In Situ Growth of CuWO4 Nanospheres over Graphene Oxide for Photoelectrochemical (PEC) Immunosensing of Clinical Biomarker

Procalcitonin (PCT) protein has recently been identified as a clinical marker for bacterial infections based on its better sepsis sensitivity. Thus, an increased level of PCT could be linked with disease diagnosis and therapeutics. In this study, we describe the construction of the photoelectrochemical (PEC) PCT immunosensing platform based on it situ grown photo-active CuWO4 nanospheres over reduced graphene oxide layers (CuWO4@rGO). The in situ growth strategy enabled the formation of small nanospheres (diameter of 200 nm), primarily composed of tiny self-assembled CuWO4 nanoparticles (2–5 nm). The synergic coupling of CuWO4 with rGO layers constructed an excellent photo-active heterojunction for photoelectrochemical (PEC) sensing. The platform was then considered for electrocatalytic (EC) mechanism-based detection of PCT, where inhibition of the photocatalytic oxidation signal of ascorbic acid (AA), subsequent to the antibody–antigen interaction, was recorded as the primary signal response. This inhibition detection approach enabled sensitive detection of PCT in a concentration range of 10 pg·mL−1 to 50 ng.mL−1 with signal sensitivity achievable up to 0.15 pg·mL−1. The proposed PEC hybrid (CuWO4@rGO) could further be engineered to detect other clinically important species.


Introduction
The recent up-gradation in photoelectrochemical (PEC) sensors based on their synergic combination with advanced nanomaterial has proven effective in achieving robust signal sensitivities for low-concentration clinical species. In particular, the detection of cancer biomarkers, whose precise detection at a low concentration is crucial for timely diagnosis and therapeutics [1,2]. Despite the rapid progress in PEC engineering, the fast charge-carrier recombination and limited immobilization area, particularly in the case of bio immunosensors, are major challenges that restrict the construction of an efficient PEC biosensor [3]. In this context, various combinations of materials, including metal-organic (0.1 M) were from Thermo Fisher Scientific. Ascorbic acid and cocamidopropyl betaine (CAPB) was purchased from Sigma Aldrich (Darmstadt, Germany). The electrochemical experiments were performed using the CHI760D electrochemical workstation of Chenhua Co., Ltd. (Shanghai, China), equipped with a PLS-SXE 300 xenon lamp serving as a light source for PEC analysis. To avoid cell heating, the distance between the lamp and photo-cell was maintained at 12 cm. The morphological evaluation was obtained using field emission scanning electron microscopy (FE SEM) analysis (Oberkochen, Zeiss Sigma). X-ray powder diffraction (XRD) was performed with a D8 Advance X-ray diffractometer (Bruker AXS, Bremen, Germany). X-ray photoelectron spectroscopy (XPS; Scienta ESCA200) and raman spectroscopy (WITech alpha300) were carried for the phase confirmation analysis. In this study, a modified glassy carbon electrode (GCE), a saturated calomel electrode (SCE), and a platinum wire electrode (PE) were used as working, reference, and counter electrodes, respectively.

In Situ Growth of CuWO 4 Nanospheres over Graphene Oxide (rGO)
Initially, reduced graphene oxide (rGO) flakes were synthesized using a well-known modified Hummers method [15]. The obtained rGO flakes were then dispersed (0.1 mg) in an aqueous solution of 0.1 M CuCl 2 .2H 2 O (50 mL) previously containing (0.1 M) of CAPB, used as a template. The mixture was then gradually introduced with 0.1 M of Na 2 WO 4 .2H 2 O under constant stirring condition. The suspension was later transferred to a Teflon lined autoclave and subjected to heat treatment at 90 • for 12 h, to facilitate the growth of CuWO 4 nuclei. The final precipitates were thoroughly washed and dried at 60 • before utilizing them for the construction of PEC platform. The formed hybrid nanostructures are designated as CuWO 4 @rGO throughout the manuscript. Figure 1 shows the stepwise process adopted for developing an immunosensing platform using CuWO 4 @rGO hybrids. The surface of the GCE was initially polished with alumina powders (0.03 and 0.05 µm, respectively) and then washed with de-ionized water. First, a transducing platform was constructed by depositing dispersion (0.1 mg/5 mL methanol) of CuWO 4 @rGO hybrid over the pre-polished GCE surface. This layer was then allowed to dry under the nitrogenous atmosphere. To integrate the bio-recognition element, the electrode was then incubated with 5 µL of optimum 1.0 µ gmL −1 capture antibodies of PCT (Ab 1 -PCT) and then by 2 µL of 1% BSA to block the available active sites, followed by gently rinse before usage ( Figure S1a). This electrode was designated as Ab 1 /CuWO 4 @rGO-GCE and stored at 4 • C before using it for immunosensing. To detect the PCT antigen, the Ab 1 /CuWO 4 @rGO-GCE was incubated with different concentration of PCT in the range of 10 pg·mL −1 to 50 ng·mL −1 separately. purchased from Sigma Aldrich (Darmstadt, Germany). The electrochemical experiments were performed using the CHI760D electrochemical workstation of Chenhua Co., Ltd. (Shanghai, China), equipped with a PLS-SXE 300 xenon lamp serving as a light source for PEC analysis. To avoid cell heating, the distance between the lamp and photo-cell was maintained at 12 cm. The morphological evaluation was obtained using field emission scanning electron microscopy (FE SEM) analysis (Oberkochen, Zeiss Sigma). X-ray powder diffraction (XRD) was performed with a D8 Advance Xray diffractometer (Bruker AXS, Bremen, Germany). X-ray photoelectron spectroscopy (XPS; Scienta ESCA200) and raman spectroscopy (WITech alpha300) were carried for the phase confirmation analysis. In this study, a modified glassy carbon electrode (GCE), a saturated calomel electrode (SCE), and a platinum wire electrode (PE) were used as working, reference, and counter electrodes, respectively.

In Situ Growth of CuWO4 Nanospheres over Graphene Oxide (rGO)
Initially, reduced graphene oxide (rGO) flakes were synthesized using a well-known modified Hummers method [15]. The obtained rGO flakes were then dispersed (0.1 mg) in an aqueous solution of 0.1 M CuCl2.2H2O (50 mL) previously containing (0.1 M) of CAPB, used as a template. The mixture was then gradually introduced with 0.1 M of Na2WO4.2H2O under constant stirring condition. The suspension was later transferred to a Teflon lined autoclave and subjected to heat treatment at 90° for 12 h, to facilitate the growth of CuWO4 nuclei. The final precipitates were thoroughly washed and dried at 60° before utilizing them for the construction of PEC platform. The formed hybrid nanostructures are designated as CuWO4@rGO throughout the manuscript. Figure 1 shows the stepwise process adopted for developing an immunosensing platform using CuWO4@rGO hybrids. The surface of the GCE was initially polished with alumina powders (0.03 and 0.05 μm, respectively) and then washed with de-ionized water. First, a transducing platform was constructed by depositing dispersion (0.1 mg/5 mL methanol) of CuWO4@rGO hybrid over the prepolished GCE surface. This layer was then allowed to dry under the nitrogenous atmosphere. To integrate the bio-recognition element, the electrode was then incubated with 5 μL of optimum 1.0 μ gmL −1 capture antibodies of PCT (Ab1-PCT) and then by 2 μL of 1% BSA to block the available active sites, followed by gently rinse before usage ( Figure S1a). This electrode was designated as Ab1/CuWO4@rGO-GCE and stored at 4 °C before using it for immunosensing. To detect the PCT antigen, the Ab1/CuWO4@rGO-GCE was incubated with different concentration of PCT in the range of 10 pg·mL −1 to 50 ng·mL −1 separately.

PCT Detection from Simulated Blood Plasma
To ensure the analytical workability of the designed PEC sensors, the detection of PCT was also performed in simulated blood plasma (SBP). To simulate the real plasma-like characteristics, abundant proteins such as 40 mg of albumin, 10 mg of immunoglobulins, and 2.5 gm of fibrinogen were diluted in 10 mL of PBS solution (pH 7.0) to mimic their average plasma concentration. The SBP was then used in the quantification of different PCT concentrations in the range of 5 to 25 ng·mL −1 using designed PEC biosensors. To further validate the performance, the spiked samples were also quantified using the standard ELISA technique.  Figure 2f, confirms the growth of nanospheres along the edges of the flakes. Unlike conventional hybrids, where aggregation usually results in a complete mishmash of the particles. The nanospheres, in this case, have maintained their structural integrity without any structural collapse. Such structural features are highly beneficial in achieving enhanced electro-catalytic redox characteristics based on their exposed active sites and higher surface area. In addition, the nanospheres could be seen to be connected at the edge of rGO flakes, which is beneficial in achieving faster charge-transportation at the electrode interface. Figure 3a shows the XRD pattern of CuWO 4 @rGO hybrids, in reference to its compositional counterparts.

PCT Detection from Simulated Blood Plasma
To ensure the analytical workability of the designed PEC sensors, the detection of PCT was also performed in simulated blood plasma (SBP). To simulate the real plasma-like characteristics, abundant proteins such as 40 mg of albumin, 10 mg of immunoglobulins, and 2.5 gm of fibrinogen were diluted in 10 mL of PBS solution (pH 7.0) to mimic their average plasma concentration. The SBP was then used in the quantification of different PCT concentrations in the range of 5 to 25 ng·mL −1 using designed PEC biosensors. To further validate the performance, the spiked samples were also quantified using the standard ELISA technique.

Characterization of CuWO4@rGO
Hybrid  Figure 2f, confirms the growth of nanospheres along the edges of the flakes. Unlike conventional hybrids, where aggregation usually results in a complete mishmash of the particles. The nanospheres, in this case, have maintained their structural integrity without any structural collapse. Such structural features are highly beneficial in achieving enhanced electro-catalytic redox characteristics based on their exposed active sites and higher surface area. In addition, the nanospheres could be seen to be connected at the edge of rGO flakes, which is beneficial in achieving faster charge-transportation at the electrode interface. Figure 3a shows the XRD pattern of CuWO4@rGO hybrids, in reference to its compositional counterparts.  The XRD pattern for rGO, CuWO 4 , and its hybrid is shown in Figure 3a. In resemblance to the rGO with a representative 10.3 • , the XRD pattern for CuWO 4 @rGO hybrids also consist of this peak with a slightly declined intensity. The characteristic peaks for CuWO 4  The formation of the hybrid was further confirmed from Raman analysis. The corresponding spectra (Figure 3b) consists of the typical CuWO 4 peaks along with prominent D and G bands at 1350 and 1605 cm −1 , respectively [23]. The sharp peak around 900 cm −1 is attributed to the W-O vibration from the tungstate of CuWO 4 . The compositional purity of the hybrid was assessed from XPS analysis. The high-resolution de-convoluted profiles for the major components are shown in Figure 4a-d. The Cu 2p spectrum consists of two major peaks at 932.8 and 952.9 eV attributed to the core Cu 2+ of the CuWO 4 @rGO hybrid. The presence of the satellite peak confirms the presence of copper as in Cu (II) form, whereas the intense binding energy of Cu 2p 3  The XRD pattern for rGO, CuWO4, and its hybrid is shown in Figure 3a. In resemblance to the rGO with a representative 10.3°, the XRD pattern for CuWO4@rGO hybrids also consist of this peak with a slightly declined intensity. The characteristic peaks for CuWO4 were observed at 15.1°, 19.2°, 23.8°, 24.9°, 29.2°, 31.6°, 36.7°, and 43.8°, indexed to the (010), (001), (110), (0 11), (111), (111), (200), and (121) planes of CuWO4 triclinic phase, respectively, as standardized against (JCPDS No 80-1918) [22]. The formation of the hybrid was further confirmed from Raman analysis. The corresponding spectra (Figure 3b) consists of the typical CuWO4 peaks along with prominent D and G bands at 1350 and 1605 cm −1 , respectively [23]. The sharp peak around 900 cm −1 is attributed to the W-O vibration from the tungstate of CuWO4. The compositional purity of the hybrid was assessed from XPS analysis. The high-resolution de-convoluted profiles for the major components are shown in Figure 4a-d. The Cu 2p spectrum consists of two major peaks at 932.8 and 952.9 eV attributed to the core Cu 2+ of the CuWO4@rGO hybrid. The presence of the satellite peak confirms the presence of copper as in Cu (II) form, whereas the intense binding energy of Cu 2p3/2 suggests that the Cu 2+ ions are surrounded by W-O from WO4 −2 ions. The W4f spectrum exhibits two major peaks at 37.1 eV and 34.8 eV, indicating the presence of tungsten as in the W 6+ state. The O1s spectrum is resolved into three peaks (531.6, 530.1, and 529.3 eV) related to surface adsorbed hydroxide, Cu-O, and W-O bonds. The C1s spectrum confirms the presence of carbon (284.2 eV), epoxy carbon (285.2 eV), and carboxyl (286.1 eV) species from the rGO counterpart of the CuWO4@rGO hybrid [24]. The XPS analysis in support of XRD and Raman confirms the successful formation and compositional purity of CuWO4@rGO hybrids.

Photoelectrochemical Performance of CuWO4@rGO Hybrids
To evaluate the electro-catalytic performance of CuWO4@rGO hybrids, CV measurements were recorded using 0.1 mM of ascorbic acid (AA). Figure 5a shows the measured CV curves, where the CuWO4@rGO hybrid exhibits a relatively higher current response at lower potential value compared with its pristine counterparts. The obtained response of CuWO4@rGO hybrid against AA was further

Photoelectrochemical Performance of CuWO 4 @rGO Hybrids
To evaluate the electro-catalytic performance of CuWO 4 @rGO hybrids, CV measurements were recorded using 0.1 mM of ascorbic acid (AA). Figure 5a shows the measured CV curves, where the CuWO 4 @rGO hybrid exhibits a relatively higher current response at lower potential value compared with its pristine counterparts. The obtained response of CuWO 4 @rGO hybrid against AA was further recorded with different scan rates in the range of 5 to 50 mVs −1 . As seen (Figure 5c), the anodic peak could still maintain peak-shape characteristics at higher scan rates, reflecting the electrochemically reversible charge-transfer process of the diffused redox species, that is, AA. The observed linearity between measured current response and the square root of the scan rate (inset of Figure 5c) further defines this interfacial redox process to be diffusion-limited. To ensure maximum signal throughput, the pH of AA was optimized. Figure S1b indicates that the maximum current output was achieved when AA was maintained at pH 7. The photocatalytic activity of CuWO 4 @rGO hybrid was also evaluated under the illumination condition ( Figure 5b). As evident, CuWO 4 @rGO hybrid exhibits significantly high photocurrent response (∆I = 20 µA) under light. This increased current response could be explained using the EC-mechanism. In this case, the scavenger (AA), could readily oxidize over the surface of the CuWO 4 @rGO hybrid under the illumination condition, consuming the photo-generated holes, while at the same time promoting the electron transfer to the conduction band of CuWO 4 , and subsequently to the rGO layer. Here, rGO serves as a charge collector, facilitating the interfacial charge-transfer process. The increased photocurrent, in this case, confirms the oxidation of AA as a favourable process for the designed electrode, and thus AA could be anticipated as a potential redox mediator in the PEC immunosensor designed for the PCT biomarker. The current versus time measurements were carried to confirm the immobilization of biorecognition elements (PCT antibodies (Ab1)) over CuWO4@rGO/GCE (Figure 5d). The measurements were recorded in 0.1 mM AA with 0.1 M PBS (pH 7) with 30 s light on/off cycles for about 150 s. Here, Ab1/CuWO4@rGO/GCE exhibited a relatively lower current response compared with its protein-free counter-part. This decline is the consequence of active site blockage subsequent to the immobilization of insulating species (Ab1). Figure 6a shows the variation of photocurrent response with different concentrations of PCT standards. The measurements were carried under constant illumination, with a fixed potential of 0.15 V, 0.1 mM of The increased photocurrent, in this case, confirms the oxidation of AA as a favourable process for the designed electrode, and thus AA could be anticipated as a potential redox mediator in the PEC immunosensor designed for the PCT biomarker. The current versus time measurements were carried to confirm the immobilization of biorecognition elements (PCT antibodies (Ab 1 )) over CuWO 4 @rGO/GCE (Figure 5d). The measurements were recorded in 0.1 mM AA with 0.1 M PBS (pH 7) with 30 s light on/off cycles for about 150 s. Here, Ab 1 /CuWO 4 @rGO/GCE exhibited a relatively lower current Sensors 2020, 20, 148 7 of 10 response compared with its protein-free counter-part. This decline is the consequence of active site blockage subsequent to the immobilization of insulating species (Ab 1 ). Figure 6a shows the variation of photocurrent response with different concentrations of PCT standards. The measurements were carried under constant illumination, with a fixed potential of 0.15 V, 0.1 mM of AA as a mediator, and 0.1 M PBS (pH 7.0) as an electrolyte. This decline of the photoresponse with increasing concentration of PCT is the result of the immuno-complex formed between the PCT antibody-antigen at the CuWO 4 @rGO interface. In this case, the difference of photocurrent recorded before and after incubating the electrode with a specific concentration of PCT was taken as the primary signal. Figure 6b shows the corresponding calibration for the Ab 1 /CuWO 4 @rGO/GCE against the log concentration of PCT in the concentration range of 50 ng·mL −1 to 10 pg·mL −1 . The detection limit of the devised PEC sensor was estimated to be 0.15 pg·mL −1 of PCT (3x S/N). The selectivity of the designed immunosensing platform was evaluated against different biomarkers including prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSA). The measurements were carried using the same conditions as mentioned in Section 2.2. The negligible current variation in the presence of interferents supports the robust immunoreactivity of the devised sensor towards the PCT antigen. The photo-response of six independently fabricated electrodes was also measured to evaluate signal reliability of the sensor. Figure 6d shows the normalized response Ab 1 /CuWO 4 @rGO/GCE against standard 10 ng·mL −1 of PCT, with 0.1 mM AA, in a 0.1 M PBS electrolyte. As expected, a negligible current variation was observed, which anticipates high signal reliability each time a new CuWO 4 @rGO/GCE is fabricated. The shelf-life of the devised sensor was evaluated for a period of seven days with measurements recorded at an interval of one day ( Figure S1c). As evident, the photocurrent response of Ab 1 /CuWO 4 @rGO/GCE during the storage period does not change significantly, justifying the sensor's capability to endure a longer service life. The practical workability of the designed PEC biosensor was evaluated by sensing PEC biomarkers from simulated blood plasma. The simulated blood plasma (SBP) comprised of human albumin solution, immunoglobulins, and fibrinogen maintained at their typical human blood plasma levels. The SBP was spiked with 5, 15, 20, and 25 ng·mL −1 of PCT and applied to ab 1 /CuWO 4 @rGO/GCE for detection. Figure S1d compares the obtained values with those determined using the ELISA technique. The close proximity between the measured values evidently supports the capability of the designed sensor to selectivity detect PCT without any effect from the complex biological medium, ensuring its workability in real-blood applications. Table 1 compares the analytical capability of the devised sensors with other electrochemical driven sensors previously reported for the quantification of PCT. As evident, the designed sensors offers competitive performance with additional merit of sophisticated integration within portable PEC devices, designed particularly for point-of-care clinical applications. sensor to selectivity detect PCT without any effect from the complex biological medium, ensuring its workability in real-blood applications. Table 1 compares the analytical capability of the devised sensors with other electrochemical driven sensors previously reported for the quantification of PCT. As evident, the designed sensors offers competitive performance with additional merit of sophisticated integration within portable PEC devices, designed particularly for point-of-care clinical applications.

Conclusions
In this study, we describe a sensitive PEC platform devised for the detection of PCT clinical biomarkers. The platform comprises of highly photo-active CuWO 4 , grown in situ over conductive rGO layers. The CuWO 4 with its template-based in situ growth approach adopts the morphology of 3D nanospheres, which are composed of tiny self-assembled nano-boulders with the size range of 2-5 nm. The in situ growth enables excellent interfacial contact between CuWO 4 and rGO, allowing the construction of highly photoactive platform sensitive towards PCT biomolecules. The designed PEC sensor signal relies on the EC-mechanism, where signal inhibition observed for the photo-catalytic oxidation of AA, subsequent to the protein-antibody interaction, was taken as the primary PEC response. This strategy enabled sensitive inhibition signal linearity in the concentration range of 10 pg·mL −1 to 50 ng·mL −1 with a limit of detection of 0.15 pg.mL −1 for the PCT biomarker. Moreover, the devised platform, when tested in simulated blood plasma (SBP), demonstrated excellent reliability, anticipating its future potential in practical clinical applications.