Pegylated, steptavidin-conjugated quantum dots are effective detection elements for reverse-phase protein microarrays

被引:98
作者
Geho, D
Lahar, N
Gurnani, P
Huebschman, M
Herrmann, P
Espina, V
Shi, A
Wulfkuhle, J
Garner, H
Petricoin, E
Liotta, LA
Rosenblatt, KP [1 ]
机构
[1] SW Texas State Univ, Div Translat Pathol, Dept Pathol, Dallas, TX 75390 USA
[2] NCI, Pathol Lab, NIH, FDA Clin Proteom Program, Bethesda, MD 20892 USA
[3] US FDA, Ctr Biol Evaluat & Res, Off Cellular & Gene Therapy, Bethesda, MD 20892 USA
基金
美国国家科学基金会;
关键词
D O I
10.1021/bc0497113
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Protein microarray technologies provide a means of investigating the proteomic content of clinical biopsy specimens in order to determine the relative activity of key nodes within cellular signaling pathways. A particular kind of protein microarray, the reverse-phase microarray, is being evaluated in clinical trials because of its potential to utilize limited amounts of cellular material obtained through biopsy. Using this approach, cellular lysates are arrayed in dilution curves on nitrocellulose substrates for subsequent probing with antibodies. To improve the sensitivity and utility of reverse-phase microarrays, we tested whether a new reporter technology as well as a new detection instrument could enhance microarray performance. We describe the use of an inorganic fluorescent nanoparticle conjugated to streptavidin, Qdot 655 Sav, in a reverse-phase protein microarray format for signal pathway profiling. Moreover, a pegylated form of this bioconjugate, Qdot 655 Sav, is found to have superior detection characteristics in assays performed on cellular protein extracts over the nonpegylated form of the bioconjugate. Hyperspectral imaging of the quantum dot microarray enabled unamplified detection of signaling proteins within defined cellular lysates, which indicates that this approach may be amenable to multiplexed, high-throughput reverse-phase protein microarrays; in which numerous analytes are measured in parallel within a single spot.
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收藏
页码:559 / 566
页数:8
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