Neutral polymers as coatings for high resolution electrophoretic separation of Aß peptides on glass microchips.

This study reports a comparison of the performances of two neutral polymers, poly ethylene-oxide (PEO) and poly(dimethylacrylamide-co-allyl glycidyl ether) (EpDMA), in glass microchips to achieve zone electrophoresis separation of several truncated forms of beta amyloid (Aß) peptides, sharing very similar structures. The peptides were derivatized by FluoProbes 488 NHS to allow their fluorescence detection. Two protocols based either on PEO or EpDMA led to good pH stabilities in addition to a significant reduction of the electroosmotic flow. These two polymer coatings allowed repeatable analyses and high resolution for the simultaneous analysis of three Aß peptides, Aß 1-38, Aß 1-40 and Aß 1-42, considered as potential biomarkers of Alzheimer's disease. A recovery study showed that EpDMA was superior in reducing the adsorption of the Aß peptides on the coated inner wall. Finally, the separation method relying on the EpDMA coated microchips was validated as linear using a calibration curve and the LOD was estimated to be close to 200 nM. Despite very short migration distances, different N-terminal or C-terminal truncated Aß peptides, corresponding to promising biomarker combinations for the future diagnostic, were fully resolved. The method was successfully applied to detect these peptides in spiked cerebrospinal fluid and has provided a first achievement towards the development of a microsystem that would integrate preconcentration and separation steps.

Analysis of amyloid-ß peptides in cerebrospinal fluid samples by capillary electrophoresis coupled with LIF detection.

We report a CE-LIF method for the separation and detection of five synthetic amyloid-ß peptides corresponding to an important family of CSF-biomarkers in the context of Alzheimer disease (AD). The presumed most relevant peptides (Aß1-42, Aß1-40, and Aß1-38) that may support the differentiation between AD and healthy patients or other dementias were successfully detected in CSF by incorporating an immunoconcentration step prior to CE analysis of derivatized peptides. We labeled the Aß peptides with a fluoroprobe dye before CE-LIF analysis. This reagent reacts with the amino groups of lysine residues and produced mostly ditagged Aß peptides under the proposed experimental conditions. The labeling reaction displayed similar efficiency with each one of the five different synthetic Aß peptides that were tested. The limit of detection of the CE-LIF method approached 280 attomoles of injected synthetic labeled Aß peptides. We obtained excellent correlation between peak areas and peptide concentrations from 35 nM to 750 nM. For the detection of Aß peptides in human CSF samples, we enriched the peptides by immunoprecipitation prior to the CE-LIF analysis. The comparison of the CE-LIF profiles obtained from CSF samples from 3 AD patients and 4 non-demented control subjects indicated noticeable differences, suggesting that this method, which relies on a multibiomarker approach, may have potential as a clinical diagnostic test for AD.

New frontiers in Alzheimer's disease diagnostic: Monoamines and their derivatives in biological fluids.

Current diagnosis of Alzheimer's disease (AD) relies on a combination of neuropsychological evaluations, biomarker measurements and brain imaging. Nevertheless, these approaches are either expensive, invasive or lack sensitivity to early AD stages. The main challenge of ongoing research is therefore to identify early non-invasive biomarkers to diagnose AD at preclinical stage. Accumulating evidence support the hypothesis that initial degeneration of profound monoaminergic nuclei may trigger a transneuronal spread of AD pathology towards hippocampus and cortex. These studies aroused great interest on monoamines, i.e. noradrenaline (NA), dopamine (D) ad serotonin (5-HT), as early hallmarks of AD pathology. The present work reviews current literature on the potential role of monoamines and related metabolites as biomarkers of AD. First, morphological changes in the monoaminergic systems during AD are briefly described. Second, we focus on concentration changes of these molecules and their derivatives in biological fluids, including cerebrospinal fluid, obtained by lumbar puncture, and blood or urine, sampled via less invasive procedures. Starting from initial observations, we then discuss recent insights on metabolomics-based analysis, highlighting the promising clinical utility of monoamines for the identification of a molecular AD signature, aimed at improving early diagnosis and discrimination from other dementia.

Microchip electrophoresis profiling of Aß peptides in the cerebrospinal fluid of patients with Alzheimer's disease.

The preferential aggregation of Aß1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This is accompanied by a relative reduction of the concentration of Aß1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer's disease. Here, we describe a microchip gel electrophoresis method in polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aß peptides in cerebrospinal fluid. To control the electroosmotic flow (EOF) in the PDMS channel and also to reduce the adsorption of the peptides to the surface of the channel, a new double coating using poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) and methylcellulose-Tween-20 was developed. With this method, separation of five synthetic Aß peptides (Aß1-37, Aß1-38, Aß1-39, Aß1-40, and Aß1-42) was achieved, and relative abundance of Aß1-42 to Aß1-37 could be calculated in different standard mixtures. We applied our method for profiling of Aß peptides in CSF samples from nonAlzheimer patients and patients with Alzheimer's disease. Aß peptides in the CSF samples were captured and concentrated using a microfluidic system in which magnetic beads coated with anti-Aß were self-organized into an affinity microcolumn under the a permanent magnetic field. Finally, we could detect two Aß peptides (Aß1-40 and Aß1-42) in the CSF samples.

New method based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) to monitor interaction between nanoparticles and the amyloid-ß peptide.

A novel application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was proposed to efficiently detect and monitor the interaction between polymeric nanoparticles and the ß-Amyloid peptide (Aß(1-42)), a biomarker for Alzheimer's Disease (AD), at concentrations close to physiological conditions. The CE-LIF method allowed the interaction between PEGylated poly(alkyl cyanoacrylate) nanoparticles (NPs) and the soluble Aß(1-42) peptide monomers to be highlighted. These results were confirmed by surface plasmon resonance (SPR) and confocal laser scanning microscopy (CLSM). Whereas SPR showed an interaction between the NPs and the Aß(1-42) peptide, CLSM allowed the formation of large aggregates/assemblies at high NP and peptide concentrations to be visualized. All these results suggested that these nanoparticles could bind the Aß(1-42) peptide and influence its aggregation kinetics. Interestingly, the non-PEGylated poly(alkyl cyanoacrylate) NPs did not alter the aggregation kinetics of the Aß(1-42) peptide, thus emphasizing the high level of discrimination of the CE-LIF method with respect to NPs.

Correction: Correlation between cognition and plasma noradrenaline level in Alzheimer's disease: a potential new blood marker of disease evolution.

A Correction to this paper has been published: https://doi.org/10.1038/s41398-020-01102-y.

Correlation between cognition and plasma noradrenaline level in Alzheimer's disease: a potential new blood marker of disease evolution.

Recent evidence showing degeneration of the noradrenergic system in the locus coeruleus (LC) in Alzheimer's disease (AD) has motivated great interest in noradrenaline (NA) as a potential brain hallmark of the disease. Despite the current exploration of blood markers for AD, the deregulation of the plasma NA concentration ([NA]plasma) in AD is currently not well understood. This retrospective study includes a cohort of 71 patients (32 AD patients, 22 with other dementia and 17 without dementia) who were given consultations for memory complaints in the Cognitive Neurology Center of Lariboisière (Paris) between 2009 and 2014. As previously described in brain tissue, we show for the first time a linear correlation between [NA]plasma and Mini Mental State Examination (MMSE) score in AD patients. We observed that high [NA]plasma in AD patients was associated with higher [Aß1-42]CSF than in other AD patients with [NA]plasma similar to NC patients. In parallel, we observed a lower (p-Tau/Tau)CSF in AD patients with low [NA]plasma than in non-AD patients with [NA]plasma similar to [NA]plasma in NC patients. Our data suggest that [NA]plasma could be a potential biomarker of disease evolution in the context of AD and could possibly improve early diagnosis.

"Microchip Electrophoresis," with Respect to "Profiling of Aß Peptides in the Cerebrospinal Fluid of Patients with Alzheimer's Disease".

Aggregation of beta-amyloid peptides especially Aß1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This event is normally accompanied by a relative reduction of the concentration of Aß1-42 in the cerebrospinal fluid (CSF) of patient developing the signs of Alzheimer's disease. Here, we describe methods for isolation and for microchip gel electrophoresis of Aß peptides in polydimethylsiloxane (PDMS) microfluidic chip. The method was applied to compare the relative concentration of Aß1-42 with other Aß peptides, for example, Aß 1-40 in CSF. In order to increase the sensitivity of detection, Aß peptides in the CSF samples were first captured and concentrated using magnetic beads coated with specific anti-Aß antibodies.

Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer's disease.

We report here a CE method for the separation and quantitation of five amyloid peptides (Abeta1-42, 1-40, 1-39, 1-38, and 1-37) considered as potential biomarkers of Alzheimer's disease. These amyloid peptides have very similar structures. Sample preparation and storage conditions are critical parameters to ensure their solubility and to avoid the aggregation process in particular for Abeta1-42. Their solubility was found fully dependent on the NH(4)OH concentration that was employed initially to dissolve the lyophilized amyloid peptides. Conditions to achieve a full separation of these peptides were found using a dynamic coating with 1,4-diaminobutane (DAB). The linear decrease of their electrophoretic mobility highlighted an ion-pairing phenomenon between the peptides and DAB. The optimal background electrolyte was a 40 mM borate buffer, pH 9 containing 3 mM of DAB. Under these conditions, resolutions ranged from 1.3 to 2.4 with theoretical plates reaching 300,000. Under the retained conditions, we showed that adsorption of peptides to silica was negligible (recovery over 94.5%) and depletion effect of the background electrolyte was overcome. The method was finally validated in terms of linearity and repeatability and the limits of detection for the five Abeta peptides were estimated. The inter-day repeatability of the migration times was very satisfactory with RSDs less than 1.55%. The RSDs of the peak areas were below 5%. With this CE-UV method, limits of detection of the peptides ranged from 300 to 500 nM. We finally demonstrated that this method can be applied to real biological samples such as CSF.

Contribution of CE to the analysis of protein or peptide biomarkers.

Biomarker analysis is pivotal for disease diagnosis and one important class of biomarkers is constituted by proteins and peptides. This review focuses on protein and peptide analyses from biological fluids performed by capillary electrophoresis. The various strategies that have been reported to prevent difficulties due to the handling of real samples are described. Innovative techniques to overcome the complexity of the sample, to prevent the adsorption of the analytes on the inner capillary wall, and to increase the sensibility of the analysis are summarized and illustrated by different applications. To fully illustrate the contribution of CE to the analysis of biomarkers from human sample, two detailed protocols are given: the analysis from CSF of five amyloid peptide, biomarkers of the Alzheimer disease, and the analysis of sialoforms of transferrin from human serum.

Microchip electrophoresis, with respect to "profiling of Aß peptides in the cerebrospinal fluid of patients with Alzheimer's disease".

Aggregation of beta amyloid peptides especially Aß1-42 in amyloid plaques is one of the major -neuropathological events in Alzheimer's disease. This event is normally accompanied by a relative reduction of the concentration of Aß1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer's disease. Here, we describe a microchip gel electrophoresis method in a polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aß peptides. The method was applied to compare the relative concentration of Aß1-42 with other Aß peptides, for example, Aß 1-40 in CSF. In order to increase the sensitivity of detection, Aß peptides in the CSF samples were first captured and concentrated using magnetic beads coated with specific anti-Aß antibodies.

Development of a magnetic immunosorbent for on-chip preconcentration of amyloid ß isoforms: Representatives of Alzheimer's disease biomarkers.

Determination of amyloid ß (Aß) isoforms and in particular the proportion of the Aß 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer's disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aß peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (µIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aß antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aß antibodies, as well as optimizing the immobilization technique and µIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aß IS for µIP and specific capture of 5 Aß peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aß peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aß peptides in human CSF sample was performed.

PEGylated nanoparticles bind to and alter amyloid-beta peptide conformation: toward engineering of functional nanomedicines for Alzheimer's disease.

We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aß(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the interaction with monomeric and soluble oligomeric forms of Aß(1-42) peptide was demonstrated by capillary electrophoresis, surface plasmon resonance, thioflavin T assay, and confocal microscopy, where the binding affected peptide aggregation kinetics. The capture of peptide by NPs in serum was also evidenced by fluorescence spectroscopy and ELISA. Moreover, in silico and modeling experiments highlighted the mode of PEG interaction with the Aß(1-42) peptide and its conformational changes at the nanoparticle surface. Finally, Aß(1-42) peptide binding to NPs affected neither complement activation in serum nor apolipoprotein-E (Apo-E) adsorption from the serum. These observations have crucial implications in NP safety and clearance kinetics from the blood. Apo-E deposition is of prime importance since it can also interact with the Aß(1-42) peptide and increase the affinity of NPs for the peptide in the blood. Collectively, our results suggest that these engineered long-circulating NPs may have the ability to capture the toxic forms of the Aß(1-42) peptide from the systemic circulation and potentially improve Alzheimer's disease condition through the proposed "sink effect".

Selegiline-functionalized, PEGylated poly(alkyl cyanoacrylate) nanoparticles: Investigation of interaction with amyloid-ß peptide and surface reorganization.

Alzheimer's disease (AD) is a neurodegenerative disorder for which the research of new treatments is highly challenging. Since the fibrillogenesis of amyloid-ß peptide 1-42 (Aß(1-42)) peptide is considered as a major cause of neuronal degeneration, specific interest has been focused on aromatic molecules for targeting this peptide. In this paper, the synthesis of selegiline-functionalized and fluorescent poly(alkyl cyanoacrylate) nanoparticles (NPs) and their evaluation for the targeting of the Aß(1-42) peptide are reported. The synthetic strategy relied on the design of amphiphilic copolymers by tandem Knoevenagel-Michael addition of cyanoacetate derivatives, followed by their self-assembly in aqueous solutions to give the corresponding NPs. Different cyanoacetates were used: (i) hexadecyl cyanoacetate (HDCA) to form the hydrophobic core of the NPs; (ii) rhodamine B cyanoacetate (RCA) for fluorescent purposes; (iii) methoxypoly(ethylene glycol) cyanoacetate (MePEGCA) for stealth properties and (iv) selegiline-poly(ethylene glycol) cyanoacetate (SelPEGCA) to obtain the desired functionality. Two different amphiphilic copolymers were synthesized, a selegiline-containing copolymer, P(MePEGCA-co-SelPEGCA-co-HDCA), and a rhodamine-labelled counterpart, P(MePEGCA-co-RCA-co-HDCA), further blended at variable ratios to tune the amount of selegiline moieties displayed at the surface of the NPs. Optimal formulations involving the different amphiphilic copolymers were determined by the study of the NP colloidal characteristics. Interestingly, it was shown that the zeta potential value of the selegiline-functionalized nanoparticles dramatically decreased, thus emphasizing a significant modification in the surface charge of the nanoparticles. Capillary electrophoresis has then been used to test the ability of the selegiline-functionalized NPs to interact with the Aß(1-42) peptide. In comparison with non functionalized NPs, no increase of the interaction between these functionalized NPs and the monomeric form of the Aß(1-42) peptide was observed, thus highlighting the lack of availability of the ligand at the surface of the nanoparticles. A mechanism explaining this result has been proposed and was mainly based on the burial of the hydrophobic selegiline ligand within the nanoparticles core.

Colloidal properties of biodegradable nanoparticles influence interaction with amyloid-ß peptide.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the extracellular deposition of amyloid-ß peptides (Aß). During the past few years, promising approaches based on nanotechnologies have emerged to alter Aß aggregation and its related toxicity. This study aims to investigate the influence of the nanoparticle colloidal properties over the interaction with Aß peptide 1-42 (Aß(1-42)). Using capillary electrophoresis with laser-induced fluorescence detection, it was shown that biodegradable poly(ethylene glycol)-block-polylactide (PEG-b-PLA) nanoparticles were able to interact with Aß(1-42) peptide leading to its uptake in rather short time periods. In addition, we highlighted the crucial role of the nanocarrier colloidal properties on the uptake kinetics. Whereas nanoparticles stabilized by sodium cholate (lower size and higher negative surface charge) gave optimum uptake kinetics, nanoparticles stabilized with others surfactants presented lower interactions. In contrast, PEG density seemed to have no influence on the interaction when sodium cholate was used for the preparation. This study intends to give new insights into Aß(1-42) peptide interaction with nanoparticulate systems by helping to determine suitable nanoparticle characteristics regarding forthcoming therapeutic strategies against AD.