Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein-DNA interactions in nuclear extracts from breast cancer cell.

The large number of estrogen receptor (ER) binding sites of various sequence patterns requires a sensitive detection to differentiate between subtle differences in ER-DNA binding affinities. A self-assembled monolayer (SAM)-assisted silicon nanowire (SiNW) biosensor for specific and highly sensitive detection of protein-DNA interactions, remarkably in nuclear extracts prepared from breast cancer cells, is presented. As a typical model, estrogen receptor element (ERE, dsDNA) and estrogen receptor alpha (ERα, protein) binding was adopted in the work. The SiNW surface was coated with a vinyl-terminated SAM, and the termination of the surface was changed to carboxylic acid via oxidation. DNA modified with amine group was subsequently immobilized on the SiNW surface. Protein-DNA binding was finally investigated by the functionalized SiNW biosensor. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were employed to characterize the stepwise functionalization of the SAM and DNA on bare silicon surface, and to visualize protein-DNA binding on the SiNW surface, respectively. We observed that ERα had high sequence specificity to the SiNW biosensor which was functionalized with three different EREs including wild-type, mutant and scrambled DNA sequences. We also demonstrate that the specific DNA-functionalized SiNW biosensor was capable of detecting ERα as low as 10 fM. Impressively, the developed SiNW biosensor was able to detect ERα-DNA interactions in nuclear extracts from breast cancer cells. The SAM-assisted SiNW biosensor, as a label-free and highly sensitive tool, shows a potential in studying protein-DNA interactions.

[1]  A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts. , 2008, Analytical biochemistry.

[2]  Zhiqiang Gao,et al.  Highly sensitive sensors for alkali metal ions based on complementary- metal-oxide-semiconductor-compatible silicon nanowires , 2007 .

[3]  I. Ohdomari,et al.  Patterning of DNA nanostructures on silicon surface by electron beam lithography of self-assembled monolayer. , 2004, Chemical communications.

[4]  Dominique Vuillaume,et al.  Nano-field effect transistor with an organic self-assembled monolayer as gate insulator , 1998 .

[5]  Zhiqiang Gao,et al.  Silicon nanowire arrays for label-free detection of DNA. , 2007, Analytical chemistry.

[6]  George M. Whitesides,et al.  Structure and reactivity of alkylsiloxane monolayers formed by reaction of alkyltrichlorosilanes on silicon substrates , 1989 .

[7]  Jihoon Shin,et al.  Formation of Uniform Aminosilane Thin Layers: An Imine Formation To Measure Relative Surface Density of the Amine Group , 1996 .

[8]  Russell Hilf,et al.  The Effects of Estrogen-Responsive Element- and Ligand-Induced Structural Changes on the Recruitment of Cofactors and Transcriptional Responses by ERα and ERβ , 2002 .

[9]  Fred J Sigworth,et al.  Importance of the Debye screening length on nanowire field effect transistor sensors. , 2007, Nano letters.

[10]  C. Klinge,et al.  Footprint analysis of estrogen receptor binding to adjacent estrogen response elements , 1996, The Journal of Steroid Biochemistry and Molecular Biology.

[11]  Mark E. Thompson,et al.  Label-free, electrical detection of the SARS virus N-protein with nanowire biosensors utilizing antibody mimics as capture probes. , 2009, ACS nano.

[12]  Ajay Agarwal,et al.  Label-free direct detection of MiRNAs with silicon nanowire biosensors. , 2009, Biosensors & bioelectronics.

[13]  Martha L Bulyk,et al.  DNA microarray technologies for measuring protein-DNA interactions. , 2006, Current opinion in biotechnology.

[14]  I. Haller.  Covalently attached organic monolayers on semiconductor surfaces , 1978 .

[15]  C. Lieber,et al.  Nanowire Nanosensors for Highly Sensitive and Selective Detection of Biological and Chemical Species , 2001, Science.

[16]  Ajay Agarwal,et al.  Label-free electrical detection of cardiac biomarker with complementary metal-oxide semiconductor-compatible silicon nanowire sensor arrays. , 2009, Analytical chemistry.

[17]  Gengfeng Zheng,et al.  Detection, Stimulation, and Inhibition of Neuronal Signals with High-Density Nanowire Transistor Arrays , 2006, Science.

[18]  Gengfeng Zheng,et al.  Multiplexed electrical detection of cancer markers with nanowire sensor arrays , 2005, Nature Biotechnology.

[19]  Iwao Ohdomari,et al.  Nanoscale patterning of protein using electron beam lithography of organosilane self-assembled monolayers. , 2005, Small.

[20]  Xuema Li,et al.  Sequence-Specific Label-Free DNA Sensors Based on Silicon Nanowires , 2004 .

[21]  N Balasubramanian,et al.  Highly sensitive measurements of PNA-DNA hybridization using oxide-etched silicon nanowire biosensors. , 2008, Biosensors & bioelectronics.

[22]  J. Wood,et al.  Interaction of estrogen receptors α and β with estrogen response elements , 2001, Molecular and Cellular Endocrinology.

[23]  Chin-Yo Lin,et al.  Combinational application of surface plasmon resonance spectroscopy and quartz crystal microbalance for studying nuclear hormone receptor-response element interactions. , 2006, Analytical chemistry.

[24]  Marc Tornow,et al.  Organophosphonate-based PNA-functionalization of silicon nanowires for label-free DNA detection. , 2008, ACS nano.

[25]  N Balasubramanian,et al.  DNA sensing by silicon nanowire: charge layer distance dependence. , 2008, Nano letters.

[26]  Xiaodi Su,et al.  Characterization of protein--DNA interactions using surface plasmon resonance spectroscopy with various assay schemes. , 2007, Biochemistry.

[27]  Guo-Jun Zhang,et al.  Morpholino-functionalized silicon nanowire biosensor for sequence-specific label-free detection of DNA. , 2010, Biosensors & bioelectronics.

[28]  Li Zhang,et al.  Silicon nanowire biosensor for highly sensitive and rapid detection of Dengue virus , 2010 .

[29]  Charles M. Lieber,et al.  Direct ultrasensitive electrical detection of DNA and DNA sequence variations using nanowire nanosensors , 2004 .

[30]  Po-Jen Hsieh,et al.  Label-free detection of protein-protein interactions using a calmodulin-modified nanowire transistor , 2009, Proceedings of the National Academy of Sciences.

[31]  Gengfeng Zheng,et al.  Electrical detection of single viruses. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[32]  James R Heath,et al.  Quantitative real-time measurements of DNA hybridization with alkylated nonoxidized silicon nanowires in electrolyte solution. , 2006, Journal of the American Chemical Society.

[33]  Y. Lyubchenko,et al.  AFM for analysis of structure and dynamics of DNA and protein-DNA complexes. , 2009, Methods.

[34]  Mark A. Reed,et al.  Label-free immunodetection with CMOS-compatible semiconducting nanowires , 2007, Nature.

[35]  E. Kobatake,et al.  On-chip biosensing of estrogen receptor-α at single molecular level , 2004 .