This work was supported by the National Institutes of Health (grant 08UDE017788-05). E.B.P. also acknowledges support from the Spanish Foundation for Science and Technology (FECYT). The authors thank Shonda T. Gaylord and Pratyusha Mogalisetti for critical reading of the manuscript.

<img alt="Figure 1" fo:content-width="5in" width="500" src="/files/ftp_upload/50726/50726fig1highres.jpg" /> 
Figure 1. Workflow for applying fiber-optic microsphere antibody array to saliva profiling.
 (1) Microspheres are internally encoded with two fluorescent dyes; (2) the encoded microspheres are externally modified with protein-specific monoclonal antibodies; (3) the multiplexed microspheres are mixed, and (4) randomly deposited in microwells etched at the proximal end of a fiber-optic bundle; (5) salivary proteins are captured by microspheres through sandwich immunoassay, and (6) quantified using fluorescence microscopy.
1. Microspheres Encoding
<ol>
	<li>Weigh sodium europium (III) thenoyltrifluoroacetonate trihydrate (Eu-TTA, MW = 869.54 g/mol) in an amber glass vial and prepare a 200 mM stock solution in tetrahydrofuran (THF). Mix gently by pipetting; visually verify the dye is completely dissolved.</li>
	<li>Weigh coumarin 30 (C30, MW = 347.41 g/mol) in an amber glass vial and prepare a 12 mM stock solution in THF. Mix gently by pipetting; visually verify the dye is completely dissolved.</li>
	<li>Prepare 700 &#956;l of working solution for each microsphere type using the stock solutions and THF to reach final concentration listed in Table 1.</li>
	<li>Ensure microspheres (10% w/v) are well suspended by vortexing, and then transfer 60 &#956;l suspension to a 1.5 ml microcentrifuge tube.</li>
	<li>Add 600 &#956;l PBS to microsphere suspension and mix by pipetting 20x. Centrifuge the tube at 10,000 rpm for 3 min and carefully remove the supernatant. Repeat this process another 2x to wash the microspheres.</li>
	<li>Wash the microsphere pellet 3x with THF using a similar procedure as step 1.5.</li>
	<li>Centrifuge the microsphere/THF suspension at 10,000 rpm for 3 min, remove the supernatant, and re-suspend the pellet in 600 &#956;l working solution listed in Table 1. Transfer the mixture to a new 1.5 ml microcentrifuge tube.</li>
	<li>Seal the microcentrifuge tube with Parafilm and place it on a shaker. Shake at 3,000 rpm and incubate for 24 hr, protect from light by covering the setup using a box covered with aluminum foil.</li>
	<li>Centrifuge the microsphere suspension at 10,000 rpm for 3 min to form a pellet.</li>
	<li>Remove the supernatant dye solution, wash the microspheres 6x with 600 &#956;l methanol and then with 600 &#956;l PBS containing 0.01% Tween-20 another 6x, as described in step 1.5.</li>
	<li>Store the encoded microspheres in 600 &#956;l PBS containing 0.01% Tween-20 at 4 &#176;C until use, protect from light.</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Microsphere type name:</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
		</tr>
		<tr>
			<td>Eu-TTA (mM)</td>
			<td>100</td>
			<td>100</td>
			<td>10</td>
			<td>100</td>
			<td>10</td>
			<td> </td>
			<td> </td>
		</tr>
		<tr>
			<td>C30 (mM)</td>
			<td> </td>
			<td>1</td>
			<td>1</td>
			<td>6</td>
			<td>6</td>
			<td>1</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Table 1. Concentrations of Eu-TTA and C30 working solutions.
2. Preparation of Protein-capture Microspheres
<ol>
	<li>Prepare MES buffer [2-(N-morpholino)ethanesulfonic acid (MES) 0.1 M, NaCl 0.9%, SDS 0.01%, pH 5.7] and PBS/SDS buffer (Sodium Phosphate 0.01 M, NaCl 0.154 M, SDS 0.01%, pH 7.4) in advance at room temperature.</li>
	<li>Add 200 &#956;l encoded microsphere suspension (containing ~2 mg microspheres) into a Safe-Lock 1.5 ml microcentrifuge tube. It is important to use this type of microcentrifuge tube to avoid spillage during the incubation in step 2.6.</li>
	<li>Wash the microspheres with 600 &#956;l MES buffer 3x as described in step 1.5. Add 700 &#956;l MES to the microsphere pellet and mix well by pipetting.</li>
	<li>Prepare 0.105 g/ml 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, MW = 191.7 g/mol) and 0.163 g/ml sulfo-N-hydroxysulfosuccinimide (sulfo-NHS, MW = 217.13 g/mol) solutions in MES buffer in separate tubes.</li>
	<li>Add 150 &#956;l freshly prepared EDC solution drop by drop into the microsphere suspension. The EDC hydrolyzes very quickly, thus it is essential to use freshly made solution to ensure the immobilization efficiency. Immediately after the addition of the EDC, add 150 &#956;l sulfo-NHS solution to the microspheres, mix well by pipetting and verify the suspension is homogenous.</li>
	<li>Cap the tube, seal it with Parafilm, and place it on a shaker. Shake at 1,500 rpm for 4 hr, protect from light using a box covered with aluminum foil.</li>
	<li>Centrifuge the microsphere suspension at 10,000 rpm for 3 min to form a pellet and remove the supernatant. Wash the microspheres with 500 &#956;l PBS/SDS buffer 3x as described in the step 1.5.</li>
	<li>Add 200 &#956;l PBS/SDS buffer to the pellet, mix the microspheres well and transfer the solution to 300 &#956;l PBS/SDS buffer containing 60 &#956;g capture antibodies. It is important to suspend the microsphere pellet first and then add the microsphere suspension into the antibody solution.</li>
	<li>Incubate the antibody-microspheres mixture by shaking at 1,500 rpm for 4 hr on a shaker, protect from light using a box covered with aluminum foil.</li>
	<li>Centrifuge the microsphere suspension at 10,000 rpm for 3 min to form a pellet and remove the supernatant. Wash the microspheres with 500 &#956;l StartingBlock (TBS) blocking buffer 3x, as described in step 1.5.</li>
	<li>Mix microspheres with 1 ml of TBS blocking buffer and incubate microspheres at 1,500 rpm for 1 hr on a shaker, protect from light using a box covered with aluminum foil.</li>
	<li>Centrifuge the microsphere suspension at 7,000 rpm for 3 min to form a pellet and remove the supernatant. Wash the pellet 3x with 500 &#956;l TBS blocking buffer, as described in step 1.5.</li>
	<li>Centrifuge the microsphere solution at 7,000 rpm for 3 min to form a pellet and remove the supernatant. Add 500 &#956;l TBS blocking buffer and mix by pipetting.</li>
	<li>Store the modified microsphere suspension at 4 &#176;C until use, protect from light.</li>
	<li>Repeat this procedure to make other types of microspheres using corresponding antibodies.</li>
</ol>
3. Fiber-optic Microarray Assembly
<ol>
	<li>Mix 50 &#956;l of each type of protein-capture microspheres into a new autoclaved microcentrifuge tube. Centrifuge the stock microspheres pool at 7,000 rpm for 3 min, and remove the supernatant so that the remaining volume is approximately 100 &#956;l.</li>
	<li>Hand-cut fiber-optic bundles to approximately 5 cm in length and sequentially polish both ends on a polishing machine using 30, 15, 9, 6, 3, 1, 0.5, and 0.05 &#956;m-sized diamond lapping films. Sonicate the polished bundle in deionized water for 2 min to remove any particles.</li>
	<li>Put a small magnetic stirring bar into a 0.5 ml microcentrifuge tube, add 400 &#956;l of protein-free PBS buffer and stir on a magnetic stirring plate for 30 min to remove air bubbles.</li>
	<li>Etch one end of the polished fiber bundle in a 0.025 N HCl solution for 150 sec10,16. Immediately submerge and sonicate the etched end in deionized water for 1 min. Dry the fiber bundles with compressed air.</li>
	<li>Mount the fiber bundle on a fiber holder and block the etched end in the bubble-free, Protein-free PBS buffer for 1 hr.</li>
	<li>Deposit 1 &#956;l aliquot of the stored microsphere suspension on the etched end of the fiber-optic bundle. Protect the setup from light with a box with aluminum foil, and wait for 15 min. The volume of the suspension will decrease by evaporation and force the microspheres into the etched microwells. Repeat this loading step one more time.</li>
	<li>Use a swab saturated with sample solution to remove microspheres that are not trapped in the microwells. The protein microarray is now ready to use. Go immediately to step 4.1 for saliva analysis.</li>
</ol>
4. Saliva Sample Analysis Using Microspheres Microarray
<ol>
	<li>Add 200 &#956;l sample solution (100 &#956;l saliva supernatant diluted with equal volume of StartingBlock T20 (PBS) blocking buffer. The collection protocol for saliva supernatant has been published previously10). into a 0.5 ml autoclaved microcentrifuge tube. Dip the protein microarray loaded on the fiber into the solution and incubate on the shaker at 600 rpm for 2 hr, protect from light using a box covered with aluminum foil.</li>
	<li>Prepare 20 ml wash buffer by adding 200 &#956;l 10 % BSA solution and 200 &#956;l 10% Tween-20 into 19.6 ml PBS, mix well by gently shaking.</li>
	<li>Dip the protein microarray into a new autoclaved microcentrifuge tube containing 200 &#956;l wash buffer and shake at 600 rpm for 2 min. Repeat this wash 3x.</li>
	<li>Incubate the microarray in 100 &#956;l solution of a detection antibody cocktail containing 5 &#956;g/ml of anti-VEGF, IP-10, IL-8, EGF, MMP-9, IL-1&#946; biotinylated detection antibodies in StartingBlock T20 (PBS) blocking buffer for 30 min at room temperature on the shaker at 600 rpm, protect from light using a box covered with aluminum foil.</li>
	<li>Wash the microarray 3x with wash buffer as described in step 4.3.</li>
	<li>Incubate the microarray in 200 &#956;l of 20 &#956;g/ml streptavidin R-phycoerythrin conjugate (SAPE) solution in PBS at 600 rpm on the shaker for 10 min, protect from light using a box covered with aluminum foil.</li>
	<li>Wash the microarray 5x with wash buffer as described in step 4.3.</li>
	<li>Clean the distal end of the fiber (i.e., the end away from the microspheres) bundle with a swab saturated with absolute ethanol.</li>
	<li>Dry both ends of the fiber with compressed air. Mount the fiber on an epifluorescent microscope and image through the distal end of the fiber.</li>
	<li>Acquire microsphere images corresponding to Eu-TTA, C30, and SAPE fluorescence emission intensities. Optical filters setup and exposure times for fluorescence imaging of Eu-TTA, C30, and SAPE are depicted in Table 2.</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Channel</td>
			<td>Eu-TTA</td>
			<td>C30</td>
			<td>SAPE</td>
		</tr>
		<tr>
			<td>Exciter</td>
			<td>365/10x</td>
			<td>350/50x</td>
			<td>546/10x</td>
		</tr>
		<tr>
			<td>Beamsplitter</td>
			<td>525DCLP</td>
			<td>400DCLP</td>
			<td>560LP</td>
		</tr>
		<tr>
			<td>Emitter</td>
			<td>620/60m</td>
			<td>460/50m</td>
			<td>580/30m</td>
		</tr>
		<tr>
			<td>Exposure time</td>
			<td>1 sec</td>
			<td>0.3 sec</td>
			<td>1 sec</td>
		</tr>
	</tbody>
</table>
Table 2. Parameters for the three fluorescent images of the microarray.

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The authors declare that they have no competing financial interests.

Researchers should pay extra attention to the following steps: for better decoding accuracy, it is necessary to verify the microspheres were homogeneously suspended in all incubation and wash steps during the microspheres encoding procedure. In addition, the encoded microspheres need to be protected from light throughout the entire experiment. Following proper encoding and storage procedures, we found that overall decoding accuracy was above 99%. The encoded microspheres should be stored at 4 &#176;C. Avoid freez...

Since the first microarray reported by Mark Schena and coworkers in the mid-1990s, this powerful tool has been utilized in many fields of biological research1. Antibody microarrays capable of simultaneously detecting multiple proteins in diagnostic fluids, such as blood, have important applications in clinical diagnostics and biomarker screening2-10. Saliva, containing many of the same analytes as blood, has been considered as a preferable alternative to blood because saliva collection is safe, noninvasive, and can be carried out by minimally-trained medical personnel11-13. Currently, multiplexed protein analysis using saliva samples is limited by several important factors, including the low concentration of target analyte14 and the wide concentration range of different biomarkers15..
Herein, we demonstrate the analysis of six proteins: human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metallopeptidase 9 (MMP-9), and interleukin-1 beta (IL-1&#946;). The performance of the method was initially verified using standard solutions constituting recombinant analyte proteins and blocking buffer. Real saliva samples collected from patients of different chronic respiratory diseases as well as healthy controls were also tested with satisfactory performance. The protocol should be applicable to other protein analytes and other microsphere-based assays. This platform offers considerable advantages to the Analytical Chemistry field as it enables fast, accurate, and reproducible simultaneous analysis of low concentrations of several proteins with a broad dynamic range, minimal non-specific interactions, reduced sample consumption, and low cost in comparison to an analogous Enzyme-Linked Immunosorbent Assay (ELISA).

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Eu-TTA dye</td>
			<td>Fisher Scientific</td>
			<td>AC42319-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>THF</td>
			<td>Sigma-Aldrich</td>
			<td>34865-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber glass vial</td>
			<td>Fisher Scientific</td>
			<td>03-339-23B</td>
			<td></td>
		</tr>
		<tr>
			<td>Coumarin 30 dye</td>
			<td>Sigma-Aldrich</td>
			<td>546127-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspheres</td>
			<td>Bangslabs</td>
			<td>PC05N/6698</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma-Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tw-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BupH MES buffered saline</td>
			<td>Thermo Scientific</td>
			<td>28390</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>05030-500ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-lock microcentrifuge tube</td>
			<td>VWR labshop</td>
			<td>53511-997</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS</td>
			<td>Thermo Scientific</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB293</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IP-10 capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB266</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB208</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB636</td>
			<td></td>
		</tr>
		<tr>
			<td>Human MMP-9 capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB936</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-1&#946; capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 isotype control antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB002</td>
			<td></td>
		</tr>
		<tr>
			<td>StartingBlock (TBS) buffer</td>
			<td>Thermo Scientific</td>
			<td>37542</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl standard solution 1.0 N</td>
			<td>Sigma-Aldrich</td>
			<td>318949-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein-free (PBS) buffer</td>
			<td>Thermo Scientific</td>
			<td>37572</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGF 165</td>
			<td>R&#38;D Systems</td>
			<td>293-VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IP-10</td>
			<td>R&#38;D Systems</td>
			<td>266-IP</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human MMP-9</td>
			<td>R&#38;D Systems</td>
			<td>911-MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td></td>
		</tr>
		<tr>
			<td>StartingBlock T20 (PBS) buffer</td>
			<td>Thermo Scientific</td>
			<td>37539</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocker BSA in PBS</td>
			<td>Thermo Scientific</td>
			<td>37525</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated VEGF detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF293</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated IP-10 detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF266</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated IL-8 detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF208</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated EGF detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF236</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated MMP-9 detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF911</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated IL-1&#946; detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF201</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin, R-phycoerythrin</td>
			<td>Invitrogen</td>
			<td>S-21388</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (200 proof)</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Fluorescence images from three channels showing a small section of the fiber-optic bundle are shown in Figures 2A-C. These images were analyzed using an algorithm written in MATLAB (as described in more detail in the Discussion section). The analysis employs both information from the Eu-TTA encoding image (Figure 2A) and the C30 encoding image (Figure 2B) to decode the microspheres, and the fluorescence intensities of different microspheres in the signal image (F...

Watch this Scientific Journal Video about Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles at JoVE.com

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

multiplexed-fluorescent-microarray-for-human-salivary-protein

Research

JoVE Journal

Chemistry

11.6K Views.  Tufts University. We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 μm polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Video: Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Gyroid Nickel Nanostructures from Diblock Copolymer Supramolecules

Nanoporous metal foams possess a unique combination of properties - they are catalytically active, thermally and electrically conductive, and furthermore, have high porosity, high surface-to-volume and strength-to-weight ratio. Unfortunately, common approaches for preparation of metallic nanostructures render materials with highly disordered architecture, which might have an adverse effect on their mechanical properties. Block copolymers have the ability to self-assemble into ordered nanostructures and can be applied as templates for the preparation of well-ordered metal nanofoams. Here we describe the application of a block copolymer-based supramolecular complex - polystyrene-block-poly(4-vinylpyridine)(pentadecylphenol) PS-b-P4VP(PDP) - as a precursor for well-ordered nickel nanofoam. The supramolecular complexes exhibit a phase behavior similar to conventional block copolymers and can self-assemble into the bicontinuous gyroid morphology with two PS networks placed in a P4VP(PDP) matrix. PDP can be dissolved in ethanol leading to the formation of a porous structure that can be backfilled with metal. Using electroless plating technique, nickel can be inserted into the template&#39;s channels. Finally, the remaining polymer can be removed via pyrolysis from the polymer/inorganic nanohybrid resulting in nanoporous nickel foam with inverse gyroid morphology.

This article describes the preparation of well-ordered nickel nanofoams via electroless metal deposition onto nanoporous templates obtained from self-assembled diblock copolymer based supramolecules.

Nanoporous metal foams possess a unique combination of properties - they are catalytically active, thermally and electrically conductive, and furthermore, ...

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 ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

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 ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of the data and determines which properties of the film can be quantified. The quartz crystal microbalance offers a uniquely sensitive platform for measuring fine changes in mass and/or mechanical properties of an applied film by observing the changes in mechanical resonance of a quartz crystal oscillating at high frequency. The advantages of this approach include its experimental versatility, ability to study changes in properties over a wide range of experimental time lengths, and the use of small sample sizes. We demonstrate that, based on the thickness and shear modulus of the layer deposited on the sensor, we can acquire different information from the material. Here, this concept is specifically exploited to display experimental parameters resulting in mass and viscoelastic calculations of adsorbed collagen on gold and polyelectrolyte complexes during swelling as a function of salt concentration.

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of ...