Surface enhanced Raman spectroscopy for multiplex detection and quantification of BRCA1 splice variants

Lan Sun, Purdue University

Abstract

Alternative splicing is a crucial mechanism for generating protein diversity. Breast cancer susceptibility gene 1 (BRCA1) has displayed cell- and tissue-specific alternative splicing patterns. It has been suggested that BRCA1 alternative splicing could be used as a potential cancer marker. Currently available techniques for alternative splicing profiling are limited in multiplexing capability, sensitivity and quantification ability. In this research a surface enhanced Raman scattering (SERS) based platform was designed and tested to perform multiplex and quantitative detection of BRCA1 splice variants capitalizing on the strong multiplexing capability and excellent sensitivity of SERS technology. A novel DNA probe utilizing nonfluorescent Raman tags and gold nanoparticles was developed with strong multiplexing capability demonstrated by simultaneous differentiation of eight different probes in a mixture. Good long-term stability was obtained. Then in a proof-of-concept study, these probes were applied in an array-format for multiplex detection of DNA sequences specific to BRCA1 splice variants. Four-plex detection was realized with excellent specificity. Detection sensitivity of 1 fM was demonstrated. Commonly, SERS technology could only be utilized for qualitative or semi-quantitative detection of biological materials due to the heterogeneous surface enhancement. Tremendous efforts have been taken to generate reproducible and tunable SERS substrates using advanced fabrication technologies. However, control of surface enhancement remains a challenge. A plasmon-phonon band representing surface features of metallic nanostructures was explored in this research and a novel methodology was proposed to utilize this Raman band as a self-referencing standard for quantification. This approach circumvents the need to produce SERS substrates that can enhance the Raman signal with a high degree of uniformity so that quantification of biomolecules is possible. A strategy integrating S1 nuclease digestion with SERS detection was developed to quantify BRCA1 splice variants directly from cancer cells (MCF-7 and MDA-MB-231). The SERS quantification protocol not requiring any amplification step was cross tested by two different nonfluorescent Raman labels and validated by reverse transcription polymerase chain reaction (RT-PCR). This strategy was also demonstrated for the duplex detection of BRCA1 splice variants Δ(9, 10) and Δ(5). The variants can be quantified effectively in presence of other splice variants.

Degree

Ph.D.

Advisors

Irudayaraj, Purdue University.

Subject Area

Molecular biology|Analytical chemistry|Biomedical engineering

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