an-automated-sampling-system-tailored-data

Research

JoVE Journal

Chemistry

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The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

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The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

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An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

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Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

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In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

A number of different procedures for preparing nanoparticles for surface analysis are presented (drop casting, spin coating, deposition from powders, and cryofixation). We discuss the challenges, opportunities, and possible applications of each method, particularly regarding the changes in the surface properties caused by the different preparation methods.

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, ...

ARL Spectral Fitting as an Application to Augment Spectral Data via Franck-Condon Lineshape Analysis and Color Analysis

The ARL Spectral Fitting application provides a free, publicly accessible, and fully transparent method for performing Franck-Condon Lineshape Analysis (FCLSA) on spectral data, in addition to CIE color coordinate determination and basic spectral processing. While some of the features may be found in commercial software or in programs made by&#160;academic research groups, we believe that ARL Spectral Fitting is the only application that possesses all three of the aforementioned attributes.
This program is intended as a standalone, GUI-based application for use by an average laboratory researcher without requiring any coding knowledge or proprietary software. In addition to the standalone executable hosted on ARL GitHub, the associated MATLAB files are available for use and further development.
FCLSA augments the information found in luminescence spectra, providing meaningful insight into the relationship between the ground and excited states of a photoluminescent species. This insight is achieved by modeling spectra with two versions (modes) of an equation that are characterized by either four or six parameters, depending on which mode is used. Once optimized, the value of each of these parameters can be used to gain insight into the molecule, as well as to perform further analysis (for example, the free energy content of the excited-state molecule). This application provides tools for easy by-hand fitting of imported data, as well as two methods for optimizing this fit-damped least-squares fitting, powered by the Levenberg-Marquardt algorithm, and derivative-free fitting utilizing the Nelder-Mead simplex algorithm. Furthermore, estimations of sample color can be performed and reported in CIE and RGB coordinates.

This protocol introduces Franck-Condon Lineshape Analyses (FCLSA) of emission spectra and serves as a tutorial for the use of ARL Spectral Fitting software. The open-source software provides an easy and intuitive way to perform advanced analysis of emission spectra including excited state energy calculations, CIE color coordinate determination, and FCLSA.

The ARL Spectral Fitting application provides a free, publicly accessible, and fully transparent method for performing Franck-Condon Lineshape Analysis ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...