Hybrid Graphene Oxide Based Plasmonic-Magnetic Multifunctional Nanoplatform for Selective Separation and Label-Free Identification of Alzheimer's Disease Biomarkers

Abstract

Despite intense efforts, Alzheimer's disease (AD) is one of the top public health crisis for society even at 21st century. Since presently there is no cure for AD, early diagnosis of possible AD biomarkers is crucial for the society. Driven by the need, the current manuscript reports the development of magnetic core-plasmonic shell nanoparticle attached hybrid graphene oxide based multifunctional nanoplatform which has the capability for highly selective separation of AD biomarkers from whole blood sample, followed by label-free surface enhanced Raman spectroscopy (SERS) identification in femto gram level. Experimental ELISA data show that antibody-conjugated nanoplatform has the capability to capture more than 98% AD biomarkers from the whole blood sample. Reported result shows that nanoplatform can be used for SERS "fingerprint" identification of β-amyloid and tau protein after magnetic separation even at 100 fg/mL level. Experimental results indicate that very high sensitivity achieved is mainly due to the strong plasmon-coupling which generates huge amplified electromagnetic fields at the "hot spot". Experimental results with nontargeted HSA protein, which is one of the most abundant protein components in cerebrospinal fluid (CSF), show that multifunctional nanoplatform based AD biomarkers separation and identification is highly selective.

Keywords: Alzheimer’s disease biomarkers; fingerprint identification of β-amyloid and tau protein; hybrid graphene oxide; plasmonic-magnetic multifunctional nanoplatform; surface enhance Raman spectrosocpy.

Figure 1

(A) High-resolution TEM image using JEM-2100F transmission electron microscope showing the morphology of iron magnetic core–gold plasmonic shell nanoparticles; (inset) high-resolution SEM picture confirming the core–shell morphology of freshly prepared nanoparticles. (B) EDX mapping data of freshly prepared core–shell nanoparticle showing the presence of Fe and Au. (C) High-resolution TEM picture showing the morphology of freshly prepared core–shell nanoparticle attached multifunctional hybrid graphene oxide. (D) High-resolution SEM picture showing the three-dimensional view of hybrid graphene oxide, which clearly shows the formation of core–shell nanoparticle assembly on graphene oxide surface. (E) FTIR spectrum from freshly prepared core–shell nanoparticle attached multifunctional hybrid graphene oxide showing the existence of amide A, I and II bands, as well as –CN band, which indicate the formation of amide bond. The stretches –OH and –C–OH groups due to the graphene oxide also be seen on the FTIR spectra. (F) EDX data of freshly prepared multifunctional hybrid graphene oxide showing the presence of Fe, Au, C, and O. We have also observed Cu and Al peaks in the EDX data, which originate from the support grid. (G) Extinction spectra of core–shell nanoparticle and nanoparticle attached hybrid graphene oxide. Due to the formation of core–shell nanoparticle assembly on graphene oxide surface, the excitation spectra is very broad for hybrid material. (H) Photograph showing that the core–shell nanoparticle attached hybrid graphene oxide is highly magnetic, which allows them to be separated by using a bar magnet.

Figure 2

(A) ELISA results showing β amyloid capture efficiency from infected blood samples using anti-β amyloid antibody attached plasmonic-magnetic hybrid graphene oxide based nanoplatform. Plots also show that separation efficiency is less than 3% in the absence of anti-β amyloid antibody. (B) Spectrum showing SERS intensity from β amyloid conjugated nanoplatform after magnetic separation. Observed SERS signal is directly from the β amyloid. Other than D and G bands, no SERS signal was observed when whole blood without β amyloid was used. (C) Plot showing how SERS amide I band intensity from β amyloid conjugated nanoplatform changes with concentration between 0 and 6 pg/mL. Our experimental data show that the detection efficiency can be as low as 500 fg/mL.

Figure 3

(A) ELISA results showing tau protein capture efficiency from an infected blood sample using anti-tau antibody attached plasmonicmagnetic hybrid graphene oxide based nanoplatform. Plots also show that separation efficiency is less than 4% in the absence of anti-tau antibody. (B) Spectrum showing SERS intensity from tau protein conjugated nanoplatform after magnetic separation. Observed SERS signal is directly from the tau protein. (C) Concentration dependent SERS spectra from tau protein conjugated nanoplatform after magnetic separation. Our experimental data show that the detection efficiency can be as low as 100 fg/mL. (D) Plot showing SERS enhancement of Raman signal from 50 ng tau protein in the presence of core–shell nanoparticle and from 500 ng tau protein in the presence of core–shell nanoparticle attached graphene oxide hybrid.

Figure 4

ELISA results showing selective capturing of tau protein and β amyloid from infected blood sample using anti-tau and anti β amyloid antibody attached nanoplatform. Plots also show that separation efficiency is less than 1% in for HSA.

Scheme 1

(A) Schematic Representation Showing the Synthetic Pathway for the Development of Core-Shell Nanoparticle Attached Hybrid Graphene Oxide Based Multifunctonal Nanoplatform and (B) Schematic Representation Showing Plasmonic-Magnetic Hybrid Graphene Oxide Platform for Label-Free SERS Detection of AD Biomarkers