Aptamer‐based biosensors and application in tumor theranostics

Abstract An aptamer is a short oligonucleotide chain that can specifically recognize targeting analytes. Due to its high specificity, low cost, and good biocompatibility, aptamers as the targeting elements of biosensors have been applied widely in non‐invasive tumor imaging and treatment in situ to replace traditional methods. In this review, we will summarize recent advances in using aptamer‐based biosensors in tumor diagnosis. After a brief introduction of the advantage of aptamers compared with enzyme sensors and immune sensors, the different sensing designs and mechanisms based on 3 signal transduction modes will be reviewed to cover different kinds of analytical methods, including: electrochemistry analysis, colorimetry analysis, and fluorescence analysis. Finally, the prospective advantages of aptamer‐based biosensors in tumor theranostics and post‐treatment monitoring are also evaluated in this review.


| INTRODUC TI ON
The latest statistics from the American Cancer Society found that the average number of people dying from cancer in the United States each year is as high as 600 000. 1 Fear of cancer is a common phenomenon at this stage. The limitation of effective treatment and delay in diagnosis are the main reasons for the high incidence of cancer mortality. 2 In view of this, timely and accurate diagnosis of early cancer is particularly important for improving the cure rate of cancer. Currently, common cancer diagnosis methods include tissue biopsy, proteomics, tumor imaging, and biomarker detection. 3 Compared with the first 3, biomarker detection is more common in clinical screening and diagnosis due to its characteristics of less invasive damage and lower cost. However, the detection of tumor markers at this stage has low sensitivity and cannot be used for low-level concentration screening in the early stages of cancer, resulting in a certain missed diagnosis rate. Designing a low detection limit and high affinity detection strategy is particularly important for the detection of early cancer marker proteins. 4 Recently, due to the application of various signal amplification technologies in biosensors, such as enzyme catalysis, nucleic acid chain reaction, biotin-streptavidin, click chemistry, cascade reaction, nanomaterials, etc., biosensors have high reproducibility and sensitivity to effectively circumvent the limitations of traditional methods. 5 With a very low detection limit, the biological signal is converted to a visual signal that can be used to measure the level of specific proteins on or secreted by tumor cells. The biosensor was defined as "an independent integrated device." 6 Usually, they mainly include 3 major components: biometric identification component, signal conversion component, and information reading component.
Most of the biological recognition elements are macromolecules such as antibodies, aptamers, and enzymes, which have the characteristics of specifically recognizing target analytes to facilitate quantitative or semi-quantitative analysis of a certain target by biosensors. 7 At present, the recognition element of the most applicable aptasensor is a short oligonucleotide chain separated from a random library in vitro by SELEX, 8,9 using different screening strategies and manipulation of the selection conditions to closely control the aptamer-target binding affinity and specificity. 10 Generally, the size of the aptamer sequence can be ~30-70 nucleotides in average length, folded into a three-dimensional structure, and connected to specific biological elements through specificity and affinity, such as metal ions, tumor marker proteins, small molecules, or even viruses, circulating tumor cells, etc. [11][12][13][14][15] For example, Li et al used aptamers and nanomaterials to assemble fluorescent aptasensors to detect tumor-associated proteins on exosomes derived from prostate cancer and breast cancer, and successfully used them in the clinical differentiation of healthy specimens from tumor specimens with the advantage of high sensitivity. 16 Based on the characteristics of rapid response and portability, biosensors using enzymes as identification elements have been used for immediate detection of tumor patients, such as detecting circulating tumor cells, prostate antigen, etc. 17,18 However, enzymes are sensitive to temperature and pH, having a short shelf life. For antibody sensors, the generation and characterization of new antibodies are time consuming and difficult, 19,20 such as induction by target preparations, animal immunity, antibody purification, and other operations are complicated and require time and material costs. [21][22][23] For some small molecules and proteins with low immunogenicity, it is also difficult for newly generated antibodies to control their binding properties and bind to similar structures to cause non-target interference, 24 and are easily affected by immunosuppressive agents. 25 Different from enzyme and antibody sensors, the most notable feature of aptamers is their ease of modification and low immunogenicity. The specific aptamer sequence can be synthesized in vitro with low cost, reproducible mass production, and is easily modified by nanomaterials for tumor marker analysis and treatment. In addition, they can tolerate various pH and salt concen-

| ELEC TRO CHEMIC AL AP TA S ENSOR
Electrochemical aptasensors are currently the most widely used biosensors in tumor imaging, which was first proposed in 2004, and they can provide low-power and ultra-low detection limits of target analytes. 31,32 Due to its high accuracy and good reproducibility, this type of sensor is often used as a minimally invasive device for POCT. 33 Generally, in the electrochemical aptasensor, the aptamer is fixed on the electrode surface as a biological recognition element.
Through the specific binding of the aptamer to the target, the capacitance change caused by binding of the analyte or the current or potential response generated by the oxidation and reduction reactions on the electrode surface is evaluated. According to the type of response signal, it can be divided into ampere method, cyclic voltammetry, electrical impedance method, etc. [34][35][36] This section discusses the design schemes of several common electrochemical aptasensors for tumor marker monitoring, including direct fixation, sandwich, and immobilization free.

| Direct immobilization
In most cases, the aptamer probe can be immobilized on the electrode surface to capture the target protein by electrostatic adsorption, covalent attachment, and affinity. 37 Several electrochemical aptasensors based on direct induction of target have been developed, with paper, ion-exchangeable polymer membrane, and

| Sandwich format
The design strategy of the sandwich electrochemical aptasensor comes from the structure of the immunosensor, including antibody-aptamer sandwich, aptamer-antibody sandwich, and aptamer-aptamer sandwich sensing layer. 39

| Immobilization free
For the above 2 types, the process of fixing the aptamer to the electrode surface is time consuming. The aptamer assembled on the electrode sometimes hinders the effective recognition between the target and the aptamer. Unlike the conventional strategy of fixing aptamers on electrodes, in Figure 1C, this design strategy is usually to form a dsDNA conformation by complementing the hybridized strand with MB or Fc-labeled aptamer. The dsDNA modified with electroactive substances is easily adsorbed by the electrode modified with specific nanomaterials, and then the electrical signal is turned on. When the target marker is present, the aptamer preferentially binds to the target, the complex falls off, and the electrical signal is turned off. In the study by

| AuNPs
At present, the most common AuNPs are used as transducers of colorimetric aptasensors in various studies. 49 Due to the surface plasmon resonance of gold nanoparticles, they have strong distance-dependent optical properties. 50 Once the differently modified gold nanoparticles are close to each other, their absorption spectra will shift, and the scattering profile will change, even- cells that expressed high NCL from normal cells not expressing NCL, and the detection limit of this method was estimated to be ~10 cells/mL, which gave sufficient sensitivity and selectivity. In addition, the operation was simpler than the more costly fluorescence and electrochemical measurements. 53

| H 2 O 2 oxidation
Another commonly used detection strategy is to simulate intrinsic enzymes, 54 such as modifying nanozymes with aptamers to increase the activity of peroxidase-like enzymes, to enhance the oxidation of

| FLUORE SCENT AP TA S EN SOR
The design of fluorescent aptasensors mainly involves fluorophores, dyes, or fluorescent nanomaterials, aptamers, and quenchers. 62

| Fluorescence resonance energy transfer
The most typical way is to modify the 3' or 5' end of the aptamer with a fluorophore or an aggregation-inducing luminescence agent, and then introduce a fluorescence quencher through electrostatic adsorption or complementary structure. The fluorescence signal is turned off based on FRET between the luminescent agent and the quencher. 16,66 When the target marker is present, the conformation switch shifts, the quencher and the fluorophore are separated, and the fluorescent signal is turned on again to quickly detect the target protein 67 ( Figure 4A). This method of turning off and then turning on the fluorescent signal effectively reduces the background signal.

| Fluorescence signal amplification
Common nucleic acid signal amplification reactions include RCA, HCR, HD-CHR, and SDR. 3 As shown in Figure 4B, Huang Figure 4B shows the principle of signal amplification in common HCR-based sensor designs.

| Fluorescence polarization
Compared with the former, the design based on fluorescence polarization is more simplified and requires no quenchers or donoracceptor pairs. As shown in Figure 4C, when the aptamer detaches from the polymer and forms a specific three-dimensional structure with the target protein, the fluorescence signal is restored. 36 Bao et al reported that the Apt-PFN+ complex was used to detect tumor markers AFP and CEA. 73

| Prospects for theranostics
In addition to early screening and diagnosis, timely treatment of pre-cancer is the top priority to reduce the mortality rate of cancer.
At present, aptasensors are mature in various cancer screening and detection technologies. However, being able to report and trigger targeted killing of tumor cells at the same time is still a challenge we need to face. 76 At this stage, some researchers have reported that aptasensors can be combined with photodynamic, photothermal, and chemotherapy to diagnose and treat tumors. 77

| CON CLUS I ON AND OUTLOOK
In this review, we focused on the detection strategies of aptamer biosensors with 3 signal transduction modes. Each type of aptamer sensing has unique advantages and corresponding limitations, which must also be considered before determining the purpose of detection. For example, the fluorescent aptasensor is easier to use for multi-channel simultaneous detection of different tumor cells than the other 2; the colorimetric aptasensor is the most intuitive and simplest of the 3 to distinguish between cancer and normal samples; the electrochemical aptasensor is more focused on non-invasive, small, portable, and low detection limits to screen tumor markers.
In addition, the article also mentions the expanded application of aptasensors in treatment and post-treatment monitoring. At present, many studies have been successfully applied in the clinic to detect cancer patient samples. We have reason to believe that the application prospect of aptasensors will become more available in the clinical practice in the foreseeable future.