Enrichment and detection of rare proteins with aptamer-conjugated gold nanorods

Abstract

Rare protein enrichment and sensitive detection hold great potential in biomedical studies and clinical practice. This work describes the use of aptamer-conjugated gold nanorods for the efficient enrichment of rare proteins from buffer solutions and human plasma. Gold nanorod (AuNR) surfaces were modified with a long PEG chain and a 15-mer thrombin aptamer for protein enrichment and detection. Studies of the effect of surface modification on enrichment efficiency of thrombin showed that a change of only one EG(6) linker unit, i.e., from 2EG(6) to 3EG(6), could increase thrombin protein capture efficiency by up to 47%. Furthermore, a 1 ppm sample of thrombin in buffer could be enriched with around 90% efficiency using a low concentration (0.19 nM) of gold nanorod probe modified with 3EG(6) spacer, and with the same probe, effective capture was achieved down to 10 ppb (1 ng) thrombin in plasma samples. In addition to α-thrombin enrichment, prothrombin was also efficiently captured from plasma samples via gold nanorods conjugated with 15-mer thrombin aptamer. Our work demonstrates efficient enrichment of rare proteins using aptamer-modified nanomaterials, which can be used in biomarker discovery studies.

Figure 1

(a) TEM image of the gold nanorods with the dimensions 77.3 ± 5.6 nm and 17.3 ± 1.1 nm; (b) Absorption spectrum of gold nanorods with two absorption maxima at 530 and 860 nm.

Figure 2

(A) Surface modification design of gold nanorods; (B) Scheme of the α-thrombin capturing protocol: 1, 2, 3 represent 0.19, 0.48 and 0.96 nM gold nanorods conjugated with 3EG₆- or 2EG₆-modified thrombin aptamers, respectively.

Figure 3

The fluorescence intensities of (A) 2EG₆-modified aptamers, (B) 3EG₆-modified aptamers, (upper figures) aptamer-conjugated AuNRs, (lower figures) unbound aptamers, while varying the concentrations of SH-PEG (MW 5000) in the surface modification of AuNRs.

Figure 4

Upper figures: Gel electrophoresis of enriched α-thrombin (A) from 338 ng of α-thrombin-spiked activation buffer: lane 1, α-thrombin standard; lanes 2, 3, 4, α-thrombin captured, respectively, via 0.19, 0.48 and 0.96 nM aptamer-2EG₆-AuNRs; lanes 5, 6, 7, α-thrombin captured, respectively, via 0.19, 0.48 and 0.96 nM aptamer-3EG₆-AuNRs; lane 8, α-thrombin captured via 0.48 nM of PEGylated-only gold nanorods and (B) from 100 ng of α-thrombin-spiked activation buffer: lane 1, α-thrombin standard; lanes 2, 3, 4, α-thrombin captured, respectively, via 0.96, 0.48 and 0.19 nM aptamer-2EG₆-AuNRs; lanes 5, 6, 7, α-thrombin captured, respectively, via 0.19, 0.48 and 0.96 nM aptamer-3EG₆-AuNRs; lane 8, α-thrombin captured via 0.48 nM of PEGylated-only gold nanorods. Figures on the right: Pixel intensity peaks of each band drawn by ImageJ software. Lower Figures: Relative band intensities calculated by ImageJ software for the captured α-thrombin compared to α-thrombin standards to demonstrate the thrombin capture percentage.

Figure 5

Left Figure: Gel electrophoresis of enriched α-thrombin from 1 ng α-thrombin-spiked human plasma: lane 1, α-thrombin standard; lanes 2, 3, 4, α-thrombin captured, respectively, via 0.19, 0.48 and 0.96 nM aptamer-2EG₆-AuNRs; lanes 5, 6, 7, α-thrombin captured, respectively, via 0.19, 0.48 and 0.96 nM aptamer-3EG₆-AuNRs. Right Figure: Pixel intensity peaks of each lane drawn by ImageJ software; blue dashed lines show the upper bands (72 kDa), and red dashed lines show the lower bands (37kDa).