Electrochemical (Bio) Sensing of Amyloid Beta Peptide (Aβ) using Metallic Nanoparticles

Amyloid-β (Aβ) peptides, the dominant factor of the aged plaques in brain of Alzheimer’s disease (AD) patient, have been considered as the main biomarkers and curative targets for the diagnosis and prognosis of AD. The aggregation of Aβ is concluded to be a precarious point in the pathogenesis of AD. Latterly, electrochemical systems have been strongly employed to selectively detect sorts of Aβ moieties and monitor the oligomerization and assembly of Aβ due to their high sensitivity, simplicity, brisk response, and compatibility with miniaturization. Hence, in this minireview, we outlined the advance in electrochemical (bio)sensing capped with metallic nanoparticles of Aβ peptides species. Abbreviations: long-term Potentiation; CSF: Cerebro- Spinal Fluid; MMSE: Mini-Mental State Examination; ER: Endoplasmic Reticulum; AICD: APP Intracellular Realm; GNPs: Gold Nanoparticles; AAO: Anodic Aluminum Oxide; SPCE: Screen Printed Carbon Electrode; CNTs: Copper Nanoparticles; SWV: Square Wave Voltammetry; ACSF: Artificial Cerebrospinal Fluid; EIS: Electrolyte–Insulator–Semiconductor; FETs: Field-Effect Transistors;

external motivator to control the size of NPs, which would provide sufficient energy to get the particle of the required size. In addition, sunlight is nontoxic, nonpolluting and simple in chemical processes [16]. In comparison to stabilizers such as plant leaves [17], fruit extracts [18], plant roots [19], glucose [20] and carbonate materials [21] used in the NPs composition, organic and natural dyes [14,[22][23][24][25][26] have specific ionic, polar, non-bond functional groups (-azo dyes, -sulfites, -hydroxyl and -nitro groups) and are usually systems that have π -conjugates [27] capable of being polymerized. Amyloid beta peptide (Aβ) is a natural product of low abundance in the healthy brain and is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by β-and γ-secretases [28]. Aβ accumulation in the brain is proposed to be an early toxic event in the pathogenesis of Alzheimer's disease (AD) [29], which is the most common form of dementia associated with plaques and tangles in the brain. Currently, it is unclear what the physiological and pathological forms of Aβ are and by what mechanism Aβ causes dementia.
Meanwhile, there is currently no cure for AD and no reliable method of diagnosis other than post-mortem brain examination.
Therefore, the development of appropriate detection methods for AD s an urgent requirement, in order to provide earlier diagnosis and, thus, useful therapeutic intervention. As a severe neurological disorder, Alzheimer's disease (AD) leads to progressive memory loss and cognitive impairment [30]. Although many details associated with AD pathology are still unknown, the deposition of extracellular beta-amyloid (Aβ) peptides residues was regarded as one of the main hallmarks in the brain [29]. Aβ is a natural product of low abundance in the healthy brain, where it is cleaved out of the neuronally-expressed amyloid precursor protein (APP) by sequential action of beta and gamma secretase enzymes [31]. After the cleavage of the APP induced by β-and γ-secretases, Aβ forms with a largely hydrophilic N-terminal domain and a C-terminal hydrophobic domain [32]. Of these Aβ species, Aβ1-40 and Aβ1-42 composed 40 and 42 amino acid residues dominate. In contrast to Aβ1-40, the amount of Aβ1-42 decreases significantly but it displays more neurotoxic resulting from the presence of the two additional hydrophobic amino acids [33]. Thus, the low-level Aβ1-42 is always considered as a promising biomarker for AD diagnosis. Aβ1-42 monomers tend to aggregate into the oligomers or fibrils under different external stimuli including oxidation stress and metal ions.
The aggregated ones of Aβ1-42, especially Aβ1-42 oligomers, are believed as major toxic effects in AD which could be considered as promising biomarkers or aggregation inhibitor-based drug targets for AD therapy [34]. Of the monomeric, oligomeric and fibrillar forms of Aβ, a plethora of evidence now indicates that soluble Aβ oligomers (AβO) are the major neurotoxic species in AD. AβO bind to neurons, particularly at the post-synaptic membrane, causing synaptic dysfunction, blocking key processes which under-lie memory and learning (e.g. long-term potentiation; LTP) and causing neurotoxicity and cell death at low concentrations [35].
The level of AβOs in the brain, particularly those with a "fibrillar" conformation, correlate with AD onset and severity much more strongly than the insoluble fibrillar load [36]. Importantly, AβO have been detected in animal models of AD before the phenotypic presentation of disease [37]. The development of more sensitive diagnostic tests to detect very low levels of biologically relevant AβO could ultimately lead to clinically useful tests for pre-symptomatic diagnosis and monitoring of AD progression, either before or during disease onset and throughout therapeutic intervention. Measuring AβO in a patient, reflects that the AβO count in patient fluids (i.e. blood and cerebro-spinal fluid; CSF) reflects the most direct and relevant biomarker for AD [38]. Levels of AβO in plasma and CSF samples have been shown, by ELISA and flow cytometry, to be elevated in AD and correlate with Mini-Mental State Examination (MMSE) scores [39]. However, many ELISA-type assays have not been able to detect AβO in CSF, although AβO were detected in AD brain samples [40]. Crucially, it was shown that levels of AβO in serum correlate directly with the CSF AβO load, demonstrating that a blood test type assay for AβO is relevant to the AβO content in CSF [41].
Measuring AβO levels in patient fluids by conventional laboratory techniques such as ELISA-type assays is time consuming and expensive. Problems related to the diagnosis and monitoring of AD underscore the need of developing new diagnostic methods using easier-to-use, low-sample volume approaches. These requirements are met by electrochemical methods, which are a reliable and profitable appliance for the determination of Aβ, in order to facilitate their use in the diagnosis and monitoring of this disease, due to their ease of use, simplicity, selectivity, sensibility and low cost [42,43]. As it can be seen by the lack of literature, electrochemical sensors have been rarely used in the detection of Aβ peptide and AD diagnostics. Therefore, electrochemical (bio) sensors capped with metallic nanoparticles for the determination of Aβ are summarized in this short review.

Alzheimer's Disease (AD)
Alzheimer's disease is outlined by the irregular accumulation of Aβ protein, which is crucial for recollection and perception, in the brain sectors. Aβ is a regular product of the cellular metabolism obtained from the amyloid precursor protein (APP). APP is manufactured in the endoplasmic reticulum (ER) and then carried to the Golgi complex, where it matures and is finally transferred to the plasma membrane. Mature APP at the plasma membrane is cleaved by the consecutive activity of the β-secretase and γ-secretase to produce Aβ [44]. The freshly developed Aβ either is delivered to the extracellular space or lingers correlated with the plasma membrane and lipid raft networks. The wind up of Aβ to ganglioside GM1 in the lipid rafts heavily promotes Aβ aggregation [45]. The binding of ApoE to Aβ is taken up by the cells through receptor-mediated endocytosis arbitrated by LRP (LDL receptorrelated protein), and LDLR controls aggregation but further the cellular uptake of Aβ [46]. Endocytosed Aβ still has entry to more subcellular containers through the vesicular transport system. Previous investigations led to Aβ fibrils as the neurotoxic promoter forcing cellular downfall, recollection failure, and additional AD attributes [47]. Over the last two decades, farther research has recommended that oligomeric or prefibrillar species of the Aβ peptide are the most harmful to neuronal cells. Soluble Aβ can tie up to numerous particles in the extracellular zone, incorporating cell surface receptors, metals and cellular membranes [47]. In that sense, AD pathogenesis incorporates both Aβ-dependent and Aβ-independent procedures.
There are still countless uncertainties about the actual pathogenesis of AD and the β-amyloid beneficence to the outbreak of the disease. Aβ or Aβ oligomers or plaques are not exclusively at fault for the outbreak of the disease [47]. The "type London" amyloid precursor protein (APP) mutations, inducing simply a slight rise in β-amyloid generation, provoke the outbreak of the pathology sooner than the "type Swedish" mutations, prompting a more immense raise of the protein [48], proposing tha there are alternative processes engaged in the outbreak of AD.
Indeed, the Aβ-independent procedures are interceded through APP, intracellular parts and PS1 through the cellular processes, such as inflammation, oxidative stress and Ca2+ dysregulation, affected in AD pathogenesis [49]. Cdk5 may be influenced by or collaborate with both paths, and its activation brings about DNA corruption, cell cycle activation and neurodegeneration [50]. Non-Aβ elements such as Tau and ApoE also assist to AD pathology [51]. All these paths can prompt synaptic dysfunction, neurodegeneration and AD.
The aged plaques, likewise, are not implied to be a sole component of Alzheimer's disease. They broaden with senility, even in healthy people, and the number of plaques in healthy controls is usually proportionate with the count located in age-matched affected persons [52]. Besides, β-amyloid is physiologically formed in healthy brains during neuronal activity and is mandatory for synaptic flexibility and recollection [53]. Along, in the AD culture, there is particularly a powerless interrelationship among the count of aged plaques and the severity of the pathology. The cleavage of APP by γ-secretase provides some portions named AICD (APP intracellular realm), which turns up to present an influential function in the outbreak of AD. In fact, it is experienced that transgenic mice for AICD exhibit tau phosphorylation and aggregation and decreased cell proliferation/survival, even in the absence of endogenous APP [54]. Large elevations of AICD may further present an influential aspect in the pathology of human brain [55].
There are considerable alternative challenging assumptions, such as the cholinergic hypothesis, the tau hypothesis, and the hypothesis that some extra environmental hazard aspects, may grant to supplementary origins of the disease 145 [56].
The cholinergic assumption recommends that AD is induced by cholinergic effects similar to the decreased synthesis of the neurotransmitter acetylcholine, or to the induction of substantial aggregation of amyloid and to the neuroinflammation [57,58]. The vast majority of ready for use therapies lie on this supposition [59]. The tau assumption posits that tau protein irregularities set up the disease avalanche as hyperphosphorylated tau forms neurofibrillary tangles, forcing fragmentation of microtubules in brain cells [60], emerging in malfunctioning of the biological activity among neurons and later in the downfall of the cells. Alternative suppositions incorporate environmental liability aspects like smoking and infection, as well as a neurovascular assumption, which implies that the blood-brain barrier is precarious for brain Aβ homeostasis and controls Aβ carriage through the LRP receptor and RAGE [61].

Electrochemical (bio)sensing using metallic nanoparticles
So far, many attempts have been made for detection of Aβ, using other analytical methods such as mass spectrometry, capillary electrophoresis, surface plasmon resonance (SPR) and so on [38,62].
However, most of these methods suffered from expensive, timeconsuming, labor intensive. Neuro imaging methodologies namely MRI, PET, SPET, NIR, SPR, and SPRi, are capable to detect Aβ in biofluids to monitor AD progression and pathogenesis. Nevertheless, these methods are limited to clinics due to requirement of sophisticated equipment and high expertise to operate. Biosensors offer a much more rapid, cost-effective highly sensitive method of analyte detection at the point-of-care. Several efforts to generate a laboratory based Aβ biosensor have been made recently although none of these systems is specific for AβO due to the nature of the bioreceptors employed. For example, Stravalaci and colleagues sought to develop an SPR-based assay that recognizes specifically AβO, however, their use of the pan-Aβ antibody 4G8 as bioreceptor would also recognize other Aβ aggregation states as well as APP and its metabolites in patient samples [63]. An electrochemical biosensor which utilized a ferrocene-conjugated peptide as bioreceptor was shown recently to detect synthetic AβO, however there was also some recognition of monomeric Aβ and the system was not tested using biologically relevant species [64]. To overcome these short comings, electrochemical sensing methodologies are being investigated for rapid, selective and sensitive detection of Aβ.
Moreover, the challenges to determine Aβ

in plasma is
in what manner the detection limit will be lowered down to 10 pg mL−1 since Aβ (1-42) levels in plasma in patients with AD and controls were detected from 25 to 85 pg mL−1. The sensitivity of a biosensor counts on the amount of analytes that can be adhered on the sensor's electrode. Late advances in micro/nanotechnologies have raised the linkage process [65,66]. Nanomaterials contribute a considerably enormous surface area than that of bulk material or thin film and have been handled to enhance the detection signal of biomedical devices [67,68]. On this basis, Wu et al. [69]   Aβ(1-16) was tested. We believe that the method would offer a useful means for quantifying Aβ in a biological matrix, and be valuable in the design of new types of electrochemical biosensors for the detection of peptides and proteins. A noticeable study [74], peptides, containing a single tyrosine (Tyr) residues, was monitored at a peak potential of 0.6 V (vs. Ag/AgCl within this sensor) in connection with differential pulse voltammetry. The peak current intensities were concentration dependent. The proposed process provides new routes for analysis of saccharide-protein interactions and electrochemical biosensor development.
Another study [76]  with -amyloid, leading to pronounced colour switch from yellow to red. In comparison to previous practices that engaged antibodies, this system showed great merits of visualization, appliance, and simplicity. By adopting the immunosensing technique, the A(1-40/1-42) could be detected with a detection limit of 86 pM. An excessively sensitive and profoundly straightforward aptasensor was constructed for the quantitation of amyloid beta (Aβ) by electrochemical transduction of a fern leaves-like gold nanostructure [79]. The gold nanostructure was incorporated by electrodeposition employing polyethylene glycol 6000 as a shapedirecting agent, and characterized electrochemically and by field DOI: 10.26717/BJSTR.2020.32.005274 25128 emission scanning electron microscopy. A specific RNA aptamer was immobilized on the fern leaves-like gold nanostructure, and binding with Aβ was detected by the ferro/ferricyanide redox marker. The aptasensor was skilled to detect Aβ in a linear range of 0.002-1.28 ng mL−1 and a limit of detection of 0.4 pg mL−1.
The aptasensor was interference-free, and for manifestation of its viability for Aβ determination in real samples, the human blood serum and artificial cerebrospinal fluid containing Aβ were tested.
An outstanding example of Aβ peptide biosensors is describing the general idea of designing electrochemical biosensors with peptide probes as the receptors of targets and the inducers of gold nanoparticles (AuNPs) assembly on electrode surface [80]. To prove the usefulness of this sensor, human chorionic gonadotropin various samples and marker identification. The detection limit for amyloid-beta (Aβ) 1-40, Aβ 1-42, and τ protein was 34.9 fM, 26 fM, and 23.6 fM, respectively (in mimicked blood under physiological condition). It has been proposed that gelsolin ties up to both Aβ  and Aβ  in a concentration dependent aspect [87].
Thus, Shi's group described two "sandwich-like" electrochemical biosensors for the detection of Aβ (1-40/1-42) peptides in the CSF and varied brain regions with gelsolin as the biorecognition item [88,89]. Screen-printed carbon electrodes modified with multiwalled carbon nanotubes (MWCNTs) and AuNPs were used for the immobilization of gelsolin and the followup capture of Aβ(1-40/1-42). In their prime work, the gelsolin-Au-thionine bioconjugates were utilized to notice the captured Aβ(1-40/1-42) moieties by the gelsolin-Aβ interaction [88]. The concentrations of Aβ(1-40/1-42) peptides were detected by screening the electrochemical reduction of thionine (Th). In the second work, the HRP-Au-gelsolin nanohybrid formed by onepot modification of AuNPs with horseradish peroxidase (HRP) and gelsolin was occupied as the nanoprobe for the realization of the captured Aβ species [89]. The binded HRP then catalyzed the oxidization of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the presence of H2O2, which formed a quantitative electrochemical signal. The detection limits of these two methods were 50pM and 28 pM, respectively. Varying from the past assays for Aβ detection, these practices counteracts the handling of antibodies for the apprehension and identification of Aβ.

Conflicts of Interest
The authors declare no conflict of interest and the funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish this review".