Biosensors for Alzheimer's disease biomarker detection: A review

Accepted Manuscript Biosensors for Alzheimer's disease biomarker detection: A review Bingqing Shui, Dan Tao, Anca Florea, Jing Cheng, Qin Zhao, Yingying Gu, Wen Li, Nicole Jaffrezic-Renault, Yong Mei, Zhenzhong Guo PII:

S0300-9084(18)30001-4

DOI:

10.1016/j.biochi.2017.12.015

Reference:

BIOCHI 5344

To appear in:

Biochimie

Received Date: 18 July 2017 Accepted Date: 29 December 2017

Please cite this article as: B. Shui, D. Tao, A. Florea, J. Cheng, Q. Zhao, Y. Gu, W. Li, N. JaffrezicRenault, Y. Mei, Z. Guo, Biosensors for Alzheimer's disease biomarker detection: A review, Biochimie (2018), doi: 10.1016/j.biochi.2017.12.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Review

Biosensors for Alzheimer’s disease biomarker detection: A review Bingqing Shui 1,†, Dan Tao 1,†, Anca Florea 2, Jing Cheng 1, Qin Zhao 1, Yingying Gu 1, Wen Li 3, Nicole Jaffrezic-Renault 4,*, Yong Mei 1,* and Zhenzhong Guo 1,* 1

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Hubei Province Key Laboratory of Occupational Hazard Identification and Control, Medical college, Wuhan University of Science and Technology, Wuhan 430065, P.R.China; [email protected] (B.S.); [email protected] (D.T.); [email protected] (J.C.); [email protected] (Q.Z.); [email protected] (Y.G.); [email protected] (Y.M.); [email protected] (Z.G.) 2 Analytical Chemistry Department, Faculty of Pharmacy, Iuliu Ha¸tieganu University of Medicine and Pharmacy, Cluj-Napoca 400012, Romania; [email protected](A.F.) 3 School of Arts, Wuhan Business University, Wuhan 430056, P.R.China; [email protected] (W.L.) 4 Institute of Analytical Sciences, UMR-CNRS 5280, University of Lyon, 5, Rue de La Doua, Villeurbanne 69100, France; [email protected] (N.J.R.) * Author to whom correspondence should be address: [email protected] (Z.G.); Tel.: +86-27-68893436; [email protected] (Y.M.); Tel.: +86-27-68893436; [email protected] (N.J.R.); Tel.: +33-437423558 † These authors contributed equally to this work.

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Abstract: Alzheimer’s disease (AD) is a chronic disease amongst people aged 65 and older. Increasing evidence has illustrated that early diagnosis holds the key to effective treatment of AD. A variety of detection techniques have been developed. Biosensors are excellent analytical tools which have applications in detecting the biomarkers of AD. This review includes appropriate bioreceptors to achieve highly sensitive and selective quantification of AD biomarkers by using transducers. AD biomarkers such as tau protein, amyloid β peptides and apolipoprotein E4, are firstly summarized. The most commonly used bioreceptors, including aptamers and antibodies, are also reviewed. We introduce aptamers specific to AD biomarkers, list the sequences of aptamers designed to capture AD biomarkers and compare the properties of aptamers with those of antibodies with regard to their efficiency as bio-recognition elements. We discuss the recent progress of aptamer systems’ applications in AD biomarkers in biosensing. The review also discusses novel strategies used for signal amplification in sensing AD biomarkers.

1. Introduction

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Keywords: Alzheimer’s disease; biomarkers; aptamers; electrochemical biosensors

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AD was discovered as a severe neurodegenerative disorder characterized by progressive memory, cognitive impairment and personality changes, with evolution to dementia and death [1,2]. The symptoms of AD are impairment of memory and other cognitive skills and a gradual loss of much capacity to carry out daily life activities. With accelerated speed of population aging, AD has become an increasingly serious public health concern all over the world. At present, more than 35 million people have been diagnosed with AD, worldwide. The number is expected to double every 20 years in large aging populations: it will reach 65.7 million in 2030, and 115.4 million in 2050 [3]. Nowadays, three best recognized and described biomarkers are considered for routine diagnosis of AD in human cerebrospinal fluid (CSF) and blood: tau protein, amyloid β peptides (Aβ) and apolipoprotein E4 (APOE4). Because biomarker studies will help to better understand the early stages of disease, early detection of AD is the key to taking timely caring measures to avoid the disease and help prevent deterioration of the patient. To date, AD biomarker-based expression techniques include mass spectrometry (MS) [4], magnetic resonance imaging (MRI), enzyme linked immunosorbent assay (ELISA) [5], Western-blot, immunohistochemistry (IHC), flexible Multi-Analyte Profiling (xMAP) [6] and position emission tomography (PET) [7]. Rong Wang et al. [4] determined the Aβ variants by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight MS measurements. They

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determined the relative peak heights of Aβ variants by comparing with the internal standards of known concentrations in MS, thus determining the levels of Aβ variants. The repeatability and sensitivity of Aβ were evaluated with these peak intensities. Thomas McAvoy and colleagues [8] based their study on a highly sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry (LCMS/MS) technology for the quantitative detection of tau protein in CSF. The technology applied a monoclonal antibody (mAb) to selectively enrich tau protein in CSF, followed by tryptic digestion to produce proteotypic peptide fragments. A diagrammatic representation of LCMS/MS technology is described in Figure 1.

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Figure 1. Diagrammatic representation of LCMS/MS technology. Tau protein and added 13C-15N Arg/Lys-labeled tau (412-AA isoform) internal standard protein (IS) were captured using tau-selective monoclonal antibodies conjugated to magnetic beads. Unbound sample components were washed away before bound components were released. Bound proteins were dried, and then enzymatically digested by trypsin into peptides. Unique peptides derived from tau protein were quantified in the sample by LCMS/MS. Adapted with permission from [8].

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To address the challenges of AD early detection, Sarah N. Fontaine et al. [9] modified and adapted a manganese-enhanced magnetic resonance imaging (MEMRI) approach that offers a powerful tool to repeatedly measure neuronal function in vivo to detect changes in broad neuronal function in transgenic mice. They measured significant changes in broad neuronal function before the deposition of bona fide tangles. Ting Yang et al. [5] developed two sandwich ELISAs for the detection of low concentrations of natural Aβ oligomers in human brain samples. They performed immunoprecipitation of Aβ in CSF with antibodies and transferred the immunodepleted solutions to new tubes for further experiments. They also excluded some negative results by spiking experiments, because of molecules in CSF that interfere with the assays. These assays provided a useful, sensitive method to quantify Aβ oligomers in multiple biological samples. Ju-Hee Kang et al. [6] described an ELISA or xMAP assay to measure AD biomarkers in CSF (Aβ1-42, total tau(T-tau), and tau hyperphosphorylated at threonine 181(P-tau181)). Based on xMAP technology, Luminex xMAP technology was used to design a prototype multiparametric bead-based assay for the simultaneous quantification in CSF of Aβ1-42, T-tau, and P-tau181 [10]. PET techniques of fibrillar Aβ deposition had contributed to AD research by enabling in vivo measurement of fibrillar amyloid in CSF. PET imaging with 18F-labelled florbetaben has been demonstrated to accurately detect Aβ neuritic plaques in the brains of patients with AD [7]. This

ACCEPTED MANUSCRIPT agent specifically binds to the amyloid β-pleated sheet structure. Most of these methods are related to clinical severity, neurasthenia and neuronal loss. MS, Western-blot, IHC, xMAP and PET are highly sensitive, MRI is a rapid assay, and ELISA is simple to use. Despite all these techniques helping to advance the detection of AD, they have some limitations, as shown in Table 1.

Technique

Limitations

MS

Expensive

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Table 1. Techniques for AD diagnosis and their limitations.

Strict low-pressure requirements

Depend strongly on energy, collision gas, pressure, and other factors MRI

Expensive Motion artifacts Insensitive to calcifications

ELISA

Time consuming and inefficient False positives

Western-blot

Low stability

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Insensitive to low level markers

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Low scanning velocity

An imbalance in any step of the procedure may skew the entire process IHC

Variable antibody reactivity

xMAP

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Interpretation is often subjective Expensive

The results are low to medium resolution A small number of heterozygous ambiguities Expensive

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PET

Poor spatial resolution

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Artifacts of movements

Many of these measurements are relatively expensive, time-consuming, inefficient and not very sensitive [11]. Electrochemical biosensors are therefore a rapid, sensitive, specific and cost effective alternative to detect AD biomarkers. In recent years, because of their high sensitivity, simple use, rapid response, and compatibility with miniaturization,electrochemical biosensors have been widely applied in clinical diagnosis, biomedical study, food quality control, environmental monitoring and other fields [12]. Biosensors are low cost and rapid tools for clinical research. They provide a potential alternative to advanced bioanalytical systems, on account of their simplicity and portability in relatively complex samples. A biosensor consists of two components: a bioreceptor and a transducer. The bioreceptor identifies the target analyte, while the transducer transforms the recognition element into a measurable signal identification [13]. A bioreceptor with selectivity for the biomarker is mainly considered. Further, some signal enhancers are introduced to achieve the desired signal amplification. In this review article, AD biomarkers are firstly described. The second major part deals with various bioreceptors that can be integrated as selective recognition elements. Then, several signal amplification strategies are discussed. Finally, some personal views on future study directions are put forward combined with current research results.

ACCEPTED MANUSCRIPT 2. Biomarkers for AD diagnosis

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AD develops slowly, the first symptom which is cognitive dysfunction does not begin before decades; therefore it’s difficult to identify pathological processes based on the clinical phenotype alone. For this reason, biomarkers are applied to early diagnosis of AD. In prodromal AD, diagnostic biomarkers can play a major role in guiding management and decision-making. A biomarker is defined as an indicator of normal biological processes, pathological processes or therapeutic responses to drugs [14]. Nowadays, there are three CSF biomarkers that are used to help diagnose AD: P-tau181, T-tau (6 isoforms), and Aβ (Aβ1-42 and Aβ1-40) [15]. APOE4 is also a major etiological factor for AD [16], and there are other biomarkers. In early-onset AD, identified by the mild cognitive impairment stage, these CSF biomarkers are diagnostically accurate, with 85-90% sensitivity and specificity [17]. According to the structure of these biomarkers, the best bioreceptor and/or format of bioassay can be chosen for high sensitivity and selectivity; this is to link it a bit to biosensing. However, insufficient knowledge of the pathology and heterogeneity of AD and other challenges may hinder the implementation of these biomarkers in clinical practice, especially in the early diagnostic stages. Thus, the present review concentrated more on the technical measurements applied to develop a highly specific and sensitive AD biosensor. 2.1 Tau proteins and derived isoforms

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Tau protein is one of the validated biomarkers for AD and tauopathy diagnosis in general [18]. It is a microtubule-associated protein that forms intracellular aggregates in several neurodegenerative diseases collectively termed tauopathies. Abnormal phosphorylation of tau proteins is the characteristic of tau pathology in AD [19], but it also undergoes all kinds of post-translational modifications, including proteolytic cleavage (truncation), glycation, nitration, acetylation, O-GlcNAcylation, ubiquitination, and other abnormal post-translational modifications [20]. A recent work suggests a physiological role for tau in dendrites. Hyperphosphorylated tau and oligomeric tau are both drawn into synaptic loss. Proteolytically stable tau oligomers are able to propagate between neurons and initiate the cascade of self-propagating misfolded proteins that cascade between neurons [21]. Alterations of the CSF concentrations of Aβ1-42 and of P-tau181 reflect cerebral amyloid and tau pathology in AD, which have been applied to early diagnosis of AD as biomarkers [22]. Tau protein is a significant pathological substrate for patients with AD and is a potential therapeutic target because tau tangles are more closely related to the severity of dementia than Aβ plaques [23]. It is known that with the development and progression of AD, T-tau and phosphorylated tau (P-tau) levels increase in CSF. A variety of the polypeptide chain, which has the elements of a transient secondary structure (e.g. β-structures, α-helices and poly-Pro helices), is unfolded. The tau structure is composed of four regions, namely the amino-terminal region (N-terminal), the proline-rich region, repeat domain region, and the carboxy-terminal region (C-terminal) [24]. The projection domain is the N-terminal region that projects away from microtubules. The assembly domain in the C-terminal region consists of the repeat domain regions and flanking regions, which combines with microtubules and make tau aggregate. The middle region of tau (amino acids 151-243) is a proline-rich region that includes multiple Thr-Pro or Ser-Pro motifs with targets of proline-directed kinases. These motifs, in the development process of AD and other tauopathies, become hyperphosphorylated and thereby are able to be identified by the majority of well-characterized tau phosphorylation-dependent antibodies (such as AT8, AT180, AT100, 12E8 and PHF1) [25]. Six main tau isoforms are expressed in the adult human brain, and these are generated by the alternative splicing of the microtubule-associated protein tau (MAPT) gene [25, 26] and lead to different expression in the progression of the disease [27]. These tau isoforms differ by the presence or absence of one or two N-terminal sequences (exon 2 encoding 29 residues or exons 2 and 3 encoding 58 amino acid residues) ; also by the inclusion or exclusion of a second 31-residue-long microtubule splicing of the repeat domain encoded by exon 10 in the C- terminal half of the protein (Figure 2) [28].

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Figure 2. Six tau isoforms expressed in an adult human brain. Tau proteins present in six isoforms that differ in the number of binding domain repeats and intron repeats with 352 to 441 amino acids. The six different tau isoforms (amino acids 352-441) are generated by alternative mRNA binding to exon 2 (yellow), exon 3 (green), and exon 10 (blue) as shown in the cube. These isoforms are depicted in terms of the number of C-terminal repeat sequences (3R or 4R) and the number of N-terminal repeats (0N, 1N, or 2N). Six different tau isoforms referred to as 4R2N (=tau40 or tau-441), 4R1N (=tau46 or tau-412), 4R0N (=tau24 or tau-383), 3R2N (=tau39 or tau-410), 3R1N (=tau37 or tau-381) and 3R0N (=tau23 or tau-352). In a healthy brain, the quantities of 3R and 4R tau are about equal. Adapted with permission from [28].

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Of the various phosphorylation sites, P-tau181 and tau hyperphosphorylate at threonine 231 (P-tau231) are AD biomarkers, which are applied widely to the diagnosis, treatment and prevention of disease. Thus, attention has been focused on these two, since measuring them proved to be favorable to the classification of AD from correlational differential diagnoses: P-tau181 intensifies the differentiation between AD and dementia with Lewy bodies and P-tau231 is also able to differentiate between AD and frontotemporal dementia (FTD) [29].

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2.2 Amyloid β peptides and derived species One of the characteristics of AD is the presence of sticky plaques of Aβ, which form around neurons. The amyloid precursor protein (APP) is an I type transmembrane single glycoprotein; it is located at chromosome 21q and composed of 695 ~ 770 amino acid residues. APP is widely expressed in almost all neuronal and non-neuronal tissues. There are two mechanisms of APP degradation, namely non-amyloidogenic and amyloidogenic pathways [30, 31]. Figure 3 depicts the latter pathways of amyloidogenic processing of APP by the β-site APP-cleaving enzyme (BACE1) and the γ-secretase complex. In this pathway, full-length APP is first processed by BACE1, and the large ectodomain is secreted. The remaining membrane-retained stub binds to a docking site on the surface of the γ-secretase complex. Depending on the different cleavage sites (γ38 γ40 γ42) of the γ-secretase at the carboxyl terminus of Aβ, this produces 39-42 amino acid residues in human brain. The amino acid sequence of amino acids 1 to 28 of Aβ is a hydrophilic amino terminus, and behind the 29-position is a hydrophobic carboxyl terminal. Therefore, the longer they are, the more easily they lead to deposition. It can be seen that γ-secretase is a key enzyme to determine the production of Aβ. The γ-secretase cleavage takes place in the middle of the membrane, liberates Aβ and the APP intracellular domain (AICD). Residues 12-19

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form a β strand in all known Aβ40 structures, with side chains of even-numbered residues exposed on the exterior surface, whereas residues 30-40 are buried in the fibril core. Close contacts between F19 and L34 side chains occur in both in vitro Aβ40 and in vitro Aβ42 fibrils [32]. However, the function of the AICD is still uncertain.

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Figure 3. The schematics of APP cleavage. Two types of Aβ peptides, namely Aβ42 and Aβ40, are produced by cleavage of the APP by the action of β-secretase and γ-secretase. Golde et al. found that the major 4-kDa species in both conditioned medium and human CSF is Aβ40 (>60-70%), although some Aβ42 (<15%) is also present along with minor amounts of other peptides (e.g. Aβ28, Aβ33, Aβ34, Aβ3-34, Aβ37, Aβ38, and Aβ39). Although Aβ40 is the major species produced, the principal species deposited within the parenchyma of the AD brain are species ending at Aβ42. Thus, species ending at Aβ42, which are a minor component of the Aβ that is normally secreted by cells, in many cases constitute the bulk of the Aβ that is deposited in the AD brain. Adapted with permission from [33-35].

2.3 APOE4 and derived isoforms

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Several potential risk factors for AD have also been identified. Human apolipoprotein E (APOE) is a 299 residue protein that mediates lipoproteins binding to the low density lipoprotein receptors around the periphery and the central nervous system (CNS). APOE binding to APOE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase (MAP3K or MKKK) that then activates MKK7 and extracellular signal-regulated kinases 1/2(ERK1/2) MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances the transcription of APP and thereby increases Aβ levels [36]. The most common isoforms of APOE are: APOE2, APOE3 and APOE4. These three isoforms differ from each other by cysteine to arginine changes at 112 and 158 positions, of which only APOE4 is considered a risk factor of AD. Individuals with two APOE4 alleles result in an increased risk of AD patients by more than seven times as compared with APOE3 alleles [37]. APOE4 is present in approximately 15% of the normal population ; however, it occurs in 50% of those developing AD [38]. Therefore, APOE4 is a strong risk gene related to sporadic AD. 2.4 APP, ADDLs, AAT, BACE1 and AD7c-NTP Apart from the three main biomarkers discussed above, other markers have been put forward to facilitate the effective diagnosis of AD. APP [39], amyloid-β-derived diffusible ligands (ADDLs) [40], α-1 antitrypsin (AAT) [41], BACE1 [42], and Alzheimer-associated neuronal thread protein (AD7c-NTP) [43] have been recognized as effective biomarkers to early diagnosis of AD. Table 2 summarizes the biomarkers known to be related to AD discussed here.

ACCEPTED MANUSCRIPT Table 2. The role of AD biomarkers in the disease. Role in AD

Biomarkers Tau

Reference

Tau is hyperphaspharylated in neurofibrillary tangles. Tau exists as six

[28]

spliced isoforms depending upon inclusion of N- terminal exons 2 and 3, and the exon 10 microtubule binding domain. The tau haplatype is associated with AD. Aβ

The monomer form of Aβ is not neurotoxic while the oligomers and

[30]

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fibrils formed via the nucleation-dependent complex process exhibit

neurotoxicity and block long-term potentiation to affect synaptic

plasticity. AD takes place due to tangles of neurofibrils including

P-tau, Aβ plaque and aggregation of insoluble hydrophobic Aβ. APOE4

APOE4 is transported with cholesterol; APOE isoforms have differing

[36]

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transport efficiencies. APOE4 is associated with increased AD risk.

APOE4 alleles are associated with increased amyloid burden and cholinergic dysfunction.

APP is a membrane protein cleaved by secretases. APP degrades via two

[39]

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APP

mechanisms namely amyloidogenic and non amyloidogenic pathways. The first is the Amyloidogenic mechanism which produces Aβ via APP cleaving by β-secretase i.e. BACE1 and γ-secretases. The Aβ produced in this manner form toxic oligomeric species and lead to plaque formation via aggregation. In the second pathway, the cleavage of APP takes place via α-secretase i.e. a disintegrin and metalloproteinase domain-containing protein 10 in neurons situated within the Aβ

ADDLs

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sequence.

The soluble pools of ADDLs that exist in the human brain extend to the

[40]

CSF, and elevated levels of ADDLs correlate with the presence of AD. AAT

As one of the protease inhibitors, AAT (normal 150–350 mg/dl in

[41]

serum) is the major protease inhibitor of human plasma protein, and it

BACE1

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has been increasingly proposed as an important biomarker of AD.

The initial cleavage to generate Aβ is BACE1. BACE1 is a type I

[42]

transmembrane aspartyl protease which represents a valuable target in

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AD therapy.

AD7c-

A novel Alu sequence-containing cDNA designated AD7c-NTP that is

NTP

expressed in neurons, and overexpressed in brains with AD. The

[43]

1,442-nucleotide AD7c-NTP cDNA encodes a~41-kD protein.

3. Bioreceptors Bioreceptors are part of the biosensor. They involve some interaction between the biomarkers and produce a signal by biological reaction. A key point of the bioreceptor is high selectivity towards the analyte among a matrix of other biological entities. Among the different types of bioreceptor, those most commonly employed in the development of AD sensors are aptamers and antibodies, as discussed below. 3.1 Aptamers

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Nucleic acid aptamers are short, single-stranded DNA (ssDNA) or RNA oligonucleotides which adopt stable three-dimensional sequence-dependent structures [44]. Nowadays, aptamers are used in biosensors as prior bioreceptors, including small ssDNA, RNA oligonucleotide fragments and small peptides, which can combined with high affinity and specificity to targets. In general, nucleic acid aptamers are selected through an effective chemistry technique, also called the systematic evolution of ligands by exponential enrichment (SELEX), screening specific ligands by repeated rounds of partition and amplification from a large nucleic acid library containing 1014-1016 different candidates [45, 46]. Aptamers have found clinical applications as desirable materials because of their specificity, non-immunogenic and nontoxic character. AD is threatening patients and has a certain influence on their lives. Although the mechanism of AD is unclear, its unique characteristic is the accumulation of misfolded proteins in the CNS [47]. Therefore, it is necessary to develop possible measures to mitigate or prevent the occurrence of AD. The search for aptamers has become a new challenge in this field. Aptamers combine with these associated target proteins in case of their accumulation, which can prevent the progression of AD. Fewer aptamers specific to tau protein have been developed and proposed. These aptamers inhibit the oligomerization propensity of tau both in vitro and in cultured cell models of tauopathy without affecting the half-life of tau. In addition, the tau aptamers significantly reduced synthesis of tau oligomer-mediated neurotoxicity and loss of dendritic spine in hippocampal neurons [48]. Close attention has been paid to Tau 381, as attested by the extensive research of tau-ssDNA interactions by Krylova et al., who also reported similar interaction for tau 410 [49]. Kim et al. [47] described novel RNA aptamers specific to the human tau 441 isoform which were screened out by an in vitro selection process. Table 3 lists the sequences of aptamers employed to capture AD biomarkers based on abundant research. Besides their use for diagnostic and visualization purposes, aptamers may also have therapeutic potential. In December 2004, Pegaptanib (Macugen) was the first aptamer approved to cure ocular neovascularization by the US Food and Drug Administration (FDA). It has been successfully used as a therapy in human beings, and as such it is of significant reference value for AD diagnosis [50]. In future, more aptamers will be employed as recognition elements to diagnose biomarkers of AD. Aptamers are called “chemical antibodies” because the functions of aptamers and conventional antibodies are similar. But compared with the properties of classical antibodies, aptamers have better characteristics including low molecular weight, quick and reproducible synthesis in vitro, easy modification, good stability, low toxicity, low immunogenicity and rapid tissue penetration (see Table 4 for details). Nonetheless, the absence of analytical applications and the observed cross-reactivity suggest the need for further efforts in selecting aptamers for AD biomarker detection [9].

Target

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Table 3. Aptamer sequences designed to capture AD biomarkers. Aptamer Sequences

Reference

ssDNA: 5'-GCGGAGCGTGGCAGG-3'

[49]

Tau-410

dsDNA: 5'-CTTCTGCCCGCCTCCTTCC-3'

[49]

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Tau-381

3'-GAAGACGGGCGGAGGAAGG-5'

Tau-441

RNA:5'-CCGUGUCUUCGUGAGGUCGGUGUCGGCUUGGCAGAAAG

[48]

GG-3'

Aβ40

DNA: 5'-HS-GCCTGTGTTGGGGCGGGTGCG-3'

[51]

Aβ42

DNA: 5'-HS-GCCTGTGTTGGGGCGGGTGCG-3'

[51]

RNA:5'-UUUACCGUAAGGCCUGUCUUCGUUUGACAGCGGCUUGUU

[52]

Aβ1-40

GACCCUCACACUUUGUACCUGCUGCCA-3' ADDLs

DNA:5'-CGCATTCAGGATTGCATGATTGCCTCGTCTTAACGGTCTCA

[40]

ACTCGTA-3' BACE1

DNA:5'-GCAATGGTACGGTACTTCCGTCATCAGCTTGTGATGTGG ATGCGAACTGCAAAAGTGCACGCTACTTTGCTAA-3'

1

[53]

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[42]

5'-GTACACGTCGGCCACCTACGCGAAGTGGAAGCCTCATTTG-3' 1

Bold underlined letter represents random sequences of 30 nt in length.

3.2 Antibodies

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At present, the scientific application of antibodies ranges from basic studies to applied researches [44]. mAb was developed by Kohler and Milstein [54] in the mid-1970s and nowadays, mAbs are used for multiple research purposes (or fields). The specificity, sensitivity, and homogeneity of antibodies have aroused the interest of researchers. John Hardy et al. injected anti-Aβ antibody to transgenic AD mice and found that the Aβ was significantly reduced and the amyloid plaques were removed [39]. Angela L. Guillozet-Bongaarts et al. described a mAb (tau-C3) specific to tau cleaved at aspartic acid 421 [55]. Scarlet and co-workers developed a novel, very sensitive electrochemical biosensor to detect the full-length tau-441 based on a microelectrode array with four gold micro band electrodes coated with oriented antibodies [56]. The biosensor used a layer of protein G to ensure that antibody Fab binding domains were oriented away from the biosensor surface and free to react with target antigens. One of the characteristics of the antibody is to increase the load capacity of the biosensor and its sensitivity to the antigen. There are two limitations of antibodies: their thermal or physical instability, and complex in vitro and in vivo generation models. Table 4. Comparison of aptamer with antibody.

Aptamers

Antibodies

Higher affinity

Lower affinity

Less expensive

Reasonable at large scale

Smaller size

Larger size

Almost all kinds of targets

Only those targets with high immunogenicity Dry ice transportation requirements

Lower chance of causing immunogenic response

Higher chance of causing immunogenic

when used in vivo

response when used in vivo

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Ambient temperature transportation requirements

Lower ability to conjugate to surfaces/resins

Produced chemically in a readily scalable process

Produced biologically in a difficult process

Can usually be reversibly denatured

Susceptible to irreversible denaturation

4. Transducers

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Higher ability to conjugate to surfaces/resins

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A biosensor transduces the interaction of the analyte with the detector element into a signal that can be easily measured and quantified. The transducer or the biological element is the part of the biosensor that works in a physicochemical, optical, piezoelectric or electrochemical way. Biosensors can be classified according to biotransducer type. The compatibility of the biotransducer with the bioreceptor is an important requirement of the successful biosensor. The major types of transducers used in biosensors include electrochemical, optical, electronic, piezoelectric, gravimetric and pyroelectric. Electrochemical and optical transducers have been used in biosensors to detect markers of AD. Thus, this review focuses on the two main biotransducers. 4.1 Electrochemical transducers Electrochemical biosensors have been considered for a long time. Detection of AD has been coupled with electrochemical transducers, due to their high sensitivity, specificity, ease of use and fast response for the analyte of interest. Electrochemical biosensors can transform chemical signals into a measurable amperometric signal by using potentiometric, amperometric and impedimetric transducers. A key point for the detection system is the selection of the most suitable electrochemical measurements.

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The most common electroanalytical techniques used for the detection of AD biomarkers are cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) [57]. EIS is a great choice of technique because it provides insight into the bimolecular interaction by its effect on electron transfer resistance (Ret) [58], and it is a very powerful tool for surface interface characterization and detection of very small changes occurring at biosensor surfaces. A variety of electrochemical sensors for the detection of AD biomarkers have been reported. Esteves-Villanueva JO et al. [59] developed a biosensor to detect tau misfolding based on electrochemical assay. The linear relationship between the charge-transfer resistance (Rct) and the tau concentration varied from 0.2 to 1.0 µM. EIS and CV were employed for the detection and quantitation of the tau protein binding to the biosensing interface based on the immobilized tau protein on the Au surface (tau-Au). Their research qualitatively and quantitatively analyzed the interactions of tau-tau. A lower impedance and higher capacitance were attributed to an increased positive charge within the film and the conformational change of protein caused by tau-tau binding. Thus, the binding of tau to a tau-Au recognition element adjusted the charge transfer resistance. The electrochemical approach may be extended to inhibitor screening in order to identify tau misfolding in search of AD therapeutic targets. Figure 4 showed the electrochemical assay for detection of tau protein binding to the immobilized tau-Au interface. The surface area of the gold disk electrodes was 0.0314 cm2. A conventional three-electrode system consists of a gold electrode (the working electrode), platinum wire (the counter electrode) and Ag/AgCl/1.0 M KCl (the reference electrode).

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Figure 4. Illustration of the tau-based biosensor and tau-tau-Au interface. The tau-Au surface (a) was exposed to the protein solution under conditions forming the tau-tau-Au surface (b). Adapted with permission from [59].

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Scarlet and co-workers have developed a very sensitive electrochemical biosensor to detect tau-441, and the limit of quantification for tau-441 was 0.03 pM [56]. EIS was used to analyze impedance change, which revealed a linear response with increasing tau concentrations. The Rct values for each analyte concentration were calculated by fitting semi-circle spectra to Randles' equivalent circuit model. The Rct values following incubation with increasing concentrations of tau and bovine serum albumin were shown in their study. The technology could also be adapted for the detection of other AD biomarkers to provide a multiple assay to diagnose AD. The GO/pPG/anti-MBP/anti-tau nanoimmunosensor was developed and had detection limits of 0.15 nM for tau proteins [60]. It was tested, optimized and characterized using EIS and DPV techniques. Yifan Dai et al. [61] designed a single-use, in vitro biosensor to detect the T-tau in phosphate-buffer saline (PBS) and undiluted human serum over the concentration range from 1000 pg/mL to 100,000 pg/mL. The antibody of T-tau concentration was 500,000 pg/mL. The bio-recognition mechanism of this biosensor system is based on the antibody and antigen interaction and the effect of a [Fe(CN)6]3-/4- redox probe through the interaction. The electrochemical DPV technique was used in the transduction mechanism. DPV applied a series of regular potential pulses superimposed on the potential stair steps and then the current was promptly measured prior to each potential change. The charging current could therefore be minimized, leading to higher sensitivity. Figure 5 shows the biosensor structure and its actual dimensions used in their research. The working and counter electrodes were both thin gold films 10 nm in thickness. Different from thick

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film printing, the laser ablation technique was applied to determine the size and dimensions of the biosensor and its electrode elements. Thin gold film was deposited on PET by the sputtering technique. Thus, the gold film was deposited at an atomic level, and without any binder, resulting uniform and reproducible working and counter electrodes. By depositing thin gold film on the polymeric substrate of a roll-to-roll fabrication process, the biosensor developed was very cost effective and of practical single use.

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Figure 5. Structure and dimensions of the thin film gold-based T-tau biosensor prototype. This biosensor consisted of three electrodes: reference, working and counter electrodes fabricated on a PET sheet. 100 individual biosensors in 4 rows were fabricated on each PET sheet (355×280 mm2). The overall dimensions of an individual biosensor were 33.0×8.0 mm2. The working electrode area was 1.54 mm2 accommodating 10-25 µL of liquid test sample. Adapted with permission from [61].

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Biosensor technology could be further optimized and applied for the detection of other AD biomarkers. Soha Ahmadi et al. [62] designed a surfaced-based electrochemical method to monitor the interactions of 2N3R tau (tau-410) with Fe (II) and Fe (III). CV and DPV were employed to monitor the current response as a function of applied potential. EIS was utilized to monitor the resistance of tau film on the gold surface and the changes in tau film properties upon iron ion interaction. They followed putative structural changes in surface bound tau protein by monitoring changes in current density. The involvement of iron in the generation of reactive oxygen species (ROS) led to oxidative stress and neuronal damage. Thus, there might be a connection between iron accumulation and tau aggregation in AD. This may set up new approaches to the development of therapeutic agents or even diagnostics devices for AD. Jahnke et al. [63] developed a new 384-multiwell microelectrode array (MMEA) to detect tau protein based on a measurement system for the sensitive label-free real-time monitoring through electrochemical impedance spectroscopy. They introduced immersive counter electrodes; all measurement electrodes within the MMEA could be connected to a single contacting point. The individual addressable immersive counter electrodes were integrated into a pin-holder plate that could be attached permanently to the kinematic module they developed. Each immersive electrode in the pin-holder plate is contacted on the top side by a spring contact pin that is connected to a multiplexer electronic circuit board under the pneumatic carriage. This assay could be used to test and screen for novel potential tauopathy therapeutics. Thus this novel tauopathy screening system could be a wonderful approach to recognize and study novel therapeutics in the field of tau related neurodegenerative diseases. Yu et al. [64] described a novel electrochemical and specific biosensor to detect soluble β-amyloid Aβ (1-40/1-42) peptides sensitively in a rat model of AD. Lin Liu and colleagues [34] designed a simple and sensitive electrochemical biosensor for the detection of total Aβ peptides using gold nanoparticles modified with Aβ (1-16)-heme (denoted as Aβ (1-16)-heme-AuNPs). The method offers a useful tool for quantifying Aβ, and may be valuable in the design of new types of electrochemical biosensors for the

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detection of Aβ. The principle of measurement of Aβ with Aβ (1-16)-heme-AuNPs using the voltammetric assay is illustrated in Figure 6

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Figure 6. Schematic overview of the strategy for Aβ detection. The anchored Aβ (1-16)-heme-AuNPs showed strong electrocatalytic O2 reduction. The monoclonal antibody specific to the common N-terminus of Aβ was immobilized onto a gold electrode for the capture of Aβ (1-16)-heme-AuNPs. The voltammetric responses were found to be proportional to the concentrations of Aβ ranging from 0.02 to 1.50 nM with a detection limit of 10 pM. Adapted with permission from [34].

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A new procedure based on coupling DNA electrochemical sensors with PCR-amplified DNA extracted from human blood has been designed by Marrazza et al. for the detection of APOE [65]. The DNA electrochemical biosensors are combined with single-stranded oligonucleotides immobilized on graphite screen printed electrodes (SPEs) at controlled adsorption potential. DNA was extracted from the actual samples and amplified by conventional methods. Gangbing Zhu et al. [41] described a novel electrochemical biosensor to detect α-1 antitrypsin (AAT) based on a sandwich-type assay featuring PTCA-CNTs as a sensing platform and ALP-AAT Ab-Ag NPs as a signal amplification strategy. The CNTs not only promoted electrons transfer, but also provided a large surface area for the immobilization of a large number of AAT aptamers. The developed biosensor played an important role in AAT detection for care diagnostics of AD. A sandwich-type electrochemical immunosensor was designed by Yibiao Liu et al [66] to provide quantitative and quantitative methods to detect APOE4 based on fractal gold (FracAu) nanostructures which were directly electrodeposited by HAuCl4 on polyelectrolyte modified indium tin oxide (ITO) electrodes afterenzyme amplification. The sensing performance of the modified interface was assessed by CV. After the HRP-labeled APOE4 antibody was functionalized, the human APOE4 could be quantitatively detected by current reaction. The results advance the detection of APOE4 and contribute to early prevention and diagnosis of AD. Table 5 lists the specifications, linear range and detection limits of electrochemical biosensors developed for AD biomarkers. Table 5. Specifications of electrochemical biosensors for AD biomarker diagnosis. Target

tau-441

Biosensor specification

Tau–Au surface

Electrochemical

Linear rang of

Detection

techniques

detection

limit

CV and EIS

0.2-1.0 µM

0.2 µM

Reference

[59]

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EIS and CV

0.01 pM-10 nM

0.03 pM

[56]

Anti-Tau nanoimmunosensor

DPV and EIS

0.5-15.1 nM

0.15 nM

[60]

_

[61]

0.01 pM

[41]

1000 pg/mL- 100,000 T-tau

Anti-T-tau (T-tau antibody)

DPV pg/mL CV and DPV

0.05- 20.0 pM

CV

1.0 - 10,000 ng/mL

0.3 ng/mL

[66]

SWV

480 pM-12 nM

240 pM

[67]

10pM

[34]

FracAu nanostructure, APOE4 HRP-labeled anti-APOE4 Fine-tuning the scan pulse frequency of Aβ1-42 soluble square wave voltammetry (SWV) to oligomer

synchronize with the surface electron transfer (ET)

Aβ1–40/1–42/

Alkaline phospahte-cysteinPrp (95–115) peptide onto Au Aβ oligomers

EIS and CV

5 pM - 2nM

3 pM

[68]

EIS and CV

10−12 -10−6M

~ 0.5 pM

[69]

UV and DPV

0.1-50 nM

28pM

[70]

CV

0.5 - 500 ng/mL

0.1ng/mL

[71]

EIS

1 pg/mL-10 ng/mL

1 pg/mL

[72]

CV

0.05 -5 nM

5 pM

[73]

CNT-MESFET

I-V curves

10-12-10-9 g/mL

1 pg/mL

[74]

SAM of AuNPs

EIS

2.65 nM- 2.04 µM.

0.57 nM

[75]

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PrPc-residues 95–110

HRP-NPs–gelsolin Aβ1–40/1–42

Anti-Aβ1–42, gold electrode Aβ1–42

0.02-1.50nM

electrode,electrochemical–chemical–che mical redox cycling.

Soluble

CV

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ALP-AAT Ab-Ag NPs

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AAT

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Aβ antibody onto AuNPs sputtered onto AAO nano-hemisphere array

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SA-ALP and TCEP

Total Aβ

4.2 Optical Optical biotransducers are applied to optical biosensors for signal transduction, and collect information about the analyte by using photons. Nanoparticles provide virtuous photostable synthesis and a noise free fluorescence signal in biocompatible environments. Surface Plasmon resonance (SPR) biotransducers can be developed for real time sensing of bimolecular interaction because they are rapid, sensitive and label-free. Suhee Kim et al. [76] introduced a novel DNA aptamer/antibody sandwich assay pairing and applied it to detect human tau-381 in undiluted plasma at concentrations as low as 10 fM via a multichannel SPR platform (Figure 7). Min Kyung Kang et al. introduced effective technology to

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detect the APOE4-mediated Aβ aggregation in physiological conditions using a single gold nanoparticle based on localized surface plasmon resonance (LSPR) [77].

5. Signal amplification strategies

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Figure 7. Schematic representation of tau detection. The SPR gold chip was first modified with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanol (MUD). Tau specific adsorption measurements were performed in buffer to establish the binding affinity of the DNA aptamer before progressing to measurements in plasma followed by the subsequent binding of anti-tau. Adapted with permission from [76].

The selection and proper immobilization of bioreceptors at the surface of transducers play an important role in the sensitivity of the developed biosensor. However, signal amplification

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strategies are also of great importance to achieve low detection limits for trace analysis. Several signal amplification strategies are used to enhance biosensor efficiency: nanomaterials (NMs), redox mediators and enzymes are commonly employed for signal amplification. In this review, we focus on the use of NMs as signal amplification strategies, as the field of nanotechnology has developed rapidly in recent years. Nanoparticles exhibit unique properties which are found to differ from their bulk materials, because of their small size (usually in the range of 1-100 nm) [78]. Biosensors have shown great promise for the detection of chemical markers and biomarkers when NMs are integrated either as signal transducing elements, to mediate current flow, or as tags in recognition elements, to indicate the detection of the analyte [79]. There are a lot of active sites and a variety of functional groups on the surface of NMs, which result in high activity for adsorption and catalysis [80]. Therefore, NMs can be used to build novel and improved sensing devices, especially electrochemical sensors and biosensors. NMs can be classified by their chemical composition including organic or inorganic. Some of the most used NMs include AuNPs, multiwall carbon nanotubes (MWCNTs) and graphene. AuNPs have fundamental and practical significance. Nanoparticles have unique physical and chemical properties, with potential applications ranging from quantum electronics to biomedicine [81]. AuNPs are inert, biocompatible, and can be modified for targeting easily. They can react with molecules containing various functional groups such as thiols or amines, allowing their biofunctionalization with different ligands (peptides, proteins, and aptamers) for specific recognition of biological targets [82]. Thus, AuNPs have been widely used in various biological analysis and biomedical detection technologies. Lucie Stegurová et al. [83] developed the suitability of AuNPs functionalized with tau-specific mAb and an oligonucleotide template for immunopolymerase chain reaction (Nano-iPCR) quantification of tau protein in human CSF samples and compared it with ELISA, either commercial or newly developed with tyramide signal amplification. AuNP-based colorimetric platforms also have been widely applied to detecting a large variety of targets, such as nucleic acids, proteins, and small molecules. Tao Hu et al. [84] proposed a colorimetric sandwich immunosensor for Aβ1-42 based on dual antibody-modified AuNPs. Figure 8 depicts the strategy employed for the development of these colorimetric immunosensors for Aβ1-42.

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Figure 8. Mechanism of the colorimetric immunosensor strategy for Aβ1-42. C-terminal antibody and N-terminal antibody were conjugated onto the surface of AuNP (C-Aβ1-42-AuNP and N-Aβ1-42-AuNP), where the C-terminal antibody or/and N-terminal antibody employed display excellent specificity to Aβ1-42. When a certain concentration of Aβ1-42 was added into the prepared AuNPs @C/N-Aβ1-42 (C-Aβ1-42-AuNP: N-Aβ1-42-AuNP=1:1), this simultaneously resulted in aggregating owing to the high specificity of C-terminal antibody and N-terminal antibody for Aβ1-42, accompanied with obvious color change from red to blue. Adapted with permission from [84].

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Even though AuNPs are mostly employed in sensing strategies of biomarkers, great interest is also emerging for MWCNTs [85]. Lisi et al. reported on a MWCNT based sensor for the detection of tau protein. For this purpose MWCNTs are modified and then decorated with the secondary antibody for tau protein. The MWCNTs-antibody conjugate is then integrated in a sandwich-based bioassay with the capacity to increase the SPR signal by about 102-fold compared to direct detection and a conventional unconjugated sandwich [86]. Graphene is an atomically thin film that is composed of hexagonally arranged carbon atoms with sp2 hybridization in two dimensions [87]. The unique properties of graphene, such as large specific surface area (2630m2g-1), high intrinsic mobility (200,000cm2v-1s-1), high Young’s modulus (~1.0TPa), thermal conductivity (~5,000Wm-1K-1), optical transmittance (~97.7%) and good electrical conductivity, have attracted attention for a wide application range [88]. Graphene oxide (GO), the oxidized derivative of graphene with unique properties, has also received a great deal of attention for various biomedical applications including biosensors. GO possesses both the graphene-like 2D carbon sheet structure and various oxygen containing functional groups such as epoxy, carboxyl, carbonyl, and hydroxyl groups, which may be placed at either the basal plane or edge of the sheet structure [89]. GO could be synthesized by oxidizing graphite with a strong acid. In GO-based biosensors, GO may serve as a material for dynamic interaction with the probe and/or for transduction of a specific response towards the target molecule into a signal. Lin Liu et al. reported a GO-based fluorescence approach to detect Aβ oligomers selectively based on the specific interaction between Aβ oligomers and the PrP (95-110) peptide. More concretely, fluorescein isothiocyanate (FITC)-labeled PrP (95-110), called FITC-PrP (95-110), was adsorbed onto the GO surface by electrostatic and π-π interactions, leading to effective fluorescence quenching. However, in the presence of Aβ oligomers, the competitive binding of Aβ oligomers with GO for the peptide probe prevented the interaction between GO and FITC-PrP (95-110), which led to the fluorescence signal. As a result, a detection limit of Aβ oligomers (equivalent monomers) was 1 nM [90]. With the increasing interest in AD biomarkers, there is an urgent need for highly sensitive and specific diagnostic platforms for its quantification. However, there are very few reports in the literature on novel signal amplification strategies for the detection of AD biomarkers, thus there is a pressing demand for more intensive studies on NMs in the detection of AD biomarkers. 6. Current Trends and Future Perspectives

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7. Conclusions

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AD biomarkers have important early diagnostic value in routine clinical practice and research. Recently, more studies have been conducted for diagnosis of AD based on tau, Aβ, APOE4 and other biomarkers; however studies on the multiplexed detection of several biomarkers are limited. Detection of multiple biomarkers is essential for correct diagnosis and prognosis. Accurate and reliable detection of biomarkers in body fluid is also of paramount importance. Most research in this field is focused on the detection of AD biomarkers in cerebrospinal fluid. There are only a few studies on biomarker detection in human blood and urine. V. Ovod et al. [91] analyzed the turnover kinetics and concentrations of Aβ biomarker (Aβ38, Aβ40, and Aβ42 ) in human plasma. With the development of effective treatments for AD, the blood test could serve as a diagnostic test to screen millions of individuals at risk of amyloidosis and AD. We expect more studies to emerge regarding the detection of AD biomarkers in blood or urine; this research can be beneficial in terms of easing the pain of lumbar puncture. Screening for more high affinity aptamers specific to AD biomarkers is a great challenge. Studies related to aptamers are limited to only 5% screening, with 95% application. Aptamer investigations on the application of AD biomarkers are scarce. Unfortunately, few aptamers have successfully been approved for clinical diagnosis of AD. There are still no answers as to why aptamers have not yet been included within clinical laboratories and only a few aptasensors have been used for clinical diagnosis. If only more aptamers were developed and specified, aptasensors would play a significant role in the development of future diagnostic technologies [92]. Thus, it is necessary to review the recent research progress of aptamers for AD biomarkers to design better aptamer systems for the early diagnostic stages of AD. Molecularly imprinted polymers (MIPs) have the following advantages: high affinity and selectivity, resistance to harsh environments, great stability, long service life and wide application range. Aptamer-MIP sensors have been applied to detect prostate cancer and other diseases, but not AD. We believe that aptamers and MIP based AD biomarkers will begin entering clinical practices in the near future. The utilization of aptamers and antibodies of differing specificities would enable the development of a multiplex device where multiple biosensors are functionalized with different bioreceptors, thus multiple AD biomarkers could be detected simultaneously. The detection of multiple AD biomarkers can save time and improve the accuracy of the diagnosis. The challenge is to establish multiplex detection of several biomarkers within the same sample solution.

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In this review, the most recent developments of AD biomarkers and biosensors for biomarker detection have been summarized. Biosensors play a crucial role in the detection of biomarkers as they are easy to use, portable, and can do analysis in real time. Among bioreceptors, aptamers have attracted special attention as their thermal stability and the fact that they can also be regenerated easily within a few minutes of denaturation make them the perfect candidates for detecting biomarkers. NMs through their inherent advantages have been of much interest in signal amplification strategies. There is a lack of specific early stage biosensing AD tools, so it is an area that is currently under-utilized in the early diagnosis of AD and its potential warrants further exploration. Acknowledgments: Zhenzhong Guo thanks the “ChuTian Scholar” Project Award of Hubei Province (P.R.China) for its support. Author Contributions: Bingqing Shui, Dan Tao, Anca Florea, Jing Cheng, Qin Zhao, Yingying Gu and Wen Li (Bibliography, Drafting the article), Nicole Jaffrezic-Renault, Yong Mei and Zhenzhong Guo (Revising the article for intellectual content). Conflicts of Interest: The authors declare no conflict of interest.

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Highlights Various biomarkers of Alzheimer’s disease employed for biosensors development have been discussed.




The technical evaluation of biosensors detecting biomarkers of AD has been described.




Details of bioreceptors including aptamers and antibodies have been discussed.




Transducers and some signal amplification strategies have also been introduced in the review.

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