This research was supported by the National Natural Science Foundation of China (32002094, 32102444), the China Agriculture Research System of MOF and MARA (CARS-19), and the Innovation Project for Chinese Academy of Agricultural Sciences (CAAS-ASTIP-TRI).

1. Preparation of the internal standard and tea infusion
<ol>
	<li>Stock solution: Dissolve 10.0 mg of paraxylene-d10 (see Table of Materials) in 10.0 mL of anhydrous ethanol to prepare a 1,000 ppm stock solution of the internal standard.</li>
	<li>Working solution: Dilute 1 mL of the stock solution (step 1.1) to 100 mL with pure water to prepare a 10 ppm working solution of the internal standard. 
	NOTE: The working solution must be prepared on the same day as the analysis.</li>
	<li>Place 3 g of tea leaves (both for green tea and black tea, see Table of Materials) into an Erlenmeyer flask, and add 150 mL boiling water. Cover the flask with a glass stopper.</li>
	<li>After 5 min, quickly filter out the tea infusion through a 300-mesh sieve.</li>
	<li>Wash the spent tea leaves twice with 30 mL of water, and combine the wash solution with the tea infusion.</li>
	<li>Cool the tea infusion to room temperature quickly in an ice water bath.</li>
	<li>Add 1.00 mL of working solution (step 1.2) into the tea infusion, and mix them well.</li>
</ol>
2. Distillation of the tea infusion by SAFE and liquid-liquid extraction of the distillate
<ol>
	<li>Prepare the SAFE assembly following the steps below.
	<ol>
		<li>Install the SAFE assembly (Figure 1), and connect the distillation bottle at the lower left (Figure 1[3]) and the collection bottle at the lower right (Figure 1[4]). Connect the circulating water tube at the rear of the SAFE glass assembly. Install the cold trap (Figure 1[5]), and connect the tube to the vacuum pump (see Table of Materials) at the upper right of the glass assembly. 
		NOTE: Check the connection of the circulating water tube; ensure that the inlet enters the top and the outlet exits from the bottom. Use deionized water for the circulation to prevent the scale from blocking the white tube in the SAFE assembly, which would result in poor circulation of the circulating water and the eventual explosion of the SAFE assembly.&#160;The distillation bottom (Figure 1[3]) can be stirred by a stirring bar to facilitate the evaporation of the sample.</li>
		<li>Set the temperature of the circulating water to 50 &#176;C and that of the water bath for the sample flask to 40 &#176;C. Close the vacuum valve (Figure 1[2]).</li>
	</ol>
	</li>
	<li>Perform the vacuum pump operation.
	<ol>
		<li>Power on the vacuum pump.</li>
		<li>Gradually increase the speed to the maximum speed of 100%. 
		NOTE: If the speed does not reach 100%, check whether the system is airtight and whether there is solvent residue inside the system.</li>
		<li>After reaching a high vacuum (preferably 10-3 Pa) 
		NOTE: The vacuum will improve when the liquid nitrogen is added to the cold trap.</li>
	</ol>
	</li>
	<li>Perform sample distillation.
	<ol>
		<li>Start the water circulation.</li>
		<li>Add liquid nitrogen to the cold trap to cover the outside of the collecting bottle.</li>
		<li>Pour the tea infusion into the sample funnel at the top left (Figure 1[1]), and then cover it with a glass stopper.</li>
		<li>Introduce the sample into the distillation flask dropwise. Control the sample drop speed so that the vacuum is kept in the proper range of around 10&#8722;3 Pa. 
		NOTE: Add liquid nitrogen during the process to ensure that the right collecting bottle is always submerged in liquid nitrogen. Try to avoid condensate formation in the cold trap.</li>
	</ol>
	</li>
	<li>Turn off the vacuum pump after the distillation is completed.
	<ol>
		<li>Press the power switch. When &#34;STOP&#34; flashes, press the Enter key to confirm.</li>
		<li>Unplug the power cord when the speed of the molecular pump decreases to &#34;0&#34;. 
		NOTE: Restart only when the speed decreases to &#34;0&#34;.</li>
	</ol>
	</li>
	<li>Restore the system to atmospheric pressure.
	<ol>
		<li>Remove the grinding plug above the sampling bottle.</li>
		<li>Unscrew the knob of the vacuum valve slowly to restore the system to atmospheric pressure.</li>
	</ol>
	</li>
	<li>Take down the collecting bottle with the sample.
	<ol>
		<li>Remove the liquid nitrogen outside the collection bottle after recovering the system to atmospheric pressure.</li>
		<li>Unscrew the collection bottle slowly. Take down the collecting bottle with the sample carefully.</li>
		<li>Close the circulating water.</li>
	</ol>
	</li>
	<li>Perform liquid-liquid extraction of the SAFE distillate.
	<ol>
		<li>Let the SAFE distillate in the bottle warm to room temperature.</li>
		<li>Extract the SAFE distillate thrice with 50 mL of dichloromethane (see Table of Materials).</li>
		<li>Combine the dichloromethane layers. Dry the extract with anhydrous sodium sulfate (see the Table of Materials). 
		NOTE: The anhydrous sodium sulfate in the solvent is considered dry enough when it is no longer cemented and can flow freely.</li>
		<li>Concentrate the extract to about 2 mL using a gentle nitrogen stream.</li>
		<li>Transfer to a sample vial of 1-2 mL, and further concentrate to 200 &#181;L using a gentle nitrogen stream.</li>
	</ol>
	</li>
</ol>
3. GC-MS analysis and data processing
<ol>
	<li>Analyze the aroma concentrates prepared in protocol section 2 using a GC-MS system (Figure 2) equipped with fused silica capillary columns (see the Table of Materials).</li>
	<li>Use helium as carrier gas with a linear velocity of 40 cm/s.</li>
	<li>Inject 3 &#181;L of the concentrate in splitless injection mode.</li>
	<li>Set the GC oven temperature program: (1) hold at 40 &#176;C for 5 min; (2) increase to 200 &#176;C at 5 &#176;C/min; (3) increase to 280 &#176;C at 10 &#176;C/min; (4) hold at 280 &#176;C for 10 min.</li>
	<li>Operate the mass selective detector in positive EI mode13 with a mass scan range from 30 m/z to 350 m/z at 70 eV.</li>
	<li>Deconvolute the GC-MS data using the Automated Mass Spectral Deconvolution and Identi&#64257;cation System (AMDIS, see the Table of Materials).</li>
	<li>Match and qualify the data after deconvolution using the NIST (National Institute of Standards and Technology) 17 mass spectrometer search program3.</li>
	<li>Calculate the retention index of the compounds14 based on the result of a set of n-alkanes (C5-C25, see the Table of Materials) under the same GC conditions.</li>
	<li>Identify the GC peaks using the NIST mass spectrometry library and the retention index database based on the simultaneous matching of the mass and retention indexes.</li>
	<li>Calculate the concentration of each volatile component in the SAFE sample relative to the internal standard using the TIC (total ion chromatography) peak area.</li>
	<li>Repeat the analysis three times, starting from the tea infusion preparation.</li>
</ol>

Unveiling Tea Aroma Complexity with Solvent-Assisted Flavor Evaporation Method

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C., Wei, G., Yang, X., Schieberle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+key+aroma+compounds+in+aLongjinggreen+tea+infusion+(Camellia+sinensis)+by+the+sensomics+approach+and+their+quantitative+changes+during+processing+of+the+tea+leaves.">Characterisation of the key aroma compounds in aLongjinggreen tea infusion (Camellia sinensis) by the sensomics approach and their quantitative changes during processing of the tea leaves.</a> European Food Research and Technology. 246 (12), 2411-2425 (2020).</li><li>Feng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tea+aroma+formation+from+six+model+manufacturing+processes.">Tea aroma formation from six model manufacturing processes.</a> Food Chemistry. 285, 347-354 (2019).</li><li>Wang, J. -. 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Part I: Comparison of headspace-solid phase microextraction and solvent assisted flavour evaporation.</a> Lwt-Food Science and Technology. 94, 178-189 (2018).</li><li>Li, Z. W., Wang, J. 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The authors have nothing to disclose.

This article describes an efficient method for analyzing volatile compounds in tea infusions using SAFE and GC-MS analysis.
Tea infusions have a complex matrix with a high content of non-volatile components. Several methods have been described in the literature for isolating the volatile components from tea infusions. A common method is simultaneous distillation extraction (SDE)15,16. However, it is not suitable for the analysis of tea...

Tea is a preferred beverage of many people all over the world1,2. The aroma of the tea is a quality criterion as well as a price-determining factor for tea leaves3,4. Thus, the analysis of the aroma composition and content of tea is of great significance for molecular sensory studies and the quality control of tea. As a result, aroma composition analysis has been an important topic in tea research in recent years5,6,7.
The content of aroma components in tea is very low, as they generally only account for 0.01%-0.05% of the dry weight of the tea leaves8. Furthermore, the large amount of non-volatile components in the sample matrix significantly interferes with analysis by gas chromatography9,10. Therefore, a sample preparation procedure is essential to isolate the volatile compounds in tea. The key consideration for the isolation and enrichment method is minimizing the matrix interference and, at the same time, maximizing the preservation of the original odor profile of the sample.
Solvent-assisted flavor evaporation (SAFE), originally developed by Engel, Bahr, and Schieberle, is an improved high-vacuum distillation technique used to isolate volatile compounds from complex food matrixes11,12. A compact glass assembly connected to a high-vacuum pump (under a typical operating pressure of 5 x 10&#8722;3 Pa) can efficiently collect volatile compounds from solvent extracts, oily foods, and aqueous samples.
This article described a method that combines the SAFE technique with solvent extraction to isolate volatile substances from a black tea infusion, followed by analysis using GC-MS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alkane mix (C10-C25)</td>
			<td>ANPEL</td>
			<td>CDAA-M-690035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkane mix (C5-C10)</td>
			<td>ANPEL</td>
			<td>CDAA-M-690037</td>
			<td></td>
		</tr>
		<tr>
			<td>AMDIS</td>
			<td>National Institute of Standards and Technology</td>
			<td>version 2.72</td>
			<td>Gaithersburg, MD</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>OHAUS</td>
			<td>EX125DH</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous sodium sulfate</td>
			<td>aladdin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black tea</td>
			<td>Qianhe Tea</td>
			<td></td>
			<td>Huangshan, Anhui province, China</td>
		</tr>
		<tr>
			<td>Concentrator</td>
			<td>Biotage</td>
			<td>TurboVap</td>
			<td></td>
		</tr>
		<tr>
			<td>Data processor</td>
			<td>Agilent</td>
			<td>MassHunter</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>TEDIA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GC</td>
			<td>Agilent</td>
			<td>7890B</td>
			<td></td>
		</tr>
		<tr>
			<td>GC column</td>
			<td>Agilent</td>
			<td>DB-5MS</td>
			<td></td>
		</tr>
		<tr>
			<td>Green tea</td>
			<td>Qianhe Tea</td>
			<td></td>
			<td>Huangshan, Anhui province, China</td>
		</tr>
		<tr>
			<td>MS</td>
			<td>Agilent</td>
			<td>5977B</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Xylene-d10</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SAFE</td>
			<td>Glasbl&#228;serei Bahr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure deionized water</td>
			<td>Milipore</td>
			<td>Milli-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Edwards</td>
			<td>T-Station 85H</td>
			<td></td>
		</tr>
	</tbody>
</table>

tea aroma analysis based on solvent-assisted flavor evaporation enrichment

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the volatile components of tea extract. This study presents a method for obtaining and analyzing the volatile components of tea extract with odor preservation using solvent-assisted flavor evaporation (SAFE) and solvent extraction followed by gas chromatography-mass spectrometry (GC-MS). SAFE is a high-vacuum distillation technique that can isolate volatile compounds from complex food matrices without any non-volatile interference. A complete step-by-step procedure for tea aroma analysis is presented in this article, including the tea infusion preparation, solvent extraction, SAFE distillation, extract concentration, and analysis by GC-MS. This procedure was applied to two tea samples (green tea and black tea), and qualitative as well as quantitative results on the volatile composition of the tea samples were obtained. This method can not only be used for the aroma analysis of various types of tea samples but also for molecular sensory studies on them.

The analytical procedure described above is illustrated in this section using the example of the aroma analysis of black tea and green tea samples.
A representative GC-MS chromatogram is shown in Figure 3. Figure 3A shows a set of n-alkanes, and Figure 3B shows the profile of an internal standard. The evaluation results for the extracts from the green tea and black tea samples are shown in <strong class="...

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Author Spotlight: Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique 

Presented here is a method for enriching and analyzing the volatile components of tea extracts using solvent-assisted flavor evaporation and solvent extraction followed by gas chromatography-mass spectrometry, which can be applied to all types of tea samples.

Tea Aroma Analysis Based on Solvent-Assisted Flavor Evaporation Enrichment

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the ...

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2.9K Views.  Tea Research Institute Chinese Academy of Agricultural Sciences. Presented here is a method for enriching and analyzing the volatile components of tea extracts using solvent-assisted flavor evaporation and solvent extraction followed by gas chromatography-mass spectrometry, which can be applied to all types of tea samples.

Video: Author Spotlight: Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique 

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

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 ...

Scale-up Chemical Synthesis of Thermally-activated Delayed Fluorescence Emitters Based on the Dibenzothiophene-S,S-Dioxide Core

We report a procedure to linearly scale-up the synthesis of 2,8-bis(3,6-di-tert-butyl-9H-carbazol-9-yl)dibenzothiophene-S,S-dioxide (compound 4) and 2,8-bis(10H-phenothiazin-10-yl)dibenzothiophene-S,S-dioxide (compound 5) using Buchwald-Hartwig cross-coupling reaction conditions. In addition, we demonstrate a scaled-up synthesis of all non-commercially available starting materials that are required for the amination cross-coupling reaction. In the present article, we provide the detailed synthetic procedures for all of the described compounds, alongside their spectral characterization. This work shows the possibility to produce organic molecules for optoelectronic applications on a large scale, which facilitates their implementation into real world devices.

Scale-up synthesis of highly efficient thermally activated delayed fluorescence emitters is described in the presented article.

We report a procedure to linearly scale-up the synthesis of 2,8-bis(3,6-di-tert-butyl-9H-carbazol-9-yl)dibenzothiophene-S,S-dioxide (compound 4) and ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

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

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

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 ...