A Label-free Electrochemical Immunosensor Based on Gold Nanoparticles-poly(ferriporphyrin-co-acrylamide)-reduced Graphene Oxide and the Application in Prostate Specific Antigen Detection

Abstract

Prostate specific antigen (PSA), as a biomarker, plays important roles in early diagnosis of male prostate diseases, especially prostate cancer. In this study, a novel electrochemical probe polymer of poly(ferriporphyrin-co-acrylamide) was synthesized, by further combined with reduced graphene oxide and gold nanoparticles, and oriented immobilization of PSA antibody, a label-free electrochemical immunosensor was obtained, and then successfully applied in the determination of PSA in 10 % non-antigen serum system. The prepared biosensor expressed high selectivity toward PSA, with a LOD of 0.001 ng mL⁻¹, a linear range of 0.01–110 ng mL⁻¹, and a sensitivity of 15.78 µA mL ng⁻¹. What’s more, it showed good stability, well repeatability and high selectivity in the real PSA sample detection. This work provided certain references for the design of the new electrochemical probes and the detection of tumor markers.

1. Introduction

Cancer has always been a major threat to human public health. It accounts for about 25 percent of all deaths each year. Prostate cancer is the second most commonly diagnosed cancer and the sixth leading cause of cancer death among men worldwide, with an estimated 1.5 million new cancer cases and 0.5 million deaths in 2019. The incidence of prostate cancer is obviously regional in the world, and there are certain ethnic differences.^1,2 In recent years, there has been an increasing trend, which should be taken seriously. With the enhancement of people's health consciousness, how to effectively control the occurrence and development of malignant tumors has attracted more and more attention from the medical community and the society. In the medical field, the “three early” principle of early detection, early diagnosis and early treatment of malignant tumors is very important. Early diagnosis is the key part. It is urgent to strengthen the research on early diagnosis of malignant tumor.

The early diagnosis method of tumor should meet the criteria of fast, convenient, cheap, safe and reliable. Compared with the traditional imaging examination and histopathological examination, the detection of tumor markers detection has shown significant advantages in its speed, efficiency and minimally invasive. The detection of tumor markers is not only reflected in screening and early diagnosis, but also has important applications in prognosis judgment, efficacy evaluation and recurrence monitoring. Tumor markers refer to substances in the body fluids, tissues or cells of patients that are different from or have higher contents than normal people. It is the biological and Molecular characteristics that distinguish tumor cells from normal cells, also known as biomarker and molecular marker. It is a kind of substance synthesized and released by tumor cells or host cells in the process of tumor genesis and development. Common tumor markers include proteins, nucleic acids, carbohydrates, polyamines, hormones and viruses. Tumor markers can be specific genes or their products in tumor cells, or they can be genes or their products that are present in some normal cells and tissues, but are abnormally expressed in a particular part of the tumor, which are characterized by abnormal amounts. Chemical, immunological and molecular biology techniques can be used for qualitative or quantitative analysis, providing a reliable basis for the study of tumor occurrence and development mechanism, tumor screening, early diagnosis, efficacy monitoring and prognosis judgment, as well as the discovery of new targets and the study of new therapeutic methods.³

Prostate Specific Antigen (PSA) is a single-stranded glycoprotein specifically synthesized and secreted by epithelial cells of prostate acinar and duct. It is functionally a serine protease belonging to kinin-like releasing enzymes. It is normally secreted into prostatic fluid or semen in an active free form called f-PSA. While the PSA in serum is mainly in the form of binding proteins, usually the sum of f-PSA and binding PSA, named t-PSA, which represents the total PSA level in serum. The detection accuracy of total PSA in serum is high, stable and repeatable, so it has great advantages in prostate cancer early diagnosis and efficacy monitoring. The American academy of clinical biochemistry, the national institute of health and clinical practice in the United Kingdom all gave similar recommendations for PSA as a marker for prostate cancer detection.^4,5 Recently published recommendations have conducted a systematic review of the most effective evidence, PSA remains the only marker recommended for the diagnosis and treatment of prostate cancer based on the current available evidence.^6–8 There have been numerous reports of prostate cancer being diagnosed in 25–35 % of men with a small increase in serum PSA concentration on further prostate histopathological examination. When the serum PSA concentration exceeds 10 ng mL⁻¹, the specificity of cancer detection can reach 40–50 % or even higher. Therefore, the detection of serum PSA is of great significance for the diagnosis and treatment of prostate cancer.^9–15

The detection of tumor markers mainly adopts genomic and proteomic methods, such as DNA Sequencing, Southern Blotting, ligand binding, immunohistochemistry by ELISA, etc.¹⁶ In the detection of tumor markers, compared with the traditional detection methods, biosensor has shown unique advantages. Some common biosensors for tumor marker detection are mainly affinity immunosensors. As the reaction between tumor markers and the corresponding antibody is an affinity, no REDOX reaction, some application optical biosensor and electrochemical biosensor often need to tag of antibody, such as with protease and some fluorescent or electrochemical probe, labelled antibody, this is obviously increased the cost and complexity of the testing process. In recent years, many scientists have turned their interest to the study of simple unlabeled electrochemical immunosensors and achieved certain results.¹⁷ Jang team build an unmarked electrochemical immunosensor with potassium ferricyanide as electrochemical probe, the LOD can reach 0.59 ng mL⁻¹.¹⁸

The design of electrochemical probe is the core of the construction of unlabeled electrochemical immunosensor and the key to the performance of the sensor. Electrochemical probe is able to provide instructions in the electrochemical detection of electrical material, they can be transition metal ions¹⁹ or its complex,^20,21 some small organic molecules,^22,23 even proteins²⁴ and some metal nanomaterials.²⁵ In general, the electrochemical probe is directly dissolved in the detection system, or modified by embedding method, cross-linking method, adsorption method in the surface of the biological sensitive film. So that the electrode and probe can achieve good electron transfer. Immobilized antibodies, as molecular recognition elements, themselves act as non-conductive proteins that impede electron transport. The specific binding between the antigen and the antibody will further hinder the transmission of electrons. Therefore, the quantitative analysis of antigen can be realized by detecting the weakening degree of electrical signals caused by antigen-antibody binding.

Ferric porphyrin (FPP) is a complex of ferric protoporphyrin, formed by hydroxyl group replacing the chloride group bound with iron atoms. Its reductive form, ferrous protoporphyrin, also known as heme, is an auxiliary group of much oxygen metabolization-related enzymes involved in many REDOX processes in the body. In the field of biosensors, ferric porphyrins are often used to simulate enzymes in the construction of many types of enzyme-free biosensors.²⁶ Porphyrin and its metal complexes play an important role in the process of life, and most of the natural porphyrin molecules involved in oxidation and photosynthesis are hydrophobic, which makes it difficult to study their REDOX action in aqueous phase. In addition, in the construction of many biosensor interfaces, the interface materials are often required to have good hydrophilicity and biocompatibility. Therefore, many types of hydrophilic porphyrin derivatives have been synthesized, their functions in natural life processes have been simulated and studied through their special thermal, photochemical and electrochemical properties, and the applications of porphyrin and metalloporphyrin in biosensors have been expanded.²⁷ Zhang's synthesis of bifunctional water-soluble porphyrin derivatives using hydrophilic core groups and zinc ion coordination functional groups. The porphyrin ring without metal can be used as a specific zinc ion fluorescence probe. This probe is of great significance for the study of zinc biological function in vivo and the diagnosis of zinc homeostasis abnormalities.²⁸

In the construction of immunosensor, the antibody is immobilized by forming s-Au bond between the thiol group (-SH) and the gold atom, so that the antibody can directly be fixed on the surface of the gold nanoparticles. The combination of antibody and antigen is dependent on its fragments antigen binding (Fab), and through the antibody Fab combined with gold nanoparticles are not has the antigen binding activity, which reduces the effective rate of immobilized antibody. Therefore, in order to improve the efficiency of immobilized antibodies, directional immobilization of antibodies is required. Among many modification methods, Staphylococcal protein A (SPA) has A good effect on the targeted capture modification of antibodies. It has been widely used in immunology research because of its characteristic of binding fragment crystallization (Fc) with a variety of animal IgG antibodies. For example, Lee using SPA self-assembled gold surface membrane efficient orientation of the antibody is fixed. Highly effective orienting modification of white antibody on gold surface.²⁹

In this paper, a novel electrochemical probe of ferric porphyrin was proposed. It is then copolymerized with acrylamide to form a polymer. A metal-organic nanocomposite was synthesized by further modifying reductive graphene oxide and gold nanoparticles. The composites were modified with gold electrode and the PSA antibody was immobilized by SPA orientation. Lastly, an electrochemical immune sensor was constructed and used to detect PSA in 10 % serum system.

2. Materials and Methods

2.1 Chemicals and reagents

Ferriporphyrin, Acrylamide, 3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), DL-Dithiothreito (DTT), TWEEN^® 20, Bovine Serum Albumin (BSA), HAuCl₄·3H₂O, Hexadecyltrimethylammonium bromide (CTAB), Ascorbic Acid (AA), and Ammonium persulfate (AP) were obtained from Sigma Aldrich (U.S.A.). Prostate specific antigen (PSA) and Prostate specific antigen antibody were purchased from R&D system. Graphene oxide (diameter: 0.5–5 µm, thickness: 0.8–1.2 nm, purity: 99 %) were obtained from Alpha Nano Technology Co. (China). Sodium borohydride, hydrogen peroxide, Na₂HPO₄·12H₂O, K₃FeC₆N₆ were supplied by Tianjin DAMAO chemical reagent factory. Pyridine and Sodium borohydride were obtained from Aladdin Industril Corporation. Human blood serum was obtained from Beijing Solarbio technology co. LTD and Staphylococcal protein A (SPA, Number 22181, from Staphylococcus aureus) was obtained from Thermo Scientific company. In this work all the other chemicals (99 %, Merck) were of analytical reagent. Millipore milli-Q ultrapure water was used during the experiments.

2.2 Apparatus and measurements

Talos F200X instrument (FEI U.S.A.) was used for Transmission electron microscopy (TEM) and Energy-dispersive X-ray spectroscopy (EDX). We used TENSOR 37 FT-IR (BRUKER, Germany) and got result of FT-IR spectra. Working electrode (Au electrode; φ = 3 mm), counter electrode (Ag/AgCl; saturated KCl), reference electrode (platinum wire; φ = 1 mm) constitute a conventional three-electrode system. The 283 Potentiostat-Galvanostat electrochemical workstation (EG&G PARC with M270 software) provided all the electrochemical experiments. J26XP (Beckman Coulter, U.S.A.) high speed refrigerated centrifuge for cryogenic centrifuge.

2.3 Preparation of the AuNPs-poly(FPP-AM)-RGO nanocomposite

An aqueous solution of 1 mg mL⁻¹ Go was fully dispersed and mixed by ultrasonic processing. Another 0.5 g mL⁻¹ sodium borohydride solution was prepared. After mixing, the solution was magnetically stirred at room temperature for 12 h until the color of the solution changed from light brown to pure black. After centrifugation and washed with deionized water for three times, and the precipitate was suspended in the deionized water to obtain RGO. 0.1 M (mol L⁻¹) CTAB solution was prepared and 1 M HAuCl₄ solution was added. Under the condition of magnetic stirring, ascorbic acid was added drop by drop, gradually the color of the solution changed from colorless to purple red. After being washed with ionic water, AuNPs were obtained.

1 mg mL⁻¹ AP solution and water-pyridine mixture (6 : 1, V/V) were configured, and the air was evacuated by vacuum treatment. Weigh 15 mg FPP and 50 mg AM and dissolve into 30 mL water-pyridine mixture. 1 mg mL⁻¹ AP solution was added and stirred magnetically at 60 °C for 24 h. AuNPs-poly(FPP-AM)-RGO was obtained by adding the RGO and AuNPs dispersions, followed by ultrasonic centrifugation. Other nanocomposites [poly(AM)-RGO, poly(FPP-AM)-RGO] were prepared using the same methods.

2.4 Construction of the AntiPSA(BSA)-SPA-AuNPs-poly(FPP-AM)-RGO/AuE

The Au electrodes were polished and polished into mirrors by using nano-alumina powder with particle size of 0.3 µm, 0.1 µm and 0.05 µm respectively, and soaked in Piranha solution (30 % H₂O₂ : 98 % H₂SO₄ = 1 : 3) for 10 min. Then they were washed by ultrasonic in deionized water and anhydral ethanol alternately for 2 minutes. Take 10 µL above AuNPs-poly(FPP-AM)-RGO drop to coat with Au electrode and dry at room temperature. Then you get AuNPs-poly(FPP-AM)-RGO/AuE. The electrodes modified by other composite materials were prepared by the same steps, denoted as RGO/AuE and poly(FPP-AM)-RGO/AuE.

The specific steps of directed immobilization of AntiPSA are as follows: The 0.1 M PBS solution (pH 7.4) of 1 mg mL⁻¹ SPA was configured, and the 10 µL SPDP solution was added. After being thoroughly mixed, the reaction was conducted at 4 °C for 3 hours. After the reaction, ultrafiltration centrifugation was performed to remove the excess SPDP, and the trapped components were carefully blown off the filter membrane with 1 mL PBS, centrifuged again, and transferred to 1 mL PBS. Add 10 mM DTT to the above solution, mix well and react at 4 °C for 3 h, and repeat the above centrifugal washing process. At this point, you get the fully functional SPA of β sulfydryl propionic acid. The newly cleaned Au electrode was immersed into the above functionalized SPA solution and reacted at 4 °C for 12 h. After the reaction, the electrode surface was carefully rinsed with PBS and deionized water successively. After that, the electrode was immersed in 0.05 % Tween 20 (0.1 M PBS dilution) for 2 h. The electrode was dried at 4 °C, and SPA-AuNPs-poly(FPP-AM)-RGO/AuE was obtained.

SPA-AuNPs-poly(FPP-AM)-RGO/AuE was immersed in 1 µg mL⁻¹ AntiPSA PBS solution and incubated at 4 °C for 12 h. After the reaction, the electrode surface was carefully rinsed with PBS and deionized water successively to remove the unfirmly bound AntiPSA. The electrode was dried at 4 °C for later use, and AntiPSA-SPA-AuNPs-poly(FPP-AM)-RGO/AuE was obtained. Then it was immersed in 1 g mL⁻¹ BSA PBS solution and incubated at 4 °C for 12 h to avoid non-specific adsorption that might exist in the detection system, and AntiPSA(BSA)-SPA-AuNPs-poly(FPP-AM)-RGO/AuE was obtained. The construction process is shown in Scheme 1.

Scheme 1.

Illustration for the establishment of AntiPSA(BSA)-SPA-poly(FPP-AM)-RGO/AuE.

3. Results and Discussion

3.1 The performance analysis of electrochemical probe

The electrochemical properties of the electrochemical probe nanocomposite constructed in this study were investigated in an oxygen-saturated PBS buffer system (0.1 M pH 7.4) by cyclic voltammetry (CV) and differential pulse voltammetry (DPV), respectively. The results are shown in Fig. 1. As you can see from Fig. 1A, there was no electrochemical response at the bare gold electrode in the PBS system, its CV and DPV were almost linear. RGO/AuE and poly(AM)-RGO/AuE also did not have any electrochemical response in the PBS system. However, due to the presence of RGO, both electrodes had good electrical conductivity, and their CV maps had a high baseline. Poly(AM)-RGO/AuE has a higher baseline than RGO/AuE, which also indicates that poly(AM) modified RGO has a higher conductivity. In CV results of poly(FPP-AM)-RGO/AuE and AuNPs-poly(FPP-AM)-RGO/AuE, a pair of reversible REDOX peaks appeared near −180 mV and −370 mV, respectively. This is just the cathodic oxygen reduction process of dissolved oxygen in solution under the catalysis of ferric porphyrins. This process can be concluded from Eqs. 1 and 2 (ferric protoporphyrin IX, Fe^IIIPPIX).

  

\begin{equation} \text{Fe$^{\text{III}}$PPIX} + e^{ - } \rightleftharpoons \text{Fe$^{\text{II}}$PPIX}^{ + } \end{equation} (1)

  

\begin{equation} \text{Fe$^{\text{II}}$PPIX}^{ + } + 1/2\text{O}_{2} + \text{H}^{ + } \rightleftharpoons \text{Fe$^{\text{III}}$PPIX} + 1/2\text{H$_{2}$O$_{2}$} \end{equation} (2)

Figure 1.

(A) Cyclic voltammograms of different modified gold electrodes in 0.1 M PBS (pH 7.4) (Scan rate: 50 mV s⁻¹, scan range: −800–600 mV); (B) Differential pulse voltammograms of different modified gold electrodes in 0.1 M PBS (pH 7.4) (Scan range: 200–−600 mV).

Equation 2 occurs between the reduced form of iron porphyrin (Fe^IIIPPIX) and the oxygen molecule, and the electrode potential of oxygen reduction is determined by the REDOX over potential of Eq. 1. Studies have shown that the primary product of this reaction is hydrogen peroxide.^30–32

Figure 1B shows the curve of negative sweep (200–−600 mV) by DPV. The gold electrode modified by each material exhibited the same electrochemical behavior as the CV. Poly(FPP-AM)-RGO/AuE and AuNPs-poly(FPP-AM)-RGO/AuE showed a reduction peak near −270 mV due to the presence of ferric porphyrins, and the latter showed better electrical conductivity due to the presence of gold nanoparticles. Because the ions chelated in the composite are easily transformed from Fe(II) to Fe(III) in a uniform weakly alkaline oxygen saturated environment (pH 7.4), the reduction peak during DPV negative scanning was used as a reference to investigate the binding reaction between PSA and its antibody.

3.2 Morphological characterization of AuNPs-poly(FPP-AM)-RGO

The above analysis indicates that the metal organic nanocomposite synthesized in this study based on ferric porphyrin has a sensitive and stable electrochemical response at the electrode interface and is qualified for the interface material of electrochemical immunosensor. Fourier transforms infrared spectroscopy (FTIR) was used to characterize the feature functional groups in the material, so as to analyze the combination of each component. In Fig. 2, 3416 cm⁻¹ represents the stretching vibration of the hydroxyl group. Compared with GO, the peak value of RGO decreased significantly, which means that GO was fully reduced to form RGO. After modification of poly(AM) and poly(FPP-AM), the peak value of the hydroxyl group of the bound water and Fe–OH was added. At 1690, 1634, 1550, 1435 and 1060 cm⁻¹, respectively, the stretching vibration of N–C=O, C=O, N–H, C–N and C–H was represented, which was mainly from poly(AM) and poly(FPP-AM). The introduction of AuNPs may have a certain influence on the spectral absorption of the above peaks, showing a decrease of the peaks.^33–35

Figure 2.

FTIR spectra of RGO, poly(AM)-RGO, poly(FPP-AM)-RGO and AuNPs-poly(FPP-AM)-RGO.

Figure 3A shows the TEM image of RGO, from which the lamellar structure of graphene can be clearly observed. The RGO dried samples combined with poly(AM) showed a certain degree of adhesion (Fig. 3B), which is mainly due to the high viscosity of poly(AM). Poly(FPP-AM)-RGO was precipitated in the dried samples to a certain extent, and its amorphous crystal structure could be observed under electron microscopy (Fig. 3C). AuNPs were uniformly dispersed in the composite material. Figure 3D showed a single AuNPs magnification image. The average diameter of the AuNPs synthesized in this experiment was around 50–80 nm.

Figure 3.

TEM images of RGO (A), poly(AM)-RGO (B), poly(FPP-AM)-RGO (C), AuNPs-poly(FPP-AM)-RGO (D) and EDX spectrum of AuNPs-poly(FPP-AM)-RGO (E) (Inset shows the scan area).

Figure 3E shows the AuNPs-poly(FPP-AM)-RGO EDX scanning result (the scanning region is shown in the illustration), in which the Au peak is from gold nanoparticles, the Fe peak is from ferric porphyrins, the C and N peaks from RGO and poly(FPP-AM) are relatively low in this field, while the Cu peak with higher peak is provided by the copper support membrane for TEM sampling. Characterizations indicated that AuNPs-poly(FPP-AM)-RGO metal organic nanocomposites have been successfully prepared in this study.

3.3 Electrochemical activity of AntiPSA(BSA)-AuNPs-poly(FPP-AM)-RGO/AuE

Figure 4 shows the CV results of different modified electrodes in 0.1 M KCl solution containing 10 mM K₃[Fe(CN)₆]. Each electrode showed a characteristic REDOX peak of [Fe(CN)₆]^4−/3−. Curve a is the CV of bare gold electrode graph, compared to all other materials, REDOX peak of the bare gold electrode is minimum, this reflects the institute sensor has excellent electric conductivity. Even modify the biological molecules (SPA, AntiPSA, BSA and PSA), the electrode itself still showed good electrical conductivity, this characteristic determines the high sensitivity of this electrochemical immunosensor. Curves b and c represent RGO/AuE and poly(AM)-RGO/AuE. The latter's good electrical conductivity is mainly due to two aspects. One is that poly(AM)-modified RGO has good dispersion, which avoids the aggregation between graphene sheets and improves the specific surface area. Secondly, the poly(AM) has a strong adsorbability to [Fe(CN)₆]^4−/3−, which contributes to the direct transfer of electrons on the electrode surface. Therefore, poly(FPP-AM)-RGO of FPP was modified, and Fe(III)–OH was chelated. Due to its conjugated planar structure and its REDOX properties, the conductivity of the electrode modified by it was further improved (curve d). It is well known that AuNPs has excellent electrical conductivity, so the AuNPs-poly(FPP-AM)-RGO modified electrode (curve e) has a maximum electrical conductivity. The effective active area of each modified electrode was determined by Randles–Sevcik equation (3).³⁶

  

\begin{equation} i_{p} = 2.69 \times 10^{5}n^{3/2}D^{1/2}C_{0}v^{1/2}A_{\textit{eff}} \end{equation} (3)

Figure 4.

Cyclic voltammograms of 10 mM K₃[Fe(CN)₆] at different modified electrodes recorded in 0.1 M KCl aqueous (Scan rate: 50 mV s⁻¹, scan area: −300–900 mV).

Where $i_{p}$ stands for the peak current (µA); n represents the number of electron transfer in the REDOX reaction, which is 1; D represents the molecular diffusion coefficient in the solution, which is (6.70 ± 0.02) × 10⁻⁶ cm² s⁻¹. C₀ represents the concentration of probe molecules in the solution, that is, 10 mM. v represents the scanning rate, which is now 50 mV s⁻¹; A_eff corresponds to the effective active area (cm²) of the modified electrode. The effective active area of AuNPs-poly(FPP-AM)-RGO/AuE was 0.16 cm², which was 2.27 times that of the bare electrode. Such a large effective active area ensures the high sensitivity and wide detection range of the sensor constructed in this study.

The electrodes of biomolecules (SPA, AntiPSA, BSA, and PSA) were modified, and the REDOX peak was reduced to a certain extent due to the resistance effect of the protein itself. The peak value of curve f was significantly lower than that of curve e, indicating that sulfydryl functionalized SPA was effectively modified on AuNPs gold electrode surface. Curve g and curve h represent the electrode after directional modification of AntiPSA and the further sealed with BSA. The difference in conductivity between the two was not significant, which indicated that the amount of BSA bonded to the electrode surface was relatively small. It is speculated that SPA can effectively capture the antibody and avoid the non-specific adsorption at the same time, which also plays the role of blocking reagent to some extent. AntiPSA(BSA)-AuNPs-poly(FPP-AM)-RGO/AuE was incubated at room temperature for 20 min in a PSA solution containing 1 ng mL⁻¹, and its CV curve was measured again. Then the REDOX peaks were reduced, indicating that antigen PSA had been bound to AntiPSA antibody.

Electrochemical impedance spectroscopy (EIS) was also used to investigate the affinity reaction between the assembly process of the sensor interface and the antigen and antibody in K₃[Fe(CN)₆] system. The results are shown in Fig. 5. The illustration shows the equivalent circuit used in the impedance spectrum of the system, including the solution resistance (Rs), the transfer resistance (Rct), the equivalent element (W) and the double-layer capacitance (Cdl). The Rct value of [Fe(CN)₆]^4−/3− REDOX system can be calculated by fitting the experimental data into the model. The bare gold electrode Rct value is about 140 Ω (curve a). When the nanocomposite was modified, the sensor interface resistance decreased and the Rct value decreased. AuNPs-poly(FPP-AM)-RGO/AuE Rct values reached minimum of about 50 Ω (curve e); Then, after modify the different biological molecules, sensing interface resistance increased again. The Rct value AntiPSA(BSA)-AuNPs-poly(FPP-AM)-RGO/AuE can reach 190 Ω (curve I), indicating that biological sensing interface has excellent electric conductivity.

Figure 5.

Electrochemical impedance spectra of 10 mM K₃[Fe(CN)₆] at different modified electrodes recorded in 0.1 M KCl aqueous (Frequency range is 0.1 to 10 kHz and the constant potential is 240 mV, inset shows the equivalent circuit used in the present system).

Figure 6 shows the results of the electrochemical immunosensor used to analyze and detect PSA in 10 % antigen-free serum system with CV (Fig. 6A) and DPV (Fig. 6B). It can be seen from the figure that the modified electrode showed the same electrochemical behavior in the 10 % serum system as in the PBS system, and the peak current of CV and DPV showed a consistent decrease before and after the combination of AntiPSA(BSA)-SPA-AuNPs-poly(FPP-AM)-RGO/AuE, indicating that the electrochemical immunosensor constructed in this study can be used for the analysis and detection of PSA in the 10 % serum system.

Figure 6.

Cyclic voltammograms (A) and differential pulse voltammograms (B) of different modified electrodes recorded in 10 % serum.

3.4 Optimization of the antigen incubation time

The affinity reaction between antigens and antibodies is a dynamic balancing process, so the incubation time of antigens has a certain influence on the signal detection of the electrochemical sensor constructed in this study. In order to determine the optimal antigen incubation time and obtain a stable electrochemical signal, 10 ng mL⁻¹ PSA was added to the 10 % serum system in this experiment, and the DPV response currents of AntiPSA(BSA)-SPA-AuNPs-poly(FPP-AM)-RGO/AuE under different incubation times were investigated. The results are shown in Fig. 7. As can be seen from Fig. 7A, the peak reduction current gradually decreased with the increase of incubation time, and was basically stable for 20 min. Figure 7B shows the line diagram of the peak current. Therefore, the optimal incubation time of this study was 20 min for PSA detection.

Figure 7.

(A) Differential pulse voltammograms at AntiPSA(BSA)-SPA-AuNPs-poly(FPP-AM)-RGO/AuE toward 10 ng mL⁻¹ PSA recorded in 10 % serum; (B) Broken-line graph of the DPV reduction peak current (Error bar = ±standard deviation, n = 5).

3.5 Detection of PSA in the serum system

In this experiment, the detection performance of the constructed electrochemical immunosensor on PSA was investigated in the 10 % serum system, and the results are shown in Fig. 8. As can be seen from the DPV response curve in Fig. 8A, with the increase of PSA concentration, the peak reduction current gradually decreases. After 5 independent experiments, the average value of the reduced peak current was used for linear fitting with the PSA concentration. The results were shown in Fig. 8B. Within the range of 0.01–110 ng mL⁻¹, there was a good linear relationship between the response current and the PSA concentration. The linear fitting equation was y (mA) = 1.578 × 10⁻⁴x (ng mL⁻¹)−0.042 (R² = 0.995). According to the slope of the equation, the sensitivity is 15.78 µA mL ng⁻¹. According to the Signal Noise Ratio of the response current, the lowest detection limit (LOD) of the immunosensor for PSA can be calculated to be 0.001 ng mL⁻¹ (S/N = 3).

Figure 8.

(A) Differential pulse voltammograms at present electrochemical immunosensor toward a series concentration of PSA recorded in 10 % serum; (B) Linear fitting curve between the DPV reduction peak current and PSA concentration (Error bar = ±standard deviation, n = 5).

Table 1 compares the performance parameters of the sensor constructed in this study with those of the immunosensor reported by predecessors. It can be seen that the sensor constructed in this study has higher sensitivity, lower detection limit and wider detection range.

Table 1. Analytical parameters for different PSA label-free electrochemical immunosensors.

Electrode	Electrochemical probe	Sensivity (mA mL ng−1)	LOD (ng mL−1)	Linear range (ng mL−1)	Ref.
3D GR–Aua/GCE	K3[Fe(CN)6]	5	0.59	0–10	18
PSA/BSA/Ab/NPGb/GCE	K3[Fe(CN)6]	18	0.003	0.05–26	37
GS–CoNP–PBSEc/GCE	CoNP	—	0.01	0.02–2	38
AuPd@Au NCsd/gce	H2O2	1.65	0.078	0.1–50	39
AuNPs-poly(FPP-AM)-RGO/AuE	FPP	15.78	0.001	0.01–110	This work

^aThree-dimensional graphene (GR)-based Au nanoparticles; ^bNanoporous gold; ^cGraphene sheet-cobalt hexacyanoferrate nanoparticles-1-pyrenebutanoic acid succinimidyl ester; ^dBimetallic dendritic core-shell AuPd@Au nanocrystals.

3.6 Reproducibility, stability and selectivity of the PSA immunosensor

Repeatability is an important parameter to ensure the application and development of immunosensor. Although the affinity between antigens and antibodies can be interrupted under many intense conditions, immobilized immune agents and immune absorbents. 5 M urea solution was selected for the repeatability of the immunosensor. After the modified electrode was used for the detection of 10 ng mL⁻¹ PSA, the modified electrode was immersed in 5 M urea solution for 10 min, and then used again for the detection of 10 ng mL⁻¹ PSA. Repeated for five times, the response current of the electrode could still reach 104 % of the initial current and the standard deviation (RSD) was 3.73 %. The stability of the immunosensor constructed in this study was evaluated by examining the electrochemical response of 10 ng mL⁻¹ PSA at the same electrode every 7 days. When not in use, the electrodes were stored in 4 °C 0.1 M PBS (pH 7.4), and the standard deviation (RSD) of the response current results was 5.41 % after an interval of 28 days.

The selectivity of this sensor for PSA detection was observed when several other tumor markers (AFP, CA125, ca19-9) and interfering protein (glucose oxidase, GOx) co-existed with PSA. The electrochemical evaluation of PSA by this electrode was investigated, and the response current was recorded in Fig. 9. It can be seen from the figure that in the presence of 10-fold concentration of interfering protein, the maximum loss value of the sensor's response current to PSA is only 2.16 %, indicating that the immunosensor constructed in this study has a high specificity for PSA detection.

Figure 9.

Response currents for the assay of 10 ng mL⁻¹ PSA without and with 100 ng mL⁻¹ AFP, CA125, CA19-9 and GOx (Error bar = ±RSD, n = 5).

3.7 Analysis of PSA in real samples

Accuracy is the key technical index to measure the practical application value of a sensor. In this experiment, the serum of prostate cancer patients was obtained from the cooperative research group and diluted by a factor of 10. The accuracy of the sensor constructed in this study was evaluated by PSA plus standard recovery test compared with ELISA, a traditional standard detection method. The results are shown in Table 2.

Table 2. Recovery test of PSA in real-life samples by the present immunosensor.

Samplea	ELISA detectedb	Present methodb	PSA addedb	PSA foundb	Recovery (%)c	RSD (%)c
Serum 1	1.87	1.90	2	3.87	98.50	4.51
Serum 2	1.96	2.02	4	6.11	102.25	3.82
Serum 3	2.01	1.94	6	8.03	101.50	4.13
Serum 4	2.11	2.09	8	10.12	100.38	2.84
Serum 5	1.92	1.89	10	11.78	98.90	3.79

^aDiluted to 10 % by 0.1 M PBS (pH 7.4); ^bRecorded as ng mL⁻¹; ^cn = 5.

According to the analysis of T test (Student's T test), there was no significant difference between the test results of this method and ELISA method, and the recovery rate of PSA test was 98.50–102.25 % and RSD was 2.84–4.51 %, indicating that the immunosensor constructed in this study had good accuracy.

4. Conclusion

In this paper, FPP was used as electrochemical probes in an innovative way and a metal organic nanocomposite was designed and synthesized. FPP had the potential to be used in high performance electrochemical probes. This provided the infinite possibility for the design of the high polymer and the application of biosensors will have a broad prospect. With the help of SPA to capture the antibody, the directional fixation of the antibody on sensor interface is realized. Thus, a label-free electrochemical immunosensor was constructed and was used to detect the sensitivity of PSA in serum successfully. The AntiPSA(BSA)-SPA-AuNPs-poly(FPP-AM)-RGO/AuE label-free electrochemical immunosensor presented a high sensitivity of 15.78 µA mL ng⁻¹ and a better LOD of 0.001 ng mL⁻¹ (S/N = 3). What’s more, the constructed immunosensor had good stability, repeatability and anti-interference performance, and could be used for PSA detection in real samples. We created the strategy of constructing a label-free electrochemical immunosensor for tumor markers using ferric porphyrins for the first time. This study provides a new technique and method for the early diagnosis of prostate cancer. It also provides technical possibilities for the diagnosis of other cancer markers.

Acknowledgments

This work is supported by the Natural Science Foundation of China (Grant Nos. 81671779 and 81871451). Also supported by Tianjin Education Commission (Grant Nos. 202110071014 and 202110071008) and Tianjin Science and Technology Commission (Grant No. 22JCQNJC00150).

CRediT Authorship Contribution Statement

Bingkai Han: Conceptualization (Lead), Methodology (Lead), Writing – original draft (Lead), Writing – review & editing (Lead)

Yuan Chen: Conceptualization (Equal), Writing – original draft (Equal)

Haotian Wang: Formal analysis (Equal), Methodology (Equal)

Wenying Zhao: Methodology (Equal)

Yuhan Hu: Data curation (Equal)

Yayun Guo: Formal analysis (Equal)

Jilong Yan: Formal analysis (Equal)

Shixiong Jia: Investigation (Equal)

Conflict of Interest

The authors declare no conflict of interest in the manuscript.

Funding

Tianjin Science and Technology Program: 22JCQNJC00150

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