The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for financial support within the Sonderforschungsbereich SFB 985 "Functional Microgels and Microgel Systems".

1. Microgel Synthesis
NOTE: N-isopropylacrylamide (NIPAM) was recrystallized from n-hexane. Other reagents were used as received.
<ol>
	<li>Conventional Batch Synthesis of Poly(NIPAM) Matrix Microgels
	<ol>
		<li>Dissolve 1.8 g NIPAM and 24 mg N,N&#39;-bisacrylamide (BIS) in 245 ml filtered (0.2 &#181;m regenerated cellulose (RC) membrane filter) double distilled water in a 500 ml three-neck round bottom flask equipped with a reflux condenser, a stirrer and a rubber septum....

<ol>
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Addition of small amounts of functional comonomer can have a significant effect on the particle size and structure of the PNIPAM derived microgels. Simultaneous small-scale test tube polymerization is a good method to account for such changes, and helps to rapidly find the right reactant compositions for target particle size for upscaling the reaction as needed. The mass of the particles is approximately exponentially dependent on the ...

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels 1,2 have attracted continuous interest over the past two decades due to their potential in various smart applications. Demonstrated use cases include switchable emulsion stabilizers 3-8, microlenses 9, cell culture substrates for easy cell harvesting 10,11, and smart carriers for low molecular weight compounds and other biomedical uses 12. From a fundamental research point of view these particles have been proven to be useful for investigating subjects such as colloidal interactions 13-15 and polymer-solvent interactions 16-18.
Successful use of PNIPAM microgels and their derivatives in any given application typically requires knowledge on the mean particle size and width of the particle size distribution. For the correct interpretation of the experimental results involving PNIPAM microgels, the particle structure, which can be affected by functional comonomers, has to be known. Dynamic and static light scattering (DLS and SLS, respectively) are uniquely suited for acquiring this information because these methods are fast and relatively easy to use; and they probe the particle properties non-invasively in their native environment (dispersion). DLS and SLS also collect data from vast number of particles avoiding the bias arising from small sample sizes, typical for microscopy methods. Therefore, the first aim of this work is to introduce good practice regarding light scattering for practitioners new to colloidal characterization.
Typically, precipitation polymerization is carried out in laboratory scale and finding the right reaction conditions for specific particle properties can be laborious and require many repetitions of the synthesis. In contrast to large batch synthesis, non-stirred precipitation polymerization 19,20 is a rapid procedure in which batches of different reactant composition can be polymerized simultaneously yielding particles of narrow size distribution. Simultaneous polymerization minimizes experimental variation and large output means that right reaction conditions can be found fast for upscaling the reaction. Hence, our second aim is to demonstrate the usefulness of non-stirred precipitation polymerization in prototyping and in applications that do not require a large amount of product.
Different aspects of synthesis and characterization come together in the example of application of fluorescent labeled PNIPAM microgels in colloidal interaction research. Here we use highly accurate single particle tracking to investigate the diffusion of labeled tracer microgels in dispersion of unlabeled matrix microgels over a wide matrix concentration range and resolve the cage effect in concentrated colloidal dispersion. Wide-field fluorescence microscopy is well suited for this purpose as it can characterize the specific behavior of a few tracer molecules among a large number of potentially different matrix species. This is in contrast to techniques such as DLS, SLS and rheology, which measure the ensemble average properties of systems and therefore cannot resolve behavior of small number of probe particles in a large system. Furthermore, in this specific example conventional light scattering methods cannot be utilized also due to high particle concentration, which leads to strong multiple scattering invalidating any standard analysis. Use of automated data processing and statistical methods enable analysis of overall system behavior also for single particle tracking, when averaged over large sample sizes.

<table border="0" cellpadding="0" cellspacing="0" width="741">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Acetone</td>
			<td style="width:199px;">VWR Chemicals</td>
			<td style="width:155px;">KRAF13455</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Bisacrylamid</td>
			<td style="width:199px;">AppliChem</td>
			<td style="width:155px;">A3636</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">n-Hexane</td>
			<td style="width:199px;">Merck</td>
			<td style="width:155px;">104374</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">N-Isopropylacrylamide</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:155px;">AC412785000</td>
			<td>recrystallized from n-hexane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Methacryloxyethyl thiocarbamoyl rhodamine B</td>
			<td style="width:199px;">Polysciences</td>
			<td style="width:155px;">23591</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Potassium peroxodisulfate</td>
			<td style="width:199px;">Merck</td>
			<td style="width:155px;">105091</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Silicone oil 47 V 350</td>
			<td style="width:199px;">VWR Chemicals</td>
			<td style="width:155px;">83851</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Toluene</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:155px;">244511</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">F12 Refrigerated/heating circulator</td>
			<td style="width:199px;">Julabo</td>
			<td>9116612</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">XY(Z) Piezo System</td>
			<td>Physik Instrumente</td>
			<td>P-545.3R7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100x Oil immersion objective</td>
			<td>Olympus</td>
			<td>UPLSAPO</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QuadLine Beamsplitter</td>
			<td>AHF Analysentechnik</td>
			<td>F68-556T</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Cobolt Jive 150 laser</td>
			<td>Cobolt</td>
			<td>0561-04-01-0150-300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multimode Fiber</td>
			<td>Thorlabs</td>
			<td>UM22-600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iXON Ultra 897 EMCCD camera</td>
			<td>Andor</td>
			<td>DU-897U-CS0-BV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser goniometer</td>
			<td>SLS Systemtechnik</td>
			<td>Mark III</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CF40 Cryo-compact circulator</td>
			<td>Julabo</td>
			<td>9400340</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser goniometer system&#160;</td>
			<td>ALV GmbH</td>
			<td>ALV / CGS-8F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multi-tau corretator</td>
			<td>ALV GmbH</td>
			<td>ALV-7004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light scattering electronics</td>
			<td>ALV GmbH</td>
			<td>ALV / LSE 5004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photon counting module</td>
			<td>PerkinElmer</td>
			<td>SPCM-CD2969</td>
			<td>2 units in pseudo cross-correlation mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">633 nm HeNe Laser</td>
			<td>JDS Uniphase</td>
			<td>1145P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">F32 Refrigerated/heating circulator</td>
			<td>Julabo</td>
			<td>9312632</td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled synthesis and fluorescence tracking of highly uniform poly(n-isopropylacrylamide) microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

The number of PNIPAM microgel particles in the batch, and thus the final particle volume, is determined early in the reaction during the nucleation phase 20 Hydrophobic co-monomer dye methacryloxyethyl thiocarbamoyl rhodamine B influences the nucleation by reducing the particle number density in the batch. The decrease in particle concentration for two different initial NIPAM concentrations can be seen as increase in...

Watch this Scientific Journal Video about Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels at JoVE.com

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

The overall goal of this work is to investigate the dynamic behavior and interaction of soft colloidal particles in dispersions of various particle densities in the vicinity of glass transition. Microgels are soft objects that can adapt their size, shape, and mobility according to their environments. In contrast to rigid colloidal particles, there are many open questions concerning the behavior of soft microgels.The main advantage of wide field fluorescence microscopy is the possibility to localize single microgels with high accuracy, directly follow their motion in C2, and to analyze the amounts of diffusion. To investigate the dynamics of single microgels in dense systems, only a small number of microgels must be fluorescently labeled. Non-stirred precipitation polymerization allows for parallel synthesis of microgel with variable reaction conditions to conveniently match the size of labeled and unlabeled microgels.Dynamic light-scattering provides the average hydrodynamic radii of the microgels within an accuracy of a few nanometers. Dissolve 1.8 grams of NIPAM and 24 milligrams of BIS in 245 milliliters of filtered double-distilled water in a 500 milliliter, three-neck round bottom flask equipped with reflux condenser, a stirrer, and a rubber septum. Insert a thermometer and a 120-millimeter needle for the nitrogen input through the septum.Heat the solution to 60 degrees Celsius while stirring. Then, deoxygenate the solution by purging with nitrogen for 40 minutes. Simultaneously prepare an initiator solution of 155 milligrams of potassium persulfate, or KPS, in five milliliters of filtered double-distilled water, and bubble the solution with nitrogen to remove oxygen.Next, wash a syringe with nitrogen, and transfer the complete five milliliter KPS solution to a nitrogen-washed syringe equipped with a 120-millimeter needle. Lift the nitrogen needle above the solution level in the three-neck flask, and add the KPS solution rapidly through the rubber septum into the reactor. Let the polymerization proceed for one hour under nitrogen flow and slow stirring at 60 degrees Celsius.Use a Buchner funnel and filter paper to filter the hot reaction solution, and discard any big aggregates. After letting the dispersion cool down, centrifuge and redisperse the dispersion three times for 40 minutes at 257, 000 times G.Finally, redisperse the sediment in a minimum viable amount of double-distilled water before lyophilizing the dispersion for storing. Weigh 257.7 milligrams of NIPAM, 3.5 milligrams of BIS, and 1.5 milligrams of methacryloxyethyl thiocarbamoyl rhodamine B dye in a glass vessel, and add 10 milliliters of filter double-distilled water.Prepare the same solution without the dye in a separate glass vessel. Ultrasonicate the dye solution for 15 minutes to dissolve the dye in water. Prepare various dilutions of the monomer solution with the dye to obtain a concentration series.Here, dye in the concentration range of 02 to 0.1 millimole per liter is used. Next, dissolve 8.4 milligrams of KPS in 10 milliliters of filtered double-distilled water to get the initiator solution. Transfer 0.5 milliliters of the concentration series and 05 milliliters of the KPS solution to test tubes with a 10 millimeter diameter to obtain the final reaction solutions.Seal the tubes with rubber septa. Preheat an oil bath in a double-walled glass vessel connected to a heating circulator to 63 degrees Celsius. Deoxygenize the reaction solutions by purging with nitrogen through 120-millimeter needles for 20 minutes.Then, insert the tubes into a floating platform, and immerse the platform in the pre-heated oil bath. Set the temperature to 60 degrees Celsius. For high-precision particle size tuning, the temperature control during the initial reaction has to be rigorous, typically plus or minus 0.1 degrees Celsius.Let the reaction proceed for an appropriate amount of time. Typically, one hour is enough. Following the reaction, transfer the reaction tubes rapidly to an oven at 60 degrees Celsius.Put one drop of the hot dispersion in 10 milliliters of filtered double-distilled water, pre-heated over the poly-NIPAM volume phase transition temperature in order to measure the hydrodynamic radii of the particles in the collapsed state. Let the rest of the dispersions cool down to room temperature, and transfer them into centrifuge tubes. After centrifuging the solution, dilute the microgels in two milliliters of filtered double-distilled water to use as tracer particles.For hydrodynamic radius determination in the collapsed state by dynamic light scattering, first wash the cuvettes and glassware with acetone vapor. Transfer approximately one milliliter of diluted particle dispersion to a measurement cuvette. Temper the dynamic light-scattering goniometer index match bath to 50 degrees Celsius, and transfer the sample to the instrument without letting it cool down.Insert the sample cuvette, and move the detector arm to the small scattering angle. After checking the beam profile and count range as described in the text protocol, move the goniometer arm to the highest scattering angle. Check that the count rate is still high enough for the measurement.If the intensity is too low, move the arm to a lower scattering angle. Then, check the beam visually through the toluene bath glass at the lowest scattering angle. If glow around the incident beam is observed, multiple scattering takes place.Acquire 20 correlograms between the minimum and maximum scattering angle with a minimum acquisition time of 60 seconds. Increase the acquisition time for weak intensity large-scattering angles if necessary. Use an appropriate objective lens of the desired magnification and aperture for excitation of the tracers and simultaneous fluorescence collection from the sample.In this work, a 100X, 1.3 numerical aperture oil-immersion objective lens is used. Place the moisture chamber onto an XYZ pi-SO table that fits into a microscope. To prevent the sample from drying, place a plasma-cleaned cover slip into the moisture chamber, and pipette 10 microliters of the desired concentration of poly-NIPAM dispersion.Depending on the excitation and emission spectra of the fluorescent dye, use a suitable laser for excitation, and adjust the laser power appropriately. A 561-nanometer diode-pump solid state laser is used here at a constant laser power of 16 milliwatts. To obtain homogeneous sample illumination, couple the laser into a multimode fiber, shake the fiber using a vortexer to temporarily average out laser speckles, and project the fiber end into the sample plane.Calibrate the Z-distance from the back reflection of the cover slip, and focus several micrometers into the sample by moving the objective slightly up, and fix the Z-position using a Z-compensator. This avoids any interface effects with the cover slip. Adjust the detector parameters, such as exposure time, to the strength of the fluorescent signal.In this case, an EMCCD camera is used with an exposure time of 0.1 seconds in electron-multiplying mode and a gain of 50. Acquire several movies with the appropriate number of frames to obtain adequate lag time to calculate the mean-squared displacement of the microgels in different regions of the sample. Eight labeled microgel batches were synthesized.For six batches, correlogram decay rates linear rise against the square of the scattering vector magnitude, which indicates narrow size distribution. A fit through the origin indicates only translational diffusion. For two batches, nonlinear behavior is observed.The deviation is caused by broad size distribution and a particle form factor minimum, which coincides with the scattering vector range. To obtain a better estimate for the mean diffusion coefficient of the particles, exclude these points from the fit. High signal to noise ratio enables tracking of fluorescent microgels.Their diffusion is restricted by the the unlabeled microgel matrix. Higher concentrations of unlabeled microgel matrix on the right leads to a pronounced cage effect, where the fluorescent microgels are trapped in an unlabeled matrix of microgels. At low concentrations of unlabeled mircogel matrix, the tracer microgels exhibit normal diffusion, which is inferred from the linear increase of mean-square displacement with lag time.However, at high concentrations close to the colloidal glass transition, they exhibit nonlinear diffusion, depicted by nonlinear temporal evolution of mean-square displacements. At higher concentration of unlabeled microgel matrix, the tracer microgels are trapped by unlabeled neighbors into transient cages, as illustrated by clustered tracks for 12 microgels. Parallel small-scale synthesis enables fast experimentation with different reaction parameters.This also minimizes any undesired experimental variation due to reaction temperature differences. In combination with the careful dynamic light-scattering characterization, the reaction conditions for the right microgel size can be found fast. In this video, we demonstrate how controlled microgel synthesis allows us to investigate the diffusion of single microgels in concentrated microgel solutions by wide field fluorescence microscopy.Following this procedure, it will be possible to understand how the structure of microgels controls their properties. This will open the door to designing new soft interactive matter.

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JoVE Journal

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10.1K visualizações.  RWTH Aachen University. The overall goal of this work is to investigate the dynamic behavior and interaction of soft colloidal particles in dispersions of various particle densities in the vicinity of glass transition. Microgels are soft objects that can adapt their size, shape, and mobility according to their environments. In contrast to rigid colloidal particles, there are many open questions concerning the behavior of soft microgels.The main advantage of wide field fluorescence microscopy is the possibilit...