The authors would like to aknowledge the colleagues and people involved in the different steps of the project. The colleagues in the JRC Karlsruhe design office, Messrs. Dietrich Knoche and Volkmar Ernest, in the JRC Karlsruhe Workshop Messrs. Christian Diebold and Joachim K&#252;st for the design and manufacturing of the lab on valve (LOV) manifold and Ringwald GmbH for the software implementation of the stopped-flow detection mode technique.

Note: Solutions were prepared from high-purity water (18.2 M&#937; cm) using devices designed for ultratrace analysis. Nitric acid was purified using a quartz sub-boiling distillation unit. Both the water purification system and the sub-boiling distillation unit were operated in a clean room.
Caution: 90Sr is acutely toxic and carcinogenic. Appropriate safety practices are essential when performing the experiments, including both engineering control systems and personal protective equipment.
1. Experiment Preparation
Note: A detailed description regarding the software architecture and its functionalities can be found elsewhere23.
<ol>
	<li>Connect the Modular Valve Position exit port to the entry port of the radio flow detector.</li>
	<li>Connect the Trigger line to selected detector.</li>
	<li>Ensure that the LOV ports are properly connected. Ensure that adequate volumes of working solutions are available for the entire protocol and that liquid sampling tubing will remain submerged.</li>
	<li>Ensure that the Autosampler is on, start the autosampler software, and via the software interface, initialize the autosampler. Click the Initialize button to generate the communication between the apparatus and the PC.</li>
	<li>Ensure the AutoRAD software has been started, check the communication ports using the options tab and initialize the software via the user interface. Refer to Discussion section for more details regarding the software graphical interface.</li>
	<li>Ensure that the analysis sequence is programmed in the detector Software. Program vew sequences using the Method editor tab in the AutoRAD software, by entering the number of steps needed and the tasks and speed for each device. 
	Note: A detailed description of the software operation can be found in previous publication23.</li>
	<li>Consult the local radioprotection officer and employ the radioprotection measurements needed for the complete procedure using 90Sr.</li>
</ol>
2. System Cleaning
<ol>
	<li>Load 10 mL of 18.2 M&#937; cm water from the flask to the syringe at 90 mL&#183;min-1. Ensure that the syringe valve position is set to the In position for this step. In this position, loading occurs directly to the syringe and not through the LOV.</li>
	<li>Drop the water to the waste via the holding coil at a flow rate of 90 mL&#183;min-1.</li>
	<li>Load 3 mL of ethanol to the holding coil at a flow rate of 3 mL&#183;min-1. Set the syringe valve position to Out.</li>
	<li>Drop the ethanol to the detector coil at a flow rate of 3 mL&#183;min-1.</li>
</ol>
3. Load Resin to the LOV
<ol>
	<li>While stirring the resin suspension in water (12 mg&#183;mL-1), pick up 3 mL via the LOV. Set the flow to 3 mL&#183;min-1</li>
	<li>Drop the resin suspension into the column channel at a flow rate of 1.2 mL&#183;min-1</li>
	<li>Clean the holding coil and drop the resin residues onto the column channel. For this, load 9 mL of 18.2 M&#937; cm water from the flask to the syringe at a flow rate of 90 mL&#183;min-1. Ensure that the syringe valve position is in the In position. Drop the water to the waste via the holding coil at a flow rate of 3 mL&#183;min-1.</li>
</ol>
4. Analysis Sequence
<ol>
	<li>Column conditioning
	<ol>
		<li>Load 2 mL of HNO3 (4 M) via the LOV to the holding coil. Set the flow to 6 mL&#183;min-1.</li>
		<li>Drop the HNO3 onto the column at a flow rate of 1.2 mL&#183;min-1.</li>
	</ol>
	</li>
	<li>Sample loading and elimination of interferences
	<ol>
		<li>Load sample (1.3 mL) from the autosampler to the holding coil at a flow rate of 6 mL&#183;min-1.</li>
		<li>Drop the sample onto the column at a flow rate of 1.2 mL&#183;min-1.</li>
		<li>Load 0.5 mL of HNO3 (4 M) to the holding coil at a flow rate of 6 mL&#183;min-1.</li>
		<li>Rinse the column with 0.5 mL of HNO3 (4 M) to elute matrix interferences at a flow rate of 1.2 mL&#183;min-1.</li>
	</ol>
	</li>
	<li>Elution of the sample and measurement
	<ol>
		<li>Load 5 mL of 18.2 M&#937; cm water to the holding coil at a flow rate of 6 mL&#183;min-1.</li>
		<li>Trigger the detector. Set the flow rate of the scintillation liquid to 2 mL&#183;min-1 in the detector software. Set the dwell to 10 s. The sample coil has a volume of 2 mL.</li>
		<li>Rinse the column.</li>
	</ol>
	</li>
	<li>rinsing of the sample probe and holding coil
	<ol>
		<li>Load 0.6 mL of HNO3 (1%) to the holding coil at a flow rate of 6 mL&#183;min-1.</li>
		<li>Load 0.6 mL of air to the holding coil at a flow rate of 6 mL&#183;min-1.</li>
		<li>Discharge 1.2 mL of the mixture to the waste.</li>
	</ol>
	</li>
	<li>Resin replacement
	<ol>
		<li>Load 0.2 mL of ethanol to the holding coil at a flow rate of 3 mL&#183;min-1.</li>
		<li>Rinse the column with 0.2 mL of ethanol at a flow rate of 1.2 mL&#183;min-1.</li>
		<li>Flush the column channel with 0.5 mL of water at a flow rate of 0.45 mL&#183;min-1.</li>
		<li>Discharge the used resin to the waste.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Radiological characterization and protection are critical issues during all phases of a nuclear facility life cycle. The need for radionuclide determination during the decommissioning of nuclear facilities requires continuous improvement of analytical procedures. This entails the enhancement of selectivity and sensitivity, and shortening the analysis time . The AUTORAD prototype fulfils these requirements. In addition, the prototype is portable and allows on-site determination. The proposed automated on-line method was successfully applied for the determination of 90Sr activity in aqueous samples.
Figure 1 shows a schematic diagram of the AutoRAD system. Communication with the prototype was achieved using the commercial graphical programming language LabVIEW 2014. The Virtual Instrument Software Architecture (VISA) mode for configuring, programming and controlling interfaces was deployed. Figure 2 shows the graphical user interface screen where the port configuration can be verified prior to system initialization. The VISA mode is independent on the operating system and program environment, so it provides exceptional versatility. The basic structure and features of the developed software have been widely discussed in previous publication24. Improvements have been achieved by controlling the burettes by the PC using an RS232 interface, thus allowing enhanced control of both burettes simultaneously and the possibility to perform measurements in stop flow mode. In contrast, the RS232 connection reacts quickly when the system backpressure increases. This can lead to system error and measurement stop. Therefore, special attention has to be paid in the process of resin loading and ionic strength of the samples.
The optimization of the experimental conditions was achieved in a set of experiments using the strontium stable isotope 86Sr as a surrogate for the radioactive 90Sr, and by coupling the AUTORAD system to an ICP-MS rather than a beta detctcor system. Figure 3 shows the ICP-MS 86Sr elution profiles. The obtained 86Sr elution profiles are in full agreement with previously reported results for total strontium using low pressure separation devices25, and have been blank subtracted. The least squares linear regression method was used to generate a fit for the purpose curve which does not introduce an extra uncertainty component. The linearity obtained was 0.995, with p-values less than the significance level. The LOD of 2 pg&#183;g-1was determined by repeated measurements of the blank according to Currie26. The reproducibility of the method, based on the relative standard deviation of the peak area calculated on the basis of three repeated runs, was always less than 4% in the range of 10 to 120 pg&#183;g-1. The shoulder witnessed in the elution profiles is most likely an artefact due to non-optimal packing of the column in the automated AUTORAD configuration.
Figure 4 shows the 90Sr profile using the the radio flow detector. The AUTORAD system is able to effectively separate 90Sr in aqueous samples.
Figure 5 shows the dependence of the 90Sr concentration with the signal is linear in the range of interest. The derived detection limit is in the fg. g-1 range, enabling, without further modification, the determination of radiostrontium in nuclear decommissioning and waste characterization samples. Reproducibility of the method, using the flow scintillation detector and based on the relative standard deviation of the peak area, is around 30% in the studied concentration range. The current set-up of the prototype, however, limits its application to environmental samples, essentially due to the short time counting in the detector. Furthermore, complex matrices might saturate the resin in the mini column.
The stopped-flow technique, where both Hamilton and radio flow detector pump are stopped during the measurement, will be implemented. This feature will improve the sensitivity by extending the residence time in the largest part of the sample zone within the flow cell. Thus, a statistically meaningful signal is acculated before the sample exits the detector. This approach will improve both the counting statistics and the detection limits. In addition, a new application is being developed including an additional ion-exchange mini-column for the removal of matrix components that could affect the retention and separation of different radionuclides in complex matric samples.

About 150 commercial nuclear power plants (NPP) are undergoing decommissioning, but the total number of nuclear facilities is much greater if research and reprocessing facilities are taken into consideration1. Decommissioning of nuclear facilities is very costly and involves transport and off site measurement of contaminated material. Savings are possible by adopting on-site and flexible measurement techniques1. There is therefore an urgent need for rapid on site analytical methods supporting the decommissioning of old nuclear facilities. Gamma-emitters can be easily and selectively determined using gamma-spectroscopy, but there is a deficiency in on-site analytical methods for the hard-to-measure (HTM) radionuclides2. Among the HTM, 90Sr is of great interest due to its toxicity and high energy emission. Its determination is time consuming and requires the separation from interferences and quantification using LSC or spectroscopic methods3,4,5,6,7,8,9,10,11,12,13.
Standard radiochemical methods are time-consuming and often require repetitions to obtain an adequate yield of strontium. Therefore, there is an urgent need for faster and accurate methods. Besides the standard separation protocols, flow injection techniques are applied in the purification and/or pre-concentration of radioisotopes5,14,15. Lab-on-Valve (LOV) devices are a further development in flow techniques. They are programmable, flow-based platforms with different coupling modes and exhibit high versatility16. Such devices allow the automatic separation and pre-concentration of the analytes prior to the detection, thus increasing reproducibility and repeatability8,17,18,19. LOV systems coupled to multisyringe flow pumps have been widely used for the detection of radionuclides, due to the minimization of the reagent consumption and waste generation8,10,17,18,19,20. Nevertheless, reported studies regarding online detection at ultra-trace levels are sparse8,17.
On-site measurements of radioactive materials have many benefits and advantages, but there is no application of flow scintillation to 90Sr monitoring. Basically, quantification is achieved using an online detector that mixes the eluate coming from the LOV device with the scintillating cocktail11,21,22. The mixture is then pumped through the counting cell and measurement is made using paired photomultipliers. Due to the small volume of the cell, the measurement time is in the seconds scale.
The aim of this research is the development of a fully automated method for on-line strontium detection over a large working range, covering both environmentally relevant concentrations and also those found in nuclear industry waste streams. The platform is mobile and can be mounted in a vehicle to perform on-site analysis of aqueous samples.

<table><tbody><tr><td>microLAB 600 series</td><td>Hamilton</td><td>ML600EE6910</td><td>Two dual syringe pump system, equiped with two 10 mL glass syringes. The instruments are interconnected using the CAN port (daisy chain).</td></tr><tr><td>FlowLogic U</td><td>LabLogic</td><td>SG-BXX-05</td><td>Liquid scintillator with high flash point</td></tr><tr><td>&amp;szlig;-RAM 5</td><td>LabLogic</td><td></td><td>flow detector, 2000 &amp;mu;L coiled Teflon flow cell.Software Laura 4.2.8 (LabLogic, England) run on desktop PC and connected to the detector via USB</td></tr><tr><td>SC-&amp;mu; DX Autosampler</td><td>Elemental Scientific Instruments (ESI)</td><td></td><td></td></tr><tr><td>Cheminert selector</td><td>Valco Instruments Co. Inc.</td><td></td><td>in-house made Lab-on-Valve has been mounted on this selector</td></tr><tr><td>Modular Valve&lt;br/&gt; Positioner (MVP)</td><td>Hamilton</td><td></td><td></td></tr><tr><td>mini magnetic stirrer</td><td>IKA</td><td></td><td></td></tr><tr><td>Nitric Acid Suprapur 65%</td><td>Merck</td><td>1.00441.1000</td><td>purified using quartz sub-boiling distillation unit</td></tr><tr><td>Sr-resin</td><td>Eichchrom Tecnologies, Inc</td><td>SR-B100-A</td><td>particle size 100-150 &amp;micro;m</td></tr><tr><td>Water system Elix 3 in combination with Mili-Q Element A10</td><td>Millipore</td><td></td><td>high-purity water (18.2 M&amp;Omega; cm)</td></tr><tr><td>Sr-90 standard</td><td>Eckert &amp;amp; Ziegler</td><td>7090</td><td>Sr-90 concentration 1.915 kBq/g &amp;plusmn; 3.0%, reference date 15-May-2016 12:00 PST</td></tr><tr><td>MLS quartz sub-boiling distillation unit</td><td>MLS GmbH</td><td></td><td>Subboiling unit for the purification of HCl and nitric acid</td></tr></tbody></table>

automated 90sr separation and preconcentration in a lab-on-valve system at ppq level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg&#183;g-1 (1.7 Bq/g).

The fully automated AUTORAD platform operated by homemade LabVIEW-based software has been developed and implemented (Figure 1). The software allows sufficient flexibility for daily operation in a user-friendly environment (Figure 2). The versatility of the platform has been also demonstrated by coupling it to different detectors (Figure 3). The applicability of the method has been demonstrated in aqueous samples spiked with a 90Sr standard (Figure 4). The parameters linearity, linear range, limit of detection (LOD) and repeatability have been evaluated (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57722/57722fig1.jpg" /> 
Figure 1. Schematic representation of the AutoRAD system showing the port configuration and reagents used. The central port is connected to the syringe pump via the holding coil (10 mL); the methacrylate LOV was fabricated in house. <a href="/files/ftp_upload/57722/57722fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57722/57722fig2.jpg" /> 
Figure 2. AutoRAD Software options menu. The bottom right corner area shows the assigned ports. Via the scroll down menu, the user is able to make changes in the assignment. <a href="/files/ftp_upload/57722/57722fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57722/57722fig3.jpg" /> 
Figure 3. 86Sr elution peaks using an Inductively coupled plasma mass spectrometry (ICP-MS) as a detector. Strontium is eluted quantitatively from the column during the first 100 s of the elution. <a href="/files/ftp_upload/57722/57722fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57722/57722fig4.jpg" /> 
Figure 4. 34.5 &#177; 1 Bq 90Sr (6.6 pg&#183;g-1) elution peak using &#223;-RAM 5 as detector. The residence time in the coil was 40 s. The overall recovery rate of the proposed methodology for 90Sr is 70% &#177; 5%. <a href="/files/ftp_upload/57722/57722fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57722/57722fig5.jpg" /> 
Figure 5. Calibration curve for the count rate versus the concentration of radiostrontium. A good linearity was obtained (R2 = 0.997). The detection limit was calculated as 320 fg&#183;g-1 &#177; 5 (1.7 Bq) by three point three times standard deviation of the blank. <a href="/files/ftp_upload/57722/57722fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level at JoVE.com

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

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7.9K Views.  European Commission, DG Joint Research Centre - JRC. Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

Video: Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

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 ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Cancer Research

Preparing an Isotopically Pure 229Th Ion Beam for Studies of 229mTh

A methodology is described to generate an isotopically pure 229Th ion beam in the 2+ and 3+ charge states. This ion beam enables one to investigate the low-lying isomeric first excited state of 229Th at an excitation energy of about 7.8(5) eV and a radiative lifetime of up to 104 seconds. The presented method allowed for a first direct identification of the decay of the thorium isomer, laying the foundations to study its decay properties as prerequisite for an optical control of this nuclear transition. High energy 229Th ions are produced in the &#945;&#160;decay of a radioactive 233U source. The ions are thermalized in a buffer-gas stopping cell, extracted and subsequently an ion beam is formed. This ion beam is mass purified by a quadrupole-mass separator to generate a pure ion beam. In order to detect the isomeric decay, the ions are collected on the surface of a micro-channel plate detector, where electrons, as emitted in the internal conversion decay of the isomeric state, are observed.

Preparing an Isotopically Pure 229Th Ion Beam for Studies of 229mTh

We present a protocol for the generation of an isotopically purified low-energy 229Th ion beam from a 233U source. This ion beam is used for the direct detection of the 229mTh ground-state decay via the internal-conversion decay channel. We also measure the internal conversion lifetime of 229mTh as well.

A methodology is described to generate an isotopically pure 229Th ion beam in the 2+ and 3+ charge states. This ion beam enables one to investigate the ...

Engineering

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Bioengineering

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Automated ⁹⁰Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

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