Anti-pollution electrochemical saliva sensor based on fluorinated side chain polypeptide as well as preparation method and application of anti-pollution electrochemical saliva sensor

Abstract

The invention belongs to the technical field of electrochemical detection, and discloses an anti-pollution electrochemical saliva sensor based on fluorinated side chain polypeptide as well as a preparation method and application of the anti-pollution electrochemical saliva sensor. The fluorinated side chain polypeptide disclosed by the invention not only has excellent hydrophilicity, can form a dense hydration layer with water molecules to repel pollutants, but also has low surface energy, and the interaction between the pollutants and an interface is remarkably reduced. Through the synergistic effect, the fluorinated side chain polypeptide has better anti-pollution performance compared with traditional zwitterionic polypeptide. The anti-pollution electrochemical sensor constructed on the basis of the fluorinated side chain polypeptide can be used for directly detecting CA15-3 in saliva, the saliva does not need to be pretreated, and the detection accuracy can be comparable with that of a commercial ELISA (Enzyme-Linked Immunosorbent Assay) method.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology, specifically relating to a method for preparing an anti-fouling electrochemical saliva sensor based on fluorinated side-chain peptides and its application in the quantitative detection of carbohydrate antigen 15-3 (CA15-3) in saliva. Background Technology

[0002] While blood analysis remains the gold standard for disease diagnosis, providing a comprehensive biomarker profile including proteins, lipids, and metabolites, its clinical application is limited by inherent limitations. In contrast, saliva has gained widespread attention as an alternative diagnostic medium, offering significant advantages in clinical practice. Its completely non-invasive collection method eliminates patient discomfort while maintaining the significant stability of its molecular components. Crucially, numerous studies have demonstrated a significant correlation between saliva and serum biomarker concentrations, validating the reliability of saliva-based diagnostics. Therefore, developing highly sensitive, user-friendly detection methods to accurately quantify biomarkers in saliva is essential for early tumor detection and improved patient prognosis.

[0003] Electrochemical biosensors have attracted widespread research interest in the field of saliva biomarker detection due to their high sensitivity, rapid detection, and real-time response. However, electrodes often become contaminated when in contact with physiological fluids, with non-specific protein adsorption reducing signal specificity. Therefore, developing anti-fouling electrochemical sensors that can effectively reduce non-specific adsorption is essential. Surface modification with zwitterionic peptides is an effective anti-fouling strategy—they form a protective layer through charge neutralization and strong hydration. However, this hydration layer can fail due to long-term contaminant deposition, so the rational design of zwitterionic peptides with excellent anti-fouling properties remains a fundamental challenge. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an anti-fouling electrochemical saliva sensor based on fluorinated side-chain peptides, its preparation method, and its application.

[0005] It should be noted that in marine ecosystems, shark skin possesses excellent antifouling properties, effectively preventing the attachment of organisms such as barnacles and sea anemones. This property stems from its unique multilayered structure, which combines superhydrophilic dermal denticles with an outer low-surface-energy mucus layer. Inspired by this, this invention introduces low-surface-energy fluorinated alkyl chains into zwitterionic peptide sequences. The hydrophilic regions of this peptide can form a hydration layer, thereby creating steric hindrance and an energy barrier to inhibit the interaction between contaminants and the surface; while the low-surface-energy regions weaken interfacial bonding, reducing the contact area and adhesion of contaminants to the surface. Therefore, synthesizing fluorinated side-chain peptides can effectively improve the antifouling properties of traditional peptides. Consequently, this invention develops a method for preparing an antifouling electrochemical sensor based on fluorinated side-chain peptides. This method can also effectively improve the accuracy of detecting disease biomarkers in real biological samples, which has profound significance for the in vitro diagnosis of cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first technical objective of this invention is to provide an antifouling electrochemical saliva sensor based on a fluorinated side-chain peptide. The sensor uses a conductive polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), and gold nanoparticles (AuNPs) as electrode modification materials, a self-designed fluorinated side-chain peptide as the antifouling material, and is further modified with a CA15-3 specific aptamer (Apt). The sequence of the fluorinated side chain polypeptide (FSP) is: C(Hyp)4K(G-Perfluoropropionic acid)EKEKEK; the sequence of the CA15-3 aptamer is: 5′-HS-(CH2)6-GCA GTT GAT CCT TTG GAT ACC CTG G-3′. The FSP and the CA15-3 aptamer are connected to the electrode via a gold-sulfur bond.

[0007] The second technical objective of this invention is to provide a method for preparing an anti-fouling electrochemical saliva sensor based on fluorinated side-chain peptides as described above, comprising the following steps: I. Preparation of Au / PEDOT modified electrode: The sensor is based on a glassy carbon electrode (GCE), and PEDOT thin film and AuNPs are deposited on the surface using a constant potential method to prepare Au / PEDOT / GCE. II. Preparation of the anti-fouling electrochemical saliva sensor: Au / PEDOT / GCE was incubated in a mixed solution of FSP and Apt to prepare the anti-fouling electrochemical saliva sensor.

[0008] Furthermore, the specific method of step I is as follows: On the GCE, a PEDOT film was electrodeposited for 20-60 s using an amperometric method in a mixed solution of 0.02-0.04 M 3,4-ethylenedioxythiophene (EDOT) and 0.02-0.04 M phytic acid (PA) at a potential of 1.1 V. The PEDOT film electrode was then subjected to cyclic voltammetry scanning for 2-6 cycles in an aqueous solution containing 5.0-10.0 mM HPtCl4 and 0.5-1.0 M KNO3 at a voltage range of –0.5-1.2 V to obtain Au / PEDOT / GCE.

[0009] Furthermore, the specific method for step II is as follows: The FSP solution, Apt solution, and TCEP solution were mixed to obtain a final FSP concentration of 0.2–0.4 mg / mL. -1 A mixed solution with a final Apt concentration of 3-6 μM and a final TCEP concentration of 0.1-0.2 mM was prepared. The Au / PEDOT / GCE mixture was incubated in the mixed solution overnight. After the reaction was completed, the mixture was rinsed with deionized water to remove unfixed FSP and Apt, thus obtaining the sensor.

[0010] Furthermore, the sequence of the fluorinated side chain polypeptide (FSP) is: C(Hyp)4K(G-Perfluoropropionicacid)EKEKEK; the sequence of the CA15-3 aptamer is: 5′-HS-(CH2)6-GCA GTT GAT CCT TTG GAT ACCCTG G-3′; that is, the sequence of the CA15-3 aptamer is: 5′-GCA GTT GAT CCT TTG GAT ACC CTG G-3′, with HS-(CH2)6 modified at the 5′ end.

[0011] Notably, this invention is the first to design and synthesize a zwitterionic peptide modified with a fluorinated alkyl chain. This peptide exhibits a "Y" configuration, with its backbone composed of a hydroxyproline-rich repeating sequence (-(Hyp)4-), forming a robust α-helical support region that ensures stable anchoring of the molecule at the interface. The two functional arms consist of a zwitterionic peptide segment (EKEKEK) and a fluorinated alkyl chain, respectively. The former forms a dense hydration layer to block contaminants, while the latter reduces interfacial adhesion through its extremely low surface energy. The synergistic effect of these three functional units ultimately achieves superior ultra-low dirt adhesion performance on the sensing surface.

[0012] The third technical objective of this invention is to claim protection for the application of an anti-fouling electrochemical saliva sensor prepared by the method described above in the quantitative detection of CA15-3 in saliva.

[0013] Specifically, the quantitative detection operation is as follows: An antifouling electrochemical saliva sensor based on fluorinated side-chain peptides was placed in solutions of the target compound CA15-3 at different concentrations and incubated at a constant temperature. The electrode interface was then rinsed with PBS buffer to remove uncaptured CA15-3, and the resulting electrode was subjected to electrochemical detection. The current signal changes at the sensing interface after incubation in the target compound solution were recorded using differential pulse voltammetry within the range of -0.2 to 0.6 V to achieve the detection of the target compound CA15-3.

[0014] Furthermore, the constant temperature incubation is at room temperature, and the incubation time is 60-120 min, to ensure the full binding of the target compound CA15-3.

[0015] Furthermore, the linear detection range of CA15-3 is 0.01 - 1000 U·mL. -1 The detection limit was 2.85 mU·mL. -1 .

[0016] It is worth noting that carbohydrate antigen 15-3 (CA15-3) is the most important specific biomarker for breast cancer. 30%-50% of breast cancer patients have significantly elevated CA15-3 levels, and changes in its levels are closely related to treatment efficacy, making it the optimal indicator for diagnosing breast cancer, monitoring postoperative recurrence, and observing treatment effectiveness. Dynamic measurement of CA15-3 is helpful for the early detection of recurrence after treatment in stage II and III breast cancer patients. Therefore, rapid and accurate measurement of CA15-3 is of great significance for the early diagnosis of breast cancer. This invention uses CA15-3 as the target detectable substance, which is representative.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention discloses for the first time the use of fluorinated side chain peptides as anti-fouling materials in sensors.

[0018] (2) The fluorinated side chain peptide disclosed in this invention is inspired by the excellent anti-fouling effect of shark skin. It is the first to introduce a low surface energy fluorinated alkyl chain into the zwitterionic peptide sequence, which achieves synergistic anti-fouling effect of hydrophilicity and low surface. Compared with traditional zwitterionic peptides, it has a better anti-fouling effect and a better application potential.

[0019] (3) Most existing electrochemical sensors cannot be directly used to detect target substances in biological fluids. The sensor of this invention can directly measure CA15-3 in saliva without the need for saliva pretreatment, and has strong practical application capabilities. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 This is a schematic diagram (a) of the preparation process of the antifouling electrochemical saliva sensor based on fluorinated side chain peptides of the present invention, and a schematic diagram (b) of the synergistic antifouling mechanism and chemical structure of FSP.

[0022] Figure 2 Scanning electron microscope images of different modified electrodes provided for embodiments of the present invention.

[0023] Figure 3 X-ray photoelectron spectra of different modified electrodes provided in embodiments of the present invention.

[0024] Figure 4 DPV response curves of different modified electrodes provided in embodiments of the present invention.

[0025] Figure 5 Contact angle diagrams (a) and surface energy diagrams (c) of aqueous phase (b) and oil phase (c) of different modified electrodes provided in embodiments of the present invention; wherein I, II, III, and IV correspond to bare GCE, Au / PEDOT / GCE, ZIP / Au / PEDOT / GCE, and FSP / Au / PEDOT / GCE, respectively.

[0026] Figure 6 This is a diagram showing the optimized peptide concentration provided in an embodiment of the present invention.

[0027] Figure 7 Fluorescence imaging and quantitative image (e) of unmodified interface (a), Au / PEDOT modified interface (b), FSP / Au / PEDOT modified interface (c), and Apt-FSP / Au / PEDOT modified interface (d) after fluorescent protein adsorption, provided for embodiments of the present invention.

[0028] Figure 8 DPV curves of bare GCE (a), Au / PEDOT / GCE (b), FSP / Au / PEDOT / GCE (c) and Apt-FSP / Au / PEDOT / GCE (d) after immersion in BSA solutions of different concentrations provided for embodiments of the present invention; (e) signal suppression rates of different modified electrodes in BSA solutions of different concentrations.

[0029] Figure 9DPV curves of bare GCE (a), Au / PEDOT / GCE (b), FSP / Au / PEDOT / GCE (c) and Apt-FSP / Au / PEDOT / GCE (d) after immersion in mucin solutions of different concentrations provided for embodiments of the present invention; (e) signal suppression rates of different modified electrodes in mucin solutions of different concentrations.

[0030] Figure 10 DPV curves of bare GCE (a), Au / PEDOT / GCE (b), FSP / Au / PEDOT / GCE (c) and Apt-FSP / Au / PEDOT / GCE (d) after immersion in artificial saliva of different concentrations provided for embodiments of the present invention; (e) signal inhibition rate of different modified electrodes in artificial saliva of different concentrations.

[0031] Figure 11 DPV curves of bare GCE (a), Au / PEDOT / GCE (b), FSP / Au / PEDOT / GCE (c) and Apt-FSP / Au / PEDOT / GCE (d) after immersion in human saliva of different concentrations provided for embodiments of the present invention; (e) signal inhibition rate of different modified electrodes in human saliva of different concentrations.

[0032] Figure 12 Line graph comparing the antifouling performance of the ZIP-FSP interface in BSA solution (a), mucin solution (b), artificial saliva (c), and human saliva (d) according to embodiments of the present invention.

[0033] Figure 13 Bar chart showing the long-term anti-fouling properties of different modified electrodes provided in embodiments of the present invention in undiluted human saliva over a period of three weeks.

[0034] Figure 14 An optimized incubation time diagram for CA15-3 provided in an embodiment of the present invention.

[0035] Figure 15 The sensor provided in this embodiment of the invention shows the DPV response and linear curve (c) of the sensor to different concentrations of CA15-3 in PBS (a) and artificial saliva (b).

[0036] Figure 16 Selectivity test diagram of the sensor provided in an embodiment of the present invention.

[0037] Figure 17 This is a repeatability test diagram of the sensor provided in an embodiment of the present invention.

[0038] Figure 18 The 100 U·mL provided in the embodiments of the present invention -1CV scans of CA15-3 before (a) and after (c) incubation for 50 cycles; 100 U·mL -1 DPV curves of CA15-3 before (b) and after (d) incubation were measured seven times using an FSP-based biosensor.

[0039] Figure 19 The storage stability test diagram of the sensor provided in the embodiment of the present invention in PBS (10 mM, pH 7.4).

[0040] Figure 20 Bland-Altman plot showing the consistency between the FSP-based sensor method and ELISA provided in this embodiment of the invention. Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] The following are embodiments of certain implementations of the present invention, which are not intended to limit the scope of the present invention.

[0043] In the following examples, the details of the instruments, equipment, and reagents are as follows.

[0044] Equipment: All electrochemical tests were performed at room temperature using a CHI 660E electrochemical workstation (Shanghai Chenhua). The test system contained 5.0 mM [Fe(CN)6]. 3- / 4- The sample was prepared with 0.1 M KCl in phosphate buffered solution (PBS, 10.0 mM, pH 7.4). A three-electrode system was used: a glassy carbon electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The water contact angle was measured using a JC2000D1 measuring system (Shanghai Zhongchen). Sample morphology was observed using a Hitachi S-4800 scanning electron microscope (Japan). Fluorescence analysis of the antifouling performance of different modified electrodes was performed using a TCS-SP5 confocal laser scanning microscope (Leica, Germany). Elemental analysis of the material surface was conducted using an ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo Fisher Scientific, UK).

[0045] Reagents: The fluorinated side-chain modified peptide (FSP, sequence C(Hyp)4K(G-perfluoropropionic acid)EKEKEK) and the zwitterionic peptide (ZIP, sequence C(Hyp)4EKEKEK) used in this study were designed by our team and synthesized by Shanghai Guotai Technology Co., Ltd. The thiol-modified CA15-3 aptamer (sequence: 5′-HS-(CH2)6-GCA GTT GAT CCT TTG GAT ACC CTG G-3′) was provided by Shanghai Sangon Biotech Co., Ltd. Phytic acid (PA), 3,4-ethylenedioxythiophene (EDOT) monomer, diiodomethane, tris(2-carboxyethyl)phosphine (TCEP), and chloroauric acid (HAuCl4) were purchased from Aladdin (Shanghai, China). Potassium nitrate (KNO3) was provided by Bolinda. Trypsin was purchased from RHAWN Chemicals (Shanghai, China). Matrix metalloproteinase-9 (MMP-9) was sourced from MedChemExpress (Shanghai, China). FITC-BSA and BSA were provided by Solarbio Science & Technology Co., Ltd. (Beijing, China). Mucin was purchased from Maclean Biotechnology Co., Ltd. (Shanghai, China). Artificial saliva was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Saliva samples from healthy volunteers were collected by our laboratory; saliva samples from breast cancer patients were collected from Qingdao Central Hospital (Qingdao, China) after obtaining informed consent. All studies involving human saliva in this experiment were approved by the Ethics Committee of Qingdao University of Science and Technology (Approval No.: QKDLL2025027). Human epidermal growth factor receptor 2 (HER2), uric acid (UA), cortisol, immunoglobulin G (IgG), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA) were provided by Solarbio Science & Technology (Beijing, China). The CA15-3 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Enzyme-Linked Immunosorbent Assay (Changzhou, Jiangsu). Ultrapure water (resistivity >18 MΩ·cm) was prepared using the MilliQ ultrapure water system (Bedford, USA).

[0046] Example 1 The construction process of the antifouling electrochemical saliva sensor based on fluorinated side-chain peptides is as follows: Figure 1 As shown in a: (1) Preparation of Au / PEDOT modified electrode: On GCE, PEDOT film was electrodeposited for 20 s at a potential of 1.1 V in a mixed solution of 0.02 M 3,4-ethylenedioxythiophene (EDOT) and 0.02 M phytic acid (PA) using the amperometric method; Au / PEDOT / GCE was obtained by scanning the PEDOT film electrode for 2 cycles in an aqueous solution containing 5.0 mM HPtCl4 and 0.5 M KNO3 within a voltage range of –0.5 to 1.2 V using cyclic voltammetry.

[0047] (2) Sensor preparation: FSP solution, Apt solution and TCEP solution were mixed to obtain a final FSP concentration of 0.2 mg·mL. -1 A mixed solution with a final Apt concentration of 3 μM and a final TCEP concentration of 0.1 mM was prepared. The Au / PEDOT / GCE mixture was incubated overnight at room temperature in the above mixed solution. After the reaction was completed, the mixture was rinsed with deionized water to remove unfixed FSP and Apt, thus obtaining the sensor.

[0048] The antifouling mechanism and chemical structure of FSP are as follows: Figure 1 As shown in b: A zwitterionic peptide modified with a fluorinated alkyl chain was designed and synthesized. This peptide exhibits a "Y" configuration, with its backbone composed of a hydroxyproline-rich repeating sequence (-(Hyp)4-), forming a robust α-helical support region that ensures stable anchoring of the molecule at the interface. The two functional arms consist of a zwitterionic peptide segment (EKEKEK) and a fluorinated alkyl chain, respectively. The former forms a dense hydration layer to block contaminants, while the latter reduces interfacial adhesion through its extremely low surface energy. The synergistic effect of these three functional units ultimately achieves superior ultra-low dirt adhesion performance on the sensing surface.

[0049] Example 2 Characterization of Au / PEDOT and PEDOT modified electrodes: Depend on Figure 2 (a) The scanning electron microscope image of PEDOT shows that when PEDOT and PA are co-deposited on the electrode surface, an irregular three-dimensional network structure is formed at the electrode interface. Figure 2 (b) After further modification with gold nanoparticles, many small particles with uniform particle size (about 10 nm in diameter) appeared on the surface of the polymer composite material, indicating that Au nanoparticles were successfully modified onto the PEDOT interface to obtain an Au / PEDOT modified electrode.

[0050] Example 3 Characterization of antifouling electrochemical sensors based on fluorinated side-chain peptides (1) XPS characterization of the sensor assembly process: XPS characterization was performed on the assembly interface to monitor the sensor fabrication process, such as... Figure 3 As shown, the PEDOT electrode is mainly composed of carbon (C), oxygen (O), sulfur (S), and phosphorus (P). After depositing AuNPs, a distinct gold (Au) peak appeared. After FSP modification to the interface, new nitrogen (N) and fluorine (F) peaks appeared, while the phosphorus (P) peak intensity decreased, indicating that the FSP was successfully immobilized on the electrode surface. Further modification with Apt resulted in a further enhancement of the phosphorus (P) peak, confirming the successful immobilization of Apt.

[0051] (2) Electrochemical characterization of the sensor assembly process: The assembly process of the biosensor was documented using an electrochemical workstation. For example... Figure 4 As shown, in the negatively charged probe [Fe(CN)6] 3- / 4- DPV curves for different modified interfaces were recorded in solution. Clearly, the DPV peak current of the bare GCE (black curve) is approximately 195 μA. When PEDOT (red curve) was modified onto the electrode, the peak current increased significantly to 223 μA due to the good conductivity of the conductive polymer-modified interface and the significantly increased specific surface area. After depositing AuNPs (blue curve), the DPV peak current increased further. Simultaneously, the abundant loading of AuNPs laid the foundation for subsequent biomolecule linkage. When FSP and Apt were connected to the electrode (green curve), the current signal of the modified electrode further decreased. This is attributed to the poor conductivity and steric hindrance effect of the biomolecules, which hindered electron transfer between the electrode interface and the solution. Finally, when the aptamer specifically bound to the target (purple curve), the current further decreased, indicating the successful construction of the sensor.

[0052] Example 4 Property characterization based on FSP interface: Surface hydrophilicity is a key indicator for evaluating antifouling performance, and its essence depends on the hydrophilic / hydrophobic properties of the modified material. Hydrophilic materials can form a hydration layer at the interface, which creates an energy and spatial barrier against the adsorption of contaminants, effectively preventing the adsorption of biomolecules such as proteins. Therefore, we first characterized the water contact angle and underwater oil contact angle of different modified interfaces. Figure 5 As shown in Figure a, the static water contact angle of the ZIP-based interface is 19.68±0.79°, significantly lower than that of the bare interface (60.31±0.89°) and the Au / PEDOT interface (42.10±1.48°). Simultaneously, the ZIP-based interface also exhibits superior oleophobicity, with an underwater oil contact angle of 164.58°. Figure 5b). Correspondingly, the contact radius of the ZIP-based interface was significantly reduced by approximately 59.60% compared to the Au / PEDOT-based interface. After introducing the fluorinated alkyl chain, the initial water contact angle increased to 28.65 ± 0.93°. Figure 5 a), while the underwater oil contact angle decreased to 160.06±1.21° (a). Figure 5 (b) Furthermore, the contact area of ​​the oil droplets was reduced by nearly 50% compared to the original interface. This is because the introduction of perfluoroalkyl chains at the molecular level prevents the formation of continuous hydrophobic regions on the surface. Therefore, the adverse effects of hydrophobic segments on interfacial hydration are effectively mitigated, thus maintaining the excellent hydrophilicity and underwater oleophobicity of the interface.

[0053] Furthermore, reducing the adhesion of biofouling to the surface is also a key factor in enhancing its antifouling performance. Interfaces with lower surface energy typically exhibit weaker binding capacity and are therefore less likely to adsorb other fouling substances. The two-liquid Owens–Wendt–Kaelble model was used to calculate surface energy. Figure 5 As shown in Figure c, the surface energy dispersion portion (γ) of the FSP-based interface. d The lower value of γ and the lower value of the polar component (γ) p The higher concentration of FSP-based electrodes compared to bare and Au / PEDOT-modified electrodes indicates a weakened van der Waals interaction between the FSP-based interface and nonpolar biofouling, while the interaction with polar water molecules is enhanced. This variation creates a surface with minimal contact area and low contaminant adhesion, allowing biofouling to detach easily.

[0054] Example 5 Anti-fouling performance test: To obtain optimal antifouling performance, the concentration of FSP was optimized (Figure 6). First, a fluorescent protein attachment experiment was conducted using fluorescein-labeled bovine serum albumin (FITC-BSA) to visually evaluate the antifouling performance of the FSP-based biosensor (Apt-FSP / Au / PEDOT) (Figure 7). Under the same testing conditions, bare, Au / PEDOT, and FSP / Au / PEDOT-modified glass electrodes served as controls. Fluorescence analysis showed that the fluorescence intensity of the FSP / Au / PEDOT and Apt-FSP / Au / PEDOT-modified surfaces was almost zero compared to the unmodified and Au / PEDOT-modified surfaces. ImageJ quantitative analysis further clarified these differences. The fluorescence coverage of peptide-modified surfaces was significantly reduced, with FSP / Au / PEDOT and Apt-FSP / Au / PEDOT surfaces showing coverage of only 0.13% and 0.24%, respectively, while the coverage of unmodified substrates and Au / PEDOT-modified surfaces reached as high as 68.26% and 55.88%, respectively. Subsequently, the antifouling performance of the FSP-based biosensor was quantitatively analyzed by measuring its DPV signal before and after exposure to biological media and calculating the signal inhibition rate. Figure 8 - Figure 11 As shown, compared with the unmodified FSP interface, the FSP-modified electrode exhibited significantly improved antifouling ability in biological media containing single protein solutions (BSA and mucin), artificial saliva, and human saliva. Notably, aptamer grafting did not significantly affect the antifouling performance of the FSP-based interface. Building on this, we further compared the antifouling performance of the FSP and ZIP-based interfaces to verify the superiority of the former (FSP interface).

[0055] like Figure 12 As shown, the FSP-based interface exhibits significantly better resistance to biofouling than the ZIP-based interface: at 20 mg / mL -1 BSA (4.9% vs 11.0%), 20 mg·mL -1 Lower signal inhibition rates were observed in mucin (6.8% vs 17.3%), undiluted artificial saliva (4.5% vs 7.8%), and human saliva (5.1% vs 10.3%). The study also compared the long-term antifouling properties of the two interfaces. Figure 13 As shown, after soaking in undiluted saliva for three weeks, the FSP-modified electrode still maintained excellent antifouling performance, with a signal inhibition rate of only 9.4%, which was significantly lower than that of the ZIP-modified electrode (20.5%), and much lower than that of the Au / PEDOT-modified electrode (33.9%) and the bare electrode (90.7%), demonstrating excellent long-term antifouling effect.

[0056] Example 6 Sensor performance test: Under optimal experimental conditions ( Figure 14 The developed biosensor was used to detect different concentrations of CA15-3, and the output signal was monitored by DPV measurement, such as... Figure 15 As shown in Figure a, with the increase of CA15-3 concentration, the peak current of DPV decreases, and the current change (ΔI) basically follows a linear relationship with the logarithm of the concentration. Figure 15 c). The CA15-3 sensor has a wide linear range (0.01 - 1000 U·mL). -1 ) and a low limit of detection (2.85 mU·mL) -1 Notably, the developed biosensor maintained a good linear response to CA15-3 even in undiluted saliva (Figure 15b). Given the presence of the target analyte in real saliva, the linearity assessment was performed using artificial saliva.

[0057] The selectivity of the sensor was tested. The selectivity of the biosensor was validated by measuring the signals of CA15-3 along with a series of other non-target analytes (carbohydrate antigen 125 (CA125), immunoglobulin G (IgG), cortisol, carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), uric acid (UA), and mixtures thereof). Figure 16 As shown, even at much higher concentrations than CA15-3, the interfering substance (CA125: 1000 U·mL) -1 Other: 1 μg·mL -1 It also produces almost no current response, which highlights the excellent selectivity of the fabricated biosensor.

[0058] To evaluate the reproducibility of the FSP-based biosensor, we used seven independently fabricated sensing electrodes to detect concentrations of 100 U·mL. -1 CA15-3. For example... Figure 17 As shown, the differential pulse voltammetry (DPV) signal exhibited high consistency across all repeatable experiments, with a relative standard deviation (RSD) as low as 3.4%. These results confirm the excellent repeatability of the FSP-based biosensor. Stability is a critical prerequisite for reliable biosensor performance. We evaluated the operational stability of the FSP-based biosensor using sequential cyclic voltammetry (CV) and repeated differential pulse voltammetry scans. Figure 18 As shown, the CV and DPV curves for 50 consecutive cycles and 7 repeated tests almost completely overlap, indicating high stability. Meanwhile, the FSP-based biosensor exhibits good storage stability within 15 days, with a retention rate of 95.5%. Figure 19 ).

[0059] Example 6 Real-world performance testing of the sensor: To evaluate the clinical applicability of the CA15-3 electrochemical biosensor, 28 human saliva samples were analyzed in parallel using the developed biosensor and a commercial ELISA kit, 14 from healthy individuals and 14 from patients. Bland-Altman analysis was used to plot the mean values ​​of the FSP-based biosensor and ELISA against their differences (…). Figure 20 This indicates a strong consistency between the two methods, with an average difference of 0.19 U·mL. -1 96% of the data points are within the 95% consistency range.

[0060] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorinated side chain polypeptide based anti-fouling electrochemical salivary sensor, characterized in that, The sensor is modified with conductive polymer poly 3,4-ethylenedioxythiophene (PEDOT) and gold nanoparticles (AuNPs) as electrode modification materials, with self-designed fluorinated side-chain polypeptide as anti-fouling material, and further modified with CA15-3 specific aptamer (Apt); and, The sequence of the fluorinated side-chain polypeptide (FSP) is: C(Hyp)4K(G-Perfluoropropionic acid)EKEKEK; the sequence of the CA15-3 aptamer is: 5'-GCA GTT GAT CCT TTG GAT ACC CTG G-3', with HS-(CH2)6 modification at the 5' end; the FSP and the CA15-3 aptamer are connected to the electrode through gold-sulfur bond.

2. A method for the preparation of a fluorinated side chain polypeptide based anti- fouling electrochemical salivary sensor according to claim 1, characterized in that, The method comprises the following steps: I. Preparation of Au / PEDOT modified electrode: the sensor is based on glassy carbon electrode (GCE), and PEDOT thin film and AuNPs are deposited on the surface of the electrode by constant potential method to prepare Au / PEDOT / GCE; II. Preparation of anti-fouling electrochemical saliva sensor: the Au / PEDOT / GCE is incubated in a mixed solution of FSP and Apt to prepare the anti-fouling electrochemical saliva sensor.

3. The method for preparing a fluorinated side chain polypeptide-based anti- fouling electrochemical salivary sensor according to claim 2, characterized in that, The specific method of step I is as follows: PEDOT thin film is obtained by depositing 3,4-ethylenedioxythiophene (EDOT) and phytic acid (PA) mixed solution with a concentration of 0.02-0.04 M and a potential of 1.1 V on the GCE for 20-60 s; and Au / PEDOT / GCE is obtained by scanning the PEDOT thin film electrode in an aqueous solution containing 5.0-10.0 mM HPtCl4 and 0.5-1.0 M KNO3 at a voltage of -0.5-1.2 V for 2-6 cycles by cyclic voltammetry.

4. The method for preparing a fluorinated side chain polypeptide-based anti- fouling electrochemical salivary sensor according to claim 2, characterized in that, The specific method of step II is as follows: The FSP solution, Apt solution, and TCEP solution were mixed to obtain a final FSP concentration of 0.2–0.4 mg / mL. -1 A mixed solution with a final Apt concentration of 3-6 μM and a final TCEP concentration of 0.1-0.2 mM was prepared. The Au / PEDOT / GCE mixture was incubated overnight at room temperature in the mixed solution. After the reaction was completed, the mixture was rinsed with deionized water to remove unfixed FSP and Apt, thus obtaining the sensor.

5. Use of the anti-fouling electrochemical saliva sensor based on fluorinated side-chain polypeptide in quantitative detection of CA15-3 in saliva according to claim 1.

6. Use according to claim 5, characterized in that, The quantitative detection is as follows: The anti-fouling electrochemical saliva sensor based on fluorinated side-chain polypeptide is placed in a solution of target CA15-3 with different concentrations, and then incubated at constant temperature, followed by washing the electrode interface with PBS buffer to remove the un-captured target CA15-3, and then electrochemically detecting the obtained electrode; the current signal change of the sensing interface in the target solution after incubation is recorded by differential pulse voltammetry in the range of -0.2-0.6 V to realize detection of the target CA15-3.

7. Fluorinated side chain polypeptide based anti-fouling electrochemical salivary sensor application according to claim 6, characterized in that, The incubation temperature is room temperature, and the incubation time is 60-120 min to ensure sufficient binding of the target CA15-3.

8. Fluorinated side chain polypeptide based anti-fouling electrochemical salivary sensor application according to claim 6, characterized in that, The linear detection range of the CA15-3 is 0.01 - 1000 U·mL -1 , and the detection limit is 2.85 mU·mL -1 .

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