Multiplex detection of breast cancer biomarkers using plasmonic molecular sentinel nanoprobes

Abstract

We have demonstrated for the first time the feasibility of multiplex detection using the surface-enhanced Raman scattering-based molecular sentinel (MS) technology in a homogeneous solution. Two MS nanoprobes tagged with different Raman labels were used to detect the presence of the erbB-2 and ki-67 breast cancer biomarkers. The multiplexing capability of the MS technique was demonstrated by mixing the two MS nanoprobes and tested in the presence of single or multiple DNA targets.

Figure 1

The operating principle of the SERS-based molecular sentinel (MS) nanoprobes. The MS nanoprobe is composed of a Raman-labeled DNA hairpin probe and a silver nanoparticle. In the absence of the complementary target DNA, a strong SERS signal is observed due to the hairpin conformation adopted by the MS nanoprobe (left: closed state). In the presence of the complementary target DNA, the hairpin conformation of the MS nanoprobe is disrupted and the SERS signal is quenched due to the physical separation of the Raman label from the surface of the silver nanoparticle (right: open state).

Figure 2

SERS spectra of the ERBB2-MS (a) and KI-67-MS (b) nanoprobes in 20 mM tris-HCl buffer (pH 8.0) containing 5 mM MgCl₂. The major Raman bands are labeled as follows: ERBB2-MS: (1) 1197 cm⁻¹, (2) 1393 cm⁻¹, (3) 1468 cm⁻¹, (4) 1590 cm⁻¹. KI-67-MS: (1*) 1218 cm⁻¹, (2*) 1354 cm⁻¹, (3*) 1508 cm⁻¹, (4*) 1535 cm⁻¹, (5*) 1650 cm⁻¹.

Figure 3

The effect of MgCl₂ concentration on the SERS intensity of one of the major Raman bands (peak #2: 1393 cm⁻¹) from the ERBB2-MS nanoprobes (a) and (peak #2*: 1354 cm⁻¹) from the KI-67-MS nanoprobes (b). (●): 5 mM, (▵): 4 mM, (+): 3 mM, (−): 0 mM.

Figure 4

SERS spectra of the ERBB2-MS nanoprobes in the presence or absence of complementary DNA targets. Upper spectrum: blank (no target DNA present). Middle spectrum: in the presence of 0.5 μM non-complementary DNA (negative control). Lower spectrum: in the presence of 0.5 μM complementary target DNA (positive diagnostic).

Figure 5

SERS spectra of the KI-67-MS nanoprobes in the presence or absence of complementary DNA targets. Upper spectrum: blank (no target DNA present). Middle spectrum: in the presence of 0.5 μM non-complementary DNA (negative control). Lower spectrum: in the presence of 0.5 μM complementary target DNA (positive diagnostic).

Figure 6

SERS spectra of the composite MS nanoprobes (ERBB2-MS + KI-67-MS) in the presence or absence of target DNA. The major Raman bands from ERBB2-MS are marked with black number, and the major Raman bands from KI-67-MS are marked with red number with (*) sign. Upper spectrum: blank (in the absence of any target DNA). Lower spectrum: in the presence of two target DNA complementary to both MS nanoprobes.

Figure 7

SERS spectra of the composite MS nanoprobes (ERBB2-MS + KI-67-MS) in the presence or absence of single target DNA. The major Raman bands from ERBB2-MS are marked with black number, and the major Raman bands from KI-67-MS are marked with red number with (*) sign. Upper spectrum: blank (in the absence of any target DNA). Middle spectrum: in the presence of single target DNA complementary to the ERBB2-MS nanoprobes. Lower spectrum: in the presence of single target DNA complementary to the KI-67-MS nanoprobes. The arrow signs illustrate the decreased SERS intensity of the major Raman bands in the presence of corresponding target DNA.