Name: an aptamer-based sensor for unchelated gadolinium(iii)
Uploaded: 2017-01-09T15:00:00.000+00:00
Duration: 5 min 15 s
Description: Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

We would like to gratefully acknowledge Dr. Milan N Stojanovic from Columbia University, New York, NY for valuable scientific input. This work is supported by funding from the California State University East Bay (CSUEB) and the CSUEB Faculty Support Grant-Individual Researcher. O.E., T.C., and A.L. were supported by the CSUEB Center for Student Research (CSR) Fellowship.

NOTA: água de qualidade para biologia molecular é utilizado em todas as preparações de amortecimento e de solução. Todos os tubos descartáveis ​​(microcentrífuga e PCR) e pontas de pipeta são DNase e RNase. Por favor consultar a folha de dados de segurança (FDS) para todos os produtos químicos antes da utilização. Uso de equipamento de proteção individual (EPI) é fortemente recomendado. 1. Preparação de Soluções de Stock aptâmero <ol><li> Comprar 2 fios de polidesoxinucleótido comercialmente. Ordenar ambas as cadeias com purificação através de cromatografia líquida de alto desempenho (HPLC). Vertente 1 (Gd-aptâmero): 5 &#39;- / 56-FAM / AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3&#39; Strand 2 (QS): 5&#39;-GTCCCGAGAGCCT / 3Dab / -3 &#39; </li><li> Dissolver cada vertente na água para fazer 100 mm soluções stock individuais do Gd-aptâmero e o QS. </li><li> Armazenar as soluções à temperatura de -20 ° C. As soluções são estáveis ​​até agora, durante 3 anos. </li><li> Para minimizar Freezciclos e-descongelamento, armazenar as soluções de estoque em 10 mL alíquotas. </li></ol> 2. Preparação da Solução 2x Gd-Sensor <ol><li> Prepara-se o tampão de ensaio (HEPES 20 mM, MgCl 2 2 mM, NaCl 150 mM, KCl 5 mM). Ajustar o pH para ~ 7,4 com NaOH e HCl, e filtrar através de filtros para frascos estéreis descartáveis ​​com 0,2 m de membrana PES. Armazenar em garrafas estéreis. Se filtrada e armazenada correctamente (à temperatura ambiente), o tampão é estável até agora, durante 2 anos. </li><li> Dilui-se 1 mL da solução de estoque de Gd-aptâmero (a partir do passo 1.2) e 2 uL da solução de estoque QS (a partir do passo 1.2) em 497 uL de tampão de ensaio. Misturar bem utilizando um vortex. Suas concentrações na solução de Gd-sensor de 2x são 200 nm e 400 nm, respectivamente. NOTA: O volume da solução de Gd-sensor de 2x preparado neste passo é de 500 mL, o que é suficiente para testar 6 - 7 concentrações variáveis de Gd3 + para a curva de calibração e as soluções de agente de contraste(passo 3). Cada amostra irá dar poços duplicados de uma placa de 384 poços. <ol><li> Ajustar o volume da solução de Gd-sensor de 2x de acordo com o número de soluções Gd 3+, que necessita de ser testado. </li></ol></li><li> Transferir a solução de Gd-sensor de 2x em 9 tubos de PCR, com 50 uL a cada tubo. Colocar os tubos num ciclizador térmico. </li><li> Definir o programa no termociclador para aquecer a solução nos tubos a 95 ° C, mantida durante 5 min, e em seguida arrefecer lentamente a solução até 25 ° C ao longo de ~ 15 min (a uma taxa de ~ 0,05-0,1 ° C / s). O ciclo de aquecimento e arrefecimento é assegurar a hibridação óptima entre o Gd-aptâmero e QS. resultados parciais de hibridação em têmpera incompleta e um fundo mais elevado de fluorescência do sensor. Se um termociclador não está disponível, realizar este processo utilizando um banho de água quente em vez disso. </li><li> Uma vez arrefecida a 25 ° C, utilizar imediatamente a solução, ou manter-se no termociclador (até cerca de 2 h) até que esteja pronto para serusava. Quando um banho de água é utilizado para o aquecimento, deixam os tubos no banho como a água arrefece-se lentamente até à temperatura ambiente. </li></ol> 3. a obtenção da curva de calibração de fluorescência e detectar a presença de não quelatada Gd3 + em solução de Gd agente de contraste <ol><li> Dissolve-se 3 GdCl sólido no tampão de ensaio (o mesmo tampão como no passo 2.1). </li><li> Através da diluição em série, preparar 100 mL cada um de 6 soluções de Gd 3+ diferentes em tubos de microcentrífuga com o dobro das concentrações finais desejadas para a curva de calibração (2x soluções). <ol><li> Por exemplo, para construir uma calibração para 0 (apenas tampão sem GdCl 3), 50, 100, 200, 400, e 800 nM de Gd3 +, preparar soluções contendo 0, 100, 200, 400, 800 e 1600 nM do ião. Certifique-se sempre incluir o &quot;vazio&quot; com 0 nM Gd 3+ como controle negativo. </li></ol></li><li> Dissolve-se o agente de contraste a ser testeed no tampão de ensaio. Preparar 2 ou 3 diferentes concentrações das soluções de agente de contraste, por diluição em série. Nota: Os testes 3 diferentes concentrações da solução de agente de contraste é recomendado. Isto é para assegurar que estas concentrações estão dentro do intervalo linear. Se as amostras testadas não exibem uma relação linear, reduzir as concentrações do agente de contraste utilizado. </li><li> Tome os tubos de PCR contendo a solução 2x Gd-Sensor do passo 2.5 do termociclador. </li><li> Adicionar 50 mL de cada solução Gd 3 + a partir do passo 3.2 em 6 dos tubos de PCR 9 contendo a solução 2x Gd-sensor. Misturar por pipetagem cima e para baixo. Cada tubo de PCR agora contém a concentração desejada de Gd3 + a ser testado, com 100 nM de Gd-aptâmero e QS 200 nm. </li><li> Para os restantes tubos de PCR contendo a solução de Gd-sensor de 2x, adicionar 50 uL das soluções de agente de contraste a partir do passo 3.3. Misture bem, pipetando para cima e para baixo algumas vezes. </li><li> Incubar as solutions nos tubos de PCR de cerca de 5 min a temperatura ambiente. Eles podem ser deixados em repouso durante até 30 min. </li><li> Transferir 45 uL de cada tubo numa placa de 384 poços. Cada tubo de PCR dará poços duplicados. </li><li> Gravar a fluorescência de cada poço num leitor de placas. O fluoróforo (FAM) usada no projeto Gd-sensor tem excitação e emissão máximos de 495 e 520 nm, respectivamente, conforme listado no site do fornecedor. Escolha comprimentos de onda ou filtros de excitação e emissão adequados, dependendo se o leitor de placas é monochromator- ou baseado em filtro. </li><li> Traçar o gráfico da fluorescência em unidades de fluorescência arbitrárias (AFU) contra concentração de Gd 3 +. </li><li> Traçar o gráfico como a mudança de fluorescência vezes contra a concentração de Gd 3 +. Calcule a variação de fluorescência vezes dividindo o AFU de cada concentração pela AFU da solução &quot;vazio&quot; (com 0 nM de D&#39;us 3+). A alteração de fluorescência de dobragem vai permitir a Normalization dos resultados, caso a exibição AFU alguns periódica (dias diferentes, etc.) variações. </li><li> Comparar a emissão de fluorescência da solução que contém o agente de contraste e o &quot;branco&quot;, que é a solução contendo 0 nM GdCl 3 (tampão apenas). NOTA: A maior fluorescência da solução GBCA implica a presença de não quelatada Gd3 +, o que pode necessitar de purificação adicional do agente de contraste. A quantidade de não quelatada Gd 3+ presente pode ser estimada utilizando a curva de calibração construída na etapa 3.10 ou 3.11. </li></ol>

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The authors declare that they have no competing financial interests.

Usando o Gd-sensor baseado em aptâmero, um aumento na emissão de fluorescência que é proporcional à concentração de não quelatada Gd3 + é observada. Para minimizar a quantidade de amostra utilizada, o ensaio pode ser executado numa microplaca de 384 cavidades com um volume total de amostra de 45 uL por poço. Neste design, a escolha de fluoresceína (FAM) e dabcil (DAB) baseou-se principalmente sobre o custo dos reagentes; para modificar o comprimento de onda de emissão, um emparelhamento diferente ...

A importância crescente das imagens de ressonância magnética (MRI) em diagnóstico clínico, que é limitada pela sensibilidade inerente da técnica, resultou no rápido crescimento de investigação para o desenvolvimento de novos agentes de contraste à base de gadolínio (GBCAs 1). GBCAs são moléculas que são administrados para melhorar a qualidade da imagem, e eles tipicamente têm a estrutura química de um ião trivalente gadolínio (Gd 3+) coordenado a um ligando polidentado. Esta complexação é de importância crítica como D&#39;us não quelatada 3+ é tóxico; tem sido implicado no desenvolvimento de fibrose sistémica nefrogénica, em alguns pacientes com doença renal ou falha 2. Consequentemente, a detecção do íon livre aquosa é fundamental para garantir a segurança dos GBCAs. A presença de não quelatada Gd3 + em soluções GBCA é muitas vezes o resultado de uma reacção incompleta entre o ligando e o ião, a dissociação do complexo, ou displacement por outros cátions metálicos biológicos 3. Entre as várias técnicas usadas atualmente para determinar a presença de D&#39;us 3+, aqueles que dependem de cromatografia e / ou classificação espectrometria mais alto em termos de versatilidade e aplicabilidade 4. Entre as suas vantagens são de alta sensibilidade e precisão, a capacidade de analisar diferentes matrizes de amostras de soro humano (incluindo 5, urina e cabelo 6, 7 de águas residuais, e formulações de agentes de contraste 8), e a quantificação simultânea de múltiplos complexos Gd 3+ (um perfil de estudos antes de 2013 é descrito em uma revisão abrangente por Telgmann et al.) 4. O único inconveniente é que vários destes métodos requerem instrumentação (tais como espectrometria de massa de plasma indutivamente acoplado) 4 que alguns laboratórios não podem ter acesso. Dentro do contexto da nova descoberta GBCA na investigação e níveis prova-de-conceito, ARmétodo baseado em espectroscopia elatively mais conveniente, rápido e de baixo custo (por exemplo, absorção de UV-Vis ou de fluorescência) pode servir como uma alternativa valiosa. Com esses aplicativos em mente, um sensor baseado em aptâmero fluorescente para aquosa Gd3 + foi desenvolvido 9. O aptâmero (Gd-aptâmero) é uma molécula de ADN de 44 bases de comprimento de cadeia simples com uma sequência específica de bases que foi isolado por meio do processo de evolução sistemática de ligandos por enriquecimento exponencial (SELEX) 9. Para adaptar o aptâmero em um sensor fluorescente, um fluoróforo está ligado ao terminal 5 &#39;da cadeia, que é então hibridado com uma cadeia de têmpera (QS), através de 13 bases complementares (Figura 1). O QS é marcado com uma molécula quencher escuro no terminal 3 &#39;. Na ausência de Gd3 +, o sensor (Gd-sensor), composta de uma mistura 1: 2 proporção molar de Gd-aptâmero e QS respectivamente, terá de emissão de fluorescência mínima devido tO transferência de energia do fluoroforo para o inibidor. A adição de solução aquosa Gd3 + irá deslocar o QS do Gd-aptâmero, resultando num aumento da emissão de fluorescência. <img alt="figura 1" src="/files/ftp_upload/55216/55216fig1.jpg" /> Figura 1. O sensor (Gd-sensor) que consiste no longo aptâmero 44-base (Gd-aptâmero) marcados com fluoresceína (um fluoróforo) e a cadeia de 13 bases de comprimento têmpera (QS) etiquetada com DABCYL (um inibidor escuro) . Na ausência de não quelatada Gd3 +, a fluorescência do sensor é mínima. Com adição de Gd3 +, o deslocamento do QS ocorre e um aumento na emissão de fluorescência é observado. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55216/55216fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Não existe, actualmente, um método baseado em espectroscopia vulgarmente utilizado para detectaring aquosa 3+ D&#39;us. Este ensaio utiliza o alaranjado de xilenol molécula, que sofre uma mudança no comprimento de onda máximo de absorção 433-573 nm após a quelação com o ião 10. A razão entre estes dois valores máximos de absorvância pode ser usado para quantificar a quantidade de não quelatada Gd3 +. O sensor de aptâmero é uma alternativa (também pode ser complementar) para o ensaio de laranja de xilenol, como os dois métodos têm diferentes condições de reacção (tais como o pH e a composição das soluções tampão utilizadas), selectividades alvo, gamas lineares de quantificação, e as modalidades de detecção 9.

<table><tbody><tr><td>Gd-aptamer</td><td>IDTDNA</td><td>Input sequence and fluorophore modification in the order form</td><td>A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company.</td></tr><tr><td>Quenching strand</td><td>IDTDNA</td><td>Input sequence and quencher modification in the order form</td><td>A different quencher for optimal energy transfer from the fluorophore may be used. The aptamer may also be ordered from another company.</td></tr><tr><td>Molecular biology grade water</td><td></td><td></td><td>No specific manufacturer, both DEPC or non-DEPC treated work equally well</td></tr><tr><td>Gadolinium(III) chloride anhydrous</td><td>Strem</td><td>936416</td><td>Toxic</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Magnesium chloride anhydrous</td><td>MP Biomedicals</td><td>0520984480 - 100 g</td><td></td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P333-500</td><td></td></tr><tr><td>Sodium hydroxide, pellets</td><td>Fisher Scientific</td><td>BP359</td><td>Corrosive</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>SA49</td><td>Toxic and corrosive</td></tr><tr><td>384-well low flange black flat bottom polystyrene NBS plates</td><td>Corning</td><td>3575</td><td>Plates which are suitable for fluorescence reading are required.</td></tr><tr><td>Nalgene Rapid-Flow sterile disposable bottle top filter</td><td>Thermo Scientific</td><td>5680020</td><td>The bottle top is fitted with 0.2 micron PES membrane</td></tr><tr><td>Disposable sterile bottles 250 mL</td><td>Corning</td><td>430281</td><td>A larger or smaller bottle may be used</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>0.2 mL PCR tubes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>Micropipets</td><td></td><td></td><td>No specific manufacturer</td></tr><tr><td>Pipet tips (non filter) of appropriate sizes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek Synergy H1</td><td></td><td>Plate readers from other manufacturers would work equally well</td></tr></tbody></table>

an aptamer-based sensor for unchelated gadolinium(iii)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

Uma mudança de fluorescência típica da solução de Gd-sensor na presença de não quelatada Gd3 + é mostrada na Figura 2. A emissão pode ser representada graficamente como a mudança de fluorescência vezes (Figura 2A) ou a fluorescência em bruto leitura (Figura 2B) em unidades arbitrárias (AFU). Ambos os lotes se obter curvas de calibração muito semelhantes, com um intervalo linear de concentrações de Gd 3+</...

Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

Um sensor baseado em Aptamer para não quelatada gadolínio (III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

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Chemistry

An Aptamer-based Sensor for Unchelated Gadolinium(III)

7.2K visualizações.  California State University, East Bay. The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor. 

Vídeo: An Aptamer-based Sensor for Unchelated GadoliniumIII

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

As investigações sobre o Complexo Ga (III) de EOB-DTPA e sua<sup&gt; 68</sup&gt; Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Química

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

preparação e<em&gt; In Vitro</em&gt; Caracterização de agentes de contraste à base de dendrímeros para Ressonância Magnética

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR Imagem Molecular do cancro da próstata com um pequeno Molecular CLT1 Peptide alvejado agente de contraste

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicina

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Bioativa de Prussian Blue Nanopartículas para Multimodal Imagem Molecular Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Bioengenharia

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Customizado aparelho de aquecimento à base de laser para lançamento acionado de cisplatina de termossensível lipossomas com Ressonância Magnética Orientação Imagem

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Levitação magnética Juntamente com imagem Portátil e Análise para a doença Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

<meta charset="utf8"></head><body>Ensaio fluorescente para detecção de novos quelantes de ferro

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

<meta charset="utf8"></head><body>Autor em destaque: avaliando o impacto de novos quelantes de ferro no metabolismo das células cancerígenas

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Método fluorescente sem células quantificável e barato para confirmar a capacidade de novos compostos de quelar ferro 

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Pesquisa do câncer

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Um sensor baseado em Aptamer para não quelatada gadolínio (III)

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Bioativa de Prussian Blue Nanopartículas para Multimodal Imagem Molecular Applications

Customizado aparelho de aquecimento à base de laser para lançamento acionado de cisplatina de termossensível lipossomas com Ressonância Magnética Orientação Imagem

Levitação magnética Juntamente com imagem Portátil e Análise para a doença Diagnostics

Método fluorescente sem células quantificável e barato para confirmar a capacidade de novos compostos de quelar ferro

Método fluorescente sem células quantificável e barato para confirmar a capacidade de novos compostos de quelar ferro

Método fluorescente sem células quantificável e barato para confirmar a capacidade de novos compostos de quelar ferro

Automated Preparation of [⁶⁸Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment