High Sensitive Immunoelectrochemical Measurement of Lung Cancer Tumor Marker ProGRP Based on TiO2-Au Nanocomposite

Progastrin-releasing peptide (ProGRP), which is known to be highly specific and sensitive to small cell lung cancer (SCLC), has been proven to be a valuable substitute for neuron-specific enolase in SCLC diagnostics and monitoring, especially in its early stages. The detection of ProGRP levels also facilitates a selection of therapeutic treatments. For the fabrication of our proposed biosensor, titanium (IV) oxide microparticles were first used, followed by dispersing gold nanoparticles into chitosan and immobilizing them onto a carbon paste electrode (CPE) surface. The developed immunosensor exhibits a much higher biosensing performance in comparison with current methods, when it comes to the detection of ProGRP. Therefore, the proposed CPE/TiO2/(CS+AuNPs)/anti-ProGRP/BSA/ProGRP is excellent for the development of a compact diagnostics apparatus.


Introduction
As a substance that can be found in blood, urine, or body tissues, tumor markers can be found in elevated levels in cancer, among other tissue types. Tumor markers are classified into different groups that indicate different disease processes. Therefore, these tumor markers have been applied in the detection of cancer occurrence in oncology. Elevated tumor marker levels are known to indicate that a patient has a tumor. Therefore, the determination of tumor marker levels is of vital significance for disease screening, diagnostics and prognostics [1,2]. Several main tumor markers have been extensively analyzed for the diagnosis of hepatocellular carcinoma, epithelial ovarian tumors, pancreatic cancer, colorectal cancer, and others, including human chorionic gonadotropin (hCG), prostate specific antigen (PSA), alphafetoprotein (AFP), carcinoma antigen 125 (CA125), carbohydrate antigen (CA19-9, CA15-3), and carcinoembryonic antigen (CEA). Around the world, studies have been carried out for the development and the improvement of clinical bioassays via affordable and portable diagnostic apparatuses [3][4][5][6]. It is essential for the protein biomarkers to be detected quantitively and sensitively, since their detection is significant across many fields, including biomedical research and diagnostics [7], systems biology [8] and proteomics [9]. Traditional methods of protein detection are enzyme-linked immunosorbent assays (ELISA) [10], radioimmunoassay (RIA) [11], electrophoretic immunoassay [12] and mass spectrometry-based proteomics [13]. However, they are lacking in

Results and Discussion
The electrochemical properties of each assembly step of the modified electrode surface were investigated using CV and EIS measurements. Figure 1 showed the CV characterizations of the modified CPEs in a 1 mM [Fe(CN) 6 ] 3−/4− solution, whereas Figure 2 displays the EIS curves. As shown in Figure 1, the plain CPE/TiO 2 /(CS+AuNPs) exhibited the maximal peak current. An obvious decrease in the peak current was observed after the anti-ProGRP was immobilized onto the surface of the electrode (CPE/TiO 2 /(CS+AuNPs)/anti-ProGRP), which indicated that the active sites and effective area for the charge transfer were decreased. The decrease in active sites and effective area was due to the surface coverage of anti-ProGRP, which hid the exposure of TiO 2 and AuNPs. In addition, the nonconducting state of the anti-ProGRP also decreased in response to [Fe(CN) 6 ] 3−/4− , during the CV scan. Albumin from bovine serum (BSA) was used to block the remaining active sites of the electrode surface. The interaction between the antibody and the antigen was completed after immersing the proposed immunosensor into the ProGRP solution (CPE/TiO 2 /(CS+AuNPs)/anti-ProGRP). Considering the immunocomplex reaction of this configuration, the peak current was dramatically decreased. The schematic diagram of the sensor preparation is illustrated in Scheme 1.
Molecules 2019, 24, x 3 of 9 measurements. Therefore, the developed immunosensor could be used for the determination of ProGRP in synthetic serum specimens.

Results and Discussion
The electrochemical properties of each assembly step of the modified electrode surface were investigated using CV and EIS measurements. Figure 1 showed the CV characterizations of the modified CPEs in a 1 mM [Fe(CN)6] 3−/4− solution, whereas Figure 2 displays the EIS curves. As shown in Figure 1, the plain CPE/TiO2/(CS+AuNPs) exhibited the maximal peak current. An obvious decrease in the peak current was observed after the anti-ProGRP was immobilized onto the surface of the electrode (CPE/TiO2/(CS+AuNPs)/anti-ProGRP), which indicated that the active sites and effective area for the charge transfer were decreased. The decrease in active sites and effective area was due to the surface coverage of anti-ProGRP, which hid the exposure of TiO2 and AuNPs. In addition, the nonconducting state of the anti-ProGRP also decreased in response to [Fe(CN)6] 3-/4-, during the CV scan. Albumin from bovine serum (BSA) was used to block the remaining active sites of the electrode surface. The interaction between the antibody and the antigen was completed after immersing the proposed immunosensor into the ProGRP solution (CPE/TiO2/(CS+AuNPs)/anti-ProGRP). Considering the immunocomplex reaction of this configuration, the peak current was dramatically decreased. The schematic diagram of the sensor preparation is illustrated in Scheme 1. The EIS spectra recorded for the different electrodes consisted of semicircle segments and a linear segment. The electron-transfer resistance (Ret), referred to the diameter of the former segment at higher frequencies whereas the diffusion process denoted the latter segment at lower frequencies.
The EIS experiment was used to investigate the stepwise immunosensor fabrication. The calculation of the semicircles was based on the Randle's cirquit. The phase angle of linear segments fitted well with the Warburg model. The EIS measurements of different modified CPE at various stages were shown in Figure 2, and the inset showed the optimized fitting circuit model [38]. An insignificant semicircle at high frequencies and a linear segment at low frequencies were observed in the Nyquistic diagram for the CPE/TiO2/(CS+AuNPs), which indicated an extremely decreased Ret to redox probe [Fe(CN)6] 3−/4− . An apparent increase in resistance for the redox probe was observed after the anti-ProGRP was immobilized, which demonstrated the distinctive property of the anti-ProGRP-electric insulation (the formation of a barrier for the charge transfer on the surface of the electrode), along with the completion of the modification process of CPE/TiO2/(CS+AuNPs)/anti-ProGRP. This was followed by immersing the BSA-blocked The EIS spectra recorded for the different electrodes consisted of semicircle segments and a linear segment. The electron-transfer resistance (R et ), referred to the diameter of the former segment at higher frequencies whereas the diffusion process denoted the latter segment at lower frequencies. The EIS experiment was used to investigate the stepwise immunosensor fabrication. The calculation of the semicircles was based on the Randle's cirquit. The phase angle of linear segments fitted well with the Warburg model. The EIS measurements of different modified CPE at various stages were shown in Figure 2, and the inset showed the optimized fitting circuit model [38]. An insignificant semicircle at high frequencies and a linear segment at low frequencies were observed in the Nyquistic diagram for the CPE/TiO 2 /(CS+AuNPs), which indicated an extremely decreased R et to redox probe [Fe(CN) 6 ] 3−/4− . An apparent increase in resistance for the redox probe was observed after the anti-ProGRP was immobilized, which demonstrated the distinctive property of the anti-ProGRP-electric insulation (the formation of a barrier for the charge transfer on the surface of the electrode), along with the completion of the  In spite of the convincing results provided by CVs and EIS spectra for the electrode modification, the CV measurement offered a more obvious drop than the EIS experiment. The results showed that CV exhibited a better performance in the characterization of modification in comparison with EIS, for the proposed CPE. As shown in Figure 3, the impedimetric response of our proposed electrode was recorded as a function of the ProGRP concentration over a range of 10 to 500 ng/mL in PBS, that contained [Fe(CN)6] 3−/4− for an incubation period of approximately 5 min. As the concentration of ProGRP was increased, a capacitance decrease was observed, possibly due to the varied dielectric/blocking features of the electrode-electrolyte interface, caused by the interaction between the antigen and the antibody. Considering the interaction between ProGRP and the anti-ProGRP, the capacitance decrease was expected to result from the distance increase between the electrolyte and the electrode. Furthermore, the capacitance magnitude decreased due to the interaction between ProGRP and the anti-ProGRP, which in turn was caused by the decrease in polar ProGRP protein molecules that replaced the water molecules on the surface of the electrode. The variation of capacitance with the concentration  In spite of the convincing results provided by CVs and EIS spectra for the electrode modification, the CV measurement offered a more obvious drop than the EIS experiment. The results showed that CV exhibited a better performance in the characterization of modification in comparison with EIS, for the proposed CPE. As shown in Figure 3, the impedimetric response of our proposed electrode was recorded as a function of the ProGRP concentration over a range of 10 to 500 ng/mL in PBS, that contained [Fe(CN)6] 3−/4− for an incubation period of approximately 5 min. As the concentration of ProGRP was increased, a capacitance decrease was observed, possibly due to the varied dielectric/blocking features of the electrode-electrolyte interface, caused by the interaction between the antigen and the antibody. Considering the interaction between ProGRP and the anti-ProGRP, the capacitance decrease was expected to result from the distance increase between the electrolyte and the electrode. Furthermore, the capacitance magnitude decreased due to the interaction between ProGRP and the anti-ProGRP, which in turn was caused by the decrease in polar ProGRP protein molecules that replaced the water molecules on the surface of the electrode. The variation of capacitance with the concentration In spite of the convincing results provided by CVs and EIS spectra for the electrode modification, the CV measurement offered a more obvious drop than the EIS experiment. The results showed that CV exhibited a better performance in the characterization of modification in comparison with EIS, for the proposed CPE.
As shown in Figure 3, the impedimetric response of our proposed electrode was recorded as a function of the ProGRP concentration over a range of 10 to 500 ng/mL in PBS, that contained [Fe(CN) 6 ] 3−/4− for an incubation period of approximately 5 min. As the concentration of ProGRP was increased, a capacitance decrease was observed, possibly due to the varied dielectric/blocking features of the electrode-electrolyte interface, caused by the interaction between the antigen and the antibody. Considering the interaction between ProGRP and the anti-ProGRP, the capacitance decrease was expected to result from the distance increase between the electrolyte and the electrode. Furthermore, the capacitance magnitude decreased due to the interaction between ProGRP and the anti-ProGRP, which in turn was caused by the decrease in polar ProGRP protein molecules that replaced the water molecules on the surface of the electrode. The variation of capacitance with the concentration of analyte was plotted for the calibration of the capacitive immunosensor, with the linear relationship, obtained as 10-500 ng/mL. The variation in the capacitance of our proposed electrode was used as a function of the ProGRP concentration under similar conditions, with a linear regression coefficient (r 2 ) of 0.991, shown for this sensor. The developed sensor was highly sensitive, possibly due to the increased surface area and the functional features of the CPE/TiO 2 /(CS+AuNPs). The biosensor provided an excellent and effective performance, due to the increased loading capacity for antibody molecules, caused by the available functional groups present on the TiO 2 /(CS+AuNPs). Furthermore, the developed sensor showed a low LOD of 0.133 ng/mL (3σ b /m). of analyte was plotted for the calibration of the capacitive immunosensor, with the linear relationship, obtained as 10-500 ng/mL. The variation in the capacitance of our proposed electrode was used as a function of the ProGRP concentration under similar conditions, with a linear regression coefficient (r 2 ) of 0.991, shown for this sensor. The developed sensor was highly sensitive, possibly due to the increased surface area and the functional features of the CPE/TiO2/(CS+AuNPs). The biosensor provided an excellent and effective performance, due to the increased loading capacity for antibody molecules, caused by the available functional groups present on the TiO2/(CS+AuNPs). Furthermore, the developed sensor showed a low LOD of 0.133 ng/mL (3σb/m). The specificity of CPE/TiO2/(CS+AuNPs)/anti-ProGRP/BSA/ProGRP was investigated by incubating this immunoelectrode with 15 μL of pathogens and metabolites, including 300 ng/mL of ProGRP, 5 mM of ascorbic acid, 5 mM of uric acid, and 5 mM of glucose. As shown in Figure 4, after these pathogens were added into the proposed electrode, the electrochemical response showed no variations during the experiments, which suggested that the probe for ProGRP was highly specific.  The specificity of CPE/TiO 2 /(CS+AuNPs)/anti-ProGRP/BSA/ProGRP was investigated by incubating this immunoelectrode with 15 µL of pathogens and metabolites, including 300 ng/mL of ProGRP, 5 mM of ascorbic acid, 5 mM of uric acid, and 5 mM of glucose. As shown in Figure 4, after these pathogens were added into the proposed electrode, the electrochemical response showed no variations during the experiments, which suggested that the probe for ProGRP was highly specific.
For the investigation of the reproducibility of the immunoelectrode, ProGRP (300 ng/mL) was used with the proposed electrode, while the variation in the current magnitude was recorded. As shown in Figure 5A, no apparent variation was observed, which suggested that this electrode was highly precise. The CVs ( Figure 5B) of our proposed immunoelectrode were recorded for ca. 30 d (10 d intervals) to study its storage stability. During the 30 days, no apparent current variation was observed; afterwards, the current showed a 4% variation from the original value. These results indicated a remarkable storage stability of our proposed electrode, for as long as 30 d.
The specificity of CPE/TiO2/(CS+AuNPs)/anti-ProGRP/BSA/ProGRP was investigated by incubating this immunoelectrode with 15 μL of pathogens and metabolites, including 300 ng/mL of ProGRP, 5 mM of ascorbic acid, 5 mM of uric acid, and 5 mM of glucose. As shown in Figure 4, after these pathogens were added into the proposed electrode, the electrochemical response showed no variations during the experiments, which suggested that the probe for ProGRP was highly specific.  For the investigation of the reproducibility of the immunoelectrode, ProGRP (300 ng/mL) was used with the proposed electrode, while the variation in the current magnitude was recorded. As shown in Figure 5A, no apparent variation was observed, which suggested that this electrode was highly precise. The CVs ( Figure 5B) of our proposed immunoelectrode were recorded for ca. 30 d (10 d intervals) to study its storage stability. During the 30 days, no apparent current variation was observed; afterwards, the current showed a 4% variation from the original value. These results indicated a remarkable storage stability of our proposed electrode, for as long as 30 d.
For the investigation of the practical performance, three serum samples were analyzed in both the enzyme-linked immunosorbent assay (ELISA, Cusabio) method and the proposed electrochemical immunosensor. ELISA is a recommended technology, commercially used for the ProGRP test. An antibody specific to ProGRP was first coated on the microplate. Then, samples were added to the microplate with a biotin-conjugated antibody. The antigen was combined with the antibodies in the microplate by a competitive binding test. Next, avidin conjugated to horseradish peroxidase (HRP) and tetramethylbenzidine (TMB) were added, and formed a color change. The result was measured spectrophotometrically at a wavelength of 450 nm. For a comparison test, the standard addition method was used. The results are summarized in Table 1. As shown in the table, the ELISA could not detect ProGRP above 2 ng/mL, while the proposed immunosensor showed excellent performance at high-concentration conditions.

Chemicals
ProGRP and anti-ProGRP were commercially available in Shengyuan Biotech Inc., Shenzhen, China. All test reagents were of analytical grade. For all experiments, the supporting electrolyte was a pH 7 KH2PO4 buffer solution (PBS), where 1 M NaOH was added to adjust the pH value. The following were purchased from XFNANO Co. Ltd (Nanjing, China): Acetic acid; commercial For the investigation of the practical performance, three serum samples were analyzed in both the enzyme-linked immunosorbent assay (ELISA, Cusabio) method and the proposed electrochemical immunosensor. ELISA is a recommended technology, commercially used for the ProGRP test. An antibody specific to ProGRP was first coated on the microplate. Then, samples were added to the microplate with a biotin-conjugated antibody. The antigen was combined with the antibodies in the microplate by a competitive binding test. Next, avidin conjugated to horseradish peroxidase (HRP) and tetramethylbenzidine (TMB) were added, and formed a color change. The result was measured spectrophotometrically at a wavelength of 450 nm. For a comparison test, the standard addition method was used. The results are summarized in Table 1. As shown in the table, the ELISA could not detect ProGRP above 2 ng/mL, while the proposed immunosensor showed excellent performance at high-concentration conditions.

Chemicals
ProGRP and anti-ProGRP were commercially available in Shengyuan Biotech Inc., Shenzhen, China. All test reagents were of analytical grade. For all experiments, the supporting electrolyte was a pH 7 KH 2 PO 4 buffer solution (PBS), where 1 M NaOH was added to adjust the pH value. The following were purchased from XFNANO Co. Ltd (Nanjing, China): Acetic acid; commercial AuNPs; CS monomer, for the preparation of TiO 2 NPs, Ti(OCH(CH 3 ) 2 ) 4 ; absolute ethanol, for synthetic serum preparation of NaCl; CaCl 2 ; N-Ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC); N-hydroxysuccinimide (NHS); KCl; urea, which was commercially available in Sigma-Aldrich (Shanghai, China); and Au nanoparticles with an average size of 20 nm (0.05 mg/mL).

Nanocomposite Preparation
For the preparation of the TiO 2 /(CS+Au)-modified CPE, first TiO 2 (20 %), mineral oil (30 %), and graphite powder (50 %) were mixed together to prepare TiO 2 /CPE. The yielded paste was filled into the hole of a Teflon body (radius: 2 mm), and the electrical contact for this CPE was a copper wire. The CS flakes were dissolved into an acetic acid solution (50 mL, 2 M) which was added with the commercial AuNPs (2 µL) and was stirred for 60 min. This was followed by a 15 min sonication, so as to uniformly disperse the AuNPs into the CS. Subsequently the CS/AuNPs solution (25 µL) was uniformly spread onto the TiO 2 /CPE. After drying in a desiccator for 45 min, the TiO 2 /(CS+AuNPs)/CPE was yielded.

Immunosensor Fabrication
The immunosensor was prepared by firstly immersing TiO 2 /(CS+AuNPs)/CPE into the anti-ProGRP solution for 60 min at 4 • C. The unspecific bindings were prevented through blocking the remaining active sides on the CPE surface with 0.25 % (w/w) BSA for 20 min. This was followed by washing the electrode surface using distilled water. The TiO 2 /(CS+AuNPs)/anti-ProGRP/BSA/CPE immunosensor was stored at 4 • C in pH 7 phosphate buffer solution (PBS), prior to use.

Measurement
The electrochemical experiment was carried out with an Autolab Potentiostat/Galvanostat (PGSTAT302/N, Metrohm), where a three-electrode configuration was used. The references and the auxiliary electrode were Ag/AgCl and a platinum wire, respectively. The EIS characterization was performed using a multi-impedance test system with a frequency range of 10 kHz-10 mHz and an AC amplitude of 10 mV.

Conclusions
In the present work, ProGRP was successfully detected through the interactions between antibodies and antigens using a CPE/TiO 2 /(CS+AuNPs) nanohybrid-based immunosensor. The electrochemical response for the CPE/TiO 2 /(CS+AuNPs)/anti-ProGRP/BSA/ProGRP immunoelectrode, as a function of the ProGRP concentration, indicated that this electrode was highly sensitive, displayed long-term stability, and possessed a low detection limit. Therefore, the proposed immunosensor could be potentially used in the medical diagnostic field.