Electrochemical Immunosensor Using Electroactive Carbon Nanohorns for Signal Amplification for the Rapid Detection of Carcinoembryonic Antigen

In this work, a novel sandwich-type electrochemical immunosensor was developed for the quantitative detection of the carcinoembryonic antigen, an important tumor marker in clinical tests. The capture antibodies were immobilized on the surface of a gold disk electrode, while detection antibodies were attached to redox-tagged single-walled carbon nanohorns/thionine/AuNPs. Both types of antibody immobilization were carried out through Au-S bonds using the novel photochemical immobilization technique that ensures control over the orientation of the antibodies. The electroactive SWCNH/Thi/AuNPs nanocomposite worked as a signal tag to carry out both the detection of carcinoembryonic antigen and the amplification of the detection signal. The current response was monitored by differential pulse voltammetry. A clear dependence of the thionine redox peak was observed as a function of the carcinoembryonic antigen concentration. A linear detection range from 0.001–200 ng/mL and a low detection limit of 0.1385 pg/mL were obtained for this immunoassay. The results showed that carbon nanohorns represent a promising matrix for signal amplification in sandwich-type electrochemical immune assays working as a conductive and binding matrix with easy and versatile modification routes to antibody and redox tag immobilization, which possesses great potential for clinical diagnostics of CEA and other biomarkers.


Introduction
Cancer is a life-threatening disease with worldwide significance for the healthcare systems and a huge economic impact. Tumor biomarkers are important tools for the detection of cancer diseases, which either originate from tumor cells or emerge from the organism as a response to it. Alterations of their concentration in the body fluids may correlate qualitatively or quantitatively with the presence of cancer cells and therefore possess important clinical value for the early detection and diagnosis of the cancer diseases and thus the prognosis of the patient [1]. In fact, some biomarkers have been routinely used in clinical diagnosis including carcinoembryonic antigen (CEA), alpha-fetoprotein, prostatespecific antigen, carbohydrate antigen 125, carbohydrate antigen 153, carbohydrate antigen 199, and so on [2]. Among them, CEA, which is a set of glycoproteins of great relevance for cell adhesion during fetal development, has been considered a common cancer biomarker in clinical diagnosis since its expression declines after birth. CEA overexpression in blood serum in adult humans is usually related to the presence or progression of different types of cancer such as colorectal, liver, breast, ovarian or lung. In addition, CEA levels can also be monitored during chemotherapy to assess the progress and result of the treatment [3]. In healthy individuals, the concentration of CEA in blood serum should be less than electrode configuration. While electrochemical impedance spectroscopy was measured on a BioLogic potentiostat (SP-300, BioLogic Systems, Grenoble, France. The gold electrode (Au-disk, 2 mm diameter) was used as the working electrode, and a Pt wire and saturated Ag/AgCl electrode were used as the counter electrode and reference electrode, respectively. All potentials in this work are quoted with respect to the potential of the Ag/AgCl reference electrode.

Fabrication of SWCNH/Thionine/AuNPs Nanocomposite (SWCNH/Thi/AuNPs)
In this method, 2 mL of a SWCNH (I) dispersion (1 mg/mL) was mixed with 2 mL of thionine (4 mg/mL), stirring vigorously for 24 h at room temperature. The product was purified with Milli-Q water by centrifugation (12,000 rpm) to remove unbound thionine molecules. The SWCNH/Thi (II) was dispersed in 2 mL of Milli-Q water and then, 8 mL of AuNPs dispersion was added to the dispersion. The mixture was allowed to react for 48 h under magnetic stirring. Subsequently, the mixture was washed several times by centrifugation (12,000 rpm); the recovered solid was redispersed in 2 mL of 0.01 M PBS and stored at 4 • C.

Preparation of Detection Antibody
Labeled SWCNH/Thi/AuNPs/AntiCEA 2 AntiCEA 2 was immobilized on SWCNH/Thi/AuNPs by covalent interaction between AntiCEA 2 and AuNPs. Briefly, 350 µL of AntiCEA 2 (121.42 µg/mL) was irradiated with a UV lamp (Trylight ® , Promete Srl. Naples, Italy) for 30 s; afterward, it was mixed with 500 µL of SWCNH/Thi/AuNPs (III) by gently stirring for 15 min. The UV source consisted of two U-shaped low-pressure mercury lamps (6 W at 254 nm) in which a standard quartz cell could be easily housed. Considering the envelope geometry of the lamps and the cell proximity, the irradiation intensity used to produce the thiol group was approximately 0.3 W/cm 2 [12].

Assembly Process of the Immunosensor
First, the Au-disk was polished on a micro cloth using 0.3 µm and later 0.05 µm alumina. Then, it was electrochemically annealed by 100 cyclic voltammetry scans using H 2 SO 4 0.5 M at a potential sweep of 0.35 to 1.5 V at 1 V s −1 (VI). The CV with H 2 SO 4 did not only clean the Au surface, but also worked as a pretreatment to improve the electroactive area of the Au electrode, helping with the sensitivity of the immunosensor.
The immobilization of the AntiCEA 1 antibody, on the surface of the gold electrode, was carried out using the photochemical immobilization technique (PIT). Briefly, 350 µL of AntiCEA 1 (15 µg/mL) was irradiated with a UV lamp (Trylight ® , Promete Srl) using a quartz cell for 30 s. The irradiated AntiCEA 1 was transferred to an Eppendorf tube, and the gold electrode was immediately dipped in the solution for 15 min. Subsequently, the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA 1 (VII). Afterward, 25 µL of BSA (50 µg/mL) was deposited on the Au-disk/AntiCEA 1 and incubated for 30 min at room temperature to avoid non-specific absorption. Subsequently, the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA 1 /BSA (VIII). Then, 25 µL of CEA antigen was deposited at different concentrations and incubated for 45 min at room temperature. Afterward, the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA 1 /BSA/CEA. Finally, 30 µL of NaPro was deposited and incubated for 45 min at room temperature, and the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA 1 /BSA/CEA/NaPro. Figure 1 shows the assembly steps of the immunosensor.

Assembly Process of the Immunosensor
First, the Au-disk was polished on a micro cloth using 0.3 µ m and later 0.05 µ m alumina. Then, it was electrochemically annealed by 100 cyclic voltammetry scans using H2SO4 0.5 M at a potential sweep of 0.35 to 1.5 V at 1 V s −1 (VI). The CV with H2SO4 did not only clean the Au surface, but also worked as a pretreatment to improve the electroactive area of the Au electrode, helping with the sensitivity of the immunosensor.
The immobilization of the AntiCEA1 antibody, on the surface of the gold electrode, was carried out using the photochemical immobilization technique (PIT). Briefly, 350 µ L of AntiCEA1 (15 µ g/mL) was irradiated with a UV lamp (Trylight ® , Promete Srl) using a quartz cell for 30 s. The irradiated AntiCEA1 was transferred to an Eppendorf tube, and the gold electrode was immediately dipped in the solution for 15 min. Subsequently, the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA1 (VII). Afterward, 25 μL of BSA (50 μg/mL) was deposited on the Au-disk/AntiCEA1 and incubated for 30 min at room temperature to avoid non-specific absorption. Subsequently, the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA1/BSA (VIII). Then, 25 μL of CEA antigen was deposited at different concentrations and incubated for 45 min at room temperature. Afterward, the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA1/BSA/CEA. Finally, 30 μL of NaPro was deposited and incubated for 45 min at room temperature, and the electrode was rinsed with PBS, obtaining Au-disk/AntiCEA1/BSA/CEA/NaPro. Figure  1 shows the assembly steps of the immunosensor. Immobilization of the AntiCEA2 on the Au-disk electrode by PIT and assembly of the electrochemical immunosensor. The electrochemical detection is achieved by a dependence on the thionine redox peak as a function of the CEA concentration.

CEA Biomarker Detection
The modification step-wise process of the working electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in [Fe(CN)6] 3-/4-10 mM in PBS 0.01 M. CEA detection was carried out by differential pulse voltammetry (DPV) in PBS 0.01 M at pH 7.4, monitoring the redox peak of thionine around −0.25 V. (VI-X) Immobilization of the AntiCEA 2 on the Au-disk electrode by PIT and assembly of the electrochemical immunosensor. The electrochemical detection is achieved by a dependence on the thionine redox peak as a function of the CEA concentration.

CEA Biomarker Detection
The modification step-wise process of the working electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in [Fe(CN) 6 ] 3-/4-10 mM in PBS 0.01 M. CEA detection was carried out by differential pulse voltammetry (DPV) in PBS 0.01 M at pH 7.4, monitoring the redox peak of thionine around −0.25 V.

SWCNH/Thi/AuNPs Characterization
The morphology of SWCNH and SWNH/Thi/AuNPs were characterized by HRTEM and STEM. Figure 2A,B shows that a single carbon nanohorn is around 2-5 nm in diameter and 40-50 nm in length. The individual nanohorns tend to aggregate forming the typical dahlia-like nanostructure with an approximate diameter of 80-100 nm [8,13]. The STEM images of SWCNH also confirmed the dahlia-like assemblies. Figure 2C-F show the SWCNH/Thi/AuNPs, which demonstrated the AuNPs were homogeneously distributed and anchored on the pristine SWCNH surface, providing uniform binding sites for the attachment of antibodies to the nanohorns. The average size of the AuNPs was 5-10 nm. Notably, the structure of the SWCNH was not altered during the incorporation of the AuNPs. dahlia-like nanostructure with an approximate diameter of 80-100 nm [8,13]. The STEM images of SWCNH also confirmed the dahlia-like assemblies. Figure 2C-F show the SWCNH/Thi/AuNPs, which demonstrated the AuNPs were homogeneously distributed and anchored on the pristine SWCNH surface, providing uniform binding sites for the attachment of antibodies to the nanohorns. The average size of the AuNPs was 5-10 nm. Notably, the structure of the SWCNH was not altered during the incorporation of the AuNPs. To verify the presence of thionine and AuNPs in SWCNH an EDS mapping was performed, and the elemental distribution within the SWNHs was verified using STEM-EDS ( Figure 3). The SWCNH/Thi/AuNPs is mainly composed of carbon ( Figure 3B), the presence of oxygen was also observed as a consequence of the oxidation treatment of SWCNH ( Figure 3C), which likely contains a variety oxygen associated functional units such as hydroxyl and carboxyl groups [14]. Besides, the element sulfur was also observed ( Figure  3D), which is attributed to the presence of thionine in the surface of the SWCNH since thionine contains a thiazinium group [15]. The presence of gold nanoparticles can be clearly seen on the SWCNH surface, which indicates that the nanoparticles observed by To verify the presence of thionine and AuNPs in SWCNH an EDS mapping was performed, and the elemental distribution within the SWNHs was verified using STEM-EDS ( Figure 3). The SWCNH/Thi/AuNPs is mainly composed of carbon ( Figure 3B), the presence of oxygen was also observed as a consequence of the oxidation treatment of SWCNH ( Figure 3C), which likely contains a variety oxygen associated functional units such as hydroxyl and carboxyl groups [14]. Besides, the element sulfur was also observed ( Figure 3D), which is attributed to the presence of thionine in the surface of the SWCNH since thionine contains a thiazinium group [15]. The presence of gold nanoparticles can be clearly seen on the SWCNH surface, which indicates that the nanoparticles observed by TEM and STEM are certainly gold nanoparticles, Figure 3E. These results prove that thionine and AuNPs were firmly tethered to the SWCNH with uniform distribution and cannot be washed off be rinsing.
Possible explanations for strong interaction between these components are on the one hand that thionine has a planar aromatic structure that facilitates strong π-π stacking interactions to the likewise aromatic SWCNH surface [16]. In addition to πstacking interactions, also coupling via (electro)activated functional groups (-C=O-and COOH) of SWCNH can be involved in thionine linking via its amino-groups [17]. Additionally, the AuNPs were attached mainly to the SWCNH surface predominantly via unspecific adsorption, which could involve physisorption, π-π stacking, hydrophobic and electrostatistic interactions [18].
A glassy carbon electrode was modified with SWCNH/Thi/AuNPs and characterized by cyclic voltammetry (CV). Figure 3F shows an anodic peak around 0.21 V and a cathodic peak at 0 V, which are characteristic potentials of the reversible two-electron transfer process of thionine at acidic pH [19]. The difference between the anodic and cathodic peak is 210 mV, and the current ratio I pa /I pc is 2.41 mA. Therefore, the thionine redox reaction can be considered as quasi-reversible process [20]. 0.9 V. This peak is characteristic for the reduction of gold in the reverse potential sweep after a considerable electro-oxidation in the forward sweep to potentials higher than 1.2 V, confirming the presence of the AuNPs [21].
The SWCNH/Thi/AuNPs nanocomposite showed distinct redox activity, supporting the incorporation of Thi on the SWCNH surface. Furthermore, it should be pointed out that the high conductivity of the SWCNH facilitated the electron transfer across this carbon material and thus the redox reaction of Thi molecules at the distal side of SWCNH, which enhanced the electrochemical current [22,23]. Likewise, the multitude of SWCNH associated AuNPs offers multiple sites for antibody immobilization, promising all in all high potential as signal tag for the fabrication of the sandwich-type immunosensor. Noteworthy, the redox properties of thionine were not affected by the incorporation into the SWCNH. Moreover, a prominent cathodic peak was observed at approximately 0.9 V. This peak is characteristic for the reduction of gold in the reverse potential sweep after a considerable electro-oxidation in the forward sweep to potentials higher than 1.2 V, confirming the presence of the AuNPs [21].
The SWCNH/Thi/AuNPs nanocomposite showed distinct redox activity, supporting the incorporation of Thi on the SWCNH surface. Furthermore, it should be pointed out that the high conductivity of the SWCNH facilitated the electron transfer across this carbon material and thus the redox reaction of Thi molecules at the distal side of SWCNH, which enhanced the electrochemical current [22,23]. Likewise, the multitude of SWCNH associated AuNPs offers multiple sites for antibody immobilization, promising all in all high potential as signal tag for the fabrication of the sandwich-type immunosensor.

Optimization Test
To verify that the immobilization of AntiCEA 1 antibodies on the surface of the Audisk by PIT was successful, the Au electrode was characterized by CV before and after immobilization of the antibodies, using a redox probe Fe(CN) 6 3-/Fe(CN) 6 4- (Figure 4). The PIT method includes an exposure of the antibodies to UV irradiation, which leads to selective photoreduction of the typical disulfide bond of the antibodies in specific cysteine-cysteine/tryptophan triads (Cys-Cys/Trp). The breaking of these Cys-Cys bonds produces free thiol groups, which can interact with the proximal gold surface, resulting in a covalent Au-S bond between the antibody and the Au surface [12]. disk by PIT was successful, the Au electrode was characterized by CV before and after immobilization of the antibodies, using a redox probe Fe(CN)6 3-/Fe(CN)6 4- (Figure 4).
The PIT method includes an exposure of the antibodies to UV irradiation, which leads to selective photoreduction of the typical disulfide bond of the antibodies in specific cysteine-cysteine/tryptophan triads (Cys-Cys/Trp). The breaking of these Cys-Cys bonds produces free thiol groups, which can interact with the proximal gold surface, resulting in a covalent Au-S bond between the antibody and the Au surface [12]. The Au-disk electrode showed well-defined anodic and cathodic peaks, due to the reversible oxidation and reduction of the solution phase Fe(CN)6 3-/Fe(CN)6 4-redox molecule, with a peak-to-peak difference (ΔEP) of 112 mV (±2. 16) and an anodic current intensity (IP) of 54.1 µ A ± (1.87). After antibody immobilization and as the AntiCEA1 concentration increased, ΔEP increased and IP decreased, confirming that the antibodies were immobilized on the Au-disk since their covalent immobilization on the surface is acting as an insulating layer, causing slower electron transfer [24]. Besides, at the concentration of 30 µ g/mL of AntiCEA1, the surface of the Au-disk was practically saturated, since the change of IP from 30 to 50 µ g/mL was minimal. It should be noted that the higher the concentration of immobilized capture antibodies, the higher the impact on the available electroactive surface area of the Au-disk. In other words, there could be a tradeoff between receptor density for binding the target molecule and the efficiency of the charge transfer between the electroactive surface and thionine redox probes. Hence, it is important to find an optimal immobilization concentration that leads to a receptor surface coverage at which the biosensor generates the highest analytical signal. Consequently, the electrodes were modified with different concentrations of AntiCEA1 and exposed to the complete detection system (CEA antigen, and the nanoprobe NaPro) at a constant concentration.
In Figure 4B, a DPV voltammogram is shown. A redox peak around −0.22 V can be observed, which is characteristic for the Thiox/Thred redox couples [20]. This demonstrates that Thi was present on the NaPro and it underwent electron transfer reactions [25]. Since the amount of attached NaPro is related with the amount of CEA present in the electrode surface, Thi works as a redox tag for the electrochemical detection of CEA. The intensity of the redox peak of Thi is related to the concentration of the biomarker CEA. The highest The Au-disk electrode showed well-defined anodic and cathodic peaks, due to the reversible oxidation and reduction of the solution phase Fe(CN) 6 3-/Fe(CN) 6 4redox molecule, with a peak-to-peak difference (∆E P ) of 112 mV (±2. 16) and an anodic current intensity (I P ) of 54.1 µA ± (1.87). After antibody immobilization and as the AntiCEA 1 concentration increased, ∆E P increased and I P decreased, confirming that the antibodies were immobilized on the Au-disk since their covalent immobilization on the surface is acting as an insulating layer, causing slower electron transfer [24]. Besides, at the concentration of 30 µg/mL of AntiCEA 1 , the surface of the Au-disk was practically saturated, since the change of I P from 30 to 50 µg/mL was minimal. It should be noted that the higher the concentration of immobilized capture antibodies, the higher the impact on the available electroactive surface area of the Au-disk. In other words, there could be a tradeoff between receptor density for binding the target molecule and the efficiency of the charge transfer between the electroactive surface and thionine redox probes. Hence, it is important to find an optimal immobilization concentration that leads to a receptor surface coverage at which the biosensor generates the highest analytical signal. Consequently, the electrodes were modified with different concentrations of AntiCEA 1 and exposed to the complete detection system (CEA antigen, and the nanoprobe NaPro) at a constant concentration.
In Figure 4B, a DPV voltammogram is shown. A redox peak around −0.22 V can be observed, which is characteristic for the Thi ox /Th red redox couples [20]. This demonstrates that Thi was present on the NaPro and it underwent electron transfer reactions [25]. Since the amount of attached NaPro is related with the amount of CEA present in the electrode surface, Thi works as a redox tag for the electrochemical detection of CEA. The intensity of the redox peak of Thi is related to the concentration of the biomarker CEA. The highest current intensity was obtained with an AntiCEA 1 concentration of 15 µg/mL, therefore, this concentration was chosen for the further implementation of the immunosensor.
Moreover, the immobilization of the AntiCEA 2 on the SWCNH/Thi/AuNPs was also carried out by the PIT. To verify the immobilization effectivity, a glassy carbon electrode (GCE) was modified with the SWCNH/Thi/AuNPs/AntiCEA 2 , testing AntiCEA 2 concentrations of 50 and 100 µg/mL. Figure 5B shows the DPV plots of the SWCNH/Thi/AuNPs. At around −0.22 V a peak was observed that can be attributed to the redox reactions of the present thionine. The redox peak possessed a current intensity of 14.79 µA for the antibody-free SWCNH/Thi/AuNPs. This current intensity decreased after immobilization of AntiCEA 2 to 5.44 µA (50 µg/mL) and 0.37 µA (100 µg/mL), confirming that AntiCEA 2 was successfully immobilized. Since the concentration of 100 µg/mL significantly decreased the redox peak of thionine by blocking the charge transfer, 50 µg/mL of AntiCEA 2 was chosen as the optimal concentration to maintain high analytical sensitivity.
At around −0.22 V a peak was observed that can be attributed to the redox reactions of the present thionine. The redox peak possessed a current intensity of 14.79 µ A for the antibody-free SWCNH/Thi/AuNPs. This current intensity decreased after immobilization of AntiCEA2 to 5.44 µ A (50 µ g/mL) and 0.37 µ A (100 µ g/mL), confirming that AntiCEA2 was successfully immobilized. Since the concentration of 100 µ g/mL significantly decreased the redox peak of thionine by blocking the charge transfer, 50 µ g/mL of AntiCEA2 was chosen as the optimal concentration to maintain high analytical sensitivity.

Electrochemical Characterization by Fabrication Steps
CV and EIS were used to corroborate the immunosensor assembly process at each modification stage and to verify the binding of the biomarker CEA and the NaPro. Both characterizations provide information on the electron transfer process and specifically, the changes in charge transfer resistance caused by anchoring the insulating biomolecules on the gold electrode. The height of the redox peaks consecutively decreases after the addition of AntiCEA1, BSA, and CEA antigen, Figure 6A. This behavior is attributed to the fact that these biomolecules do not possess conductive properties, which on the one hand do not contribute to the transport of electrons and on the other hand block the diffusion of solution-phase redox probes to the surface of the electrode [26]. In the last step, where the NaPro is incorporated, the IPa increased and ΔEP decreased, indicating that the addition of the NaPro improves the electroactivity, due to the good conductive properties of the SWCNH and AuNPs, similar effect was found in previous reports [27,28]. The corresponding changes

Electrochemical Characterization by Fabrication Steps
CV and EIS were used to corroborate the immunosensor assembly process at each modification stage and to verify the binding of the biomarker CEA and the NaPro. Both characterizations provide information on the electron transfer process and specifically, the changes in charge transfer resistance caused by anchoring the insulating biomolecules on the gold electrode.
The height of the redox peaks consecutively decreases after the addition of AntiCEA 1 , BSA, and CEA antigen, Figure 6A. This behavior is attributed to the fact that these biomolecules do not possess conductive properties, which on the one hand do not contribute to the transport of electrons and on the other hand block the diffusion of solutionphase redox probes to the surface of the electrode [26]. In the last step, where the NaPro is incorporated, the I Pa increased and ∆E P decreased, indicating that the addition of the NaPro improves the electroactivity, due to the good conductive properties of the SWCNH and AuNPs, similar effect was found in previous reports [27,28]. The corresponding changes observed at each stage confirm that each component was successfully implemented in the system.
Electrochemical impedance spectroscopy (EIS) is an effective tool to characterize the electrode-electrolyte interface properties. The charge transfer resistance (R ct ) can be calculated from the semicircular section of the Nyquist plot with the axis for the real part of the impedance in EIS at low frequencies [29].
Fe(CN) 6 3− /Fe(CN) 6 4− was used as a redox couple for the EIS experiments, Figure 6B. According to the Nyquist plot, the R ct values were Au-disk (204.65 Ω ± 5.8), Au-disk/AntiCEA 1 (1679.19 Ω ± 15), Au-disk/AntiCEA 1 /BSA (8361.51 Ω ± 84), Audisk/AntiCEA1/BSA/CEA (22861.61 Ω ± 116), and Au-disk/AntiCEA 1 /BSA/CEA/NaPro (2360.66 Ω ± 44). The addition of AntiCEA 1 , BSA, and CEA antigen increased the diameter of the semicircle consecutively, indicating that these biomolecules enhanced the blocking of the charge transfer at the electrode interface. Interestingly, R ct decreased with the incorporation of the NaPro due to the highly conductive nature of the carbon nanohorns. The result of EIS coincided with the characteristics observed for CV; which demonstrates the successful implementation of a sandwich electrochemical immunosensor for the carcinoembryonic antigen detection.
Biosensors 2023, 13, 63 9 of 14 observed at each stage confirm that each component was successfully implemented in the system. Electrochemical impedance spectroscopy (EIS) is an effective tool to characterize the electrode-electrolyte interface properties. The charge transfer resistance (Rct) can be calculated from the semicircular section of the Nyquist plot with the axis for the real part of the impedance in EIS at low frequencies [29].  Figure 6B. According to the Nyquist plot, the Rct values were Au-disk (204.65 Ω ± 5.8), Au-disk/An-tiCEA1 (1679.19 Ω ± 15), Au-disk/AntiCEA1/BSA (8361.51 Ω ± 84), Au-disk/An-tiCEA1/BSA/CEA (22861.61 Ω ± 116), and Au-disk/AntiCEA1/BSA/CEA/NaPro (2360.66 Ω ± 44). The addition of AntiCEA1, BSA, and CEA antigen increased the diameter of the semicircle consecutively, indicating that these biomolecules enhanced the blocking of the charge transfer at the electrode interface. Interestingly, Rct decreased with the incorporation of the NaPro due to the highly conductive nature of the carbon nanohorns. The result of EIS coincided with the characteristics observed for CV; which demonstrates the successful implementation of a sandwich electrochemical immunosensor for the carcinoembryonic antigen detection.

Analytical Performance of the Immunosensor
The performance of the immunosensor for the CEA biomarker detection was investigated using DPV. The CEA antigen detection was carried out in PBS 0.01 M at pH 7.4. Figure 7A shows the immunosensor response at different concentrations of CEA. The DPV signals increased as the CEA concentration rose.
The sensing mechanism is attributed to the Thi used as a redox tag, since a multitude of these redox molecules was attached to the large surface area of the SWCNH. The amount of attached NaPro is related with the amount of CEA present in the electrode surface due to the formation of immunocomplex between CEA and AntiCEA2. Therefore, the change in the current intensity of the redox peak of Thi is related to the concentration of the biomarker CEA.
The calibration curve ( Figure 7B) showed a linear relationship between the current intensity of the thionine redox peak and the logarithm of the CEA concentration. The linear detection range extended from 0.001 to 200 ng/mL for CEA. The calibration curve equation was IP (nA) = 24.726 log CCEA (ng/mL) + 363.24 (R 2 = 0.964) and the limit of detection was calculated to be 0.1385 pg/mL defined as the mean of the blank signal and 3 times the relative standard deviation. It should be noted that the concentration of CEA in blood

Analytical Performance of the Immunosensor
The performance of the immunosensor for the CEA biomarker detection was investigated using DPV. The CEA antigen detection was carried out in PBS 0.01 M at pH 7.4. Figure 7A shows the immunosensor response at different concentrations of CEA. The DPV signals increased as the CEA concentration rose.
Biosensors 2023, 13, 63 10 of 14 serum is typically 3 ng/mL [4] in healthy individuals; therefore, the proposed immunosensor covers the medical relevant concentration range of the CEA biomarker and potentially facilitates practical application to monitor this biomarker. The promising performance of this sensor could be attributed to the high signal amplification capabilities of the highly conductive SWCNH/Thi/AuNPs and their decoration with a high number of redox active thionine. Compared with other previously reported methods in the literature (Table 1), our immunosensor advanced current detection technology in the combination of exhibiting a wider detection range and lower detection limits. It should also be noted that the preparation time of the previously reported systems is typically quite long because the incubation times for the immobilization of the antibodies can take several hours while here it required only 15 min thanks to the PIT activation and enhanced the immobilization via the thiol groups of the cysteines proteins. In addition, before immobilization, other methods require a modification of the electrode surface. Meanwhile, the PIT technique used in the present work does not require any additional surface modification steps, which decreases the total fabrication time to only 2.25 h, notably less than that of other reported techniques.
Besides, the immobilization of the antibodies by the PIT is very effective since it ensured control over the orientation of the immobilized Ab, with their binding sites exposed for the formation of the antigen-antibody immune complex [12,24,30]. Indeed, Funari et The sensing mechanism is attributed to the Thi used as a redox tag, since a multitude of these redox molecules was attached to the large surface area of the SWCNH. The amount of attached NaPro is related with the amount of CEA present in the electrode surface due to the formation of immunocomplex between CEA and AntiCEA 2 . Therefore, the change in the current intensity of the redox peak of Thi is related to the concentration of the biomarker CEA.
The calibration curve ( Figure 7B) showed a linear relationship between the current intensity of the thionine redox peak and the logarithm of the CEA concentration. The linear detection range extended from 0.001 to 200 ng/mL for CEA. The calibration curve equation was I P (nA) = 24.726 log C CEA (ng/mL) + 363.24 (R 2 = 0.964) and the limit of detection was calculated to be 0.1385 pg/mL defined as the mean of the blank signal and 3 times the relative standard deviation. It should be noted that the concentration of CEA in blood serum is typically 3 ng/mL [4] in healthy individuals; therefore, the proposed immunosensor covers the medical relevant concentration range of the CEA biomarker and potentially facilitates practical application to monitor this biomarker. The promising performance of this sensor could be attributed to the high signal amplification capabilities of the highly conductive SWCNH/Thi/AuNPs and their decoration with a high number of redox active thionine.
Compared with other previously reported methods in the literature (Table 1), our immunosensor advanced current detection technology in the combination of exhibiting a wider detection range and lower detection limits. It should also be noted that the preparation time of the previously reported systems is typically quite long because the incubation times for the immobilization of the antibodies can take several hours while here it required only 15 min thanks to the PIT activation and enhanced the immobilization via the thiol groups of the cysteines proteins. In addition, before immobilization, other methods require a modification of the electrode surface. Meanwhile, the PIT technique used in the present work does not require any additional surface modification steps, which decreases the total fabrication time to only 2.25 h, notably less than that of other reported techniques.
Besides, the immobilization of the antibodies by the PIT is very effective since it ensured control over the orientation of the immobilized Ab, with their binding sites exposed for the formation of the antigen-antibody immune complex [12,24,30]. Indeed, Funari et al. [11] investigated the immobilization and orientation of antibodies (Abs) photoactivated by PIT. In their experiments, the photoactivated antibodies were immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. They found smaller contact area and larger heights measured in the surfaces with the antibodies immobilized by PIT than the ones immobilized by physisorption. Therefore, the activated antibodies tend to be more upright compared with nonirradiated ones, thereby providing better exposure to the binding sites. The immobilization and orientation of antibodies photoactivated by PIT enhance the binding capabilities of antibody receptors, which is a critical aspect of immunosensor development because both the number and the orientation of the immobilized biomolecules are closely related to the detection efficiency of the device [31].

Selectivity
The high and evolutionary evolved specificity of antibodies is one advantage of immunoassays over competing biosensor concepts. To evaluate the specificity of the electrochemical immunosensor, a selectivity analysis was performed, spiking possible interfering agents such as bovine serum albumin (BSA), human serum albumin (HSA), or CA15-3 antigen to the blank sample solution (containing the nanoprobe without the presence of CEA). The tests were performed separately by incubating the Au-disk/AntiCEA 1 /BSA electrode surface in 50 ng/mL CEA, 50 ng/mL BSA, 50 ng/mL HSA, 50 U/mL CA15-3, and blank solution (0 ng/mL CEA). Although the interfering substances were applied under the same conditions as the real analyte, the response currents were much lower compared to the response toward CEA (Figure 8). This result indicates that these substances do not interfere with the target detection and the high selectivity of the antibodies was conserved during the implementation of the immunosensor, resulting in an immunosensor with excellent selectivity for CEA.

Selectivity
The high and evolutionary evolved specificity of antibodies is one advantage of immunoassays over competing biosensor concepts. To evaluate the specificity of the electrochemical immunosensor, a selectivity analysis was performed, spiking possible interfering agents such as bovine serum albumin (BSA), human serum albumin (HSA), or CA15-3 antigen to the blank sample solution (containing the nanoprobe without the presence of CEA). The tests were performed separately by incubating the Au-disk/AntiCEA1/BSA electrode surface in 50 ng/mL CEA, 50 ng/mL BSA, 50 ng/mL HSA, 50 U/mL CA15-3, and blank solution (0 ng/mL CEA). Although the interfering substances were applied under the same conditions as the real analyte, the response currents were much lower compared to the response toward CEA (Figure 8). This result indicates that these substances do not interfere with the target detection and the high selectivity of the antibodies was conserved during the implementation of the immunosensor, resulting in an immunosensor with excellent selectivity for CEA.

Real Sample Testing
To investigate the performance of the immunosensor for detection in real clinical samples, human serum samples with known CEA concentrations were analyzed, Table 2. The standard addition method was used to corroborate electrochemical detection. CEA concentrations were calculated from the calibration curve and the tests were repeated three times for each sample. Table 2 shows the recovery (%) of the serum samples found in the range of 95 to 113%. The successful results demonstrate high accuracy and the feasibility of using the immunosensor for the electrochemical detection of CEA in real clinical samples. Therefore, the results confirm the potential of the proposed method to be implemented in the clinical field for the detection and monitoring of the carcinogenic biomarker CEA in patients.

Real Sample Testing
To investigate the performance of the immunosensor for detection in real clinical samples, human serum samples with known CEA concentrations were analyzed, Table 2. The standard addition method was used to corroborate electrochemical detection. CEA concentrations were calculated from the calibration curve and the tests were repeated three times for each sample. Table 2 shows the recovery (%) of the serum samples found in the range of 95 to 113%. The successful results demonstrate high accuracy and the feasibility of using the immunosensor for the electrochemical detection of CEA in real clinical samples. Therefore, the results confirm the potential of the proposed method to be implemented in the clinical field for the detection and monitoring of the carcinogenic biomarker CEA in patients.

Conclusions
In this work, a sandwich-type electrochemical immunosensor was developed for the quantitative determination of the CEA biomarker using a signal amplification strategy based on carbon nanohorns. The fast photochemical immobilization technique (PIT) was employed for both capture and detection antibodies to tether them onto the gold electrode and the SWCNH/Thi/AuNPs, respectively, which facilitated short assay assembly times of less than three hours. The immunosensor showed a low detection limit of 0.1385 pg/mL, a linear detection range from 0.001-200 ng/mL, and high selectivity. The remarkable performance was attributed on the one hand to the antibodies being covalently bound to the gold surfaces by PIT, controlling the orientation of their active sites. On the other hand, the large surface area, high conductivity, and manifold thionine redox activity of the SWCNH/Thi/AuNP nanocomposite enhanced the amperometric sensor signal, which resulted in a high sensitivity of the device. Therefore, the proposed strategy of PIT antibody immobilization and SWCNH/Thi/AuNP nanocomposite-based signal amplification can be used as a versatile strategy for the clinical detection of the CEA biomarker and could potentially be extended for the clinical detection of other relevant biomarkers.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Kommission für Ethik in der Forschung (KEF) of the Research Center Jülich (protocol code Humanserum and date of approval: 06.Jan.2022).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.