Recent advance in electrochemical immunosensors for lung cancer biomarkers sensing

2.1 Carbon-based nanomaterials

Carbon nanomaterials such as graphene, CNTs, and biochar have become very popular for electrochemical immunosensor fabrication owing to their exceptional electrical, mechanical, and chemical properties. Graphene is a 2D sheet of sp² hybridized carbon atoms arranged in a honeycomb lattice. It has very high specific surface area, excellent conductivity, good mechanical strength and flexibility, ease of functionalization, and biocompatibility. Graphene can be synthesized via chemical vapor deposition or solution-based oxidation-reduction methods to produce graphene oxide which is then reduced. Graphene promotes electron transfer kinetics and provides a suitable microenvironment for immobilizing biomolecules. For example, Chen et al. [25] developed an electrochemical immunosensor for sensitive detection of CEA. Graphene was used as the sensing material due to its excellent properties such as large surface area, high electrical conductivity and good biocompatibility. The researchers functionalized carboxyl graphene nanosheets with toluidine blue to form CGS-TB nanocomposites (Figure 1a). Toluidine blue acted as an electrochemical mediator to enhance the sensing signal. The CGS-TB nanocomposites were then conjugated with anti-CEA antibodies to form CGS-TB-Ab bioconjugates. These bioconjugates acted as signal tags for detecting CEA. Chitosan-AuNPs were used as the sensing platform. They were immobilized on the electrode surface and the capture anti-CEA antibodies were attached. This allowed the capture antibodies to capture CEA antigens from the sample solution. In the immunosensor, the CGS-TB-Ab bioconjugates bound to the captured CEA antigens, forming a sandwich immunoassay. The toluidine blue molecules on the graphene nanosheets generated an electrochemical signal that was proportional to the CEA concentration.

Figure 1

(a) TEM image of CGS-TB nanocomposites. (b) SEM image of N-rGO@CMWCNTs/CS@AuNPs. Reproduced with permission from refs. [25,27].

Carbon nanotubes are 1D hollow tubular structures made of rolled up graphene sheets. They have very high tensile strength, surface area, and electrical conductivity. Single-walled CNTs and multi-walled CNTs are commonly used. CNTs can be functionalized to attach capture biomolecules. The high conductivity of CNTs enhances electron transfer and signal transduction. For example, Xu et al. [26] focused on developing a label-free electrochemical immune microelectrode array for the sensitive detection of CEA. The researchers successfully synthesized a chitosan-multi-walled carbon nanotube-thionine (CS-MWCNTs-THI) hybrid film through a one-step electrochemical deposition process. This hybrid film was then employed to immobilize anti-CEA antibodies, enabling the fabrication of a simple and highly sensitive immune microelectrode array (MEA). The detection mechanism was based on the formation of antigen-antibody immunocomplexes leading to decreased response currents, with the reduction directly proportional to CEA concentration. The immune MEA exhibited a wide linear range for CEA detection (1 pg·mL⁻¹ to 100 ng·mL⁻¹) and a low limit of detection (0.5 pg·mL⁻¹ with signal noise ratio = 3). The hybrid film’s nano-porous structure and the synergistic effect of MWCNTs and THI contributed to the enhanced electrochemical performance.

Both graphene and CNTs facilitate electrochemical biomarker sensing with lower overpotentials, higher sensitivity, and lower detection limits compared to traditional electrode materials. However, graphene offers better mechanical stability and ease of surface modification compared to CNTs which tend to aggregate. Combining graphene and carbon nanotubes is also a strategy. Gu et al. [27] developed an innovative electrochemical immunosensor for the detection of CA125. The sensor, composed of nitrogen-doped reduced graphene oxide (N-rGO), carboxylated MWCNTs (CMWCNTs), CS, and AuNPs on a glassy carbon electrode (GCE) (Figure 1b), exhibited exceptional sensitivity and specificity. Through careful optimization, the sensor achieved a wide linear detection range spanning from 0.1 pg·mL⁻¹ to 100 ng·mL⁻¹ of CA125, with a low detection limit of 0.04 pg·mL⁻¹. Notably, the immunosensor showcased robust reproducibility, stability, and selectivity, demonstrating its potential for clinical applications. The sensor’s effectiveness was validated by successful analysis of spiked serum samples and real serum samples from lung cancer patients, with recovery experiments yielding accurate results and minimal variability.

Biochar is an excellent carbon material for the design of electrochemical immunosensors. Biochar is a porous carbon material produced from the pyrolysis of biomass waste such as wood, agricultural residues, and manure. It has a high surface area, stable structure, good electrical conductivity, and abundant surface functional groups. These properties make biochar very suitable for use as an electrode material in electrochemical immunosensors [28,29,30]. For example, Cancelliere et al. [31] presented a new electrochemical immunosensor based on screen-printed electrodes modified with biochar for the sensitive detection of interleukin-6 (IL-6). The use of biochar, produced from brewers’ spent grain, provides both an electrochemical enhancement of the electrode’s electron transfer kinetics and a protein binding substrate for antibody immobilization. Two IL-6 immunosensors were fabricated using different IL-6 antibodies. The layer-by-layer assembly of the immunosensors was characterized electrochemically and optimized. The biochar-modified electrodes showed improved conductivity compared to bare electrodes. The immunosensors demonstrated good analytical performance in buffer and human serum, with limits of detection as low as 4.8 pg·mL⁻¹, linear ranges from 26 to 138 pg·mL⁻¹, and recoveries from 82% to 95%. They also showed selectivity for IL-6 over other cytokines and stability for up to 14 days of storage.

2.2 Metal NPs

Metal NPs made from Au, Ag, platinum (Pt), etc., have great potential for electrochemical immunosensor applications because of their high conductivity, biocompatibility, catalytic properties, and high surface-to-volume ratio.

AuNPs are the most widely used. AuNPs can be synthesized in different shapes and sizes and are stable, non-toxic, and easily functionalized. AuNPs can adsorb proteins without altering their biological activity. Furthermore, AuNPs enhance the immobilization of capture antibodies and act as electrochemical labels when functionalized with secondary antibodies or enzymes for signal amplification. For example, Hu et al. [32] aimed to develop a biosynthesis method for AuNPs using an extract from Eucalyptus leaves. The synthesized AuNPs demonstrated significant potential as a natural antioxidant, displaying excellent free radical scavenging activity. The NPs were found to be roughly spherical in shape with sizes ranging from 10 to 30 nm. The biosynthesized AuNPs exhibited strong antioxidant activity, outperforming the standard ascorbic acid antioxidant in a DPPH assay with an IC50 value of 7.25 µg·mL⁻¹. Additionally, the AuNPs demonstrated notable antibacterial effects against several pathogenic bacterial strains. The results highlight the potential of Eucalyptus leaf extract-synthesized AuNPs for their application in the biomedical and pharmaceutical fields due to their antioxidant and antibacterial properties. Nguyen and Jen [33] reported a novel microfluidic chip designed to capture A549 human lung adenocarcinoma cells by utilizing a self-assembled monolayer (SAM) of AuNPs functionalized with cell-specific aptamers. The chip’s fabrication involves creating a SAM area on a glass substrate using photolithography and AuNP attachment through aminosilane treatment. Thiol-labeled aptamers are then self-assembled onto the AuNP layer. The success of this modification is confirmed through electrical impedance spectroscopy (EIS), showing enhanced impedance responses with the presence of the SAM of AuNPs. Our previous publication also used a glucose oxidase-functionalized gold nanorod probe that can catalyze the deposition of AuNPs [34]. An immunosensor was constructed by immobilizing capture antibodies on a carbon nanotube-modified screen-printed carbon electrode (SPCE). After the sandwich immunoassay, the gold nanorod probe is captured on the immunosensor surface. Then, the glucose oxidase catalyzes the deposition of AuNPs from a developer solution containing glucose, mediator, and gold salt. The electrochemical stripping analysis of both the gold nanorods and AuNPs produces an amplified signal response, resulting in ultrahigh sensitivity (Figure 2). When detecting CEA as a model analyte, the method achieved an excellent performance. It had a wide linear detection range from 0.01 to 100 ng·mL⁻¹ with a detection limit as low as 4.2 pg·mL⁻¹.

Figure 2

Schematic of sandwich immunoassay based on the signal tracing of the glucose oxidase-functionalized nanoprobe. Reproduced with permission from ref. [34].

AgNPs have high electrical conductivity and exhibit electrocatalytic activity towards hydrogen peroxide, allowing their use in enzyme-based amperometric sensors. AgNPs can also bind amino groups in proteins through Ag-NH₂ linkage. Asadzadeh-Firouzabadi and Zare [35] focused on developing an electrochemical nanogenosensor for the sensitive detection of miR-25 using AgNPs/single-walled carbon nanotubes (SWCNTs) nanohybrid as an electroactive label. The nanohybrid was characterized through TEM and electrochemical methods, revealing successful decoration of AgNPs on SWCNT surfaces. The nanosensor’s preparation steps were monitored using cyclic voltammetry and electrochemical impedance spectroscopy, confirming the immobilization of the probe and subsequent hybridization with miR-25. The nanogenosensor demonstrated excellent selectivity and sensitivity, successfully distinguishing between complementary, one-base mismatch, and non-complementary targets. The sensor exhibited a linear range for miR-25 concentration from 1 pM to 0.1 nM, with a detection limit of 0.313 pM. Moreover, the nanogenosensor’s reliability was confirmed by analyzing miR-25 in human plasma samples through spiking experiments, showcasing recovery rates between 97.58% and 105.91% with a low relative standard deviation. Jafari-Kashi et al. [36] reported a novel electrochemical DNA biosensor designed to detect the lung cancer biomarker CYFRA21-1. The biosensor, constructed on a modified glassy carbon electrode with layers of rGO, PPy, and AgNPs, achieves remarkable sensitivity and selectivity. Through systematic optimization of experimental conditions, the biosensor demonstrates a wide linear detection range (10 fM to 1 μM) and a low detection limit (2.4 fM). The use of AgNPs serves to enhance the biosensor’s performance, contributing to its excellent reproducibility and enabling the specific recognition of the target biomarker.

PtNPs similarly enhance electron transfer kinetics and increase the sensor sensitivity. The high surface area and biocompatibility of metal NPs ultimately allow more target analyte binding and improved detection limits. For example, Lv et al. [37] described the development of a highly sensitive electrochemical immunosensor for the detection of the lung cancer biomarker CYFRA21-1. The sensor utilizes a conjugated nanosheet called TPAPCN that exhibits aggregation-induced electrochemiluminescence (AIECL) as the signal label. When aggregated, TPAPCN produces a much stronger ECL emission signal compared to when dispersed. PtNPs were synthesized on the TPAPCN nanosheets to improve electron transfer and boost the ECL signal. The TPAPCN was conjugated to a signal antibody. In addition, three-dimensional graphene decorated with PtNPs and the electroactive molecule THI (3D-GN/PtNPs/Th) was used to immobilize the capture antibody and provide a stable internal electrochemical signal. By measuring the ratio of the ECL signal from the TPAPCN label and the electrochemical signal from the THI, the immunosensor eliminates noise and interference, improving detection accuracy. The sensor exhibited excellent sensitivity with a wide linear detection range of 50 fg·mL⁻¹ to 1 ng·mL⁻¹ and a low limit of detection of 16 fg·mL⁻¹ for CYFRA21-1.

2.3 Metal oxides

Metal oxides including iron oxide (Fe₃O₄), titanium oxide (TiO₂), zinc oxide (ZnO), copper oxide (CuO), etc., have also been incorporated into immunosensor electrode materials. They offer biocompatibility, high stability, and good electrocatalytic behavior.

Fe₃O₄ NPs in particular improve electron transfer and amplification of signal response. Wang et al. [38] described the development of a sensitive electrochemical immunosensor for detecting CEA levels. The immunosensor used a sandwich technique, with the antibody immobilized on the surface and magnetic Ag/MoS₂@Fe₃O₄ NPs used as a label. The Fe₃O₄ in the NPs allows them to be firmly attached to the electrode surface due to its magnetic properties, avoiding loss of the label. Using differential pulse voltammetry, the immunosensor was able to detect CEA in the range of 0.0001 to 20 ng·mL⁻¹, with a low detection limit of 0.03 pg·mL⁻¹. The immunosensor also demonstrated excellent stability, selectivity, and reproducibility. The use of the magnetic Fe₃O₄ NPs was key to the sensitive detection and performance of the immunosensor by keeping the label immobilized on the electrode surface.

TiO₂ NPs have been used to enhance antibody immobilization and to develop enzyme-linked immunoassays. Mavrič et al. [39] provided an overview of electrochemical biosensors based on TiO₂ nanomaterials for early detection of cancer biomarkers. TiO₂ nanostructures like nanotubes, NPs, and nanowires can enhance sensitivity by increasing surface area for binding events. Important characteristics for biosensing are high surface area, appropriate surface chemistry for biomolecule attachment, and efficient electron transfer, which can be tuned in TiO₂ by creating oxygen vacancies and Ti³⁺ defects. Sensors are often designed with additional nanomaterials like AuNPs, graphene, or semiconductor quantum dots to amplify signals and improve electron transfer. Reported detection limits are very low, down to the fg·mL⁻¹ range for some protein biomarkers, showing high sensitivity.

ZnO NPs and CeO₂ NPs also display strong protein adsorption ability to enable detection of biomarkers. For example, Che et al. [40] reported the development of an electrochemical sensor using ZnO/porous graphene oxide (ZnO/HGO) nanocomposites for the detection of the lung cancer biomarkers CEA and CA153. The ZnO NPs were grown on HGO sheets using a hydrothermal method. The ZnO/HGO composite was used to modify a glassy carbon electrode. Antibodies for CEA and CA153 were then immobilized on the electrode surface to enable specific binding to the target antigens. The sensor showed good linearity for CEA detection from 0.1–20 ng·mL⁻¹ with a detection limit of 0.07 ng·mL⁻¹. For CA153, the linear range was 0.5–70 U·mL⁻¹ and the detection limit was 0.22 U·mL⁻¹. The ZnO NPs provided good biocompatibility and a high surface area, while the HGO increased material transport and active sites. Yu et al. [41] described the development of an electrochemical immunosensor for detecting the biomarker NSE. The immunosensor uses a sandwich structure with antibodies and nanocomposite materials to amplify the detection signal. A key component is the use of Au/Cu _x O@CeO₂ nanocomposites as a label material. CeO₂ NPs were deposited onto Cu _x O nanocubes along with AuNPs to form a spiny surface. CeO₂ helped provide favorable catalytic activity and stability when combined with Cu _x O. The rough nanocomposite surface increased surface area to carry more antibodies. When tested, the immunosensor showed high sensitivity with a detection limit of 31.3 fg·mL⁻¹, a wide linear range of 50 fg·mL⁻¹ to 100 ng·mL⁻¹, and good performance in selectivity and stability. The inclusion of CeO₂ in the nanocomposite label helped enhance catalytic activity and signal amplification in the sensor.

2.4 Conducting polymers

Conducting polymers such as polyaniline (PANI), polypyrrole (PPy), and poly(3,4-ethylenedioxythiophene) (PEDOT) have also garnered interest for fabricating immunosensor electrodes owing to their electrical conductivity, biocompatibility, environmental stability, and ease of synthesis. These polymers can be chemically or electrochemically polymerized to form uniform thin films on electrode surfaces. Their conductive backbone facilitates electron transfer, while their side chains can be modified with functional groups for covalent antibody immobilization. Conducting polymers enhance surface area, improve electrochemical reactivity, and lower electrochemical impedance.

For example, PANI with good biocompatibility and electrical conductivity has been widely employed. The amine groups on PANI enable covalent binding of antibodies. PANI also helps maintain biological activity of immobilized antibodies. Park et al. [42] demonstrated an electrochemical aptasensor using a PANI/CNT nanocomposite for detecting vascular endothelial growth factor (VEGF165). The nanocomposite was synthesized by polymerizing aniline on the surface of acid-treated CNTs. Adding PANI to the CNTs increased the sensor’s sensitivity and stability compared to CNTs alone, due to PANI’s high conductivity and large surface area. The VEGF165 aptamer was covalently attached to the PANI/CNT surface (Figure 3). The sensor detected VEGF165 with high sensitivity from 0.5 pg·mL⁻¹ to 1 μg·mL⁻¹, with a detection limit of 0.4 pg·mL⁻¹. This is better performance than other reported VEGF sensors. The sensor also showed good selectivity for VEGF165 against other proteins. The use of the PANI/CNT nanocomposite and aptamer binding provided excellent sensitivity and selectivity for detecting the VEGF165 biomarker.

Figure 3

(a) Schematic illustration of the preparation of the PANI/CNT nanocomposite and the assembled VEGF165 aptamer on the sensor surfaces for the VEGF sensors. (b) Fabrication and modification process of graphene-PEDOT:PSS modified paper-based aptasensor. Reproduced with permission from refs. [42,44].

Similarly, PPy provides a biocompatible microenvironment to maintain antibody binding activity. Kivrak et al. [43] described the development of an electrochemical aptasensor using PPy nanofibers for the detection of NSCLC cells. The aptasensor uses a pencil graphite electrode modified with composite nanofibers made of polyacrylonitrile (PAN) and 10% PPy by weight. The high conductivity and porosity of PPy enhances the performance of the nanofiber-modified electrode. The aptamer specific to the target A549 cancer cells is immobilized on the surface. When the target cells bind to the aptamer, it causes an increase in charge transfer resistance measured by electrochemical impedance spectroscopy. The aptasensor shows high sensitivity and selectivity for detecting A549 cells down to 1.2 × 10³ cells·mL⁻¹, with human cervical cancer HeLa cells used as a negative control. The PPy-containing nanofiber electrode interface enables sensitive impedimetric detection of the aptamer-cancer cell interactions.

Copolymers combining PSS and PEDOT have also been synthesized to further improve electrochemical properties and leverage synergistic effects. The high surface area and permeability, good electrical conductivity, and biocompatibility of conducting polymers ultimately result in higher loading of capture antibodies and lower detection limits. Yen et al. [44] described the development of a low-cost paper-based electrochemical aptasensor for detecting CEA. The sensor uses a graphene and PEDOT:PSS nanocomposite coating on paper to create a conductive and sensitive substrate for aptamer immobilization. Modifying the paper with PEDOT:PSS helped improve the conductivity and sensing performance of the electrode. The aptasensor showed a good linear detection range of 0.77–14 ng·mL⁻¹ for CEA and a limit of detection around 1 ng·mL⁻¹ in serum samples. The key benefit of PEDOT:PSS modification was enhancing the conductivity and sensitivity of the paper-based electrode in a cost-effective and accessible way.

4.1 Enzyme labels

Enzyme labels like HRP and glucose oxidase (GOx) are widely coupled to secondary antibodies. The enzyme catalyzes the breakdown of substrates like hydrogen peroxide, producing an electroactive product that amplifies the current or potential response.

For instance, HRP can act as an electron transfer agent to amplify the electrochemical signal [64]. The HRP is conjugated to streptavidin-coated magnetic beads, which allows high loading of HRP on the sensor surface. HRP catalyzes the oxidation of hydroquinone (HQ) by hydrogen peroxide. This reaction generates an electrochemical signal (reduction peak of HQ) that can be measured by voltammetric techniques like square wave voltammetry. The level of HRP on the sensor surface, and consequently the electrochemical signal, depends on the cleavage of the Asp-Glu-Val-Asp (DEVD) peptide by caspase 3. When caspase 3 is present, it cleaves the DEVD peptide, releasing HRP from the surface and decreasing the electrochemical signal. So, by measuring the reduction peak current of HQ, the authors are able to detect caspase 3 activity down to 100 pM. The decrease in peak current correlates to the amount of caspase 3 present.

GOx was used to label the biomarker proteins Annexin II and MUC5AC [65]. The proteins are conjugated to GOx using glutaraldehyde chemistry. When the GOx-labeled proteins bind to their specific antibodies on the sensor surface, the GOx can catalyze glucose oxidation, generating H₂O₂. The H₂O₂ generated is then reduced by the hydrazine catalyst on the sensor surface, producing a measurable current response. So, the GOx provides an electrochemical signal to detect binding of the biomarker proteins. More GOx-labeled protein bound to the sensor surface leads to more H₂O₂ generation and higher current. Less GOx binding causes lower current. This forms the basis of a competitive assay – unlabeled biomarker proteins compete with GOx-labeled proteins for antibody binding sites. More unlabeled protein leads to less GOx binding and lower current. So, the concentration of unlabeled biomarker protein can be quantified by measuring the reduction in current compared to a baseline with just GOx-labeled protein. GOx amplification of the binding event into a detectable electrochemical signal allows sensitive detection of the biomarker proteins down to pg·mL⁻¹ levels. In another work [66], GOx catalyzes the oxidation of glucose to generate hydrogen peroxide H₂O₂. H₂O₂ acts as a coreactant to enhance the ECL reaction of luminol. So, the presence of GOx leads to in situ production of H₂O₂ which amplifies the ECL signal. GOx is conjugated to the Ab2. So, when the immunoreaction occurs between the antigen and Ab1, the GOx labeled Ab2 binds as well, bringing GOx to the electrode surface. This increases the amount of GOx near the electrode, generating more H₂O₂ and enhancing the ECL signal further. GOx is loaded onto graphene along with the ZnO NPs and Ab2. Graphene provides a larger surface area to load more GOx and Ab2, further amplifying the ECL signal.

4.2 NP labels

Nanomaterials like AuNPs, CNT, graphene, Co₃O₄, etc., can be functionalized with detector antibodies to serve as labels for signal enhancement. Owing to their high conductivity, catalytic properties, and large surface area, they act as nanocarriers to load a high number of detector antibodies and effectively amplify immunosensor response.

For example, Co₃O₄@CeO₂-Au@Pt nanocomposite has been used as enzyme-mimetic labels [67]. Co₃O₄ nanocubes have excellent electrocatalytic activity towards the reduction of H₂O₂. CeO₂ NPs further enhance the catalytic performance of Co₃O₄ due to a synergistic effect. Au@Pt NPs also have good electrocatalytic properties for H₂O₂ reduction. Combining Au@Pt with Co₃O₄@CeO₂ results in a nanocomposite with superior catalytic activity compared to the individual components. The Co₃O₄@CeO₂ core-shell structure provides a large surface area for loading the Au@Pt NPs. This increases the amount of nanocomposite that can bind to the secondary antibody and thus enhances the catalytic current signal. The nanocomposite improves electron transfer efficiency on the electrode surface, leading to higher sensitivity. Co₃O₄@CeO₂-Au@Pt exhibits enzyme-mimetic catalytic activity similar to HRP towards H₂O₂ reduction. Using the nanocomposite instead of HRP helps avoid instability issues associated with enzyme labels. The synergetic effect and enhanced catalytic properties of the Co₃O₄@CeO₂-Au@Pt nanocomposite allow it to act as an enzyme-mimetic label to amplify the electrochemical signal and improve detection sensitivity for the immunosensor.

4.3 Dendrimers

Dendrimers are highly branched globular macromolecules with numerous surface groups that can conjugate to signal molecules like enzymes, metals, NPs, and redox markers. The three-dimensional architecture provides a high loading capacity. For example, Idris et al. [68] reported that immunosensor is based on an exfoliated graphite electrode modified with a nanocomposite of carbon nanodots (CNDTs) and polypropylene imine dendrimer (PPI) (Figure 5). The dendrimer plays an important role in the sensor. The PPI dendrimer was electrodeposited onto the electrode surface modified with CNDTs. It increases the surface area of the electrode for higher antibody loading. Its spherical morphology and surface functional groups facilitate immobilization of the antibody through both electrostatic and host-guest interactions. It improves conductivity and accelerates electron transfer kinetics at the electrode interface. Its biocompatibility helps maintain the bioactivity of the antibody for improved analyte binding. Together with the CNDTs, the dendrimer-CNDT nanocomposite synergistically enhances the electrochemical signal. This results in higher sensitivity with a low detection limit of 0.00145 ng·mL⁻¹ for CEA.

Figure 5

Processes involved in the preparation of the immunosensor. Reproduced with permission from ref. [68].

4.4 DNA nanostructures

Self-assembled DNA nanoarchitectures such as tetrahedrons, nanoflowers, nanoribbons, and nanocages can integrate numerous enhancer moieties like redox reporters, enzymes, and NPs onto their large surface area. Hybridization chain reaction or strand displacement amplification using functional DNA structures can also produce a cascading signal enhancement effect. For example, Liu et al. [69] described the development of an electrochemical biosensor for detecting lung cancer-related microRNAs using 3D DNA origami nanostructures. The sensor uses a 3D DNA tetrahedron structure as the base. This tetrahedron has three thiol groups that allow it to self-assemble on a gold electrode surface. A DNA strand with a stem-loop structure is attached to the top of the tetrahedron. The loop portion is complementary to the target microRNA. The stem part contains a ferrocene tag for electrochemical signaling. When the target microRNA hybridizes to the loop, it opens up the stem-loop structure. This moves the ferrocene tag closer to the electrode surface, increasing the electrochemical signal. The 3D tetrahedron structure provides a scaffold to correctly orient the signaling stem-loop probe on the surface. It also provides some distance between the ferrocene tag and the electrode, making the “signal-on” sensing mechanism possible. Without the 3D nanostructure, the stem-loop probe would lie flat on the surface. The ferrocene would already be close to the electrode, so target hybridization would not change the signal as much. This amplifies the current or potential response by several orders of magnitude. However, DNA nanoassembly requires optimization and the structures may degrade under harsh experimental conditions.

5.1 CEA

CEA is an important broad-spectrum tumor marker upregulated in lung cancer. Electrochemical immunosensors have been developed targeting CEA for early diagnosis. For instance, Wang et al. [70] reported an electrochemical immunosensor for detecting the CEA. The immunosensor is constructed on a fluorine-doped tin oxide electrode modified with a composite of PANI-loaded few-layered MXene (MXene@PANI). AuNPs and β-cyclodextrin (Au-β-CD) are further electrodeposited on the surface to immobilize anti-CEA antibodies (Figure 6a). This sensor exhibits excellent performance for CEA detection with a wide linear detection range of 0.5–350 ng·mL⁻¹ and a low limit of detection of 0.0429 ng·mL⁻¹. The high conductivity of the MXene@PANI composite and the capability of Au-β-CD to efficiently immobilize antibodies contribute to the sensor’s sensitivity. The immunosensor also demonstrates good selectivity, reproducibility, and stability. Furthermore, it shows feasibility for detecting CEA in human serum samples, with recovery ratios of 97.52–103.98%. Gan et al. [71] described a similar electrochemical immunosensor for detecting CEA. The immunosensor uses magnetic DNA-tagged nanoprobes made of zirconia/iron oxide NPs bound to DNA and antibodies. When the sensor detects CEA, it forms a sandwich structure with capture antibodies on the sensor surface, CEA, and the antibody-bound nanoprobes (Figure 6b). The large amount of enzyme horseradish peroxidase on the nanoprobes amplifies the signal. This sensor was able to detect CEA at concentrations as low as 5 pg·mL⁻¹, which is 100 times more sensitive than conventional ELISA methods. Testing on human serum samples showed good accuracy compared to ELISA.

Figure 6

Several electrochemical sensors for CEA detection. (a) PANI-loaded MXene and gold-decorated β-cyclodextrin. (b) DNA/(ZMPs-HRP-CEA Ab2)n. Reproduced with permission from ref. [70,71].

5.2 NSE

NSE is an important biomarker upregulated in lung cancer tissues. Wang et al. [72] developed a sensitive electrochemical immunosensor using a MoS₂@Au nanozyme label for detecting the biomarker NSE. The MoS₂ nanoflowers provided abundant sites to load Au nanoclusters which catalyzed H₂O₂ decomposition to amplify the amperometric signal. The NSE antibody was coupled to the MoS₂@Au nanozyme while the capture antibody was immobilized on reduced graphene oxide (Figure 7). The immunosensor detected NSE from 0.1 pg·mL⁻¹ to 10 ng·mL⁻¹ with a low detection limit of 0.05 pg·mL⁻¹. It showed good selectivity against other proteins, reproducibility of 1.51% RSD, and stability for 48 hours. The sensor was successfully applied to NSE detection in spiked serum samples with recoveries from 97.6% to 99.9%. The MoS₂@Au nanozyme label enhanced sensitivity and the performance indicates that this electrochemical immunosensor has potential for clinical diagnosis of NSE.

Figure 7

MoS₂@Au-Ab2 bioconjugate for NSE detection. Reproduced with permission from ref. [72].

Karaman et al. [73] described the development of a highly sensitive electrochemical immunosensor for detecting NSE. The immunosensor uses a GCE modified with AuNPs-decorated graphene and MoS₂ nanocomposites as the sensor platform. This provides a large surface area for antibody binding. The signal is amplified using a cobalt iron oxide-silver nanocomposite, which binds to secondary antibodies. The immunosensor showed excellent performance for NSE detection with a wide linear range from 0.01 to 1 pg·mL⁻¹ and a low limit of detection of 3 fg·mL⁻¹. It also demonstrated high selectivity against interfering proteins and stability over a 7-week period. Han et al. [74] used a signal amplification strategy based on the electrocatalytic properties of nickel hexacyanoferrate nanoparticles (NiHCFNPs) towards dopamine oxidation. The sensor is fabricated by modifying a glassy carbon electrode with a film of porous gold nanocrystals, followed by NiHCFNPs and then a layer of AuNPs-functionalized graphene nanosheets (Au-Gra). This provides a large surface area for antibody loading. In the presence of dopamine, the NiHCFNPs greatly enhance the oxidation current response. This amplification allows sensitive detection of NSE down to 0.3 pg·mL⁻¹, with a wide linear range from 0.001–100 ng·mL⁻¹. The immunosensor exhibits high selectivity, reproducibility, and stability. Testing on clinical samples shows good agreement with standard ELISA results.

5.3 CYFRA 21-1

CYFRA 21-1 is an important NSCLC biomarker. A novel immunosensor was reported based on a hydroxyapatite-chitosan nanocomposite platform and dendritic CuS NPs label. Kumar et al. [75] reported on the development of a highly sensitive electrochemical biosensor for the detection of the oral cancer biomarker CYFRA-21-1. The biosensor uses a nanostructured yttrium oxide (nY₂O₃) platform functionalized with antibodies specific for CYFRA-21-1. Structural characterization shows successful synthesis of oval-shaped nY₂O₃ NPs around 80 nm in size. The nY₂O₃ was electrophoretically deposited onto an ITO electrode and the antibodies were immobilized using EDC-NHS chemistry (Figure 8). Testing shows that the biosensor exhibits a wide linear detection range from 0.01 to 50 ng·mL⁻¹ CYFRA-21-1, with a remarkably low limit of detection of 0.01 ng·mL⁻¹. The biosensor demonstrates high sensitivity of 226.0 Ω·mL·ng⁻¹. Analysis of saliva samples shows good correlation between the biosensor and ELISA for detecting CYFRA-21-1 levels.

Figure 8

Stepwise fabrication process of the BSA/anti-CYFRA-21-1/APTES/nY₂O₃/ITO biosensor. Reproduced with permission from ref. [75].

Aydın et al. [76] presented a new electrochemical biosensor for detecting the lung cancer biomarker CYFRA 21-1. The biosensor uses an ITO electrode modified with a multilayer film consisting of electrodeposited AuNPs and an electropolymerized amino-substituted pyrrole polymer (P(PyAmn)). The AuNPs provide high conductivity and the P(PyAmn) polymer provides a large surface area for antibody immobilization. The biosensor detects CYFRA 21-1 through specific binding to anti-CYFRA 21-1 antibodies on the surface, which causes an increase in impedance measured by EIS. The biosensor demonstrated a wide linear detection range from 0.015–90 pg·mL⁻¹, a low detection limit of 4.59 fg·mL⁻¹. It showed high selectivity for CYFRA 21-1 over other proteins. The biosensor was successfully applied to detect spiked CYFRA 21-1 in human serum samples with recoveries of 95.47–106.70%. Lu et al. [77] utilized a ring-opening polymerization (ROP) amplification strategy to greatly improve detection sensitivity. The immunosensor is fabricated by immobilizing capture antibodies onto a gold electrode modified with a self-assembled monolayer. After CYFRA 21-1 binds to the capture antibodies, detection antibodies are added, which initiates the ROP reaction. This results in a large number of electroactive ferrocene molecules becoming attached to the detection antibodies, amplifying the detection signal. Using this ROP amplification strategy, the immunosensor demonstrated a low detection limit of 9.08 fg·mL⁻¹ for CYFRA 21-1, which is 100–1,000 times lower than other reported methods. The immunosensor also showed excellent selectivity against other proteins, a wide linear detection range of 1 pg·mL⁻¹ to 1 μg·mL⁻¹, and good accuracy for the detection of clinical serum samples.

5.4 Emerging biomarkers

In addition to conventional markers, immunosensors have also targeted emerging lung cancer biomarkers. For instance, progastrin-releasing peptide (ProGRP) is an important biomarker for lung cancer diagnosis and monitoring, especially in early stages [78]. The immunosensor utilizes antibodies specific to ProGRP immobilized on the electrode surface. The interaction between ProGRP and the antibodies causes measurable changes in electrical signals that correlate with ProGRP concentration. The modified electrode provides a large surface area and optimal environment for antibody binding. The immunosensor demonstrated high sensitivity with a detection limit of 0.133 ng·mL⁻¹ ProGRP, along with excellent reproducibility and storage stability over 30 days.

VEGF165 plays a key role in tumor angiogenesis and metastasis in lung cancer. Amouzadeh Tabrizi et al. [79] described the development of a novel electrochemical aptasensor for the sensitive and selective detection of VEGF165. The aptasensor is based on an anti-VEGF165 aptamer immobilized on a screen-printed electrode modified with ordered mesoporous carbon-gold nanocomposite. The aptasensor detected VEGF165 through changes in interfacial electron transfer resistance measured by electrochemical impedance spectroscopy. The sensor exhibited a wide linear detection range from 10.0 to 300 pg·mL⁻¹ and a low limit of detection of 1 pg·mL⁻¹.

Detecting microRNAs (miRNAs) like miR-21 holds immense importance in lung cancer diagnostics due to their stability and regulatory roles. Liu et al. [80] reported a novel electrochemical biosensing platform based on a hybrid DNA hydrogel, which was developed for the sensitive detection of lung cancer-specific miR-21. The biosensor utilized ferrocene-tagged DNA recognition probes that, when hybridized with miR-21, led to the dissolution of the DNA hydrogel, causing a reduction in current detectable by cyclic and differential pulse voltammetry. Notably, the biosensor exhibited a remarkable detection limit of 5 nM for miR-21, along with a linear range of detection spanning from 10 nM to 50 μM. Importantly, the biosensor showcased high specificity for miR-21 over interfering sequences, demonstrating its potential for selective clinical diagnosis.

The study by Xu et al. [81] revolved around the significance of detecting the EGFR exon 19 status in lung cancer, as patients with exon 19 deletions often exhibit greater sensitivity to EGFR tyrosine kinase inhibitors than those with other EGFR mutations. By analyzing the conversations, it is evident that the utilization of deep learning techniques, particularly the Bidirectional Long Short-Term Memory model, proves to be effective in automating the detection of EGFR exon 19 deletions. This model, trained on a substantial dataset of patient genomic sequences, demonstrates excellent performance in terms of accuracy, sensitivity, and specificity, outperforming previous methods such as Amplification Refractory Mutation System PCR.