Can Electrochemical Aptasensors Achieve the Commercial Success of Glucose Biosensors?

Enzymes and antibodies are widely available biorecognition elements in bioanalytical tools such as personal glucose monitoring (PGM) devices and lateral flow assays (LFA). Meanwhile, electrochemical aptamer‐based (EAB) sensors are promising affinity‐based bioanalytical tools with potential advantages over such conventional bioassays. However, several critical factors affect the stability of EAB sensors, pivotal for their commercialization including 1) electrode defects due to surface treatment methods, 2) hampering effects of redox molecules, 3) electrical potential‐induced aptamer detachment, 4) thermal‐induced monolayer solubilization, 5) biochemical/enzymatic degradation, 6) biofouling, and 7) inadequate statistical design and analysis in EAB sensor fabrication. Herein, antidotes for the obstacles are proposed by applying novel surface treatment methods, adapting redox molecule, tuning electrochemical tests, tweaking backfilling agents, and anti‐bio‐fouling coatings. Nonetheless, the obstacles are a driving force to clear pathways toward bringing EAB sensors to the market for therapeutic drug and metabolite monitoring, point of care sensors, macromolecule detection, and pathogen diagnostics.

DOI: 10.1002/adsr.202300132sensitivity.Glucose oxidase is particularly effective at targeting glucose molecules and oxidizing them into gluconic acid and hydrogen peroxide. [3]Detection of hydrogen peroxide can be achieved using amperometric or voltammetric methods, or indirectly by using substrates such as horseradish peroxides to produce electroactive compounds. [3]On the other hand, there are other class of POC-based bioanalytical tools that are commonly used for measuring blood glucose levels.The emergence of more sensitive and advanced bioanalytical tools for individuals with diabetes, including continuous glucose monitoring (CGM) devices, has been observed. [4]These devices feature wireless data communication, long-term usability, user-friendliness, minimally invasive sampling, high sensitivity, and broad shelf-life. [4]esearch on antibodies commonly used in LFAs traces back over eight decades and was developed by Berson and Yalow, earning them the Nobel Prize in Medicine in 1977. [5]Since its introduction, immunoassays have undergone significant evolution, becoming an established and commercialized technology.Rapid detection kits based on LFAs have become a well-established solution for pointof-care (POC) detection of biochemicals and pathogens in nonclinical settings. [6]LFAs were first introduced in the late 1980s for rapid pregnancy testing and later for the detection of sexually transmitted diseases. [6]Nowadays, they are commonly used for the detection of SARS-CoV-2 and drugs of abuse. [6]The mechanism of sensing in a conventional LFA is based on the strong and selective affinity between the immobilized antibodies on gold nanoparticles, i.e., detection probes, and the target antigen to form an immune complex. [7]The immune complex wicks along a paper-based strip via capillary effects into the detection zone in which the antigen is sandwiched between the detection probe and a secondary antibody, i.e., capture probe, which consequently form a line that can be visualized by naked eye. [7]hile other types of biorecognition elements such as aptamers, nucleic acids, and molecularly imprinted polymers can be used in biosensors, enzymes and antibodies have remained the predominant choices in biosensing platforms, despite their inherent limitations. [8]Particularly, there are limited numbers of oxidoreductase enzymes with narrow a target pool capable of undergoing oxidation or reduction.Regarding antibodies, their target pool is restricted to targets  [ 14] Temperature/pH induced degradation [ 15] High but prone to nuclease degradation [ 9] TARGET POOL Limited to oxidoreductase reactions [ 9 ] Limited to immunogenic compounds [ 1,8] Wide range but limited with hydrophobic/ negatively charged targets [ 16] AVERAGE SIZE ≈3-7 nm [ 17] ≈10-15 nm [ 18] ≈1-2 nm [ 18] DEVELOPMENT PROCESS In vitro by microbial fermentation In vivo by recombinant technologies [ 19] In vivo, requires immune response and animals [ 8] In vitro, SELEX [1] PRODUCTION TIME Days to weeks [ 19] Weeks to months [ 8] Days to weeks [ 1] that produce an immune response. [8,9]Also, the production of antibodies requires lengthy and expensive in vivo procedures. [10]n the contrary, artificially-produced bioreceptors, particularly aptamers can be produced cost-effectively with a consistent structural form. [10]Meanwhile there is an acute need to detect and monitor other biochemicals, biomarkers, pathogens, and macromolecules related to human healthcare such as disease management, clinical diagnosis, and therapeutic drug monitoring. [9]erein, we will focus on aptamers as an emerging biorecognition element with potential advantages over the conventional biorecognition elements.Aptamers are synthetic, singlestranded DNA, RNA, or peptide molecules that can bind to specific target molecules with high affinity and specificity. [11]ptamers are selected through a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment), where a random library of nucleic acid sequences is screened for binding to a particular target molecule. [12]The aptamer sequence can be optimized through SELEX to selectively bind to a particular target molecule. [13]Aptamers can offer potentially large target pool that can bind to non-immunogenic molecules.Their in vitro production can be cost-effective with higher stability and shelf life compared to antibodies. [1]Table 1.provides a comparison between antibody, enzyme, and aptamer as biorecognition elements in biosensors.
A literature survey reveals that enzymes and antibodies exhibit significantly high sensitivity and selectivity, making them suitable as highly specific biorecognition elements. [1,20]Meanwhile, aptamers can be produced with high selectivity and sensitivity during the SELEX process and also through post modificaton by biochemical tags or internal modification.However, stability of aptamers can be superior to that of enzymes and antibodies. [8]They can naturally and reversibly fold at lower temperature and unfold at high temperatures however they can be prone to catalytic degradation in biofluids consisting DNAzymes and nucleases but can be prevented by chemical modification. [9]ntibodies and enzymes are more prone to non-reversible conformational change in high temperature which reduces their performance. [14,15]The target pool of enzymes are limited to oxidoreductase reactions meanwhile antibodies can only be effective in targeting analytes which produce immune responses. [9]eanwhile, aptamers can be produced for wide ranges of tar-gets that are not necessarily immunogenic.16b] Since optical-based detection in aptasensors has been reviewed extensively, [21] herein only electrochemical aptamer-based (EAB) sensors will be evaluated.The EAB sensors typically consist of three components: the aptamer, the electrode, and the signal transduction system. [22]The aptamer is immobilized on the surface of the electrode, which serves as the biorecognition element.The target molecule in the sample solution binds to the aptamer, causing a change in the electrochemical properties of the electrode surface.The signal transduction system, a potentiostat, detects and converts this change into a measurable electrical signal. [22]n a typical EAB sensor, each end of an aptamer strand can be modified with a desired functional group for two primary purposes in oligonucleotide EAB sensors: a) chemical immobilization on an electrode by modifying the 5′ end with thiol or amine groups and b) covalent attachment of a redox molecule such as methylene blue (MB) or Ferrocene (Fc) on the 3′ end. [22]There are several mechanisms by which the binding event between the aptamer and the target can be transduced into an electrical signal.One common mechanism is based on using a redox-active probe, i.e., [Fe(CN) 6 ] 3/4− in the measurement solution. [23]In the presence of a target analyte, aptamer-target complex would impede the redox molecules to migrate from the bulk solution to the electrode's surface; thus, a decrease in the produced current is anticipated which can be monitored by direct current (DC) voltammetry techniques. [23]Alternative current (AC) electrochemical impedance spectroscopy (EIS) technique can also be used to measure the charge transfer resistance increase caused by spatial hindrance of target-aptamer complex. [23,24]nother mechanism is based on the use of redox probes that are covalently attached to the aptamer.The binding event between the aptamer and the target molecule can induce a conformational change, which causes the aptamer's end (either 3′ or 5′) to get closer or further to/from the electrode, therefore causing a shift in the redox activity and the produced current. [25]  ments, i.e., antibody, enzyme, and aptamer, in electrochemical biosensors.
As mentioned earlier, enzymes and antibodies have been the predominant and successful biorecognition elements in commercially available biosensing platforms.However, a critical question persists: Can other formats of electrochemical sensors, such as EAB sensors, achieve a comparable level of success in detecting molecules beyond oxidoreductase targets and immunogens?Several reasons challenge the commercial viability of EAB sensors, as discussed below.

Electrode Pre-Treatment can Lead to Undesired Defects
The quality of self-assembled monolayers (SAMs) on polycrystalline gold is dependent on the reliable removal of inherent contaminants on ambient surfaces and exposure of a pristine gold surface. [30]In the past, acidic piranha solution was commonly used to remove organic contaminants during gold electrode production.However, piranha can create uncontrollable defects on the gold electrode, causing extensive etching and oxidation.As a safer alternative, sulfochromic acid was proposed to eliminate organic residues but it causes permanent etch pits and is not recommended for repeated use. [31]26a] X-ray photoelectron scattering (XPS) provided an accurate picture of surface composition of gold surfaces treated with different methods. [32]Although all surface pre-treatment methods significantly decreased contamination and increased SAMs coverage, they induced structural changes that affected functionalization characteristics.Different pre-treatment methods did not result in significant differences in surface area or molecule coverage, but some induced structural changes to the gold surface and affected the morphology of the thiolated molecule layer.Pre-treatment methods which contain chloride anions were found to cause changes to the structure of the gold surface, leading to surface redistribution and possible introduction of defects. [32]26c] Anion adsorption can result in a passivating layer of Au 2 SO 4 or AuCl, which is formed through a redox reaction on the Au electrode.It is recommended to avoid chloride leakage and platinum counter electrodes when preparing gold electrodes using CV.Surface roughness is another debated topic, as recent studies have shown that alkanethiolate monolayers on rough gold surfaces can act as stronger barriers to electron transfer than those on smooth surfaces. [25]However, the defects on smooth surfaces were larger than those on rough surfaces, leading to an increased rate of heterogeneous electron transfer.

Redox Molecules can Change the Surface Properties of EAB Sensors
Understanding electron transfer at electrode-electrolyte interfaces is crucial for probing biomolecular interactions on the surface of heterogenous aptasensors.[Fe(CN) 6 ] 3/4− is a common redox probe to test the electron transfer on gold-based electrochemical aptasensors.However, the interaction between gold electrodes and [Fe(CN) 6 ] 3/4− are prone to unwanted reactions.Early studies have focused on measuring intermediate species and byproducts such as Prussian blue [33] and recent studies show the evolution of Au(CN) 2 − on the gold electrode's surface during voltametric techniques such as CV. [34]27d] Such disruptive and reactive nature of [Fe(CN) 6 ] 3/4− could result in detachment of aptamers from the gold electrode which significantly drift sensor's signals and reduces its performance over time.Other redox probes such as covalently attached Ferrocene and methylene blue are introduced as alternative probes in analysing electrochemical aptasensors. [25,35]owever, the synthesis of covalently attached redox tags are cumbersome and more expensive than conventional redox probes.Moreover, fluctuations in the charge transfer resistance in redoxtagged aptasensors are very common due to inherent sensitivity of aptamers to their media as well as sensitivity of electrodes to the solution's ionic strength which can result in false positives during sensing experiments.Loss of thiolated nucleic acid probes due to electrochemical interrogation can alter the 2D and 3D structures of the ssDNA probes, exacerbating signal changes. [23,25]n conclusion, the use of [Fe(CN) 6 ] 3/4− as a redox probe in electrochemical aptasensors can lead to unwanted reactions and negatively impact the sensor's performance over time.While alternative redox probes have been introduced, they come with their own limitations and challenges.A comprehensive study is needed to develop more effective and reliable redox probes for analyzing biomolecular interactions on the surface of heterogeneous aptasensors.

Aptamers Detach from the Electrode During Electrochemical Measurement
Even though thiolated DNA SAMs are meant to form a robust covalent bond with a gold electrode, they can be detached from the surface during electrochemical tests.For instance, aptamers electrochemical potentials versus Ag/AgCl more negative than −0.4 V and more positive than −0.1 can oxidize the thiol bonds which desorb them from the surface. [28]Moreover, SAMs are prone to desorption and structural change during multiple CV scans. [23]Thus, it is critical to define a safe potential window during electrochemical tests and avoid repeated CV scans to reduce SAM detachment.

Elevated Temperature Leads to Monolayer Solubilization
Thermal stability of DNA SAMs on gold surfaces is a critical parameter correlated with the sensor's lifetime.Thermal desorption of DNA SAMs are still a significant challenge particularly for monitoring of therapeutic drugs and metabolites. [9]29a] Meanwhile, the crystallography of the underlying surface is crucial for thermal stability.29a] Moreover, there is a trade-off between sensitivity and stability in DNA SAMs.29b]

Enzymes in Body Fluids can Degrade Aptamers
DNA SAMs face significant challenges when introduced into the bloodstream.The presence of both endogenous nucleases and catalytic DNAzymes poses a threat to the stability and integrity of these aptamers. [9,28]Blood nucleases actively target extracellular DNA, including aptamers.These nucleases cleave phosphodiester bonds within the DNA backbone, leading to the fragmentation and degradation of aptamer molecules.DNAzymes, designed for specific catalytic activities, may inadvertently target DNA aptamers.If a DNAzyme recognizes a sequence within an aptamer as its substrate, it can initiate cleavage, causing the fragmentation of the aptamer structure. [9,28]

Biofouling Reduces Electron-Transfer Rates and Aptamer's Mobility
One of the primary concern in EAB sensors for long term monitoring is protein biofouling. [9,28]Biofouling leads to an initial reduction in redox-tag current when scanned in protein-rich fluids like serum.The mechanism involves hydrophobic protein domains fouling at defect sites in the monolayer.Additionally, the reorganization of biofouled protein molecules and their interactions with immobilized aptamer molecules can impact sensor response over multiple days.Overall, protein biofouling has significant implications, affecting electron-transfer rates and limiting the freedom of mobility of aptamers.

Statistical Design of Experiments and Analysis are Necessary for EAB Sensor Optimization
In analytical chemistry, statistical data analysis and statistical DoE are fundamental topics covered in textbooks.These concepts are also essential in biosensors, a novel branch of bioanalytical techniques.It is important to utilize fundamental statistical concepts in every step of biosensor design and fabrication, including error calculations, significant tests, and statistical DoE.Textbooks such as Statistics and Chemometrics for Analytical Chemistry by Miller and Miller can provide valuable guidance in this area. [36]egarding the aptamer-based biosensors, experimental conditions such as salt concentration, buffer, and pH value have been found to affect the selectivity, affinity, and 3D structure of aptamers. [23,37]Despite this, high-resolution structures of aptamer complexes have been determined under different experimental conditions with no apparent relationship between them.The DoE approach considers all factors simultaneously and provides an empirical correlation between these factors on the chosen response, unlike the conventional method of changing one factor at a time. [38]

Proposed Methods to Alleviate the Constraints in EAB Sensor Stability
Herein, we overview the antidotes for the obstacles by applying novel surface treatment methods, adapting redox molecule, tuning electrochemical tests, tweaking backfilling agents, aptamer chemical modification, anti-bio-fouling coatings, and statistical design of experiments.

Plasma Treatment is a Highly Effective Electrode Cleaning Technique
Highly purified gold surfaces are essential for the successful formation of firmly attached SAMs on the electrode surface.Gold surfaces commonly capture impurities during post-processing, storage, and chip transportation. [30]This contamination evidently impacts both the SAM's quality and the sensor's efficiency.Plasma cleaning methods using H 2 and O 2 have shown to be more effective compared to electrochemical cleaning methods to remove contamination which would result in a higher SAM density. [32,39]

Tuning Electrochemical Test Parameters Reduce Electric Field Induced Aptamer Desorption
Meanwhile, redox tagged aptamers at a narrow potential window (partial scans) reduce the electrochemical oxidation and desorption of thiolated SAMs. [9,28]In case of label-free EAB sensors, EIS technique has minimal effect on surface density of SAMs due to the small potential biases used in this technique. [23]Moreover, to reduce the etching of gold electrodes caused by [Fe(CN) 6 ] 3/4− redox-pair the introduction of Hexaammineruthenium (III) as an alternative redox-probe during EIS)measurements, with the application of a DC-bias, has proven successful. [40]The in situ generated Hexaammineruthenium (III) redox-probe enhances the stability of the sensor, reducing the risk of false signals and allowing for the impedimetric detection of biomolecules. [40]

Optimizing the Alkylthiolates Length Chain may Increase the Stability of Aptamers
Conventional electrochemical aptamer sensors mostly utilize 6mercapto-1-hexanol (MCH), a hydroxyl-terminated alkylthiolate, as the passivating blocking layer molecule. [41]Extending the alkylthiolate chain length increases the intermolecular forces in self-assembled monolayers on gold substrates, leading to improved stability in buffer conditions. [42]Longer-chain alkylthiolates offer enhanced stability due to increased van der Waals interactions, resulting in more orderly packed monolayers. [9]However, for electrochemical aptamer sensors, which rely on electron transfer, longer alkylthiolate chains present challenges.They increase the electron-tunneling distance and reduce defects in the monolayer, hindering efficient electron transfer.While attempts have been made to use longer alkylthiolate monolayers to improve stability, issues with impeded electron transfer and potential toxicity arise. [12]Nonetheless, In contrast to the commonly used MCH in EAB sensors, employing 8-mercapto-1-octanol (MCO) demonstrated significantly more consistent redox-tag currents and voltammograms with only slight elevations in oxygen reduction currents. [9]

Nanocomposite Electrodes and Hydrogel Membranes Reduce Biofouling
Despite the alterations to sensor signaling properties caused by biofouling, implementing membrane protection and surface chemistry modifications can alleviate these adverse changes, preserving sensor performance during extended multiday operations. [9]Electrochemical protein biosensors capable of functioning in whole blood for an extended period, exceeding one month, are achieved through the utilization of composite electrodes comprising nanowires and bovine serum albumin. [43]t consists of a 3D porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials, including gold nanowires, gold nanoparticles, or carbon nanotubes.These nanocomposites prevent non-specific interactions, enhance electron transfer to the electrode surface.Moreover, zwitterionic polybetaine hydrogel membranes can be used to minimize biofouling. [9]

Conclusion
Addressing technological challenges in EAB sensors, particularly SAMs on gold electrodes, is crucial for improving stability and performance.Identified bottlenecks include electrode defects, redox molecule interference, aptamer detachment, thermal instability, enzymatic degradation, biofouling, and statistical design issues.Proposed solutions involve novel surface treatments, optimized redox molecules, tailored electrochemical tests, modified backfilling agents, aptamer chemical enhancements, anti-biofouling coatings, and robust statistical design.Specific solutions include effective electrode cleaning with plasma treatment, fine-tuning electrochemical parameters, optimizing alkylthiolate chain lengths, and using nanocomposite electrodes and hydrogel membranes to reduce biofouling.Overall, overcoming these challenges through proposed strategies is essential for enhancing EAB sensor reliability and extending operational lifetimes in diverse applications.

Figure 1 .
depicts the most common mechanisms of biosensing in the three types of introduced biorecognition ele-

Figure 1 .
Figure 1.Schematic representation of electrochemical biosensors based on different biorecognition elements including a) antibody: in a label-free approach increase in the thickness of the biorecognition layer causes steric hindrance which reduces electron transfer rate of redox mediator.In a sandwich assay, the signals originate from the catalytic reaction of an enzyme molecule, designated as a signal tracer, with the detection antibody.The electrode can then detect the electroactive products.b) enzyme: the substrate undergoes catalytic conversion into an electroactive product, which can be monitored using the amperometry technique.In labeled enzymatic sensors transfer of electron from enzyme to electrode needs an external chemical mediator.c) aptamer: in a label-free EAB steric hindrance reduces electron transfer rate of redox mediator.In redox-tagged aptamers, reducing the distance between the redox mediator and the electrode can increase the electron transfer rate.Created with BioRender.com.
Ardalan is a Ph.D. student in the chemistry department of the University of New Brunswick under the supervision of Dr. Anna Ignaszak.He is the recipient of the Dr. William S. Lewis Doctoral Fellowship.He obtained his B.Sc. from the Sharif University of Technology and his M.Sc.from Chemistry and Chemical Engineering Research Center of Iran.His current research focuses on the design and fabrication of electrochemical biosensors for rapid detection of infectious diseases.Anna Ignaszak is a Professor at the University of New Brunswick (Canada), before that she was a Junior Professor (W1) at the Friedrich-Schiller University in Jena (Germany).She obtained a PhD in her native Poland in 2006 and moved to Canada to take on a position as a research associate at The University of British Columbia, and at the National Research Council of Canada.She has a diverse background in materials for electrochemical sensors and electrochemical energy applications.Her group at UNB currently develops electrochemical sensing platforms for early detection of Covid-19, Lyme bacteria, and cancer biomarkers.

Table 1 .
Comparative analysis of enzyme, antibody, and aptamer as common biorecognition elements in biosensors.