1. Introduction

Each year, tens of millions of people around the world are diagnosed with NDs including Alzheimer’s disease (AD), Parkinson’s disease (PD) or prion diseases. These are known as “protein misfolding disorders” and are caused by proteins that are prone to aggregation. In NDs, there is a slow, progressive loss of the structure or dysfunction of neurons in the central nervous system, leading to deficits in specific brain functions. They are considered incurable due to the lack of a known way to reverse the progressive degeneration of neurons. Most NDs begin years before symptoms are present, and the first symptoms appear when a significant number of neurons are damaged or the damage affects a specific part of the central nervous system. For many of these illnesses, diagnosis and treatment are limited. Early diagnosis of age-related disorders is an increasingly pressing health challenge worldwide and is crucial in the management of the disease and determines the patient’s living conditions ^[1][2][1,2]. The detection of NDs can be accomplished by determining the concentration of specific biomarkers including α-synuclein (α-syn), amyloid β peptide (Aβ), tau protein or human cellular prion protein (PrPC) in body fluids. Therefore, non-invasive, simple, fast and real-time methods of detecting the biomarkers of NDs are particularly sought after. To fulfill these requirements, electrochemical biosensors offer potential diagnostic and theranostic applications. Biosensors are analytical devices that convert the result of a biological reaction into an analytical signal. Each biosensor consists of two basic components: an analytically active layer (the receptor part) and a transducer. The recognition elements include molecules constituting biological material, e.g., nucleic acids (DNA or RNA aptamers), proteins, enzymes, antibodies or whole cells, immobilized on a suitable carrier. In the analytically active layer, the process of intermolecular recognition (receptor–analyte) takes place. During this process, a physicochemical signal is generated, and a transducer converts it into an analytically useful signal. The most commonly used transducers are optical, piezoelectric or electrochemical systems [3]. In the last few years, special emphasis has been placed on aptamers as the molecular recognition elements in electrochemical biosensors (aptasensors), which has been confirmed by the rapid increase in the number of scientific papers in this field ^[4][5][6][7][4,5,6,7]. According to Web of Science, publications concerning electrochemical aptamer-based biosensors represent about 28% of the total number of publications on electrochemical biosensors [3]. Aptamers are short, single- or double-stranded DNA or RNA nucleotides or peptides [8]. They have emerged as good alternatives to antibodies in the design of electrochemical biosensors. They are distinguished by a low immunogenicity, longer shelf life, easy storage and the possibility of transport at ambient temperature. The advantages of aptamer manufacturing processes include their lower cost, batch-to-batch variability, simple upscaling and purification. These processes do not require experiments with animals or cells. Additionally, they can be selected under non-physiological conditions ^[9][10][9,10,]. They also exhibit a high binding affinity and specificity for a broad range of targets (such as proteins, amino acids, peptides, drugs, small metal ions and small organic molecules, as well as bacteria and viruses and even whole cells) due to their specific three-dimensional structures ^{[11][12][13][14][15]}[12,13,14,15,16]. Moreover, aptamers display a specificity for some targets (e.g., small molecules or ions) that cannot be recognized by antibodies. They are also able to distinguish forms and isoforms of the same protein, which is an important advantage for biomarker detection. Furthermore, it is possible to manipulate the binding reaction conditions and optimize the aptamers’ recognition sequences to fine-tune their affinities ^[16][18]. Aptamers are also good candidates for conjugation, labeling or functionalization, which is often achieved in the step of aptamer synthesis ^[17][19,20^[18][19]21,22]. With each new aptamer–protein couple, a systematic analysis of their binding process, conditions and structure of the created complexes, as well as their interfacial behavior, is necessary ^[19][20][22,23]. In this framework, a suitable method for aptamer immobilization on a solid surface is critical for the successful elaboration of the biosensor. The selection of the aptamer deposition strategy strongly relies on the modification of the aptamer and the physicochemical features of the electrode surface ^[8][20][8,23]. The electrode material used for the deposition of aptamers should allow the precise control of the packing density and spatial orientation of the aptamers in the deposited layer via their controlled immobilization on the surface. This bind should be strong and stable, providing the conformational flexibility required for the process of the specific recognition of the analyte. The aptamer immobilization procedure should ensure the formation of a stable, reproducible receptor layer and the sensitive detection of the analyte, and it should limit the non-specific adsorption of matrix components. An important issue to be addressed in the development of protein aptasensors is the occurrence of false-positive signals due to the non-specific adsorption of proteins found in biological fluids ^[21][24]. The most frequently described methods of assembly of aptamers on solid surfaces are physisorption, chemisorption and affinity conjugation (avidin–biotin). Physical absorption based on electrostatic interactions is the simplest method for depositing of aptamers on solid surfaces. However, the low stability of the electrostatically deposited layer caused by the rapid desorption of the aptamers from the surface is problematic in this method. The solution may be additional stabilization by covalent bonding ^[21][24]. The second very common method of attaching aptamers to solid surfaces is chemisorption. Aptamers are covalently deposited on the surfaces of the solid electrodes depending on the type of substrate and its functionalization. However, they do not spontaneously form covalent bonds with the electrode surfaces. Therefore, it is necessary to functionalize either the aptamer or the electrode surface ^[22][25]. Possibly the most popular approach is the chemisorption of a thiol-modified aptamer on a gold surface ^[23][24][26,27]. However, this method is unstable over time. Generally, aptamers are co-immobilized on the gold electrode surface with alkanethiols ^[25][26][28,29], dithiols ^[27][28][30,31] or anti-biofouling agents ^[29][30][32,33] to fill unmodified areas. One of the most effective approaches to aptamer immobilization is based on the strong affinity of biotin to avidin ^[31][34], streptavidin ^[32][35] and neutravidin ^[33][36]. Compared to other aptamer immobilization methods, the high affinity of biotin to streptavidin increases the potential number of aptamers that can be deposited on the surface of the detection platform. This is because each streptavidin molecule can bind to two biotinylated aptamers. This implies a reduction in non-specific adsorption and an improvement in the signal-to-noise ratio (S/N) of the biosensor ^[34][37]. The appropriate preparation of the detection platform ensures the low sensitivity and high selectivity of the constructed aptasensor. Therefore, it is important to implement a signal amplification strategy through the use of functional nanomaterials in the detection layer ^[35][36][38,39]. A wide range of nanomaterials, including silica nanoparticles ^[37][40] and precious metals ^[38][39][41,42], carbon nanomaterials ^[40][41][43,44], nanocomposites and nanohybrids ^[42][43][45,46], polymers ^[44][45][47,48] and metal oxides ^[46][49], can be used in the construction of electrochemical aptasensors for biomedical applications. Advances in electrochemical aptasensors have resulted not only from the use of nanomaterials but also from the interdisciplinary combination of nanotechnology, engineering, nanomaterial chemistry and related fields ^{[3][47][48][49][50][51][52]}[3,50,51,52,53,54,55]. Recently, there is a huge demand for new portable analytical devices with better selectivity and specificity for health-care applications, mainly for addressing the point-of-care (POC) detection of biomarkers for chronic and emergent diseases such as cancer, neurodegenerative disorders, cardiovascular diseases or chronic respiratory diseases [3]. This represents the main reason for the development of innovative techniques for the modification and immobilization of aptamers as biorecognition molecules with unique properties in the field of electrochemical aptasensors ^[53][54][56,57]. Nevertheless, their numerous assets and significant advances have not yet resulted in their entry to the market of electrochemical aptasensors ^[55][56][57][58,59,60]. Electrochemical aptasensors are widely used in the detection of ND biomarkers due to their numerous advantages such as their speed of analysis, low cost, easy operation and high miniaturization potential.

2. Electrochemical Aptasensors for the Detection of Neurodegenerative Diseases Biomarkers

A measurable change arising in biological surroundings such as human tissues or body fluids is defined as a biomarker. This change should arise from the pathological condition or response of the body to treatment when assessing the effectiveness of pharmacological therapy ^[58][59][61,62]. Thus, biomarkers are compounds that need to reflect the basic neuropathological features of a disease, and their determination should be conducted by means of a safe, easy and quick diagnostic test ^[60][63].

There has recently been great progress in the determination of biomarkers aimed at detecting these diseases in the phase preceding the appearance of distinctive clinical symptoms ^[61][62][66,67]. Enzyme-linked immunosorbent assay (ELISA) ^[63][64][65][68,69,70], magnetic resonance imaging (MRI) ^[66][71], manganese-enhanced magnetic resonance imaging (MEMRI) ^[67][72], mass spectrometry (MS) ^[68][69][73,74], flexible multi-analyte profiling (xMAP) ^[70][75], positron emission tomography (PET) ^[71][72][76,77], surface plasmon resonance ^[73][78], Western blotting ^[74][75][79,80], scanning tunneling electron microscopy ^[76][81], immunohistochemistry ^[77][78][79][82,83,84] and fluorescence ^[80][81][82][85,86,87] are some of the major techniques used for the detection of biomarkers of NDs. Despite their numerous advantages, these methods are relatively expensive, time-consuming and difficult to access, and they often require specialized equipment, qualified personnel and large volumes of samples. Moreover, they are not yet suitable for POC diagnostics.

The development of easy-of-use electrochemical aptamer-based platforms with a high sensitivity and specificity is probably one of the most promising methods to solve some of the problems regarding fast and cost-effective measurements appropriate for POC diagnostics due to their potential in terms of their portability and novel microfabrication technologies ^{[83][84][85][86]}[88,89,90,91].

Recently developed sensing platforms for electrochemical detection of biomarkers of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and prion diseases are summarized in Table 1.

Biomarker	Biorecognition Element	Techniques	Linear Range of Detection	LOD	Ref.
α-syn oligomer	DNA Apt	DPV	6 × 10−11–1.5 × 10−7 M	10−11 M	[87][102]
	SH-DNA Apt	EIS	–	10−12 M	[88][103]
	DNA Apt-MB	DPV	10−15–10−9 M	6.4 × 10−16 M	[89][104]
	NH2-DNA Apt	EIS	10−19–10−14 M	7 × 10−20 M (buffer) 9 × 10−20 M (plasma)	[90][105]
Total α-syn	DNA Apt-AuNPs conjugate	Vm	10−11–10−6 M	10−11 M	[91][106]
Aβ	RNA Apt	DPV	0.002–1.28 ng/mL	0.4 pg/mL	[92][114]
AβOs	Ab-DNA Apt sandwich	DPV	–	10−10 M	[93][115]
	DNA Apt-AuNFs	DPV	10−9–2 × 10−6 M	4.5 × 10−10 M	[94][116]
	DNA Apt	EIS	10−10–5 × 10−7 M	3 × 10−11 M	[95][119]
	Apt-Poly T-CuNPs	DPV	10−11–2.2 × 10−9 M	3.5 × 10−12 M	[96][117]
	DNA Apt1 Apt2	LSV	10−12–10−8 M	4.3 × 10−13 M	[97][118]
	SH–stem-loop DNA Apt-Fc	ACV	10−12–2 × 10−7 M	3 × 10−13 M	[98][121]
	NH2-DNA Apt	DPV	4.43 × 10−14–4.43 × 10−6 M	10−14 M	[99][122]
	DNA Apt–Fc	ACV	10−13–1.5 × 10−6 M	2 × 10−15 M	[100][120]
	THAS	DPV	10−15–10−11 M	5 × 10−16 M	[101][123]
	SH-DNA Apt	DPV	5 × 10−16–5 × 10−13 M	2.5 × 10−16 M	[102][124]
	SiO2Ag- DNA Apt bioconjugate	DPV	5 pg/mL–10 ng/mL	1.22 pg/mL	[103][125]
	SH-DNA Apt	DPV	0.5–10 pg/mL –	160 fg/mL (buffer) 900 fg/mL (serum)	[104][126]
	DNA Apt–SnS2 NSs	EIS	10−4–103 ng/mL –	238.9 fg/mL (PB) 56.9 fg/mL (HS)	[105][127]
AβOs(40)	DNA Apt	SWV	0.100–1.00 µM	9.3 × 10−11 M	[106][128]
Tau-381 protein	NH2-DNA Apt	DPV	1.0–10−10 M	7 × 10−13 M	[107][136]
	Anti-tau Ab + tau-381 DNA Apt	DPV	0.5–10−10 M	4.2 × 10−13 M	[108][137]
	NH2-DNA Apt–MWCNTs	EIS	10−15–1 × 10−9 M	10−15 M	[109][138]
	SH-DNA Apt–VG@Au	DPV	0.1 pg/mL–1–1 ng/mL	0.034 pg/mL	[110][139]
PrPC	Biot DNA Apt with dT15 spacer	DPV	10−12–10−9 M	8 × 10−13 M	[111][147]
	Biot DNA Apt with dT15 spacer	CV	10−12–10−5 M	5 × 10−13 M	[112][150]
	MB DNA Apt and FcA	SWV	2 × 10−13–10−5 M	1.6 × 10−13 M	[113][142]
	DNA1 Apt and DNA2 Apt	SWV	2 × 10−14–2.8 × 10−13 M	7.6 × 10−15 M	[114][151]