DNA/RNA-based electrochemical nanobiosensors for early detection of cancers

Abstract

Nucleic acids, like DNA and RNA, serve as versatile recognition elements in electrochemical biosensors, demonstrating notable efficacy in detecting various cancer biomarkers with high sensitivity and selectivity. These biosensors offer advantages such as cost-effectiveness, rapid response, ease of operation, and minimal sample preparation. This review provides a comprehensive overview of recent developments in nucleic acid-based electrochemical biosensors for cancer diagnosis, comparing them with antibody-based counterparts. Specific examples targeting key cancer biomarkers, including prostate-specific antigen, microRNA-21, and carcinoembryonic antigen, are highlighted. The discussion delves into challenges and limitations, encompassing stability, reproducibility, interference, and standardization issues. The review suggests future research directions, exploring new nucleic acid recognition elements, innovative transducer materials and designs, novel signal amplification strategies, and integration with microfluidic devices or portable instruments. Evaluating these biosensors in clinical settings using actual samples from cancer patients or healthy donors is emphasized. These sensors are sensitive and specific at detecting non-communicable and communicable disease biomarkers. DNA and RNA's self-assembly, programmability, catalytic activity, and dynamic behavior enable adaptable sensing platforms. They can increase biosensor biocompatibility, stability, signal transduction, and amplification with nanomaterials. In conclusion, nucleic acids-based electrochemical biosensors hold significant potential to enhance cancer detection and treatment through early and accurate diagnosis.

Keywords:

• Biosensors
• nucleic acids
• cancer detection
• electrochemical sensing

1. Introduction

Nucleic acid-based electrochemical biosensors identify cancer-related compounds using their electrical characteristics [1]. Nucleic acids like DNA and RNA contain genetic information and may recognize target molecules with high specificity and affinity [1]. Nucleic acids binding to targets can modify electrochemical biosensors’ current, voltage, or impedance. DNA aptamers with graphene oxide detect prostate-specific antigens, DNAzymes on gold nanoparticles detect microRNA-21, and RNA aptamers with magnetic beads detect carcinoembryonic antigens [2,3]. Despite their low cost, sensitivity, quick response, and simple operation, these biosensors struggle with stability, repeatability, interference, and standardization [4]. Nucleic acid aptamers are DNA or RNA single strands that precisely bind target molecules. After identification, aptamers can be used in cancer biosensors [4]. When mounted on a transducer surface, aptamers bind to their target molecule, changing the signal and allowing quantification. Aptamers have many advantages over antibodies, including more straightforward and faster production and modification, lower immunogenicity and toxicity, improved stability and re-foldability, and the ability to connect to targets antibodies cannot or cannot recognize [5]. Their shortcomings include poorer binding affinity and specificity than antibodies, susceptibility to nuclease degradation and clearance in vivo, and difficulties penetrating biological barriers or reaching intracellular targets [6–9]. Figure 1 depicts a schematic representation of genosensors based on nucleic acids for detecting cancer [10]. It also highlights the significant prevalence of cancer among various diseases and the most well-known types of cancer [10]. Electrochemical genosensors are devices capable of quantifying cancer-related genetic material and identifying its specific types [10]. Some advantages that they possess in comparison to other cancer detection methods include superior sensitivity, selectivity, simplicity, and affordability. These are merely a few of the characteristics. These instruments also use additional components such as electrodes, probes, labels, and signal amplifiers [10]. The objective of these components is to produce an electrical signal directly proportional to the concentration of the targeted DNA or RNA [10]. This signal can be quantified through various techniques, including impedance, voltammetry, amperometry, and field-effect transistors, among others [10]. In Figure 2A, a schematic illustration vividly portrays the distinctive features of electrochemical genosensors. These advanced genosensors play a pivotal role in monitoring the entire spectrum of cancer progression, spanning from the initial stages of tumor formation to the infiltration of adjacent tissues, and ultimately, the dissemination of metastasis to distant organs (Figure 2B) [11]. Genosensors can identify alterations in the expression of specific genes throughout these stages of biological development. For instance, certain genes play a role in cell proliferation, cell death, blood vessel formation, and immune response. These genes have been suggested as potential biomarkers for diagnosing and predicting the outcome of cancer [11].

Figure 1. Schematic illustration of genosensors based on nucleic acids for detecting cancer, highlighting the significant tumor prevalence [10].

Figure 2. (A) Schematic presentation for depicting advantages, parts, and diverse measurement methods of electrochemical genosensors. (B) Cancer genosensors originate from tumorigenesis, cancer cell migration, and colonization to a secondary site [11].

Aptamers hold great promise in cancer therapy owing to their remarkable specificity and affinity for target molecules. These versatile molecules find application in various aspects, independently or in combination with other agents, for targeted drug delivery, cancer diagnosis, treatment, and immunotherapy [12]. AS1411, for instance, has demonstrated the ability to induce apoptosis and inhibit angiogenesis in cancer cells, undergoing clinical trials to treat diverse cancers. Another noteworthy example is NOX-A12, an RNA aptamer that binds to CXCL12, a chemokine implicated in cancer progression and metastasis. Clinical testing of NOX-A12 reveals its potential to disrupt the CXCL12-CXCR4 interaction, bolstering immune responses against cancer cells in haematological malignancies and solid tumors. (ApDCs) represent a cutting-edge approach in cancer therapy, encompassing various forms such as aptamer-nucleotide analogue conjugates, aptamer-drug intercalation conjugates, and aptamer-chemical linker conjugates. These (ApDCs) contribute to heightened drug specificity and efficacy, reduced toxicity and side effects, and improved pharmacokinetics and biodistribution. For example, the conjugation of AS1411 with doxorubicin results in AS1411-Dox, a selectively targeted formulation that releases doxorubicin in response to nucleolin binding, thereby enhancing cytotoxicity while minimizing systemic toxicity [13].

Aptamers and antibodies, as molecular recognition elements, exhibit precise and high-accuracy connections to target molecules. However, size, immunogenicity, stability, synthesis, binding affinity, and specificity distinctions can impact their performance and applicability in cancer therapy. Electrochemical genosensors, leveraging nucleic acids and aptamers, emerge as promising tools with substantial potential in cancer detection and therapy. Ongoing research in this domain promises to unveil novel diagnostic and therapeutic strategies for the benefit of cancer patients. In contrast, antibodies, immune system proteins, play a crucial role by recognizing and binding to specific antigens [14]. These variations in molecular design and functionality underscore the diverse avenues through which molecular recognition elements contribute to advancing our understanding and treatment of cancer [15–19]. Antibiotics are employed extensively in cancer treatment as valuable diagnostic and therapeutic tools, independently or in conjunction with other modalities. Despite their widespread use, antibodies have inherent limitations, including elevated costs, intricate production processes, immunogenicity, toxicity concerns, instability, and challenges in penetrating tissues and tumors [20].

In contrast, aptamers, single-stranded DNA/RNA molecules, offer a promising alternative. Capable of adopting distinctive three-dimensional configurations, aptamers exhibit heightened affinity and specificity in binding to specific target molecules. This unique ability positions aptamers as a potential solution to antibody-associated challenges, presenting a more streamlined and practical approach to cancer diagnostics and therapeutics [19]. They are produced by an in vitro selection progression named (SELEX), which does not need animals or cell cultures. Aptamers have many benefits over antibodies: they are cheap, easy to synthesize, low/not immunogenic, stable, easy to modify, and trim. These features make aptamers fit for various uses in cancer diagnosis and therapy [21].

The main electrochemical mechanisms driving DNA- or RNA-based sensing can be classified into direct and indirect detection [22]. Direct detection relies on the intrinsic electrochemical properties of nucleic acids, such as their ability to mediate electron transfer or generate electroactive signals. Indirect detection involves using additional labels or probes to produce electrochemical changes upon hybridizing or recognizing the target nucleic acids [22]. Both direct and indirect detection methods can be further enhanced by employing various signal amplification strategies, such as nanomaterials, enzymes, catalytic reactions, or chain reactions. Electrochemical biosensors based on these mechanisms offer several advantages over other techniques, such as high sensitivity, selectivity, simplicity, low cost, fast response time, and minimal sample consumption. In this review, we will discuss the recent advances and challenges in developing and applying electrochemical biosensors for DNA- or RNA-based sensing [22,23].

1.1. DNA-based genosensing for cancer detection

DNA-based bioelectrochemical sensors represent an innovative technology designed to detect diverse disease biomarkers, with a particular focus on applications in cancer diagnosis. DNA, the molecule responsible for encoding the hereditary information of living organisms, is structured as a pair of intertwined strands in these sensors [24–26]. DNA serves multifunctional roles in biosensors that leverage electricity and chemistry, acting as a recognition component, signal converter, or signal enhancer. DNA can function as a recognition element, utilizing its inherent complementary base pairing property, demonstrating high specificity in hybridizing with target nucleic acids like DNA or RNA. This distinctive capability enhances the precision and specificity of biosensor reactions [27–29]. DNA is a versatile biosensor component, functioning not only as a recognition element but also as a signal transducer and amplifier. DNA can generate an electrical signal when interacting with a target by exploiting its electrochemical properties, including redox activity, charge transfer, and conductivity [30].

Additionally, DNA's enzymatic properties, such as polymerization, cleavage, or ligation, enable it to act as a signal amplifier. This enzymatic functionality amplifies the electrical signal by producing additional DNA molecules, thereby enhancing the sensitivity and responsiveness of the biosensor [31–34]. DNA-based bioelectrochemical sensors can achieve high sensitivity, selectivity, and rapidity for detecting cancer biomarkers, such as mutations, RNA expressions, microRNAs, or (ctDNA). DNA-based bioelectrochemical sensors can also be coupled with different nanomaterials, such as gold nanoparticles, graphene, or quantum dots, to enhance the performance of the sensors [31, 35,36].

Illustrative instances of DNA-based bioelectrochemical sensors for cancer detection include (i) a sensor designed to identify alterations in the (KRAS) gene associated with colorectal cancer. This sensor employs a gold electrode coated with thiolated capture probes and gold nanoparticles labeled with signal probes to facilitate detection [37–39]. The sensor identifies high-accuracy and precision (KRAS) changes in clinical samples [40]. (ii) Another noteworthy DNA-based bioelectrochemical sensor for cancer detection focuses on assessing telomerase activity, a key indicator of cancer cell growth. This sensor features a glassy carbon electrode coated with ferrocene-labeled telomeric repeats and methylene blue-labeled hairpin probes. With exceptional accuracy and precision, the sensor effectively identifies telomerase activity in cancer cell extracts [41]. (iii) A DNA-based bioelectrochemical sensor designed to detect (ctDNA), indicative of tumor cell release into the bloodstream, is notable. The sensor incorporates a gold electrode coated with capture probes and employs exonuclease III (Exo III) as a multiplication enzyme. Demonstrating high accuracy and precision, this sensor successfully identifies (ctDNA) in plasma models derived from lung cancer cases [42,43]. DNA-based bioelectrochemical sensors are not only valid for cancer diagnosis, but also for monitoring the treatment response and the prognosis of the patients [44]. For example, a sensor based on DNA hybridization detects (EGFR) gene mutations, which are associated with lung cancer and affect the response to tyrosine kinase inhibitors [45]. The sensor used a gold electrode coated with capture probes and silver nanoparticles marked with signal probes [46,47]. The sensor identified (EGFR) changes in plasma samples from lung cancer cases before and after therapy with high accuracy and precision [46]. A different DNA multiplication sensor detects p53 gene changes, which are linked to colorectal cancer and affect the survival rate [48]. The sensor used a gold electrode coated with capture probes and (RCA) as a multiplication method [49,50]. The sensor identified p53 changes in stool samples from colorectal cancer patients with high accuracy and precision [51]. However, some obstacles and limitations still need to be solved to create and apply DNA-based biosensors that use electricity and chemistry for cancer detection [52]. Some of these obstacles are fine-tuning the DNA sequences and structures to achieve high precision for the target and low toxicity and interference by other biomolecules or substances present in complex biological samples [53–55]. The enhancement of the electrode materials and architectures increases the surface area, electrical conductivity, biocompatibility, and stability of the sensors [56]. Creating novel nanomaterials and multiplication strategies can improve the sensors’ sensitivity and dynamic range without compromising specificity and simplicity [57]. The validation of the sensors in clinical settings with large numbers of samples from different sources and conditions. The prospects of DNA-based bioelectrochemical sensors for cancer detection are bright and promising [58]. With the advances in DNA synthesis and modification techniques, nanomaterials science, electrochemical methods, and bioinformatics tools, more novel and efficient DNA-based bioelectrochemical sensors will be designed and fabricated for various cancer biomarkers. These sensors will provide rapid, accurate, sensitive, selective, portable, and low-cost tools for cancer patients’ early diagnosis, treatment monitoring, and prognosis evaluation [59].

1.2. RNA-based genosensing for cancer detection

RNA-based biosensors that use electricity and chemistry are a new technique for identifying various disease indicators, especially cancer [60]. RNA is a single-stranded molecule transferring genetic information from DNA to proteins [61]. RNA can also act as a recognition element, a signal transducer, or a signal amplifier in bioelectrochemical sensors. RNA can be used as a recognition element by exploiting its complementary base pairing property, which allows it to hybridize with target nucleic acids, such as DNA or RNA, with high specificity. RNA can also be used as a signal transducer by exploiting its electrochemical properties, such as redox activity, charge transfer, or conductivity, which can generate an electrical signal upon interaction with the target [62]. RNA can also be used as a signal amplifier by exploiting its enzymatic properties, such as polymerization, cleavage, or ligation, which can amplify the electrical signal by producing more RNA molecules [63]. Figure 3 shows a simplified illustration of a bioelectrochemical genosensor that can detect adenocarcinoma, a cancer that arises from glandular cells. The genosensor uses nucleic acids, molecules that store genetic information, to identify specific DNA or RNA sequences related to adenocarcinoma. The genosensor operates by fixing the nucleic acid probes on the electrode surface, enabling target DNA or RNA molecules to be recognized and bound. The subsequent hybridization event produces an electrochemical signal that can be quantified and associated with the sample’s presence and amount of adenocarcinoma. Employing nucleic acid-based genosensors offers several benefits, such as high sensitivity, specificity, and accuracy [64]. RNA-based bioelectrochemical sensors can achieve high sensitivity, selectivity, and rapidity for detecting various cancer biomarkers, such as RNA expressions, microRNAs, or long non-coding RNAs. RNA-based bioelectrochemical sensors can also be coupled with different nanomaterials, such as gold nanoparticles, graphene, or quantum dots, to enhance the performance of the sensors [36, 65].

Figure 3. An illustration of a bioelectrochemical genosensor detecting adenocarcinoma. (A) Illustration depicting the electrochemical (PNA) platform on paper for miRNA-492 detection. (B) Fine-tuning the (PNA) concentration involved testing concentrations within the 25–200 nM range, with a probe immobilization time set at 1 h. The inset displays the current signal on the paper-based screen-printed electrode (SPE), indicated by a black dashed line in the absence and a blue solid line in the presence of 50 nM miRNA-492 with 100 nM () immobilized on the working electrode surface. (C) The optimization of PNA immobilization time explored intervals of 30 min, 1, 2, and 4 h using 100 nM PNA and 100 nM miRNA-492. The inset illustrates the current signal at the paper-based SPE, showing a black dashed line without and an orange solid line with 100 nM miRNA-492, where 100 nM (PNA) had been immobilized on the working electrode surface for 1 h. (D) Examination of the temporal impact on the binding between (PNA) and miRNA-492 involved testing various binding durations within 10 min to 2 h. The conditions included 100 nM (PNA), a (PNA) immobilization time of 1 h, 100 nM miRNA-492, and five mM Ru(NH₃)₆³⁺. The inset illustrates the current signal on the paper-based screen-printed electrode (SPE), with a black dashed line representing the absence and a purple solid line representing the presence of 100 nM miRNA-492, where 100 nM (PNA) had been immobilized on the working electrode surface with a binding time of 20 min. (E) The response curve was generated by testing different concentrations of miRNA-492 within the range of 10 to 1000 nM. The solutions were prepared in a 50 mM phosphate buffer with 100 mM NaCl, pH = 7, using 100 nM (PNA), a (PNA) immobilization time of 1 h, 5 mM Ru(NH₃)₆³⁺, and a binding time of 20 min (n = 3). The inset depicts the response curve within the linear range of 50 to 100 nM, maintaining the conditions of 100 nM (PNA), 1-h (PNA) immobilization time, 5 mM Ru(NH₃)₆³⁺, and a binding time of 20 min (n = 3). (F) The response curve was obtained by testing different concentrations of miRNA-492, prepared in undiluted serum, within the range of 10 to 1000 nM. The conditions included 100 nM (PNA), a (PNA) immobilization time of 1 h, 5 mM Ru(NH₃)₆³⁺, and a binding time of 20 min (n = 3). The inset shows the response curve within the linear range of 50 to 100 nM, maintaining the same conditions of 100 nM (PNA), 1-h () immobilization time, 5 mM Ru(NH₃)₆³⁺, and a binding time of 20 min (n = 3). All measurements were conducted using a paper-based screen-printed electrode with a printed Ag/AgCl pseudoreference. (F) The impact of 0.435 mM albumin, 16.7 mM urea, 27.8 mM glucose, and 0.595 mM uric acid was assessed. This evaluation utilized 100 nM (PNA), a (PNA) immobilization time of 1 h, 100 nM miRNA-492, 5 mM Ru(NH₃)₆³⁺, and a binding time of 20 min (n = 3) [64].

Some examples of RNA-based bioelectrochemical sensors for cancer detection are sensors based on RNA hybridization that detect mRNA expression of VEGF-A, a gene that promotes angiogenesis in tumors. The sensor used a gold electrode coated with capture probes and silver nanoparticles marked with signal probes [66]. The sensor identified VEGF-A expression in serum models from lung cancer cases with high accuracy and precision. A sensor that uses RNA electricity detects (miR-21), a slight non-coding RNA that controls gene expression and is involved in cancer progression [67]. The sensor used a glassy carbon electrode coated with ferrocene-labeled hairpin probes and methylene blue-labeled capture probes [68]. The sensor identified (miR-21) with high accuracy and precision in serum trials from breast cancer cases [20, 69]. An RNA multiplication-based sensor detects long non-coding RNA HOX (HOX) transcript antisense intergenic RNA), a large non-coding RNA that controls gene expression and is linked to cancer metastasis [70]. The sensor used a gold electrode coated with capture probes and (RCA) as a multiplication method [49]. The sensor identified HOX in plasma samples from colorectal cancer patients with high accuracy and precision. RNA-based biosensors that use electricity and chemistry help diagnose cancer and monitor the treatment response and the prognosis of the patients [71,72]. For example, a sensor that uses RNA pairing detects mRNA expression of HER2, a gene overexpressed in many forms of cancer, and is a target for several anticancer drugs [73]. The sensor used a gold electrode coated with capture probes and gold nanoparticles marked with signal probes [46, 74]. The sensor identified HER2 expression in plasma samples from breast cancer patients at all times of therapy with high accuracy and precision [75]. A different sensor that uses RNA multiplication detects mRNA expression of (KRAS), a gene changed in many forms of cancer and affects the response to targeted therapy [76–78]. The sensor used a gold electrode modified with capture probes and (EXPAR) as an amplification method. The sensor detected (KRAS) mutations in plasma samples from colorectal cancer patients before and after treatment with high sensitivity and selectivity [79]. However, some challenges and limitations still need to be overcome to develop and apply RNA-based bioelectrochemical sensors for cancer detection [80,81]. Some of these challenges are fine-tuning the RNA sequences and structures to achieve high accuracy for the target, and low toxicity and interference by other biomolecules or substances present in complex biological samples [53,54]. The enhancement of the electrode materials and architectures to increase the surface area, electrical conductivity, biocompatibility, and stability of the sensors. Developing novel nanomaterials and amplification strategies can enhance the sensors’ sensitivity and dynamic range without compromising specificity and simplicity [56, 82].

The validation of the sensors in clinical settings with large numbers of samples from different sources and conditions. The prospects of RNA-based bioelectrochemical sensors for cancer detection are bright and promising [83,84]. With the advances in RNA synthesis and modification techniques, nanomaterials science, electrochemical methods, and bioinformatics tools, more novel and efficient RNA-based bioelectrochemical sensors will be designed and fabricated for various cancer biomarkers. These sensors will provide rapid, accurate, sensitive, selective, portable, and low-cost tools for cancer patients’ early diagnosis, treatment monitoring, and prognosis evaluation [59]. In Figure 4, the paper explores the transformative influence of contemporary electrochemical affinity biosensors, emphasizing significant advancements in (Figure 4A) (i) materials manufacturing and technology, including nanomaterials; (ii) bio(nano)materials and receptors inspired by nature; (iii) technologies for gene editing and amplification; and (iv) techniques for signal detection and processing. Additionally, the figure delves into the integration of electrochemical biosensors into daily life, as illustrated in (Figure 4B) (a), and investigates the complexities of electrochemical methods and materials used in microfluidic fabrication in (Figure 4B) (b). It elucidates the principles governing the incorporation of electrodes into lateral flow strips (LFS) and the diverse types of electrodes and modifications propelling the development of electrochemical lateral flow assays (eLFAs) in (Figure 4B) (c). The narrative further unfolds by introducing a "dispersible electrode" in (Figure 4C), specifically designed to detect the TP53 gene mutation in blood. Finally, the figure sheds light on antifouling/protective strategies for electrochemical biosensing in (Figure 4D), reinforcing the scientific landscape against interference [85]. Identifying microRNA in blood is a promising method for diagnosing and monitoring various diseases, including cancer. A novel microRNA identification method uses an electrically reconfigurable gold-coated magnetic nanoparticle network, enabling nucleic acid pairing and signal multiplication [85].

Figure 4. (A) Explores the dynamic world of modern electrochemical affinity biosensors. The landscape is adorned with innovative manufacturing and technology for (i) (nano)materials, offering many possibilities. (ii) Bio(nano)materials synthesis and nature-inspired receptor design elevate biomolecular interactions to an art form. (iii) Gene editing and amplification technologies shape the future of precision medicine at the frontier. (iv) Signal detection and processing techniques orchestrate a symphony of discovery [85]. (B) Electrochemical biosensors blend into our daily lives, effortlessly integrating with portable, wearable, and implantable devices. A seamless integration of technology and lifestyle. The delicate dance of electrochemical procedures and the alchemy of microfluidic fabrication materials reveal the magic behind the production of microfluidic electrochemical devices. Illustrated graphic showcasing the exploration of knowledge regarding the incorporation of electrodes into lateral flow strips (LFS) and the evolution of electrochemical lateral flow assays (eLFAs) [86–88]. (C) Schematic depicting the captivating journey of discovery, featuring the "dispersible electrode" as the central protagonist in the exploration of the TP53 gene mutation coursing through the bloodstream [89]. (D) Strengthening comprehension in the realm of electrochemical biosensing. Antifouling techniques act as guardians, safeguarding signal purity amidst interference challenges—figures serving as a shield against distorting elements, preserving our scientific gaze [90].

2. Electrochemical nucleic acids-based genosensing

Electrochemical genosensors use nucleic acids to detect biomolecules related to cancer-related diseases. They have three main parts: a recognition part that binds to the target, a transducer that converts the binding into an electrical signal, and a signal amplifier that enhances the signal. They can measure different electrical changes when nucleic acids interact with their targets. They have benefits such as low cost, high sensitivity, fast response, and easy operation [91]. However, they also have stability, reproducibility, interference, and standardization challenges. They need more research and development to improve their performance and applicability. Figure 5 shows the electrical test for measuring gene-specific DNA methylation-based on adenine-enriched unmethylated ss-DNA and guanine-enriched methylated ss-DNA. The two kinds of DNA are attached on a solid-phase extraction (SPE) gold electrode, with more adenine-enriched unmethylated ss-DNA than guanine-enriched methylated ss-DNA. The electrical signal in the test is produced by the (CV) interrogation of DNA-bound [Ru(NH₃)₆]³⁺ complexes [92]. The CV method involves applying a voltage to the DNA-bound [Ru(NH₃)₆]³⁺ complexes and measuring the resulting current. The current obtained is related to the amount of the DNA on the electrode and can be used to measure the relative amounts of the adenine-enriched unmethylated ss-DNA and guanine-enriched methylated ss-DNA in the sample. The inset in the figure shows a typical CV signal, with the adenine-enriched unmethylated DNA giving a higher CV charge than the guanine-enriched methylated DNA. This shows that the test can distinguish between the two kinds of DNA based on their attachment features and generate a measurable electrical signal. The electrical test for measuring gene-specific DNA methylation has several benefits [93].

Figure 5. The underlying principle of quantifying a gene-specific DNA methylation assay involves the differential adsorption of adenine-enriched unmethylated single-stranded DNA (ss-DNA) compared to guanine-enriched methylated ss-DNA on the SPE-Au electrode. The unmethylated ss-DNA, being adenine-enriched, exhibits relatively more significant adsorption on the electrode surface. The interrogation of DNA-bound Ru(NH₃)₆]³⁺ complexes through constant current (CC) generates significant electrochemical signals. The inset displays typical CC signals, illustrating that adenine-enriched unmethylated DNA produces a higher CC charge than guanine-enriched methylated DNA [92].

2.1. Examples of electrochemical genosensing nucleic acids based on cancer detection

Electroanalytical methods have recently achieved significant progress in detecting and measuring cancer biomarkers through electrochemical DNA genosensors [94]. These genosensors depend on the hybridization events between a DNA or (PNA) capture probe and the target nucleic acid sequence for detection, with the hybridization event causing a noticeable change in the electrochemical signal. These genosensors have shown high sensitivity and specificity, making them a desirable option for early cancer detection [95,96]. In particular, several studies have developed electrochemical genosensors based on nucleic acids for biomarker detection in breast cancer. For example, researchers technologically advanced label-free electrochemical genosensors that employ DNAzyme to detect the bladder cancer biomarker miRNA-21, demonstrating high selectivity against other miRNAs [97–100]. Likewise, other scientists reported on electrochemical genosensors that utilize the (HCR) to detect the breast cancer biomarker miRNA-21, exhibiting high specificity against other miRNAs.

• Choosing the right nucleic acid receptors, such as aptamers, DNAzymes, or ribozymes, is essential for ensuring optimal performance and selectivity of the biosensors. Nevertheless, the establishment of these receptors remains complex and time-intensive, necessitating the use of high-throughput screening and optimization techniques.

• Several parameters, including temperature, pH, ionic strength, enzymatic degradation, and interference from nonspecific binding or fouling, can influence the stability and reproducibility of nucleic acid receptors and biosensors. Hence, it is essential to implement efficient measures to augment the biosensors’ biocompatibility and longevity through adequate protection and immobilization procedures.

• Combining nucleic acids with nanomaterials, such as carbon nanotubes, graphene, quantum dots, or gold nanoparticles, can enhance the transmission and amplification of signals in biosensors. Nevertheless, achieving the most efficient design and production of these nanocomposites necessitates meticulous regulation of the dimensions, configuration, chemical properties, and modification of the nanomaterials, along with the interplay and alignment of the nucleic acids on the nanomaterials.

• The successful implementation and utilization of biosensors in clinical environments necessitate the utilization of intricate biological specimens, such as blood, serum, urine, or saliva, which may include diverse interfering components, such as proteins, salts, or metabolites. Consequently, the biosensors must possess exceptional selectivity and durability to overcome the influence of the surrounding environment. Additionally, they should exhibit remarkable sensitivity and precision to detect the minute quantities of the desired biomarkers.

• Before their extensive implementation in clinical practice, it is imperative to address the crucial matters of standardization and regulation of biosensors. The biosensors must adhere to regulatory bodies’ quality and safety standards, such as the Food and Drug Administration (FDA) or the European Medicines Agency (EMA). Furthermore, it is necessary to compare and evaluate the biosensors with established techniques like polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA) to prove their dependability and usefulness.

Table 1 provides a concise overview of recent research projects investigating the application of nanomaterials in detecting several biomarkers associated with cancer, Alzheimer’s disease, and microRNA expression. The table provides a comprehensive overview of each study, including details such as the title, authors, year, publication, biomarker, nanomaterial, sensitivity, and reference. The nanomaterials comprise gold nanoparticles, quantum dots, graphene, carbon nanotubes, and a double-tetrahedral DNA framework. The sensitivity of the detection approach varies based on the specific biomarker being targeted.

Table 1. Current research focuses on the utilization of nanomaterial-based biosensors to detect biomarkers.

Title	Authors	Year	Journal	Biomarker	Nanomaterial	Sensitivity	Reference
The recent advancements in the early detection of cancer biomarkers by DNAzyme-assisted aptasensors	Hossein Kamali et al.	2022	Journal of Nanobiotechnology	Various proteins	Gold nanoparticles, quantum dots, graphene, etc.	Varies depending on the target	[101]
Electrochemical Biosensors Based on Nanomaterials for Early Detection of Alzheimer’s Disease	Celia Toyos-Rodríguez et al.	2020	Sensors	Amyloid-beta, tau protein, etc.	Carbon nanotubes, gold nanoparticles, graphene, etc.	Varies depending on the target	[102]
Recent Progresses in Electrochemical DNA Biosensors for MicroRNA Detection	Zhenzhen Li et al.	2021	Journal of Analysis and Testing	Various microRNAs	Gold nanoparticles, carbon nanotubes, graphene, etc.	Varies depending on the target	[22]
A double-tetrahedral DNA framework based electrochemical biosensor for ultrasensitive detection and release of circulating tumor cells	C. Wang et al.	2021	Analyst	Circulating tumor cells	Double-tetrahedral DNA framework	0.1 cell mL^−1	[103]
Nanogenosensors based on aptamers and peptides for detection of cancer biomarkers	S. M. Taghdisi et al.	2024	SN Applied Sciences	Various proteins and nucleic acids	Gold nanoparticles, carbon nanotubes, graphene, etc.	Varies depending on the target	[104]

These studies showcase the potential of electrochemical genosensors based on nucleic acids for detecting cancer biomarkers [97–99, 105]. Figure 6 delves into the fascinating world of voltammetric affinity biosensing, unraveling the intricate principles governing electrodes modified with nanoporous membranes and specific bioreceptors [85]. Figure 6A provides insights into the dynamic "On" and "Off" responses in the absence and presence of target biomolecules, showcasing the artistry of molecular recognition [85]. Figure 6B explores the brilliance of a biosensor chip, a technological marvel harmonizing diverse immunoassay formats and enzymatic tracers. This chip enables the simultaneous determination of cortisol and insulin, introducing readers to a multifaceted analytical landscape and a symphony of insights [85]. Immerse yourself in the schematics of electrochemical lateral flow assays (eLFAs) in (Figure 6C), where scientific creativity meets practical application. Unravel the complexities of eLFAs designed for determining protein (a) and genetic (b) biomarkers, offering a dual perspective on biochemistry and genetics and contributing to the article’s comprehensive exploration of diagnostic solutions [85].

Figure 6. (A) Engage in an investigation of the dynamic principles that govern voltammetric affinity biosensing, where electrodes undergo a transformation through nanoporous membranes, each containing particular bioreceptors. Observe the fascinating interaction between the "On" response when the target biomolecule is not there and the subtle "Off" response when it is, vividly illustrating the molecular recognition process [106]. (B) Discover the cleverness of a biosensor chip, a sophisticated fabric that effortlessly combines several immunoassays and enzymatic tracers. This chip enables the simultaneous measurement of cortisol and insulin while facilitating the integration of many analytical variables, resulting in a comprehensive and insightful analysis [107]. (C) Immerse yourself in the intricate details of electrochemical lateral flow assays (eLFAs), where the combination of scientific expertise and practical implementation is showcased. Explore the complexities of eLFAs, specifically created to identify protein (a) and genetic (b) biomarkers. These assays provide a unique insight into biochemistry and genetics and contribute to developing comprehensive diagnostic solutions [108,109].

3. DNA aptamer-based genosensors in PSA identification

DNA aptamer-based genosensors have emerged as a promising approach for identifying (PSA), a commonly used indicator for prostate cancer identification. (PSA) is a glycoprotein spread by the prostate gland, and its blood levels can indicate prostate cancer [110]. However, current (PSA) identification methods, such as (ELISA), have limitations in terms of sensitivity and specificity. In contrast, DNA aptamer-based genosensors have shown high sensitivity and specificity, making them a potential solution to these limitations [111,112]. The study presents a range of electrochemical affinity biosensors that accurately detect bacterial genetic material using single (a) and multiblock (b) polyA capture probes in (A) [85]. Explore the complex field of immune response profiling using a bioplatform in (B), which involves using magnetic beads (MBs) decorated with N and in-house-expressed S ectodomains of SARS-CoV-2 variants. The amperometric detection using screen-printed carbon electrodes (SPCEs) provides a detailed analysis of N- and S-specific immunoglobulin isotypes (IgG, IgM, and IgA). Experience the innovative multiplexed microfluidic bioplatform in (C), a remarkable technological advancement that enables the simultaneous detection of viral load and ß-lactam antibiotic levels in nasal swabs and serum samples from individuals infected with COVID-19 [85]. This index functions as a navigational tool, leading readers to the text’s various and wide-ranging insights. These insights cover bacterial genetics, immune response dynamics, and sophisticated COVID-19 diagnostics [85].

One example of such a genosensor is the electrochemical biosensor designed by Zhang et al. (2016) for (PSA) identification using a DNA aptamer [113]. The biosensor used a gold electrode improved with a self-assembled monolayer of thiolated DNA aptamer against (). The biosensor’s detection principle was based on the change in the electrical signal upon attachment of (PSA) to the aptamer probe. The biosensor showed a linear response to (PSA) concentrations ranging from 0.1 pg/mL to 15 ng/mL, with (LOD) of 0.052 pg/mL. The biosensor also demonstrated high specificity against other proteins, such as human serum albumin and lysozyme, and correlated with the results obtained by a commercial ELISA kit in clinical serum samples [114–116]. This statistic provides insights into advanced techniques used in genetic diagnostics and COVID-19 detection. It also paves the way for the future of biosensor technology, where artificial intelligence and smart devices will play vital roles in shaping healthcare.

Other cases have also reported the design of DNA aptamer-based biosensors for identifying (PSA) and other cancer indicators, including (CEA), (HER2), and (AFP). The biosensors can be designed using various identification principles, such as colourimetric, electrical, and fluorescent, and can be optimized for sensitivity and specificity [117]. However, there are challenges in developing DNA aptamer-based biosensors, such as selecting aptamers with high accuracy for the target biomolecule and optimizing biosensor performance. Nevertheless, the benefits of DNA aptamers, such as ease of synthesis, high accuracy, and stability, make them attractive candidates for biosensor development [118,119]. Therefore, DNA aptamer-based biosensors offer a promising strategy for identifying cancer indicators, including (PSA). They offer several advantages over traditional antibody-based biosensors and can be designed to identify cancer indicators using various techniques. The development of DNA aptamer-based biosensors for cancer diagnosis and treatment could create sensitive and specific diagnostic tools for early cancer detection and personalized treatment [120]. Figure 7 shows improvement in the sensitivity and accuracy of the genosensor, Fe-MOF is used as a signal enhancer. Fe-MOF is a metal-organic framework that can multiply the electrical signal by catalyzing the reduction of hydrogen peroxide. The Fe-MOF is incorporated into the genosensor to improve the electrical signal generated by the pairing event, resulting in a more sensitive and accurate identification of the (ctDNA) [121].

Figure 7. Identifying (ctDNA) in blood is becoming an increasingly important tool for diagnosing and monitoring cancer. A new electrical genosensor has been developed for sensitively detecting non-small cell lung cancer (ctDNA) using a unique sensing platform and signal enhancement method. The genosensor uses NG-PEI-COFTAPB-TFPB as the sensing platform, designed to capture and match with the target (ctDNA) in the sample. The platform is functionalized with a specific probe that can recognize and bind to the (ctDNA) sequence with high accuracy. The pairing event changes the electrical signal, which can be measured and correlated with the presence and concentration of the (ctDNA) in the sample [121,122].

4. RNA aptamer-based genosensors for (CEA) detection

RNA aptamer-based biosensors have emerged as a promising technique for identifying cancer indicators such as (CEA) [123]. RNA aptamers are short, single-stranded RNA molecules that selectively attach to specific targets, allowing for the detection of (CEA) with high sensitivity and specificity. Several studies have developed RNA aptamer-based biosensors for (CEA) detection, each with benefits and drawbacks [46, 124,125]. One example of such a biosensor is the SPR biosensor developed by Xia et al. (2015) for (CEA) identification, which is label-free and easy to use. Other examples include the EIS biosensor developed by Zhang et al. (2018), the FP biosensor developed by Yang et al. (2019), the GO biosensor developed by Wu et al. (2020), and the microcantilever biosensor developed by Mirzaei et al. (2021). Figure 8 provides a thorough summary of the essential construction components used in DNA nanomachines for early cancer detection. The figure illustrates the process of aptamer selection using (SELEX) under the aptamer methods category [126]. It also demonstrates the ability of aptamers to recognize and attach to their specific targets accurately. The image delves into classical molecular beacons’ underlying architecture and operating concepts [126]. Furthermore, it explores the complexities of signal amplification accomplished through (HCR) (b, ii) and (CHA) (b, iii).

Figure 8. (A) Key construction ingredients utilized in DNA nanomachines for early cancer diagnosis. (a, i) The aptamer selection technique based on (SELEX). (ii) The concept of aptamers specifically identifying and binding to their intended targets. (b, i) This article discusses traditional molecular beacons’ fundamental structures and operational concepts. (ii) The process by which signal amplification occurs via (HCR). (iii) The process by which signal amplification occurs via CHA. (c, i) RNA-cleaving DNAzymes can cleave specific ribonucleic acid sites within a DNA strand facilitated by metal ions. (ii) The DNAzymes that imitate peroxidase can facilitate the catalysis of ABTS2− for colourimetric detection or interact with luminol for chemiluminescence detection. Reproduced with permission from ref. [126]. (B) This is a schematic representation of the process of biofunctionalizing the immunosensor. Reproduced with authorization from the ref. [127]. (b) The composition of the peptide probe labeled with methylene blue and the underlying principle of detection for the electrochemical biosensor based on this peptide. Used with authorization from the reference [128]. (C) This illustration describes using aptamer/electroactive species-loaded AuNPs to amplify the signal probes and detect target CTCs in whole blood. The process involves capturing, isolating, amplifying, and multiplexing the detection of the target CTCs. Used with authorization from the reference [129].

Furthermore, the diagram depicts the function of RNA-cleaving DNAzymes in specifically cleaving ribonucleic acid sites with the assistance of metal ions (c, i), as well as the ability of DNAzymes to imitate peroxidase activity for colourimetric or chemiluminescence detection (c, ii) [127]. The following part (B) concentrates on the biofunctionalization procedure of immunosensors, providing a detailed schematic depiction of the process and the composition of a peptide probe tagged with methylene blue for electrochemical biosensor detection (b) [128]. Section (C) explains the use of gold nanoparticles (AuNPs) loaded with aptamer/electroactive species to enhance signal probes for detecting circulating tumor cells (CTCs) in whole blood. It focuses on capturing, isolating, amplifying, and multiplexing steps in this novel approach [129].

The long single-stranded DNA products that result can be detected using an electrical signal produced by the oxidation or reduction of the electroactive probe [130]. The dual electrical genosensor offers several advantages, including high sensitivity, specificity, and selectivity. The genosensor incorporates two different electrical identification methods, enabling the simultaneous identification of two different lncRNAs. This feature enhances the accuracy and reliability of the genosensor for the early detection of prostate cancer. Using the dual electrical genosensor to detect lncRNAs offers a noninvasive and cost-effective method for the early detection of prostate cancer. The genosensor can be used in clinical settings to screen and monitor prostate cancer cases.

5. Road to the future

There is still space for innovation and enhancement in nucleic acid-based electrochemical biosensors, notwithstanding their significant potential for cancer diagnosis. In order to accomplish the ultimate objective of creating dependable, precise, and user-friendly biosensors for cancer detection and, eventually, to enhance cancer prevention, therapy, and management, the subsequent research avenues should be pursued:

Enhancing the stability and consistency of electrochemical biosensors that rely on nucleic acids: The stability and reproducibility of these biosensors are essential for their clinical application and dependability. Hence, it is imperative to employ efficient tactics to safeguard and maintain the integrity of nucleic acids against degradation, oxidation, or alterations in pH. This can be achieved by utilizing nuclease-resistant analogues, introducing protective groups to modify the nucleic acids, or inserting them into nanocarriers. Furthermore, it is necessary to standardize and optimize these biosensors’ fabrication and performance parameters to guarantee their uniformity and precision across various batches, operators, and laboratories. Moreover, it is necessary to implement quality control and validation protocols to assess the durability and dependability of these biosensors when tested with actual samples obtained from cancer patients or healthy individuals.

To improve the sensitivity and specificity of nucleic acids-based electrochemical biosensors, enhancing their capability to detect low-abundance and mutant cancer biomarkers in complex and diverse biological materials is crucial. Hence, employing efficient tactics to diminish or eradicate the disruptive impacts caused by different substances that can adhere to the transducer surface, contend with the desired molecules, or produce extraneous signals is imperative. This can be achieved by employing selective capture probes, blocking agents, washing procedures, or signal amplification techniques. In addition, innovative techniques for enhancing signal strength, such as rolling circle amplification, exponential amplification reaction, hybridization chain reaction, or (CHA), can augment the quantity of nucleic acids or electroactive substances on the transducer surface, leading to improved signal-to-noise ratios. In addition, biosensors can achieve even greater signal intensity and specificity by employing various signal amplification techniques or utilizing multifunctional amplifiers, such as nanocomposites, enzymes, or aptamer-conjugates.

Investigating novel nucleic acid recognition elements and transducer materials for electrochemical biosensors that rely on nucleic acids: The selection and configuration of the nucleic acid recognition components and the transducer materials can significantly influence the effectiveness and capabilities of these biosensors. Hence, investigating novel nucleic acids with unique capabilities, such as catalytic activity, structural alterations, self-organization, or movement, can amplify the accuracy and selectivity of biosensors. Some potential options for nucleic acid recognition elements include ribozymes, DNA or RNA switches, DNA or RNA origami, and DNA or RNA nanomachines. In addition, the advancement of novel nanomaterials, such as carbon nanotubes, graphene, quantum dots, metal nanoparticles, or metal-organic frameworks, has the potential to enhance the electrical conductivity, biocompatibility, surface area, and catalytic activity of the transducers. In addition, developing novel transducer structures, such as nanowires, nanofibers, nanosheets, or nanoflowers, can improve the ability of the transducers to carry a load, transmit signals, and transfer mass. Moreover, incorporating various transducer materials or employing numerous signal-measuring methodologies can enhance the biosensor’s ability to perform multiplexing and multimodal functions.

Combining nucleic acid-based electrochemical biosensors with microfluidic devices or portable tools for point-of-care testing: The incorporation of these biosensors into microfluidic devices or portable instruments can provide numerous benefits, including size reduction, automation, simultaneous analysis of multiple targets, and on-site testing. Microfluidic devices offer accurate and effective sample volume, flow rate, and mixing manipulation. They also allow for integrating several functionalities, including sample preparation, nucleic acid extraction, amplification, hybridization, and detection, all on a single chip. Portable technologies, such as cellphones, wearable gadgets, or paper-based tools, provide wireless, immediate, and user-friendly use and display of biosensors. They also facilitate data storage, transfer, and analysis. These characteristics can enhance the accessibility, convenience, and affordability of biosensors for cancer diagnosis in settings with low resources or remote areas.

6. Discussion

Electrochemical biosensors that utilize nucleic acids have emerged as a highly promising tool for diagnosing cancer. These biosensors have the advantages of fast, sensitive, and specific detection of different cancer biomarkers in liquid biopsy samples. These biosensors utilize the distinctive characteristics of nucleic acids, such as their capacity to combine with complementary sequences, create secondary structures, facilitate processes, or attach to specific targets. By combining nucleic acids and electrochemical transducers, such as electrodes, nanomaterials, or microfluidic devices, these biosensors can produce detectable electrical signals that indicate the existence and amount of cancer biomarkers in the sample.

This study provides a concise overview of the latest developments in electrochemical biosensors for cancer diagnosis that utilize nucleic acids as recognition components. Specifically, it focuses on three primary types of nucleic acids: DNA aptamers, DNAzymes, and RNA aptamers. We have also compared these biosensors and antibody-based alternatives, emphasizing their strengths and weaknesses. We have shown explicit instances of biosensors that selectively detect crucial cancer biomarkers, including (PSA), (miR-21), and (CEA), and thoroughly examined their efficacy and practical uses.

Nevertheless, despite the notable advancements achieved in this domain, various obstacles and constraints that necessitate resolution before the widespread adoption of these biosensors in clinical environments remain. Several of these challenges encompass:

6.1. Factors affecting the performance of nanogenosensors

6.1.1. Stability

Nucleic acids are susceptible to deterioration caused by nucleases, oxidation, or alterations in pH, which might impact their functionality and dependability. Hence, it is imperative to implement effective measures to improve these biosensors’ longevity and functional reliability through appropriate safeguarding and stabilization techniques. By employing nuclease-resistant analogues like (PNAs) or (LNAs), modifying nucleic acids with protective groups such as (PEG) or (PVA), or incorporating them into nanocarriers like liposomes or polymersomes, their stability and bioavailability can be enhanced.

Various factors, including the quality and quantity of the nucleic acid recognition elements, the type and modification of the transducer materials, the conditions for immobilization and hybridization, the preparation and handling of the samples, and the measurement and analysis of the signals, can influence the reproducibility of nucleic acid-based electrochemical biosensors. Hence, it is crucial to standardize and optimize these parameters to guarantee these biosensors’ reproducibility and precision. Furthermore, it is necessary to implement quality control and validation processes to assess the uniformity and durability of these biosensors when used in various batches, by different operators, and in different laboratories.

6.1.2. Interference

The intricate and diverse composition of biological samples, such as blood, serum, or urine, can create difficulties for the accuracy and sensitivity of nucleic acids-based electrochemical biosensors. The samples may contain many compounds that can interfere with the experiment, such as proteins, salts, metabolites, or other nucleic acids. These substances can stick to the surface of the transducer, compete with the target molecules, or create background signals. Hence, employing efficient tactics to diminish or eradicate the disruptive impacts is essential, such as discerning capture probes, obstructive compounds, cleansing procedures, or signal enhancement techniques.

6.1.3. Uniformization

The absence of established and proven reference methods and materials for identifying and measuring cancer biomarkers in liquid biopsy samples can restrict the capacity to compare and apply nucleic acids-based electrochemical biosensors. The detection ranges, limits of detection, calibration curves, and accuracy levels of biosensors might vary based on the selection of nucleic acid recognition elements, transducer materials, and signal measurement methodologies. Consequently, it is necessary to harmonize and normalize these characteristics to guarantee these biosensors’ compatibility and interoperability. Furthermore, it is necessary to create consensus guidelines and criteria for selecting, validating, and reporting cancer biomarkers and biosensors. This will help to facilitate the translation and integration of these biosensors into clinical practice.

Nucleic acid-based electrochemical biosensors can detect changes in target molecules’ electrical characteristics due to the nucleic acid’s unique interactions with those molecules. The biosensors can accomplish this in various ways, depending on the nucleic acid type. Here, we will take a quick look at DNA aptamers, DNAzymes, and RNA aptamers, the three primary nucleic acid types utilized in electrochemical biosensors for cancer diagnostics.

6.1.4. DNA aptamers

Aptamers are short, single-stranded DNA molecules with a high affinity and specificity for binding to tiny molecules, proteins, and metal ions, among other potential targets. The (SELEX) algorithm is used to pick them out of vast databases of random sequences. Target molecule electrochemical behavior can be changed by conformational changes brought about by binding to DNA aptamers. When bound to target molecules, aptamers can provide electrochemical signals; alternatively, they can mediate electron transfer between target molecules and electrode surfaces. When the aptamer interacts with its target, the signals are amplified. Alternatively, the aptamers can be connected with additional electroactive molecules, such as enzymes, redox indicators, or nanoparticles.

6.1.5. DNAzymes

DNAzymes are molecules with DNA that can accelerate certain chemical events, including phosphorylation, cleavage, and ligation. Their structure consists of a substrate strand and a catalytic core. The enzyme’s catalytic action occurs in the catalytic core, while the substrate strand has a cleavage site and a target molecule recognition site. The substrate strand cleaves when the target molecule attaches to the recognition site and triggers the catalytic core. Voltammetry, impedance, and chronocoulometry are a few electrochemical techniques that can identify substrate strand cleavage. As an illustration, the substrate strand can be imbued with an electroactive molecule—a redox indicator, an enzyme, or even a nanoparticle—that can provide a signal when detached from the catalytic core. Another option is to bond the substrate strand to the surface of the electrode; then, the cleavage can alter the capacitance or electrical resistance.

6.1.6. RNA aptamers

Though they differ in building material from DNA aptamers, RNA aptamers serve a similar purpose. In addition, they can undergo conformational changes when bound to targets with great affinity and specificity. RNA aptamers’ structural variety, thermal stability, and catalytic activity are superior to DNA aptamers. Another role for RNA aptamers is that of ribozymes, which are RNA molecules that can catalyze particular events like phosphorylation, cleavage, or ligation. Methods like electron mediators, electroactive labels, or signal amplifiers can be employed to electrochemically detect RNA aptamers, much as they do for DNA aptamers.

Electrochemical biosensors that rely on nucleic acids primarily work by these principles. These processes allow the biosensors to identify cancer biomarkers in liquid biopsy samples with remarkable speed and sensitivity. However, as we will see in the next section, several obstacles and restrictions must be overcome. These include issues with the biosensors’ stability, reproducibility, interference, and standardization.

7. Conclusion

This study provides a comprehensive overview of the most recent developments and applications of nucleic acid-based electrochemical biosensors for cancer detection. These biosensors utilize the unique properties of nucleic acids, such as their ability to bind with complementary sequences, form secondary structures, catalyze reactions, or bind to specific targets. This enables them to generate measurable electrical signals that indicate the presence and concentration of cancer biomarkers in liquid biopsy samples. These biosensors offer numerous advantages over conventional methods and antibody-based electrochemical biosensors, such as cost-effectiveness, enhanced sensitivity, quick response, and easy operation. We have presented visual representations of biosensors designed to selectively identify essential cancer biomarkers, such as (PSA), (miR-21), and (CEA), and evaluated their efficacy and applications. Furthermore, we have identified and emphasized these biosensors’ challenges and limitations, such as stability, repeatability, interference, and standardization. We have also implemented practical strategies and improvements to address these issues effectively.

The subsequent paragraphs will provide a concise overview of the primary accomplishments of nano-scale biosensors utilizing DNA and RNA, focusing on their detection targets, sensing methods, and signal amplification strategies.

Targets for detection: Nano-scale biosensors utilizing DNA and RNA can accurately identify various biomarkers associated with non-communicable and communicable diseases, exhibiting exceptional sensitivity and specificity. The biomarkers encompass nucleic acids, proteins, metal ions, and small biological molecules. These biomarkers are associated with diagnosing, prognosis, and treating many malignancies, infectious diseases, genetic abnormalities, and metabolic diseases. For instance, biosensors that rely on DNA and RNA can identify (PSA) to detect prostate cancer, (miR-21) to detect colorectal cancer, (CEA) to detect colorectal and breast cancer, (HPV) to detect cervical cancer,(HBV) to detect liver cancer, and glucose to detect diabetes.

Sensing mechanisms: Nano-scale biosensors that utilize DNA and RNA can take advantage of the self-assembly, programmability, catalytic activity, and dynamic behavior of these molecules to provide versatile and flexible sensing platforms. The platforms can be categorized into two distinct groups: direct detection and indirect detection. Direct detection is based on the inherent electrochemical characteristics of nucleic acids, such as their capacity to facilitate electron transfer or produce electroactive signals. Indirect detection entails employing supplementary labels or probes to induce electrochemical alterations when the target nucleic acids are hybridized or recognized. Both direct and indirect detection methods can be improved by utilizing diverse signal amplification methodologies, including nanomaterials, enzymes, catalytic processes, or chain reactions.

Signal amplification solutions involve the integration of DNA- and RNA-based nano-scale biosensors with nanomaterials. This integration enhances the biosensors’ ability to transmit and amplify signals and improves their biocompatibility and stability. Nanomaterials, such as gold nanoparticles, carbon nanotubes, graphene, quantum dots, and magnetic nanoparticles, possess significant surface area, excellent conductivity, powerful adsorption capabilities, and distinctive optical features. These attributes can enhance the biosensors’ sensitivity, selectivity, and response time. DNA and RNA can also serve as templates or frameworks for organizing nanomaterials into specific configurations, such as nanowires, nanorods, nanoflowers, and nanostars. This arrangement can then be used to manipulate the electrochemical signals of biosensors.

To summarize, electrochemical biosensors utilizing nucleic acids have numerous benefits compared to alternative methods, including heightened sensitivity, specificity, ease of use, affordability, rapid reaction time, and minimal sample usage. This study focuses on the latest progress and difficulties in creating and utilizing electrochemical biosensors for detecting DNA or RNA. In addition, we have put forward other possible domains for further investigation and prospects to advance the field of electrochemical biosensors that rely on nucleic acids. These activities include exploring new nucleic acid recognition components, developing advanced transducer materials and designs, formulating novel signal amplification techniques, integrating biosensors with microfluidic devices or portable instruments, and validating biosensors in clinical settings using actual cancer patients or healthy donors. This review is anticipated to stimulate additional investigation and advancement in this field, resulting in the development of more reliable, accurate, and user-friendly biosensors for cancer detection. Ultimately, this will improve cancer prevention, treatment, and management.

Some of the hot questions remaining for future research in electrochemical biosensors based on nucleic acids:

• What strategies can be employed to enhance the stability and repeatability of nucleic acid aptamers, particularly when exposed to complex biological matrices and fluctuating environmental conditions?

• What steps should be taken to create new nucleic acid structures and patterns that can improve biosensors’ ability to recognize and catalyze reactions and allow them to combine different functions and sensing modes?

• In order to improve signal transduction, amplification, and modulation, how can the interface between nucleic acids and, carbon nanotubes and other nanomaterials be optimized?

• The question is, how can we expand biosensors’ dynamic range and sensitivity by creating novel signal amplification mechanisms such as feedback loops, enzymes, catalytic events, or chain reactions?

• Compared to more traditional methods, how can we ensure that biosensors work as intended in real-world clinical situations by analyzing samples from patients or healthy donors?

Abbreviations
ApDCs	=	Aptamer-drug conjugates
CEA	=	carcinoembryonic antigen
CHA	=	Catalytic Hairpin Assembly
ctDNA	=	Circulating tumor DNA
EGFR	=	epidermal growth factor receptor
EXPAR	=	exponential amplification reaction
HBV	=	hepatitis B virus
HCR	=	Hybridization Chain Reaction
HOX	=	Homeobox
HPV	=	human papilloma virus
KRAS	=	Kirsten rat sarcoma virus
LNAs	=	locked nucleic acids
LOD	=	Limit of detection
miR-21	=	microRNA-21
PEG	=	polyethene glycol
PNAs	=	peptide nucleic acids
PSA	=	prostate-specific antigen
PVA	=	polyvinyl alcohol
RCA	=	rolling circle multiplication
SELEX	=	Systematic evolution of ligands by exponential enrichment

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Funding

The author(s) reported there is no funding associated with the work featured in this article.