Highly sensitive and selective detection of the pancreatic cancer biomarker CA 19-9 with the electrolyte-gated MoS2-based field-effect transistor immunosensor

Since evaluating CA 19-9 antigen level in human serum is crucial for the early diagnosis of a vast range of diseases, especially pancreatic cancer, applying a simple, rapid, and sensitive detection method is essential. We employed an electrolyte-gated field-effect transistor with MoS2 nanosheets channel as an immunosensor to recognize CA 19-9 tumor marker. In order to obtain MoS2 nanosheets and use them as a semiconducting channel, the liquid phase exfoliation method was performed. Later, the MoS2 channel surface was modified by covalent immobilization of antibody 19-9. Electrical measurements revealed the depletion mode n-type behavior of MoS2 nanosheets with the FET mobility of 0.02 cm2 V-1 s-1, current on/off ratio of 883.96, and the subthreshold swing of 795.54 mV/decade. Due to the n-type behavior of the MoS2-based FET immunosensor, with increasing the concentration of the CA 19-9 antigen at a wide linear concentration range from 1.0×10-12 U/ml to 1.0×10-4 U/ml, the source-drain current decreased and low detection limit of 2.8×10-13 U/ml was obtained. The designed MoS2-based FET immunosensor, owning high selectivity, performed accurately for trace amounts of real human serum samples. The remarkable properties of this immunosensor enable the diagnosis of pancreatic cancer in the early stages, which increases the chance of curing this disease.


Introduction
Pancreatic cancer is a highly invasive disease with a high mortality rate and the early detection of it is an important issue [1]. Only 8% of patients with pancreatic cancer can live more than five years after diagnosis [2], and only 15% of patients can undergo tumor resection at the time of diagnosis [1,3]. This is due to the flaw in the early detection of the disease since the symptoms appear only when the cancer is in its final stages or has metastasized [4]. For the early detection of pancreatic cancer, several biomarkers have been studied in diagnostic methods. However, because of their low sensitivity and specificity, these biomarkers could not identify whether the disease is benign or malignant [5]. But studies have clarified the significant role of carbohydrate antigen  in the diagnosis and treatment management of pancreatic cancer [6][7][8]. Based on the reports, tumor size, stage, burden [9], resection capability, postoperative relapse, and prognosis are associated with CA 19-9 level [10,11]. Besides, the impacts of chemotherapy or chemoradiotherapy on the tumor can be deduced from CA 19-9 level during the treatment [12][13][14]. So far, several techniques including fluorescence [15], chemiluminescence [16,17], electrochemiluminescence [18,19], enzyme-linked immunosorbent assay (ELISA) [20], and surface plasmon resonance (SPR) biosensors [21] have been used to detect and measure CA 19-9 in serum. Nevertheless, the time-consuming and complicated measurement processes, high-cost equipment, and low sensitivity are the drawbacks of many mentioned techniques [22]. Therefore, new methods are required to be able to provide accurate results in the shortest time with high sensitivity and selectivity and also be able to detect the lowest levels of CA 19-9 antigen in small amounts of serum samples.
Biosensors are constructed of a transducer that converts the changes in the physical or chemical properties of the substance into a measurable signal. Among different kinds of transducing mechanisms, electrochemical transducers are preferred [23]. Lately, electrochemical biosensors have replaced conventional techniques due to their simplicity and accuracy, and low material consumption and high efficiency [24]. The surface of the electrochemical immunosensors can be modified with a variety of nanomaterials to restrict nonspecific interactions between molecules and analytes [25]. These modified electrochemical immunosensors are worthy for detecting many diseases such as acute myocardial infarction [24], acute kidney injury [26], small cell lung cancer (SCLC) [27], Alzheimer's [28], COVID-19 [25], endometriosis [29] and different types of cancers [30] due to the selective binding of antigen with its specific antibody along with other advantages that were mentioned earlier. Electrochemical transducers can be varied based on their electrical interface with substance. Among these electrical interfaces, field-effect transistor (FET)-based biosensors are famous for their beneficial features like small size, low cost, high sensitivity, and selectivity [23].
FET-based biosensors are gaining much more attention for their noble properties, such as fast response time, label-free and in-situ detection, simple apparatus configuration, and low power utilization [23,31]. FET-based biosensors have been employed in various fields, including the food industry, agriculture, clinical analysis, and the environment, also for the detection of many types of target analytes sensitively and selectively [32], such as PBASE-modified graphene FET biosensor for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [33], single-walled carbon nanotubes (SWNTs)-based field-effect transistor biosensor for detecting human serum albumin (HSA) [34], NiO-FET biosensor for lactate and HIV-1 gene detection [35]. One of the most substantial components of biosensors is the bioassay element, which defines the type of biosensor. Catalytical FET-based biosensors comprise enzymes or tissues as bio-recognition elements, and affinity FET-base biosensors consist of antibodies, membrane receptors, or nucleic acids functioning as bio-recognition elements [36]. Since the FET sensors function via conductance variation of the FET channel, the type of channel material has a tremendous impact on enhancing the sensor performance. Semiconductor nanomaterials have exhibited superior performance as channel materials than bulk semiconductors. Among semiconductor nanomaterials, two-dimensional nanomaterials possess remarkable properties that make them a supreme candidate for FET channel material. In contrast one-dimensional semiconductors, 2D semiconductors are capable to generating a better connection with electrodes and providing a better surface for target molecules due to their tunable broad nanosheets [31]. Transition metal dichalcogenides (TMDs) are a new generation of 2D layered materials owning a transition metal atom such as Mo, W, etc., and two chalcogen atoms like Se, Te, or S in their formula [37,38]. These atoms are placed together in a layer via a covalent bond [39]. These layers are held together through weak Van der Waals interactions, so layers can be exfoliated easily to attain few-layered thin TMDs [40][41][42][43]. Unique crystal structures, atomic-scale thickness, strong spin-orbit coupling, and remarkable electronic and mechanical properties of TMDs make them qualified nominees for application in numerous fields, including electronics/optoelectronics [44], catalysis [45], energy storage and conversion [46,47], flexible electronics, spintronics [48] and sensing [49,50].
MoS 2 can be cited as a good example of TMD semiconductors. MoS 2 synthesis techniques can be categorized into two groups; top-down techniques that include mechanical exfoliation, liquid-phase exfoliation, and sputtering, and bottom-up techniques e.g., physical vapor deposition (PVD), chemical vapor deposition (CVD), atomic layer deposition, and chemical solution [51]. The amount, form, and scale of the acquired MoS 2 vary in each method [52]. Exfoliation procedures (e.g., mechanical exfoliation [40,53] or liquid phase exfoliation [54]) can be followed to delaminate bulk MoS 2 crystals, which are comprised of layers packed together through fragile Van der Waals forces [55]. Due to excellent optical, electrical, chemical properties, and biocompatibility of MoS 2 , it has been widely applied in various fields such as environmental applications [56][57][58], electrocatalysis (especially for hydrogen-evolution reaction (HER)), photocatalysis, batteries, biological applications [54] (for detecting and curing diseases such as curing cancer and Alzheimer [52]), electronic devices and sensors. One of the advantages of MoS 2 is a tunable band gap that varies depending on the thickness of the MoS 2 . Bulk MoS 2 demonstrates an indirect band gap of 1.3 eV. However, on the contrary, single-layer MoS 2 exhibits a direct band gap of 1.8-1.9 eV [54], notable mobility up to 200 cm 2 V -1 s -1 at 250 K and excellent current on/off ratio more than 1×10 8 at room temperature for field-effect transistors based on single-layer MoS 2 [55]. Furthermore, theoretical measurements have predicted the mobility of monolayer MoS 2 in the range of 10-1000 cm 2 V -1 s -1 at room temperature and up to 10 5 cm 2 V -1 s -1 at low temperatures, depending on several factors, namely the impurity density and the dielectric environment [59][60][61].
In this research, our goal was to integrate the positive characteristics of field-effect transistors with MoS 2 nanosheets (as channel material) which leads to the extraordinary performance of the MoS 2 -based field-effect transistor immunosensor for the detection of CA 19-9 antigen. To this end, dispersed MoS 2 nanosheets were dropped and dried on the channel surface. To strengthen the bond between MoS 2 and CA 19-9 antibody, the MoS 2 channel surface was modified by applying 1-naphthylamine (NA) and glutaraldehyde (GA). Subsequently, CA 19-9 antibody was immobilized on the MoS 2 channel surface for later measurements. Eventually, the MoS 2 -based FET immunosensor was employed for detecting and measuring the amount of CA 19-9 antigen in the prepared samples. The outcomes verified the high sensitivity and selectivity of the introduced biosensor and its excellent performance for low quantities of real serum samples. MoS 2 -based FET immunosensor provides an important opportunity for diagnosing pancreatic cancer at early stages or precise monitoring of the tumor treatment process.

Fabrication of MoS 2 -based FET immunosensor
The procedure of constructing the field-effect transistor on a glass substrate has been demonstrated in Fig. 1. First, to increase the adhesion of the gold electrode to the substrate, the glass substrate was washed and cleaned with acetone, alcohol, and deionized water (Fig. 1a). Then, the glass substrate was immersed in a solution of hydrogen peroxide and sulfuric acid in a ratio of 3 to 7 overnight to form hydroxyl functional groups on its surface. After that, by applying thermal evaporation of gold granules (99.99% pure) and passing it through a patterned mask (Fig. 1b), Au source and drain electrodes with a thickness of 30 nm are deposited on the glass substrate to create FET channels with a length of 30 μm and width of 1.0 mm. According to Fig. 1c, four pairs of Au source and drain electrodes are formed on the glass substrate. Each pair of source and drain electrodes act as an independent field-effect transistor. Therefore, by cutting each pair of source-drain electrodes, eventually, four FETs will be obtained. To facilitate the connection of the FETs to the PGSTAT device, two copper wires were attached to the source-drain electrodes via silver glue (Fig. 1d). In this work, Ag/AgCl reference electrode was applied as the gate electrode.
To prevent direct interaction of the electrolyte and Au electrodes, and also to reduce the current leakage between the source and gate electrodes during the measurements, Au electrodes were covered with silicone sealant (Fig. 2a). To prepare MoS 2 nanosheets, 1.0 mg of MoS 2 crystal was added to 1.0 ml of DMF and sonicated for 2 hours. Then, 20 μl of dispersed MoS 2 nanosheets were placed in the 20 μl cell on the electrodes and dried at 70 °C (Fig. 2a). The MoS 2 -channel surface must be modified to improve the antibody-MoS 2 junction. For this purpose, 20 μl of 1-naphthylamine solution (20 μM in methanol) was placed on MoS 2 for 3 hours and then washed with phosphate buffer solution (0.01 M and pH 7.4). 1-naphthylamine was stacked on the MoS 2 surface through van der Waals interactions between the naphthalene group of the 1-naphthylamine and the MoS 2 plane (Fig. 2b). Afterward, 10 μl of 2% (v/v) glutaraldehyde was added and after 3 hours it was washed with PBS. Addition of glutaraldehyde results in a Schiff-base reaction between the aldehyde group of glutaraldehyde and the amine group of 1-naphthylamine (Fig. 2c). 20 μl of CA 19-9 antibody was incubated at 4 °C for 20 hours. Again, a Schiff-base reaction took place between antibody and glutaraldehyde ( Fig. 2d). After stabilization of the antibody, the MoS 2 FET immunosensor is ready to detect CA 19-9 antigen. The time required for the antigen to bind to the antibody is 20 minutes (Fig. 2e). The schematic of the MoS 2 -based FET immunosensor equipped with a liquid gate electrode is illustrated in Fig. 2f.

Results and discussion
Characterization Figure 3a shows the SEM image of bulk MoS 2 crystals in which the size of flakes is relatively large, around 3 to 40 micrometers. The SEM image displayed in Fig. 3b is related to the dispersed MoS 2 which demonstrates the formation of large and thin MoS 2 nanosheets. The advantages of the large surface area of nanosheets are that they can be well established between the source and drain electrodes, and they also enhanced the charge transfer. The optical properties of dispersed MoS 2 nanosheets were investigated by employing UV-Vis spectroscopy. The UV-Vis spectrum, which is illustrated in Fig. 3c, consists of four characteristic absorption peaks. The marked absorption peaks at 615 and 676 nm are attributed to excitonic transitions at the K-point of the Brillouin zone of few-layer molybdenum disulfide, and the peaks at 410 and 450 nm are related to the direct excitonic transition from the deep valence band to the conduction band [37,[62][63][64]. The energy band gap of MoS 2 nanosheets was calculated via the Tauc plot (Fig. 3d). The value of 1.89 eV indicated the presence of single-layer MoS 2 nanosheets with a direct band gap [65,66].

Electrical characterization
To investigate the electrical properties of the molybdenum disulfide channel, source-drain current vs. source-drain voltage (I DS -V DS ) diagrams were plotted under different gate voltages (V G ) ranging from 0.0 to 1.0 V (with steps of 0.1 V) (V DS from 0 to +0.4 V). According to Fig. 4a, as the gate voltage increases from zero to more positive voltages, the source-drain current increments, indicating an n-type behavior of the transistor channel. The positive voltage of the gate electrode increases the number of electrons in the channel via an electrostatic field, and thus, current is transmitted through electrons. Therefore, by controlling the density of electron carriers at the MoS 2 surface, the current passing through the channel can be adjusted.
The transfer diagram (I DS -V G ) of the proposed MoS 2 -based FET immunosensor was drawn at a constant V DS of 0.4 V (Fig. 4b). Here, W and L stand for the width and length of the FET channel, respectively. C is the specific capacitance of the oxide layer of the transistor, which, in liquid-gated FET, it is equivalent to the double-layer capacitance (C dl ). EIS measurements were employed for calculating the C dl . The Nyquist plot and the corresponding equivalent electrical circuit used for fitting are demonstrated in Fig. 4c. A frequency range from 100 kHz to 0.1 Hz and V G of 0.8 V (dc bias voltage) was applied to the circuit. The obtained specific capacitance (C dl ) value was 5.57 μF/cm 2 , and the FET mobility was calculated to be 0.02 cm 2 V -1 s -1 . Figure 4d exhibits the results of output current measurements before and after MoS 2 modification, after stabilization of CA 19-9 antibody, and after incubation of CA  (1) antigen with a concentration of 1.0×10 -5 U/ml. As can be seen, the slope of the source-drain current-voltage diagram decreases after each step. This is due to the electrostatic gating effect of NA, GA, CA 19-9 antibody, and CA 19-9 antigen. These compounds reduce the effective gating field of the gate electrode with positive voltage because of a negative charge in their structures. So, the channel conductivity was decreased due to electron carrier decrements.

Sensitivity of the immunosensor towards detection of CA 19-9 antigen
The binding of the CA 19-9 antigen to the CA 19-9 antibody changes the output signal of the immunosensor. To identify the sensing performance of the MoS 2 -based FET immunosensor, I DS -V DS diagrams (V DS from zero to 0.4 V) at a constant gate voltage of 1.0 V, after adding different concentrations of CA 19-9 antigen from 1.0×10 -12 U/ml to 1.0×10 -4 U/ml were drawn (Fig. 5a). According to the diagram, the source-drain current decreases when the concentration of CA 19-9 antigen increases. CA 19-9 antigen will have a negative charge due to owning an isoelectric point lower than the pH of PBS used (7.4), hence because of the negative gating effect and consequently the discharge of negative charge carriers (electrons) at the MoS 2 surface, it acts as a gate electrode with a negative voltage and reduces the conductivity of MoS 2 . Fig. 5b represents the calibration curve corresponding to this downward trend. The detection limit of the procedure used in this work for CA 19-9 detection was measured by applying the 3σ/S method, which was 2.8×10 -13 U/ml. As shown in Table 1, compared to other CA 19-9 detection methods, the FET-based immunosensor with MoS 2 channel has a much lower limit of detection (LOD) and a wide linear range.

Real sample measurements
The application of the proposed immunosensor to analyze real samples was also assessed. For this purpose, a negative serum sample was used. As previously mentioned, the normal amount of CA 19-9 antigen in a healthy blood sample is between 0 U/ml to 37 U/ml. CA 19-9 antigen concentration in the used negative serum sample was determined via the ELISA method by the medical laboratory and was reported as 4.96 U/ml. Thereupon, to perform the measurements using the MoS 2 -based FET immunosensor, the serum was first diluted step by step (4.96×10 -8 U/ml) to bring the response within the linear range of the proposed technique. According to the standard addition method, four samples were prepared. They consist of the real sample and standard solutions with concentrations of 0, 4, 8, and 12 (×10 -8 U/ml), respectively. I DS -V DS diagrams were plotted for each of these prepared solutions (Fig. 5d). Next, the magnitude of current was measured for each one at a specific sourcedrain voltage (0.35 V), and the corresponding calibration diagram was drawn (inset of Fig. 5d). The concentration of CA 19-9 antigen in the negative serum sample was extracted through the calibration diagram (4.59 U/ ml), which is very close to the value determined via the ELISA test. Furthermore, the recovery and relative standard deviation percentages for the proposed liquidgated MoS 2 -based FET immunosensor were calculated, the values of which are 95.64-103.35% and 2.85-3.57%, respectively ( Table 2). These values reveal that employing a FET immunosensor with the MoS 2 channel to detect CA 19-9 antigen in human serum samples is a highly effective and reliable approach.

Conclusion
In summary, we offered an immunosensor for CA 19-9 detection and measurement by utilizing a field-effect transistor and employing semiconducting MoS 2 nanosheets as channel material. Molybdenum disulfide nanosheets are very beneficial for this application due to their superior electrical properties as well as the large specific surface area of the nanosheets. Therefore, they can establish an adequate connection between source-drain terminals and represent a desirable surface for biomolecules to bind. MoS 2 nanosheets exhibited a depletion mode n-type behavior with a current on/off ratio of 883.96, a subthreshold swing of 795.54 mV/decade, and the mobility of 0.02 cm 2 V -1 s -1 at room temperature in the proposed FET equipped with liquid gate. The superiorities of the MoS 2 -based FET immunosensor are its very low detection limit of 2.8×10 -13 U/ml and wide linear range (1.0×10 -12 to 1.0×10 -4 U/ml) compared to other methods. This immunosensor was able to detect CA 19-9 marker with high accuracy and rapidity in a human serum sample. It also had excellent recovery with relatively low standard deviation percentages. Thus, the proposed MoS 2 -based FET immunosensor is very promising for rapid and early detection of pancreatic cancer using scarce amounts of human serum samples.