Whole‐Cell Electrochemical Aptasensors for Cancer Diagnosis: Current Advances and Prospects

Cancer is one of the most life‐threatening diseases worldwide. Numerous diagnostic and therapeutic techniques, such as nanomaterials‐based cancer detection and imaging‐guided focal therapy, are successfully developed to achieve highly precise cancer theranostic strategies. However, due to unpredictive alterations occurring in cancer cell morphology, a lack of sensitivity and selectivity is a critical challenge among most biomarkers’ detection. To address this issue, instead of targeting proteins or biomarkers, the detection of cancer using whole cell‐based biosensors are ubiquitously discovered through a selective oligonucleotide, so‐called aptamer. Aptamer‐based whole‐cell detection is concurrently engaged with various nanomaterials, such as magnetic beads, gold (Au) nanoparticles, and graphene family to establish a better performance of the biosensor. In this research review, the recent strategies and prospects on whole‐cell cancer detection platform based on electrochemical aptasensors integrated with nanomaterials are thoroughly discussed and summarized. Finally, future challenges and prospects are also provided.


Introduction
Cancer is one of the major causes of death worldwide. [1]The incidence of cancer has increased to ≈8.2 million mortality cases, DOI: 10.1002/adsr.20230015132.6 million patients suffering from cancer, and 14.1 million new cases reported, with less developed countries experiencing more evidence in the last 5 years. [2]n the US, 43% of cancers case in men include prostate, lung-bronchus, and colorectal cancer, with prostate cancer dominating the figure.Meanwhile, breast, lung-bronchus, and colorectal cancers are highly prevalent among the female population with breast cancer alone accounting for 30% of all cancer cases in women. [3]umerous studies have been actively conducted to develop and introduce effective techniques for cancer diagnostics down to the molecular differential stage. [4]Indeed, understanding cancer at the molecular level is such a critical issue to characterize cancer malignancy among the disease stages successfully.In cancer-related biomarkers, proteins expressed by the specific cancer cells could provide the fundamental screening of diagnostic as well as treatment. [5]However, the time-consuming and intricate purification processes of a single protein from its complex biological matrix create challenges to obtain a potential biomarker beneficently.Due to the effect of genetic alteration, cancer phenotype also varies with the species, which somehow creates another issue of discrepancy in biomarkers expression. [6]These reasons further point out that most of the biomarkers commonly used in cancer assays still lack sensitivity and selectivity. [7]iosensors have successfully opened a new pathway in medical technology due to their proficient devising method to monitor blood glucose levels for diabetes patients. [8]These findings have convinced scientists to develop cancer diagnostics devices that are as simple as blood glucose sensor.To confront the challenges and achieve effective cancer treatment, some strategies are currently being considered, which include the use of whole cells as the "real" target.The genetic signatures appearing on the cancer cell surface comprise consequential "marker" types, which are beneficial to magnify the detection process.As a consequence, various detection techniques have been developed by utilizing specific antibodies, aptamer, DNA hybridization, or other biomolecules extracted from a complex biological matrix.These particular elements are further immobilized on the supporting surface thus, probing an excellent binding affinity toward the abnormal cells. [9]7c,10] Aptamer offers great choices in generating an ultra-sensitive result via proper integration with electrochemical sensors. [11]Interestingly, aptamers are vastly compatible with various electrochemical biosensor platforms, thereby contributing to the global market for rapid medical diagnostics. [12]The global market for blood cancer molecular diagnostics kits is $ 335.9 million in 2016 and is predicted to reach $ 6980 million in 2026, with a 32.9% annual growth rate. [13]10c,14] NPs in biosensors have significantly advanced the device performance and application diversity, especially for sensorrelated technology. [15]For example, the unique properties of nanoscale materials integrated with biosensors can provide an excellent platform to obtain ultrasensitive detection and remarkable stability. [16]Besides, a combination of nanomaterials and wholecell specific recognitions yields high selectivity and sensitivity for cancer cell detection.An efficient recognition in this regard could be achieved by functionalizing the nanomaterials. [17]For instance, graphene and its analogous 2D materials have sparked a great interest in cancer cell detection in recent years. [18]The implementation of such materials could enhance the biosensor performance due to the large surface-to-volume ratio, ultrathin planar surface, distinctive electronic bandgap, rich electrochemically active site, and remarkable physicochemical property. [19]19b,20] To the best of our knowledge, the review determining wholecell biosensing for each certain cancerous cells type has not yet been disseminated.Herein, the latest development of biosensor configuration for whole-cell cancer detection incorporated with nanomaterial, type of biorecognition, particularly aptamer, and a wide-range analytical performance are discussed.Besides, the electrochemical approaches along with the sensitivity, limit of detection (LOD), stability, linear detection range, as well as selectivity, are thoroughly highlighted.In their applications, nanomaterials usage in whole-cell biosensors offers clinical benefits, especially in designing a high-intelligent probe for various cancer detection strategies (Figure 1).

Multistage Carcinogenesis of Cancer
Generally, cancer could be attributed to numerous causes, such as unhealthy lifestyle, consumption of preservative foods, excessive alcohol consumption, and inhalation of tobacco (smoking).These behaviors affect the immune system by damaging and modulating multistage carcinogenesis.The four stages of carcinogenesis (Figure 2) are explained below.Each stage is defined by modifications in morphology, biochemical genetic changes, and epigenetic changes. [21]t the beginning, carcinogenesis starts from a repetitive exposure to one of the factors above or potential chemical compounds, which alters the metabolism of healthy cells, and thus initiates cancer growth.This initiation progresses when the nucleic acid arrangement of healthy cells is genetically altered.Second, promotion, the affected cells secrete carcinogenic metabolites, leading to a mutation in the nucleic acids of other neighboring cells, causing them to become cancerous.Subsequently, transformation, the affected cells that have been transformed begin to accumulate.Thereby, progression, the amount of clonal expansion and the changes in target cells milieu, provide a specific environment for the affected cells to grow as malignant and immortalized cells. [22]umor promoters may not induce the malignant stage since the cancer cells already have self-promoter potency.Interestingly, this hallmark enables the expansion of malignant cells to be broader, reversible, and comprehensive with anti-programmed death cells, which could impact the physiological alteration at the tissue level. [23]However, the expression of aggressive malignant cells, called tumor progression, are unstable genomic and have uncontrolled growth characteristics.The malignant phenotype of cells is physiologically heterogeneous and able to metastasize as well as to support their expansion as the primary tumors.23a,24]

Urgency for Whole-Cell Cancer Detection
The cell membrane has a critical purpose in regulating cellular life, functioning as growth, proliferating, signaling, metabolism processing, and apoptosis.The specific cause explaining how a mutation leads to cancer with physiological disorders is due to the abnormalities of cellular expression, that is, malfunction of cell membrane receptors. [25]Several reports studied certain receptors present on cell membrane as biomarkers for specific cancer investigation, such as in drug treatment purpose.However, most of these biomarkers resulted in low selectivity after being examined among types of cancers. [26]s compared to normal cells, cancer cells possess six biological characteristics acquired during the multistage development of tumors.23b] Most of the studies start from the molecular stage by first classifying the disease into proteomic or genomic level.The conventional approaches provide significant outcomes, yet it is time-consuming and laborious as it connects the abundant library with patients' medical records. [7]Meanwhile, there are polymerase chain reaction (PCR) and nucleic acid amplification-(NAATs) methods, which are commonly applied to sequence the gene of interest then sort the genetic and molecular information of the target gene.However, the complex steps which start from RNA's expression stage, long isolation control, and checking variability status remain less effective. [27]osensor is simplified and integrated version of a complex analytical instrument compiled into one compartment of a sensing system.The device instrumentation contains three important components: to respond specific biological substances or analytes as the target, viz., the biological receptor (bioreceptor/biorecognition element), as a signal converter/quantifier (transducer), and as signal processor or interpreter (display) (Figure 3).Bioreceptors are commonly extracted either from biological resources (e.g., antibodies, antigens, cells, and enzymes) or the environment (e.g., bacteria cells, glucose, and any other organic compounds).As opposed to the conventional methods (e.g., HPLC, LC-MS, spectrophotometry), a biosensor embraces the bioreceptor as an agent to identify the nature of the analyte as the target based on its physical-chemical properties. [28]The application of biosensors has been used widely to various fields, such as medical diagnostics, pharmaceutical drug discovery, food safety, environmental waste control, and military defense. [29]merging biosensors for early-stage cancer detection are beneficial to turning the complicated cancer screening procedures into simplified and reliable one.In terms of early detection, biosensors usually target the cancer biomarker as it represents the existence of a particular substance produced by cancerous cells and corresponds to the dynamic changes in the physiology of cancer cells. [30]7b,31] Thus, biomarkers are also essential in monitoring the drug delivery for cancer therapy as well as the progression of cancer. [32]

Antibody and Specific Biomolecules
Molecular targeting in cancer detection is associated with the recognition of cell surface properties, that is, cellular signatures (protein, enzyme, and metabolite). [33]Generally, the principle of antibodies as biorecognition to target cells is through binding the active sites of those surface signatures over-expressed on the cell surface, commonly known as antibody-antigen interaction. [34]esides targeting specific cell signatures, the antibody could recognize its target selectively. [35]Therefore, antibodies are widely used in several biomedical fields, especially in escorting the anticancer agent to the abnormal cells to promote the induction of anti-tumor immune responses. [36]Similarly, some biomolecules are selected to define a high-affinity binding in targeting receptors, that is, folic acids for folate receptors. [37]They are involved in the recognition of the target cells for diagnostics purposes as well as assembling the drugs to be delivered at the particular disease site.For example, the folate receptors on the target cell surfaces will be immediately induced to initiate self-receptor endocytosis after activation by folic acids. [37,38]However, there are only a few

Nucleic Acids
Recently, cancer diagnostics strategies are merged into DNA sequences (genomic) analysis for molecular analysis suggesting a highly sensitive method to identify genetic mutation of organisms.Harnessing the Witson-Crick idea, the hybridization of nucleic acids are visible for the biosensor targeting method, as a probe tool to detect mutation or distinguish types of diseases.The duplex formation undergone between the capture probe and the target oligonucleotides could be recognized due to an appropriate hybridization process or through other related changes initialized from the binding evidence.The hybridized state indicating the target oligonucleotides will then appear as a readable signal. [39]he capture probe is commonly generated from various kinds of materials that have been designed to recognize the conformational body of the target.However, it is still limited for short oligonucleotides used as a probe.Generally, to obtain a higher specificity, the capture probe length should be ≈18-25 nucleotides.Extremely long probes could potentially affect the analysis as it may turn to unexpected hybridization. [40]Further, a biosensor-based labeling approach, so-called microarray, has been taken into account using hybridization of short nucleic acids as a new targeting agent.The hybridization technique is useful in design of sequencing libraries, genome screening, and pathogen detection. [41]

Aptamers
Aptamers are synthetic oligonucleotides (either RNA or DNA) populated from several iterative sequencing processes of SELEX against specific targets involving the control of biomolecules to retain the selectivity of the oligonucleotides.Aptamers cover a large number of biological target types, ranging from small to large molecules, including toxins, proteins, and cells. [42]Furthermore, the most common targets of aptamers to detect cancer are proteins present on the cancerous cell surface.Unfortunately, not all the proteins have readily been purified to allow them to be recognized.The proteins in a complex membrane (such as raft membrane) are more likely to be shielded and difficult to access.
Cell-based SELEX is used to generate aptamers that own greater specificity toward specific binding interactions with whole-cells as the target. [43]Cell-based SELEX maintains better aptamer selectivity as compared to the conventional SELEX in targeting cell proteins.Cell-SELEX principally works according to the type of cell-body or whole-cells being introduced, which naturally preserves their original targets including their essential proteins or other biomarkers.10b] The primary process of cell-SELEX involves synthesizing the nucleotides of single-strand DNA/RNA (ssDNA/RNA) (10 15 -10 16 ) sequences from random oligonucleotides, which consists of the primary combination of nucleic acids.Under selected environments, the nucleotide library is incubated with the target cells until the sequences are bound to the target cells.The unbound sequences are removed, and the restricted sequences are eluted from the target binding to get into the next process.The selected oligonucleotides are then introduced to the target and control cells in an event, to interrogate a binding possibility toward the control cells.Subsequently, the enriched selected sequences are separated from the unrestricted cells.The enriched aptamers will be incriminated to the next round of counters as well as further selection processes until the most enriched sequences are collected.The resulting aptamers are ready for use in selective binding with the target cells (Figure 4). [42,44]he iterative processes are controlled by flow cytometry investigating the fluorophore-labeled primers to show the binding affinity. [43]Once the high-affinity aptamers are obtained, the cloning and sequencing clones will vindicate the specific sequences and conformation of selected aptamers. [45]To better understand the ability of aptamer recognition of target cells, the selected aptamers have been reported to distinguish the real cells from other look-alike-target cells, by intercalating their complex structures into the aptamers. [46]ince the cell-SELEX process only involves the capability of oligonucleotides in binding the target cells directly, the information on the target cell morphology, conformation, and appearance, is not further required at the prior stage.However, these conformational analyses are required to manage and retain the natural conformation of biomolecules composed on the target cells from an ineffective protein cell purification.15a,47] Table 2 presents the recent aptamers commonly used for cancer cell recognition and their electrochemical aptasensing strategies.

Electrochemical Biosensing of Cancer Biomarker
The electrochemical technique is a well-known method to generate prompt and reliable results for biological and clinical sample detections.Various applications of electrochemical biosensor yield tremendous benefits, especially from a user perspective, for example, low-cost, ease of handling, portability, and lowsample volume requirement. [61]This technique, in particular, was proposed in several studies engaging non-biological and biological analytes for detection at tissue and cell levels analysis, [62] particularly in cancer marker detection (Table 2).Therefore, selecting major platforms to construct an excellent electrochemical biosensor encourages a specific knowledge about the type of (bio)molecular recognition that would be employed to interact with the target interest.61b,c] In this review, the electrochemical biosensors are classified into amperometric/voltammetric, impedimetric, photoelectrochemical (PEC), and electrochemiluminescence (ECL) techniques.The amperometric method determines the presence of electronic differences of electroactive substrates due to the reduction-oxidation mechanism at a constant potential.Each current measured is linear to each concentration of the substrate applied.Whereas, voltammetry is a subclass of amperometry and remains the most popular electrochemical technique, which occupies the benefits of amperometric and potentiometric in a single type of measurement.61b,c] Contrarily, the impedimetric permits the conductivity interactions toward the target biomolecules on the electrode surface. [63]During the impedance measurements, the redox-active agent is commonly employed to notify any circumstance change of electrical properties upon the complex formation between the recognition agent and analyte.The change will then be transformed into electronic transfer resistance (R et ) signal with respect to the impedance behavior. [64]The PEC process is a promising low-cost method for converting chemical energy to electricity when exposed to light and applied potential.PEC biosensing has received great attention in recent years because of its capacity to detect biomolecules using photocurrent generated by biomolecule oxidation. [65]In addition, ECL is another electrochemical mechanism for a wider analyte detection target.As this method is a combination of electrochemistry and visual luminescence measurement, the actual substrate reading will first be done electrochemically, monitoring the potential until the electron leaps are produced.Subsequently, in the presence of light and the support of additional precursors, the current is converted into reactive species by releasing the measurable light. [66]

State-of-the-Art on Whole-Cell Electrochemical Aptasensors for Cancer Detection
The early diagnosis of cancer is required as the cancerous cells exhibit a gradual growth so that they can migrate and turn to be     Reproduced with permission. [70]Copyright 2019, American Chemical Society.For interpretation of the references to color in these figures, the reader is referred to the web version of this article.
invasive.The electrochemical technique has been a well-known method to produce a prompt and reliable result in clinical sample detections for decades.An example of this is the glucose-meter which is intently based on the amperometric principle.However, to achieve cellular detection capabilities in a such complex samples, the selection of nanomaterial could be decisive.Therefore, this is worth noting that understanding how nanomaterial is integrated with the biological element in an electrical composite is important so that it could elicit a great level of sensitive diagnostic.The recent advances of different electrochemical aptasensors for identification of whole-cancer cells have been summarized in this section, based on the method principles.

Lymphoma
Lymphoma is a diverse group of lymphoid neoplasms with marked differences in clinical course and response to treatment. [67]Lymphomas are classified according to their postulated normal cell type of origin (B-cell vs T-cell), morphology (Hodgkin vs non-Hodgkin), and level of cellular differentiation. [68]In 2010, Ding and co-workers demonstrated TD05 aptamers by using electrochemical and ECL techniques to investigate the best LOD from different modified treatments of the aptamers against Ramos cells (a human B-cell lymphoma). [69]First aptamers treatment analyzed with square wave voltammetry (SWV) was modified with magnetic Au NPs and cadmium NPs (Cd NPs).In contrast, the second treatment modified with magnetic beads with Ruthenium-based complex (without NPs) was analyzed with ECL.The LOD for SWV and ECL obtained were 67 and 89 cells mL −1 , respectively.From the reported results, the use of bifunctionalized NPs provides excellent sensitivity of the aptamers, and the electrochemical method may contribute to better signal amplification than ECL for Ramos cell detection. [69]ecently, a multiplexed whole cell-aptasensor employing Au NPs functionalized with magnetic graphene nanosheet (Au NPs-Fe 3 O 4 -GS) to recognize some circulating tumor cells (CTCs) including CCRF-REM and Ramos cells (Figure 5A) in clinical sample. [70]A mixture of different aptamers previously obtained from the cell-SELEX (i.e., Td05 and Sgc08) was conjugated to the NPs complex through Au-thiol bond as the recognizing agent.Upon the sample incubation, the presence of Fe 3 O 4 allowed to separate the recruited analyte complex with the NPs from the unbound mixture, such as human whole blood sample.Thionine and 6-ferrocenyl-1-hexanethiol were selected as the redox probe to encounter the redox potentials of the samples on screenprinted carbon electrode (SPCE), followed by the SWV measurement.The results showed an increasing pattern from both cells subsequently, with excellent linearity ranging from 5 to 500 cells mL −1 and LOD of 3 to 4 cells mL −1 , for CCRF-CEM and Ramos cells, respectively (Figure 5B-D).Detection of CTCs in real blood samples from healthy volunteers and cancer patients was also performed demonstrating the clinical feasibility of the developed nanobiosensor for practical analysis of rare cancer cell detection. [70]

Hepatocellular Cancer
Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and is the primary cause of cancer death globally. [71]A progress in the electrochemical sandwich architecture was fabricated by Sun and co-workers based on the catalytic enzyme and hybrid nano-electrocatalysts for the detection of HepG2 cells. [72]HepG2 secretomes have previously been identified as hepatocellular carcinoma biomarkers. [73]The thiolated-TLS11a was used as a cell-targeted aptamer and immobilized onto Au NPs deposited on the glassy carbon electrode (GCE) surface.In principle, the nano-electrocatalysts were composed of the horseradish-peroxidase (HRP), a nanoprobe complex, and surface-metallic nanocomposite assembled onto the captured HepG2 cell on the electrode surface.Then, a complex of DNA-zyme was employed as an effective electrocatalytic bioprobe to induce signal amplification, benefitting from the aptamer sequence-integrated G-quadruplex and hemin interaction to form an HRP-like catalytic reaction.The catalytic reactions were occurred due to the presence of the original HRP and the nanoprobe complex.In this study, three combinations of hybrid metals, including bimetallic nanomaterial with core-shell NPs (i.e., AuPd), superparamagnetic NPs (i.e., Fe 3 O 4 ), as well as metal oxide (i.e., MnO 2 ), were also conjugated on the nanoprobe via an Au-thiol bond to exhibit the catalytic performance of the peroxidase-like probe activity as well as be the nanoprobe-carrier (Figure 6A).The cytosensor strategy was started by capturing the target cells selectively on the surface-immobilized aptamer.Nanoelectrocatalysts were applied to oxidize hydroquinone and H 2 O 2 into benzoquinone, which was directly associated with the amount of the electrochemical species and linearly reflected the amount of the captured HepG2 cells (Figure 6B).A wide detection range was yielded between 1 × 10 2 to 1 × 10 7 cells mL −1 along with the LOD down to 15 cells mL −1 (Figure 6C).The selectivity study showed that the developed aptasensor was highly selective for HepG2, not for other cell lines (Figure 6D).Interestingly, sensor regeneration is possible by transferring the negative voltage pulse to break the Au-thiol bond during the construction. [72]everal published data have mentioned the use of microdevices incorporated with the anti-EpCAM antibody to specifically bind to circulating tumor cells (CTC).The presence of the antibody was steadily bound to magnetic beads [74] or the Au NPs in order to achieve an amplified signal. [74,75]The application of real clinical samples was also reported to be further tested on micrographene aerogel sensor for early detection of liver cancer.The folic acid and octadecylamine were described to be incorporated on the graphene microdevice in a covalent linkage thus, the binding to cancer cell resulted in high efficiency. [76]

Colorectal Cancer
Colorectal cancer (CRC) is one of the most lethal and prevalent cancers accounting for 1.9 million new CRC cases and 930 000 deaths in 2020 worldwide. [77]Early diagnosis of CRC or precancerous alteration allows for early intervention to reduce or prevent cancer progression and lethality. [78]Recently, a selfassembled monolayer (SAM) aptasensor using an electrochemical technique was developed for the detection of CRC. [79]The SAM solution of 11-mercaptoundecanoic acid (11-MUA) was applied to introduce tighter and more stable binding to the aptamers owing to the covalent bonding as compared to the selfbinding aptamers on the Au electrode.The selective binding was observed in HCT-116 cells as the target cells among the nontarget cells, which showed ≈40% of signals higher than the nontarget cells.The currents of the different concentrations were examined using CV resulting in a higher current peak along with an increased cell concentration with a linearity range from 6 to 1000 cells mL −1 , and as low as 7 cells mL −1 of LOD. [79]nother aptasensor was recently developed to detect CRC cells (i.e., CT26) based on Cr-metal-organic frameworks (MOF) and cobalt phthalocyanine NPs (denoted as Cr-MOF@CoPc) in human serum samples. [80]The CT26-targeted DNA aptamer of 5′-GAAGTGAAAATGACAGAACACAACA-3′ was used as the main bioreceptor in this study.The stepwise synthesis and modification step of the biosensor is depicted in Figure 7A.EIS and DPV were used to quantify the sensing response of the aptasensor toward the analyte resulting in LOD of 36 and 8 cells mL −1 , respectively (Figure 7B).The linear dynamic range was revealed at the concentration range 50 to 1 × 10 7 cells mL −1 of the CT26 cell suspension (Figure 7C). [80]

Leukemia
Leukemia is a major hematological cancer that causes death and morbidity at various ages. [81]For diagnosis, leukemia is prominently characterized by circulating malignant white blood cells. [82]A simple aptamer cytosensor was proposed for direct detection of K562 cells based on aptamer-specific K562 cells (i.e., T2-KK1B10 [83] ) and a biotin-conjugated concanavalin A (bio-ConA) resulting in a linearity response at K562 cell concentration from 1 × 10 2 to 1 × 10 7 cells mL −1 with a low LOD at 79 cells mL −1 . [84]The stepwise procedure of biosensor fabrication is depicted in Figure 8A including the modification of Au electrode surface with a series of biomolecules such as SH-aptamer, 6-mercapto-1-hexanol (MCH), bovine serum albumin (BSA), bio-ConA, and streptavidin-conjugated alkaline phosphatase (ST-ALP).The DPV response of the aptasensor against increasing cell concentration and its linearity are shown in Figure 8B,C with appreciable selectivity, stability, and reproducibility.The cytosensor was applied to the human blood sample indicating its potential to detect leukemia cells marker in a real clinical environment. [84]igure 6.A) The fabrication process of layer-by-layer nanoprobe and B) re-utilization of self-assembled cytosensor.C) DPV response over various concentrations of HepG2 cells (a-g; 0-1 × 10 7 cells mL −1 ).The inset figure shows a linear plot of peak current versus the log.value of the HepG2 cell concentration.D) Peak current in response to different cell lines at a concentration of 1 × 10 5 cells mL −1 .Error bars: SD, n = 3. Reproduced with permission. [72]Copyright 2016, Elsevier.

Breast Cancer
Breast cancer is considerably curable through an early diagnosis, while local-regional therapies (i.e., surgery and radiotherapy) serving as the basis. [85]A CV-based aptasensor was developed by Zhu and colleagues [86] to detect the MCF-7 cells.MCF-7 cells are estrogen responsive and are frequently employed in vitro to examine estrogen receptor positive breast cancers. [87]MUC1 aptamer was employed to capture the target cells, and HRP was used as an initiator of catalytic current.The sensing method is based on the sandwich model to improve the signal amplification.The measurement is complete when the catalytic process occurs between HRP and the redox probe resulting in an oxi-dized current, which is equal to the cell concentration.The linear range achieved was from 1.0 × 10 2 to 1.0 × 10 7 cells mL −1 with 100 cells mL −1 of sensitivity. [86]urthermore, the authors advanced the detection technique by using a dual detection strategy (i.e., aptamers and antibodies) on a reusable electrode.The device was constructed by functionalizing the magnetic field to split the recognition part from the transducer.Initially, MUC1 aptamer labeled with HRP and anticarcinoembryonic-antigen antibody (anti-CEA antibody) were attached to the magnetic NPs. [88]The role of magnetic NPs was to facilitate a magnetic restriction for collecting and measuring target cells.After performing the sensing measurement, the working electrode was restored by washing off the biorecognition and ).Reproduced with permission. [80]Copyright 2020, Elsevier.
the targets altogether from the electrode surface.This sensor reached LOD of 100 cells mL −1 with respect to linear range from 1.0 × 10 2 to 1.0 × 10 6 cells mL −1 . [88]CF-7 cell detection was reported by Wang and co-workers using a sandwich-label-free biosensor. [89]The construction was initiated by electrodepositing the thiolated-MUC1 aptamer colinked polyadenine (polydA) onto the Au electrode.PolydA was suggested as a linker since its multi-consecutive adenines sequence could facilitate an intrinsic affinity toward the Au electrode and aptamer.Hybrid NPs, consisting of Au NPs, and GO, were then conjugated with the modified aptamer as the label-free probe, which was applied to the captured cells.The characterization and detection were carried out using DPV, CV, as well as EIS along with Fe(CN) 6 3-/4− as a redox probe.The selectivity study showed that the sensor device could differentiate MCF-7 cells among other cancer cell and normal cells in diluted human serum indicating its potential clinical applicability.The reported biosensor could produce a wide linear range of 10-10 5 cells mL −1 and a low LOD down to 8 cells mL −1 . [89] ultra-low detection limit was achieved by designing doubletetrahedral DNA framework-based amperometric biosensor system for the detection and release of CTCs (i.e., MCF-7 cells). [90] screen-printed Au electrode was modified using an upright tetrahedral DNA framework, and an inverted tetrahedral DNA framework offered three vertex chains to multivalently connect with aptamers.The linear range of the sensor device was 1 to 10 5 MCF-7 cells, with an LOD of 1 cell.The developed sensor was also used to detect the target analyte in mimic whole blood samples, indicating that the sensor system has clinical diagnostics potential.[90]

Ovarian Cancer
Ovarian cancer is often detected late in its progression, and there is no effective screening approach in place. [91]Despite significant advances in chemotherapy and surgery, ovarian cancer remains one of the most lethal gynecological malignancies in Figure 8. A) Scheme of the electrochemical cell-based aptasensor fabrication for the detection of chronic myelogenous leukemia K562 cells.B) DPV response of aptasensor with target cell concentrations of 0-2 × 10 7 cells mL −1 (from a to h).C) Linear regression plot of DPV peak currents versus log.K562 cell concentration.Reproduced with permission. [84]Copyright 2016, Wiley-VCH.the world. [92]In 2013, Liu and colleagues conducted a layerby-layer strategy for the detection of HeLa cells, biomarker for ovarian cancer. [93]The Au electrode was first immersed with 3mercaptopropionic acid (MPA) to obtain a negative charge.The surface was then submerged into poly (ethylene imine) and assembled with the SWCNTs to produce the positive and negative charges.In this step, the folic acid substrate was conjugated with SWCNTs and acted as a biorecognition agent for binding capacity toward the folate receptor on the target cell surface.This strategy resulted in an amplified signal for the constructed sensor due to high electrons promoted on the cytosensor surface.An improved sensitivity was achieved in cell measurement using DPV with a wide linear range from 10 to 10 6 cells mL −1 and a low LOD of 10 cells mL −1 .In addition, this study demonstrated the presence of SWCNTs could promote the electron transfer capabilities of redox probes. [93]ore recently, another layer-by-layer electrode-based aptasensing was observed by Wang and co-workers. [94]In this report, clinically recognized AS1411 aptamer was associated with 2Dgraphene fabricated with ferrocene-appended poly(allylamine hydrochloride) (PAH) and poly(sodium-p-styrene sulfonate) (PSS) nanocomposites to target HeLa cells.The target cells were captured based on G-quadruplex conformation by the receptor.The functionalization of the nanocomposites and graphene on the electrode promotes the enhancement of electronic signals from the detected target cells.The presence of the target cells on the electrode surface was interpreted by the decrease of DPV peak as a result of electric currents blockade.The linear range and LOD were indicated from 10 to 1.0 × 10 6 and 10 cells mL −1 , respectively. [94]

Prostate Cancer
Emerging aptamers in the biosensor system were developed by Min and co-workers for PSMA (prostate-specific membrane antigen)-expressed prostate cancer cell detection. [95]Through streptavidin-coated quantum dots as a linker and biotin functionalization, the authors combined dual aptamers which have different targets to enhance the binding complex in targeting PSMAexpressed prostate cancer cells (LNCaP and PC3 cells), in which A10 RNA aptamer is used to recognize the PSMA (+) prostate cancer cells, and DUP-1 aptamer is used to recognize the PSMA (−) prostate cancer cells.Several preparations were considered toward the aptamers on the electrode surface before evaluation with the EIS technique.The exciting part of these stages is to determine the mixing ratio of both aptamers, which is a significant step since it affects the sensitivity and selectivity in targeting the cells.The analytical signal was increased once the prostate cancer cells were introduced onto the Au electrode surface, indicating a higher resistance of electron transfer (R et ).The presence of the biological elements blocked the working electrode, thereby hindering the redox probe from accessing the surface.Consequently, this biosensor satisfied the LOD as low as 100 cells mL −1 for both LNCaP and PC3 cells. [95] recent study has reported the development of a chronoimpedimetric aptasensor for the detection of circulating prostatic tumor cells (CpTC), that is, LNCaP cells, with the use of immobilized RNA aptamer selective PSMA on the Au NPs modified SPCE.[96] The extremely low LOD was achieved at 0.62 cells mL −1 with a linear concentration range of 1 to 40 cells mL −1 emphasizing the potential application of the developed sensor toward the sensitive detection of PSMA on CpTCs as part of future prostate cancer diagnosis approach.[96] The large surface area of the 2D nanomaterial may benefit higher receptor loading for whole-cell cancer detection.[97] The tunable surface strategy of poly(N-isopropylacrylamide) (PNI-PAM) covalently grafted onto graphene oxide (GO) was computationally investigated to trigger the sensing system interactions with prostate cancer cell proteins around lower critical solution temperatures.The system deployed aptamer as the recognition element of the target protein.The study suggested the unique behavior of PNIPAM polymer to enable the temperature-controlled detection of whole-cell cancer protein.[97]

Hepatocellular Cancer
11d] The authors immobilized TLS11a aptamers on the Au electrode surfaces through two functional groups (amino and carboxylic acid groups) to induce a stable bio-deposition.Interestingly, the biosensor was crafted based on the sandwich architecture, which consists of the aptamers as biorecognition, cancer cells as the target, and other aptamers on the outermost construction.This label-free device was able to advance the sensing specificity toward the HepG2 cells.Electrochemical quartz crystal microbalance and EIS were carried out to investigate the sensor performance toward the target cells.In contrast, cyclic voltammetry (CV) was utilized to examine the electrochemical profiles of the sensor surface.Herein, the authors benefitted from the use of the flanked aptamers to exhibit the electron charge transfer (R ct ) as well as an excellent sensitivity.11d]

Leukemia
11a] The reported electrochemical method has shown enhancement in reproducibility and precision due to the use of carbon nanofiber-doped chitosan colloidal solution onto GCE (CNF-CS/GCE) which accumulates conductivity during cytosensing.In addition, the chitosan hydrogel attached to the electrode retains the natural properties of cancer cells.The electrode modification was characterized using CV analysis, while EIS was employed to investigate the method's performance.11a] Similarly, Ding and co-workers have investigated nanocomposite gel-modified chitosan to detect K562 cells.The developed nano-gel benefits not only in the immobilization process, but also in the characterization of the cell's behavior. [98]The concentration profiles were characterized using the EIS method and resulted in high electron transfer resistance in greater cell concentration.The linear range was observed from 1.34 × 10 4 to 1.34 × 10 8 cells mL −1 with a low LOD of 8.71 cells mL −1 .This study suggests that the use of nanocomposite gel improves cell entrapment capacity and preserves excellent biocompatibility toward the cells. [98]Further, an EIS-based aptasensor was also proposed by utilizing GO and poly-L-lysine (PLL) film to profile K562 living cells adhesion. [99]The study aimed to approach the fundamental cell analysis of PLL/GO film as a biocompatible interface and to examine the electrochemical behavior in cell multiplication and apoptosis processes.As a result, the assembled films showed a wide linear range from 1.0 × 10 2 to 1.0 × 10 7 cells mL −1 , suggesting that the immobilized living cells are compatible with PLL/GO film along with LOD of 3.0 × 10 2 cells mL −1 .This work demonstrated that GO offers a promising sensing platform to advance the immobilization capacity of living cells. [99]lectrochemical aptasensors for different leukemia-associated cells, that is, CCRF-CEM cells [100] and human promyelocytic leukemia cells (HL-60 cells), [38] were developed.In CCRF-CEM cells-targeted sensor, the authors took advantage of the use of thiol conjugated aptamer for physical adsorption to the Au electrode surface.The binding interaction between thiolterminated sgc8c aptamer and CCRF-CEM cells was investigated by CV and EIS techniques.To figure out the selectivity of the aptamers, fluorescent microscopy was also employed.A wide linear range obtained between R ct and the logarithmic concen- ).C) Linear regression plot of ΔR ct versus the concentration of MCF-7 cells (n = 3).Reproduced with permission. [103]Copyright 2020, Elsevier.
tration values of the target cells was observed from 1.0 × 10 4 to 1.0 × 10 7 cells mL −1 , with an LOD of 6.0 × 10 3 cells mL −1 . [100]or the HL-60 cells sensing platform, carboxyl methyl chitosan (CMC)-modified GO and indium tin oxide-modified GCE was used for the detection of cell markers.An electrochemical impedance study employed folic acid as a biorecognition element against the folic acid receptor which are overexpressed on the cancer cell's surface.The presence of GO enhanced the selectivity and sensitivity of biosensor, while CMC helped in the binding mechanism and dispersity of folic acid.The linear range obtained was from 5.0 × 10 2 to 5.0 × 10 6 cells mL −1 , with a sensitivity of ≈5.0 × 10 2 cells mL −1 . [38]

Breast Cancer
Shen and co-workers developed a label-free and reusable aptasensor for the detection of MCF-7 cells. [101]A hybrid DNA between the EpCAM aptamer and capture probe was designed to specifically bind to the MCF CTCs membranes.CTCs are essential prognostic biomarkers for the diagnosis of cancer. [102]6mercapto-1-hexanol (MCH) was immobilized onto the electrode surface as a capture probe interface for uniform distribution and as affluence for cell collection.The aptasensor performance was initiated by the hybridization process of aptamer with its capture probe and thus, selective binding could be set toward the CTCs.The analytical changes were quantitatively recorded employing EIS that exhibited a wide linear range from 30 to 1.0 × 10 6 cells mL −1 and LOD of 10 cells mL −1 .Due to the hybrid concept, this proposed aptasensor could be reutilized by assembling the Uracil DNA Excision Mix, to stop the sensing process and cleave the hybridized aptamer-probe for further reuse and cells collection purposes. [101]ore recently, Li and co-workers developed a breast cancer aptasensor comprising carbon dots loaded with zirconium in MOF with high amounts of amino substrates. [103]MOF is one of the hybrid nanomaterials possessing a tunable porous surface, high stability, and wide surface area, allowing any ligand and conjugate function to bind through various chemical bonds, including van der Waals, electrostatic, hydrophobic, and covalent bonding.To target the ligand, HER2 aptamer was covalently bonded to phosphate groups to be assembled over the MOF skeleton.The amino groups present within the composite further help promote the binding between the aptamer and MOF recognizing two different cell lines, MCF-7 and HER-2 cells.The schematic procedure of this aptasensor is depicted in Figure 9A below.Through the EIS method, the MOF composite gained a low LOD of 31 cells mL −1 of MCF-7 with a three-order of linear range from 1.0 × 10 2 to 1.0 × 10 5 cells mL −1 (Figure 9B,C). [103]raphene families have been ubiquitously applied in cancer biosensors owing to their versatile properties, such as large surface area, excellent electrical conductivity, and catalytic reactivity. [104]Incorporating graphene family nanomaterials, an impedimetric aptasensor was developed on the basis of reduced graphene oxide (rGO)-chitosan-Au NPs composite to detect MCF-7 cancer cells with AS1411 aptamer as the bioreceptor. [105]he biosensor produced a wide linear range of 1 × 10 1 -1 × 10 6 cells mL −1 , and a low LOD of 4 cells mL −1 . [105]The detection limit is lower than previous study [103] due to the advantage of 2D nanomaterials which can provide high aptamer loading on the material surface.Interestingly, a lower LOD was further achieved by employing a composite composed of rGO nanosheets and rhodium NPs for the detection of HER2 + circulating tumor cells. [106]The sensor could yield LOD as low as 1.0 cell mL −1 with a linear dynamic range of 5-10 × 10 4 cells mL −1 .The remarkable analytical performances including detection limit, specificity, selectivity, stability, and reproducibility indicated that the developed aptasensor could be applied for clinical diagnosis of breast cancer patients. [106]2.5.Ovarian Cancer Feng and co-workers fabricated a label-free impedimetric aptasensor to detect HeLa cells through functionalizing 3,4,9,10-perylene tetracarboxylic acid (PTCA) onto the graphene surface.[107] The use of PTCA substrate was to induce more carboxylic groups on the graphene surface and protect graphene from aggregation.AS1411 aptamer was electrodeposited on the modified and formed a G-quadruplex structure to bind to the target cells.The sensing performance was completed by hybridizing the aptamer's complement, which allows disruption in the binding event for the reusable applications.The impedance measurement resulted in a linear range from 1.0 × 10 3 to 1.0 × 10 6 cells mL −1 with the LOD of 794 cells mL −1 .[107]

Ovarian Cancer
A recent study was carried out to design a PEC-based aptasensor to detect HeLa cancer cells by using 2D graphite-like carbon nitride nanosheets (g-C 3 N 4 NSs), as visible light-sensitive material. [108]The linear dynamic range was observed from 10 to 10 −6 cells mL −1 with 5 cells mL −1 LOD.This study showed an excellent approach based on the successful conjugation of visiblelight-induced 2D nanocomposite with aptamer and target cancer cells. [108]nother PEC aptasensor was constructed benefitting from of the excellent PEC activity of Bi nanocrystal, N,O-co-doped carbon core-shell nanohybrids (Bi@NOC), and the signal amplification effect of thioflavin-T for the selective detection of telomerase activity in cancer cell. [109]The illustration for the sensor development is shown in Figure 10A.The sensor could detect telomerase activity with the linear dynamic range from 5.0 × 10 2 to 1.0 × 10 6 HeLa cells and LOD at 60 cells (yielded from the linear tendency of photocurrent vs cell concentrations), as depicted in Figure 10B,C. [109]

Lymphoma
The latest study in Ramos cell detection by using ECL technique was proposed by Yang and co-workers employing the polyamidoamine dendrimers as a signal inducer of Ru(bpy) 3 2+ -tripopylamine catalytic label system. [110]In their study, the dendrimers act as an interactant to amplify the ruthenium signal and as a nanomaterial to provide a high contact surface for the loaded ruthenium.The constructed biosensor was a sandwich model employing a couple of TD05 aptamers.One was linked covalently to the single-wall carbon nanotubes (SWNTs)-modified carbon electrode and another was linked after the cells were captured.This architecture benefits the efficiency of the binding event between the biorecognition and the target cells as well as widens the molecules' surface area.Interestingly, the authors also studied the binding affinity of the aptamers by varying the length of polyethylene glycol (PEG) spacers, which resulted in six units of PEG that generated the highest affinity for binding aptamers thus, resulting in a quenched signal.The selectivity study showed that the TD05 was capable of distinguishing the Ramos cells among the other cancer cell types with a LOD observed at ≈55 cells mL −1 . [110]

Leukemia
Recently, the bipolar-electrodes on Au NPs-modified indium tin oxide chips in combination with the ECL measurement were developed for leukemia cell (HL-60) detection. [111]Zhang and coworkers fabricated the electrode consisting of anode and cathode layers, which allowed the abundant transfer of electrons to be multiplied as readable signals by the ECL.Interestingly, the authors used two types of reservoirs, Ru(bpy) 3 2+ and luminol, which sustained the on-off signal performance.Apart from the respective electrodes, the ECL-tandem bipolar electrodes enhanced the intensity of the peroxide signal produced by the enzymatic process of DNAse.This phenomenon results in the electronic transfer, amplification on the cathode to give an electron barrier effect.The use of Au NPs was not only to help induce more oxygen releases for signal improvement, but also to protect the indium tin oxide from electrodeposition.With the linear range between 3.2 × 10 2 and 2.5 × 10 5 cells mL −1 , the LOD was observed down to 80 cells mL −1 enabling differentiation of the target cells selectively among the control cells. [111]

Conclusions and Prospects
We briefly summarized the recent advancements of whole-cell aptasensors in cancers (Table 2).Most of the projected cancer types discussed include lymphoma, prostate, hepatocellular, colorectal, leukemia, breast, and ovarian cancer.Lung cancer was not included in this review owing to limited reports on the use of electrochemical aptasensor to detect the whole-cell biomarkers.However, several developments in this field are shown in Table 2.In general, recent reports showed that the need for a preliminary study of the cancer stage is still highly essential to extract major evidence that is in accordance with the cancer type, status, and correlated therapy.Utilizing whole-cell as the real target in recognition strategy offers more opportunities to generate a promising and reliable biosensor for future cancer theranostic.Due to its recognition on the specific cell-surface, whole-cell targeting is appropriate in determining the status of the molecular stage associated with cancer malignancies and linked to altered-protein expression.On the other hand, the fabrication of nanomaterialelaborated biosensors can generate great benefits, in terms of signal amplification, selectivity, and detection sensitivity.responses of the cytosensor over different HeLa cell concentrations, (a-j; 0-1 × 10 6 cells mL −1 ).C) Linear calibration plot of photocurrent intensity versus concentration of HeLa cells.Reproduced with permission. [109]Copyright 2018, Wiley-VCH.
Moreover, the selection of aptamers as cancer biorecognition has attracted vast attention for its fast tissue penetration, low toxicity, ease-to-modify, and highly selective binding to the cell of interest.In combination with NPs (e.g., magnetic NPs and quantum dots), the aptamers' performance could potentially improve the targeted cell colocalization.For instance, assembling two or multi-different selective molecular probes, such as antibodyaptamer, aptamer-aptamer, and multi-aptamers, would be beneficial to exhibit the performance of biosensors.The biosensor's LOD is not necessary in an extremely low concentration, as long as the LOD enables one to cope with the marker concentration in real physiological circumstances.Nevertheless, some cancer cells slough off primary solid tumors, extravasate into and circulate in the circulation, and therefore show great potential in liquid-biopsy-based cancer diagnostics and monitoring. [112]112a] However, this challenge triggers great opportunities for single-cell detection platforms via biosensing techniques.More emphasis should also pinpoint the selectivity and practical ability of the device to detect a clinically relevant sample of the cancers.In the end, an intense effort to advance the biosensor-based performance complemented with computational studies should be expanded and revolutionized as a sturdy platform for pursuing highly sensitive and futuristic whole-cell cancer biosensors.

Figure 1 .
Figure 1.Illustration of whole-cell cancer detection with various output parameters based on electrochemical aptasensor strategies toward the common types of cancer disease.The incorporation of inorganic/organic nanomaterials and immobilized bioreceptors could provide a highly sensitive method for the cancerous marker's detection.

Figure 5 .
Figure 5. A) Scheme of the development of aptamer-functionalized Au NPs array-decorated magnetic graphene nanosheet recognition probe to electrochemically capture and isolate rare circulating tumor cells from the human whole blood sample.B) SWV responses of the biosensors over various concentrations of Ramos and CCRF-CEM cells (a-j; 0-500 cells mL −1 ).The calibration plots of peak current versus concentrations of C) the CCRF-CEM cells and D) the Ramos cells.Error bars: SD, n = 3. Reproduced with permission.[70]Copyright 2019, American Chemical Society.For interpretation of the references to color in these figures, the reader is referred to the web version of this article.

Figure 7 .
Figure 7. A) Procedure of the fabrication of electrochemical aptasensor based on the Cr-MOF@CoPc nanohybrid.B) DPV response for CT26 cells detection with various concentrations (50-1 × 10 7 cell mL −1 ).C) The calibration curves between the peak current density (ΔI) and the concentrations of the CT26 cell suspension.The inset figure shows the linear fit plot of ΔI versus log. of CT26 concentration (error bars: SD, n = 3).Reproduced with permission.[80]Copyright 2020, Elsevier.

Figure 10 .
Figure10.A) Scheme of the PEC aptasensor for telomerase detection based on Bi@NOC NHs and the signal amplification effect of Th-T.B) PEC responses of the cytosensor over different HeLa cell concentrations, (a-j; 0-1 × 10 6 cells mL −1 ).C) Linear calibration plot of photocurrent intensity versus concentration of HeLa cells.Reproduced with permission.[109]Copyright 2018, Wiley-VCH.

Table 1 .
Comparison of the biorecognition molecules in whole-cell detection.

Table 2 .
List of aptamers commonly used for cancer cell recognition and their whole-cells detection platform.