Coronary Au@Bi Nanospheres for Highly Impedance‐Sensitive Immunosensing toward CEA

Novel coronary Au@Bi nanospheres (NSs) are simply synthesized by sparkling gold nanoparticles (Au NPs) on the surface of bismuth (Bi) NSs and successfully used as highly impedance‐sensitive immunosensing platform to develop a new label‐free electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA). Firstly, Bi NSs are prepared by one‐pot hydrothermal method, showing uniform sphere morphology with a dense oxide film formed outside Bi NSs due to the easy oxidation of Bi in the air. The oxide film significantly hinders the transmission of electrons and effectively improves the sensitivity of the proposed electrochemical impedance immunosensor. The surface functionalization of Bi NSs with Au NPs enhances the biocompatibility, stability, as well as active surface area of the produced Au@Bi NSs, preferable for the efficient loading of antibody molecules. The electrochemical impedance spectroscopy (EIS) is selected as the optical technique to measure CEA with the constructed immunosensor based on Au@Bi NSs. Because of the excellent impedance‐responsive performance, this new immunosensor displays highly sensitive detection toward CEA in a linear range of 50 fg mL−1–100 ng mL−1 with an extremely low detection limit of 9.83 fg mL−1 and could be potentially used in early clinical detection of cancer.


Introduction
Cancer is known as one of the diseases with the highest mortality worldwide, which endangers the health of countless people, DOI: 10.1002/adsr.202200050 greatly reducing the family happiness index. [1] The early diagnosis and treatment of cancer is of high significance to save people from cancer. The determination of tumor markers plays an important role in screening and diagnosis of cancer in the early stage. [2] Carcinoembryonic antigen (CEA) exists in human blood as a broad-spectrum tumor marker, and is usually used to determine the occurrence of cancer according to its existing level in human blood. [3] A few CEA molecules mostly trigger disease processes through sufficiently regulating the cells' biological functions, as a consequence, numerous pathological biomarkers are present at very low levels during the early stages of the disease development. [4] The need to develop analytical methods with high sensitivity is quite urgent for detection of biomolecules, especially coupling with the specificity of biological recognition events. [5] Based on this view, immunoassay as a hopeful procedure has shown its outstanding availability for such detection by combining the unique advantages of highly specific immunological recognition with the convenience of biosensing devices, [6] which provides a high possibility to solve the bottlenecks in detecting biomolecules with low abundance and the ultraweak biological signal. [5] Consequently, immunoassay procedures are considerably employed as valid diagnosis procedures in biochemical, environmental and clinical evaluations as well as in food industries. [7] In the past decades, chemiluminescence immunoassay, [8] enzyme-linked immunosorbent assay, [9] fluorescence immunoassay, [10] electrochemical biosensor, [11] radioimmunoassay, [12] etc. have been extensively explored to detect a variety of protein analytes with high sensitivity. [13] However, these methods except for electrochemical biosensor are somehow limited by the cumbersome professional operation, high cost and time-consuming process, significantly hampering their wide application in clinical detection. [14] Considerable interest has been attached to electrochemical immunosensors owing to their intrinsic advantages over other existing methods, such as better portability, higher sensitivity, lower cost and simpler operation, for the initial detection of tumor markers, including ones with low abundance. [15] The corresponding electrochemical immunoassay usually involves in the techniques of amperometry, impedance, and voltammetry. [16] The electrochemical impedance spectroscopy (EIS) stands out for its advantages such as no labeling, real-time monitoring, high sensitivity, and simple operation. [17] Particularly, EIS provides a non-destructive way to characterize the electrical properties of biological interfaces. [18] The increasing demand for early-disease screening and diagnosis makes methods with more ultra-sensitivity urgent so as to meet the requirements of clinical analysis. For the purpose to achieve the excellent sensitivity and high selectivity of biosensing strategy, a suitable method should be introduced to functionalize nanomaterials with biomolecules as specific recognition elements for signal triggering, as nanomaterialbased signal generation holds great promise and potential in exerting excellent sensitivity and high selectivity to biosensors for in situ or online detection of tumor markers benefiting from both short analysis procedure, easily miniaturizable scale and some other extraordinary physicochemical properties. [19] In recent years, nanocomposites have been widely used to construct various ultra-sensitive immunosensor with obviously improved sensitivity, [20] especially bismuth (Bi) based nanocomposites, because of their extremely low biological toxicity, favorable electrochemical properties and preferable biocompatibility. [19] The application of novel Bi nanomaterials in the construction of electrochemical immunosensors has great advantages. [19] The synthesized small-size Bi nanomaterials possess large specific surface area, which provides adequate active sites for efficient surface modification and biological attachment. Since Bi is easy to be oxidized in the air, the surface of Bi nanomaterials prepared by thermal reduction method is usually oxidized to form a stable oxide film, and thus hinders the electron transfer of the electrode surface, which is the key feature for the construction of EIS immunosensor and the enhancement of the sensor's sensitivity. [20] The Bi nanomaterials can effectively improve the sensitivity and stability of the EIS sensor due to the easiness of being oxidized. [21] Gold nanoparticles (Au NPs) are often selected to functionalize Bi nanomaterials for the effective immobilization of antibody (Ab) because of their high stability and excellent electrochemical performance. [22] This is fairly helpful to improve the biocompatibility of nanomaterials and the stability of sensing platform. As a result, the sensitivity of the EIS immunosensor can be enhanced for CEA detection. [23] Commonly, Au NPs are attached to the aminated surface of Bi NPs, [24] where the exposed amino groups on the surface provide empty orbitals, and lone pair electrons to form stable coordination bonds with Au. Compared with other Bi-based materials, Au@Bi nanocomposites are obviously featured by uniform morphology and good dispersion, good for producing low detection limit through offering effective surface area of the electrode. [25] Herein, coronary Au@Bi nanospheres (NSs) were synthesized as the sensing platform to develop a new label-free EIS immunosensor for the first time, and realized the ultra-sensitive detection of CEA, as shown in Scheme 1. As an impedimetric immunosensing platform, the Au@Bi NSs synthesized by a thermal reduction method were endowed with a large specific surface area owing to the unique coronary structure good for efficient antibody immobilization. As expected, the biocompatibility of Au@Bi NSs was greatly enhanced with excellent electrochemical properties, particularly the EIS property. The electron transfer over the electrode interface was effectively hindered as the surface oxidation of Au@Bi NSs easily occurred in the air, which shows great potential to construct the Au@Bi based EIS immunosensor for the clinical CEA detection. Figure 1A shows the scanning electron microscope (SEM) image of Au@Bi NSs where the nano-sized Bi synthesized by thermal reduction method was featured by uniform sphere morphology with smooth surface in small particle size. Au NPs were evenly distributed on the surface of Bi NSs via stable chemical bonds with no shedding phenomenon of Au NPs around, suggesting that Au NPs successfully grew on the surface of Bi NSs. Through the characterization of single Bi NSs by transmission electron microscope (TEM) ( Figure 1B,D), the spherical morphology and uniform particle size of Bi NSs could be clearly observed with good dispersibility. The subsequently as-prepared Au@Bi NSs displayed a unique coronary structure with excellent aqueous dispersibility helpful for the effective loading of biomolecules, like CEA, as shown in the TEM images of Au@Bi NSs ( Figure 1C,E). Under the heating operation, the morphology of Bi NSs was well kept without damage, revealing the excellent mechanical stability and production repeatability of Au@Bi NSs. Also, Au NPs grew evenly on the surface of Bi NSs ( Figure 1C). Figure 1F shows the lattice stripe of Au@Bi NSs, which corresponded to the X-ray powder diffraction (XRD) result of Au@Bi NSs in Figure 1G. As seen from XRD, the synthesized Bi NSs completely correspond to the standard card of the elemental Bi, and the Au NPs outside Bi NSs matched well the standard card of gold, which indicates that Au@Bi NSs were successfully prepared. Figure 1H analyzed the ultraviolet-visible (UV-vis) absorption spectra of Bi NSs and Au@Bi, where there was an obvious UV absorption peak at 535 nm belonging to the special UV absorption peak of Au NPs, further proving the existence of Au NPs and the successful synthesis of Au@Bi NSs. In Figure 1I, the main FT-IR vibration bands were obviously observed for both Bi NSs and Au@Bi NSs. Since the surface of Au@Bi NSs was modified by aminopropyl triethoxysilane (APTES), the characteristic peak of amino group (−NH 2 ) on the Au@Bi surface was visualized in the FT-IR spectrum. At the wavelength of 3130 cm −1 , the broadband of amino stretching motion partially coincided with that of hydroxyl stretching motion, and the broadband at 1637 cm −1 corresponded to the amino deformation motion, indicating the successful surface modification of Au@Bi with −NH 2 .

Characterization of Au@Bi NSs
Furthermore, the electrochemical properties and the structure of the novel Au@Bi NSs were well characterized (Figure 2). According to the mapping test of the Au@Bi NSs (Figure 2A-D), Bi element was evenly distributed throughout the whole sphere, and the O element partly covered on the surface of Bi NSs owing to the formation of bismuth oxide by surface oxidation during the synthesis of Bi NSs in the air. Au element sparkled evenly outside the Bi NSs, which indicates the successful modification of Au NPs. XPS was carried out to analyze the composition of the obtained Au@Bi NSs. Figure 2E,F shows the fitted XPS spectra of elements Bi and O. The characteristic peaks in both figures belonged to the characteristic peak of Bi─O bonds, indicative of the existence of a layer of bismuth oxide film forming on the surface of Bi NSs, which might effectively hinder the electron transfer, the key factor for the construction of the EIS immunosensor using Au@Bi NSs. After the Ab was immobilized on the surface of the Au@Bi NSs, the contact angle decreased significantly (Figure 2G), resulting from the formation of the stable covalent bond between Au NPs and amino groups, which also reveals the success of Ab modification. The preferred dispersion of Au@Bi NSs was found by standing still in phosphate buffered solution (PBS) for 30 min ( Figure 2H).

Electrochemical Characterization of the Au@Bi-Based EIS Immunosensor
The preparation process of the Au@Bi based EIS immunosensor was analyzed by cyclic voltammetry (CV) and EIS, and the layer-by-layer modification of the proposed immunosensor was determined as well by this electrochemical test. Firstly, CV curves during the preparation process of the sensor were obtained in 0.1 m KCl solution containing 2.5 mm [Fe(CN) 6 ] 3-/4− , as shown in Figure 3A. After the bare glassy carbon electrode (GCE) was modified with Au@Bi NSs (Au@Bi/GCE), the redox peak current of curve b was significantly lower than that of bare GCE (curve a), which indicates that Au@Bi NSs effectively hindered the transmission of electrons over the electrode surface, and the redox peak might be caused by the presence of Au NPs. While the subsequent modification of Ab decreased the redox peak current in curve c with the potential shift observed. The possible reason is that the immobilized biomolecules blocked the diffusion of the signal probe [Fe(CN) 6 ] 3-/4− to the electrode surface. Few electrons could be then transferred in the electrode interface, and the potential shifts might be caused by the different structure of the electric double layer, suggesting that Ab was easily attached to the surface of Au@Bi NSs. Bovine serum albumin (BSA) was selected to cover the unbound nonspecific active site on the electrode (curve d), and further decreased the peak current. The peak current was significantly reduced after CEA was specifically combined with Ab immobilized on the electrode surface. The uniform presence of Au NPs on the surface of Bi NSs considerably increased the loading number of biomolecules, resulting in an obviously decreasing current response. The Au@Bi based EIS immunosensor was thus fabricated using Au@Bi as an ideal carrier and confirmed through layer-by-layer modification. The EIS curves were utilized to jointly determine the successful construction of the immunosensor with CV analysis (Figure 3B), where the Nyquist plot curve consists of a semicircle and a linear part. The semicircle corresponds to the charge transfer resistance (R ct ) and the illustration is an equivalent circuit diagram. Here, the label-free impedance sensor was constructed using the layer-by-layer self-assembled electrode to detect CEA. The impedance change caused by the layer-by-layer surface modification was recorded and used to verify the successful construction  of the as-proposed immunosensor. Figure 3B shows that the radius of the semicircle increased when the electrode surface was successively modified. The corresponding R ct values were calculated by using Zview software and listed in

Optimization of the Experimental Conditions
In order to maximize the performance of the developed EIS immunosensor, the experimental conditions were optimized in detail so as to ensure the stability and sensitivity of the sensor. With the aim to carry out the accurate quantitative analysis of CEA, four aspects were assessed such as pH value, the concentration of Au@Bi NSs, incubation time as well as incubation temperature. The concentration of CEA was set as 1.0 ng mL −1 in the whole experiment process. When the pH value of the detection media was 7.4 ( Figure 3C), the resistance value measured with the sensor reached the optimal value, since pH values higher or lower than 7.4 are regarded to affect the activity of biomolecules. When the concentration of the Au@Bi NSs was 5 mg mL −1 , the sensor performed the best. The concentration higher than 5 mg mL −1 could cause the falling off of Au@Bi NSs from the electrode surface, resulting in the decrease of resistance value ( Figure 3D). When the incubation between CEA and Ab occurred at the temperature of 37°C, the resistance of the obtained sensor went up to the peak value. This might be because the optimal incubation temperature between antigen and antibody is commonly 37°C ( Figure 3E). Also, their incubation time affects the resistance value. The incubation time of 30 min led to the peak value ( Figure 3F). After 30 min, the R ct value slightly decreased and kept stable with longer incubation time applied owing to the saturated incubation, so 30 min was set as the optimal incubation time between CEA and Ab for the sensor development.

Detection Performance of the Au@Bi-Based Immunosensor
Under the optimal experimental conditions, the detection performance of immunosensor was studied by EIS method toward different concentrations of CEA. In the presence of CEA, the electron transfer rate significantly decreased, resulting in the increase of R ct value. As shown in Figure 4A, the radius of EIS Label-free g-C 3 N 4 /CdSe 10 ng mL −1 -100 μg mL −1 0.21 ng mL −1 [17] Label-free Au@Ce 2 Sn 2 O 7 0.001-70 ng mL −1 0.53 pg mL −1 [26] Sandwich-type CPS@PANI@Au 0.006-12 ng mL −1 1.56 pg mL −1 [27] Impedance-type Ion sensitive field effect transistor 1-50 pg mL −1 1 pg mL −1 [10] Impedance-type mucilage-GNPs-SNPs 100 ng mL −1 -0.1 pg mL −1 0.078 pg mL −1 [28] Impedance-type Au@Bi 50 fg mL −1 -100 ng mL −1 9.83 fg mL −1 This work curve was enlarged with the increasing CEA concentration, and the increase of R ct was directly proportional to the CEA concentration (c). In the concentration range of 50 fg mL −1 -100 ng mL −1 , a good linear relationship was achieved between R ct and the concentration of CEA ( Figure 4B) with the limit of detection (LOD) calculated to be 9.83 fg mL −1 according to 3 /S, where represents the standard deviation of the lowest signal and S the slope of the obtained linear calibration curve. These results show that the immunosensor using Au@Bi NSs as EIS sensing platform possessed lower detection limit and wider detection range, compared with other previously reported immunosensors listed in Table 2, Better performance and higher sensitivity were thus found for the newly developed EIS immunosensor. The formation of oxide film on the surface of Bi NSs majorly contributed to high impedance-sensitive property of this immunosensor and the effective improvement of the sensor stability.

Selectivity, Reproducibility, and Stability
The selectivity, reproducibility, and stability play an important role in the practical application of the immunosensor. To fully verify the selectivity of the constructed sensor toward CEA, four interfering substances of immunoglobulin G (IgG), BSA, alpha fetoprotein (AFP) and porcine serum albumin (PSA) were selected to coexist with CEA (1 ng mL −1 ), where the concentration of each interferant was 10 ng mL −1 . As shown in Figure 4D, no obvious impedance response was found to single interfering substance without CEA because of the quite low R ct values. Compared with the R ct measured for single CEA solution, the R ct value did not obviously change after adding interfering substances due to the highly specific binding between CEA and Ab, indicating the good selectivity of the constructed immunosensor. To test the reproducibility, six immunosensors were separately constructed to detect CEA at three different concentrations of 1, 0.1, and 0.01 ng mL −1 . As shown in Figure 4C, the R ct values were not significantly affected for each concentration, thus verifying the sensor's favorable reproducibility. Moreover, the constructed immunosensor was stored at 4°C for 15 days in order to evaluate its long-term stability. Figure 4E shows, during 15 days, CEA in three levels same as abovementioned was detected with the stored immunosensor every three days and no obvious decrease in R ct value was found, which was likely attributed to the stable state of Bi NSs after being sparkled with Au NPs, the good stability of the immunosensor was thus verified ( Figure 4F).

Analysis of Human Blood Samples
Blood samples from two patients were tested to judge the accuracy and reliability of the constructed EIS immunosensor (Figure 4F). The CEA concentration tested in the blood samples of patient I was lower than the normal value, and the one in the blood samples of patient II was higher than the normal value. CEA with concentrations of 1, 5, and 10 ng mL −1 was added based on a standard method to the blood samples of the two groups, respectively, for reliable detection, where the relative standard deviation (RSD) and recovery were calculated. As listed in Table 3, the calculated average recovery was between 98.0% and 101.3% with RSD falling in the range of 0.38-2.21 (n = 3), indicating that the immunosensor could accurately detect the CEA content in human body. The results demonstrate that the unmarked immunosensor constructed with Au@Bi NSs performed quite well in the detection of the actual samples. Therefore, a conclusion could be made that the as proposed Au@Bi-based EIS immunosensor holds great potential in the early sensitive detection of tumor markers and practical clinical application.

Conclusions
In this work, we synthesized Bi NSs by thermal reduction method and sparkled Au NPs for the purpose to immobilize antibodies. The synthesized Au@Bi NSs were successfully used to construct a novel label-free EIS immunosensor for the sensitive detection of CEA. The novel Au@Bi NSs was featured by a unique coronary morphology with a large specific surface area. After being modified with Au NPs, both the biocompatibility and the stability of the Au@Bi NSs were greatly improved, which was preferred to fix a large number of antibodies and improve the sensor sensitivity. The novel immunosensor performed very well in wide linear range with LOD, and displayed excellent properties of great antiinterference, high reproducibility and desired stability. The detection of actual samples was proved to be fairly reliable with this new immunosensor, which offers high possibility for its practical clinical application.
Apparatus: All electrochemical performance parameters were tested on an Autolab PGSTAT 100 potentiostat/galvanostat (Shanghai Nano Industrial Co., Ltd, China). A three-electrode system was applied including a platinum wire electrode as counter electrode, a saturated calomel electrode as reference electrode and a GCE as the working electrode. The water contact angle was measured by the contact angle system OCA (Beijing Eastern-Dataphy Instruments Co., Ltd, China). SEM images and energy dispersive spectroscopy (EDS) spectrum were obtained using a field emission SEM (Zeiss, Germany). TEM images were obtained by a JEOL JEM 2100F TEM. XRD was tested using a Rigaku Ultima IV X-ray diffractometer from 5°to 80°. UV-vis absorption spectra were recorded on UV-1900 spectrophotometer (Shimadzu, Japan). The hydrodynamic diameter was measured by a NanoBrook 90Plus Particle Size Analyzer.
Synthesis of Bi NSs: Dissolve 162 mg of BiCl 3 , 100 mg of sodium hydroxide and 500 mg of polyvinylpyrrolidone in 25 mL ethylene glycol, respectively, under ultra-sonication and stirring, until the solution became milky white without solid precipitation observed. Afterward, vacuumize and remove water from the device under argon protection throughout the whole process. Transfer the device to the oil bath heating device, and raise the temperature to 80°C first and then to 180°C for 1 h when the solution changed to light yellow and finally to black. Pour the reaction solution into the ice bath prepared in advance to quench the reaction. After the solution cooled to room temperature, collect Bi NSs followed by washing with absolute ethanol for three times. The finally obtained Bi NSs dispersed in 10 mL absolute ethanol solution for subsequent use.
Synthesis of Au NPs: Add 200 μL of potassium chloroaurate (10 mm) into 10 mL ultra-pure water, and fully ultrasonically stir the solution. Add 200 μL trisodium citrate (10 mm) to the above solution which was stirred magnetically for 10 min, followed by the addition of 200 μL sodium borohydride (0.1 m). The resultant solution was stirred for 30 min and changed to orange red. If the solution is purple, it means that the particle size of gold seed is too large due to the overtime reaction. Finally, the prepared Au NPs were stored at 4°C.
Synthesis of Au@Bi NSs: The Bi NSs ultrasonically dispersed in anhydrous ethanol with 200 μL APTES added under vigorous stirring. Then the reaction was carried out in an oil bath at 70°C for 1.5 h. 1.0 mL of the surface aminated Bi NSs dispersed in 10 mL of absolute ethanol which was mixed with 60 μL nano gold seed. Subsequently, 300 μL tetrachloro auric acid (10 mm) and 300 μL trisodium citrate (10 mm) were added together under stirring. 5 min later, 100 μL AA (0.1 m) was added for another 30 min reaction to form the coronary Au@Bi NSs.
Assembly of the Au@Bi-Based EIS Immunosensor: Scheme 1 expresses both the preparation process of the coronary Au@Bi NSs as the EIS immunosensing platform and the specific preparation process of the Au@Bi based EIS immunosensor for CEA detection. Firstly, after GCE was pretreated using aluminum oxide powder (0.05 μm in diameter), 6.0 μL of 10 μg mL −1 Au@Bi NSs was dropped on the smooth electrode surface which was thoroughly dried at room temperature. Afterward, 6.0 μL of 10 μg mL −1 Ab was added dropwise on the surface of Au@Bi/GCE for combining incubation between Ab and Au@Bi through forming a stable chemical bond. The unbound Ab was removed with PBS (pH = 7.4). 3.0 μL 1% BSA solution was used to block the nonspecific binding sites, and the excess BSA was washed off with PBS. In the last step, different concentrations of CEA were cultured on the modified electrode surface with the nonspecific bound CEA removed with PBS. The constructed Au@Bi based EIS immunosensor was stored at 4°C for subsequent use.
Analysis of Human Blood Samples: All human blood samples were used with the approval of Biomedical Ethics Committee in University of Shanghai for Science and Technology and the informed written consent of all participants were obtained. Two human serum samples were selected for the CEA assay, one of which was from a cancer patient and the other one from a non-cancer patient. The blood samples were centrifuged at 2000 rpm for 10 min and the serum was separated for this electrochemical immunosensor assay. In addition, three concentrations of CEA (1, 5, and 10 ng mL −1 ) were added to the samples by the standard addition method, where the relative standard deviations (RSD) and recoveries were calculated to evaluate the feasibility and accuracy of this immunosensor.