This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP (Nos. 2014R1A2A1A01006527 and 2011-0030075).

1. Fabricação de um molde de mestre para a focagem hidrodinâmica dispositivo micro (DMPB) através de fotolitografia <ol><li> Projetar uma fotomáscara para o DMPB (Figura 1a), utilizando software assistida por computador (CAD) de acordo com o protocolo do fabricante. </li><li> Lavar uma bolacha 4 &#39;silício com acetona, álcool isopropílico (IPA), e água desionizada (DI) para remover o pó orgânico e inorgânico a partir da bolacha. </li><li> Limpar a bolacha de silício com O2 no plasma a 100 W de potência durante 5 min para aumentar a resistência de ligação entre a bolacha e SU-8. </li><li> Spin-revestimento de 4 ml do fotorresistente negativo, SU-8 2150, sobre a bolacha a 3.000 rpm durante 30 segundos para se conseguir uma espessura de 150 um (B1 na Figura 1b). </li><li> Coloque a pastilha revestida SU-8 numa placa de aquecimento durante 5 min a 65 ° C, ajustar a temperatura a 95 ° C, e, em seguida, deixar a bolacha sobre a placa de aquecimento durante 30 min a cozer macio. </li><li> Coloque oconcebido fotomáscara sobre a bolacha e expor à luz UV (260 mJ cm -2, 26 seg para 10 mW cm -2) em um alinhador de máscara (B2 na Figura 1b). </li><li> Realizar pós-exposição cozer numa placa de aquecimento (65 ° C durante 5 minutos e depois 95 ° C durante 12 min). </li><li> Desenvolver o wafer por imersão num banho de revelador SU-8 durante 10 min, e, em seguida, transferi-la para revelador novo durante 5 segundos para se obter uma superfície limpa. </li><li> Lavar a bolacha durante 20 segundos com água desionizada e secá-lo por 10 s com gás N2 (B3 na Figura 1b). Use o wafer fabricado como um molde mestre para polidimetilsiloxano (PDMS) fundição na Seção 2. </li></ol> 2. Fabricação do DMPB através Fundição PDMS <ol><li> Use o wafer padronizada obtida na Seção 1 como o molde mestre para fundição PDMS. </li><li> Misturar o PDMS pré-polímero e um agente de cura de forma homogénea a uma proporção em peso de 10: 1; por exemplo, utilizar um g de agente de cura para 10 g de PDMS pré-polímerer. </li><li> Verter o PDMS pré-polímero no interior do molde mestre e desgaseificar-lo durante 1 hora numa câmara de vácuo (B4 na Figura 1b). </li><li> Coloque o molde mestre com o pré-polímero de PDMS em uma estufa a 65 ° C durante 3 h. </li><li> Cortar o PDMS curados para o tamanho de um único chip, utilizando um bisturi afiado. Retire cuidadosamente a réplica PDMS curado do molde mestre com a mão. </li><li> Repita os passos 2.2 a 2.5 para obter uma réplica idêntica PDMS. </li><li> Perfurar orifícios de entrada e saída em uma das réplicas que utilizam um furo-perfurador com um diâmetro ligeiramente menor que o diâmetro exterior da tubagem de ligação. </li><li> Aplicar tratamento com plasma de ar para a área de ligação de cada réplica usando um tratador de corona. 34 Cuidado: Use o tratador corona em uma área com boa ventilação para evitar a acumulação de ozono. </li><li> Queda de 5 mL de metanol para as áreas tratadas com plasma de ar. Finamente alinhar dois PDMS réplicas idênticas para fabricar o DMPB pela manipu lado<html> mento, e verificar o alinhamento através de um microscópio (b5 na Figura 1b). Nota: As réplicas tratados com plasma PDMS aéreos são bastante pegajoso e difícil de manipular. Assim, 5 mL de metanol é adicionado à superfície tratada por plasma a ar para funcionar como um lubrificante. </li><li> Coloque a DMPB num forno regulado para 65 ° C durante a noite para reforçar a ligação entre duas réplicas de PDMS (B6 na Figura 1b). Bond dois PDMS réplicas idênticas para aumentar a altura do microcanal do DMPB e evitar o entupimento das microgotículas no canal microfluídico durante a operação. </li></ol><img alt="figura 1" src="/files/ftp_upload/52813/52813fig1.jpg" /> Figura 1: Visão Geral do Procedimento DMPB Fabrication (a) Projeto parâmetros da fotomáscara para o DMPB.. (B) Ilustração do processo de fabricação para o DMPB.ftp_upload / 52813 / 52813fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. 3. Preparação de NIPAAm-ricos (N-rico) e NIPAAm-pobres (N-pobres) Fases por separação de fases de Supersaturated NIPAAm <ol><li> Dissolver monómero NIPAAm em água desionizada a razão de W / W de 1: 1 utilizando um misturador de vórtice; por exemplo, dissolver-se 10 g de NIPAAm em 10 ml de água Dl (primeira imagem da Figura 2a). Observação: Uma vez que o monómero NIPAAm está totalmente dissolvidos à temperatura ambiente, a solução é mais turvo (segunda imagem da Figura 2a). Este fenómeno é o primeiro sinal de que a separação de fases induzida pela solubilidade do monómero NIPAAm supersaturada ocorreu com sucesso. </li><li> Permitir que a solução de monómero a descansar numa posição vertical à temperatura ambiente durante pelo menos 15 min. A fase superior é a fase de N-rico, e a fase mais densa de fundo é a fase de N-pobres (terceira imagem da Figura 2a). As densidades de thfases e N-rico e N-pobres são 0,93 ± 0,01 e 0,99 ± 0,01 g cm -3, respectivamente 15. </li><li> Quando a interface de separação das duas fases se torna claro, extrair cuidadosamente 2 mL de solução de monómero a partir das fases de n-ricas e N-pobres sem perturbar esta interface usando uma pipeta. </li><li> Adicionar 4 mg de N, N &#39;-methylenebisacrylamide (MBAAm) como um agente de reticulação e 4 mg de 4- (2-hidroxietoxi) fenil- (2-hidroxi-2-propil) cetona como um fotoiniciador para a extraída N-rico e N -pobres soluções de monômero para preparar fluidos do núcleo 1 e 2 para a baixa concentração de reticulação (2 mg ml -1) de amostra (B1 e B2 na Figura 2b). </li><li> Repita a etapa anterior de 3,3 e adicionar 80 mg de MBAAm e 4 mg de 4- (2-hidroxietoxi) fenil- (2-hidroxi-2-propil) cetona, em cada uma solução de monómero de N-rico e pobre N-extraídos para preparar fluidos do núcleo 1 e 2 para a alta concentração de agente de reticulação (40 mg ml -1) amostra (B3 e B4 na Figure 2b). </li><li> Dissolve-se 10% em peso de agente tensioactivo de óleo em óleo mineral para preparar o fluido de revestimento (b5 na Figura 2b). </li></ol><img alt="Figura 2" src="/files/ftp_upload/52813/52813fig2.jpg" /> Figura 2:. Preparação de Material para Janus Microhydrogel Síntese (a) Preparação de soluções de N-N-ricas e pobres de monómero por meio de separação de fases de NIPAAm supersaturada. (B) Os detalhes dos materiais e montagem experimental utilizada no protocolo. <a href="https://www.jove.com/files/ftp_upload/52813/52813fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 4. Síntese de Janus Microhydrogels Usando o DMPB <ol><li> Carga 2 ml de fluidos do núcleo 1 e 2 (B1, B2 B3 ou B4 na figura 2b) e o fluido de revestimento (b5 na Figura 2b) separados em três seringas de 3 ml. </li&gt; <li> Montar as seringas para as bombas de seringa e ligar cada seringa para a entrada de fluido apropriado do DMPB utilizando tubagem (figura (b). Use tubagem para ligar a saída de fluido do DMPB para um reservatório de recolha. </li><li> Definir as bombas de seringa e infundir fluidos do núcleo 1 e 2 e o fluido de revestimento a velocidades de fluxo de 2, 2, e 10 ul min -1, respectivamente. </li><li> (Opcional) Ajustar a velocidade de fluxo de fluidos do núcleo 1 e 2 para ajustar os rácios de volume relativo de cada lado da microgotícula Janus. </li><li> Posicionar a fonte de luz UV na perpendicular de cerca de 1 cm de distância do reservatório de recolha. Ligar a fonte de luz UV e monitorizar visualmente a produção contínua de microhydrogels Janus. Cuidado: Use proteção UV-óculos de proteção quando o monitoramento da produção microhydrogel. </li><li> Recolher os microhydrogels Janus fabricadas em um tubo cônico e lavá-los usando IPA. Em seguida, centrifugar o tubo cônico (780 g por 5 min) para liquidar amicrohydrogels. </li><li> Repita Passo 4.6 várias vezes para remover o óleo mineral em torno das microhydrogels Janus completamente. </li><li> Repita Passo 4.6, mas o uso de água DI, com um agente tensioactivo água de 0,005% (v / v) em vez de IPA para remover o restante IPA em torno dos microhydrogels Janus. </li><li> Armazenar microhydrogels Janus completamente lavada num frasco de 10 ml contendo água Dl. </li></ol> 5. Análise da anisotrópica Thermo-responsividade de Janus Microhydrogels <ol><li> Usar uma pipeta para colocar microhydrogels Janus sintetizados a partir da Secção 4 numa placa de 24 poços. Permitir que as microhydrogels em repouso durante 15 segundos até uma monocamada é formada na superfície do fundo do poço. </li><li> Obter uma imagem do microhydrogel Janus a 24 ° C usando um microscópio óptico na posição vertical com uma lente objectiva 5X. </li><li> Definir um módulo termoeléctrico, sob a placa e assim controlar a tensão de este módulo para aumentar a temperatura da solução contendo Janus microhidrogéis a 32 ° C. </li><li> Obter uma imagem do microhydrogel Janus a 32 ° C, mais uma vez usando um microscópio óptico vertical com uma lente objetiva de 5X. </li><li> Repita os passos 5,2-5,4 24 vezes, tendo o cuidado de escolher um microhydrogel Janus diferente para análise estatística. </li><li> A partir das 25 imagens de diferentes microhydrogels Janus, de 24 e 32 ° C, medir o raio das partes PN-ricas e pobres-PN dos microhydrogels Janus usando software de análise de imagem de acordo com as instruções do fabricante. </li></ol>

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Commun. 47 (9), 2634-2636 (2011).</li><li>Hauber, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab chip. 6 (12), 1548-1549 (2006).</li><li>Nisisako, T., Torii, T., Takahashi, T., Takizawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+monodisperse+bicolored+Janus+particles+with+electrical+anisotropy+using+a+microfluidic+co-flow+system.">Synthesis of monodisperse bicolored Janus particles with electrical anisotropy using a microfluidic co-flow system.</a> Adv. Mater. 18 (9), 1152-1156 (2006).</li><li>Seiffert, S., Romanowsky, M. B., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus+microgels+produced+from+functional+precursor+polymers.">Janus microgels produced from functional precursor polymers.</a> Langmuir. 26 (18), 14842-14847 (2010).</li><li>Peppas, N. A., Hilt, J. Z., Khademhosseini, A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+in+biology+and+medicine:+from+molecular+principles+to+bionanotechnology.">Hydrogels in biology and medicine: from molecular principles to bionanotechnology.</a> Adv. Mater. 18 (11), 1345-1360 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Dois materiais de base imiscíveis são geralmente utilizados para sintetizar os microhydrogels Janus. Até recentemente, microhydrogels Janus constituídos do mesmo material de base foram raramente reportados e os microhydrogels Janus relatados não tinha uma morfologia interna clara devido à perturbação causada pela miscibilidade dos materiais componentes. 35, 36 Neste protocolo, demonstramos um método para sintetizar microhydrogels Janus compostos inteiramente de material de base única, PNIPAAm, com u...

Hydrogels are a network of hydrophilic polymer chains.1 An increasing amount of research in the field of hydrogels has promoted significant advances and revealed their similarity to biological tissues; the properties of hydrogels allow the uptake of large amounts of water while maintaining their structure. Environmentally responsive hydrogels have also been studied extensively because of their ability to swell or shrink reversibly in response to external stimuli.2 Several triggers, including temperature,3-5 pH,6,7 light,8,9 electric fields,10,11 and specific molecules, such as glucose,12,13 have been suggested to control the geometric shape of hydrogels. Among the many environmentally responsive hydrogels currently available, poly(N-isopropylacrylamide) (PNIPAAm), a well-known thermo-responsive hydrogel, exhibits volume shrinkage above a low critical solution temperature (LCST) of 32 &#176;C.14 A recent study by Sasaki et al.15 reported the intriguing liquid-liquid phase separation of supersaturated NIPAAm, which is the monomer of PNIPAAm. According to this report, supersaturated NIPAAm was dissolved with a 10-fold molar excess of H2O, and soon after, the solution separated into two liquid phases when allows to stand at a temperature above 25 &#176;C; by contrast, dilute NIPAAm was dissolved homogeneously under the same conditions.
Microparticles made of environmentally responsive hydrogels are fascinating candidates for application in drug delivery,16,17 catalysis,18 sensing,19,20 and photonics.21 Traditional synthetic methods including emulsion polymerization, are used to produce hydrogel microparticles with polydispersity.22,23 However, certain applications require microparticles with a narrow size distribution, for example, to stabilize the pharmacokinetics of drug delivery.24 Irregularly shaped or polydisperse embolic microparticles aggregate proximally into clusters, leading to chronic inflammatory responses in embolic particles for cancer therapeutic treatment.25,26
The microfluidic approach is at the forefront of research as a means of fabricating micro-sized particles with narrow size distributions and complex shapes.27-31 The advantages of fabricating microparticles in the microfluidic device are predicated by the small characteristic length of the microfluidic device, which results in a low Reynolds number. In contrast to traditional bulk emulsification where drops are formed in parallel, microdroplets produced in microfluidic devices are generated in series and subsequently polymerized into microparticles upon exposure to UV irradiation. The fundamental principle of droplet formation using a microfluidic device is balance between the interfacial tension and the shear force of the sheath fluid acting on the core fluid. 
Despite the obvious advantages of microfluidic fabrication of droplets/particles, Janus droplets/particles consisting of the same base material are rarely reported because the internal morphology of these droplets/particles is generally disturbed by the diffusion and perturbation of the core fluids. To circumvent this intrinsic limitation, two groups recently reported the preparation of the Janus microparticles by employing heat-induced phase separation of colloidal nanoparticles and UV-directed phase separation.32,33
To this end, we report a microfluidic approach to synthesize Janus microhydrogels entirely composed of a single base material and obtain a product with clearly compartmentalized morphology. Our approach is based on the primary concept of liquid-liquid phase separation of supersaturated NIPAAm monomer. The resulting Janus microhydrogels were found to possess unique properties including anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

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			<td style="width:119px;">EMD Millipore RIOs Essential 5</td>
			<td>Prepering&#160; deionized water</td>
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			<td height="40" style="height:40px;width:216px;">SU-8 2150 negative photoresist</td>
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			<td height="40" style="height:40px;width:216px;">Hot plate</td>
			<td style="width:88px;">Misung Scientific</td>
			<td style="width:119px;">HP330D, HP150D</td>
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			<td height="40" style="height:40px;width:216px;">SU-8 developer</td>
			<td style="width:88px;">Microchem</td>
			<td style="width:119px;">Y020100 4000L1PE</td>
			<td>Developing SU-8</td>
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			<td height="40" style="height:40px;width:216px;">Mask aligner system for photolithograpy</td>
			<td style="width:88px;">Shinu Mst Co.</td>
			<td style="width:119px;">CA-6M</td>
			<td>Photolithography</td>
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			<td height="40" style="height:40px;width:216px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:88px;">Dow Corning</td>
			<td style="width:119px;">1064891</td>
			<td>PDMS casting</td>
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			<td height="40" style="height:40px;width:216px;">N-isopropylacrylamide (NIPAAm)</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:119px;">415324-50G</td>
			<td>Monomer</td>
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			<td height="60" style="height:60px;width:216px;">N,N&#39;-methylenebisacrylamide (MBAAm)</td>
			<td style="width:88px;">Sigma-Aldrich</td>
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			<td>Crosslinker of NIPAAm</td>
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			<td height="60" style="height:60px;width:216px;">4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, Irgacure 2959</td>
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			<td style="width:119px;">55047962</td>
			<td>Photoinitiator of NIPAAm</td>
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			<td height="40" style="height:40px;">ABIL EM 90</td>
			<td style="width:88px;">Evonik Industries</td>
			<td style="width:119px;">201109</td>
			<td>Sufactant for oil</td>
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			<td height="60" style="height:60px;width:216px;">Vortex mixer</td>
			<td style="width:88px;">Scientific Industries Inc.</td>
			<td style="width:119px;">Vortex-Genie 2</td>
			<td>Mixing</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Tygon tubing</td>
			<td style="width:88px;">Saint-Gobain</td>
			<td style="width:119px;">I.D. 1/32&#34;, O.D. 3/32&#34;, Wall 1/32&#34;</td>
			<td>Connecting tube between syringes and HFMD</td>
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			<td height="40" style="height:40px;width:216px;">UV light source</td>
			<td style="width:88px;">Hamamatsu</td>
			<td style="width:119px;">Spot light source LC8</td>
			<td>Polymerization from NIPAAm to PNIPAAm</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Syringes, NORM-JECT (3ml)</td>
			<td style="width:88px;">Henke-Sass Wolf GmbH</td>
			<td style="width:119px;">22767</td>
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			<td style="width:119px;">KDS model 200</td>
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			<td height="40" style="height:40px;width:216px;">Tegitol Type NP-10</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:119px;">NP10-500ML</td>
			<td>Surfactant for water</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Oil red O</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:119px;">O0625-25G</td>
			<td>Dye for N-rich phase</td>
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			<td height="40" style="height:40px;width:216px;">Oil Blue N</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:119px;">391557-5G</td>
			<td>Dye for N-rich phase</td>
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			<td height="40" style="height:40px;width:216px;">Yellow food dye</td>
			<td style="width:88px;">Edentown F&#38;B</td>
			<td style="width:119px;">NA</td>
			<td>Dye for N-poor phase</td>
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			<td height="40" style="height:40px;width:216px;">Green food dye</td>
			<td style="width:88px;">Edentown F&#38;B</td>
			<td style="width:119px;">NA</td>
			<td>Dye for N-poor phase</td>
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			<td height="21" style="height:21px;width:216px;">Centrifuge machine</td>
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			<td height="42" style="height:42px;width:216px;">Image analysis software</td>
			<td style="width:88px;">IMT i-Solution Inc.</td>
			<td style="width:119px;">iSolutions DT</td>
			<td>Measurement of radius</td>
		</tr>
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synthesis of poly(n-isopropylacrylamide) janus microhydrogels for anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

A Figura 3a apresenta um esquema da montagem experimental utilizada para sintetizar microhydrogels Janus através do DMPB. As fases de n-ricas e pobres N-precisamente foram injectados no DMPB como fluidos do núcleo 1 e 2 e, em seguida, fundiram-se e divididos em microgotículas Janus no orifício pelo fluido de revestimento de óleo mineral por causa da instabilidade Rayleigh capilar. Consequentemente, microgotículas Janus compostas por fases de N-N-ricas e po...

Watch this Scientific Journal Video about Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability at JoVE.com

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Síntese de Poli (<em&gt; N</em&gt; -isopropylacrylamide) Janus Microhydrogels para anisotrópica Thermo-resposta e Organophilic / Carregando Capability hidrofílico

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

synthesis-poly-n-isopropylacrylamide-janus-microhydrogels-for

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Chemistry

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

9.9K visualizações.  Pohang University of Science and Technology (POSTECH). We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Vídeo: Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

Síntese, Entrega Celular e<em&gt; In vivo</em&gt; Aplicação de sensores de pH à base de Dendrímero

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Química

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

Semeado Síntese de CdSe / CdS Rod e tetrápodes Nanocristais

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Protocolo para a Síntese de Derivados de Anilina Orto-trifluorometoxilados

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Síntese e Caracterização de Supramoleculares Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Síntese de Bioconjugados de proteínas<em&gt; via</em&gt; Química cisteína-maleimida

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Hidroquinona baseada Síntese de ouro Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

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.

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.

Síntese controlada e Fluorescência de rastreamento de Poly altamente uniforme (<em&gt; N</em&gt; -isopropylacrylamide) microgéis

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

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Faceta-para-faceta vinculando de forma anisotrópica cádmio coloidal calcogênio nanoestruturas

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

Enxertia de nanotubos de carbono de Multiwalled com poliestireno para habilitar auto-montagem e anisotrópica Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...