4.5 Nanoparticles for Diagnosis of AD
4.5.3 Inorganic NPs for AD Diagnosis
immobilized on the ML surface for amyloid affi nity (A. Skouras et al, unpublished results). Ongoing in vitro and in vivo studies are currently carried out in order to demonstrate the potential of such multifunctional MLs to target Aβ plaques in the brain.
biofunctionalized with antibodies against Aβ-40 and Aβ-42 demonstrated that immunomagnetic reduction signals of Aβ-40 and Aβ-42 in plasma from normal humans and AD patients showed signifi cant diff erences.
Such results may fi nd future applications in AD diagnosis [271].
Gold NPs: Gold nanoparticles (AuNPs) have extraordinary optical, electronic, and molecular-recognition properties. Electronic microscopy is one of the areas where gold nanoparticles have been extensively used as contrast agents. Th ey can be associated with many traditional biologi- cal probes such as antibodies, lectins, superantigens, glycans, nucleic acids and receptors. Because gold particles have various sizes they can be easily spotted in electron micrographs, while it is possible for multiple experi- ments to be conducted simultaneously. Th ese advantages make AuNPs one of the main NPs used in studies for AD diagnosis, mainly for sensitive measurement of Aβ concentrations. Some examples of recently proposed techniques follow. Recently, a research group developed an ultrasensitive electrical detection method for Aβ1–42 using scanning tunneling micros- copy (STM) [297]. For this, a monoclonal antibody (mAb) fragment with high affi nity for Aβ1–42 was immobilized onto a gold surface and the sample was deposited onto the mAb-Functionalized surface, leading to its capture. Subsequently, mAb-Au NP complex was reacted and resulted in the formation of “sandwich-like” structures. Th e resulting chip was fi nally analyzed by STM. It was shown that the surface density of the Au NPs correlated with the number of Aβ-Antigen binding events and that a suc- cessful Aβ detection was achievable at a concentration 10 fg/mL. Another interesting procedure for Aβ detection was based on electrochemical sens- ing of saccharide-protein interactions [295]. Th e densely packed sialic acid domains were able to capture the Aβ peptides as a result of specifi c interac- tions, and the method enabled the detection of non-labeled Aβ down to sub-micromolar concentrations. Remarkable results towards the develop- ment of new approaches for biomarker detection have been proposed by Georganopoulou et al., who developed an ultrasensitive NPs-based bio- barcode assay capable of detecting AD soluble biomarkers in CSF [275].
Th e key feature of the system relies on the isolation of antigens by means of a “sandwich process” involving oligonucleotide (DNA-barcode) modi- fi ed Au NPs, and magnetic microparticles (MMPs), both functionalized with antibodies specifi c to the ADDLs. Practically, an excess of Au NPs and MMPs (when compared to the ADDLs concentration) are mixed in a CSF sample; the recognition of the antigen from both particles leads to the formation of sandwiches that are then purifi ed by magnetic separation.
Th e strands of a dehybridized double-stranded DNA are isolated and easily quantifi ed by a scanometric method using DNA microarray. Th e effi cient
antigen sequestration in solution and the amplifi cation process resulting from the large number of DNA strands released for each antigen recogni- tion, allowed the system to identify ADDLs at sub-femtomolar concentra- tions, thus improving the ELISA test sensitivity by 6 orders of magnitude.
Neely et al. designed Au NPs coated with mAb specifi c to τ protein and employed the NPs in a two-photon Rayleigh scattering assay, which enabled the detection of τ protein at concentrations greater than about 1 pg.mL-1.
Th is concentration was about 2 orders of magnitude lower than typical τ protein concentration values (i.e., 195 pg.mL-1) in CSF. Moreover, the two- photon Rayleigh scattering assay showed a strong sensitivity for τ protein and was able to discriminate other proteins such as bovine serum albumin [276]. El-Said et al. developed a method to detect Aβ plaques using sur- face-enhanced Raman scattering. Briefl y gold nanoparticles (Au NPs) were electrochemically deposited on an indium tin oxide (ITO) substrate [273].
Aβ antibodies were immobilized on the Au-NP-coated ITO substrate, aft er which the interactions between the antigen and the antibody were determined via SERS spectroscopy. Th e SERS responses had a good linear relationship that corresponded to the change in the concentration of the antigen with high sensitivity (100 fg/ml). Recently, Sakono et al. reported a simple method to detect Aβ aggregates. Au NPs modifi ed with the Aβ antibody were treated with bovine serum albumin to stabilize their dis- persibility in buff er [307]. Aft er adding appropriate concentrations of Aβ a red-coated precipitate could be observed by naked eye. Th e precipitate is only observed when oligomers or fi brils are added, but not in the presence of monomers. Another group fabricated a dense Au nano dot array (ca. 60 nm) on indium tin oxide (ITO). Aβ1–42 antibody is allowed to immobilize on the Au dots followed by its target protein and Au NP-antibody complex is prepared and then applied to preimmobilized protein arrays to get the pulse like current peak under STM. STM derived profi les show a logarith- mic increase of the current with an increase of the Aβ1–42 concentration, successfully detecting concentrations as low as 100 fg/mL (23 fM) [272].
Quantum Dots (QD): A nanoprobe for amyloid-β aggregation and oligomerization using PEG coated QDs as Aβ42 labels, was recently devel- oped [295]. Th e oligomerization behavior of Aβ42 in solution and on intact cells was compared, as well as the ingestion manner of microglia for Aβ42 monomers and oligomers. In order to use this diagnostic technology to monitor Aβ42 biochemical behavior in vivo, in addition to QD safety considerations, special attention should be paid to the successful passage of QD-Aβ nanoprobes through the BBB. QDs conjugated with transferrin were recently reported to be able to successfully transmigrate through an in vitro model of the BBB. Another interesting approach [308] utilized a novel
fl uorogenic nanoprobe prepared from the assembly of CdSe/ZnS QDs and gold (Au) NPs in which QD was conjugated with a specifi cally designed β-secretase (BACE1) substrate peptide. Th is coordination-mediated bind- ing of the QD with Au nanoparticles via Ni-NTA-histidine (His) interac- tion resulted in highly effi cient quenching of QD fl uorescence through a distance-dependent fl uorescence resonance energy transfer (FRET) phe- nomenon. Th e prequenched QD-Au assembly recovered the fl uorescence in the presence of the BACE1 enzyme aft er incubation in vitro. Th e high quenching effi ciency of AuNP and robust QD fl uorescence signal recov- ery upon BACE1 enzymatic digestion enabled the visualization of BACE1 activity in living cells. Th ese results show the potential application of QD-AuNP nanoparticles as an effi cient probe to identify active molecules in BACE1-related diseases such as AD. Zhang et al. reported the labeling of Aβ with QDs (QDs-Aβ) by a simple mixing-incubation strategy through which Aβ became wrapped on the surface of QDs. Such QDs inhibited the formation of β-folding, and the fi brillation of Aβ [309]. Th e QDs-Aβ retained dispersivity and fl uorescence properties. When coincubated with astrocytes, the QDs-Aβ were endocytosed, without separation of QDs and Aβ; Aβ was degraded in 24 h, indicating that labeling with QDs did not aff ect the uptake and degradation of Aβ by astrocytes, providing a possible new method for visualization of the the Aβ elimination process.