Carbon Material Hybrid Construction on an Aptasensor for Monitoring Surgical Tumors

Carcinoembryonic antigen (CEA) is a glycoprotein, one of the common tumor biomarkers, found at low levels in body fluids. Generally, overexpression of CEA is found in various cancers, including ovarian, breast, lung, colorectal, gastric, and pancreatic cancers. Since CEA is an important tumor biomarker, the quantification of CEA is helpful for diagnosing cancer, monitoring tumor progression, and the follow-up treatment. This research develops a highly sensitive sandwich aptasensor for CEA identification on an interdigitated electrode sensor. Carbon-based material was used to attach a higher anti-CEA capture aptamer onto the sensor surface through a chemical linker, and then, CEA was quantified by the aptamer. Furthermore, CEA-spiked serum was tested by using the immobilized aptamer, which was found to not affect the target validation. The limit of detection for CEA in PBS and serum is calculated from a linear regression graph to be 0.5 ng/mL with R2 values of 0.9593 and 0.9657, respectively, over a linear range from 0.5 to 500 ng/mL. This CEA quantification by the aptasensor can help diagnose various surgical tumors and monitor their progression.


Introduction
Tumor biomarkers have a great value for diagnosing and screening tumors and help enhance the efficacy of evaluation. Tumor biomarkers are substances that can be attributed to the development of carcinogenesis at different stages of cell progression [1,2]. Common tumor biomarkers are sugar lipids, protein, microRNA, RNA, and DNA. Carcinoembryonic antigen (CEA) is a glycoprotein expressed during the development of human fetuses. After birth, the expression of CEA is inhibited and found to be at a much lower level in healthy adults [3]. At the same time, the overexpression of CEA has been found in various human surgical and nonsurgical cancers, including gastric, lung, ovarian, breast, pancreatic, and colorectal cancers [4][5][6][7]. erefore, quantifying CEA levels helps significantly in diagnosing cancer and monitoring the therapeutic condition. Current CEA identification in immunoassays requires expensive instruments and complex procedures. erefore, a highly sensitive, selective, cost-effective, and rapid CEA biosensor is mandatory to detect and quantify the level of CEA.
Various biosensing techniques have been developed for the identification of CEA in human serum, such as radioimmunoassay, mass spectrometry, and enzyme-linked immunosorbent assay [8,9]. Most biosensing methods are time consuming, expensive, and require complex procedures.
us, developing a method with simple, low sensitivity, sensitivity, and speed is needed for CEA quantification. We used an electrochemical method with a carbon nanomaterial-modified electrode to detect CEA levels. Nanomaterial-applied biosensors have attracted increasing attention in the fields of medicine and diagnosis. e use of nanosized materials has led to enhanced detection of target molecules at a lower detection limit with increased sensitivity, faster detection, and lower cost [10][11][12][13]. Furthermore, the larger surface area of the nanomaterials enables the bioreceptor immobilization of an enhanced amount. Common nanomaterials, such as gold, graphene-derived materials, silver, aluminum, and silica, are more popular for various biomedical applications [14][15][16]. A biosensor basically consists of four major paths: a bioreceptor, transducer, signal processer (convert electrical to desired signal), and a display interface. In biosensor applications, nanomaterials are primarily used as transducer materials, which help to enhance the electrical signal upon the binding of biomolecules on the sensor surface. Apart from that, nanomaterialconjugated biomolecules improve the stability of the molecules with a higher number of immobilizations on the sensing surface and enhance the biomolecular interaction. In particular, one-, two-, and three-dimensional carbon-based micro/nanomaterials, such as carbon nanotubes, carbon quantum dots, graphene, nanodiamonds, nanofibers, and carbon nanohorns, have been effectively utilized in various biomedical fields due to their significant mechanical, electronic, thermal, optical, and physical properties [17][18][19]. In addition, carbon materials in electrochemical sensors have been successfully proven to show better sensitivity, higher selectivity, and biocompatibility to identify a wide range of biological and chemical biomolecules [20].
is research work used carbon material for surface functionalization of an anti-CEA aptamer attachment on a sensor surface and enhanced the electric signal upon binding of CEA with its aptamer.
Similar to antibodies, aptamers have been found to act as efficient detection probes for various biomolecules [21][22][23]. e aptamer is a recently emerged molecule generated from the randomized library of molecules by a SELEX method. Aptamers are single-stranded RNA or DNA molecules that can specifically bind to targets, including peptides, proteins, small molecules, carbohydrates, toxins, and whole cells [24,25]. Aptamers form various structures due to their tendency to form single-stranded loops and helices. Aptamers bind with the target via their secondary/tertiary structures rather than the primary sequence, which helps to bind with high sensitivity and selectivity. Due to their high binding affinity, aptamers are a focus in the field of biosensors for early diagnosis [26][27][28][29]. Aptamers have been selected against a wide range of targets, including tumor biomarkers. Circulating oligonucleotides, such as RNA and DNA, are commonly used as probes to identify various diseases, including tumors. In this research, an aptamerbased sandwich assay was conducted with specific quantitative analysis with CEA.

Activation of Carbon Material.
e carbon material (graphene) (5 mg) was mixed with 50 mL of HCl and heated under reflux with stirring for 10 h. en, the purified graphene was mixed with acids (1 : 3 v/v ratio of HNO 3 : H 2 SO 4 ) and sonicated for 10 h at room temperature. e product was further washed with distilled water until reaching neutral pH to obtain carboxylated graphene (COOHgraphene).
e working IDE was first washed with deionized water and dried under nitrogen flow. en, the surface was treated with KOH for 10 min and thoroughly washed again using distilled water. Furthermore, COOH-graphene was carefully drop cast onto the KOH-treated electrode. en, the activation of the COOH group in graphene was conducted by treating the electrode with EDC-NHS for 10 min. After the activation process, 1 μM anti-CEA aptamer (amine-terminated aptamer) was introduced onto IDE and incubated for 2 h. Subsequently, 1% BSA was drop cast onto the aptamer-modified electrode and kept for 1 h to cover the unbound sites. Finally, IDE was washed with PBS buffer to remove the excess reagents and biomolecules. e electrochemical response was recorded for each immobilization process.

Quantification of CEA by Aptamer-CEA-Aptamer Sandwich
Assay. An aptamer-based sandwich assay was conducted to quantify the level of CEA. CEA concentrations ranging from 0.5 ng/mL to 1000 ng/mL were titrated and drop cast onto the anti-CEA aptamer electrode surface. en, 1 μM of anti-CEA aptamer (detection aptamer sequence) was drop cast onto all the CEA-immobilized surfaces individually. e electrochemical response was recorded for each CEA concentration, and then, the difference in the current value was plotted in an Excel worksheet to calculate the linear regression coefficient. From this graph, the detection limit for CEA was calculated.

Quantification of CEA in Human Serum.
To identify the clinical feasibility of the developed aptasensor, experiments were conducted with CEA-spiked serum. CEA at concentrations ranging from 0.5 ng/mL to 1000 ng/mL spiked independently in diluted human serum, and the same experiments were conducted as described previously. e electrochemical responses were recorded for each concentration, and the current value was compared with CEAspiked PBS (without serum). e limit of detection was determined as the lowest concentration of an analyte (on the calibration line with low concentrations) and compared to the background signal (S/ N � 3 : 1) and limit of detection � standard deviation of the baseline + 3σ. All measurements were performed by using a picoammeter (Keithley) over a voltage range of 0-2 V with sweep intervals of 0.1 V. e whole setup was kept at room temperature, and all surface modifications and interactions were performed under wet conditions, either using double distilled water (for chemical modification) or 10 mM phosphate buffered saline at pH 7.4 (for biological interaction). Washings with ten reaction volumes of the respective solutions were carried out before the measurements were taken.

Results and Discussion
An aptasensor for the tumor biomarker CEA was generated on a graphene-modified interdigitated electrode (IDE) surface. Figure 1 shows a graphical demonstration of the CEA aptamer-based assay. A potassium hydroxide-treated electrode surface was constructed with COOH-activated graphene and then activated with EDC-NHS to activate COOH groups on graphene to be converted into NH 2 . On the activated surface, the anti-CEA aptamer was covalently immobilized onto the electrode surface by COOH with NH 2 . Furthermore, BSA was added as a blocking agent to cover the excess activated surface. Finally, CEA was detected by a sandwich assay with the detection aptamer on the capture aptamer-constructed electrode surface. A two-dimensional graphene sheet was used for the surface modification on IDE. IDE was prepared on silica materials, and the dielectrode fingers were fabricated using aluminum. e fabricated dielectrode structure had a length of 8000 μm and a width of 5000 μm. e gap width among the 18 pairs of fingers created by the dielectrode was 100 μm. Furthermore, the finger length was measured to be 4000 μm, and the electrode thickness was 55 nm. e intactness of the received electrode surface was validated under a high-power microscope (Figure 1).

Imaging and EDX Analysis of Graphene.
e shape and size of the graphene image were analyzed by FESEM and FETEM observations (Figures 2(a) and 2(b)). As shown in figure, the graphene is observed to be a flake with a uniform distribution. e EDX result confirms the presence of the major elements of carbon (74.97%) and O (15.33%) in the graphene (Figure 2(c)).

Surface Functionalization with Anti-CEA Aptamer.
Surface functionalization of the anti-CEA aptamer process was confirmed by an IDE sensor. e electrochemical response for each process was recorded and the immobilization process was confirmed (Figure 3(a)). e bare electrode shows a response of 2.04E−08 A, which is changed to 5.08E−08 A after treatment with KOH. e activated graphene further increases the response to 9.38E−08 A. is increased response clearly indicates the attachment of graphene on the electrode surface. Furthermore, the anti-CEA aptamer shows the highest electrochemical response of 1.74E−07 A. e difference in the response is observed to be much higher after the introduction of the aptamer on the sensing electrode, which confirms that the surface is modified with the anti-CEA aptamer (Figure 3(b)). Finally, BSA was added for blocking, and no significant response was noted because the activated sites on graphene are fully covered with anti-CEA aptamer. is result confirms that a higher number of anti-CEA aptamers is attached onto the electrode surface through graphene, which helps to lower the detection limit for CEA. Various studies have proven that a higher number of captured biomolecules immobilized with the right arrangement leads to a higher number of target molecules. In addition, graphene material-conjugated biomolecules are stable and help to increase the binding of biomolecules on the sensing surfaces. In this research, graphene is found to help to increase the anti-CEA aptamer immobilization on IDE and increase the electrochemical response upon the interaction of CEA with its aptamer.  Figure 1: Graphical demonstration of the CEA aptasensor. A potassium hydroxide-treated electrode was constructed using COOHactivated graphene, and then, the anti-CEA aptamer was covalently immobilized onto the electrode surface through COOH with NH2. BSA was added to cover the excess activated surface, and then, the CEA was detected by the immobilized aptamer.    Figure 3: (a) Surface functionalization for anti-CEA aptamer immobilization. e electrochemical response for each process was recorded, and the immobilization was confirmed. is increment in response clearly indicates the attachment of the anti-CEA aptamer on the electrode surface. (b) Electrochemical response for anti-CEA aptamer attachment. e difference in response is observed to be much higher after the introduction of the aptamer on the sensing electrode surface, which confirms that the surface is modified with the anti-CEA aptamer process. e averaged values obtained from the replicates are displayed by the error bars.

Sandwich Assay of Aptamer-CEA-Aptamer on Graphene-
Aptamer Immobilized Electrode Surface. Different concentrations of CEA were diluted in PBS and drop cast onto an anti-CEA aptamer-constructed electrode surface independently for CEA identification, and then, the fixed capture aptamer was added onto the surface. e lowest CEA concentration of 0.5 ng/mL was added to the electrode surface, and the current response was measured to be 3.87 E−07 A. e difference in current was determined to be 2.13 E−07 A, which confirms the binding of 0.5 ng/mL CEA with the immobilized aptamer (Figure 4(a)). By further increasing the CEA concentration to 5, 50, 500, and 1000 ng/mL, the responses were increased to 5.01 E−07, 6.62 E−07, 7.29 E−07, and 7.82 E−07 A, respectively. e electrochemical response was increased by increasing the CEA concentration ( Figure 4(b)).

CEA Identification from Spiked Serum Samples.
Various concentrations of CEA were dissolved in 1 : 100 diluted human serum and drop cast onto an anti-CEA aptamer-constructed electrode surface for CEA identification. Initially, the lowest CEA concentration at 0.5 ng/ mL was added to the electrode surface, and the current response was measured to be 3.48 E−07 A. e difference in current was determined to be E−07 A, which confirms the detection of 0.5 ng/mL CEA in the spiked serum sample assisted by the immobilized aptamer ( Figure 5(a)). Furthermore, by increasing CEA concentrations in human serum to 5, 50, 500, and 1000 ng/mL, the responses were found to be increased to 4.51 E−07, 6.03 E−07, 6.89 E−07, and 7.3 E−07 A, respectively. Electrochemical responses were increased by increasing the CEA concentrations in the diluted serum ( Figure 5(b)). is result shows the clear detection of CEA in human serum without any interferences.

Comparison of CEA Detection in Spiked PBS and Human
Serum. Similar concentrations of CEA (0.5-1000 ng/mL) were diluted in PBS or human serum and detected on the anti-CEA aptamer-constructed IDE sensor surface. e difference in response (from blank) was plotted on a linear regression graph and compared. As shown in Figure 6, CEAspiked serum shows a slightly lower response than CEAspiked serum in PBS, and the response of CEA at all concentrations increased with increasing CEA concentration. In addition, the limit of detection for CEA is found to be 0.5 ng/mL in both PBS and serum spiked samples. is result confirms the selective and effective identification of CEA in the biological sample.

Conclusion
Carcinoembryonic antigen (CEA) is a glycoprotein found to be a common biomarker for various tumors. Quantifying the level of CEA helps to diagnose the tumor and its condition at earlier stages. is research developed an aptamer-based sandwich assay for CEA identification on graphene-modified interdigitated electrodes. Higher anti-CEA capture-aptamer immobilization was achieved through graphene, and CEA was quantified by aptamer detection. Furthermore, CEA-spiked serum was found to show an increased electrochemical signal when CEA concentration was increased, which confirmed the detection of CEA in the biological fluid. e limit of detection for CEA in PBS and serum is calculated to be 0.5 ng/mL, with R 2 values of 0.9593 and 0.9657, respectively. is CEA biosensor can help to quantify CEA at its lower level and diagnose various tumors.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Disclosure e funder had no role in the design, analysis, or writing of this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  Figure 6: Limit of detection of CEA in PBS and human serum. Similar concentrations of CEA were diluted in PBS and human serum and detected on an anti-CEA aptamer-constructed IDE sensor. e difference in the response (from blank) was plotted by a linear regression graph. e limit of detection for CEA is found to be 0.5 ng/mL in both PBS and serum-spiked samples. Figure insets show the diagrammatical representations.