We acknowledge funding from NIH (NHLBI Contract No. HHSN268201000044C to R.W., S.H. and S.Y.S.).

1. Preparation of GST and Nanoparticle (NP) Conjugates
<ol>
	<li>GST-TCO preparation:
	<ol>
		<li>Add 8 &#956;l of TCO-NHS solution (50 mM in DMSO) to 100 &#956;l of GST (1 mg/ml in PBS) and shake mixture at RT for 1 hr.</li>
		<li>Remove excess reagent using a Zeba spin desalting column. The recovered filtrate containing the GST-TCO conjugate is stored at 4 &#176;C prior to use.</li>
	</ol>
	</li>
	<li>GST-Tz preparation:
	<ol>
		<li>Add 6 &#956;l of Tz-NHS solution (25 mM in DMF) to 75 &#956;l of GST (1 mg/ml in PBS) and shake mixture at RT for 1 hr.</li>
		<li>Dilute the reaction mixture with 25 &#956;l of PBS and purify using a spin desalting column. The filtrate containing the GST-Tz conjugate is stored at 4 &#176;C prior to use.</li>
	</ol>
	Note: Mass analyses (MALDI-TOF) showed that on average ~10 Tz or TCO molecules were conjugated to GST.</li>
	<li>NP-TCO preparation:
	<ol>
		<li>Add 100 &#956;l of TCO-NHS solution (50 mM in DMSO) to 150 &#956;l of aminated nanoparticle (NP-NH2, 8.7 mg Fe/ml in PBS, 3 nm iron core ~8,000 Fe/NP) and shake mixture at RT for 1 hr.</li>
		<li>Remove excess reagent by gel filtration using a NAP-10 column eluted with PBS buffer. Collect the colored band containing the NP-TCO product.</li>
		<li>Concentrate the filtrate to a final volume of ~150 &#956;l using a centrifugal filter device (100 k MWCO).</li>
		<li>Add 50 &#956;l of succinic anhydride, (0.1 M in DMSO) to the NP-TCO solution and shake at RT for 1 hr (anhydride reacts with any remaining amines to form terminal carboxylic acids. This prevents nonspecific interactions with the dextran surface).</li>
		<li>Purify the NP-TCO product using a NAP-10 column eluting with PBS. Store the solution of NP-TCO at 4 &#176;C prior to use.</li>
	</ol>
	</li>
</ol>
2. Surface Preparation
All surface plasmon resonance assays are performed on a Biacore T100 instrument (GE Healthcare) at 25 &#176;C using a CM5 sensor chip and PBS-P as the running buffer unless otherwise noted. Biacore Control and Evaluation software supplied with the instrument are employed for setting up experiments and analyzing data. Two modes of operation, application wizards and manual run will be used for surface preparation and monitoring on-chip capture-cycloaddition. The method builder mode will be used for setting up reverse orientation binding experiments and for measuring cycloaddition reaction rates. Data are double reference subtracted and kinetic analyses are performed using a 1:1 Langmuir binding model.
<ol>
	<li>Use the amine immobilization wizard template and select immobilization on Flow cells 1 (reference) and 2 (detection). Modify amine method to activate surface carboxylic groups by injection of a 1:1 solution of 0.4 M EDC: 0.1 M NHS for 480 sec at a flow rate of 10 &#956;l/min. Set the injection of ethanolamine to quench remaining activated esters to 420 sec contact time at a flow rate of 10 &#956;l/min.</li>
	<li>Input ligand name, anti-GST (18 &#956;g/ml in acetate buffer, pH 5.0) and set to inject for a contact time of 420 sec at a flow rate of 10 &#956;l/min.</li>
	<li>Place required solution vials in reagent rack 2 making sure to match the positions with the content list. Save and start immobilization. 
	Note: Running the immobilization wizard in this manner results in anti-GST immobilization levels in the range of ~14,000 to 17,000 RU.</li>
	<li>Edit the immobilization wizard to only inject GST ligand (20 &#956;g/ml) solution for 420 sec at 5 &#956;l/min over reference Flow cell 1.</li>
</ol>
3. Monitoring On-chip Capture-cycloaddition of Functionalized Molecules
<ol>
	<li>Monitoring on-chip capture-cycloaddition in real-time (Figure 2).
	<ol>
		<li>Place GST-TCO (20 &#956;g/ml), Tz-BnNH2 (10 &#956;M) and regeneration (10 mM glycine, pH = 2.0) solution vials in reagent rack 2. Select the manual run method, set the flow rate to 5 &#956;l/min and flow path to Flow cell 2.</li>
		<li>Inject solution of GST-TCO for 420 sec.</li>
		<li>Inject solution of Tz-BnNH2 for 600 sec.</li>
		<li>Regenerate the surface with two 30 sec injections of regeneration solution.</li>
	</ol>
	</li>
	<li>Monitoring on-chip assembly of multi-component structures in real-time (Figure 3):
	<ol>
		<li>Place GST-Tz (20 &#956;g/ml), NP-TCO (100 &#956;g Fe/ml), Tz-BnNH2 (10 &#956;M) and regeneration (10 mM glycine, pH = 2.0) solution vials in reagent rack 2. Select the instrument manual run method, set the flow rate to 5 &#956;l/min and the flow path to Flow cell 2.</li>
		<li>Inject a solution of GST-Tz for 60 sec.</li>
		<li>Inject a solution of NP-TCO for 60 sec.</li>
		<li>Inject a solution of Tz-BnNH2 for 60 sec.</li>
		<li>Regenerate the surface with two 30 sec injections of regeneration solution.</li>
	</ol>
	</li>
</ol>
4. Monitoring Immobilization Density and Determination of Cycloaddition Rates
<ol>
	<li>Characterization of small molecule cycloaddition reaction rates (Figure 4).
	<ol>
		<li>Select new method and input general parameters. In the assay steps panel create a new step and name it Sample. Set the Purpose and Connect base settings to Sample.</li>
		<li>In the Cycle Types panel Create a new cycle step and insert the following commands; Capture, Sample, Regeneration 1 and Regeneration 2.</li>
		<li>Select the Capture command; input ligand name (GST-TCO, 20 &#956;g/ml), contact time 120 sec at 5 &#956;l/min and set flow path to: Second. Select the Sample command; input 180 sec contact time, 60 sec dissociation time, 30 &#956;l/min flow rate and set flow path to: Both. Select the Regeneration 1 command; input regeneration solution name (10 mM glycine-HCl pH 2.0), contact time of 30 sec at 30 &#956;l/min and set flow path to: Second. Repeat the same for Regeneration 2 command.</li>
		<li>Select Setup Run and set flow path to: 2-1. Select next and fill sample list with analyte solution (Tz-BnNH2) and concentration series (0.3-10 &#956;M, 1:2 dilution). Select next to rack position panel. Place solution vials in reagent rack 2 and dilution series in 96-well plate making sure to match the positions with the content list. Save method template and start binding assay. Binding data and kinetic analysis are shown in Figure 4.</li>
	</ol>
	</li>
	<li>Characterization of NP cycloaddition reaction rates (Figure 4).
	<ol>
		<li>Open the saved method template from above and change the following parameters. Select the Capture panel; change ligand name to GST-Tz, 20 &#956;g/ml. Select the Sample panel; change contact time to 120 sec contact time and dissociation time to 120 sec.</li>
		<li>Select the sample list; change analyte to NP-TCO and concentration series (7 to 224 nM, 1:2 dilution). Place solution vials in reagent rack 2 and dilution series in 96-well plate making sure to match the positions with the content list. Save method template and start binding assay. Binding data and kinetic analysis are shown in Figure 4.</li>
	</ol>
	</li>
</ol>
5. Measuring the Binding of FKBP12 to Immobilized AP1497
Reversed-orientation binding studies employ FKBP12 as the analyte and compound AP1497 as immobilized ligand (Figure 5). The general method for this assay is set up as follows using the method builder tool:
<ol>
	<li>Select new method and input general parameters. In the assay steps panel create and name Capture, Sample and Regeneration steps. Select corresponding Purpose and Connect base settings.</li>
	<li>Select Cycle Types panel and create 3 cycle steps: Capture, Sample and Regeneration.</li>
	<li>Insert the Capture command twice under Capture cycle step. Select the Capture 1 panel and select capture solution as variable, set contact time to 300 sec at a flow rate of 5 &#956;l/min and set flow path to: Second. Select the Capture 2 panel and select capture solution as variable, set contact time to 250 sec at a flow rate of 5 &#956;l/min and set flow path to: Second.</li>
	<li>Insert the Sample command under the Sample cycle step. Select the Sample panel; set contact time to 60 sec, dissociation time to 200 sec at a flow rate of 30 &#956;l/min and set flow path to: Both.</li>
	<li>Insert the Regeneration command twice under the Regeneration cycle step. Select the Regeneration 1 panel; input regeneration solution name (10 mM glycine-HCl pH 2.0), contact time of 30 sec at 30 &#956;l/min and set flow path to: Second. Repeat the same for the Regeneration 2 panel.</li>
	<li>Select Setup Run and set flow path to: 2-1. Select next and input capture solution names (GST-TCO and AP1497-Tz), sample name (FKBP12), concentration series (0.020 to 5 &#956;g/ml, 1:2 dilution) and MW (13,000). Place solution vials in reagent rack 2 and dilution series in 96-well plate making sure to match the positions with the content list. Save method template and start binding assay. Kinetic analysis data are shown in Figure 5.</li>
</ol>
6. Measuring the Binding of FKBP12 to AP1497 Attached to Immobilized NPs
The general method for nanoparticle immobilization, small molecule derivatization and FKBP12 binding assay is set-up as follows using the method builder tool:
<ol>
	<li>Open the saved method template from above and modify. Insert an additional Capture command under Capture cycle step. Select the Capture 1 panel; deselect capture solution as variable and name capture solution 1 (GST-Tz). Set contact time to 60 sec at a flow rate of 5 &#956;l/min and set flow path to Second. Select the Capture 2 panel; deselect capture solution as variable and name capture solution 2 (NP-TCO). Set contact time to 90 sec at a flow rate of 5 &#956;l/min and set flow path to second. Select the Capture 3 panel; deselect capture solution as variable and name capture solution 3 (AP1497-Tz). Set contact time to 180 sec at a flow rate of 5 &#956;l/min and set flow path to Second.</li>
	<li>Select Setup Run and set flow path to: 2-1. Select next twice. Place solution vials in reagent rack 2 and dilution series in 96-well plate making sure to match the positions with the content list. Save method template and start binding assay. Kinetic analysis data are shown in Figure 5.</li>
</ol>

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The authors declare that they have no competing financial interests.

The capture-cycloaddition method described here enables rapid, reversible immobilization of modified nanoparticles and small molecules for label-free chip-based interaction and kinetic studies. The immobilization protocol can be performed in minutes requiring &#60;10 &#956;M concentrations of small-molecule ligands. By modulating ligand concentration and contact time immobilization densities can be closely controlled. Our data show that on-chip bioorthogonal reactions preserve the ability of in situ functionaliz...

Efficient conjugation reactions are valuable tools for attaching bioactive molecules to surfaces for a variety of biotechnology applications. Recently, the very fast bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) has been used to label cell surfaces, subcellular structures, antibodies and nanoparticles.1-7 Here, we use the [4+2] cycloaddition reaction in combination with antigen/antibody capture (GST/anti-GST) for reversible on-chip synthesis of multi-component structures for Surface Plasmon Resonance (SPR) interaction analysis and monitor the process in real-time (Figure 1).8,9 Notably, the capture-cycloaddition strategy enables surface regeneration using an established protocol.8 As a consequence, assembly of stable sensor surfaces with control over ligand orientation and density for variety of new assay formats is now possible. Using this strategy we demonstrate the immobilization of TCO/Tz-derivatized small molecules and characterize the cycloaddition rates in a variety of buffer conditions. We chose the well-known interaction between FKBP12 and a molecule AP1497 that binds FKBP1210-12 as an example to verify that the capture-cycloaddition strategy preserves the ability of the small molecule to interact with its target when either directly attached to immobilized GST antigens or to immobilized nanoparticles (NPs).
This method offers several benefits. First, the reversible immobilization of small molecules on sensor chips is now possible. Second, TCO/Tz immobilization of small molecules also enables label-free interaction studies that reverse the orientation of canonical SPR studies, and may provide a complementary view of a binding interaction. Third, this method enables the microfluidic synthesis of targeted nanoparticles, and immediate evaluation of their binding properties. This promises to improve the efficiency of evaluating or screening targeted nanoparticles, and also decrease the amounts of nanoparticles required.13-15 Fourth, this approach can measure the reaction kinetics of bioorthogonal cycloaddition reactions in real-time under continuous flow. Finally, the TCO/Tz immobilization chemistry is robust in the presence of serum. Taken together, we anticipate that this versatile approach will broadly facilitate construction of stable sensor surfaces for a wide variety of microfluidic studies with relevance to in vitro and in vivo cellular applications.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Sensor Chip CM5</td>
			<td>GE Healthcare</td>
			<td>BR-1005-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine coupling kit</td>
			<td>GE Healthcare</td>
			<td>BR-1000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>GST capture kit</td>
			<td>GE Healthcare</td>
			<td>BR-1002-23</td>
			<td></td>
		</tr>
		<tr>
			<td>NAP-10 Columns</td>
			<td>GE Healthcare</td>
			<td>17-0854-01</td>
			<td></td>
		</tr>
		<tr>
			<td>GST, lyophilized in 1X PBS</td>
			<td>Genscript</td>
			<td>Z02039</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>rhFKBP12</td>
			<td>R&#38;D Systems</td>
			<td>3777-FK</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfactant P-20</td>
			<td>GE Healthcare</td>
			<td>BR-1000-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine 2.0</td>
			<td>GE Healthcare</td>
			<td>BR-1003-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba spin desalting column</td>
			<td>Thermo</td>
			<td>89882</td>
			<td>7 K MWCO</td>
		</tr>
		<tr>
			<td>Amicon Ultra 4</td>
			<td>Fisher</td>
			<td>UFC810096</td>
			<td>100 K centrifugal filter</td>
		</tr>
		<tr>
			<td>TCO-OH</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>TCO-NHS</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house, *Commercially available from Click Chemistry Tools # 1016-25</td>
		</tr>
		<tr>
			<td>Tz-BnNH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>Tz-NHS</td>
			<td>Ref. 8</td>
			<td>764701</td>
			<td>Synthesized in-house, *Commercially available from Sigma Aldrich # 764701</td>
		</tr>
		<tr>
			<td>NP-NH2 = CLIO-NH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>AP1497, AP1497-Tz</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>SPR Biosensor</td>
			<td>GE Healthcare</td>
			<td>Biacore T100</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic on-chip capture-cycloaddition reaction to reversibly immobilize small molecules or multi-component structures for biosensor applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

Data and figures have been adapted from reference 8. 
 Efficient reversible immobilization of bioactive small molecules with control over orientation and density plays a key role in development of new biosensor applications. Using the fast bioorthogonal reaction between TCO and Tz, we describe a method for the stepwise assembly and regeneration of ligand surfaces with retention of biological activity. Figure 2 shows the real-time monitoring of Tz-BnNH2 immobil...

Watch this Scientific Journal Video about Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications at JoVE.

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

microfluidic-on-chip-capture-cycloaddition-reaction-to-reversibly

Research

JoVE Journal

Chemistry

10.7K Views.  Massachusetts General Hospital. We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Video: Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...