Trends in Analytical Chemistry 172 (2024) 117549

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Advancements in magnetic aptasensors: Recent progress and future trends

in biosensor technology
Milad Baghal Behyar a, Azadeh Nilghaz b, c, Rokhsareh Ebrahimi e, Mohammad Hasanzadeh d, *,
Nasrin Shadjou f
a
Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b
Institute of Frontier Materials, Deakin University, Waurn Ponds, VIC, 3216, Australia
c
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia
d
Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
e
Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
f
Department of Nanotechnology, Faculty of Chemistry, Urmia University, Urmia, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Biosensors have been explored for their application in analytical assays with an increasing reliance on nano­
Magnetic nanoparticles materials and biomolecule recognition units. Recently, magnetic nanoparticle-aptamer (MNPA) hybrids found
Aptamers crucial for their strengths in target binding, low limit of detection (LOD), enhanced sensitivity, exceptional
Aptasensors
stability, and superior selectivity. They have been employed for the identification of a diverse range of analytes
Biosensing
Biological samples
in biological, food, and environmental samples. However, their integration with conventional sensing technol­
Food ogies remains a challenge. Herein, the current state-of-the-art of MNPA sensors are reviewed. First, the signifi­
Environmental samples cance of using MNP and aptamers in biosensors is discussed and then the technologies incorporating MNPAs to
achieve higher sensitivity and selectivity in quantifying analytical samples are introduced. Finally, prospects and
practical approaches to address the existing drawbacks of MNPA sensors are presented.

1. Introduction the diversity and enrichment of aptamer pools [9]. The process of in
silico aptamer design involves the use of computational algorithms and
1.1. Aptamers and their significance in analytical applications modelling to predict sequences in aptamers with precise binding prop­
erties [10]. The process of structure-guided aptamer design involves
Aptamers are single-stranded DNA or RNA molecules, commonly utilizing structural information of the target molecule to strategically
synthesized through a meticulous in-vitro selection procedure known as engineer aptamers with the capability to interact with specific binding
the systematic evolution of ligands by exponential enrichment (SELEX) sites [11]. In contrast to the conventional base pairing interactions in
[1–3]. In this method, various fragments of random nucleic acids are nucleic acids, aptamers possess the ability to adopt intricate
incubated with target molecules to form specific aptamers followed by three-dimensional (3D) structures with a high degree of specificity and
washing the unbound fragments away. The bound fragments are sub­ sensitivity, driven by their inherent self-annealing characteristics [12].
sequently amplified to generate aptamers for the specific target mole­ During the selection process, the binding affinity and selectivity of
cule [4]. Non-SELEX methods —diverged from the conventional aptamers can be intentionally modified by adjusting their sequence and
SELEX— are also employed as alternatives for aptamer selection such as optimizing their 3D structure, offering a distinct advantage over anti­
a high-throughput sequencing-based selection [6], an in silico aptamer bodies [13,14]. Various techniques are frequently employed to assess
design [7], and a structure-guided aptamer design [8]. These methods the binding affinity of aptamers towards their respective target mole­
are designed to optimize the aptamer selection process and enhance the cules, offering quantitative insights into the robustness of the
diversity and specificity of the resulting aptamers while minimizing the aptamer-target interactions [15]. These techniques include surface
associated time and cost [5]. The high-throughput sequencing-based plasmon resonance (SPR) [16], isothermal titration calorimetry (ITC)
selection utilizes next-generation sequencing technologies to evaluate [17], fluorescence-based assays [18], electrophoretic mobility shift

* Corresponding author. Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
E-mail address: [email protected] (M. Hasanzadeh).

https://doi.org/10.1016/j.trac.2024.117549
Received 3 December 2023; Received in revised form 17 January 2024; Accepted 17 January 2024
Available online 26 January 2024
0165-9936/© 2024 Elsevier B.V. All rights reserved.
M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

assays (EMSA) [19], and atomic force microscopy (AFM) [20]. Aptamers characterized as magnetic, ferromagnetic (FM), or ferrimagnetic (FI)
possess inherent stability, allowing them to undergo multiple denatur­ [65–67]. N’eel first discovered that iron particles with diameters below
ation and renaturation cycles, facilitating a simple regeneration of the 32 nm exhibited remarkable resistance to external forces, revealing their
sensor surface [21,22]. This property of aptamers is attributed to the inherent ability to tolerate significant pressure [68]. MNPs were later
strong covalent bonds that connect their nucleotide subunits. Further­ employed as magnetic fluids, catalysis, biotechnology/biomedicine,
more, the secondary and tertiary structures adopted by aptamers magnetic resonance imaging (MRI), information storage, and environ­
improve their stability by enabling the formation of enduring hydrogen mental remediation [69,70], where optimal performance was achieved
bonds and other non-covalent interactions within their 3D folds. These with the particle size remaining below the critical threshold depending
characteristics made aptamers attractive recognition units for bio­ on the materials-typically ranging from 10 to 20 nm [71]. Within this
sensing applications [23–25]. Aptamers show properties comparable to range, NPs transform into a singular magnetic field, and while the
antibodies towards their target molecules [26], with further benefits temperature surpasses the designated block temperature, they demon­
such as their facile synthesis, minimal variability between batches, and strate characteristics of a super-magnetism [72]. The individual NPs
reduced immunogenicity [27,28]. Additionally, aptamers can be easily exhibit a broad and consistent magnetic moment, resembling a robust
decorated by various functional groups and/or labels to facilitate their paramagnetic atom. They show rapid responses to external magnetic
implementation in a wide range of sensing platforms, including elec­ fields, with minimal residual flux density and vorticity (the magnetic
trochemical (EC) [29,30], electrochemiluminescence (ECL) [31], pho­ field strength required to cancel out magnetization) [73]. Super­
toelectrochemical (PEC) [32], optical [33], and field effect transistors paramagnetic NPs are highly desirable for applications requiring mini­
(FETs) [34] sensors. mal mass formation at ambient temperature [74]. MNPs in general show
a wide range of potential applications in biological reserach, including
1.2. Applications of aptamers cell isolation, enzyme immobilization, drug delivery, and protein puri­
fication [75–77]. These particles display a distinct characteristic of
Aptamers are primary sensing components in aptasensors, wherein being subject to external magnetic field manipulation, while also being
they are immobilized on a transducers’ surface (e.g., electrode or optical capable of replication in the absence of such influence [78].
platform) to enable the detection of target molecules [35]. Various
methods of immobilization such as physical adsorption [36], covalent 1.4.1. Magnetic and physical characteristics of MNPs
attachment [37], and encapsulation within a matrix [38] are employed The physical stability of MNPs is primarily determined by their
to maintain the conformational integrity and binding activity of morphology. The size of the particles significantly influences their
aptamers while providing a stable interface for target recognition. The magnetic torque, thereby affecting their behavior in response to a
immobilization strategy also needs to preserve the functionality of the magnetic field [79,80]. Decreasing the particle size results in an
aptamer, ensuring its effectiveness in the target binding [39]. Upon augmentation of their surface area, which in turn can induce alterations
binding aptamers to the target molecule, a distinctive alteration in the in their non-crystalline characteristics and subsequently impact their
aptamer’s structure occurs, resulting in measurable signals and insights magnetic moment [81]. This phenomenon is more obvious in iron oxide
into the concentration of the target molecule within a sample analyte particles which are commonly used in MRI and the particle size plays a
[40,41]. Aptasensors present numerous advantages in comparison to crucial role in determining the signal strength and quality [82].
other types of biosensors [42–44]. They possess the capability to selec­ Among various MNPs, ferri, superparamagnetic, and ferro particles
tively bind to a variety of target molecules, including small molecules find broad applications in drug delivery, showing enhanced sensitivity
[45], proteins [46], and whole cells [47], which makes them suitable for to external magnetic fields due to the magnetic moment of the network
various environmental [48], medical [49], and food applications [50]. unit and the arrangement of the domains [83,84]. As a result, they
Furthermore, aptamers possess inherent stability and reproducibility experience an inert state in the absence of an external magnetic field.
due to their tightly folded 3D structure which results in limited solvent The distinctive characteristics of MNPs contribute significantly to their
exposure and subsequent long-term storage and repeated utilization unique properties including their single-domain nature and super­
[51]. paramagnetic behavior [85]. The width of the domain walls plays a
crucial role in the separation of directional spins within a coherent area.
1.3. Integration of nanomaterials in aptasensor The development and sustainability of these domains are intricately
associated with the energy consumption. However, upon reaching a
Nanomaterials are also found of great interest and importance in the critical diameter, the reduction in particle size facilitates the formation
development of aptasensors due to their unique characteristics [52–55]. of individual particles exhibiting singular amplitudes. In such cases, the
Their high surface-to-volume ratio provides an increased number of formation of walls becomes unsuitable in terms of energy efficiency. The
binding sites for aptamers, with the potential enhancement in the sen­ comprehension of magnetic characteristics in NPs holds significance in
sor’s sensitivity [56,57]. The functionalization of nanomaterials with elucidating the underlying mechanisms of MNPs, as well as facilitating
various chemical groups or biomolecules also imparts a high degree of their precise design and control. The field of MNPs shows promising
selectivity for the target analyte by introducing affinity and recognition prospects, attributed to the reduction in magnetic fields, which conse­
properties, thus reducing the chance of false-positive or false-negative quently leads to the emergence of superparamagnetic properties [86].
outcomes [58–61]. Signal amplification can be achieved through their The superparamagnetic characteristics of NPs are intricately influ­
interactions with aptamers and target molecules. For instance, gold enced by the magnetic anisotropy shown by these particles. Notably, the
nanoparticles (AuNPs), with localized SPR, undergo a substantial orientation of the magnetic moment of NPs along the easy crystal axis
alteration in their optical characteristics upon binding, resulting in the leads to the reduction of anisotropic magnetic energy (Ea). Specifically,
signal amplification and the detection of target molecules at remarkably in spherical MNPs, the magnetic crystal anisotropy is equivalent to the
lower concentrations [62,63]. Finally, nanomaterials can increase the overall magnetic anisotropy, hindering the alteration of the magnetic
stability and durability of aptasensors by improving the mechanical and orientation. As the size of the NP decreases to a critical threshold, the Ea
structural integrity of the sensors [64]. approaches a level comparable to the thermal energy required for acti­
vation. Despite the low anisotropic energy barrier, the magnetic orien­
1.4. Magnetic nanoparticles (MNPs) tation of the NPs can be readily changed through the influence of an
external magnetic field or thermal activation energy. The behaviour of
MNPs refer to a category of particles that typically range in size from MNPs closely resembles superparamagnetic atoms. Although the mag­
nanoscale to microscale and possess a magnetic structure that can be netic properties of individual NPs are relatively low, each particle

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behaves akin to a paramagnetic atom, albeit with a significantly larger and saline environments, rendering them highly appropriate for binding
magnetic moment. This phenomenon is referred to as super­ to cancer bio-markers under various extrinsic and intrinsic conditions
paramagnetism, in which the magnetic orientation of NPs undergoes [123].
rapid fluctuations, as opposed to aligning consistently in a specific di­ Various polymers, including polyvinyl pyrrolidone, polyethylene
rection. Therefore, the blocking temperature refers to the thermal glycol, polylactic-co-glycolic acid, polyethylene-co-vinyl acetate, and
threshold at which the magnetic anisotropic Ea of NPs becomes domi­ PVA, are commonly employed as coating materials in liquid suspensions
nant, surpassing the impact of the thermal activation [79,87]. [124]. Plus, NPs synthesized in a liquid medium can be coated with
various natural substances, including polylactic acid, dextran, gelatine,
1.4.2. Applications of MNPs in analytical assays starch, albumin, liposomes, chitosan, and ethyl cellulose [125]. The
MNPs possess numerous benefits including unique physicochemical choice of MNP coating is contingent upon its intended application.
properties, dimensional characteristics, and cost-effectiveness in However, it is crucial to prioritize the biocompatibility of the coating
manufacturing [88]. MNPs demonstrate optimal performance in the size materials due to their direct exposure to biological agents. Numerous
range of 10–20 nm, owing to their supermagnetic properties and the experiments have been conducted to investigate such issues, including
surface-to-volume ratio [89,90]. This makes them suitable for applica­ the utilization of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte­
tions requiring rapid responses to applied magnetic fields. As their trazolium bromide (MTT) assay for cell viability test upon exposure to
characteristics are closely linked to their dimensions, optimized tech­ NPs [126]. Dextran is more widely utilized owing to its long-lasting
niques such as controlled co-precipitation [92], thermal decomposition properties, high biocompatibility, and low molecular weight in the
[93], high-energy ball milling [94] are necessary to synthesize particles bloodstream [127]. Surface silanization is a widely employed technique
with desired physicochemical properties [91]. These MNPs with right for the functionalization of MNPs. This approach offers numerous ad­
characteristics have found diverse applications in various fields vantages, including neutrality in oxidation, low toxicity, and reduction
including wastewater treatment, sample preparation, disease therapy, reactions, good stability under acidic conditions, and facile chemical
water purification, cell labelling and imaging, disease diagnosis through modification. It can also be carried out in aqueous environments and
MRI, tissue engineering, biosensors, and other detection systems [95]. organic solvents, even under moderate temperatures, without a
Due to all these characteristics, MNPs has recently attracted significant requirement for any specific conditions or costly apparatus. Hence, this
attention in aptasensors. approach presents an optimal means of safeguarding the internal mag­
netic core. This process culminates in the establishment of a stable bond
1.4.3. Functionalization strategies of MNPs on the surface. Given the excellent chemical bonding properties of si­
MNPs undergo surface functionalization involving modifications lanes, they serve as an ideal substrate for stabilizing aptamers [128].
such as coating, conjugation, and surface chemistry adjustments, to The functionalization of MNPs provide a range of advantageous
make them suitable for various applications [96]. For instance, MNPs properties. Firstly, it enables the binding of biomolecules, including
should possess a smaller size range of 50–160 nm, uniform dispersion, proteins, antibodies, ligands, and aptamers. This modification allows the
non-toxic characteristics, stability, and high surface area to volume ratio specific identification of different target molecules. Furthermore, the
as well as magnetic values. However, under practical conditions, MNPs application of organic and inorganic molecules as coatings on the sur­
exhibit limited stability when exposed to highly acidic conditions and face of MNPs has been found to significantly enhance their half-life by
are susceptible to leaching [97]. This phenomenon significantly ham­ delaying their clearance [129]. The reticuloendothelial system,
pers their potential for reuse and diminishes their overall lifespan. Their encompassing the spleen, liver, and inner lining of the bone marrow
high surface area to volume ratio is associated with the accumulation of helps in the elimination of these particles. Uncoated MNPs are promptly
particles and a decrease in surface energy. The strong interparticle recognized and cleared by phagocytic cells [130]. However, the intro­
attraction hinders the dispersion of NPs in both aqueous solutions and duction of coatings alters the surface properties such as hydrophobicity,
substrates. Therefore, the immobilization of proteins, enzymes, and surface charge, and pH, leading to a delay in particle clearance. This
aptamers on NPs is diminished. To address these issues, surface func­ modification in surface characteristics subsequently influences the
tionalization can generate MNPs favourable and enduring characteris­ interaction between MNPs and the cleansing system, resulting in
tics [98] by incorporating diverse functional groups onto their surfaces extended retention within the biological system. Finally, the use of
[99]. This process can be accomplished through a variety of chemical surface coatings with high electron transfer capability will increase the
reactions, including silanization [100], carboxylation [101], and ami­ electrical conductivity, useful in biosensing applications [131].
nation [102] as well as polymer [98,103], silica [104], or metal oxide
coatings [105] which help with their stability, biocompatibility, and 1.5. Rationale for analyte sensing in food, biological, and environmental
functionality, while providing a platform for further conjugations [106]. samples
Conjugation serves as a means of functionalizing MNPs through the
attachment of specific biomolecules, including antibodies, peptides, or The analyte sensing in food, biological, and environmental samples is
enzymes, onto their surfaces [110–112]. crucial for various reasons [132]. Biological samples including serum,
Coating can be accomplished using techniques such as sol-gel [107], urine, blood, and tissues provide invaluable insights into the physio­
emulsion polymerization [108], or layer-by-layer assembly [109]. logical status of the body, allowing disease diagnosis, treatment
Various types of coatings such as siloxane [113], polymer [114], assessment, and understanding of biological mechanisms [133]. Food
bi-functional ligands [115], sometimes 2,3-dimercaptosuccinic acid analysis ensures the safety, quality, and authenticity of food products.
(DMSA) [116], and phospholipid micelles [117], inorganic materials The presence of contaminants, such as pesticides, pathogens, allergens,
(like gold) [118], activated carbon [119], metal oxides [120], or heavy metals, can result in foodborne illnesses or even death, posing
aluminium [121] and silica oxide [122] are used to modify the surface significant risks to public health. The detection of these analytes is
charge of particles, enhance biocompatibility, prolong their lifespan in essential in identifying and controlling potential hazards, preventing
the biological body fluids such as blood, and enhance particles perfor­ outbreaks, and ensuring compliance with food regulations [134–138].
mance [95]. For example, DMSA forms carboxylate chelates with iron Environmental samples, such as water, air, plants, and soil are essential
ions. This property enables the creation of disulfide crosslinking be­ in the evaluation of environmental integrity, identifying pollutants (e.g.,
tween the ligands involved. Furthermore, DMSA facilitates the intro­ pathogenic microorganisms, hazardous chemicals, and heavy metals),
duction of thiol groups onto the surface, which in turn enables precise and understanding their impact on human health and ecosystems [139,
and selective binding to specific biomarkers. These particles exhibit 140]. Therefore, the development of sensitive, specific, rapid, and
suitable stability in aqueous solutions, including saline phosphate buffer cost-efficient biosensors holds significant importance in addressing

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potential obstacles associated with the detection of analytes in various of aptamers modified with amino groups. This conjugation strategy
samples [141]. MNPAs possess distinctive attributes that make them aimed to enhance the selectivity and sensitivity of the nanocomposite by
highly advantageous for the development of biosensors with superior increasing the effective surface area available for aptamer binding. They
diagnostic efficiency [142–193]. In this review, we aim to explore the employed two distinct EC detection methods to assess endotoxin levels:
impact of MNPAs on the analytical efficacy of biosensors in detecting a labelled approach utilizing methylene blue (MB) and a label-free
analytes present in food, biological, and environmental samples. strategy employing ferrocyanide. The aptasensor demonstrated an
impressive LOD, reaching as low as 0.2 and 4 fg mL− 1 for MB and
2. Biosensors based on MNPAs for analyte sensing ferrocyanide, respectively. Moreover, it exhibited a linear range of
detection from 0.01 to 0.09 pg mL− 1 for MB and from 0.1 to 0.9 pg mL− 1
2.1. Electrochemical (EC) sensing for ferrocyanide. The aptasensor demonstrated a robust and reliable
response when exposed to both the serum of patients and that of healthy
EC analysis has emerged as a promising and economically feasible individuals, thereby confirming its suitability for medical applications.
technique for the detection of electroactive compounds in analyte so­ Pourmadadi and co-workers [216], also developed a sensitive apta­
lutions [194–196]. EC analysis provides the distinct advantage of sensor to identify CA 15-3. The surface of the electrode was modified
facilitating both qualitative and quantitative analysis, rendering it a with g-C3N4/Fe3O4 NPs to immobilize aptamer chains, thereby
user-friendly and straightforward approach [197,198]. Its working enhancing both the active surface area of the electrode and electrical
mechanism is based on the oxidation-reduction reactions, which occur conductivity.
at the interface between the sensor regions and the molecules of interest The developed platform was evaluated by square wave voltammetry
[199–201]. The sensing efficiency of EC has been proven in various (SWV) with a linear range of 1–9 UmL− 1 and a LOD of 0.2 UmL− 1. The
analytes, such as diclofenac, endotoxin, leukemia cancer cells, breast platform has been employed as a label-free sensor in a medium con­
cancer biomarkers (CA 15-3), and cortisol, among others [202–204]. taining potassium ferrocyanide. Additionally, it has been utilized with
The applications of MNPs have garnered significant attention in MB labelling in a phosphate buffer medium. The electrode demonstrated
biosensors due to their surface functionalization capability, facile sol­ successful application in the analysis of serum samples from both pa­
vent separation using a magnet, and extensive surface area [205]. MNPs tients and healthy individuals, thereby confirming its promising poten­
also facilitate electron transfer and thus increase the sensitivity of the EC tial for biosensing applications. Baghayeri and colleagues [217],
biosensors [206]. Furthermore, the combination of MNP with aptamer developed an EC aptasensor for label-free detection of bisphenol A
leads to the emergence of very sensitive and selective biosensors. For (BPA). They used a combination of AuNPs, functional copper MNPs
example, Rohani et al. [207], synthesized MNPs using polydopamine (CuFe2O4-SH), and MWCNTs that were modified with 6-mercapto-1-­
(PDA) — a polymer derived from the polymerization of dopamine hexanol (MCH) and aptamers before introducing them on a glassy car­
molecules — under optimized pH conditions. Subsequently, the surface bon electrode (GCE). The obtained results exhibited a satisfactory
of Fe3O4 MNPs was coated with PDA to form PDA@Fe3O4. The prepared linearity index ranging from 0.05 to 9 nM along with a low LOD of 25.2
material was utilized to modify carbon ceramic electrodes (CCEs) pM for the accurate quantification of BPA. The EC tests revealed that the
through the formation of covalent bonds. They employed an effective presence of gold NPs in conjunction with MNPs and MWCNTs within the
approach to immobilize a diclofenac-specific ssDNA aptamer onto the nanocomposite resulted in a synergistic enhancement on the modified
surface of PDA@Fe3O4 modified CCE. The developed label-free apta­ electrode’s surface. This enhancement significantly facilitated the effi­
sensor was successfully employed for trace-level detection of sodium cient detection of BPA. Furthermore, the proposed apta-platform shows
diclofenac in human blood serum. This was achieved using the differ­ significant potential for application in medical diagnostics and the food
ential pulse voltammetry (DPV) technique in a signal-off approach. The industry, where the rapid and accurate detection of BPA holds promise
estimated LOD for diclofenac detection was around 0.11 nM, exhibiting for ensuring public health. Additionally, Beiranvand et al. [218], used
notable stability and repeatability within the concentration range of NH2 functionalized Fe3O4/gold NPs-decorated CNTs for the detection of
0.5–400 nM. BPA (Fig. 1). The surface immobilization of an animated detection
A novel approach was also developed for the identification of probe, specifically a DNA aptamer, was achieved on a surface composed
thrombin biomarkers using a magnetic force-assisted EC aptamer- of NH2-functionalized Fe3O4/gold NPs. This immobilization was facili­
antibody sandwich assay [208]. The detection was based on cathodic tated through the utilization of glutaraldehyde as a linker. The apta­
currents generated by a complex formed between toluidine blue O (TBO) sensor encompasses the synergistic utilization of the precisely arranged
and thrombin antibody-modified MNPs immobilized on the electrode Fe3O4/gold NPs and the covalent immobilization of the detection probe
surface. To apply the method in a serum sample, a specific aptamer to onto the sensing interface. The analytical performance of the developed
thrombin was employed which was immobilized onto a conducting EC aptasensor was evaluated using the DPV technique. The detection of
polymer layer known as poly-(2,2’:5’,5″-terthiophene-3’-p-benzoic acid) BPA was quantitatively determined by monitoring the changes in the
(pTBA). To enhance the detection process, streptavidin, and DPV current signal at various concentrations of BPA ranging from 1 to
starch-coated MNPs were conjugated with biotinylated thrombin anti­ 600 nM. As anticipated, the current intensity exhibited a decrease as the
bodies and TBO, facilitating efficient amperometry-based detection. The concentration of the target analyte increased. This behaviour can be
results showed that there is a linear relationship between thrombin attributed to the formation of a higher number of BPA-aptamer com­
concentration in the range of 1–500 nM and current intensity. The plexes on the surface of the modified electrode. In addition, the prepared
integration of MNPs and aptamers in analytical techniques holds great platform was used to measure the target analyte in environmental water
promise, offering significant advancements in various applications. samples and was able to show recovery in the range of 95.8–103.4 %.
Specifically, carbon-based MNPs (e.g., rGO [209], graphitic carbon EC aptasensors were also developed by surface modification of a GCE
nitride (g-C3N4) [210], multi-walled carbon nanotubes (MWCNTs) using a porous carbon nanomaterial called Z-1000 with an average
[211], carbon nanotubes (CNTs) [212], carbon porous structures [213]) particle size of 70 nm [219]. The particles were synthesized through the
show a range of beneficial characteristics for use in aptasensors, carbonization process of zinc(II)-2 methylimidazole metal-organic
including exceptional electrical conductivity, substantial surface area, framework (MOF). They possess an outstanding EC property and a
remarkable chemical stability, cost-effectiveness, and favourable large specific surface. The immobilization of a thrombin-binding
biocompatibility [214]. For instance, Zamani and colleagues [215], aptamer (CP) onto MNPs was achieved through a condensation reac­
engineered a sensitive EC sensor for dual detection of lipopolysaccharide tion. Subsequently, the CP-MNPs conjugate was further coupled with a
(LPS) or endotoxin by employing thiourea as a conjugating agent to reporter probe (RP) that was modified with electroactive MB. In the
covalently attach a magnetic graphene-based nanocomposite with a pair presence of thrombin, the identification factor known as CP showed a

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Fig. 1. Schematic illustration of A) the synthesis process of NH2-functionalized Fe3O4/gold NPs and B) the various stages involved in fabricating the aptasensor
[218]. Reprint with permission from Elsevier Science, 2017.

specific binding affinity towards thrombin, leading to the formation of carbon dots (GQDs), fullerene C60, single-walled CNTs (SWCNTs), car­
the CP/MNP/thrombin complex. Consequently, the interaction between bon dots (CDs), etc in aptasensors. The incorporation of MNPAs into
the RP and MNPs was disrupted, resulting in the dissociation of RPs from composite systems alongside other nanomaterials gives them a funda­
MNPs. The released RPs were effectively captured by the modified GCE mental breach to their physicochemical properties. This breach can be
through π-stacking interactions occurring between the carbon nano­ attributed to the interactions of MNPs with other constituents of the
structure and nucleobases. The electrical signals produced by MB were composite, thereby potentially compromising the desired analysis
effectively monitored by DPV technique, wherein its peak current was sensitivity and selectivity, as well as overall performance. In addition,
directly proportional with the concentrations of thrombin. The platform under these conditions, the magnetic properties of MNPs may change.
exhibited an LOD of 0.8 fM for thrombin, along with a linear range from To deal with these possible issues, it is recommended that the properties
10 fM to 100 nM. Notably, this approach was effectively employed for of the obtained nano-hybrids be carefully investigated by the re­
the analysis of serum samples spiked with thrombin. The recoveries searchers. Additionally, techniques such as surface modification of
achieved in these experiments were ranged from 98.1 to 99.4 %, with MNPs can be very effective. It is important to acknowledge that EC
relative standard deviations (RSDs) of 3.9–4.0 %. A list of recently sensors utilizing MNPA have primarily been focused on the detection of
developed MNPA-based EC sensors was summarized in Table 1. analytes in biological samples, while comparatively less emphasis has
As shown in Table 1, MNPA-based EC sensors have been developed been placed on their application for identifying analytes in food and
for a broad range of sample analytes with the main emphasis on the environmental samples. This discrepancy highlights the need for further
voltammetric technique than amperometric or impedimetric ones. In exploration and development of EC aptasensors for the analysis of
addition, it revealed that carbon nanomaterials have the ability to analytes in these specific sample matrices.
enhance the performance of MNPAs. Therefore, it is recommended to
explore the use of other carbon-based nanomaterials such as graphene

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Table 1
Comparison of MNPA-based EC biosensors for the detection of different analytes.
Detection Technique Structure Biological Target Reale sample Detection Range LOD Ref
method Element

EC DPV Aptamer-PDA@Fe3O4- CCE Aptamer Diclofenac Human blood serum 0.5–400 nM 0.11 nM [207]
samples
EC Amperometric pTBA/aptamer/thrombin/ Aptamer, Thrombin Phosphate buffered 1–500 nM 0.49 nM [208]
MNP@antibody-TBO and saline and human
antibody serum sample
EC SWV rGO/magnetite/gold/LPS Aptamer Endotoxin Human blood serum 0.01–0.09 pg mL− 1 0.2 fg mL− 1 for MB [215]
specific ssDNA aptamer/MB/ samples for MB and 0.1–0.9 and 4 fg mL− 1 for
GCE pg mL− 1 for ferrocyanide
ferrocyanide
EC SWV Aptamer-based nano Aptamer CA 15-3 Serum samples 1 ‒ 9 UmL− 1 0.2 UmL− 1
[216]
biosensor with g -C3N4/
magnetic nano-structure
EC DPV MCH/aptamer/gold/ Aptamer BPA Mineral water, milk, 0.05–9 nM 0.025 nM [217]
CuFe2O4-Pr-SH/MWCNTs/ and juice samples
GCE
EC DPV Fe3O4/gold NPs/CNTs Aptamer BPA Environmental water 1–600 nM 300 pM [218]
samples
EC DPV Porous carbon materials and Aptamer Thrombin Human serum 10 fM – 100 nM 0.8 fM [219]
homogeneous detection samples
EC DPV Intercalation of ethidium Aptamer Leukemia Human blood plasma 10–106 cell mL− 1
10 cell mL− 1
[220]
bromide with aptamer cancer cells samples
conjugated Fe3O4@gold
1 1
EC SWV MNPA based sensor with tris Aptamer Tumor Human serum 10 pg mL− – 100 ng 10 pg mL− [221]
(2-carboxyethyl)phosphine necrosis samples mL− 1
hydrochloride -mediated factor-alpha
amplification

2.2. Optical sensing human urine and serum samples spiked with known concentrations,
yielded recovery rates ranging from 94.8 to 102 %. In another study, an
Optical assays have emerged as highly valuable analytical methods aptamer-functionalized magnetic adsorbent was integrated into mag­
for monitoring changes in light beam intensity or variations in their netic solid-phase extraction (MSPE) for specific improvement of BPA
phases upon interaction with physical systems [222,223]. Optical [242]. Silica-coated Fe3O4 microspheres (Fe3O4@SiO2) were synthe­
probes have demonstrated their advantages over conventional sensors, sized using the sol-gel method and subsequently modified with nucleic
offering cost-effectiveness, increased sensitivity, accelerated analysis, acids to create functional MNPs (Fe3O4@SiO2@aptamer). Magnetic
improved specificity [224], and suitability for a wide range of applica­ separation, followed by the elution of the aptamer/BPA composite
tions, including chemical sensing [225], environmental monitoring resulted in amplified fluorescence with the LOD of 0.05 ng mL− 1.
[226], and biomedical imaging [227]. Optical sensors are commonly Further, Yu and co-workers [243] developed a MNPA sensor for the
categorized into five distinct groups, each offering practical and valu­ detection of thrombin in bovine serum samples. The thrombin aptamer
able applications. These groups encompass colorimetric [228], fluores­ labelled with a fluorescent dye (CY3) was immobilized onto the surface
cence [229], surface-enhanced Raman scattering (SERS) [230], SPR of MNPs through the interaction between the phosphate backbone of the
[231], and Förster or fluorescence resonance energy transfer (FRET) CY3-aptamer and hydroxyl groups present on the bare MNPs. This
[232]. interaction resulted in the quenching of fluorescence. Upon the intro­
duction of thrombin, the CY3-aptamer underwent a conformational
2.2.1. Fluorescence sensing change, adopting a G-quartet structure. This structural alteration facil­
Fluorescence sensing is a technique that employs ultraviolet light to itated the binding of the CY3-aptamer to thrombin, leading to the
stimulate the electrons present in various materials such as dyes, detachment of the CY3-aptamer from the MNPs and subsequent resto­
nanomaterials, and oligonucleotides [233–235]. The fluorescence sig­ ration of fluorescence. This probe exploits the specific binding affinity
nals arising from the reversion to the base state are quantified using a between the CY3-aptamer and thrombin, thereby ensuring high speci­
fluorimeter [236–238]. Fluorescence sensors have gained widespread ficity. Furthermore, the incorporation of uncoated MNPs facilitates
popularity in analytical assays due to their enhanced sensitivity, time fluorescence quenching. The obtained signal in this report showed a
efficiency, and cost-effectiveness [239,240]. Incorporating aptamers suitable linear correlation with the concentration of the analyte within
into fluorescence sensors has been shown to further improve their per­ the range of 1–60 nM. The estimated LOD for thrombin was found to be
formance. For example, Liu et al. [241] developed a multifunctional as low as 0.5 nM.
fluorescent sensor for the monitoring of adenosine 5′-triphosphate (ATP) Niazi et al. [244], advanced this approach and developed a zear­
by immobilizing the 6-carboxyfluorescein-labelled aptamer (FAM-ap­ alenone (ZEN) aptamer conjugated with amine-functionalized MNPs as
tamer) onto the surface of PDA-coated magnetite NPs (Fe3O4@PDA) a capture probe, in conjunction with time-resolved fluorescence (TRFL)
through π-π stacking interaction. The sensor consisted of three distinct NPs labelled with complementary DNA (cDNA) as a signal probe. The
functionalities, namely recognition, magnetic separation, and fluores­ fluorescence intensity reached its peak when ZEN was absent, but
cence emission. In the presence of ATP, the FAM-aptamer located on the gradually decreased as the concentration of ZEN increased. This can be
sensor’s surface shows a binding affinity towards ATP and subsequently attributed to the binding of aptamers to ZEN, which weakens the affinity
dissociates back into the solution. Consequently, this interaction leads to between cDNA and aptamers. Consequently, some TRFL-NPs-cDNA
an amplification of fluorescence in the supernatant, allowing for a molecules were released from the TRFLNPs-MNPs composite. A better
quantification of ATP concentration. The response of the sensor exhibits correlation between the concentration of ZEN and fluorescence at a
linearity within the concentration range of 0.1–100 μM for ATP. Sub­ wavelength of 544 nm was observed. Notably, a high degree of linearity
sequent application of the sensor for the quantitative analysis of ATP in was observed within the range of 0.001–10 ng mL− 1 concentration of

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M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

ZEN. The biosensor was used to measure the target analyte in different ability to facilitate the oxidation of o-phenylenediamine, thereby
maize and wheat samples with a recovery ratio of 80.76–119.66 % and yielding o-benzoquinone diamine. In this study, aptamers and immu­
90.04–114.75 %, respectively. noglobulin Y (IgY) antibodies have been employed for the conjugation
Shi et al. [245], proposed an aptamer-based fluorescence probe using with magnetic beads and AgNCs, respectively. These conjugates have
CDs for the sensitive detection of β-lactoglobulin (β-LG). The aptamer been developed to bind Listeria monocytogenes at distinct, specific
was immobilized onto MNPs, while the CDs were utilized as a labelling binding sites. The disassembly of colloidal AuNPs leads to a discernible
agent for the cDNA. The assay relies on the occurrence of hybridization alteration in color, from blue to red. This transformation is characterized
between a cDNA and aptamer as its fundamental principle. In the by distinct peaks observed at wavelengths of 730 and 525 nm, respec­
presence of β-LG, the aptamer shows a preferential affinity towards tively. The proposed approach enables the colorimetric determination of
β-LG, resulting in a partial liberation of the CDs-cDNA into the sur­ the analyte within a dynamic range of 10 to 106 cfu mL− 1, without the
rounding solution. Following the process of magnetic separation, the need for pre-enrichment. The LOD achieved is as low as 10 cfu mL− 1.
remaining liquid fraction of the solution contains the liberated Recoveries within the range of 97.4–101.3 % were observed during the
CDs-cDNA entities. These entities are subsequently quantified using analysis of spiked food samples.
fluorometry, with optimal excitation and emission wavelengths of 354 In another attempt, a colorimetric assay was successfully developed
and 447 nm, respectively. Under optimized conditions, the fluorescence for the rapid and sensitive identification of Vibrio parahaemolyticus by
intensity exhibited a direct correlation with the concentration of β-LG employing MNPs and AuNPs [259]. Initially, the aptamer that shows a
within the linear range of 0.25–50 ng mL− 1. Notably, the LOD for β-LG response towards Vibrio parahaemolyticus was immobilized onto the
concentration is determined to be as low as 37 pg mL− 1. The proposed surface of MNPs, enabling its utilization as a highly specific magnetic
approach was effectively employed for quantifying β-LG in hypoaller­ separator. Furthermore, the aptamer was immobilized onto the surface
genic formulations, with further potential as a valuable device in of AuNPs as a colorimetric probe. In the presence of the target cell, a
ensuring food safety and quality control. Guo et al. [246], also devel­ sandwich configuration is established, consisting of MNP conjugated
oped a sandwich dual recognition unit, wherein the target analyte was with aptamer, followed by bacteria, another aptamer, and ultimately
efficiently captured using aptamer-functionalized MNPs and subse­ AuNPs. This assembly is facilitated by the specific sensing between the
quently identified through antibody recognition. The utilization of this Vibrio parahaemolyticus and the aptamer. The magnetic separation
MNPA exhibited promising advancements in addressing the method was subsequently employed to produce an identification signal.
aggregation-induced quenching phenomenon encountered during the By leveraging the unique optical characteristics of AuNPs, a discernible
fluorometric detection of Listeria monocytogenes with a LOD of 10 CFU visual signal was observed, leading to a colorimetric detection approach
mL− 1 and a linear range of 10–106 CFU mL− 1. Therefore, it can be that does not require sophisticated instrumentation. Under optimized
concluded that the incorporation of natural/synthetic biological com­ condition, the probe shows a linear correlation with analyte concen­
ponents such as cells, antibodies, enzymes, genes, and molecularly tration within the range of 10 to 106 cfu mL− 1, while demonstrating a
imprinted polymer (MIP) into aptamers contributes to achieving optimal LOD as low as 2.4 cfu mL− 1.
performance in fluorescence biosensors. MNPA-based colorimetric sensor has only been used to detect a
limited number of bacteria, so it is expected that this assay can be
2.2.2. Colorimetric sensing developed to detect more analytes. However, due to their specific ad­
Colorimetric sensing is a rapid optical method to detect a broad vantages such as simplicity, cost-effectiveness, and ease of interpreta­
range of analytes by employing chromogenic compounds/nanomaterials tion, their protentional for various applications worth more exploring.
as signal-labelling agents [247–251]. It is based on visual observation For instance, apart from AuNPs, AgNPs and quantum dots (QDs) can also
and measuring the color intensity of developed colors from a chemical be used along with MNPAs owing to their excellent optical properties. It
reaction, rendering it a valuable sensing platform for point-of-care is imperative to exercise utmost caution when combining QDs and
analysis [252–254]. Extensive research has been conducted on colori­ AgNPs with MNPAs to ensure the preservation of MNP properties
metric assays mainly using AuNPs due to their advantageous charac­ without inducing any violations.
teristics, including visual observability and straightforward
manipulation [255,256]. For instance, Wang and colleagues [257], 2.2.3. Surface-enhanced Raman scattering (SERS) sensing
introduced a colorimetric strategy based on host-guest interactions for SERS has emerged as an analytical technique with potential appli­
the specific identification of Listeria monocytogenes. The assay was cations in surface science, biomarker detection, food safety, and
developed through a self-assembly process and supramolecular in­ homeland security. The widespread adoption of SERS can be attributed
teractions between cucurbituril’s [7]carbonyl groups and AuNPs, to its inherent label-free nature, remarkable specificity, straightforward
completed by aptamer and urease-modified magnetic nanoparticles. implementation, and high-sensitivity [260,261]. The sensing method­
These MNPAs were designed to exhibit a high degree of specificity in ology employed in SERS revolves around the detection and quantifica­
recognizing and binding to Listeria monocytogenes. Additionally, they tion of Raman scattering signals emitted by analytes when they are close
were capable of hydrolyzing urea, leading to the generation of ammo­ to a textured metallic surface. Typically, this surface is fabricated using
nium ions (NH+ 4 ). This reaction facilitated the reversal of CB [7] induced noble metals like Ag or Au [262]. The combination of SERS sensors and
AuNPs aggregation. In the presence of Listeria monocytogenes, the mag­ MNPAs holds great potential for the advancement of analytical assays,
netic conjugates show a notable affinity for the bacterial surface. This offering further improvement in sensitivity and selectivity towards
interaction leads to the specific binding of the conjugates to the bacterial specific target analytes. In a simplified format, uncoated MNPAs are
cells, effectively obstructing the catalytic active sites. Consequently, the employed as biosensors. These biosensors are composed of bare MNPs
enzymatic production of ammonium ions is impeded. The ratio of that are functionalized with recognition elements. The lack of a coating
normalized absorbance at 700 nm and 525 nm was directly proportional allows a greater surface area for the recognition elements to bind to the
to the concentration of Listeria monocytogenes in the range of 10 to 106 target analyte, resulting in enhanced performance of the biosensor
cfu mL− 1. Listeria monocytogenes spiked food samples were analysed [263]. Using this format, He et al. [264], introduced a highly-sensitive
without pre-enrichment, resulting in recoveries ranging from 98.4 to aptasensor-based SERS substrate for the detection of microcystin-LR
99.3 %. Moreover, RSD of less than 10 % was obtained for these samples. (MC-LR). They employed a conjugation strategy to attach MC-LR
In another study, a colorimetric technique was developed for the aptamer and its corresponding cDNA to AuNPs and MNPs, respec­
detection of Listeria monocytogenes using de-aggregation of tively. Subsequently, to enhance the efficacy of the detection system,
AuNPs-mediated-o-phenylenediamine [258]. Silver nanoclusters they employed MC-LR aptamer-AuNPs and cDNA-MNPs as signal and
(AgNCs) have also been employed as an artificial enzyme, exhibiting the capture probes, respectively. The probe was validated by detecting

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M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

MC-LR in spiked tap water with a linear range from 0.01 to 200 ng mL− 1 Staphylococcus, using Fe3O4@gold/bacteria/SERS tags. The LODs for the
and a LOD of 0.002 ng mL− 1. platform were determined to be 25, 10, and 10 cells mL− 1 for Salmonella
Yang and colleagues [265] also introduced an aptamer-based SERS typhimurium, Listeria monocytogenes, and Escherichia coli, respectively,
substrate for the detection of prostate-specific antigen (PSA) by the when analyzing real food samples. In a similar study, Pang and
integration of MNPs and core-AuNP satellite assemblies. The strong and co-workers [268], have developed a SERS substrate for the dual recog­
specific binding affinity between the aptamer and PSA resulted in the nition of bacterial cells through the integration of aptamer and antibiotic
dissociation of Au core-satellite assemblies, wherein the concentration molecules (Fig. 2). They synthesized aptamer-Fe3O4@gold MNPs that
of functionalized AuNPs (signal probes) in the supernatant increased function as both magnetic and SERS-activated substrates for the specific
progressively upon continuous addition of PSA. The utilization of MNPA enrichment of bacteria. Additionally, they prepared vancomycin-SERS
was explored as a means of enhancing the performance of the SERS tags (Au@4-mercaptobenzoic acid (4- MBA)) to enable sensitive iden­
substrate. In this study, the MNPs were employed as both applied ma­ tification of pathogenic bacteria. The combination of Au-shell-based
terials and separation tools, thereby facilitating the concentration effects dual-SERS enhancement and aptamer/vancomycin-based dual-sensing
of the mixture. The use of a magnet enabled the efficient removal of the ability allowed to achieve a LOD of 3 cells mL− 1. This innovative
mixture from the supernatant, thereby enhancing the overall efficacy of approach also provided a wide linear range from 10 to 107 cells mL− 1
the substrate. The SERS signals obtained from the supernatant exhibited with recovery rates ranging from 95.0 to 106.4 % and a RSD below 5.3
a direct correlation with PSA concentrations across a broad range with a %.
LOD of 5 pg mL− 1. To achieve efficient analytical results, Ag-coated MNPAs were also
Although aptasensors based on uncoated MNPs have a larger surface used for highly-sensitive detection of various analytes [269]. For
area, uncoated NPs are prone to aggregation, which can negatively instance, Ag-coated MNPAs are employed for trace detection of protein
affect their performance. To mitigate this issue, they usually coat with a biomarkers. The platform was comprised of Au nano-bridged nanogap
thin layer of Au, Ag, etc., which not only prevents aggregation but also particles labelled with a reporter molecule as a SERS tag. Additionally, it
enhances the sensitivity and selectivity of the sensor [266]. Zhou and incorporated an innovative magnetic capture substrate composed of
their colleagues [267], have proposed a method for the sensitive Ag-coated Fe3O4-AuNPs. The substrate was validated for the detection of
detection of bacteria through a magnetically assisted SERS-label C-reactive protein (CRP) with an LOD of 10 fM in the range of 10 fM to
immunoassay. Their approach utilized a universal strategy by incorpo­ 10 nM. To achieve this, aptamers targeting CRP were modified on both
rating free antibody labeling and the orientation recognition of staph­ the SERS tag and magnetic capture substrate to enable specific sensing
ylococcus proteins A (PA)-SERS tags. The SERS assay was comprised of by Au nano-gap NPs-CRP-AgMNPs sandwich assay. In addition, this
two distinct nanomaterials working in tandem to quantify target bac­ approach demonstrated remarkable selectivity towards CRP, even in the
teria. Firstly, aptamer-conjugated Fe3O4@Au MNPs served as a mag­ presence of various interferences and in real human serum samples.
netic SERS system for pathogen enrichment. Secondly, PA-modified Considering the efficient analytical results of MNPA-based SERS sub­
SERS tags (AuNPs@ 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB)@PA) strates, we hope that researchers expand this type of sensors for the
were utilized as a probe for the quantitative detection of the target simultaneous detection of multiple analytes in real samples.
bacteria. Following the enrichment of the target bacteria, a free anti­
body was employed to specifically label the target bacteria, resulting in 2.2.4. Resonance-based sensing: surface plasmon resonance (SPR) and
the generation of multiple Fc fragments. These Fc fragments served as a Förster or fluorescence resonance energy transfer (FRET) sensing
means to direct the orientation-dependent binding of PA-SERS tags. This SPR is a technique for the quantification of changes in the refractive
work described a novel approach for the facile construction of sandwich index of a metal surface—usually Au— induced by the interaction with
immunocomplexes for most bacteria, except for a few species of other atoms/molecules [270]. The main advantage of SPR is its ability to

Fig. 2. Schematic illustration of A) AuNPs-vancomycin SERS tags synthesis procedure, B) aptamer-Fe3O4@Au preparation steps, and C) detection of Staphylococcus
aureus with designed sensor [268]. Reprint with permission from Elsevier Science, 2019.

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M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

facilitate label-free identification of molecular interactions at the surface size-tunable emission spectrum [274]. Using this technique, Zhu et al.
of the probe [271], which makes it suitable for a range of analytical [275], developed a novel FRET MNPA sensor with the ability for mag­
assays. For instance, Zhu et al. [272] fabricated an SPR biosensor for the netic decoration of exosomes using aptamers to facilitate the identifi­
quantitative analysis of leukocyte cell-derived chemotaxin 2 (LECT2). cation of surface proteins of cancer cells through a light-up FRET
Tyrosine kinase and Epidermal growth factor-like domains 1 (Tie1) with method. They used MNPs as a platform for incorporating fluorescent
immune globulin-like are unassigned receptors that interact with QDs and aptamers. Notably, the fluorescence emission of the QDs was
LECT2. The receptors possessed a C-terminal Fc tag, located distantly effectively modulated upon binding of the aptamers to their cDNA se­
from the binding regions of LECT2. The Fc aptamer was deliberately quences present on the surface of AuNPs. Subsequently, the expulsion of
employed to bind to the Fc tag of the Tie1 protein. This interaction AuNPs occurred as a consequence of competitive binding between
facilitated the conjugation of Fe3O4-coated AgMNPs (Ag@MNPs) to the aptamers and exosomes. This led to a linear augmentation of QD fluo­
Fc aptamer, thereby ensuring the outward exposure of the LECT2 rescence intensity, which was dependent on the concentration of exo­
binding site. The enhanced SPR signal of Ag@MNPs was observed due to somes within a wide range (500–5 × 109 particles per mL). In addition,
the orientation properties of the captured protein. By leveraging this this approach was able to successfully demonstrate a LOD of 13 particles
phenomenon, a highly sensitive sensor for detecting LECT2 was devel­ per mL. Considering the excellent optical properties, it is anticipated
oped with an LOD of 10.93 pg mL− 1. The development of MNPA sensors that more integration of QDs will occur in FRET sensors based on
is still in its infancy and further research is required to fully reveal their MNPAs.
potential in biosensensing applications. Recently developed MNPA-based optical bioassays for different
FRET is another Resonance-based sensing technique wherein the analytes are summarized in Table 2. As shown, MNPAs are mainly
electronic excitation of a donor molecule, typically a fluorophore, is employed in the development of fluorescence and SERS biosensors and
efficiently transferred to a recipient molecule via dipole-dipole in­ their applications in colorimetric, SPR, and FRET biosensors is over­
teractions [273]. Core-cell QDs are used by researchers due to their looked. Additionally, the primary focus is on biomedical applications.
excellent properties in optical research. For example, they have strong Therefore, there is a need to broaden MNPA-based biosensors for ana­
photoluminescence performance, broad absorption spectrum, and lytes in food and environmental samples. Furthermore, given the

Table 2
Comparative analysis of MNPA-based optical bioassays for sensing different analytes.
Detection Technique Structure Biological Target Reale sample Detection LOD Ref
method Element Range

Optical Fluorescence FAM-aptamer-Fe3O4@PDA Aptamer ATP Human urine and 0.1–100 μM 89 nM [241]
serum samples
− 1
Optical Fluorescence Fe3O4@SiO2@aptamer Aptamer BPA Milk samples 0.1–100 ng 0.05 ng mL [242]
mL− 1
Optical Fluorescence MNPs-CY3-aptamer Aptamer Thrombin Bovine serum 1–60 nM 0.5 nM [243]
samples
1
Optical Fluorescence avidin-conjugated TRFL-NPs Aptamer, ZEN Maize and wheat 0.001–10 0.21 pg mL− [244]
and MNPA and cDNA samples ng mL− 1
1
Optical Fluorescence Fe3O4-aptamer/cDNA-CDs Aptamer, β-LG Hypoallergenic 0.25–50 ng 37 pg mL− [245]
and cDNA formulas samples mL− 1
Optical Fluorescence MNPA and Immunoglobulin G- Aptamer, Listeria monocytogenes Water, and pork 10–106 cfu 10 cfu mL − 1
[246]
1-(4-hydroxyphenyl)-1,2,2- and meat mL− 1
triphenylethene@ bovine antibody
serum albumin
Optical Colorimetric Gold NPs-based colorimetric Aptamer Listeria monocytogenes Pork 10–106 cfu 10 cfu mL− 1
[257]
assay mL− 1
Optical Colorimetric MNPA and IgY- bovine serum Aptamer, Listeria monocytogenes Pork 10–106 cfu 10 cfu mL − 1
[258]
albumin-silver nanoclusters and mL− 1
antibody
Optical Colorimetric MNPA and aptamer-gold NPs Aptamer Vibrio Spiked raw 10–106 cfu 2.4 cfu mL− 1
[259]
parahaemolyticus shrimp samples mL− 1
1
Optical SERS MNPs-cDNA-gold NPs- 4- Aptamer, MC-LR Tap water sample 0.01–200 0.002 ng mL− [264]
MBA-aptamer and cDNA ng mL− 1
− 1
Optical SERS MNPA-gold NPs-4,4′-dipyridyl- Aptamer PSA Human serum 5–500 pg 5 pg mL [265]
PSA complementary samples mL− 1
Optical SERS Fe3O4@gold-aptamer- Aptamer, Salmonella Food, and 0–107 cell 25, 10, and 10 cells [267]
antibody, and gold NPs- and typhimurium, Listeria biological mL− 1 mL− 1 for Salmonella
DTNB@PA antibody monocytogenes, and samples typhimurium, Listeria
Escherichia coli monocytogenes, and
Escherichia coli
Optical SERS Aptamer-Fe3O4@gold/ Aptamer Staphylococcus aureus Milk, orange 10–107 cells 3 cells mL− 1 [268]
Staphylococcus aureus/gold juice, and blood mL− 1
NPs- vancomycin tags
sandwich complexes
Optical SERS 4-amino thiophenol-labelled Aptamer CRP Human serum 10 fM – 10 10 fM [269]
gold bridge with nano-gap NPs samples nM
and silver-coated MNPA
1
Optical SPR Silver@MNPs/MCH/LECT2/ Aptamer LECT2 Blood samples 0.1–100 ng 10.93 pg mL− [272]
Para-Sulfonatocalix [4] mL− 1
arene/gold electrode
Optical FRET Fe3O4-poly(ethylene imine)- Aptamer, Cancerous surface Serum samples 500–5 × 13 particles per mL [275]
QD-aptamer-gold NP-DNA and DNA proteins 109
particles per
mL

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M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

immense significance of portable sensors, it is anticipated that optical (SPCE) through the application of an external magnetic field. The in­
measurements utilizing smartphones will be advanced in the near tensity of the signal exhibited a direct correlation with the concentration
future. This technological advancement will enable individuals to of DES, ranging from 0.3 pg mL− 1 to 0.1 μg mL− 1. Notably, the LOD was
promptly detect and identify analytes within biological, food, and determined to be as low as 0.1 pg mL− 1 in fish samples with excellent
environmental samples directly at the sampling location. Plus, the use of recoveries ranging from 80 to 120 %.
machine learning technology can be very important in obtaining accu­ Although a few ECL-based MNPAs have been developed for analyte
rate results without the help of skilled personnel. detection, the integration of hybrid nanomaterials with MNPAs holds
great promise for further advancements in this domain. The integration
2.3. Electrochemiluminescence (ECL) sensing of nanohybrids, silica, and carbon nanomaterials with MNPAs holds
great potential in developing ECL biosensors with superior sensitivity
ECL functions based on the generation of light through the stimula­ and selectivity for identifying diverse analytes.
tion of various types of luminophores (such as NPs, molecular entities, or
atoms) induced by EC reactions [276]. This method provides numerous 2.4. Photoelectrochemical (PEC) sensing
benefits over conventional chemiluminescence, including enhanced
sensitivity, ease of use, stability, and a broad operating range [277]. PEC sensing relies on a photoinduced electron transfer (PET) process
Recently, ECL was used in combination with MNPAs facilitating occurring between the photoactive material, the analyte of interest, and
label-free sensing of various analytes. For instance, it was employed for the electrode [283,284]. PEC sensors offer optimal repeatability and
ultrasensitive detection of leukemia marker mRNA, in particular miR-16 sensitivity due to the segregation of detection and excitation signals,
[278] through cyclic target chain displacement polymerization, facili­ effectively mitigating background noises [285,286]. Using this tech­
tated by the Klenow fragment of DNA polymerase. This process was nique, Liu et al. [287], developed a PEC sensor based on
helped in the liberation of mRNA and initiates the subsequent cycle of immobilization-free dual-aptamers, combined with photoactive bismuth
polymerization. Subsequently, the amalgamation of AuNPs coated with oxyiodide/Au/CdS (BiOI/gold/CdS) composites for simultaneous
PDNA and ADNA, both infused with (Ru(bpy)2+ 3 ), was introduced. This nucleic acid identification and signal amplification strategies (Fig. 3)
complex referred to as AuNPs@(PDNA+ADNA-Ru(bpy)2+ 3 ), suitable for [287]. In this assay, the detection of exosomes was achieved through the
employment in the ECL intensity measurement. The amplification of the utilization of two aptamers, serving as recognition elements. Upon
detected signal was greatly enhanced due to the polymerization cycle binding to the exosomes, the aptamers initiated a cascade reaction
and aggregation phenomenon exhibited by the Ru(bpy)2+ 3 illuminator. involving deoxyribonucleotidyl transferase (TdT) enzyme. This enzy­
The experimental findings demonstrated a robust linear correlation matic process led to the polymerization of nucleotides, resulting in the
between the ECL signal and the logarithm of the target mRNA concen­ subsequent enrichment of alkaline phosphatase (ALP) on the surface of
tration within the range of 0.1 fM to 0.1 μM. The quantification of mRNA Fe3O4. Following successful magnetic separation, ALP acted as a catalyst
spiked in the human serum sample was conducted, with the recovery to produce ascorbic acid, utilizing it as an electron donor. This enzy­
ranging from 97.2 % to 102.0 %. Another novel ECL biosensor has been matic reaction initiated a subsequent redox cycle, thereby facilitating
developed to achieve remarkable sensitivity and specificity in detecting signal amplification. This solution-based assay was resulted in a superior
tumor cells using bio-barcode toehold-aptamer/DNA primer/Au–Fe3O4 recognition and signal amplification efficiency compared to heteroge­
(TA/DP/Au–Fe3O4) nanoconjugates with an optimized ratio of 1:10 neous approaches. The biosensor showed a linear range spanning from
[279]. This ratio was chosen to minimize any potential cross-linking 100 to 1.0 × 106 particles μL− 1, with an estimated LOD was 21 particles
reactions and enhance the efficiency of target cell recognition. The μL− 1.
nanoconjugates were immobilized on a substrate through the hybridi­ Active optical properties of materials such as transition metal chal­
zation of the aptamer to a capture probe consisting of an 18-mer cogenides (cadmium telluride (CdTe) [288], CdS [289], silver sulfide
sequence. Under optimized conditions, this approach was able to suc­ (Ag2S) [290], cadmium selenide (CdSe) [291], lead(II) sulfide (PbS)
cessfully detect Ramos cells in up to 16 cells. [292], molybdenum disulfide (MoS2) [293]), metal oxides (tungsten
The integration of different materials, such as NPs, nanowires, and trioxide (WO3) [294,295], nickel oxide (NiO) [296], titanium dioxide
nanotubes, with biomolecules or organic compounds has resulted in the (TiO2) [297], zinc oxide (ZnO) [298], tin(IV) oxide (SnO2) [299]) and
formation of nanohybrids with potential applications in biosensors. The carbon materials [300] make them suitable for use in PEC sensors. In
notably high surface-to-volume ratio of nanohybrids facilitates the cre­ this line, Wang et al. [301] developed a PEC sensor to detect circulating
ation of binding sites, thereby enhancing the sensitivity of the biosensor tumor cells (CTCs) by employing hexagonal carbon-nitrogen tubes
[280]. Incorporating nanomaterials, such as silica nanomaterials, QDs, (HCNT) as visible light-responsive materials. The amplification of the
etc., into the design of the biosensor enables signal amplification, detection signal was achieved through the utilization of Ag2S nano­
resulting in improved detection limits and enhanced accuracy in quan­ crystals, which possess the capability to effectively absorb visible light.
tifying analytes [281]. Using this method, a novel ECL assay, capable of This property enables them to compete with the HCNT, thereby resulting
detecting diethylstilbestrol (DES), has been developed [282]. This assay in a reduction of the photocurrent intensity. The assay designed for the
operates without the need for antibodies by adopting a signal-on detection of MCF-7 cells in human whole blood samples with an excel­
mechanism. The detection system incorporates magnetic molecular lent selectivity and a linear concentration range from 10 to 5000 cells
imprinting polymers (MMIPs) with aptamer-labelled cadmium sulfide mL− 1, showcasing its ability to accurately quantify cell concentrations
(CdS) QDs conjugated probes. The synthesis of MMIPs involved the within this specified range. Li and colleagues [302] also presented a
utilization of 4’-hydroxypropiophenone as a template molecule to create novel magnetic-optical bifunctional beacon, comprising Fe3O4@­
imprint sites on a PDA coating that encapsulated a core-shell structure of SiO2@TiO2, which has been successfully employed in a pioneering PEC
Fe3O4@SiO2. In another study, an aptamer has been selected to specif­ sensor. This sensor was able to selectively capture progesterone in
ically target the phenol group of 17β estradiol (E2) as a molecular complex biological samples as well as governing magnetic separation
recognition element wherein conjugated with CdS QDs to create a and cleaning processes. The utilization of a magnetic separation
CdS-aptamer signal tag. The purpose of using this tag was to facilitate approach has proven to be an effective means of removing complex
the detection of the phenol group of DES. When the desired target coexisting species present on the modified electrode surface, thereby
molecule was incubated in the presence of MMIPs and CdS-aptamer, a significantly augmenting the selectivity of the engineered PEC assay. In
composite material known as sandwich MMIPs-DES-CdS-aptamer was this sensor, analyte concentration was inversely proportional to the
successfully synthesized. Subsequently, the composite material was photocurrent intensity, so the photocurrent decreased with increasing
immobilized onto the surface of a screen-printed carbon electrode progesterone concentration from 1 to 6000 pM.

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M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

Fig. 3. Schematic illustration of the PEC bioassay developed for the detection of cancer exosomes [287]. Reprint with permission from Elsevier Science, 2024.

As summarized in Table 3, several MNPA-based PEC sensors have applications.

been developed for various analytical assays, however, the scope of their
applications can still be expanded. For instance, carbon nanomaterials, 2.5. Field effect transistor (FET) sensing
transition metal chalcogenides, metal oxides, and other compounds with
excellent optical properties can be employed. The current PEC-MNPA FETs are fundamental constituents of integrated circuits, serving as
sensors are primarily focused on detecting analytes in biological and three-terminal devices that operate as electric switches. The flow of
food samples and further studies are required for environmental electric current can be controlled by the application of a small electric

Table 3
Comparison of MNPA-based FET, PEC, and ECL biosensors for various applications.
Detection Structure Biological Target Reale sample Detection Range LOD Ref
method Element

ECL Gold NPs@(PDNA+ADNA-Ru(bpy)2+ 3 ))- Aptamer, and miR-16 Human serum 0.1 fM – 0.1 μM 0.043 fM [278]
Fe3O4@gold/GCE DNA sample
ECL Bio-bar-code toehold-aptamer/DNA primer/ Aptamer, and Ramos cells Complex samples, 20–500 cells 16 cells [279]
gold–Fe3O4 DNA and whole blood
samples
ECL MMIPs-QDs-aptamer Aptamer DES Fish samples 0.3 pg mL− 1 – 0.1 μg 0.1 pg [282]
mL− 1 mL− 1
PEC BiOI/Au/CdS, and aptamer-modified Fe3O4 Aptamer Cancer exosomes – 100 particles μL− 1 – 21 particles [287]
NPs 1 × 106 particles μL− 1
μL− 1
PEC GCE-HCNT-MNPs-antibody-CTCs-aptamer- Aptamer, and MCF-7 cells Human whole blood 10–5000 cells mL− 1 3 cells [301]
Ag2S nanocrystals antibody mL− 1
PEC Fe3O4@SiO2@ TiO2-NH2-aptamer-cDNA Aptamer, and progesterone Liquid milk samples 1–6000 pM 0.3 pM [302]
cDNA
PEC rGO-BiFeO3-based PEC sensing system Aptamer, and PSA Human serum 0.001 − 100 ng 0.31 pg [311]
DNA samples mL− 1 mL− 1
PEC NaYF4:Yb,Tm@ZnO-based PEC biosensor Aptamer, and Carcinoembryonic – 0.1–300 ng mL− 1 0.032 ng [312]
DNA antigen mL− 1
− 1
PEC DNA walker/aptamer-magnetic bead and Aptamer, and PSA Human serum 0.01–50 ng mL 1.5 pg [313]
hairpin DNA1/gold NPs@bismuth vanadate/ DNA samples mL− 1
fluorine-doped tin oxide
FET ITO/graphene- PBASE-aptamer-magnetic Aptamer, and CTNI – – 10 pM [309]
beads/antibody antibody
FET Aptamer- magnetic fluorescence NPs Aptamer Escherichia coli – 0–106 cfu mL− 1
100 cfu [310]
mL− 1

11
M.B. Behyar et al. Trends in Analytical Chemistry 172 (2024) 117549

field between the channel and the gate electrode, allowing the modu­ Despite the considerable potential of MNPAs in the analysis of target
lation of a weak current signal, which can subsequently be amplified. analytes in various samples, several limitations still need to be
Additionally, various environmental stimuli have the potential to in­ addressed. The primary objective is to develop cost-effective, highly
fluence channel conductivity and elicit a current response. The utiliza­ sensitive, and user-friendly sensors for the precise quantification of
tion of FETs in sensing applications shows immense potential due to diverse analytes. This can be achieved by integrating smartphones,
their inherent advantages in signal amplification [303]. Moreover, the microfluidic systems, and portable microchips into MNPA technology.
integration of multiple sensing elements on a single microchip enables In addition, the use of machine learning can be very effective in
multiplexed analysis [304]. achieving accurate results without the need for skilled operators.
Owing to special characteristics such as mechanical strength, high Finally, the incorporation of advanced nanomaterials such as dendritic
electrical conductivity, and extensive surface area [305–307], graphene fibrous nano-silica (DFNS), metal-organic frameworks (MOFs), layered
has been widely employed for the fabrication of FET sensors [308]. Zhu double hydroxide (LDH), and zeolitic imidazolate framework (ZIF), in
et al. [309] demonstrated a magnetic graphene FET sensor for the MNPAs is useful for the advancement of microchips capable of detecting
quantitative analysis of cardiac troponin I (CTNI). Graphene film was multiple analytes simultaneously.
used as a conductive channel and subsequently moved onto a glass
substrate featuring two indium tin oxide (ITO) electrodes. The immo­ CRediT authorship contribution statement
bilization of CTNI aptamer onto a graphene film was accomplished
through the use of 1-pyrene butanoic acid succinimidyl ester (PBASE) to Milad Baghal Behyar: Writing – original draft, Methodology,
effectively capture CTNI. To create a sandwich-type assembly consisting Investigation. Azadeh Nilghaz: Writing – review & editing, Methodol­
of aptamer/CTNI/antibody/magnetic nanobeads, magnetic nanobeads ogy, Investigation, Formal analysis. Rokhsareh Ebrahimi: Writing –
decorated with CTNI antibody were introduced into the chamber of original draft, Investigation, Formal analysis, Data curation. Moham­
reaction. The application of a periodic magnetic field resulted in the mad Hasanzadeh: Writing – review & editing, Supervision, Project
observation of a modified impedance in the graphene film due to the administration, Conceptualization. Nasrin Shadjou: Writing – review &
magnetic force exerted on the complex. The obtained outcomes were editing, Validation, Formal analysis.
rationalized through the application of a theoretical framework, in
which the magnetic force induces a bending effect on the CTNI aptamer Declaration of competing interest
strand. As a result of the interaction between magnetic nanobeads/CTNI
aptamer and graphene transistors, a bending phenomenon occurred, The authors declare that they have no known competing financial
which subsequently resulted in the modulation of the double conductive interests or personal relationships that could have appeared to influence
layer of the transistors. To mitigate the impact of noise interference, the work reported in this paper.
periodic sampling and integration are common, which effectively en­
hances the signal-to-noise ratio. Consequently, this approach facilitated Data availability
the detection of CTNI with exceptional sensitivity, yielding a remarkable
LOD of 10 pM. In another study, it has been shown that bacterial cells No data was used for the research described in the article.
can rapidly and effectively be enclosed within MNPs and subsequently
direct towards the sensing surface composed of reduced graphene oxide
Acknowledgments
(rGO) through the application of an external magnetic force [310]. For
instance, Escherichia coli encapsulation in MNPs was achieved using FET
We appreciate the financial support provided for this work by the
sensors functionalized with aptamers and rGO. Considering the high
Pharmaceutical Analysis Research Center at Tabriz University of Medi­
potential of MNPA-based FET sensors, the limited number of studies
cal Sciences, Tabriz, Iran under Grant No.66610.
underscores the need for more investigations in the field. In addition, the
lack of microfluidic FET sensors based on MNPAs is a deep research gap
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