Ultrasensitive Leaky Surface Acoustic Wave Immunosensor for Real-Time Detection of Alpha-Fetoprotein in Biological Fluids

We propose an ultrasensitive leaky surface acoustic wave (LSAW) immunosensor based on molybdenum disulfide @ cuprous oxide—gold (MoS2@Cu2O-Au) nanoparticles and subsequent gold staining for the detection of alpha-fetoprotein (AFP). MoS2@Cu2O-Au nanoparticles, with their large specific surface area and good biocompatibility, not only capture the secondary antibodies (Ab2) but also amplify the mass loading effect of the acoustic wave sensor in the detection of AFP. The immunosensor signals are further amplified upon injection of gold staining solution. The developed immunosensor achieved a low detection limit of 5.5 and 25.0 pg/mL with and without gold staining, respectively. The immunosensor demonstrated its efficiency for the quantitative detection of AFP in complex biological fluids, including human serum and saliva samples, with excellent selectivity and long-term stability, showing great potential for the quantification of AFP in clinical diagnosis.


Introduction
Cancer poses a serious threat to human health worldwide, and its early diagnosis has the potential to reduce mortality rates. The detection of tumor markers is an effective approach for the early diagnosis of cancer [1]. Alpha-fetoprotein (AFP), an embryo-specific glycoprotein, is a widely accepted biomarker for fetal defects and tumor progression. The level of AFP in serum of healthy humans is generally below 25 ng/mL, while its concentration increases up to 500 ng/mL in some diseases, such as the germ cell tumor, hepatocellular carcinoma (HCC), and yolk sac tumor [2]. The concentration of serum AFP is considered as a gold standard for the diagnosis of HCC in liver cancer patients. However, the use of saliva for the detection of tumor markers is noninvasive and more acceptable for screening than the serum assay. Accordingly, ultrasensitive detection of AFP in body fluids is crucial for the early diagnosis of cancer.
Numerous detection methods, including fluorescence, electrochemical, colorimetric, and enzyme-linked immunosorbent assays, have been established for the quantification of AFP [3][4][5][6]. However, several techniques have major drawbacks, such as long analysis time, proposed for the detection of AFP, as illustrated in Figure 1. Owing to their good stability, great biocompatibility, and large specific surface area, MoS 2 @Cu 2 O-Au nanoparticles not only enhance the number of captured antibodies but also increase the mass loading on the sensor surface, resulting in high sensitivity. The markedly amplified sensitivity was achieved further by injecting the gold staining solution. The immunoassay was conducted with varying concentrations of AFP from 0.01 to 50 ng/mL and achieved an ultralow limit of detection (LOD) of 5.5 and 25 pg/mL with and without gold staining, respectively. The real-sample detection capabilities of the presented biosensor were evaluated by detecting AFP in human serum and saliva samples with satisfactory results. Salivary AFP detection is a promising strategy for the detection of HCC with saliva as the diagnostic fluid alternative to the serum assay. In addition, the immunosensor exhibited acceptable selectivity and excellent long-term stability, which has promising applications for clinical diagnosis. mine hydrochloride. This reaction leads to the deposition of gold on the AuNPs as nucleation sites, increasing their mass and size, and thus resulting in remarkable signal amplification of the immunoassay.
Herein, an ultrasensitive, sandwich-type LSAW immunosensor based on MoS2@Cu2O-Au nanoparticles and subsequent gold staining as a signal amplification strategy was proposed for the detection of AFP, as illustrated in Figure 1. Owing to their good stability, great biocompatibility, and large specific surface area, MoS2@Cu2O-Au nanoparticles not only enhance the number of captured antibodies but also increase the mass loading on the sensor surface, resulting in high sensitivity. The markedly amplified sensitivity was achieved further by injecting the gold staining solution. The immunoassay was conducted with varying concentrations of AFP from 0.01 to 50 ng/mL and achieved an ultralow limit of detection (LOD) of 5.5 and 25 pg/mL with and without gold staining, respectively. The real-sample detection capabilities of the presented biosensor were evaluated by detecting AFP in human serum and saliva samples with satisfactory results. Salivary AFP detection is a promising strategy for the detection of HCC with saliva as the diagnostic fluid alternative to the serum assay. In addition, the immunosensor exhibited acceptable selectivity and excellent long-term stability, which has promising applications for clinical diagnosis.

Preparation of MoS 2 @Cu 2 O-Au-Ab2 Conjugate
The MoS 2 @Cu 2 O nanohybrid was prepared according to the procedures reported in a previous study [30]. In brief, 26 mg of ammonium tetrathiomolybdate ((NH 4 ) 2 MoS 4 ) and 80 mg of copper nitrate trihydrate (Cu(NO 3 ) 2 ·3H 2 O), each in 20 mL of DI water, were mixed after sonication for 8 min, separately. Subsequently, 200 µL of hydrazine hydrate (N 2 H 4 •H 2 O) was added dropwise into the aqueous mixture and sonicated again for 25 min. The aqueous mixture was kept at 200 • C for 10 h in a Teflon-lined autoclave in an oven. The obtained product was rinsed in succession with water and ethanol five times. Finally, the product was dispersed into 3.0 mL of water and frozen-dried to obtain solid powder.
For comparison, Cu 2 O nanocrystals were synthesized similarly to the preparation of MoS 2 @Cu 2 O nanohybrid, but with the addition of 150 µL N 2 H 4 •H 2 O and without added (NH 4 ) 2 MoS 4 . Furthermore, MoS 2 nanoflowers were also synthesized in a similar The preparation of gold nanoparticles was based on previous reports [35]. Briefly, HAuCl 4 aqueous solution (0.01%, 100 mL) was heated to boil with vigorous stirring. Afterwards, trisodium citrate solution (1%, 2.5 mL) was added into the boiling HAuCl 4 solution, resulting in a color change from light yellow to deep red. After the color change, heating was continued for another 15 min. The resultant solution was allowed to cool to ambient temperature and stored at 4 • C for further use.
In the following step, 10 mg synthesized MoS 2 @Cu 2 O powder was dispersed in 10 mL of freshly prepared AuNPs solution under constant stirring for 24 h. The resultant product was rinsed with DI water and ethanol to remove excess AuNPs. Finally, the sediments were dried in a vacuum oven and referred to as MoS 2 @Cu 2 O-Au. MoS 2 @Cu 2 O-Au nanoparticles were characterized by scanning electron microscopy (SEM, Zeiss EVO-MA) and transmission electron microscopy (TEM, Titan 3 Themis G2, ThermoFisher Scientific). The elemental mapping for SEM images was carried out by energy-dispersive X-ray spectroscopy (EDS).
Four milligrams of MoS 2 @Cu 2 O-Au conjugate was ultrasonically dispersed in 1.0 mL ultrapure water. Subsequently, 20 µg/mL Ab 2 (1 mL) was added in solution dropwise and shaken at 4 • C for 12 h. The mixture was centrifuged and rinsed with DI water to remove unbound Ab 2 . Then, the precipitates were redispersed in 1 mL DI water and stored at 4 • C.

Fabrication of SAW Device
The SAW devices were fabricated on a single-side, polished, 36 • -rotated Y-cut and X-propagation LiTaO 3 wafers. The SAW sensors were designed at a center frequency of 175 MHz. The input and output interdigital transducer (IDT) of each sensor contained 36 finger pairs with an electrode width and wavelength of 6 and 24 µm, respectively. First, the wafers were rinsed sequentially with acetone, isopropyl alcohol, followed by DI water, and dried with nitrogen gas. The IDT patterns were fabricated as follows. A negative photoresist (sunlift1303) was spin-coated on the piezoelectric wafer after solvent cleaning. After prebaking (65 • C/30 s and 95 • C/90 s), the coated wafer was exposed to a UV light source for 2.1 s, followed by another baking round (65 • C/20 s and 95 • C/20 s). Finally, the wafer was treated with the developer solution for 25 s, followed by washing with acetone.
The delay line area (space between two IDTs) with dimensions of 9.2 mm × 7.2 mm was used for the immunoreaction. The Ti/Au (20/50 nm) thin film was deposited by electron beam physical evaporation on the delay line of SAW devices.

Immobilization of Ab 1 on Delay Line of SAW
First, the Au-coated delay line area of the SAW sensor was cleaned with Piranha solution (30% H 2 O 2 : concentrated H 2 SO 4 = 1:3) for 5 min. Then, the sensor was rinsed with DI water and ethanol, followed by drying with nitrogen gas. The same cleaning steps were used after each chemical treatment. Afterwards, the delay line was soaked in mercaptoacetic acid (50 mM) for 16 h and rinsed with ethanol and DI water to remove the excess. In the following step, the delay line was treated with a 1:1 mixture solution of NHS (100 mM) and EDC (400 mM) in pure methanol for 2 h to activate the surface. After solvent cleaning and drying, the delay line was incubated with 100 µg/mL of Ab 1 at 4 • C overnight. Then, it was treated with 3% BSA in PBS for 3 h to block nonspecific adsorption. Finally, the delay line of the SAW sensor was washed with PBS, followed by drying with nitrogen gas. The immobilization of Ab 1 on delay line of SAW sensor was confirmed using contact angle measurement system (CAG100 Contact Angle Goniometer, Beijing United Test Co., Ltd., Beijing, China), QCM frequency measurements (QCM-I, MicroVacuum Ltd., Budapest, Hungary), optical microscopy (CX40 Biological Optical Microscope, Ningbo Sunny Technology Co., Ltd., Zhejiang, China), and scanning electron microscopy (SEM, Zeiss EVOMA).

SAW-Based Sandwich Immunoassay
The SAW measurement system comprises an injection pump, microfluidic chamber, network analyzer, and personal computer (PC), as shown in Figure 2. A specially designed microfluidic channel was fabricated to obtain automated quantitative sample injection. The injection pump (TS-1B syringe pump system, Longer Precision Pump Co., Ltd, Hebei, China) was used for injecting sample solutions on the surface of the SAW sensor placed in the microfluidic chamber, at a flow velocity of 25 µL/min ( Figure 2). delay line of the SAW sensor was washed with PBS, followed by drying with nitrogen gas. The immobilization of Ab1 on delay line of SAW sensor was confirmed using contact angle measurement system (CAG100 Contact Angle Goniometer, Beijing United Test Co., Ltd., Beijing, China), QCM frequency measurements (QCM-I, MicroVacuum Ltd., Budapest, Hungary), optical microscopy (CX40 Biological Optical Microscope, Ningbo Sunny Technology Co., Ltd., Zhejiang, China), and scanning electron microscopy (SEM, Zeiss EVOMA).

SAW-Based Sandwich Immunoassay
The SAW measurement system comprises an injection pump, microfluidic chamber, network analyzer, and personal computer (PC), as shown in Figure 2. A specially designed microfluidic channel was fabricated to obtain automated quantitative sample injection. The injection pump (TS-1B syringe pump system, Longer Precision Pump Co., Ltd, Hebei, China) was used for injecting sample solutions on the surface of the SAW sensor placed in the microfluidic chamber, at a flow velocity of 25 μL/min (Figure 2). The immunoreaction was performed as follows. First, 10 mM PBS was allowed to flow on the sensor surface placed in the microfluidic chamber at 25 μL/min to obtain a stable signal of the center frequency. Then, the AFP sample solution (in PBS) was incubated with MoS2@Cu2O-Au-Ab2 conjugates, and this mixture was allowed to flow on the sensor surface at 5 μL/min. After PBS washing, the gold staining solution comprising 20 mM hydroxylamine hydrochloride and 10 mM gold (III) chloride trihydrate was injected. Finally, the delay line was washed again with PBS until an equilibrate signal of center frequency was achieved.
AFP solutions of 0.01 to 50 ng/mL concentrations were tested using this immunoassay. Each concentration of AFP was tested in triplicate, and the shift in the center frequency for the immunoreaction was recorded as the mean ± standard deviation (SD) to evaluate the repeatability. The center frequency change for each test was recorded using a network analyzer, Keysight Technologies, E5071C. Data were recorded in real time by a specific program based on LabVIEW software. The immunoreaction was performed as follows. First, 10 mM PBS was allowed to flow on the sensor surface placed in the microfluidic chamber at 25 µL/min to obtain a stable signal of the center frequency. Then, the AFP sample solution (in PBS) was incubated with MoS 2 @Cu 2 O-Au-Ab 2 conjugates, and this mixture was allowed to flow on the sensor surface at 5 µL/min. After PBS washing, the gold staining solution comprising 20 mM hydroxylamine hydrochloride and 10 mM gold (III) chloride trihydrate was injected. Finally, the delay line was washed again with PBS until an equilibrate signal of center frequency was achieved.
AFP solutions of 0.01 to 50 ng/mL concentrations were tested using this immunoassay. Each concentration of AFP was tested in triplicate, and the shift in the center frequency for the immunoreaction was recorded as the mean ± standard deviation (SD) to evaluate the repeatability. The center frequency change for each test was recorded using a network analyzer, Keysight Technologies, E5071C. Data were recorded in real time by a specific program based on LabVIEW software.

Characterization of MoS 2 @Cu 2 O-Au
The morphology of MoS 2 @Cu 2 O-Au nanoparticles at various stages of synthesis was characterized by scanning electron microscopy (SEM). Figure 3A shows the SEM image of the as-synthesized Cu 2 O nanocrystals with an average size of~80-120 nm. Similarly, the SEM and TEM images of as-synthesized MoS 2 exhibited a flower-like morphology with a large surface area ( Figure 3B,E). Upon the addition of Cu 2 O nanocrystals to the solution during MoS 2 nanoflower synthesis, Cu 2 O nanocrystals were dispersed with MoS 2 nanoflowers, which results in the formation of coral-like MoS 2 @Cu 2 O nanohybrids ( Figure 3C). The uniform dispersion of the MoS 2 @Cu 2 O nanohybrid was further confirmed by TEM ( Figure 3G). When gold nanoparticles (AuNPs) were added to the MoS 2 @Cu 2 O nanohybrid, the surface morphology changed significantly ( Figure 3D,H). Numerous AuNPs were loaded on the MoS 2 @Cu 2 O nanohybrid by vigorous shaking, as observed in the TEM image of MoS 2 @Cu 2 O-Au ( Figure 3H). The diameter of AuNPs is~13 nm, measured using the TEM image ( Figure 3F). characterized by scanning electron microscopy (SEM). Figure 3A shows the SEM image of the as-synthesized Cu2O nanocrystals with an average size of ~80-120 nm. Similarly, the SEM and TEM images of as-synthesized MoS2 exhibited a flower-like morphology with a large surface area ( Figure 3B,E). Upon the addition of Cu2O nanocrystals to the solution during MoS2 nanoflower synthesis, Cu2O nanocrystals were dispersed with MoS2 nanoflowers, which results in the formation of coral-like MoS2@Cu2O nanohybrids ( Figure  3C). The uniform dispersion of the MoS2@Cu2O nanohybrid was further confirmed by TEM ( Figure 3G). When gold nanoparticles (AuNPs) were added to the MoS2@Cu2O nanohybrid, the surface morphology changed significantly ( Figure 3D,H). Numerous AuNPs were loaded on the MoS2@Cu2O nanohybrid by vigorous shaking, as observed in the TEM image of MoS2@Cu2O-Au ( Figure 3H). The diameter of AuNPs is ~13 nm, measured using the TEM image ( Figure 3F). The elemental composition of MoS2@Cu2O-Au was characterized by SEM-EDS elemental mapping, as shown in Figure S1. From these images, the presence of Au, Mo, O, Cu, and S elements in this material is clearly observed, which is further verified by the energy-dispersive X-ray (EDX) spectra of MoS2@Cu2O-Au in Figure S2. The elemental mapping indicates successful synthesis of MoS2@Cu2O-Au nanoparticles.

Preparation and Characterization of Self-Assembled Monolayer (SAM)
The scheme of a sandwich-type SAW immunosensor for the detection of AFP is presented in Figure 1. First, an oriented mercaptoacetic acid monolayer can be directly formed on the Au-coated SAW surface via strong Au-S bonds, with the tail carboxylic groups oriented at the interface [36]. Upon activation with EDC and NHS, carboxylic groups were replaced with ester groups. Finally, ester groups were replaced by amine groups of primary antibodies (Ab1), which resulted in the immobilization of Ab1 on the SAW surface as a SAM layer. The immobilization of Ab1 on the sensor surface was characterized by QCM frequency measurements based on previous reports [28]. Figure 4A  The elemental composition of MoS 2 @Cu 2 O-Au was characterized by SEM-EDS elemental mapping, as shown in Figure S1. From these images, the presence of Au, Mo, O, Cu, and S elements in this material is clearly observed, which is further verified by the energy-dispersive X-ray (EDX) spectra of MoS 2 @Cu 2 O-Au in Figure S2. The elemental mapping indicates successful synthesis of MoS 2 @Cu 2 O-Au nanoparticles.

Preparation and Characterization of Self-Assembled Monolayer (SAM)
The scheme of a sandwich-type SAW immunosensor for the detection of AFP is presented in Figure 1. First, an oriented mercaptoacetic acid monolayer can be directly formed on the Au-coated SAW surface via strong Au-S bonds, with the tail carboxylic groups oriented at the interface [36]. Upon activation with EDC and NHS, carboxylic groups were replaced with ester groups. Finally, ester groups were replaced by amine groups of primary antibodies (Ab 1 ), which resulted in the immobilization of Ab 1 on the SAW surface as a SAM layer. The immobilization of Ab 1 on the sensor surface was characterized by QCM frequency measurements based on previous reports [28]. Figure 4A shows the typical sensorgram for each step of QCM electrode fabrication. The bare electrode (Au coated) was adopted as a reference (Curve a). Curve b shows the decrease in the QCM frequency due to the binding of MPA on the sensor surface, resulting from its reaction with the gold-coated electrode. The frequency decreased further as the electrode was incubated with NHS/EDC, showing coupling of the molecules on the sensor surface (Curve c). A significant frequency shift was observed when Ab 1 was injected on the electrode surface, indicating mass loading on the sensor surface (Curve d). These results confirmed the immobilization of Ab 1 onto the electrode surface. of the SAW surface. Figure 4C clearly shows the added thin layer of SAM on the SAW surface compared to the blank control in Figure 4B. These results are in agreement with previous reports [37]. The successful formation of SAM was confirmed further by measuring the contact angle of water droplets on the delay line of the SAW surface ( Figure 5). As shown in Figure 5B, the contact angle was reduced from 79° to 26° after the formation of SAM.  SEM images were also used to verify the immobilization of the Ab 1 on the delay line of the SAW surface. Figure 4C clearly shows the added thin layer of SAM on the SAW surface compared to the blank control in Figure 4B. These results are in agreement with previous reports [37]. The successful formation of SAM was confirmed further by measuring the contact angle of water droplets on the delay line of the SAW surface ( Figure 5). As shown in Figure 5B, the contact angle was reduced from 79 • to 26 • after the formation of SAM. Optical microscopy images were further used to verify the immobilization of Ab1 on the delay line of the SAW surface. As shown in Figure S3Ab, the SAM-coated delay line distinctly shows the captured antibody molecules, which are absent from the blank control group. The capture of antibody molecules on the delay line was further confirmed using FITC-labeled primary antibody (Ab1-FITC). As presented in Figure S3Bb, the fluorescent effect was observed only after the formation of SAM, indicating the successful capture of antibodies on the SAW surface, whereas the blank control remained dark, suggesting that no antibody was captured.

Real-Time Detection Studies with SAW Immunosensor
The typical response-time curve of the SAW immunosensor is presented in Figure  6A. At first, PBS was pumped through the chamber until stabilization of the center frequency baseline. Then, the AFP antigen mixed with MoS2@Cu2O-Au-Ab2 conjugate was The contact angles were drastically reduced after the formation of the SAM bioreceptor because of the adsorption of hydrophilic antibody molecules. The adsorbed polar biological molecules attract water molecules, resulting in a reduced contact angle after the formation of SAM. These results demonstrate the successful capture of Ab 1 on the delay line of the SAW surface, as anticipated.
Optical microscopy images were further used to verify the immobilization of Ab 1 on the delay line of the SAW surface. As shown in Figure S3Ab, the SAM-coated delay line distinctly shows the captured antibody molecules, which are absent from the blank control group. The capture of antibody molecules on the delay line was further confirmed using FITC-labeled primary antibody (Ab 1 -FITC). As presented in Figure S3Bb, the fluorescent effect was observed only after the formation of SAM, indicating the successful capture of antibodies on the SAW surface, whereas the blank control remained dark, suggesting that no antibody was captured.

Real-Time Detection Studies with SAW Immunosensor
The typical response-time curve of the SAW immunosensor is presented in Figure 6A. At first, PBS was pumped through the chamber until stabilization of the center frequency baseline. Then, the AFP antigen mixed with MoS 2 @Cu 2 O-Au-Ab 2 conjugate was allowed to selectively bind with the specific antibodies in the flow cell, causing a large shift in the center frequency. The signals were amplified further, as the gold staining solution was introduced on the sensor surface, inducing gold deposition onto the captured AuNPs. The real-time detection curve indicates the successful binding of target AFP with specific antibodies and subsequent mass enhancement by gold staining. The frequency shift observed upon injection of AFP solution incubated with Ab 2 (without conjugation with MoS 2 @Cu 2 O-Au nanoparticle) is shown in Figure S4. The center frequency shifts at saturation with different concentrations of AFP were extracted from real-time curves ( Figure 6B) and normalized to fit into one plot ( Figure 6C). In this graph, the center frequency value for each concentration is blank (without AFP) subtracted. In double logarithmic coordinates, a linear relationship between the center frequency shift and AFP concentration is obtained, with a correlation coefficient of 0.9942 ( Figure 6C, solid line). The limit of detection (LOD) was estimated at 5.5 pg/mL for AFP with gold staining, calculated as the blank frequency shift by PBS + 3 × noise [38]. The LOD of the present immunosensor was significantly better than various piezoelectric immunosensors reported for the detection of AFP [39][40][41].
The analytical properties of the immunosensor were also tested without gold staining. A linear dependence was observed between the center frequency shift and AFP concentration ranging from 0.03 to 50 ng/mL on a log-log coordinate with the correlation co-

Quantification of AFP
To investigate the sensitivity of sandwich-type SAW immunosensor, various concentrations of AFP ranging from 0.01 to 50 ng/mL were tested and exposed to respective concentrations of gold staining solution. The real-time frequency shifts of the gold staining at different concentrations of AFP are presented in Figure 6B.
The center frequency shifts at saturation with different concentrations of AFP were extracted from real-time curves ( Figure 6B) and normalized to fit into one plot ( Figure  6C). In this graph, the center frequency value for each concentration is blank (without AFP) subtracted. In double logarithmic coordinates, a linear relationship between the center frequency shift and AFP concentration is obtained, with a correlation coefficient of 0.9942 ( Figure 6C, solid line). The limit of detection (LOD) was estimated at 5.5 pg/mL for AFP with gold staining, calculated as the blank frequency shift by PBS + 3 × noise [38]. The LOD of the present immunosensor was significantly better than various piezoelectric immunosensors reported for the detection of AFP [39][40][41].
The analytical properties of the immunosensor were also tested without gold staining. A linear dependence was observed between the center frequency shift and AFP concentration ranging from 0.03 to 50 ng/mL on a log-log coordinate with the correlation coefficient of 0.9958. The LOD was estimated at 25 pg/mL for AFP in the absence of gold staining ( Figure 6C, dotted line).
The excellent performance of the LSAW immunosensor might be attributed to two factors. First, MoS 2 @Cu 2 O-Au nanoparticles with high specific surface area and great biocompatibility not only increased the number of captured Ab 2 but also magnified the mass loading on the sensor surface, resulting in high sensitivity even without gold staining. Similarly, gold staining as a signal amplification strategy further improved the sensitivity of immunosensor, resulting in an extremely low LOD for AFP.

Immunosensor Selectivity
The selectivity of the immunosensor has been evaluated for its vital role in examining biological fluids in situ without separation. The selectivity analysis was conducted by testing AFP and other interfering substances, such as the carcinoembryonic antigen (CEA), tryptophan, prostate-specific antigen (PSA), and BSA, each at a concentration of 10 ng/mL. As shown in Figure 7A, the immunosensor did not exhibit any considerable affinity for the nonspecific analytes. A large frequency shift was observed only for the target AFP, while no significant frequency shift was detected corresponding to any nonspecific targets, indicating an acceptable selectivity of the developed immunosensor.

Immunosensor Selectivity
The selectivity of the immunosensor has been evaluated for its vital role in examining biological fluids in situ without separation. The selectivity analysis was conducted by testing AFP and other interfering substances, such as the carcinoembryonic antigen (CEA), tryptophan, prostate-specific antigen (PSA), and BSA, each at a concentration of 10 ng/mL. As shown in Figure 7A, the immunosensor did not exhibit any considerable affinity for the nonspecific analytes. A large frequency shift was observed only for the target AFP, while no significant frequency shift was detected corresponding to any nonspecific targets, indicating an acceptable selectivity of the developed immunosensor.

Detection of AFP in Biological Fluids
To evaluate the practical applicability of the proposed immunosensor, the results of APF detection in human serum and saliva samples were used as an effective analysis base. First, serum and saliva samples were diluted with PBS (2.5 fold); then, a standard concentration of AFP was added to each sample. Subsequently, the spiked samples were tested by the LSAW immunosensor, and the results are listed in Table 1. The obtained recovery values are in the satisfactory range of 95-108%.

Detection of AFP in Biological Fluids
To evaluate the practical applicability of the proposed immunosensor, the results of APF detection in human serum and saliva samples were used as an effective analysis base. First, serum and saliva samples were diluted with PBS (2.5 fold); then, a standard concentration of AFP was added to each sample. Subsequently, the spiked samples were tested by the LSAW immunosensor, and the results are listed in Table 1. The obtained recovery values are in the satisfactory range of 95-108%. Moreover, the commercially available ELISA method was employed as a reference method to validate the LSAW immunosensor. The data show good agreement between the two methods with high accuracy ( Table 2). These results suggest that the proposed immunosensor is reliable for the detection of AFP in complex biological fluids. The analytical properties of the LSAW immunosensor have been compared with previous reports in terms of their detection of AFP, and the results are summarized in Table S1. The LOD of the LSAW immunosensor was either comparable or superior to other detection methods, and our assay worked efficiently for different kinds of biological fluids, a factor not addressed in most of the reported methods in terms of AFP detection.
The LSAW immunosensor not only quantitatively detects the concentration of AFP but also monitors the capture of AFP by its specific antibodies in real time. Real-time detection provides information about the dynamics of antibody-antigen interaction and may facilitate real-time drug testing technology.
Moreover, the immunosensor operates under a continuous flow mode, making it particularly suitable for the direct detection of tumor markers in clinical samples, eliminating the need for pretreatment of clinical samples. This enables rapid analysis of clinical samples with a considerable reduction in the assay cost.

Long-Term Stability
Long-term stability of the LSAW immunosensor was tested for 5 weeks. The fabricated immunosensor was stored at 4 • C and tested for 50 ng/mL of AFP intermittently every week. According to Figure 7B, the center frequency response was about 70% of the initial value after 5 weeks, confirming excellent long-term storage stability of the SAW immunosensor.

Conclusions
We developed an ultrasensitive, sandwich-type leaky surface acoustic wave (LSAW) immunosensor based on coral MoS 2 @Cu 2 O-Au nanoparticles and subsequent gold staining for the real-time detection of alpha-fetoprotein (AFP) in biological fluids. A markedly enhanced sensitivity of the immunosensor was achieved by labeling the secondary antibodies with MoS 2 @Cu 2 O-Au nanoparticles. The detection sensitivity was increased further by employing gold staining as a signal amplification strategy. The proposed immunosensor exhibited long-term stability and satisfactory selectivity with an ultralow detection limit for AFP. These merits make our LSAW sensing platform prospective as an impressive novel method for ultrasensitive detection of AFP in clinical applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/chemosensors9110311/s1, Figure S1: Elemental mapping of MoS 2 @Cu 2 O-Au, Figure S2: EDX spectra of MoS 2 @Cu 2 O-Au, Figure S3: Optical microscopic images of the delay line before and after immobilization of Ab 1 and FITC-Ab 1 , Figure S4: Frequency shift observed upon injection of AFP solution incubated with Ab 2 , Table S1: Comparison of other methods for detection of AFP with SAW based immunosensor.