S.E. Duke bağış özel girişim Fonu, Wells Fargo Fonu, ilaç Vakfı yanı sıra başlangıç fonlar üzerinden University of North Carolina, desteğinden Charlotte kabul eder.

1. BSA-Au(III) kompleksinin sentezi
<ol><li>BSA 25 mg 1 ml 5 mL tepki şişe yüksek performanslı sıvı kromatografi (HPLC) sınıf suda çözülür. Not: Çözüm net görünmesi gerekir.</li>
	<li>HPLC sınıf suda 5 mM bir konsantrasyon için altın (III) klorür trihidrat (chloroauric asit) geçiyoruz. Not: Çözüm sarı görünmesi gerekir. Chloroauric asit solüsyonu bu konsantrasyonu hazırlanan bir BSA Au oranı 1:13 neden olur.<ol>
<li>Alternatif olarak, chloroauric asit bir çözüm herhangi bir yerde bir konsantrasyon ile hazırlamak 0,38 mM arasında (BSA:Au = 1:1) 20 mm (BSA:Au = 1:50) içinde HPLC sınıf su. Not: BSA farklı oranlarda altın uyarma-emisyon haritanın büyük ölçüde farklı kırmızı floresans desenleri neden olur.</li>
	</ol>
</li>
	<li>Bir 37 ° C su banyosunda BSA tepki şişe yerleştirin ve 750 bir manyetik karıştırıcı kullanarak devir / dakikada karistirin.</li>
	<li>Hemen karıştırma başladıktan sonra 1 mL chloroauric asit ekleyin. Belgili tanımlık eriyik rengini net sarıya dönüştürmek.</li>
	<li>2 min için karışımı ilave edin 37 ° C'de ve 750 rpm bir manyetik karıştırıcı kullanarak.</li>
	<li>Reaksiyon şişe 100 μL 1 M NaOH pH 12'ye getirmek için çözüm ekleyin. Not: Hemen NaOH eklendikten sonra çözüm biraz sarı-kahverengi için karartmak ve daha sonra geri dönmek için sarı.</li>
	<li>2 h için 750 rpm ve 37 ° C'de karıştırmaya devam Çözüm yavaş yavaş sarıdan koyu sarı/kahverengi renk için değiştirmeniz gerekir. Bu renk değişikliği kırmızı Floresanda BSA-Au(III) karmaşık oluşumu gösterir.</li>
	<li>Örnek oda sıcaklığında 2 gün boyunca oturmak izin ve çözüm bir kehribar Brown'a koyulaştırmak devam edecek ve floresan yoğunluğu artacaktır.<ol>
<li>Alternatif olarak, çözüm rengini bir amber kahverengi geliştikçe daha fazla h 12 37 ° C'de karıştırmaya devam örnek ver.</li>
	</ol>
</li>
</ol>2. BSA-Cu(II)-Au(III) sentezi
<ol><li>BSA 25 mg 1 mL HPLC sınıf su içinde çözülür. Çözüm net görünmesi gerekir.</li>
	<li>Bakır (II) klorür dihydrate HPLC sınıf su konsantrasyonu 5 mm geçiyoruz. Çözüm ışık görünmelidir mavi.</li>
	<li>Sulu BSA çözüm 1 mL 5 mL tepki şişe ekleyin ve bir su banyosu 37 ° C'de yer Karışımı 750 devir / dakikada ilave edin.</li>
	<li>Hemen 0,5 mL bakır (II) klorür dihydrate çözeltisi tepki flakon ve 2 min için karıştırın. Çözüm ışık kalır mavi.</li>
	<li>12'ye pH getirmek ve 2 h için karıştırmak için izin vermek için 1 M NaOH 75 μL ekleyin. Belgili tanımlık eriyik-ecek var olmak mor.</li>
	<li>Chloroauric asit konsantrasyonu 5 mm HPLC sınıf suda çözülür.</li>
	<li>0.5 mL sulu chloroauric asit reaksiyon şişeyi ekleyebilir ve pH geri 12 1 M NaOH kullanarak ayarlayabilirsiniz.</li>
	<li>2 h reaksiyon karışımı ilave edin. Not: Çözüm için kahverengi bir renk gelişmesi gerektiğini.</li>
</ol>3. BSA-Ni(II)-Au(III) sentezi
<ol><li>BSA 25 mg 1 mL HPLC sınıf su içinde çözülür. Çözüm net görünmesi gerekir.</li>
	<li>HPLC sınıf su konsantrasyonu 5 mm nikel (II) klorür hekzahidrat geçiyoruz. Çözüm ışık görünmesi gereken yeşil.</li>
	<li>Sulu BSA çözüm 1 mL 5 mL tepki şişe ekleyin ve bir su banyosunda 37 oC. heyecan 750 rpm karışımı yerleştirin.</li>
	<li>Hemen tepki flakon ve 2 min için karışımı 0.5 mL nikel (II) klorür hekzahidrat çözeltisi ekleyin. Not: Çözüm ışık kalır yeşil.</li>
	<li>12'ye pH getirmek ve 2 h için karıştırmak için izin vermek için 1 M NaOH 75 μL ekleyin. Not: Çözüm koyu sarı olacak.</li>
	<li>Chloroauric asit konsantrasyonu 5 mm HPLC sınıf suda çözülür.</li>
	<li>0.5 mL sulu chloroauric asitin reaksiyonu şişeyi ekleyebilir ve geri ile 12 pH ayarlayabilirsiniz.</li>
	<li>2 h reaksiyon karışımı ilave edin. Not: Çözüm için kahverengi bir renk gelişmesi gerektiğini.</li>
</ol>4. [Cys34-capped-BSA]-Au(III) sentezi
<ol><li>N-ethylmaleimide (NEM) 2 mg fosfat tamponlu tuz (PBS, pH 7,4) 1 mL geçiyoruz.</li>
	<li>BSA 2 mg 1 mL PBS-NEM çözeltisi içinde çözülür.</li>
	<li>Çözüm 5 mL tepki şişe aktarmak ve 20 ° c için 1 h 500 rpm'de ilave edin.</li>
	<li>PBS, 50 rpm'de unreacted NEM kaldırmak için bir manyetik karıştırıcı ile gecede karıştırarak 500 ml 12 kDa diyaliz boru kullanarak çözüm diyaliz.</li>
	<li>Chloroauric asit konsantrasyonu 0.4 mM için PBS çözülür. Not: Bir soluk sarı çözüm olacaktır.</li>
	<li>Reaksiyon şişe su banyosu 37 ° C'de transfer 750 devir / dakikada ilave edin.</li>
	<li>Reaksiyon flakon 1 mL chloroauric asit çözeltisi eklenir ve 2 min için karıştırmak için izin.</li>
	<li>75 μL 1 M NaOH pH 12'ye getirmek ve 2 h için karıştırmak için izin vermek için tepki şişe ekleyin.</li>
</ol>5. [all-thiol-capped-BSA]-Au(III) sentezi
<ol><li>2 M üre ve 50 mM Amonyum Bikarbonat (NH4HCO3, pH 8.0) HPLC sınıf su çözeltisi hazırlamak.</li>
	<li>BSA 3.3 mg 1 ml Yukarıdaki çözüm ve 5 mL tepki şişe transfer geçiyoruz.</li>
	<li>Bir hisse senedi çözüm 0.25 m olun tris(2-carboxyethyl) TCEP 62,5 mg 1 mL HPLC su içinde eriterek tarafından fosfamin (TCEP).</li>
	<li>TCEP son konsantrasyonu 8 mM olana TCEP hisse senedi çözüm tepki şişe ekleyin.</li>
	<li>Bir su banyosu için 1s 50 ° C'de çözümde kuluçkaya Manyetik karıştırıcı kullanarak 500 rpm'de karıştırın.</li>
	<li>Çözüm için tamamen oda sıcaklığında soğumaya izin verin.</li>
	<li>Bir hisse senedi çözüm 100 mm NEM NEM 12.5 mg 1 ml HPLC sınıf su çözülerek hazırlayın.</li>
	<li>NEM son konsantrasyonu 16 mM kadar NEM hisse senedi çözüm tepki şişe ekleyin.</li>
	<li>20 ° C'de 2 h için birleştirmek çözüm izin verin 500 rpm'de karıştırın.</li>
	<li>Çözüm 50 rpm'de aşırı TCEP, NEM ve üre kaldırmak için bir manyetik karıştırıcı ile geceleme 500 mL 50 mM NH4HCO3 karıştırarak bir 12 kDa diyaliz boru kullanarak çözüm diyaliz.</li>
	<li>Bir su banyosu 37 ° C'de tepki şişe taşımak ve 750 devir / dakikada ilave edin.</li>
	<li>Chloroauric asit 0,66 mM bir konsantrasyon için HPLC sınıf suda çözülür.</li>
	<li>Reaksiyon flakon 1 mL chloroauric asit çözeltisi eklenir ve 2 min için karıştırmak için izin.</li>
	<li>Çözüm pH 12 kadar 1 M NaOH ekleyin ve 2 h için karıştırmak devam etmek çözüm sağlar.</li>
</ol>

<ol>
	<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+Nanoparticles.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoparticles.</a> Journal of the American Chemical Society. 131, 888-889 (2009).</li><li>Saha, K., Agasti, S. S., Kim, C., Li, X., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles+in+Chemical+and+Biological+Sensing.">Gold Nanoparticles in Chemical and Biological Sensing.</a> Chemical Reviews. 112, 2739-2779 (2012).</li><li>Zhang, 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=New+Gold+Nanostructures+for+Sensor+Applications:+A+Review.">New Gold Nanostructures for Sensor Applications: A Review.</a> Materials. 7, 5169-5201 (2014).</li><li>Chen, L. -. Y., Wang, C. -. W., Yuan, Z., Chang, H. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Gold+Nanoclusters:+Recent+Advances+in+Sensing+and+Imaging.">Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging.</a> Analytical Chemistry. 87 (1), 216-229 (2015).</li><li>Cai, W., Gao, T., Hong, H., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Gold+Nanoparticles+in+Cancer+Nanotechnology.">Applications of Gold Nanoparticles in Cancer Nanotechnology.</a> Nanotechnology, Science and Applications. 1, 17-32 (2008).</li><li>Nune, S. K., 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=Nanoparticles+for+Biomedical+Imaging.">Nanoparticles for Biomedical Imaging.</a> Expert Opinion on Drug Delivery. 6, 1175-1194 (2009).</li><li>Dorsey, J. F., 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=Gold+Nanoparticles+in+Radiation+Research:+Potential+Applications+for+Imaging+and+Radiosensitization.">Gold Nanoparticles in Radiation Research: Potential Applications for Imaging and Radiosensitization.</a> Translational Cancer Research. 2, 280-291 (2013).</li><li>Daniel, M. -. C., Astruc, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Assembly,+Supramolecular+Chemistry,+Quantum-Size-Related+Properties,+and+Applications+toward+Biology,+Catalysis,+and+Nanotechnology.">Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology.</a> Chemical Reviews. 104 (1), 293-346 (2004).</li><li>Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Nanotechnology:+Opportunities+and+Challenges.">Cancer Nanotechnology: Opportunities and Challenges.</a> Nature Reviews Cancer. 5, 161-171 (2005).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Interesting+Optical+Properties+and+Recent+Applications+in+Cancer+Diagnostics+and+Therapy.">Gold Nanoparticles: Interesting Optical Properties and Recent Applications in Cancer Diagnostics and Therapy.</a> Nanomedicine. 2, 681 (2007).</li><li>Arvizo, R., Bhattacharya, R., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Opportunities+and+Challenges+in+Nanomedicine.">Gold Nanoparticles: Opportunities and Challenges in Nanomedicine.</a> Expert Opinion on Drug Delivery. 7, 753-763 (2010).</li><li>Doane, T. L., Burda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Unique+Role+of+Nanoparticles+in+Nanomedicine:+Imaging,+Drug+Delivery+and+Therapy.">The Unique Role of Nanoparticles in Nanomedicine: Imaging, Drug Delivery and Therapy.</a> Chemical Society Reviews. 41, 2885 (2012).</li><li>Egusa, S., Ebrahem, Q., Mahfouz, R. Z., Saunthararajah, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+Exchange+on+Gold+Nanoparticles+for+Drug+Delivery+and+Enhanced+Therapeutic+Index+Evaluated+in+Acute+Myeloid+Leukemia+Models.">Ligand Exchange on Gold Nanoparticles for Drug Delivery and Enhanced Therapeutic Index Evaluated in Acute Myeloid Leukemia Models.</a> Experimental Biology and Medicine. 239, 853 (2014).</li><li>Qu, X., 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=Fluorescent+Gold+Nanoclusters:+Synthesis+and+Recent+Biological+Application.">Fluorescent Gold Nanoclusters: Synthesis and Recent Biological Application.</a> Journal of Nanomaterials. (784097), (2015).</li><li>Chakraborty, I., Pradeep, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomically+Precise+Clusters+of+Noble+Metals:+Emerging+Link+between+Atoms+and+Nanoparticles.">Atomically Precise Clusters of Noble Metals: Emerging Link between Atoms and Nanoparticles.</a> Chemical Reviews. 117, 8208-8271 (2017).</li><li>Raut, S., 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=Evidence+of+energy+transfer+from+tryptophan+to+BSA/HSA+protected+gold+nanoclusters.">Evidence of energy transfer from tryptophan to BSA/HSA protected gold nanoclusters.</a> Methods and Applications in Fluorescence. 2, (2014).</li><li>Le Gu&#233;vel, X., Daum, N., Schneider, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+Human+Transferrin-Stabilized+Gold+Nanoclusters.">Synthesis and Characterization of Human Transferrin-Stabilized Gold Nanoclusters.</a> Nanotechnology. 22 (27), (2011).</li><li>Kawasaki, H., Yoshimura, K., Hamaguchi, K., Arakawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-Stabilized+Fluorescent+Gold+Nanocluster+for+Sensitive+and+Selective+Hg2++Detection.">Trypsin-Stabilized Fluorescent Gold Nanocluster for Sensitive and Selective Hg2+ Detection.</a> Analytical Sciences. 27 (6), 591 (2011).</li><li>Lu, D., 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=Lysozyme-Stabilized+Gold+Nanoclusters+as+a+Novel+Fluorescence+Probe+for+Cyanide+Recognition.">Lysozyme-Stabilized Gold Nanoclusters as a Novel Fluorescence Probe for Cyanide Recognition.</a> Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 121, 77-80 (2014).</li><li>Dixon, J. M., Egusa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change-Induced+Fluorescence+of+Bovine+Serum+Albumin-Gold+Complexes.">Conformational Change-Induced Fluorescence of Bovine Serum Albumin-Gold Complexes.</a> Journal of the American Chemical Society. 140, 2265-2271 (2018).</li><li>Peters, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All About Albumin. , (1996).</li><li>Masuoka, J., Saltman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc(II)+and+Copper(II)+Binding+to+Serum+Albumin.+A+Comparative+Study+of+Dog,+Bovine,+and+Human+Albumin.">Zinc(II) and Copper(II) Binding to Serum Albumin. A Comparative Study of Dog, Bovine, and Human Albumin.</a> Journal of Biological Chemistry. 269, 25557-25561 (1994).</li><li>Takeda, K., Wada, A., Yamamoto, K., Moriyama, Y., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change+of+Bovine+Serum+Albumin+by+Heat+Treatment.">Conformational Change of Bovine Serum Albumin by Heat Treatment.</a> Journal of Protein Chemistry. 8 (5), 653-659 (1989).</li><li>Klotz, I. M., Curme, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-protein+Combinations.+Copper+with+Bovine+Serum+Albumin.">The Thermodynamics of Metallo-protein Combinations. Copper with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 70, 939-943 (1948).</li><li>Fiess, H. A., Klotz, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-Protein+Combinations.+Comparison+of+Copper+Complexes+with+Natural+Proteins.">The Thermodynamics of Metallo-Protein Combinations. Comparison of Copper Complexes with Natural Proteins.</a> J. Am. Chem. Soc. 74, 887-891 (1952).</li><li>Rao, M. S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+the+Interaction+of+Nickel(II)+with+Bovine+Serum+Albumin.">A Study of the Interaction of Nickel(II) with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 84, 1788-1790 (1962).</li><li>Peters, T., Blumenstock, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Binding+Properties+of+Bovine+Serum+Albumin+and+Its+Amino-terminal+Peptide+Fragment.">Copper-Binding Properties of Bovine Serum Albumin and Its Amino-terminal Peptide Fragment.</a> Journal of Biological Chemistry. 242, 1574-1578 (1967).</li><li>Xue, Y., Li, X., Li, H., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Thiol-Gold+Interactions+towards+the+Efficient+Strength+Control.">Quantifying Thiol-Gold Interactions towards the Efficient Strength Control.</a> Nature Communications. 5, 4348 (2014).</li><li>Xu, 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=The+Role+of+Protein+Characteristics+in+the+Formation+and+Fluorescence+of+Au+Nanoclusters.">The Role of Protein Characteristics in the Formation and Fluorescence of Au Nanoclusters.</a> Nanoscale. 6 (3), 1515-1524 (2014).</li></ol>

Yazarlar ifşa gerek yok.

PH 12 hazırlanan BSA-Au(III) bileşikler kırmızı floresans bir emisyon dalga boyu λemadlı sergi 640 = ultraviyole (UV) ışık λexile heyecanlı zaman nm = 365 nm (Şekil 1A). Kırmızı floresans ortaya çıkması yavaş bir süreçtir ve bir kaç gün için maksimum yoğunluğu artırmak için oda sıcaklığında alacak. Yüksek sıcaklık kırmızı floresans daha hızlı üretmek için kullanılan rağmen tepki 37 ° C'de çalıştıran optimum sonu...

Bir ultraviyole (UV) sergileyen bir BSA-Au bileşik-dikkat çekici ile heyecanlı kırmızı floresans shift stokes, başlangıçta Xie ve arktarafından sentezlenen. 1. benzersiz ve istikrarlı kırmızı floresans alanlarda2,3,4,5,6,7görüntüleme veya nanomedicine8 algılama gibi çeşitli uygulamalar bulabilirsiniz ,9,10,11,12,13. Bu bileşik, nano-bilim son yıllarda14,15,16, alanında birçok araştırmacılar tarafından kapsamlı bir şekilde çalışılmıştır. BSA-Au bileşik Au25 nanoclusters yorumlanır. Bu bileşik ayrıntılı incelemek ve kırmızı floresans kökenini anlamak için sunulan Yöntem hedefidir. Sunulan yaklaşım takip ederek, birden çok Au bağlayıcı siteleri, varlığı ve floresan, Au25 nanoclusters, tek-site çekirdekleşme alternatif kökeni resimli. Aynı yaklaşım diğer proteinler17,18,19 Au(III) ile complexed içsel floresan özelliklerini değiştirebilirsiniz nasıl eğitim için istihdam edilebilir.
Kırmızı-floresan BSA-Au bileşik sentezi BSA floresan yoğunluğu ve uyarma-emisyon harita (EEM)20Peaks'e konumunu en üst düzeye çıkarmak için Au (BSA:Au) için molar oranları dar bir denetimi gerektirir. Bu birden çok bağlayıcı siteleri Au(III) bağlamak, mevcut Asparagine parçası (veya Asp parça, ilk dört amino asit kalıntıları, N-terminus, BSA) de dahil olmak üzere gösterilebilir21,22. BSA (Cys-34) 34th amino asit de Au(III) koordine etmek ve kırmızı fluorescence([Cys34-capped-BSA]-Au(III))20yönteminde dahil olmak gösterilir. Tüm Cys-Cys disülfür bağları fizyon ve tüm thiols, kırmızı floresans kapatma üzerine üretilen ([all-thiol-capped-BSA]-Au(III)). değil Bu kırmızı floresans üretmek için Au(III) bağlama site olarak Cys-Cys disülfür bağları gerekliliğini gösterir.
Protein Kimya teknikler yaygın olarak BSA-Au(III) kompleksleri nano-bilim toplumda eğitim için kullanılmış değil. Ancak, BSA Au(III) bağlama sitelerin detaylı bilgi sahibi olarak bu komplekslerin, de bazı yönlerini anlamak için bu teknikler istihdam için değerli olacaktır. Bu makalede bazı teknikler göstermek için tasarlanmıştır.

<table><tbody><tr><td>Bovine Serum Albumin (BSA), 96%</td><td>Sigma-Aldrich</td><td>A5611</td><td></td></tr><tr><td>gold (III) chloride trihydrate, 99.9%</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Copper (II) chloride dihydrate, 99.999%</td><td>Sigma-Aldrich</td><td>459097</td><td></td></tr><tr><td>Nickel (II) chloride hexahydrate, 99.9%</td><td>Sigma-Aldrich</td><td>654507</td><td></td></tr><tr><td>N-Ethylmaleimide (NEM), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;l</td><td>30142</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Corning</td><td>MT21040CV</td><td></td></tr><tr><td>Ammonium bicarbonate, 99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr></tbody></table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

Sunulan iletişim kurallarının amacı biçimi değişikliği indüklenen kırmızı floresans verimli BSA, Au(III) bağlama işlemi incelemektir (λem 640 = nm) BSA-Au(III) kompleksleri. Yöntem kırmızı floresans ortaya çıkması BSA biçimler denge pH kaynaklı geçişler ile ilişkili göstermek için pH ayarlar. Kırmızı floresan BSA-Au(III) kompleksleri sadece BSA "A-biçimli" biçimi için karşılık gelen pH ya da 9,7 üzerinde bir ayarlama ile oluşmuş olabilir. Au molar oranı BSA ayarlamak için ve Au(III) bağlama işlemi saat ders izlemek için iletişim kuralı açıklanmıştır. Au(III) kırmızı floresans üretmek için BSA, başına en az sayıda daha az yedi yaşında. Biz iletişim kuralı birden çok Au(III) bağlayıcı siteleri BSA içinde varlığını göstermek için adımlar açıklanmaktadır. İlk, bakır ekleyerek (Cu(II)) veya nikel (Ni(II)) Özellikler Au(III) tarafından takip, bu yöntem ortaya bir bağlama sitesi kırmızı fluorophore değil Au(III). İkinci olarak, BSA ajanlar kapatma thiol tarafından değiştirerek, başka bir nonfluorophore oluşturan Au(III) bağlama site ortaya çıkıyor. Üçüncü olarak, BSA conformation fizyon ve disülfür bağları kapatma değiştirme, mümkün Au(III) bağlama site (ler) gösterilmiştir. Diğer proteinler ve metal katyonlar etkileşimler çalışmaya, BSA biçimler ve Au(III) bağlama, kontrol etmek için açıklanan protokol genel olarak uygulanır.

BSA-Au(III) karmaşık floresans, o bu gözlenmiştir BSA içsel mavi floresans çevrimi (λem 400 = nm) kırmızı floresans için (λem 640 = nm) pH 9,7 bir denge ile ilgili oluşur geçiş (Şekil 1). EEM BSA-Au(III) Au molar oranları farklı BSA, Şekil 2' de gösterilen ve bu verileri nasıl molar oranı değiştirme aynı emisyon dalga boyu farklı uyarma dalga boylarında, verimleri...

Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

Altın katyonlar (Au(III)). Sığır serum albumin (BSA) de konformasyon bağımlı benzersiz BSA-Au floresans karakterize gelince sunulan çeşitli biçimler için bağlama eğitimi iletişim kurallarını

Sığır Serum albümin Gold(III) bağlama tarafından çeşitli biçimler Luminophore oluşumu

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

luminophore-formation-various-conformations-bovine-serum-albumin

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

6.8K Görüntüleme.  University of North Carolina. Altın katyonlar (Au(III)). Sığır serum albumin (BSA) de konformasyon bağımlı benzersiz BSA-Au floresans karakterize gelince sunulan çeşitli biçimler için bağlama eğitimi iletişim kurallarını

Video: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Malarya Protein Biyomarker Histidin Zengin Protein-II Tespiti için İridyum (III) Lüminesans Probe

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Kimya

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

Slp1 Polimerler ve Monolayer Au-etkileşimden<em&gt; Lysinibacillus sphaericus</em&gt; JG-B53 - QCM-D, biyomoleküler metal Çalışmaları Araçlar olarak ICP-MS ve AFM

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Proteom çapında Quantification maddelerinin homojenliği tek molekül düzeyinde etiketlenmesi

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Biyokimya

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

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

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 ...

Biyomühendislik

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

<meta charset="utf8"></head><body>Sığır Kolostrumunda Toplam Protein ve Biyoaktif Protein Konsantrasyonlarının Belirlenmesi

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Sığır Kolostrumunda Toplam Protein ve Biyoaktif Protein Konsantrasyonlarının Belirlenmesi

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (&#955;em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

<meta charset="utf8"></head><body>Metal Katyonların Varlığında Sınırlı Proteoliz ve Jel Elektroforezi: Serum Albüminlerinde AuIII Bağlayıcı Lüminesan Alan

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

Metal Katyonlarının Varlığında Sınırlı Proteoliz ve Jel Elektroforezi: Serum Albüminlerinde Au(III) Bağlayıcı Lüminesan Alan

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Biyolojik Uygulamalar için Yakın Kızılötesi Yayan Altın Nanokümelerin Sentezi

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

Amiloid Beta 1-40 Kaplı Altın Kolloidlerin pH'a Bağlı Tersinir Kendi Kendine Montajının Karakterizasyonu

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Sığır Serum albümin Gold(III) bağlama tarafından çeşitli biçimler Luminophore oluşumu

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Malarya Protein Biyomarker Histidin Zengin Protein-II Tespiti için İridyum (III) Lüminesans Probe

Slp1 Polimerler ve Monolayer Au-etkileşimden

Proteom çapında Quantification maddelerinin homojenliği tek molekül düzeyinde etiketlenmesi

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

Sığır Kolostrumunda Toplam Protein ve Biyoaktif Protein Konsantrasyonlarının Belirlenmesi

Sığır Kolostrumunda Toplam Protein ve Biyoaktif Protein Konsantrasyonlarının Belirlenmesi

Metal Katyonlarının Varlığında Sınırlı Proteoliz ve Jel Elektroforezi: Serum Albüminlerinde Au(III) Bağlayıcı Lüminesan Alan

Metal Katyonlarının Varlığında Sınırlı Proteoliz ve Jel Elektroforezi: Serum Albüminlerinde Au(III) Bağlayıcı Lüminesan Alan

Biyolojik Uygulamalar için Yakın Kızılötesi Yayan Altın Nanokümelerin Sentezi

Amiloid Beta 1-40 Kaplı Altın Kolloidlerin pH'a Bağlı Tersinir Kendi Kendine Montajının Karakterizasyonu