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.

Not: Moleküler biyoloji dereceli su Tüm tampon ve çözelti preparatlar kullanılır. Tüm atılabilir tüpleri (mikrosantrifüj ve PCR) ve pipet uçları DNase- ve RNaz-ücretsiz. Kullanmadan önce tüm kimyasalların malzeme güvenlik bilgi formu (MSDS) danışın. Uygun kişisel koruyucu ekipman (KKE) kullanılması önemle tavsiye edilir. Aptamer Stok çözeltiler hazırlanması 1. <ol><li> Ticari polydeoxynucleotide 2 ipliklerini satın alın. yüksek performanslı sıvı kromatografisi (HPLC) ile saflaştırma her iki hat sipariş. 1 (Gd-aptamer) Strand: 5 &#39;- / 56-FAM / AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3&#39; Dizin 2 (QS): 5&#39;-GTCCCGAGAGCCT / 3Dab / 3 &#39; </li><li> Gd-aptamer ve QS 100 uM bireysel hisse senedi çözümleri yapmak için su her telini eritin. </li><li> -20 ° C&#39;de bu çözümleri saklayın. çözümler şimdiye kadar, 3 yıl boyunca stabildir. </li><li> Freez en aza indirmek içinE-çözülme döngüleri, 10 ul alikotları stok solüsyonları saklayın. </li></ol> 2x Gd-sensör Çözüm 2. Hazırlık <ol><li> Deney tamponu hazırlayın (20 mM HEPES, 2 mM MgCI2, 150 mM NaCI, 5 mM KCI). NaOH ve HCl ile pH değeri için ~ 7.4 ayarlayın ve 0.2 um PES membran ile steril tek kullanımlık şişe üst filtreler aracılığıyla filtre. Steril şişelerde saklayın. filtre edilmiş ve (oda sıcaklığında) saklandığı takdirde, tampon, şimdiye kadar, 2 yıl stabildir. </li><li> deney tamponu 497 uL ve (adım 1.2 dan itibaren) QS stok solüsyonu 2 uL (adım 1.2 dan itibaren) Gd-aptamer stok solüsyonu 1 mcL seyreltin. bir girdap kullanarak iyice karıştırın. 2x Gd sensör çözelti içindeki konsantrasyonu, sırasıyla, 200 nM ve 400 nM. NOT: - kalibrasyon eğrisi ve kontrast madde çözümleri için 3+ Gd 7 değişik konsantrasyonlarda bu adımda hazırlanan 2x Gd-sensör çözeltisinin hacmi 6 test etmek için yeterli 500 uL olduğunu(Aşama 3). Her bir numune, 384 oyuklu plaka çoğaltma kaynaklarıdır verecektir. <ol><li> Test edilmelidir GD 3+ çözümlerin sayısına göre 2x Gd-sensör çözümünün ses seviyesini ayarlayın. </li></ol></li><li> Her tüpe 50 uL ile 9 PCR tüpleri içine 2x Gd-sensör çözüm aktarın. bir termal döngü tüpler yerleştirin. </li><li> 95 ° C&#39;ye kadar tüplere çözelti ısı termal döngü programı alan 5 dakika tutun ve daha sonra yavaş yavaş oranında (15 dakika ~ boyunca 25 ° C&#39;ye kadar soğumaya çözüm ~ 0,05-0,1 ° C / s). Isıtma ve soğutma çevrimi Gd-aptamer ve QS arasında optimal hibridizasyon sağlamaktır. eksik söndürme ve sensör daha yüksek bir eşiğe Kısmi melezleme sonuçları. Bir termal döngü cihazı kullanılabilir durumda değilse, bunun yerine sıcak su banyosu kullanılarak bu süreci yürütmek. </li><li> Bir kez 25 ° C&#39;ye kadar soğutuldu, hemen çözüm kullanmak, veya hazır olana kadar (yaklaşık 2 saat kadar) termal döngüleyici içinde tutmakKullanılmış. Bir su banyosu ısısı için kullanıldığında, su yavaş yavaş oda sıcaklığına soğurken, banyo tüpleri bırakın. </li></ol> 3. Floresan Kalibrasyon eğrisi oluşturmadan ve Gd kontrast maddesi çözeltisi içinde 3+ Unchelated Gd varlığını tespit <ol><li> Deney tamponu içinde bir katı GdCl 3 (adım 2.1 ile aynı tamponu) içinde çözülür. </li><li> Seri seyreltme yoluyla, kalibrasyon eğrisi (2x çözümleri) için nihai istenen konsantrasyonlarda iki katı 100 uL mikrosantrifüj tüpler 6 farklı Gd 3+ çözümleri her hazırlamak. <ol><li> Örneğin, 0 bir kalibrasyon oluşturmak için, 50, 100, 200, 400 (Resim GdCl 3 sadece tampon), ve Gd 3+ 800 nM, 0, 100, 200, 400, 800, 1600 nM içeren çözeltiler hazırlanması iyonu. Emin negatif kontrol olarak 0 nM Gd 3+ ile &#39;boş&#39; her zaman dahil olun. </li></ol></li><li> Test için kontrast ajanı çözülürdeney tamponu içinde ed. Seri seyreltme ile kontrast maddesi çözeltilerinin 2 ya da 3 farklı konsantrasyondan hazırlayın. NOT: kontrast madde solüsyonu 3 farklı konsantrasyonlarda test tavsiye edilir. Bu durum, bu konsantrasyon, doğrusal aralığı içinde olduğunu sağlamaktır. doğrusal bir ilişki göstermez test örnekleri, kontrast madde konsantrasyonlarını azaltmak durumunda kullanılır. </li><li> termal döngü dışına adım 2.5 den 2x Gd-sensör çözeltisi içeren PCR tüpleri atın. </li><li> 2x Gd-sensör çözeltisi içeren 9 PCR tüpleri 6 içine adım 3.2 her Gd 3+ çözeltisi 50 uL ekleyin. Yukarı ve aşağı pipetleme karıştırın. Her bir PCR tüpü artık istenen Gd 3+ konsantrasyonunu test etmek için, 100 nM Gd-aptamer ve 200 nM QS içerir. </li><li> 2x Gd-sensör çözeltisi içeren Kalan PCR tüpleri için, adım 3,3 kontrast madde çözümleri 50 mcL ekleyin. yukarı pipetleme ve birkaç kez aşağı iyice karıştırın. </li><li> s inkübeOda sıcaklığında yaklaşık 5 dakika için PCR tüplerine Çözeltileri. En fazla 30 dakika süreyle beklemeye bırakıldı olabilir. </li><li> 384 oyuklu bir plakaya her bir tüpe 45 uL aktarın. Her bir PCR tüpü yinelenen kuyu verecektir. </li><li> Bir levha okuyucu üzerinde her bir floresan kaydedin. tedarikçinin Web sitesinde listelenen Gd-sensör tasarımında kullanılan fluorofor (FAM), sırasıyla 495 ve 520 nm eksitasyon ve emisyon maxima vardır. levha okuyucu monochromator- veya filtre bazlı olmasına bağlı olarak, uygun eksitasyon ve emisyon dalga boylarını veya filtreler seçin. </li><li> Gd 3+ konsantrasyonuna karşı keyfi floresan birimlerinin (AFU) floresan grafiği çizilir. </li><li> Gd 3+ konsantrasyonuna karşı floresan kat değişikliği olarak grafik çizilir. (Gd 0 nM 3+) ile &#39;boş&#39; çözümün AFU her konsantrasyon AFU bölerek floresan katlık değişime hesaplayın. floresan kat değişim n sağlayacaktırSonuçların ormalization, AFU ekran bazı periyodik (farklı günlerde, vb) varyasyonlar gerekir. </li><li> 0 nM GdCl 3 (sadece tampon) içeren çözüm kontrast maddeyi içeren çözeltinin ve &#39;boş&#39;, floresan emisyonunu karşılaştırın. Not: GBCA çözeltisinin bir daha yüksek floresans kontrast madde, daha fazla saflandırma gerektirebilir unchelated Gd 3+ varlığını ima eder. Unchelated Gd 3+ miktarı, adımı 3.10 veya 3.11 inşa kalibrasyon eğrisi kullanılarak tahmin edilebilir. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Aptamer göre Gd-sensörü, gözlenen unchelated Gd 3+ konsantrasyonu ile orantılıdır flüoresan emisyonunda bir artışa kullanılması. Kullanılan örnek miktarı en aza indirmek için, deney, göz başına 45 uL toplam numune hacmi olan bir 384-çukurlu bir mikrolevhadaki çalıştırılabilir. Bu tasarımda, floresein (FAM) ve DABCYL (Dab) seçimi öncelikle reaktiflerin maliyeti dayanmaktadır; emisyon dalga boyu, floroforun farklı eşleştirme değiştirme ve söndürücü 11 kullanılab...

Tekniğin doğal duyarlılığı ile sınırlıdır klinik tanı, manyetik rezonans görüntüleme (MRG) artan önemi, yeni gadolinyum bazlı kontrast maddeler (GBKM&#39;lerin) 1 geliştirilmesine yönelik araştırma hızlı büyüme sonuçlandı. GBKM&#39;lerin görüntü kalitesini artırmak için tatbik edilmektedir moleküllerdir ve bunlar genellikle çok dişli bir ligand ile koordine bir üç değerli gadolinyum iyonu (Gd 3+) kimyasal yapıya sahiptir. Bu kompleksleşme toksik 3+ unchelated Gd gibi kritik öneme sahiptir; böbrek hastalığı veya yetmezliği 2 olan bazı hastalarda nefrojenik sistemik fibrozis gelişiminde rolü olduğu bilinmektedir. Sonuç olarak, sulu serbest iyon tespit GBKM&#39;lerin güvenliğini sağlamada etkili olduğunu. GBCA Çözeltilerin unchelated Gd 3+ varlığı genellikle ligand ve iyon kompleksinin ayrışma veya displacemen arasında bir tam olmayan reaksiyon sonucudiğer biyolojik metal katyonları 3 ile t. Şu Gd 3+, çok yönlülük ve uygulanabilirliği 4 açısından kromatografi ve / veya spektrometre en yüksek değerli dayalı olanlar varlığını tespit etmek için kullanılan çeşitli teknikler arasında. Onların güçlü yanları arasında yüksek hassasiyet ve doğruluk, (bir liste ve çoklu Gd 3+ komplekslerin eşzamanlı ölçümü (insan serumu 5, idrar ve saç 6, atıksu 7 ve kontrast ajan formülasyonları 8 dahil) çeşitli numune matrisleri analiz yeteneği olan çalışmaların 2013 öncesinde Telgmann ve diğ.) 4 kapsamlı bir inceleme açıklanmaktadır. Tek dezavantajı bu yöntemlerden birkaç bazı laboratuvarlar erişimi olmayabilir (örneğin indüktif eşleşmiş plazma-kütle spektrometresi gibi) instrumentations 4 gerektirir olmasıdır. araştırma ve kanıt-of-concept seviyelerinde, ar de roman GBCA keşif kapsamındaelatively daha rahat, hızlı ve uygun maliyetli spektroskopik tabanlı bir yöntem (örneğin UV-Vis absorpsiyon veya floresan gibi) değerli bir alternatif olarak hizmet verebilir. Akılda bu uygulamalar sayesinde, sulu Gd 3+ için bir floresan aptamer tabanlı sensör 9 geliştirilmiştir. Aptamer (Gd-aptamerin) üstel zenginleştirme (SELEX) 9 tarafından ligandların sistematik evrim süreci boyunca izole edildi bazların spesifik dizisi ile bir 44 baz uzunluğunda tek iplikli bir DNA molekülüdür. Floresan sensör içine aptamer uyarlamak için, bir flüorofor, sonra 13 tamamlayıcı bazlar (Şekil 1) aracılığı ile su verme telinin (QS) ile hibridize olan telin, 5 &#39;ucuna eklenir. QS 3 &#39;ucunda bir karanlık söndürücü molekülü ile etiketlenir. 1 oluşan Gd 3+, sensör (Gd-sensör) yokluğunda, sırasıyla: Gd-aptamer ve QS 2 mol oranı, en az floresans emisyon nedeniyle t olacaktırquencher için fluorofordan o enerji transferi. Sulu Gd 3+ eklenmesi floresan emisyonunda bir artışa neden, Gd-aptamer gelen QS ettirilir. <img alt="Şekil 1" src="/files/ftp_upload/55216/55216fig1.jpg" /> Floresein (bir fluorofor) ve DABCYL ile etiketlenmiş 13 baz uzunluğunda söndürme iplikçik (QS) ile etiketlenmiş 44 baz uzunluğunda aptamer (Gd-aptamer) (koyu söndürücü) oluşur Şekil 1. sensörü (Gd-sensör) . Unchelated Gd 3+ yokluğunda, algılayıcının flüoresan az. Gd 3+ eklenmesiyle, QS yerinden oluşur ve floresan emisyonunda bir artış gözlenmektedir. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55216/55216fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Günümüzde vardır, yaygın bir şekilde kullanılan spektroskopik bazlı yöntem algılama içinSulu Gd 3+ ing. Bu tahlil iyonunun 10 şelasyon üzerine 433 nm 573 ila maksimum absorpsiyon dalga boyu bir kayma uğrar molekül ksilenol turuncu kullanır. Bu iki emilimi maksimum oranı unchelated Gd 3+ miktarını belirlemek için kullanılabilir. iki yöntem, hedef seçicilik, miktarının doğrusal aralıkları ve algılama yöntemlerinin (örneğin pH ve kullanılan tampon çözeltilerin bileşiminde gibi) farklı reaksiyon koşullarına sahip olarak aptamer sensörü, ksilenol turuncu tahlil alternatif (aynı zamanda tamamlayıcı olabilir) &#39;dir 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.

Unchelated Gd 3+ mevcudiyetinde Gd sensör çözeltisi tipik bir floresans değişimi, Şekil 2 &#39;de gösterilmiştir. Emisyon flüoresan kat değişim (Şekil 2A) ya da rasgele birimlerde okuma ham floresans (Şekil 2B) olarak grafiğe edilebilir (SKB). Her iki araziler&gt; 3 uM sinyalin 1 uM ve doygunluk altında Gd konsantrasyonları için doğrusal aralık 3+ ile çok benzer kalibrasyon eğrileri elde. Saptam...

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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.

Unchelated Gadolinium için bir Aptamer tabanlı Sensörü (III)

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

an-aptamer-based-sensor-for-unchelated-gadolinium-iii

Universidad San Sebastian

Research

JoVE Journal

Chemistry

An Aptamer-based Sensor for Unchelated Gadolinium(III)

7.2K Görüntüleme.  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. 

Video: 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.

EOB-DTPA ve Ga (III) Complex Araştırmalar Onun<sup&gt; 68</sup&gt; Ga Radyoaktif Analog

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) ...

Kimya

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.

Hazırlama ve<em&gt; İn Vitro</em&gt; Manyetik Rezonans Görüntüleme için Dendrimer tabanlı Kontrast Ajanlar Karakterizasyonu

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.

Küçük Moleküler CLT1 Peptide Hedefli Kontrast Agent ile Prostat Kanseri MR Moleküler Görüntüleme

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

Tıp

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.

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

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

Biyomühendislik

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.

Manyetik Rezonans Görüntü Rehberlik ile Termosensitif Lipozomlu gelen Sisplatinin Tetikleme Release için özel tasarlanmış Lazer-bazlı ısıtma Aparatı

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.

Hastalık Teşhis için Taşınabilir Görüntüleme ve Analiz ile birleştiğinde Manyetik Levitasyonunun

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>Yeni Demir Şelatörlerinin Tespiti için Floresan Testi

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>Yazar Spotlight: Yeni Demir Şelatörlerinin Kanser Hücresi Metabolizması Üzerindeki Etkisinin Değerlendirilmesi

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.

Yeni bileşiklerin demiri şelatlama yeteneğini doğrulamak için ölçülebilir ve ucuz hücresiz floresan yöntemi 

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

Kanser Araştırmaları

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 ...

Unchelated Gadolinium için bir Aptamer tabanlı Sensörü (III)

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

EOB-DTPA ve Ga (III) Complex Araştırmalar Onun

Hazırlama ve

Küçük Moleküler CLT1 Peptide Hedefli Kontrast Agent ile Prostat Kanseri MR Moleküler Görüntüleme

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

Manyetik Rezonans Görüntü Rehberlik ile Termosensitif Lipozomlu gelen Sisplatinin Tetikleme Release için özel tasarlanmış Lazer-bazlı ısıtma Aparatı

Hastalık Teşhis için Taşınabilir Görüntüleme ve Analiz ile birleştiğinde Manyetik Levitasyonunun

Yeni bileşiklerin demiri şelatlama yeteneğini doğrulamak için ölçülebilir ve ucuz hücresiz floresan yöntemi

Yeni bileşiklerin demiri şelatlama yeteneğini doğrulamak için ölçülebilir ve ucuz hücresiz floresan yöntemi

Yeni bileşiklerin demiri şelatlama yeteneğini doğrulamak için ölçülebilir ve ucuz hücresiz floresan yöntemi

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