Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Dikkat: Kullanmadan önce ilgili tüm kimyasalların güvenlik bilgi formları (SDS) danışın. Ilaç tarama deneyi kendi toplu meslektaşı ile karşılaştırıldığında ek tehlikeler olabilir nanomateryallerin sentezini ve taşıma kapsar. Emin olun, gerekli tüm kontrol önlemleri mühendislik kontrolleri (davlumbaz) ve kişisel koruyucu donanımlar (KKD, örneğin, emniyet uzunluğu pantolon, kapalı-toe ayakkabıları, kimyasala dayanıklı eldivenler ve emniyet gözlükleri) kullanımı da dahil olmak üzere, deney boyunca uygulanan edilecek. Uyuşturucu Tarama için Kimyasal Reaktifler 1. Hazırlık <ol><li> Au NC&#39;ler sentezi için öncüleri <ol><li> Altın (III) klorür çözeltisi 15 mM hazırlamak için ultra saf su 6,9 ml altın (III) klorür çözeltisi (% 99.99 zerre metaller temelinde, seyreltik HCI içinde 30 wt.%), 30 mg eritin. Dikkat: altın klorür solüsyonu aşındırıcı ve tahriş edicidir. Göz ve deri ile doğrudan teması engellemek için uygun KKE giyin. </li><li> Diultra saf su 1 ml HSA veya BSA&#39;nın ssolve 74 mg, 74 mg / protein stok çözeltisi ml hazırlamak. </li></ol></li><li> İlaç solüsyonları <ol><li> İstenilen ilaç miktarını, örneğin, (a) ibuprofen 1.9 mg, (b) warfarin 2,8 mg, (c) fenitoin 2.3 mg ve 450 mM hazırlamak için 20 ul DMSO (d) sülfanilamid 1.5 mg çözülür İlaç stoku çözümleri. Not: Bu ilaçlar, hidrofobik ve suda zayıf çözünürlüğe sahip olan, çünkü DMSO çözücüsü olarak seçilir. </li></ol></li><li> Diğer reaktifler <ol><li> NaOH çözeltisinin 1.5 M hazırlamak için ultra saf su 10 ml NaOH peleti 600 mg çözülür. Üre çözeltisi 20 M hazırlamak için ultra saf su, 2 ml üre 2.4 g çözündürülür. </li></ol></li></ol> Protein-Templated Au NC&#39;ler 2. sentezi <ol><li> HSA şablonu Au NC&#39;ler (HSA Au NC&#39;ler) <ol><li> İki ayrı sıcaklık kontrollü manyetik karıştırma ile iki cam şişeleri, içinde bir mikro manyetik karıştırma çubuğuna sahip, her biri koyunaradaki. </li><li> 60 ° C&#39;ye kadar bir manyetik karıştırıcı sıcaklığını ayarlamak ve &quot;60 ° C&quot; olarak bunun üstüne bir cam şişe etiket; Diğer manyetik karıştırıcı bunun üstüne cam şişe &quot;RT&quot; olarak etiketlenir, oda sıcaklığında (RT) tutulurken. </li><li> Protein şablon içinde Au iyonlarının kapsülleme izin vermek için HSA çözeltisi 200 ul ultra-saf su 200 ul ve (aksi belirtilmediği sürece 360 ​​rpm olarak ayarlanır) sabit karıştırma altında, her şişeye altın (III) klorür çözeltisi 200 ul ekle. </li><li> Au NCS oluşturulmasında HSA indirgeme yeteneği etkinleştirmek ve 0 dakika reaksiyon süresi kayıt başlamak üzere 2 dakika sonra, her şişeye NaOH çözeltisi 20 ul ekle. </li><li> Her 20 dakika, 384 oyuklu siyah plaka numunesinin her birinden çözeltilerinin 50 ul çeker ve bir mikro levha okuyucusu ile emisyon spektrumu ölçmek. Tipik tarama ayarları: - 850 nm λ ex = 370 nm, λ em 410 =. </li><li> 100 dakika sonra, manyetik karıştırıcı durdurun. </li><li> Bir lavaboya akan su altında cam şişeleri serinleyin. Dikkat: cam şişe sıcak. Yanmasını önlemek için ısıya dayanıklı eldiven giyin. </li><li> Her bir numune farklı sıcaklık koşullarında Au NC&#39;ler oluşum kinetiği elde etmek için her zaman fotoemisyon spektrumları çizilir. </li></ol></li><li> BSA-şablonu Au NC&#39;ler (BSA-Au NC&#39;ler) <ol><li> Bir manyetik karıştırıcı üzerine mikro manyetik karıştırma çubuğu içeren bir cam şişe yerleştirin. Oda sıcaklığında sıcaklığına bırakın. </li><li> BSA çözeltisi 200 ul, üre 200 ul ve sabit karıştırma altında bir cam şişe içinde, altın (III) klorür çözeltisi 200 ul karıştırın. </li><li> 2 dakika sonra, Au NCS oluşturmak için BSA azaltarak yeteneği etkinleştirmek ve 0 dk reaksiyon zamanını kaydetmek başlamak için NaOH solüsyonu 20 ul ekleyin. Saatlik reaksiyon karışımının fotoemisyon spektrumunu ölçün. Not: BSA-Au NCS fotoemisyon tayfı ölçülürdaha az BSA-Au NC&#39;ler oluşumu oranı nedeniyle ürenin daha az etkinlik, aynı sıcaklıkta, HSA Au daha yavaş olduğu için protein açığa. </li><li> 7 saat sonra, manyetik karıştırıcı durdurun. Üre denatürasyonu Au NC&#39;ler oluşum kinetiği görmek için her zaman en fotoemisyon spektrumları çizilir. </li></ol></li></ol> 3. Küçük Moleküler Uyuşturucu Madde Tarama <ol><li> HSA farklı küçük molekül ilaç ekran nispi bağlanma afinitesi <ol><li> Beş cam şişeye, bir sıcaklık kontrol çoklu manyetik karıştırıcı üzerine içinde manyetik karıştırma çubuğu ile, içeren her yerleştirin. A, b, c, bu şişeleri etiketleyin ve her bir ilaç, yani ibuprofen, warfarin, fenitoin ve sülfanilamid sırasıyla d. Kontrol olarak saf HSA ile bir etiketleyin. </li><li> Her şişeye HSA 200 ul ekle. Dört uygun şişelere ilaç çözeltisi 1 ul ekleyin. Kontrol DMSO 1 ul ekle. Karıştırıcı açın ve 360 ​​spin hızını ayarlamakrpm ve HSA bağlanma ilaç tamamlanmasına izin vermek için 1 saat süre ile inkübe edilir. </li><li> 1 saat sonra, Milli-Q su içinde 200 ul ve sabit karıştırma altında, her şişeye altın (III) klorür çözeltisi 200 ul ilave edin. 10 dakika boyunca sabit karıştırma altında 60 ° C&#39;ye kadar sıcaklık ayarlayın. Her flakon 1.5 M NaOH 20 ul ekleyin ve 0 dk reaksiyon zamanını kaydetmek başlar. </li><li> Hızlı bir şekilde emisyon spektrumunu (sonuçlar 0 dakika olarak işaretlenmiş), bir 384-kuyucuklu siyah levha, her bir şişeden çözeltisi 50 ul çeker ve ölçün. Her bir örnek, her 10 dakika emisyon spektrumunu kaydetme. Dört farklı bir zamanda en az dört spektrumları toplamak, yani 0, 10, 20, ve 30 dk. </li><li> Tutarlı bir trend ile sonuçları elde etmek için 3.1.4 birkaç kez - Tekrar 3.1.1 adımlar. Manyetik karıştırıcı durdurun. </li><li> (- D, ve kontrol: a) her bir numune için zamana bağımlı fotoemisyon spektrumları çizilir. Di Au NC&#39;ler oluşum kinetiği karşılaştırmak için zamana karşı tüm numunelerin ve arsa zirve yoğunluğunu tespitfferent ilaç yüklü protein şablonları. </li></ol></li><li> HSA belirli bir ilacın bağlanması sabit ölçün <ol><li> 3.1.4 - yineleyin 3.1.1 adımlar. (Sadece ilaç yüklemesi olmadan HSA kullanılarak bir kontrol numunesi de dahil olmak üzere), dört farklı konsantrasyonda ibuprofen çözümlerle ilaç çözeltisi değiştirin. Buna ilaç konsantrasyonu göre cam şişe etiketleyin. </li><li> 10 dakika sonra, bir lavabo akan suyun altında cam şişeleri soğumasını. Dikkat: cam şişe sıcak. Yanmasını önlemek için ısıya dayanıklı eldiven giyin. </li><li> Bir 384-kuyucuklu siyah plaka her şişeye yukarıdaki çözeltilerin 50 ul çizin ve emisyon spektrumunu ölçmek. </li><li> Farklı ilaç konsantrasyonlarında sonuçların başka iki takım edinme 3.2.3 kez daha - Tekrar 3.2.1 adımlar. </li><li> Manyetik karıştırıcı durdurun. Fotoemisyon spektrumları arsa ve sonuçlarını analiz. <ol><li> Her parti elde edilen ham veriler için her fotoemisyon spektrumları arsaYazılımın örnek gibi OriginPro yazılımı gibi ve pik yoğunluğunu bulmak. </li><li> Göreceli floresan hesaplayın ve arsa [(I 0 - I) / I 0] I ve ben sırasıyla ilaç yüklü HSA ve saf HSA oluşan Au NC&#39;ler floresan yoğunluğu başvurmak 0 ilaç konsantrasyonu, karşı. </li><li> Michaelis-Menten denklemi kullanılarak tek bir site bağlanma modeline uyan verilerin bağlama sabiti hesaplanır: R max en yüksek yanıt sinyalini gösterir C / (Kd + C) x Y = R max, Cı ligandın konsantrasyonudur ve K, D bağlama sabitidir. Böyle OriginPro olarak yazılımda bu gerçekleştirin. Doğrusal Olmayan Eğri Fit takılması menü Analizi seçin, ardından Büyüme / Sigmoidal kategoriden Tepesi fonksiyonu seçin. Ayarlar: Fonksiyon Seçimi sayfasında, gömme sonuçlarını göstermek için Fit tıklayın. </li></ol></li></ol></li></ol>

<ol>
	<li>Flarakos, J., Morand, K. L., Vouros, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Solution-Based+Medicinal+Library+Screening+against+Human+Serum+Albumin.">High-Throughput Solution-Based Medicinal Library Screening against Human Serum Albumin.</a> Anal. Chem. 77, 1345-1353 (2005).</li><li>Vuignier, K., Veuthey, J. -. L., Carrupt, P. -. A., Schappler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Analytical+Strategy+to+Measure+Drug&#8211;Plasma+Protein+Interactions:+From+High-Throughput+to+In-Depth+Analysis.">Global Analytical Strategy to Measure Drug&#8211;Plasma Protein Interactions: From High-Throughput to In-Depth Analysis.</a> Drug Discov. Toda. 18, 1030-1034 (2013).</li><li>Zsila, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdomain+Ib+Is+The+Third+Major+Drug+Binding+Region+of+Human+Serum+Albumin:+Toward+The+Three-Sites+Model.">Subdomain Ib Is The Third Major Drug Binding Region of Human Serum Albumin: Toward The Three-Sites Model.</a> Mol. Pharm. 10, 1668-1682 (2013).</li><li>Dalvit, C., Fagerness, P. E., Hadden, D. T. A., Sarver, R. W., Stockman, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine-NMR+Experiments+for+High-Throughput+Screening:+Theoretical+Aspects,+Practical+Considerations,+and+Range+of+Applicability.">Fluorine-NMR Experiments for High-Throughput Screening: Theoretical Aspects, Practical Considerations, and Range of Applicability.</a> J. Am. Chem. Soc. 125, 7696-7703 (2003).</li><li>Ghuman, J., Zunszain, P. A., Petitpas, I., Bhattacharya, A. A., Otagiri, M., Curry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structural Basis of the Drug-binding Specificity of Human Serum. 353, 38-52 (2005).</li><li>Mao, H., Hajduk, P. J., Craig, R., Bell, R., Borre, T., Fesik, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+Design+of+Diflunisal+Analogues+with+Reduced+Affinity+for+Human+Serum+Albumin.">Rational Design of Diflunisal Analogues with Reduced Affinity for Human Serum Albumin.</a> J. Am. Chem. Soc. 123, 10429-10435 (2001).</li><li>Dalvit, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+NMR-Based+Screening+with+Competition+Binding+Experiments.">High-Throughput NMR-Based Screening with Competition Binding Experiments.</a> J. Am. Chem. Soc. 124, 7702-7709 (2002).</li><li>Krenzel, E. S., Chen, Z., Hamilton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correspondence+of+Fatty+Acid+and+Drug+Binding+Sites+on+Human+Serum+Albumin:+A+Two-Dimensional+Nuclear+Magnetic+Resonance+Study.">Correspondence of Fatty Acid and Drug Binding Sites on Human Serum Albumin: A Two-Dimensional Nuclear Magnetic Resonance Study.</a> Biochemistr. 52, 1559-1567 (2013).</li><li>Lee, Y., Zeng, H., Ruedisser, S., Gossert, A. D., Hilty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Magnetic+Resonance+of+Hyperpolarized+Fluorine+for+Characterization+of+Protein&#8211;Ligand+Interactions.">Nuclear Magnetic Resonance of Hyperpolarized Fluorine for Characterization of Protein&#8211;Ligand Interactions.</a> J. Am. Chem. Soc. 134, 17448-17451 (2012).</li><li>Salvi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boosting+the+Sensitivity+of+Ligand&#8211;Protein+Screening+by+NMR+of+Long-Lived+States.">Boosting the Sensitivity of Ligand&#8211;Protein Screening by NMR of Long-Lived States.</a> J. Am. Chem. Soc. 134, 11076-11079 (2012).</li><li>Zsila, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+Dichroism+Spectroscopic+Detection+of+Ligand+Binding+Induced+Subdomain+IB+Specific+Structural+Adjustment+of+Human+Serum+Albumin.">Circular Dichroism Spectroscopic Detection of Ligand Binding Induced Subdomain IB Specific Structural Adjustment of Human Serum Albumin.</a> J. Phys. Chem. 117, 10798-10806 (2013).</li><li>Navratilova, I., Hopkins, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragment+Screening+by+Surface+Plasmon+Resonance.">Fragment Screening by Surface Plasmon Resonance.</a> ACS Med. Chem. Lett. 1, 44-48 (2010).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Phase+SPR+Biosensor+for+Efficient+Targeted+Drug+Screening+with+High+Sensitivity+and+Stability.">Investigation of Phase SPR Biosensor for Efficient Targeted Drug Screening with High Sensitivity and Stability.</a> Sensor. Actuat. B-Che. 209, 313-322 (2015).</li><li>Lu, Y., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-Nanometre+Sized+Metal+Clusters:+From+Synthetic+Challenges+to+The+Unique+Property+Discoveries.">Sub-Nanometre Sized Metal Clusters: From Synthetic Challenges to The Unique Property Discoveries.</a> Chem. Soc. Rev. 41, 3594-3623 (2012).</li><li>Yu, Y., Yao, Q., Luo, Z., Yuan, X., Lee, J. Y., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursor+Engineering+and+Controlled+Conversion+for+The+Synthesis+of+Monodisperse+Thiolate-Protected+Metal+Nanoclusters.">Precursor Engineering and Controlled Conversion for The Synthesis of Monodisperse Thiolate-Protected Metal Nanoclusters.</a> Nanoscal. 5, 4606-4620 (2013).</li><li>Jin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+Sized,+Thiolate-Protected+Gold+Nanoclusters.">Quantum Sized, Thiolate-Protected Gold Nanoclusters.</a> Nanoscal. 2, 343-362 (2010).</li><li>Jiang, D. -. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Expanding+Universe+of+Thiolated+Gold+Nanoclusters+and+Beyond.">The Expanding Universe of Thiolated Gold Nanoclusters and Beyond.</a> Nanoscal. 5, 7149-7160 (2013).</li><li>Aikens, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+Structure+of+Ligand-Passivated+Gold+and+Silver+Nanoclusters.">Electronic Structure of Ligand-Passivated Gold and Silver Nanoclusters.</a> J. Phys. Chem. Lett. 2, 99-104 (2010).</li><li>Gao, Y., Shao, N., Pei, Y., Chen, Z., Zeng, X. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+Activities+of+Subnanometer+Gold+Clusters+(Au16&#8211;Au18,+Au20,+and+Au27&#8211;Au35)+for+CO+Oxidation.+ACS.">Catalytic Activities of Subnanometer Gold Clusters (Au16&#8211;Au18, Au20, and Au27&#8211;Au35) for CO Oxidation. ACS.</a> ACS Nan. 5, 7818-7829 (2011).</li><li>Negishi, Y., Nobusada, K., Tsukuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione-Protected+Gold+Clusters+Revisited:+Bridging+the+Gap+between+Gold(I)&#8722;Thiolate+Complexes+and+Thiolate-Protected+Gold+Nanocrystals.">Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)&#8722;Thiolate Complexes and Thiolate-Protected Gold Nanocrystals.</a> J. Am. Chem. Soc. 127, 5261-5270 (2005).</li><li>Zhu, M., Aikens, C. M., Hollander, F. J., Schatz, G. C., Jin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+the+Crystal+Structure+of+A+Thiol-Protected+Au25+Cluster+and+Optical+Properties.">Correlating the Crystal Structure of A Thiol-Protected Au25 Cluster and Optical Properties.</a> J. Am. Chem. Soc. 130, 5883-5885 (2008).</li><li>Yu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Highly+Luminescent+Au22(SG)18+Nanocluster.">Identification of a Highly Luminescent Au22(SG)18 Nanocluster.</a> J. Am. Chem. Soc. 136, 1246-1249 (2014).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+at+the+Core&#8211;Ligand+Interface+of+Au+Lipoic+Acid+Nanoclusters+That+Enhances+the+Near-IR+Luminescence.">Oxidation at the Core&#8211;Ligand Interface of Au Lipoic Acid Nanoclusters That Enhances the Near-IR Luminescence.</a> J. Phys. Chem. 118, 20680-20687 (2014).</li><li>Zhu, Y., Qian, H., Jin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Atomic-Level+Strategy+for+Unraveling+Gold+Nanocatalysis+from+the+Perspective+of+Aun(SR)m+Nanoclusters.">An Atomic-Level Strategy for Unraveling Gold Nanocatalysis from the Perspective of Aun(SR)m Nanoclusters.</a> Chem. Eur. J. 16, 11455-11462 (2010).</li><li>Niesen, B., Rand, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+Film+Metal+Nanocluster+Light-Emitting+Devices.">Thin Film Metal Nanocluster Light-Emitting Devices.</a> Adv. Mater. 26, 1446-1449 (2014).</li><li>Shang, L., Dong, S. J., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-Small+Fluorescent+Metal+Nanoclusters:+Synthesis+and+Biological+Applications.">Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications.</a> Nano Toda. 6, 401-418 (2011).</li><li>Wu, X., He, X., Wang, K., Xie, C., Zhou, B., Qing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+Near-Infrared+Gold+Nanoclusters+for+Tumor+Fluorescence+Imaging+in+Vivo.">Ultrasmall Near-Infrared Gold Nanoclusters for Tumor Fluorescence Imaging in Vivo.</a> Nanoscal. 2, 2244-2249 (2010).</li><li>Archana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular-Receptor-Specific,+Non-Toxic,+Near-Infrared-Emitting+Au+Cluster-Protein+Nanoconjugates+for+Targeted+Cancer+Imaging.">Molecular-Receptor-Specific, Non-Toxic, Near-Infrared-Emitting Au Cluster-Protein Nanoconjugates for Targeted Cancer Imaging.</a> Nanotechnolog. 21, 055103 (2010).</li><li>Yue, Y., Liu, T. Y., Li, H. W., Liu, Z. Y., Wu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-Assisted+Synthesis+of+BSA-Protected+Small+Gold+Nanoclusters+and+Their+Fluorescence-Enhanced+Sensing+of+Silver(I)+Ions.">Microwave-Assisted Synthesis of BSA-Protected Small Gold Nanoclusters and Their Fluorescence-Enhanced Sensing of Silver(I) Ions.</a> Nanoscal. 4, 2251-2254 (2012).</li><li>Liu, Y., Ai, K., Cheng, X., Huo, L., Lu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanocluster+Based+Fluorescent+Sensors+for+Highly+Sensitive+and+Selective+Detection+of+Cyanide+in+Water.">Gold Nanocluster Based Fluorescent Sensors for Highly Sensitive and Selective Detection of Cyanide in Water.</a> 20, 951-1907 (2010).</li><li>Liu, J., Yu, M., Zhou, C., Yang, S., Ning, X., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+Tumor+Targeting+of+Renal-Clearable+Luminescent+Gold+Nanoparticles:+Long+Tumor+Retention+and+Fast+Normal+Tissue+Clearance.">Passive Tumor Targeting of Renal-Clearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance.</a> J. Am. Chem. Soc. 135, 4978-4981 (2013).</li><li>Negishi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Loading+of+Small+Aun+Clusters+(n+=+10&#8211;39)+onto+BaLa4Ti4O15+Photocatalysts:+Toward+an+Understanding+of+Size+Effect+of+Co-Catalyst+on+Water+Splitting+Photocatalytic+Activity.">Controlled Loading of Small Aun Clusters (n = 10&#8211;39) onto BaLa4Ti4O15 Photocatalysts: Toward an Understanding of Size Effect of Co-Catalyst on Water Splitting Photocatalytic Activity.</a> J. Phys. Chem. C. , (2015).</li><li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoclusters.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters.</a> J. Am. Chem. Soc. 131, 888-889 (2009).</li><li>Celej, M. S., Montich, G. G., Fidelio, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Stability+Induced+by+Ligand+Binding+Correlates+with+Changes+in+Protein+Flexibility.">Protein Stability Induced by Ligand Binding Correlates with Changes in Protein Flexibility.</a> Protein Sci. 12, 1496-1506 (2003).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Analysis+of+Ligand-Induced+Changes+in+Protein+Thermal+Unfolding+Applied+to+High-Throughput+Determination+of+Ligand+Affinities+with+Extrinsic+Fluorescent+Dyes.">Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes.</a> Biochemistr. 49, 10831-10841 (2010).</li><li>Yu, Y., New, S. Y., Xie, J., Su, X., Tan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Based+Fluorescent+Metal+Nanoclusters+for+Small+Molecular+Drug+Screening.">Protein-Based Fluorescent Metal Nanoclusters for Small Molecular Drug Screening.</a> Chem. Commun. 50, 13805-13808 (2014).</li><li>Shortridge, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and Drug Discover. , (2010).</li></ol>

The authors have nothing to disclose.

Bu yöntemde vurgulanan gereken birkaç kritik adımlar vardır. Farklı küçük molekül ilaçların göreceli bağlanma afinitesi tarama protokolü Adımlar 3.1.2, 3.1.3, 3.1.4 ve nispi bağlanma gücü için tutarlı eğilimi gösteren iyi sonuçlar elde etmek için kritik öneme sahiptir. Bu adımlarda, ölçüm için reaksiyon çözümleri ekleyerek kimyasallar ve çizim eylemleri gecikme etkisi ve tutarlılığı sağlamak için uygulanmalıdır ölçümü için reaksiyon çözümleri ekleyerek kimyasallar ve çi...

Insan serum albümini (HSA) ve sığır serum albümini (BSA) gibi serum albüminler, plazmada en fazla protein vardır ve kan bölmesi osmotik basıncının sağlanmasında önemli bir rol oynamaktadır. Ayrıca, steroidler gibi, suda düşük çözünürlüklü, küçük moleküller, yağ asitleri, tiroid hormonları ve ilaçlar çok çeşitli taşıyıcı proteinler olarak kabul edilmektedir. Bağlama özelliği albüminler serum, bu moleküllerin (örneğin, afinite ya da bağlanma kuvvetinin, bağlanma yerleri) farmakokinetik önemli bir konu teşkil etmektedir. 1-4 Çeşitli analitik yöntemler gibi albüminler serum farklı ilaçların bağlanma özelliklerini incelemek için geliştirilmiştir X-ışını kristalografisi, 5,6 nükleer manyetik rezonans (NMR), 7-11 ve yüzey plazmon rezonans (SPR), 12,13 vs. Bununla birlikte, bu yöntemler, bir sıkıcı ve zaman alıcı bir analiz işlemi (ya da tarafından kısıtlanır, örneğin, X-ışını Crystallo tek kristal büyümeGrafik çalışması), özel ve pahalı ekipman gerekliliği (SPR), veya tespiti için pahalı izotop etiketleme (NMR) muhtaç. Bu, hızlı bir düz ileri ve maliyet-etkin bir şekilde, küçük molekül ilaç taraması için alternatif yollar oluşturmak çok arzu edilen bir durumdur. Altın nanokümeler (Au ​​NC&#39;ler), nedeniyle ayrık ve boyut bağımlı elektronik yapıya, 18 Onlar kapsamlı araştırma alanları çekmiştir 2 nm. 14-17 daha küçük boyutları ile metal atomları onlarca birkaç ihtiva nanomaterial özel bir tip vardır 20-23 Bu özel malzeme özellikleri, özellikle de güçlü bir floresan gibi biyolojik sistemlerde algılama ve görüntüleme. 24-32 ultrasmall floresan Au NC&#39;ler işlevsel proteinleri kullanılarak sentezlenebilir gibi çeşitli uygulama alanı bulmaktadır. 19 ve moleküler benzeri bir emişe ve emisyon, Böyle bir şablon olarak serum albüminleri olarak. 33 tipik bir protein şablonu sentezinde<html> Au NC&#39;ler Au tuzları bir miktar birinci protein sarmalanır ve daha sonra proteinin kendisinin azaltılır. Protein indirgeme yeteneği alkalin çözüm artan pH ile aktive edilebilir fonksiyonel amino asit kalıntılarını (örneğin, tirosin) oluşturucu atfedilir. Protein yapısının Unfolding Au NC&#39;ler oluşumu için kritik bir aşama olarak kabul edilir. Katlanmamış protein, daha fazla indirgeyici fonksiyonel gruplar kapsüllenmiş Au tuzları maruz olmasıdır. Protein denatüre edici maddelere karşı, ısı tedavisi veya maruz kalma ile elde edilebilir açılma. Küçük molekül ilaçların tanıtımı da orta denatürasyon sıcaklığı ve açılımı entalpilerini değiştirerek yani açılımı sürecini etkileyebilir. 34,35 Tüm bu faktörlerin etkisi, sırayla floresan Au NC&#39;ler oluşum kinetiği ile yansıtılabilir ve tecelli Elde edilen Au NC&#39;ler floresans yoğunluğu. 36 e_content &quot;&gt; Bu video daha yüksek bir sıcaklığa (60 ° C) &#39;de ilaç yüklü albümin proteinleri Au NCS sentezlenmesiyle, doğallık bozucu maddeler (örneğin, ürea) elde edilen Au NCS. floresan yoğunluğunun varlığında ilaç taraması yöntemini göstermektedir sinyal değerini göstermektedir. Birincisi, Au, Au NC&#39;ler NC&#39;ler. İkinci oluşumu kinetiklerini proteini (ısı işlemi veya denatüranlar ile yol açılan) açılma göstermek için 60 ° C ya da üre mevcudiyetinde muamele HSA ve BSA şablonlarında sentezlenir, Au NC&#39;ler farklı ilaçlar ile önceden yüklenmiş bir protein şablonları sentezlenir, ve elde edilen Au NC&#39;ler nispi floresan yoğunlukları ile ilaç yüklemesi etkisi nispi bağlanma mukavemeti ölçüsü sağlayan, incelenmiştir. Sonuç olarak, Au NC-ilaç tarama programı için değiştirilir sabit bir konsantrasyon protein yüklenmiş ilaç içeriğini değiştirerek, ilaç-protein bağlayıcı sabiti (KD) kantitatif ölçümü.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

Biz Au NC&#39;ler tarafından yayılan farklı flüoresans sinyaline dayalı ilaç yüklü protein içinde flüoresan altın nanoyığınlar (Au NCS) oluşturan bir hedef proteine, küçük ilaç moleküllerinin bağlanma afinitesini belirlemek için yeni bir ilaç tarama yöntemi göstermektedir. Insan serum albümini (HSA) ve sığır serum albümini (BSA) gibi albümin proteinleri modeli proteinler olarak seçilir. Dört adet küçük molekül ilaç albümin proteinleri, farklı bağlanma afiniteleri (ör ibuprofen, varfarin, fenitoin ve sülfanilamid) test edilir. Bu denatüre edici koşullar (örneğin, 60 ° C veya üre varlığında) altındaki ilaç yüklü albumin protein içindeki fluoresan Au NC&#39;ler oluşumu oranı (ilaç olmadan) bozulmamış proteininde oluşmuş daha yavaş olduğu bulunmuştur. Dahası, şekillendirilmiş NC&#39;ler floresan yoğunluğu ile ters albümin proteinleri bu ilaçların bağlanma ilgisi ilişkili olduğu bulunmuştur. Özel olarak,Au NC&#39;ler oluşum hızı daha yavaş ilaç-protein bağlanma afinitesi daha yüksektir ve bu şekilde elde edilen Au NC&#39;ler daha düşük bir floresans yoğunluğu görülmektedir. Elde edilen Au NC&#39;ler floresan yoğunluğu dolayısıyla test farklı ilaçların göreceli bağlanma gücü basit bir ölçümünü sağlar. Bu yöntem aynı zamanda, sadece sabit bir protein konsantrasyonunda protein yüklenmiş ilaç içeriğini değiştirerek spesifik ilaç-protein bağlayıcı sabiti (KD) ölçmek üzere genişletilebilir. Ölçülen sonuçlar, diğer itibar ama daha karmaşık yöntemler kullanılarak elde edilen değerlerle iyi uyum sağlar.

Bir proteinin fazla reaktif fonksiyonel gruplar (örneğin, tirosin kalıntıları) kapsüllenmiş Au iyonları azaltmak ve böylece Au NC&#39;ler oluşumu oranını hızlandırmak için maruz çünkü açılma protein, protein-şablonu Au NC&#39;ler oluşumu için önemli bir işlemdir. Isıtma ve dış denatüre maddeleri protein açılımı sürecini teşvik için iki ortak araçlardır. 1 model protein olarak HSA kullanarak, Au NC&#39;ler oluşum kinetiği üzerinde ısıtma etkisi ve eklem...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Protein-Hedefleme Küçük Molekül İlaç Tarama için hızlı ve Nicel florimetrik Yöntemi

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

Research

JoVE Journal

Bioengineering

9.9K Görüntüleme.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Video: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

İlaç Etkisi Micropatterned Hücreler Geliştirilmiş Görselleştirme ve Kantitatif Analiz

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Biyomühendislik

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Yüksek verim Antifungal İlaç Tarama Candida albicans Biyofilm Chip (Ca BChip)

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

AFM-tabanlı Tek-molekül Kuvvetleri Spektroskopisi ile cellulosome Reseptör-ligand Sistemleri Araştırılması

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Adenilat siklaz uyuşturucu kaynaklı Sensitizasyonu: Küçük Molekül ve siRNA Tarama Uygulamaları için Deney düzene ve minyatür

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Biyomedikal Uygulamalar için Biyolojik Tabanlı Akıllı Nano Hidroksiapatit Rapid Mix Hazırlanması

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Kardiyak İlaç güvenlik Taraması için insan mühendisliği kalp dokusunun Otomatik Büzülme Analizi

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Enjekte edilebilir rejeneratif terapi ve yüksek-den geçerek uyuşturucu tarama için 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Kimyasal epigenetik değiştiriciler ile hücresel makine yeniden yönlendirerek Gen transkripsiyonu bastırmak

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Otomatik bir Mikrobioreactor sistemi kullanılarak Çin hamster yumurtlama hücrelerinden bir monoklonal antikor arıtma ve analitik

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Açık Kaynak Mikroplaka Uyumlu Aydınlatma Panelleri Uygulamaları

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...