Recent Progress on Highly Selective and Sensitive Electrochemical Aptamer-based Sensors

Highly selective, sensitive, and stable biosensors are essential for the molecular level understanding of many physiological activities and diseases. Electrochemical aptamer-based (E-AB) sensor is an appealing platform for measurement in biological system, attributing to the combined advantages of high selectivity of the aptamer and high sensitivity of electrochemical analysis. This review summarizes the latest development of E-AB sensors, focuses on the modification strategies used in the fabrication of sensors and the sensing strategies for analytes of different sizes in biological system, and then looks forward to the challenges and prospects of the future development of electrochemical aptamer-based sensors.


Introduction
Biosensors have been widely used in qualitative and quantitative analysis in complexed biological systems [1] . They play vital roles in the fields of clinical medicine [2] , pathology [3] , human immunology [4] , pharmacy [5] , environmental science [6] and other fields. Particularly, the pandemic situation has further advanced the biosensors to meet the requirements of practical applications, and thus more efficient, sensitive, stable and portable biosensors have brought into the sharp focus of the field [7][8][9][10] . Usually, biosensors mainly rely on electricity [11] , heat [12] and optics [13] to transform the concentration and distribution of analytes, including neurotransmitters, metabolites, enzymes, antigens, antibodies, viruses, and cells in biological systems into detectable signal responses [14][15][16][17][18][19] .
Aptamers, including RNA aptamers and DNA aptamers are a class of single stranded oligonucleotide molecules with a specific recognition function [34][35][36] . They are nucleotide sequences with recognition ability for specific substances selected from a series of nucleotide sequences by systematic evolution of ligands by exponential enrichment(SELEX) [37][38][39][40] .
When aptamers bind to target molecules, the intermolecular interaction alters the configuration or chemical environment of the aptamer, which may change the electrochemical properties, such as electron transfer rate and interface properties [64][65][66] . Based on this, scientists have created a great panel of electrochemical biosensors that rely on aptamers for highly selective recognition [67][68][69] .
Electrochemical aptamer-based(E-AB) sensors not only merit the advantages of aptamers, but also have the advantages of fast analysis rate and high sensitivity of electrochemical analysis methods. Up to now, some E-AB sensors can reach sensitivity down to fmol/L level [70][71][72] . Together with their excellent anti-interference ability, adaptability with portable devices, E-AB sensors hold great promise for practical application [73] . In this minireview, we summarize the latest development of E-AB sensors, and focus on the modification strategies used in the fabrication of sensors and the sensing strategies for analytes of different sizes in biological system.

Fabrication of E-ABs
To build a selective and sensitive E-AB sensor, one of the most critical steps is to stably modify aptamers on the electrode surface [74] . Because the nucleic acid has many reactive sites, the H Review structure of aptamer can be easily designed to modify the reactive groups, so that the aptamer can form a stable structure through covalent or non-covalent interaction with the electrode surface [75][76][77] .

Covalent Strategy
Covalent strategies refer to the formation of covalent bonds between aptamers and electrode surface. They are the most commonly used modification strategies in the process of aptamer modification. Most E-AB sensors are fabricated by covalent strategies [78][79][80][81][82][83][84][85] . With the advantages of stable bonding and simple steps, covalent strategies can directly combine aptamers with the electrode surface stably [86,87] .
One of the most common covalent strategies is to modify aptamers on the surface of gold electrodes or on gold nanoparticles attached to the electrode surface by gold-sulfur bonds(Au-S) [88][89][90][91][92][93] . This kind of modification strategy is simple, can be carried out at room temperature, and the modification process is not limited by the shape of the electrode. It can be applied to gold micro electrodes and gold nanoparticles to meet the requirements of portable sensors.
Xiao et al. [94] reported a portable paper E-AB sensor that can be applied to in-situ detection. After modifying a single-layer carbon nanotube film on the paper in vacuum atmosphere, gold nanoparticles are deposited on it to form a "three Connecting aptamers with gold nanoparticles through Au-S bond gives this covalent strategy flexibility, which can make it obtain greater advantages by combining gold nanoparticles with other materials [95][96][97][98][99] . Xu et al. [100] reported a modification strategy of modifying the carbon electrode by combining multi-layer carbon nanotubes with gold nanoparticles, and then connecting with the aptamers through Au-S bond. This modification method increased the effective modification area and the conductivity of the electrode surface, resulting in the higher signal response and more aptamers for higher selectivity. The detection limit of long non-coding RNAs(lncRNAs) by differential pulse voltammetry(DPV) method is down to 42.8 fmol/L, and the linear range is 0.01 pmol/L-10 nmol/L. Compared with the interfering substance at 50 pmol/L, the target substance at 1 pmol/L can produce a significant signal response.
Stability, flexibility, cost and adaptability of electrodes are several factors that must be considered in the selection of modification strategy in the fabrication process. With the development of electrochemistry, carbon electrodes show excellent performance and gradually become one of the main electrodes. In this system, the application of Au-S bond is greatly limited. For carbon electrode system, it is a common modification strategy to generating stable amide bonds (-CO-NH-) by using 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride(EDC) and N-hydroxy succinimide(NHS) activated carboxyl groups and amino modified aptamers [78][79][80] . Amide bond has the advantages of strong stability, diverse generation strategies, flexible design and wide application system [101][102][103] . It can be applied to the modification of carbon electrodes, emerging carbon materials and metal nanoparticles [104][105][106] . New materials, such as graphene and metal nanoparticles have the advantages of good conductivity, stable adhesion, strong chemical reactivity and large surface area. When they are combined with aptamers, improved amounts of aptamers are modified on electrodes, offering higher selectivity and sensitivity of sensors [107][108][109] .
Liang et al. [79] reported a sensor combining aptamer with graphene oxide(GO) on the surface of glassy carbon electrode, which can be used for detecting α-fetoprotein(AFP). Firstly, the oxygen-containing groups on the surface of GO were oxidized to carboxyl group through the mixed solution of chloroacetic acid and sodium hydroxide, and then they could bond with the aptamers modified with amino group through amide bond. The 20-folds interfering proteins PSA and CEA have not caused obvious signal response, demonstrating its high selectivity. The detection limit is 3 pg/mL[ Fig.1(B)]. In another example, Upan et al. [80] reported a sensor, in which PtNP/GO-COOH nanoparticles were generated from GO and platinum by the aqueous phase reduction method, modified on the surface of glassy carbon electrode and formed amide bond with the AFP aptamers. This sensor showed high selectivity and sensitivity with a detection limit achieving 1.22 ng/mL by square wave voltammetry(SWV) method.
When covalent strategies carried out on the electrode surface, it is difficult to ensure that aptamers completely cover the whole electrode surface. In order to prevent electrochemical active substances from reacting on the unmodified electrode surface and producing false-positive electrical signals, it is usually necessary to add blocking agents to passivate the unmodified area on the electrode surface [110] .
There are three types of passivation strategies, a) blocking the Mercaptohexanol(MCH) is a blocking agent often used in gold electrode system because of its strong Au-S binding ability and mild reaction conditions [111][112][113][114][115] . Moreover, MCH is difficult to be protonated or deprotonated in the detection system, thus reduces the impact of electrostatic interaction and provides a better passivation effect [116] . Recently, increasing studies have shown that the ratio of aptamer to MCH also significantly affects the performance of sensors. When fabricating the sensor of tenofovir(TFV), Aliakbrinodehi et al. [117] found that the sensor showed the highest signal response when the ratio of aptamer to MCH was 1:100 [ Fig.1(D)]. Higher or lower ratio will lead to the decrease of response value. This is probably because at a lower ratio, aptamers cannot fully capture the targets, while at a higher ratio, not all aptamers on the electrode surface change their configuration completely when capturing the targets due to the influence of steric resistance and electrostatic repulsion, which hinders the rate of electron transfer. Other than MCH, bovine serum albumin(BSA) is also a widely used blocking agent [118][119][120][121][122] . BSA has a large number of amino and carboxyl groups on the surface, so it can combine with both hydrophilic and hydrophobic surfaces to inhibit the nonspecific binding on the electrode surface [123] .

Noncovalent Strategy
Noncovalent strategies refer to the connection of aptamers and electrode surface through electrostatic adsorption, hydrogen bonding, van der Waals interactions, etc. Compared with covalent strategies, noncovalent strategies exhibit unique advantages, such as less strict requirements for aptamer modification, wider application field, and more flexible design [124] . They are relatively straightforward and can quickly realize the assembly and functionalization of sensors.
Streptavidin is a protein composed of four peptide chains.
It can form a very strong interaction with biotin through noncovalent interaction. The extremely low dissociation constant(Kd=10 -15 mol/L) demonstrates that this interaction is very stable [125] . By modifying streptavidin on the electrode surface and biotin on the 5ʹ end of the aptamers, the aptamers can be efficiently modified on the electrode surface. On this basis, the strategy can be further developed by combining with nano materials to obtain better signal response. Gopinath et al. [126] reported a sensor using streptavidin and biotin to modify the complex of aptamers and gold nanoparticles on the electrode, and verified that the signal response of the sensor employing gold nanoparticles was higher than that of the platform without gold nanoparticles. Compared with the platform without gold nanoparticles, the detection limit decreased to 10 times lower.
The application of various materials also provides more possibilities for the improvement of the selectivity and sensitivity of sensors. Cai et al. [127] reported a method of combining GO, thionine, chitosan and streptavidin modified gold nanoparticles to form a modified electrode, and then combined with biotin modified aptamers to form a sensor. This strategy enhanced the loading efficiency of the aptamers, leading to a great sensitivity with a detection limit of 10 pg/mL for estrogen(17β-e2).
Though noncovalent strategies have a wide range of applications, they are prone to be affected by different types and structures of aptamers. Zhang et al. [128] reported that few or single-layer nano disulfide transition metal layers, such as aptamers on the surface of carbon fiber electrode [130] .
Specifically, we pre-modified the CFE surface with nhexamine chain by electrooxidation method, and then the aptamers with cholesterol-modification at its 5ʹ end can spontaneously assemble on the alkyl chain layer through hydrophobic interaction to form an aptCFE sensor. We implanted this micro sensor into the brain of mice. After

Applications of E-ABs
The application scope of the biosensor mainly depends on its sensitivity, selectivity, and stability, which requires the detection limit of the sensor to be lower than the actual concentration of the detection environment, quick response, and not to be disturbed or affected by interfering substances [31,132,133] . Up to date, E-ABs have been applied for the sensing of a variety of analytes, including small molecules, macromolecules and large analytes such as cells and viruses.

Sensing of Small Molecules
Small biological molecules, such as neurotransmitters, metabolites and cell secretions play a vital regulatory role in the biological system [134][135][136] . The detection of biological small molecules is of great significance for understanding the mechanism of biological activities and disease monitoring.
Due to the high selectivity and sensitivity of aptamers, E-AB sensors have been widely used in the actual detection of a variety of biological small molecules [137][138][139] . Based on the electrochemical properties of these small molecules, they can be divided into electroactive or electroinactive analytes.

Electroactive Analytes
For the sensing of electroactive analytes, the flexible aptamer chain captures the target, wraps around the small molecule, and makes the small molecule close to the electrode surface for electron transfer. In fact, few electroactive substances, such as dopamine are suitable for sensing in this direct way [140][141][142] .
However, in many cases, when the aptamer wraps the targeted molecule, it shields the signal of the analyte. In addition, this direct sensing strategy has no amplification effect. Thus, the signal response is limited to the amount of modified aptamers and the concentration of the analyte, and the signal response is usually not as good as indirect sensing [143][144][145] . The detection limit of the sensor sensing in a direct way by the oxidation of dopamine is usually nmol/L level, which could be further improved with the assistance of nano materials with high conductivity [146,147] .
Because the electrochemical signal is generated from the analyte itself in the direct sensing based approach, the fabrication steps of the sensors for electrochemical active substance are relatively simple and can be applied to a variety of systems, especially biological systems. For example, Ferapontova et al. [148] reported a sensor that could be directly

Electroinactive Analytes
In biological systems, many small molecules, such as gas molecules and metabolic substances do not have electrochemical activity or require a high potential to generate electrical signals, so they need the assistance of other signal substances to generate signals through the changes of electrode interface chemical environment [149][150][151][152][153] . Indirect sensing strategies of electroinactive analytes have the advantage that the signal intensity does not directly depend on the number of aptamers modified on the electrode, so the detection limit can be further improved, and can be applied to complex environments with higher selectivity. However, compared with direct sensing, the fabrication of the sensor is usually more complex, the cost may be higher, and more factors need to be considered in design.
Since the target substance itself does not have electrochemical activity, one of the most common strategies is to modify the electrochemically active groups on the aptamer, such as methylene blue(MB), which is usually modified at one end of the aptamer. When the aptamer does not bind to the target substance, the nucleic acid chain stretches, the electrochemical active group is far away from the electrode surface, and the electron transfer rate is slow. When the configuration of the aptamer binding target changes, the electrochemically active group is close to the electrode surface, the electron transfer rate is accelerated, and a larger current response signal is generated, which is positively correlated Review with the concentration of the target substance [154][155][156][157][158] . White et al. [82] reported a sensor that modified the gold electrode with MB-modified aptamers. When the aptamer captured the target, MB approached to the electrode and electron transfer occurred, and electrons of indicator electrode prussian white(PW) were lost on the color plate connected with the sensing electrode to make PW become prussian blue(PB) [Fig.4(A)]. Based on the process, the colorimetric method can be used to detect the concentration of ATP.
In addition to modifying electrochemical active groups, the method of inserting electrochemical indicators is also very common. The most commonly used one is ruthenium complex ions that can bind to the nucleic acid chain skeleton through non-covalent interactions and generate electrochemical signals.
Since a single aptamer can bind multiple ruthenium complex ions, the signal response significantly increases, which has a wide range of applications [159][160][161][162] . Dionysiou et al. [159] reported a sensor using aptamers to capture microcystin-LR(MC-LR) for the removal of Ru(NH3)6 3+ and obtain a response signal.
Based on the reason that after the aptamers bind to MC-LR, the Ru(NH3)6 3+ originally bound to the aptamer will be removed, resulting in the decrease of SWV peak, the reduction of Ru(NH3)6 3+ signal is positively correlated with the concentration. The selectivity for microcystin congeners with very similar structures and inference substances that may appear in the lake environment has been verified[ Fig.4(B)].
The detection limit of the sensor is 9.2 pmol/L.
As an oligonucleotide sequence with recognition ability, aptamer itself has the hybridization ability of nucleotide sequence. When aptamers bind with their targets, the interaction between them is often greater than that of the hybridization of aptamers and their complementary chain, so the hybridization ability of nucleic acid can be used to design more diverse sensing strategies, which not only provides higher flexibility for the sensing strategies of the E-AB sensor, but also further improves the selectivity and application range of the sensor.
Hybridization and dehybridization between nucleic acid sequences need appropriate temperature. Lin et al. [84] reported a sensor, which hybridizes nucleic acid by heating electrode itself rather than  pressure, which can be transformed into electrical signals by piezoelectric materials [167][168][169][170] . Wang et al. [171] reported a method of detection for okadaic acid using piezoelectric materials by E-AB sensor. After the piezoelectric materials are modified with complementary chains of aptamers, they are incubated together with the mixture of okadaic acid and okadaic acid aptamers. Through competitive bonding, the signal is correlated with the concentration of okadaic acid. Finally, gold nanoparticles modified with complementary chains are used for improving conductivity and electrode surface interaction.
The detection limit of this detection platform for okadaic acid is 0.26 ng/mL. In addition, the selectivity of the sensor was proved by analyzing with other three DSP toxins, and 10-folds interfering substances had no obvious interference.

Sensing of Macromolecule
The detection of macromolecules in biological systems, such as proteins and enzymes with complex structures, is also the focus of analytical chemistry. Biological macromolecules are very important in cancer detection and pathological research [172][173][174] . Their efficient detection is conducive to the prediction and prevention of diseases in advance. E-AB sensors can also be applied to macromolecules in biological systems with different sensing strategies [175] .
Due to the size of macromolecules, complex structures may be formed on the electrode surface when aptamers capture macromolecules, which may hinder the signal response process on the electrode surface, so it is necessary to adopt appropriate strategies to amplify the signal [176][177][178] .
Employing electrochemical indicators is an excellent strategy to enhancing the sensitivity and selectivity of E-AB sensor. Li et al. [179] reported a general strategy of using exonuclease to control the switching state of the sensor. Firstly, aptamer is modified on the electrode and combined with Ru(NH3)6 3+ .
After binding with the target, the exonuclease cannot decompose the aptamer due to the change of configuration, while the aptamer that does not bind to the target is decomposed. Therefore, the signal intensity of ruthenium hexamine is proportional to the concentration of the target, the interference of other substances is reduced obviously, and no significant signal was observed with 100-folds interfering substance. Using CV method, the detection limit of lysozyme is 2.0 nmol/L.
Another excellent strategy is amplifying the signals after the aptamer binding with target leaving the electrode.
Yuan et al. [180] reported a sensor that can be used to detect When aptamers bind to macromolecular targets, it is also possible that aptamers only recognize and bind part of the macromolecules. Therefore, macromolecular substances may have multiple recognition aptamers and can form a variety of structures with aptamers [181][182][183] . Sandwich structure is a common sensing strategy for macromolecular targets and even bigger analytes. The principle of sandwich structure is to combine the material modified on the electrode surface with the analyte, and then attract a new aptamer, so that the analyte molecules are wrapped in it to form a structure similar to sandwich, and the signals of analytes can be detected through the change of electron transfer rate after structure change or the introduction of new catalytic or electroactive groups [184][185][186] .
A variety of substances can participate in the construction of sandwich structure to form a stable sandwich structure.
Shim et al. [187] reported a sandwich structure sensor assisted by magnetic force. Firstly, the aptamer was modified on the surface of the working electrode and the thrombin in the system was captured. Then, when the electromagnet of the working electrode was turned on, the magnetic nanoparticles (MNP) modified by antibody and toluidine blue B(TBO) will be close to the target, and then the auxiliary electromagnet was used to remove the magnetic nanoparticles not binding with thrombin to obtain the sandwich structure pTBA/Apt/ thrombin/MNP@Ab-TBO. By detecting the current change, the detection limit of the sensor is 0.49 nmol/L. Nie et al. [188] also used the sandwich structure to detect thrombin. The difference is that they used two different aptamers to fabricate a new kind of sandwich structure TBA1/thrombin/TBA2-Go-RuNP. TBA2 is modified on graphene oxide attached with ruthenium nanoparticles. The detection limit of ECL method is 28.73 fmol/L[ Fig.6(B)].
New technologies, such as molecular printing, can also be applied to the fabrication of sandwich structures. He et al. [189] reported a sensor based on molecular imprinted polymer(MIP) and aptamers. After a layer of chitosan is coated on the surface of the glassy carbon electrode, the molecular imprinting process is carried out on the surface, and the special structure can be formed after removing t he template. When there is a target in the system, the target will be embed into the imprinting layer and attract the gold nanoparticles modified with aptamers to form a sandwich structure. The results show that the selectivity and the sensitivity of the sensor are significantly enhanced by molecular printing[ Fig.6(C)]. Using DPV method, the detection limit for amyloid-β oligomer is 1.22 pg/mL, and the linear range is 5 pg/mL-10 ng/mL.

Sensing of Cells and Viruses
Compared with macromolecules, cells and viruses are more complex organic bodies. Their efficient detection is conducive to the direct observation of the mechanism and development process of diseases, playing a great role in human health and environmental governance [66,87,139,190] .
In the sensing process, cells and viruses are large, and a single aptamer is difficult to wrap the whole analyte [66,89,153] .
Therefore, some strategies of macromolecular sensing cannot be directly applied to these analytes, while the selectivity and the sensitivity are still the core part of the sensor development.
When sandwich structure is used in cell or virus detection, it needs to be combined with nanomaterials to improve the signal in order to obtain higher sensitivity and selectivity.
Tabrizi et al. [97] reported a sensor for detecting adenocarcinoma gaseous cancer cell(AGS). Firstly, carbon nanotubes and primary aptamers were modified on it, capturing AGS, and then metal nanoparticles modified with secondary aptamers were immobilized. With hydrogen peroxide as a reductant, the concentration of AGS could be detected by amperometric method. The detection limit of the sensor is 6 cell/mL and the linear range is 10-5×10 5 cell/mL.
Vajhadin et al. [101] reported a sandwich structure that used aptamers to recognize specific proteins to capture HER2 positive cancer cells. The aptamers are dividedly modified on As viruses and cells are complex organisms, their surfaces have many different functional structures, and they have abundant sites for aptamer recognition [89,195,196] . Therefore, multi segment of aptamers can be used to form a winding structure for analytes, which can successfully capture viruses and cells with high sensitivity and selectivity. Xiang et al. [89] modified the primer DNA on the surface of the gold electrode, and then formed a multivalent aptamer network for capturing the target circulating tumor cells(CTCs) with the assistance of DNA template and rolling circle amplification. After the aptamer network wrapping the CTCs, gold nanoparticles modified with antibodies will be introduced into the electrode surface as a catalyst for the H2O2 oxidation to generate signals.
Except for the target cancer cells, other cancer cells cannot generate obvious signal responses[ Fig.7(B)]. Using CV method, the detection limit of the sensor in the whole blood is 5 cell/mL.
The binding and detection of aptamers with macromolecules or larger analytes require aptamers with better affinity. This can be achieved by combining multiple aptamers [197][198][199] . For instance, the detection of coronavirus(SARS-Cov-2) is the most concerned challenge since the outbreak of the epidemic.