Electrochemical detection of fusion genes: Advancing cancer diagnosis and therapy

Electrochemical detection of fusion genes: Advancing cancer diagnosis and therapy Nasim Izadi a , Aneta Fried a,b , Sarka Sevcikova a,b , Johana Strmiskova a,b , Ludmila Moranova a , Susana Campuzano c,d,** , Martin Bartosik a,* a Research Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 656 53 Brno, Czech Republic b National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic c Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain d CIBER of Frailty and Healthy Aging (CIBERFES), Instituto de Salud Carlos III, 28046 Madrid, Spain ARTICLE INFO Keywords: Fusion genes Electrochemical detection Precision oncology Cancer biomarker DNA analysis ABSTRACT Fusion genes are formed by joining segments of two different genes through translocations, deletions, chromosomal insertions, or aberrant gene splicing. These fusions, detectable at the DNA, RNA, and protein levels, are diagnostically and prognostically relevant molecular indices for the selection of targeted therapies, monitoring of minimal residual disease after treatment, and prediction of relapse. In contrast to traditional determination techniques, electroanalytical technologies provide affordable, versatile, portable, and rapid detection methods that do not compromise specificity, sensitivity, robustness, or accuracy. These advantages make them highly promising for advancing research, enabling clinical adoption, supporting pharmaceutical development, and expanding access to accurate diagnostics and effective therapies. In this context, this review article addresses the potential and opportunities offered by electroanalytical technologies for fusion gene detection. It provides a brief description of the most common fusion genes in hematological malignancies and solid tumors, an explanation of frequently used standard techniques, a critical overview of recent electrochemical (EC)-based studies targeting the most common fusion genes, and highlights the challenges faced by current EC bioassays. 1. Introduction Fusion genes arise from chromosomal rearrangements, that is, structural alterations in a chromosome that have a serious impact on gene function or expression. These are mostly translocations, where a segment of one chromosome breaks off and attaches either to different sites on the same chromosome (intrachromosomal translocation) or to a different chromosome (interchromosomal translocation) [1]. Other chromosomal rearrangements, besides translocations, include inversions (i.e., reversed orientation of a chromosome segment), duplications (extra copies of a chromosome segment), and deletions (loss of a chromosome segment). The most common examples of these rearrangements are listed in Table 1. Previously, chromosomal aberrations were thought to be the only mechanism by which fusion products are generated. Later, it became clear that some fusion products could arise without structural chromosomal rearrangements – through the aberrant splicing of genes. These genetic alterations play pivotal roles in the development and progression of a wide range of diseases. While some of these alterations are linked to rare syndromes, such as Norrie disease retinopathy, Pallister-Killian syndrome, and Potocki-Shaffer syndrome, they are especially significant in the context of cancer [2–5]. In oncology, these genetic changes often serve as both diagnostic markers and therapeutic targets. The oncogenic potential of fusion genes often arises from their ability to produce abnormally active proteins - typically tyrosine kinases (TKs) or transcription factors [6]. A prime example is the BCR::ABL1 fusion gene, best known for its association with the Philadelphia chromosome in chronic myeloid leukemia (CML). While tyrosine kinases drive uncontrolled cellular signaling, fusion transcription factors disrupt normal gene regulation, contributing to tumorogenesis. In addition to signaling and transcription, some fusion proteins affect cellular architecture by altering the cytoskeleton, thus influencing cell migration [7,8]. These structural disruptions can enhance the invasiveness of cancer cells [9]. In some cases, fusion genes promote * Corresponding author at: Research Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 656 53 Brno, Czech Republic ** Corresponding author at: Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain E-mail addresses: [email protected] (S. Campuzano), [email protected] (M. Bartosik). Contents lists available at ScienceDirect Electrochimica Acta journal homepage: www.journals.elsevier.com/electrochimica-acta https://doi.org/10.1016/j.electacta.2025.147472 Received 10 June 2025; Received in revised form 29 August 2025; Accepted 27 September 2025 Electrochimica Acta 542 (2025) 147472 Available online 27 September 2025 0013-4686/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). epithelial-mesenchymal transition (EMT), a process critical for tumor metastasis. By regulating cytoskeletal remodeling, EMT-related fusions enable cancer cells to detach from the primary tumor and invade surrounding tissues [10]. Additionally, fusion proteins may interfere with the key regulatory pathways that govern the cell cycle, differentiation, and apoptosis. When these controls break down, cancer cells can proliferate unchecked and evade programmed cell death, further contributing to tumor growth and resistance to treatment [11]. Fusion gene analysis thus plays a critical role in oncology by enabling precise molecular classification of tumors, which is essential for selecting targeted therapies. Identifying specific fusion events can directly guide the use of approved pharmaceutical agents, as described in Table 1. Given the importance and increasing number of identified fusion genes, novel methods and technologies are needed for their analysis. Recently, electrochemical (EC) techniques have emerged as promising alternatives for detecting DNA cancer biomarkers [12–16] because they offer rapid detection times, cost-effective and simple instrumentation, and the capability for parallel detection on miniaturized electrode chips. Several recent reviews have appeared [17–21], but they deal exclusively with the application of EC techniques in leukemia detection (mostly BCR::ABL1 fusion gene). To our knowledge, there is no comprehensive review of EC studies for the detection of various fusion genes and transcripts in both hematological malignancies as well as solid tumors. Moreover, no review has combined the biological relevance of fusion genes (leukemia, see Section 2, and solid tumors, Section 3), standard methods of detection (Section 4), and critical review of recent EC-based studies as powerful alternatives to these methods, targeting the most prevalent fusion genes and describing the challenges faced by current EC bioassays (Section 5). 2. Fusion genes in leukemia Fusion genes are hallmarks of many leukemias and often drive disease development through chromosomal rearrangements in hematopoietic progenitor cells. The resulting fusion proteins frequently exhibit oncogenic properties that disrupt gene expression, cell cycle regulation and DNA repair. A key example is the above-mentioned BCR::ABL1 fusion in CML, which produces abnormal TKs essential for leukemogenesis. Currently, approximately 50 recurrent fusion genes are linked to leukemia, and new ones are continually being identified. While some fusions are shared across leukemia subtypes - CML, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL) - each disease typically harbors distinct recurrent fusion genes. The most significant leukemogenic fusions are discussed below. The BCR::ABL1 fusion is an oncogene present in over 95% of CML cases [38,39], making it the defining genetic abnormality of the disease, although it also occurs in the B-cell ALL subtype (in approximately 3–6% of pediatric cases and 25–40% of adult cases) [40,41]. This fusion encodes a constitutively active TK that drives cell proliferation, survival, and apoptosis resistance by activating pathways such as RAS, PI3K, and STAT5. Its presence is crucial for diagnosis and serves as a therapeutic target for tyrosine kinase inhibitors (TKIs), such as imatinib [42]. The BCR::ABL1 fusion gene exhibits breakpoint variability, resulting in different isoforms with distinct clinical implications (Fig. 1). The most common BCR::ABL1 fusion isoform, p210, is typically associated with CML. It results from a fusion between BCR exon 13 (e13, also called b2) or exon 14 (e14, also called b3) on chromosome 22 and ABL1 exon 2 (a2) on chromosome 9. These fusions are designated as e13a2 (b2a2) and e14a2 (b3a2), respectively. This isoform generally responds well to first-generation TKIs, such as imatinib. In contrast, the shorter p190 isoform, arising from a breakpoint in BCR exon 1 (e1) fused to ABL1 exon 2 (a2), designated e1a2, is more commonly found in Philadelphia chromosome-positive ALL and is often associated with reduced sensitivity to imatinib, potentially requiring more potent or second-generation TKIs. Less common variants, such as p230 (BCR exon 19 fusion) and p180 (BCR exon 16 fusion), also exist. Identifying the specific BCR::ABL1 variant is critical for tailoring treatment, as the breakpoint location can influence oncogenic potential, therapeutic response, and prognosis [43]. Table 1 List of most common fusion genes implicated in various types of tumors. Fusion gene 1 Rearrangement Type of cancer Standard therapy Ref. BCR::ABL1 t(9;22) 2 (q34;q11) 3 CML 4 , ALL 5 TK inhibitors (imatinib, ponatinib, dasatinib) [22] ETV6::RUNX1 t(12;21)(p13;q22) 6 ALL N/A [23] PML::RARA t(15;17)(q24;q21) AML 7 (M3 8 ) all-trans retinoic acid, arsenic trioxide [24] MLL:: AF4, AF9, ENL, ELL t(4;11), t(9;11), t(11;19) (q23;p13.3), t (11;19) (q23;p13.1) ALL N/A [25] RUNX1::RUNX1T1 t(8;21)(q22;q22) AML (M2 8 ) cytarabine +daunorubicin [26] EWSR1::FLI1 t(11;22)(q24;q12) Ewing’s sarcoma N/A [27] ETV6::NTRK3 t(12;15)(p13;q25) congenital fibrosarcoma, congenital mesoblastic nephroma, secretory breast carcinoma TRK inhibitors (larotrectinib, entrectinib) [28–30] EML4::ALK inv(2) 9 (p21p23) NSCLC 10 ALK inhibitors (crizotinib, ceritinib, alectinib, brigatinib, lorlatinib) [31] KIF5B::RET inv(10)(p11q11) lung adenocarcinoma RET inhibitors (selpercatinib, pralsetinib) [32] BRAF::KIAA1549 dup 11 (7)(q34q34) pilocytic astrocytoma MEK inhibitors (selumetinib) [33] FGFR3::TACC3 dup(4)(p16p16) glioblastoma, bladder cancer FGFR inhibitors (erdafitinib, futibatinib, pemigatinib) [34,35] TMPRSS2::ERG del 12 (21)(q22q22) prostate cancer N/A [36] 1 The HUGO Gene Nomenclature Committee recommends using the double colon (::) to describe gene fusions [37]. This aligns with the The International System for Human Cytogenomics Nomenclature (ISCN), where a single colon (:) signifies a chromosome break, and a double colon (::) indicates a break and reunion 2 t - translocation 3 q - long arm of chromosomes 4 CML - chronic myeloid leukemia 5 ALL - acute lymphoblastic leukemia 6 p - short arm of chromosomes 7 AML - acute myeloid leukemia 8 M0–M7 are subtypes of AML classified according to the French-American-British classification system 9 inv - inversion 10 NSCLC - non-small-cell lung cancer 11 dup - duplication 12 del - deletion. N. Izadi et al. Electrochimica Acta 542 (2025) 147472 2 Acute promyelocytic leukemia (APL), a distinct AML subtype, is defined by the PML::RARA fusion gene resulting from t(15;17) translocation. The PML::RARA chimeric protein disrupts PML’s role in apoptosis and impairs promyelocyte differentiation, driving leukemogenesis [44,45]. Rare alternative RARA fusions, such as GTF2I::RARA and ZBTB16::RARA, can occur in APL and are typically resistant to standard therapies [46,47]. The MLL gene (11q23) is frequently rearranged in both ALL and AML, forming fusion proteins with partners such as AF4, AF9, ENL, and ELL. The MLL gene encodes a key regulator of gene expression in hematopoiesis, and its fusion with other genes disrupts transcriptional control and chromatin modification, thereby driving leukemogenesis. MLL fusion-positive leukemia is typically aggressive, therapy-resistant, and associated with a poor prognosis. While MLL fusions occur in both infants and adults, they are more common in childhood leukemia [25]. The ETV6::RUNX1 fusion gene is one of the most common genetic alterations in pediatric ALL and serves as a key diagnostic marker for this disease. This fusion disrupts normal hematopoietic differentiation and promotes leukemogenesis by altering the gene expression. RUNX1, a crucial transcription factor in blood cell development, loses its function when fused with ETV6, leading to the accumulation of abnormal lymphoid cells. Despite its role in leukemogenesis, ETV6::RUNX1-positive ALL generally responds well to chemotherapy and is associated with a favorable prognosis [23]. 3. Fusion genes in solid tumors For a long time, fusion genes were believed to be found almost exclusively in hematopoietic diseases, such as leukemia. Their presence in solid tumors has been largely overlooked, and only a few researchers have actively searched for them in solid tumors. This changed in 1992 when the EWSR1::FLI1 fusion gene was identified in Ewing sarcoma [48]. Subsequently, fusion genes have been discovered in other sarcomas, as well as in prostate, lung, breast carcinomas, and gliomas [49–52]. Ewing sarcoma is a bone and soft tissue cancer that primarily affects children and adolescents. Resistance to conventional therapies is common; thus, there is an urgent need to develop targeted treatments for this disease [53]. Approximately 85% of Ewing sarcoma cases have the EWSR1::FLI1 fusion. In the remaining cases, the EWSR1 gene is usually found to be fused with other members of the ETS family [48,54]. EWSR1::FLI1 most commonly arises from the t(11;22)(q24;q12) Fig. 1. Simplified scheme showing t(9;22) translocation forming the Philadelphia chromosome. Breakpoints in the BCR gene (e1/exon 1, b2/exon 13, b3/exon 14, e19/exon 19) and ABL1 gene (a2/exon 2) generate different BCR::ABL1 fusion transcripts. Common isoforms include e1a2, b2a2, b3a2, and a less common e19a2 variant, each associated with distinct hematologic malignancies. N. Izadi et al. Electrochimica Acta 542 (2025) 147472 3 translocation, which fuses the strong transcriptional activation domain of the RNA-binding protein EWSR1 with the DNA-binding domain of FLI. This leads to the formation of an abnormal transcription factor involved in tumorigenesis and tumor progression. Prostate cancer is the second most common solid malignancy in men and the fifth most frequent cause of cancer-related deaths worldwide [55]. For a long time, the only diagnostic biomarker employed was prostate-specific antigen (PSA). However, elevated PSA level may not necessarily indicate prostate cancer, as it may also be associated with benign prostatic hyperplasia. In 2005, Tomlins et al. detected recurrent fusions of TMPRSS2 with either ERG or ETV1 (both ETS family transcription factors) in prostate cancer tissue. TMPRSS2 is a transmembrane serine protease regulated by androgens. When fused to ERG or ETV1, its androgen-responsive promoter drives overexpression of these transcription factors [56]. TMPRSS2::ERG is present in more than half of the prostate cancer cases and in some breast cancer cases. Aberrant ERG expression has been demonstrated to lead to increased angiogenesis and, thus, cancer development [57]. Although the prognostic significance of this fusion is controversial, there is a probable association between increased ERG expression and tumor stage [58]. Another fusion gene identified in solid tumors is EML4::ALK, which is present in approximately 5% of NSCLC cases. This fusion gene was first discovered in 2007 and is predominantly found in non-smokers or light smokers [59,60]. Patients with this fusion gene typically respond well to ALK TKI, but resistance inevitably emerges, with certain variants being more strongly associated with treatment failure. Therefore, genotyping these variants is beneficial for selecting the most appropriate drug [61]. Different tumor groups involve the fusion of genes from the NTRK family (NTRK1, NTRK2, or NTRK3). Genes from this group encode neurotrophin receptors (TrkA, TrkB, TrkC, respectively), and their fusions with other genes typically result in the activation of their kinase subunit in the absence of ligand binding [62]. This phenomenon leads to the aberrant activation of signaling pathways that regulate cell growth and survival, as well as those involved in tumor development and progression [63]. A significant number of tumors with NTRK fusion respond well to TKIs, such as larotrectinib or entrectinib [64]. Resistance may arise primarily from mutations in the kinase subunit of NTRK; however, next-generation TRK inhibitors have shown promising efficacy [65]. The best-known of these is the ETV6::NTRK3 fusion found in congenital fibrosarcoma and other neoplasms, including human secretory breast carcinoma and salivary gland secretory carcinoma [29,66,67]. Additionally, other fusions involving genes from the NTRK group have been documented in various lesions, such as papillary thyroid carcinoma and gliomas [68,69]. In gliomas, BRAF (especially the BRAF::KIAA1549 fusion) is frequently identified in fusions. It may also be present in melanoma, lung adenocarcinoma, and other tumors. BRAF encodes a serinethreonine kinase that is part of the mitogen-activated protein kinase (MAPK) – extracellular signal-regulated kinase (ERK) signaling pathway [70,71]. BRAF fusions result in the constant activation of the kinase domain, leading to excessive signaling and cell proliferation [72]. This type of fusion is most commonly observed in pilocytic astrocytoma (a low-grade glioma), where it has a favorable prognostic impact [73]. 4. Standard methods of detection The detection of fusion genes is essential for the molecular stratification of tumors and the classification of their subtypes [74], with significant implications for treatment response, prognosis, and overall survival [75,76]. Advances in chromosomal banding techniques in the 1950s led to the discovery of the first chromosomal and genetic rearrangements [77]. Over the years, other methods for detecting fusion genes have emerged, such as immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). Furthermore, reverse transcription-polymerase chain reaction (RT-qPCR) has become an important tool for detecting fusion transcripts. The introduction of deep sequencing has revolutionized the field, uncovering thousands of fusion gene variants and identifying new potential drug targets that have advanced to clinical trials [74]. The advantages and disadvantages of these methods are presented in Table 2. However, the detection of fusion genes can be challenging, particularly when dealing with rare variants, promiscuous fusion partners, or atypical breakpoints [78–80]. Selecting the correct diagnostic method requires careful consideration of factors such as the type and quality of the tested sample, prior knowledge of fusion partners and their breakpoints, and the potential added benefits of identifying previously unknown fusion partners [81]. Equally important is the consideration of how real-world clinical samples are collected, processed, and prepared, as these pre-analytical steps critically influence the success and reliability of downstream diagnostic methods. In hematological malignancies such as leukemia, peripheral blood or bone marrow aspirates are commonly collected [82], whereas in solid tumors, samples are obtained by biopsy or surgical resection, or alternatively through liquid biopsy of blood, urine, or saliva for circulating tumor DNA (ctDNA) analysis [83, 84]. For molecular methods such as RT-qPCR or RNA sequencing, total RNA is extracted (e.g., using silica column–based kits or TRIzol) and its quality is assessed before reverse transcription into complementary DNA (cDNA), which serves as the template for amplification [82]. In contrast, cytogenetic approaches such as FISH or karyotyping analyze intact cells or nuclei fixed on slides, enabling direct visualization of chromosomal rearrangements without RNA extraction or amplification [85]. The first evidence pointing to the existence of fusion genes dates back to 1960, when Nowell and Hungerford observed ‘minute chromosomes’ in leukocytes of leukemia patients using early karyotyping techniques [77]. Improved resolution has allowed a better understanding of chromosomes and their rearrangements, leading to groundbreaking discoveries in the 1970s and beyond [86]. Karyotyping is a technique for visualizing chromosomes via metaphase spreads but has notable limitations [87]. This approach requires the culturing of living cells, which can be challenging in the case of tumor cells. Additionally, balanced translocations and chromosomal aberrations smaller than 5 Mb may not be detected [88]. FISH is currently considered the gold standard for detecting fusion genes because of its quick turnaround time, high sensitivity, and specificity [89]. This method is based on the hybridization of a DNA probe labeled with fluorophores to a complementary DNA region. Two FISH approaches are commonly used for the detection of fusion genes. With split-signal probes, the suspected gene is labeled with a red fluorophore at the 5’ end and a green fluorophore at the 3’ end (or vice versa). In intact cells, the acquired signals are in close proximity and can thus be observed as yellow, whereas in fusion genes, they are distinct from each other. In contrast, fusion-signal probes are based on fusion partners labeled with green and red. If a fusion gene is present, it appears yellow; otherwise, two different signals are observed [88,89]. Unlike karyotyping, FISH does not require metaphase cells, making this approach more accessible [88]. FISH also does not require prior knowledge of Table 2 Advantages and disadvantages of different approaches in detecting fusion genes. Method Biomolecule target Advantages Disadvantages Karyotyping DNA Cost-effective Low resolution and long turnaround time IHC Protein Cost-effective Limited availability of reliable antibodies FISH DNA Widely available, high sensitivity Costly, low resolution RT-qPCR RNA Rapid, low-cost, high sensitivity Necessary prior knowledge of fusion partners Sequencing DNA or RNA High accuracy, no previous knowledge of fusion partners required Costly, complex data processing N. Izadi et al. Electrochimica Acta 542 (2025) 147472 4 fusion partners but is usually restricted to the detection of the most common gene rearrangements [90]. Although traditional FISH is not suitable for whole-genome screening, its modifications, such as spectral karyotyping (SKY), multicolor FISH, comparative genomic hybridization (CGH), and array-CGH, help to overcome this limitation. However, these modifications are not widely used in clinical practice [88]. IHC is a fast and widely available technique that allows direct visualization of fusion proteins in tumor cells [91]. The most common approach involves antibodies targeting the N-terminus and C-terminus of the wild-type protein, which detects the fusion protein when only partial expression is observed [92]. IHC is cost-effective and requires minimal starting materials [93]. Its limitations include the restricted availability of specific antibodies and sensitivity to proper sample handling, particularly tissue fixation [94]. IHC is used to detect fusion proteins in solid tumors (for example, NTRK fusions) but is not applicable to leukemia [95]. RT-qPCR uses specific primers to detect fusion transcripts, primarily in leukemia samples. Primer design for RT-qPCR requires prior knowledge of both fusion partners and their exon breakpoints, which in turn leads to high sensitivity. However, this can be seen as a double-edged sword, as it limits its application to common variants [96]. While RT-qPCR enables the quantification of fusion transcripts, it should be considered a complementary method, particularly when IHC and FISH results are discordant. Both FISH and RT-qPCR can miss fusion genes with atypical breakpoints [97,98]. Next generation sequencing (NGS) is a high-throughput technique that sequences small DNA or RNA fragments in a massively parallel fashion, allowing for the simultaneous testing of multiple known or unknown genes in a single run. This has led to the identification of approximately 90% of all known fusion gene variants [74]. However, its main limitations include the high cost of the new sequencing system and the required reagents [90]. Additionally, complex data processing, long turnaround times, and the need for sufficient starting material can be challenging in clinical practice [81]. Recently, the costs of commercially available gene panels have significantly reduced, making sequencing more accessible in healthcare settings. Targeted panels typically use amplicon-based or hybrid-capture methods. Hybrid-capture approaches rely on the hybridization of biotin-labeled probes to the gene of interest, whereas amplicon-based methods involve the PCR amplification of a specific region of interest [99,100]. A recent study has shown that panels based on RNA sequencing have higher sensitivity and lead to fewer false negatives than DNA-based panels [101]. NGS can sequence RNA and DNA, each with a specific approach, and can be applied to genomes, exomes, transcriptomes, and more. RNA sequencing detects and quantifies transcriptionally active fusion genes, thus identifying driver mutations leading to cancer progression and excluding passenger fusions [93]. RNA-based probes are easier to design because of fixed fusion breakpoints [102]. RNA sequencing is not affected by intronic regions and can distinguish splicing isoforms [103], although RNA degradation (especially in formalin-fixed tissue samples) may affect the accuracy of the results [104]. However, RNA sequencing is not yet applicable to blood samples [102]. DNA sequencing is more stable than RNA approaches and can precisely identify fusion breakpoints, single nucleotide polymorphisms (SNPs), copy number variants (CNVs), and other genomic alterations [105]. Deep coverage and longer reads are necessary for precise data, as introns with similar repeated sequences can affect detection accuracy [102]. DNA probes target exonic, intronic, and intergenic regions [106, 107], whereas RNA probes specifically target exonic regions [108]. Whole-genome sequencing is the most comprehensive option; however, its high cost and time-consuming nature make its implementation in clinical practice difficult. Whole-exome sequencing significantly reduces costs; however, its potential use is severely limited because the majority of fusions occur in introns [93,109]. 5. Electrochemical methods for fusion gene analysis EC detection offers a cost-effective, rapid, and highly sensitive approach for cancer biomarker detection, with simplified instrumentation, low sample consumption, and potential for point-of-care applications. EC biosensing in cancer research has been a fruitful area of study, targeting both nucleic acids and proteins as tumor biomarkers [12, 110–118]. Regarding nucleic acids, many papers have appeared in recent years describing novel strategies for the analysis of non-coding RNAs [119–122], DNA methylation [123–125], DNA point mutations [15,126,127], oncoviral sequences [116,128,129], and ctDNA [130]. Although less frequent, several EC studies have also reported fusion gene detection, mostly of the BCR::ABL1 fusion gene, but also of other genes (Table 3 and reviewed in [17–20]). Most often, techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS) or amperometry have been used across studies to measure hybridization events or enzymatic reactions, enabling the sensitive and specific detection of various fusion genes. Each of these EC techniques has distinct advantages and limitations in biosensing applications. CV provides rapid insight into redox processes and surface modifications, although its sensitivity is limited for low-abundance biomarkers. DPV offers much higher sensitivity by reducing background currents; however, the measurements take longer and are more susceptible to variability. EIS enables highly sensitive and label-free detection, albeit with longer acquisition times and the need for careful modeling. Amperometry stands out as the most successfully translated EC technique from academia to industry, offering simple, real-time, and highly sensitive detection; however, it is generally restricted to single analyte determination and can be affected by interferents in complex samples. Here, we review recent advances in EC biosensors applied to fusion gene analysis, noting each study’s contributions, strengths, and limitations, particularly focusing on whether real samples were used. When working with real samples, an enzymatic pre-amplification step is usually required, mostly with PCR or its variants. For instance, Chen et al. [131] and Lin et al. [132] used PCR products of the BCR:: ABL1 fusion gene to evaluate their EC DNA biosensors, utilizing locked nucleic acid (LNA) probes for improved selectivity. LNA-modified probes are particularly effective in distinguishing single-base mismatches and are thus of great value in detecting mutations linked to CML. In Chen’s study [131], the biosensor featured an LNA-modified capture probe covalently attached to a glassy carbon electrode modified with 4-aminobenzenesulfonic acid. This setup promoted selective hybridization between the capture probe and target DNA, forming a double-stranded DNA (dsDNA) structure that was detectable by DPV using methylene blue (MB) as an EC indicator. Upon hybridization, the DPV peak current of MB decreased because of the restricted access to guanine bases in dsDNA. This current reduction was directly proportional to the target DNA concentration, allowing for the quantitative detection of the BCR::ABL1 fusion gene. This approach emphasizes a straightforward covalent attachment method, resulting in stable probe immobilization and sensitivity to target DNA concentration. However, the authors used only two real samples (one positive and one negative, without any details) and a single PCR product from the K562 cell line (a positive control for the b3a2 variant), limiting their study by restricting the evaluation of assay performance across a wider range of clinical samples and reducing the ability to demonstrate robustness under diverse conditions. Similarly, Lin’s biosensor [132] used a hairpin-structured LNA probe on a gold electrode, which was modified through sulfur-gold interactions to enhance probe stability. The hairpin configuration of the LNA probe adds an additional level of structural specificity, improving its ability to detect mutations with single-base precision. After hybridization, Lin’s biosensor monitored changes in the EC signals via DPV, with MB serving as the electroactive indicator. The observed decrease in the peak current signaled successful hybridization, and the hairpin structure provided a unique advantage by further N. Izadi et al. Electrochimica Acta 542 (2025) 147472 5 Table 3 Main features of EC biosensors and biossays reported for fusion genes or transcripts detection. Fundamentals Technique Fusion gene/ transcript (Disease) Linear Range/LOD 1 Application Ref. Hybridization at immobilized DNA probe on a GCE 2 DPV 3 (MB 4 )BCR::ABL1 fusion gene (CML) 1.25 ×10 −7 −6.75 ×10 −7 M/ 5.9×10 −8 M –[150] Sandwich hybridization of branched DNAamplified target, CP 5 immobilized at a SPE SWV 6 (AP/1-NP) p185 BCR::ABL1 fusion transcript (ALL) 2.2 ×10 5 −1.6 ×10 8 copies/6.1 ×10 4 copies mRNA from SUP-B15 cells [151] Hybridization on LNA 7 probe immobilized at a 4-ABSA-modified GCE DPV (MB) BCR::ABL1 fusion gene (CML) 1.0 ×10 −12 −1.1 ×10 −11 M/9.4 ×10 −13 M PCR products from a positive real sample and K562 cells [131] Hybridization at immobilized DNA probe on a GCE DPV (STS 8 )BCR::ABL1 fusion gene (CML) 2.0 ×10 −8 −2.0 ×10 −7 M/6.7 × 10 −9 M cDNA from CML patients [152] Hybridization on a thiolated hairpin LNA probe assembled on AuE 9 DPV (MB) BCR::ABL1 fusion gene (CML) –/1.2 ×10 –10 M PCR products from a positive real sample [132] Sandwich hybridization assay using a thiolated LNA probe assembled on an AuE and a biotinylated reported probe Chronoamperometry (HRP 10 /TMB 11 /H 2 O 2 ) BCR::ABL1 fusion gene (CML) –/–PCR products from K562 cells [153] Hybridization at thiolated-hairpin LNA probe assembled on a AuNPs/AuE DPV (CuR 212 )BCR::ABL1 fusion gene (CML) –/1.0 ×10 −10 M PCR products from real samples [154] Hybridization at thiolated DNA probe assembled on a GNPs 13 / MWCNTs 14 / CeO 2 -CS/GCE DPV (MB) BCR::ABL1 fusion gene (CML) 1 ×10 −9 −1 ×10 −12 M/5 ×10 −13 M PCR products from real samples [155] Hybridization at DNA probe immobilized on GNPsPANI 15 -modified AuE EIS 16 ([Fe(CN) 6 ]3 −/4− )BCR::ABL1 fusion gene (ALL) –/6.94 ×10 −17 M cDNA from ALL patients [136] Hybridization at DNA probe immobilized at a CS–CdTe/ITO 17 electrode DPV (MB) BCR::ABL1 fusion gene (CML) 1 ×10 −6 −1 ×10 −11 M/1 × 10 −12 M cDNA from CML patients [156] Hybridization at thiolated DNA probe assembled on a QCdSe-SA-LB 18 /ITO electrode DPV (MB) BCR::ABL1 fusion gene (CML) Up to 1.0 ×10⁻⁵ M/1.0 ×10⁻¹⁴ M cDNA from CML patients [157] Hybridization at thiolated molecular beacon probe assembled on an AuE, CSRP 19 combining cascade hybridization and QDs 20 tagging ASV 21 (Cd) BCR::ABL1 fusion gene (CML) 1.0 ×10⁻¹⁴ −1.0 ×10⁻¹⁰ M/2 × 10⁻¹⁵ M –[158] Hybridization at thiolated DNA probe assembled at a biosynthesized AuNPs/ poly(catechol)/GS/GCE DPV (Catechol) BCR::ABL1 fusion gene (ALL) 1.0 ×10⁻⁴−1.0 ×10⁻¹¹ M/1.0 × 10⁻¹² M PCR products from real samples [159] Hybridization at DNA probe immobilized at a FePt/ERGNO 22 /CPE EIS ([Fe(CN) 6 ] 3−/4− )BCR::ABL1 fusion gene (CML) 1.0 ×10 −14 −1.0 ×10 −9 M/2.6 ×10 −15 M PCR products from real samples [160] Hybridization at immobilized DNA probe on a GCE DPV (ISO 23 )BCR::ABL1 fusion gene (CML) 1.0 ×10⁻¹¹−5.0 ×10⁻⁷ M/3.0 × 10⁻¹² M –[161] Hybridization with thiolated and biotinylated hairpin probe at a AuNPs/ PANI/CS-GS/GCE DPV (AP+1-NP) BCR::ABL1 fusion gene (CML) 1.0 ×10⁻¹¹−1.0 ×10⁻⁹ M/2.11 ×10⁻¹² M PCR products from a positive real sample and K562 cells [162] Hybridization at biotinylated DNA probe immobilized on PANI-MoS 2 /ITO bioelectrode EIS ([Fe(CN) 6 ] 3−/4− )BCR::ABL1 fusion gene (CML) 1.0 ×10 −6 −1.0 ×10 −17 M/3 × 10 −18 M cDNA from CML patients [163] Dual detection mode, hybridization at DNA probe immobilized on Hb@AuNCs 24 -GS/ AuNPs modified GCE “Signal on”: EIS ([Fe (CN) 6 ] 3−/4− ) Signal off: DPV (MB) BCR::ABL1 fusion gene (CML) “Signal on”: 1.0 ×10⁻¹⁶−1.0 × 10⁻¹¹ M/3.7 ×10⁻¹⁷ M, “Signal off”: 1.0 ×10⁻¹⁶−1.0 ×10⁻¹¹ M/3.0 × 10⁻¹⁷ M cDNA from CML patients [164] Sandwich hybridization at thiolated capture probe assembled on AuNPs/ Ti 3 C 2 T x MXene/GCE, biotinylated reported probe and DNA walking machine (DNA-Au@Fe 3 O 4 ) DPV (AP+1-NP) BCR::ABL1 fusion gene (CML) 2.0 ×10⁻¹⁶−2.0 ×10⁻⁸ M/5.0 × 10⁻¹⁷ M Supplemented human serum samples [165] Split-type EC biosensor using enzymelinked DNA magnetic beads and duplex LCR 25 coupled with OR logic gate design Amperometry (HRP/ TMB/H 2 O 2 )BCR::ABL p210 transcript (CML) 1.0 ×10 −18 −5.0 ×10 −14 M and 1.0 ×10 −15 −1.0 ×10 −12 M/1.0 ×10⁻¹⁸ M cDNA from CML patients [133] ZnO/PICA 26 nanocomposite for DNA immobilization EIS PICA/ZnO BCR::ABL1 fusion gene (CML) 1.0 ×10⁻¹⁵−1.0 ×10⁻⁹ M/2.2 × 10⁻¹⁶ M synthetic oligonucleotide sequences that mimic the BCR:: ABL1 fusion gene [139] Fe₃O₄ nanoparticles functionalized CNTs 27 EIS using [Fe(CN)₆]³⁻/⁴⁻ as redox probe BCR::ABL1 fusion gene (CML) 1.0 ×10⁻¹³−1.0 ×10⁻⁹ M/ 3.3 ×10⁻¹⁴ M synthetic oligonucleotide sequences that mimic the BCR:: ABL1 fusion gene [138] PICA-functionalized ZnO nanostructure EIS BCR::ABL1 fusion gene (CML) Not specified/1.0 ×10⁻¹⁶ M synthetic oligonucleotide sequences that mimic the BCR:: ABL1 fusion gene [166] Hybridization at immobilized DNA probe on a GCE DPV (AE) PML::RARA fusion gene (APL) 1.5 ×10 −8 −1.5 ×10 −7 M/6.7 × 10 −8 M –[167] Hybridization at immobilized DNA probe on a poly-CCA 28 film modified GCE DPV (MB) PML::RARA fusion gene (APL) 1.0 ×10 −12 −1.0 ×10 −11 M/6.7 ×10 −13 M –[168] (continued on next page) N. Izadi et al. Electrochimica Acta 542 (2025) 147472 6 Table 3 (continued) Fundamentals Technique Fusion gene/ transcript (Disease) Linear Range/LOD 1 Application Ref. Hybridization on hairpin LNA probe dually labeled with biotin and FAM immobilized at a streptavidinpoly-CCA-GCE Chronoamperometry (HRP/TMB/H 2 O 2 ) PML::RARA fusion gene (APL) In 20% serum: 1.0 ×10⁻¹²−1.0 ×10⁻⁷ M/8.3 ×10⁻¹⁴ M PCR products from NB4 cells and a positive real sample [144] Hybridization at immobilized DNA probe on an aldehyde-agarose hydrogelmodified GCE DPV (MB) PML::RARA fusion gene (APL) 4.0 ×10 −12 −1.2 ×10 −11 M/4.0 ×10 −12 M –[169] Hybridization at thiolated DNA probe assembled on a NPG electrode DPV (MB) PML::RARA fusion gene (APL) 6.0 ×10⁻¹¹−2.2 ×10⁻¹⁰ M /6.7 ×10⁻¹² M PCR products from real samples [143] Hybridization at DNA probe immobilized on ECR 29 monolayer film modified GCE DPV (MB) PML::RARA fusion gene (APL) 5.0 ×10⁻¹²–2.0 ×10⁻¹⁰ M/9.82 ×10⁻¹³ M –[141] Sandwich hybridization with thiolated and biotinylated LNA capture and reporter probes at AuE Chronoamperometry (HRP/TMB/H 2 O 2 ) PML::RARA fusion gene (APL) 5.0 ×10⁻¹¹–1.0 ×10⁻⁸ M (synthetic target)/ and 7.9 × 10⁻¹⁴ M/100μ L hybridization system (PCR amplicons) PCR products from NB4 cells [170] Hybridization at a dual-channel EC DNA sensor array based on the “Y” junction structure and restriction endonuclease assisted cyclic enzymatic amplification at AuEs Chronoamperometry (HRP/TMB/H 2 O 2 ) PML::RARA fusion gene (APL) –/4.7 ×10⁻¹⁴ M PCR products from NB4 cells, synthetic PML::RARA fusion gene [142] Dual probe sensing strategy involving two groups of 2-F RNA-modified probes and sandwich hybridization assays using thiolated capture probes and biotinylated reporter probes at AuEs Chronoamperometry (HRP/TMB/H 2 O 2 ) PML::RARA fusion gene (APL) 5.0 ×10⁻¹³−1.5 ×10⁻¹¹ M/8.4 × 10⁻¹⁴ M –[171] Hybridization at immobilized DNA probe on a CDs 30 /GO 31 /GCE DPV (MB) PML::RARA fusion gene (APL) 2.50 ×10 −10 −2.25×10 −9 M/ 8.30×10 −11 M –[172] Double-probe sandwich hybridization (thiolated and biotinylated capture and reported probes) and enzyme-mediated multiple signal electrocatalysis on d-AuE Chronoamperometry (HRP/TMB/H 2 O 2 ) PML::RARA fusion gene (APL) 5.0 ×10⁻¹³−5.0 ×10⁻¹¹ M/7.1 × 10⁻¹⁴ M PCR products and enzymedigested PCR products from NB4 cells [173] Split-type EC sensor developed by integrating NASBA 32 , sandwich hybridization and enzyme-linked magnetic microbeads Amperometry (HRP/ TMB/H 2 O 2 )PML::RARA transcript (APL) 5.0 ×10⁻¹⁶−5.0 ×10⁻¹⁴ M and 5.0 ×10⁻¹⁴−1.0 ×10⁻¹¹ M/1.0 × 10⁻¹⁶ M Suplemented human serum samples and tRNA extracts from NB4 and K562 cells [174] Sandwich-mode EC DNA biosensor with LNA probes and biotinylated reporter probe Amperometry (HRP/ TMB/H 2 O 2 ) PML::RARA fusion gene (Acute Promyelocytic Leukemia) 1.0 ×10⁻¹³−1.0 ×10⁻¹¹ M/7.4 × 10⁻¹⁴ M synthetic PML::RARA fusion gene [140] EC biosensor based on ECR film DPV PML::RARA fusion gene (APL) 5.0 ×10⁻¹²−2.0 ×10⁻¹⁰ M/9.82 ×10⁻¹³ M synthetic PML::RARA fusion gene [141] DNA CP immobilized on GCE via CDs/ PAMAM 33 /rGO 34 nanocomposites and an assistant probe labeled with Au@Ag₂S nanoparticles ECL-RET PML::RARA fusion gene (APL) 5 ×10 −15 −5 ×10 −10 M/7.2 × 10 −16 M synthetic PML::RARA fusion gene [175] genosensor constructed from PPy 35 and graphene QD on ITO substrates CV, EIS PML::RARA fusion gene (APL) 1.0 ×10⁻¹² −1.0 ×10⁻¹⁰ M/2.14 ×10⁻¹³ M (M7) and 6.77 ×10⁻¹³ M (APLB) cDNA from APL patients, synthetic oligonucleotides for M7 and APLB (regions on chromosomes 15 and 17) [176] Hybridization at biotinylated DNA capture probe attached to streptavidin MBs and adsorption of target transcripts onto Au-SPEs DPV ([Fe(CN) 6 ] 3−/ 4 − )TMPRSS2::ERG fusion mRNA (PCa) –/10 cells Urinary samples from PCa patients [146] Integrated biochip comprised of on-chip electrical cell lysis, nanofluidic manipulation, isothermal solid-phase amplification of targets, and EC detection of immobilized amplicons via nanozymemediated redox reaction Chronoamperometry (Nanozyme/TMB/H 2 O 2 ) TMPRSS2::ERG gene fusion (PCa), PCA3, SChLAP1, and KLK2 –/–Urinary samples from PCa patients [149] 1 LOD – limit of detection 2 GCE – glassy carbon electrode 3 DPV – differential pulse voltammetry 4 MB – methylene blue 5 CP – capture probe 6 SWV – square wave voltammetry 7 LNA – locked nucleic acid 8 STS – sodium tanshinone IIA sulfonate 9 AuE – gold electrode 10 HRP – horseradish peroxidase 11 TMB – tetramethylbenzidine N. Izadi et al. Electrochimica Acta 542 (2025) 147472 7 minimizing the nonspecific interactions and enhancing the specificity. Lin’s use of a hairpin LNA probe provided a distinct structural feature that potentially enhances specificity and reduces background noise compared to the linear probe used by Chen et al. Again, the bioassay was tested on only one positive and one negative real PCR sample without specifying any details, limiting the evaluation of its clinical applicability and practical performance in biological settings. Recently, a split-type EC biosensor coupled with an OR logic gate design was developed for the sensitive detection of ultra-low levels of the BCR::ABL1 p210 transcript [133]. The biosensor utilizes enzyme-linked DNA magnetic beads based on a duplex ligase chain reaction (LCR), a PCR variant that detects specific mutations or genetic variations using two oligonucleotides that hybridize to adjacent regions on a target single-stranded DNA (Fig. 2a). When these oligonucleotides perfectly match the target, a thermostable ligase enzyme joins them, and the resulting ligated products serve as templates for subsequent amplification cycles, enabling highly specific detection. LCR can be multiplexed to detect multiple mutations simultaneously. Biotinand 6-FAM–labeled LCR products were captured on streptavidin-coated magnetic beads. Horseradish peroxidase (HRP)-conjugated anti-FAM antibodies subsequently bound to the DNA–magnetic bead complexes, which were magnetically assembled on a glassy carbon electrode. HRP then catalyzed the oxidation of tetramethylbenzidine (TMB) by H₂O₂, and the EC reduction of oxidized TMB at ~0.2 V was monitored by voltammetry and amperometry. This dual-amplification strategy enabled highly sensitive detection down to 1 aM with single-base 12 CuR 2 – benzoate binuclear copper (II) complex 13 GNP – gold nanoparticles 14 MWCNT – multi-walled carbon nanotubes 15 PANI – polyaniline 16 EIS – electrochemical impedance spectroscopy 17 ITO – indium tin oxide 18 QCdSe-SA-LB – Langmuir−Blodgett monolayers of tri-n-octylphosphine oxide-capped cadmium selenide quantum dots 19 CSRP – circular strand replacement polymerization 20 QD – quantum dots 21 ASV – anodic stripping voltammetry 22 ERGNO – electrochemically reduced graphene oxide 23 ISO – isorhamnetin 24 Hb@AuNCs – Hb and gold nanoclusters composite 25 LCR – ligation chain reaction 26 PICA – poly(indole-5-carboxylic acid) 27 CNT – carbon nanotubes 28 polyCCA – poly-calcon carboxylic acid 29 ECR – eriochrome cyanine R 30 CD – carbon dots 31 GO – graphene oxide 32 NASBA – nucleic acid sequence-based amplification 33 PAMAM – poly(amidoamine) 34 rGO – reduced graphene oxide 35 PPy – polypyrrole Fig. 2. A split-type EC biosensor coupled with a) LCR and b) OR logic gate design to detect BCR::ABL1 p210 transcript. c) Amperometric responses obtained in the analysis of cDNA samples from newly diagnosed CML and ALL patients and from CML patients who had either received or were currently undergoing TKIs treatment. Reproduced with permission from Ref. [133]. N. Izadi et al. Electrochimica Acta 542 (2025) 147472 8 specificity. Additionally, poly(A) tails or fluorescent labels can be added to oligonucleotides for product differentiation based on size or for detection using sequencing-based systems, making LCR a robust tool for analysing mutations [134,135]. Their system could detect as few as 30 copies of the DNA target across a wide linear concentration range, offering high specificity at single-base resolution. Notably, the biosensor successfully detected K562 cells, which express the BCR::ABL1 p210 isoform (b3a2), even when mixed with a 1,000-fold excess of NB4 cells, which are negative for BCR::ABL1 p210 but positive for the PML::RARA fusion gene. This highlights the high specificity and practicality of the biosensor for use in complex sample mixtures. Moreover, the biosensor successfully detected the BCR::ABL1 p210 transcript in both newly diagnosed CML patients (e14a2 and e13a2) and those who had completed TKI treatment, emphasizing its potential for the early diagnosis and monitoring of CML (Fig. 2b and c). This assay has several advantages, including low cost, ease of miniaturization, and broad applicability, making it a promising tool for clinical applications. Similar to the previously mentioned study, a significant drawback of this study is the lack of clinical samples from healthy donors. Additionally, although the authors claimed that their method could detect two different isoforms, they did not specify which clinical samples corresponded to the e14a2 and e13a2 isoforms. Moreover, important cell lines such as KCL-22, which is representative of the b2a2 isoform, are absent, limiting the scope of their findings. Nevertheless, this study represents an important step towards the clinical analysis of fusion genes using EC techniques. To further improve sensitivity, various nanomaterials are often employed, such as two biosensors developed by Avelino et al. for BCR:: ABL1 detection. In the first study [136], the authors used an AuNP-polyaniline (PANI) hybrid composite in combination with thiol-modified probes on a gold electrode, with hybridization detected via DPV. The AuNPs provided a high surface area for DNA probe immobilization, while the conductive PANI matrix enhanced electron transfer at the electrode. Upon target hybridization, the negatively charged DNA backbone increased the interfacial resistance, leading to a measurable decrease in the peak current during DPV. This label-free transduction mechanism allows sensitive detection of DNA hybridization events without the need for additional enzymes or redox labels. The biosensor achieved an ultra-low limit of detection of 69.4 aM and demonstrated high specificity and sensitivity when tested with recombinant plasmid and 8 leukemia patient cDNA samples. The genosensor exhibited strong specificity, although highly concentrated negative samples produced slight non-specific responses. Patient samples validated the effectiveness of the biosensor, with clear differentiation between CML-positive and CML-negative cases. This biosensor demonstrated rapid label-free detection within 15 min, making it a promising alternative to PCR for leukemia diagnosis and minimal residual disease monitoring. Despite its strong performance, the limited real-sample testing suggests a need for broader clinical validation. Later, the same group developed an EC DNA biosensor based on a hybrid nanostructure composed of chitosan and zinc oxide nanoparticles immobilized on a polypyrrole film, with oligonucleotide probes specific to the BCR::ABL1 sequence. Using EIS and CV, Avelino’s team achieved an impressive limit of detection (LOD) of 1.34 fM. In this design, the conductive polypyrrole film served as the backbone for efficient electron transport, while chitosan provided abundant functional groups for covalent immobilization of DNA probes. The incorporation of ZnO nanoparticles further enhanced the probe density and electrode surface area. Hybridization with the complementary target DNA introduced negatively charged phosphate groups, which hinder electron transfer between the electrode and redox probe. Validation was conducted using recombinant plasmid samples and clinical cDNA samples, which were analyzed within 15 min using only 2 µL of the sample, which is a significant improvement over traditional methods. Although the biosensor demonstrated high sensitivity and specificity in detecting the BCR::ABL1 fusion gene in patients with CML, the limited inclusion of healthy control samples might have affected the comprehensive evaluation. A larger pool of healthy samples would provide more robust data on falsepositive rates and further validate the biosensor performance in distinguishing between healthy individuals and CML patients [137]. Other nanomaterials that have been used for BCR::ABL1 analysis include Fe₃O₄-functionalized carbon nanotubes [138] and poly (indole-5-carboxylic acid)-functionalized ZnO nanocomposites [139]; however, both were tested only on synthetic oligonucleotides, again questioning their potential for clinical application. The BCR::ABL1 fusion gene is the most common type of fusion gene targeted in EC studies; however, other fusion genes have also been reported. For example, Wang et al. published several studies on EC biosensors targeting the PML::RARA fusion gene. Each study explored different electrode modifications and amplification methods to enhance biosensor sensitivity and specificity for detecting target DNA sequences. In their 2011 study, they developed a sandwich-mode amperometric biosensor using LNAs on a gold electrode [140]. This sensor employs a pair of LNA probes, one immobilized on the electrode as a capture probe and the other as a biotinylated reporter probe. Streptavidin-HRP linked to the biotin tag on the reporter probe enabled signal amplification, resulting in an enzymatically amplified EC signal. HRP catalyzed the oxidation of TMB by H₂O₂, and the reduction of oxidized TMB was measured amperometrically using a gold electrode. This approach allowed the sensor to achieve a limit of detection of 74 fM with a linear range of 0.1–10 pM, showing high specificity by effectively discriminating against mismatch sequences. Subsequently, they reported an EC DNA biosensor for detecting PML::RARA based on a glassy carbon electrode modified with an eriochrome cyanine R (ECR) film [141]. The ECR film provides a stable platform for probe immobilization through covalent binding. DPV was used to measure the hybridization of the DNA probe with the target sequences using MB as an electroactive indicator. This biosensor exhibited a linear detection range of 5–200 pM, with a limit of detection of 0.982 pM. Compared to the LNA-based approach, the ECR film-modified sensor offers good selectivity, particularly for one-base mismatches, without requiring enzymatic signal amplification. In their next study, they introduced a dual-probe EC DNA biosensor for PML::RARA detection, leveraging a “Y” junction structure and restriction endonuclease-assisted cyclic enzymatic amplification (Fig. 3) [142]. In this setup, two groups of probes were designed to bind to each strand of the dsDNA target. This design provided a limit of detection of 47 fM and exhibited excellent specificity, even for distinguishing single-base mismatches in dsDNA sequences, and was applied to the detection of PCR products from NB4 cells (a positive control for APL). Each method demonstrated strong specificity, with the dual-probe system (2015) achieving the highest discrimination of single-base mismatches. While previous studies on PML::RARA did not utilize any real samples, Zhong et al. developed an EC DNA biosensor based on a nanoporous gold (NPG) electrode for the detection of the PML::RARA fusion gene using PCR products from real samples [143]. The biosensor used MB as an electroactive indicator and DPV to monitor DNA hybridization. Upon hybridization of the probe DNA with the PML::RARA target sequence, the MB reduction signal decreased because of steric hindrance, enabling sensitive and selective detection. The biosensor demonstrated a linear detection range of 60–220 pM and an ultra-low limit of detection of 6.7 pM, highlighting its potential for detecting minimal residual disease in patients with APL. Different biosensor for PML::RARA fusion gene analysis involves an enzyme-amplified EC biosensor using a hairpin LNA probe, which is dual-labeled with biotin and carboxyfluorescein (FAM) and immobilized on a streptavidin-coated electrode [144]. Upon hybridization with the target DNA, a conformational change exposes the FAM label, allowing the binding of antiFAM-HRP for enzyme-based signal amplification. The bound HRP catalyzed the oxidation of TMB by H₂O₂, and the EC reduction of oxidized TMB at the electrode surface was recorded. Both N. Izadi et al. Electrochimica Acta 542 (2025) 147472 9 [116] G. Ozcelikay, S.I. Kaya, A. Cetinkaya, S.A. Ozkan, Recent advances in electrochemical biosensors for detection of oncoviruses, Biosens. Bioelectron.: X 12 (2022) 100260, https://doi.org/10.1016/j.biosx.2022.100260. [117] C. Erkmen, V. Sanko, B.O. Ozturk, J. Quinchía, J. Orozco, F. Kuralay, Mesoporous silica-based electrochemical biosensors in the determination of cancer biomarkers: current progress on analytical performance and future trends, TrAC Trends Anal. Chem. 179 (2024) 117876, https://doi.org/10.1016/j. trac.2024.117876. [118] A.F. Cruz-Pacheco, D. Echeverri, J. Orozco, Role of electrochemical nanobiosensors in colorectal cancer precision medicine, TrAC Trends Anal. Chem. 170 (2024) 117467, https://doi.org/10.1016/j.trac.2023.117467. [119] M.N. Islam, S. Moriam, M. Umer, H.P. Phan, C. Salomon, R. Kline, N.T. Nguyen, M.J.A. Shiddiky, Naked-eye and electrochemical detection of isothermally amplified HOTAIR long non-coding RNA, Analyst 143 (13) (2018) 3021–3028, https://doi.org/10.1039/c7an02109g. [120] X. Li, G. Peng, F. Cui, Q. Qiu, X. Chen, H. Huang, Double determination of long noncoding RNAs from lung cancer via multi-amplified electrochemical genosensor at sub-femtomole level, Biosens. Bioelectron. 113 (2018) 116–123, https://doi.org/10.1016/j.bios.2018.04.062. [121] L. Mor´ aˇ nov´ a, M. Bartoˇ sík, Long Non-Coding RNAs - current methods of detection and clinical applications, Klin. Onkol. 32 (Supplementum 3) (2019) 65–71, https://doi.org/10.14735/amko20193S65. [122] L. Moranova, M. Stanik, R. Hrstka, S. Campuzano, M. Bartosik, Electrochemical LAMP-based assay for detection of RNA biomarkers in prostate cancer, Talanta 238 (Pt 2) (2022) 123064, https://doi.org/10.1016/j.talanta.2021.123064. [123] Z. Dudov´ a, M. Bartoˇ sík, M. Fojta, Magnetic bead-based electrochemical assay for determination of DNA methyltransferase activity, Electrochim. Acta 231 (2017) 575–581, https://doi.org/10.1016/j.electacta.2017.02.104. [124] T. Hossain, G. Mahmudunnabi, M.K. Masud, M.N. Islam, L. Ooi, K. Konstantinov, M.S.A. Hossain, B. Martinac, G. Alici, N.-T. Nguyen, M.J.A. Shiddiky, Electrochemical biosensing strategies for DNA methylation analysis, Biosens. Bioelectron. 94 (2017) 63–73, https://doi.org/10.1016/j.bios.2017.02.026. [125] J. Huang, S. Zhang, F. Mo, S. Su, X. Chen, Y. Li, L. Fang, H. Huang, J. Deng, H. Liu, X. Yang, J. Zheng, An electrochemical DNA biosensor analytic technique for identifying DNA methylation specific sites and quantify DNA methylation level, Biosens. Bioelectron. 127 (2019) 155–160, https://doi.org/10.1016/j. bios.2018.12.022. [126] H. Chen, X.-J. Liu, Y.-L. Liu, J.-H. Jiang, G.-L. Shen, R.-Q. Yu, Electrochemical scanning of DNA point mutations via MutS protein-mediated mismatch recognition, Biosens. Bioelectron. 24 (7) (2009) 1955–1961, https://doi.org/ 10.1016/j.bios.2008.09.029. [127] L. Moranova, J. Strmiskova, K. Ondraskova, M. Bardelcik, M. Cwik, I. Kiss, R. Hrstka, M. Bartosik, Electrochemical detection of BRAF V600E mutation in a liquid biopsy format: LAMPing the way to an improved diagnostics and treatment, Adv. Mater. Technol. 10 (7) (2024) 2401404, https://doi.org/10.1002/ admt.202401404. [128] N. Izadi, R. Sebuyoya, L. Moranova, R. Hrstka, M. Anton, M. Bartosik, Electrochemical bioassay coupled to LAMP reaction for determination of high-risk HPV infection in crude lysates, Anal. Chim. Acta 1187 (2021) 339145, https:// doi.org/10.1016/j.aca.2021.339145. [129] N. Izadi, J. Strmiskova, M. Anton, J. Hausnerova, M. Bartosik, LAMP-based electrochemical platform for monitoring HPV genome integration at the mRNA level associated with higher risk of cervical cancer progression, J. Med. Virol. 96 (10) (2024) e70008, https://doi.org/10.1002/jmv.70008. [130] A.-M. Chiorcea-Paquim, Advances in electrochemical biosensor technologies for the detection of nucleic acid breast cancer biomarkers, Sensors 23 (8) (2023) 4128, https://doi.org/10.3390/s23084128. [131] J. Chen, J. Zhang, K. Wang, X. Lin, L. Huang, G. Chen, Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-aminobenzenesulfonic acid-modified glassy carbon electrode, Anal. Chem. 80 (21) (2008) 8028–8034, https://doi.org/10.1021/ac801040e. [132] L. Lin, X. Lin, J. Chen, W. Chen, M. He, Y. Chen, Electrochemical biosensor for detection of BCR/ABL fusion gene based on hairpin locked nucleic acids probe, Electrochem. Commun. 11 (8) (2009) 1650–1653, https://doi.org/10.1016/j. elecom.2009.06.015. [133] L.-Y. Yang, J.-L. Huang, Y. Lin, Q.-Q. Cai, Y.-J. Zheng, Y. Wu, J.-Y. Chen, X.- H. Lin, A split-type electrochemical biosensor using enzyme-linked DNA magnetic beads realizes the detection of BCR/ABLp210 fusion gene in clinical samples: duplex ligation chain reaction coupled with OR logic gate design, Chem. Eng. J. 479 (2024) 147683, https://doi.org/10.1016/j.cej.2023.147683. [134] S. Dunbar, S. Das, Amplification chemistries in clinical virology, J. Clin. Virol. 115 (2019) 18–31, https://doi.org/10.1016/j.jcv.2019.03.015. [135] C.E. Keegan, A.A. Killeen, An overview of molecular diagnosis of steroid 21-hydroxylase deficiency, J. Mol. Diagn. 3 (2) (2001) 49–54, https://doi.org/ 10.1016/S1525-1578(10)60651-4. [136] K.Y.P.S. Avelino, I.A.M. Frias, N. Lucena-Silva, R.G. Gomes, C.P. de Melo, M.D. L. Oliveira, C.A.S. Andrade, Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite, Colloids. Surf. B Biointerfaces 148 (2016) 576–584, https://doi.org/10.1016/j.colsurfb.2016.09.029. [137] K.Y.P.S. Avelino, L.S. Oliveira, M.R. Santos, N. Lucena-Silva, C.A.S. Andrade, M. D.L. Oliveira, Electrochemical DNA biosensor for chronic myelocytic leukemia based on hybrid nanostructure, Bioelectrochemistry 147 (2022) 108176, https:// doi.org/10.1016/j.bioelechem.2022.108176. [138] W. Zhang, Application of Fe3O4 nanoparticles functionalized carbon nanotubes for electrochemical sensing of DNA hybridization, J. Appl. Electrochem. 46 (5) (2016) 559–566, https://doi.org/10.1007/s10800-016-0952-2. [139] W. Zhang, Poly(indole-5-carboxylic acid)-functionalized ZnO nanocomposite for electrochemical DNA hybridization detection, J. Solid. State Electrochem. 20 (2) (2016) 499–506, https://doi.org/10.1007/s10008-015-3071-9. [140] K. Wang, Z. Sun, M. Feng, A. Liu, S. Yang, Y. Chen, X. Lin, Design of a sandwichmode amperometric biosensor for detection of PML/rar α fusion gene using locked nucleic acids on gold electrode, Biosens. Bioelectron. 26 (6) (2011) 2870–2876, https://doi.org/10.1016/j.bios.2010.11.030. [141] L. Wang, L. Lin, X. Xu, S. Weng, Y. Lei, A. Liu, Y. Chen, X. Lin, Electrochemical biosensor for detection of PML/rar α fusion gene based on eriochrome cyanine R film modified glassy carbon electrode, Electrochim. Acta 69 (2012) 56–59, https://doi.org/10.1016/j.electacta.2012.02.065. [142] K. Wang, Y. Lei, G.-X. Zhong, Y.-J. Zheng, Z.-L. Sun, H.-P. Peng, W. Chen, A.- L. Liu, Y.-Z. Chen, X.-H. Lin, Dual-probe electrochemical DNA biosensor based on the “Y” junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/rar α related fusion gene, Biosens. Bioelectron. 71 (2015) 463–469, https://doi.org/10.1016/j. bios.2015.04.071. [143] G. Zhong, A. Liu, X. Chen, K. Wang, Z. Lian, Q. Liu, Y. Chen, M. Du, X. Lin, Electrochemical biosensor based on nanoporous gold electrode for detection of PML/rar α fusion gene, Biosens. Bioelectron. 26 (9) (2011) 3812–3817, https:// doi.org/10.1016/j.bios.2011.02.039. [144] L. Lin, Q. Liu, L. Wang, A. Liu, S. Weng, Y. Lei, W. Chen, X. Lin, Y. Chen, Enzymeamplified electrochemical biosensor for detection of PML–RAR α fusion gene based on hairpin LNA probe, Biosens. Bioelectron. 28 (1) (2011) 277–283, https://doi.org/10.1016/j.bios.2011.07.032. [145] A. Marusyk, K. Polyak, Tumor heterogeneity: causes and consequences, Biochim. Biophys. Acta 1805 (1) (2010) 105–117, https://doi.org/10.1016/j. bbcan.2009.11.002. [146] K.M. Koo, L.G. Carrascosa, M.J.A. Shiddiky, M. Trau, Amplification-free detection of gene fusions in prostate cancer urinary samples using mRNA–Gold affinity interactions, Anal. Chem. 88 (13) (2016) 6781–6788, https://doi.org/10.1021/ acs.analchem.6b01182. [147] Z. Fang, L. Soleymani, G. Pampalakis, M. Yoshimoto, J.A. Squire, E.H. Sargent, S. O. Kelley, Direct profiling of cancer biomarkers in tumor tissue using a multiplexed nanostructured microelectrode integrated circuit, ACS. Nano 3 (10) (2009) 3207–3213, https://doi.org/10.1021/nn900733d. [148] K.M. Koo, E.J.H. Wee, M. Trau, Colorimetric TMPRSS2-ERG gene fusion detection in prostate cancer urinary samples via recombinase polymerase amplification, Theranostics 6 (9) (2016) 1415–1424, https://doi.org/10.7150/thno.15250. [149] K.M. Koo, S. Dey, M. Trau, A sample-to-targeted gene analysis biochip for nanofluidic manipulation of solid-phase circulating tumor nucleic acid amplification in liquid biopsies, ACS. Sens. 3 (12) (2018) 2597–2603, https://doi. org/10.1021/acssensors.8b01011. [150] X.-H. Lin, P. Wu, W. Chen, Y.-F. Zhang, X.-H. Xia, Electrochemical DNA biosensor for the detection of short DNA species of Chronic myelogenous leukemia by using methylene blue, Talanta 72 (2) (2007) 468–471, https://doi.org/10.1016/j. talanta.2006.11.015. [151] A.-C. Lee, Z. Dai, B. Chen, H. Wu, J. Wang, A. Zhang, L. Zhang, T.-M. Lim, Y. Lin, Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA, Anal. Chem. 80 (24) (2008) 9402–9410, https://doi.org/10.1021/ac801263r. [152] J. Chen, J. Zhang, Q. Zhuang, J. Chen, X. Lin, Hybridization biosensor using sodium tanshinone IIA sulfonate as electrochemical indicator for detection of short DNA species of chronic myelogenous leukemia, Electrochim. Acta 53 (6) (2008) 2716–2723, https://doi.org/10.1016/j.electacta.2007.08.059. [153] K. Wang, J. Chen, J. Chen, A. Liu, G. Li, H. Luo, X. Lin, Y. Chen, A sandwich-type electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on gold electrode, Electroanalysis. 21 (10) (2009) 1159–1166, https://doi.org/10.1002/elan.200804507. [154] L. Lin, J. Kang, S. Weng, J. Chen, A. Liu, X. Lin, Y. Chen, Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator, Sens Actuators B 155 (1) (2011) 1–7, https://doi.org/10.1016/j.snb.2010.05.069. [155] S. Li, L. Wang, Y. Li, X. Zhu, L. Zhong, L. Lu, W. Zhang, B. Liu, G. Xie, W. Feng, Electrochemical determination of BCR/ABL fusion gene based on in situ synthesized gold nanoparticles and cerium dioxide nanoparticles, Colloids. Surf. B Biointerfaces. 112 (2013) 344–349, https://doi.org/10.1016/j. colsurfb.2013.07.027. [156] A. Sharma, C.M. Pandey, G. Sumana, U. Soni, S. Sapra, A.K. Srivastava, T. Chatterjee, B.D. Malhotra, Chitosan encapsulated quantum dots platform for leukemia detection, Biosens. Bioelectron. 38 (1) (2012) 107–113, https://doi. org/10.1016/j.bios.2012.05.010. [157] A. Sharma, C.M. Pandey, Z. Matharu, U. Soni, S. Sapra, G. Sumana, M.K. Pandey, T. Chatterjee, B.D. Malhotra, Nanopatterned Cadmium Selenide Langmuir–Blodgett platform for leukemia detection, Anal. Chem. 84 (7) (2012) 3082–3089, https://doi.org/10.1021/ac202265a. [158] L. Hu, T. Tan, G. Chen, K. Zhang, J.-J. Zhu, Ultrasensitive electrochemical detection of BCR/ABL fusion gene fragment based on polymerase assisted multiplication coupling with quantum dot tagging, Electrochem. Commun. 35 (2013) 104–107, https://doi.org/10.1016/j.elecom.2013.08.004. [159] M. Mazloum-Ardakani, B. Barazesh, A. Khoshroo, M. Moshtaghiun, M. H. Sheikhha, A new composite consisting of electrosynthesized conducting polymers, graphene sheets and biosynthesized gold nanoparticles for biosensing N. Izadi et al. Electrochimica Acta 542 (2025) 147472 16 acute lymphoblastic leukemia, Bioelectrochemistry. 121 (2018) 38–45, https:// doi.org/10.1016/j.bioelechem.2017.12.010. [160] J. Yang, W. Zhang, Indicator-free impedimetric detection of BCR/ABL fusion gene based on ordered FePt nanoparticle-decorated electrochemically reduced graphene oxide, J. Solid. State Electrochem. 18 (10) (2014) 2863–2868, https:// doi.org/10.1007/s10008-014-2551-7. [161] G.-X. Zhong, J.-X. Ye, F.-Q. Bai, F.-H. Fu, W. Chen, A.-L. Liu, X.-H. Lin, Y.-Z. Chen, Electrochemical biosensor for detection of BCR/ABL fusion gene based on isorhamnetin as hybridization indicator, Sens Actuators B 204 (2014) 326–332, https://doi.org/10.1016/j.snb.2014.07.115. [162] L. Wang, E. Hua, M. Liang, C. Ma, Z. Liu, S. Sheng, M. Liu, G. Xie, W. Feng, Graphene sheets, polyaniline and AuNPs based DNA sensor for electrochemical determination of BCR/ABL fusion gene with functional hairpin probe, Biosens. Bioelectron. 51 (2014) 201–207, https://doi.org/10.1016/j.bios.2013.07.049. [163] A. Soni, C.M. Pandey, M.K. Pandey, G. Sumana, Highly efficient Polyaniline-MoS (2) hybrid nanostructures based biosensor for cancer biomarker detection, Anal. Chim. Acta 1055 (2019) 26–35, https://doi.org/10.1016/j.aca.2018.12.033. [164] M. Shamsipur, L. Samandari, L. Farzin, F. Molaabasi, M.H. Mousazadeh, Dualmodal label-free genosensor based on hemoglobin@gold nanocluster stabilized graphene nanosheets for the electrochemical detection of BCR/ABL fusion gene, Talanta 217 (2020) 121093, https://doi.org/10.1016/j.talanta.2020.121093. [165] R. Yu, J. Xue, Y. Wang, J. Qiu, X. Huang, A. Chen, J. Xue, Novel Ti3C2Tx MXene nanozyme with manageable catalytic activity and application to electrochemical biosensor, J. Nanobiotechnol. 20 (1) (2022) 119, https://doi.org/10.1186/ s12951-022-01317-9. [166] X.-y. Zhou, J.-h. Pan, Y.-n. Ma, X.-j. Peng, H.-p. Wu, Q. Zhou, S.-j. Ding, H.-x. Ju, An ultrasensitive fluorescence sensing strategy for detection and in situ imaging of chronic myeloid leukemia-related BCR-ABL1 mRNA, Sens Actuators B 273 (2018) 1456–1462, https://doi.org/10.1016/j.snb.2018.07.032. [167] X.H. Lin, H.Y. Wan, Y.F. Zhang, J.H. Chen, Studies of the interaction between Aloe-emodin and DNA and preparation of DNA biosensor for detection of PMLRAR α fusion gene in acute promyelocytic leukemia, Talanta 74 (4) (2008) 944–950, https://doi.org/10.1016/j.talanta.2007.07.038. [168] N. Wei, J. Chen, J. Zhang, K. Wang, X. Xu, J. Lin, G. Li, X. Lin, Y. Chen, An electrochemical biosensor for detection of PML/RARA fusion gene using capture probe covalently immobilized onto poly-calcon carboxylic acid modified glassy carbon electrode, Talanta 78 (4-5) (2009) 1227–1234, https://doi.org/10.1016/j. talanta.2008.12.053. [169] A. Liu, X. Chen, K. Wang, N. Wei, Z. Sun, X. Lin, Y. Chen, M. Du, Electrochemical DNA biosensor based on aldehyde-agarose hydrogel modified glassy carbon electrode for detection of PML/RARa fusion gene, Sens Actuators B 160 (1) (2011) 1458–1463, https://doi.org/10.1016/j.snb.2011.10.013. [170] Y. Lei, M.-j. Feng, K. Wang, L.-q. Lin, Y.-z. Chen, X.-h. Lin, Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RAR α fusion gene in acute promyelocytic leukemia, Anal. Bioanal. Chem. 405 (1) (2013) 423–428, https://doi.org/10.1007/s00216-012-6477-6. [171] Y. Lei, K. Wang, S.-Y. Wu, D.-D. Huang, M. Dai, Y.-J. Zheng, Z.-L. Sun, Y.-Z. Chen, X.-H. Lin, A.-L. Liu, 2 ′ -Fluoro ribonucleic acid modified DNA dual-probe sensing strategy for enzyme-amplified electrochemical detection of double-strand DNA of PML/RAR α related fusion gene, Biosens. Bioelectron. 112 (2018) 170–176, https://doi.org/10.1016/j.bios.2018.04.012. [172] Z.Y. Zhang, L.X. Huang, Z.W. Xu, P. Wang, Y. Lei, A.L. Liu, Efficient determination of PML/RAR α fusion gene by the electrochemical DNA biosensor based on carbon dots/graphene oxide nanocomposites, Int. J. Nanomed. 16 (2021) 3497–3508, https://doi.org/10.2147/ijn.S308258. [173] Y. Lei, K. Wang, J.-Y. Yang, X.-H. Lin, A.-L. Liu, Sequence-specific amperometric detection based on a double-probe mode and enzyme-mediated multiple signal electrocatalysis for the double-stranded DNA of PML/RAR α -related fusion gene, Anal. Chim. Acta 1231 (2022) 340436, https://doi.org/10.1016/j. aca.2022.340436. [174] L.Y. Yu, J.Y. Chen, H.J. Weng, H.F. Lin, C.J. Zhang, L.Y. Yang, J.Z. Lin, X.H. Lin, G.X. Zhong, Cell-free transcription amplification-based split-type electrochemical sensor using enzyme-linked magnetic microbeads for minimal residual leukemia detection, Talanta 286 (2025) 127551, https://doi.org/10.1016/j. talanta.2025.127551. [175] Z.-Y. Zhang, M.-T. Lin, Y. Zhang, Z.-J. Cheng, S.-H. Han, A.-L. Liu, Y. Lei, An electrochemiluminescence resonance energy transfer biosensor based on CDs/ PAMAM/rGO nanocomposites and Au@Ag2S nanoparticles for PML/RAR α fusion gene detection, Mikrochim. Acta 190 (10) (2023) 415, https://doi.org/10.1007/ s00604-023-05993-8. [176] L.S. Oliveira, K. Avelino, S. Oliveira, N. Lucena-Silva, H.P. de Oliveira, C.A. S. Andrade, M.D.L. Oliveira, Flexible genosensors based on polypyrrole and graphene quantum dots for PML/RAR α fusion gene detection: a study of acute promyelocytic leukemia in children, J. Pharm. Biomed. Anal. 235 (2023) 115606, https://doi.org/10.1016/j.jpba.2023.115606. [177] N. Docherty, D. Macdonald, A. Gordon, A. Dobrea, V. Mani, Y. Fu, S. Pang, M. Jimenez, D.K. Corrigan, Maximising the translation potential of electrochemical biosensors, Chem. Commun 61 (2025) 13359–13377, https:// doi.org/10.1039/D5CC02322J. N. Izadi et al. Electrochimica Acta 542 (2025) 147472 17