A novel paper biosensor based on Fe3O4@SiO2–NH2 and MWCNTs for rapid detection of pseudorabies virus

Xing Guo; Jianru Hou; Zhongyun Yuan; Hongmei Li; Shengbo Sang

doi:10.1088/1361-6528/ac002a

Abbreviations

PRV	pseudorabies virus
MWCNTs	multi walled carbon nanotubes.

1. Introduction

Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV) infection in pigs, cattle, sheep, dogs and other animals, pigs are the main host and source of infection. In January 2012, PRV was observed in pigs on several farms in northern and eastern China. In Shandong Province, more than 80 000 pigs were infected, the affected pigs had high fever (>40.5 °C), anorexia, coughing, respiratory distress, conjunctival serous, mucinous secretion, and posterior paralysis. The disease was first observed in older pigs and spread within 2–3 d to younger pigs. Duration of disease was 5–7 d. Rate of illness reached 50%, and mortality was 3%–5% (Yu et al 2014). PR is widely distributed all over the world. Since the first report of PR in 1947, with the development of pig industry scale, more than 20 provinces and cities have reported PR, and it has been reported to be outbreak in many pig farms. In 2013, the prevalence rate of PR in medium-sized pig farms in Anhui Province was 34.1% (95% confidence interval 29.3%–38.9%). In conclusion, PR can greatly harm the growth of healthy pig and cause death of pigs, which will lead to the undersupply of pork in the whole society. Therefore, we need a rapid and effective diagnostic test method to monitor PRV.

Clinical symptoms of pseudorabies are very similar to other diseases that cause porcine reproductive disorders. It is difficult to diagnose them only by clinical symptoms, so researchers at home and abroad have developed techniques that can detect PRV in laboratories. The traditional diagnostic methods include virus isolation (VI) identification and animal experiments. Many methods such as serum neutralization test (SN), enzyme linked immunosorbent assay, latex agglutination test, agar diffusion test are used in PRV detection. Molecular biological diagnostic method of PRV with polymerase chain reaction and nucleic acid probe technique, in situ hybridization technique, etc. However, the traditional methods have some limitations, such as complicated operation, high technical requirements and time-consuming, so it is very necessary to develop new sensors to detect PRV.

At present, cellulose paper has been developed as a promising platform for biochemical sensors and devices due to its thin, lightweight, and flexible properties (Martinez et al 2012), which can be integrated in a manner that is inexpensive, portable, disposable, and easy to operate compared with traditional sensors and devices (Dungchai et al 2011, Hossain et al 2011). Cellulose fiber can make liquid permeate into its hydrophilic fiber matrix (Martinez et al 2010) and be functionalized to modify their properties (e.g. hydrophilicity) and their permeability and reactivity (Bracher et al 2010). As a sensing platform, it exhibits a great potential use, compared with plastic and glass substrates, paper has many advantages such as portable, low-cost, no environmental pollution and easy to be transmitted. Paper can carry out passive liquid transmission and has good biocompatibility. The hydrophobic nitrocellulose membrane shows a high degree of nonspecific binding to biomolecules, which is suitable for immobilization of enzymes, proteins and DNA. And for meeting the needs of users, the manufacturing method and analysis technology can be adjusted. Given these advantages, paper biosensors are widely used in various fields such as health diagnosis, environmental monitoring (Chouler et al 2018) and food quality control (Russell and de la Rica 2018), etc.

In order to improve the performance of paper biosensors, metal or semiconductor materials are usually used to modify paper. By doping metal or semiconductor materials, the sensitivity and response speed of paper biosensors can be improved, and metal-semiconductor composite materials have good conductivity. In the past 20 years, the study of magnetic polymer microsphere has been very active. The basic materials of magnetic polymer microspheres are magnetic materials and polymer materials. Magnetic materials include Fe₃O₄, Fe₂O₃, Pt, Ni, Co, etc. The three-layer sandwich structure of polymer materials includes the outer shell layer and the inner core, and the middle layer composed of magnetic materials (Shuchun and Zuowan 2003). The surface of shell of magnetic polymer microsphere has diversity, which can make it coupled with the ligands in bioactive substances, so as to recognize the corresponding antigens, antibodies and nucleic acids, and realize the immune function. Under the action of magnetic field, magnetic materials make it have properties of directional movement, separation and localization and magnetic response, while the functional groups provide binding sites for antigens, antibodies, enzymes and cells. Magnetic polymer microsphere make the paper biosensor have good magnetic response characteristics, the detection of PRV can be accomplished by the change of the magnetic response characteristics.

Multi walled carbon nanotubes (MWCNTs) with excellent conductivity are used to construct the conductive network of paper biosensors. MWCNTs are composed of multilayer graphite. When the PRV antibody modified on the surface of the paper specifically combines with the PRV antigen, it will produce immune complex, which leads to the larger size of the immune magnetic beads distributed in the conductive network of MWCNTs, and then further increases the gap between the layers of MWCNTs, thus increasing the contact resistance. Therefore, the concentration of PRV antigen can be reflected by the impedance change before and after antigen detection.

In this study, we have developed a paper biosensor doped with Fe₃O₄@SiO₂–NH₂ and MWCNTs for the rapid detection of PRV. Fe₃O₄@SiO₂–NH₂ provide the magnetic response properties and MWCNTs enhance the electrical conductivity. PRV antibody is covalently bonded with Fe₃O₄@SiO₂–NH₂ to form immunomagnetic beads. Combined with the excellent conductivity of MWCNTs, the conductive network of paper biosensor is constructed to reflect the concentration of PRV antigen by impedance change. Finally, the relationship between the impedance change and the concentration of PRV antigen was established, at the same time, the performance of the sensor was evaluated. The experimental results showed that the paper biosensor has advantages of simple preparation method, low-cost, high sensitivity and strong specificity, providing a valuable method for PRV detection. The field of point-of-care (PoC) (Nayak et al 2017, Tan et al 2019) diagnostics offers the tantalizing possibility of providing rapid diagnostic results in non-laboratory settings. The potential reach of PoC diagnostics into all sectors of health care, and increasingly into daily routines of individual patients and consumers, demands that technical advances take into consideration this broader transformation. The development of paper biosensors has also contributed to the field of PoC.

2. Materials and methods

2.1. Chemicals and materials

All chemicals were commercially available as analytical reagent. Pig PRV antigen concentration of 2 mg ml⁻¹, polyclonal antibody concentration of 8 mg ml⁻¹ (Shandong Landu Biotechnology Co., LTD). 10 ml PBS suspension of Fe₃O₄@SiO₂–NH₂ with amino bonding content of 200–300 μmol g⁻¹, particle size of 400–500 nm and concentration of 5 mg ml⁻¹ (Bethler Chromatography Development Center, Tianjin). 1- (3-dimethylaminopropyl) -3-ethyl carbon diimide (EDC) and N-hydroxysuccinimide (NHS) (Sigma-Aldrich). 0.01 mol l⁻¹ phosphate buffer solution (PBS solvent with pH 7.4). Whatman chromatography paper No.1 cellulose nitrate paper (Zhengcheng Scientific Laboratory Equipment Shopping Mall). MWCNTs with diameter of 4–6 nm, length of 10–20 μm and purity of >98 wt% (chengdu organic chemistry co., Ltd, Chinese academy of sciences-zhongke times nanometer). Bovine serum albumin (BSA) 1% (W/V) BSA (Sigma Corporation, USA). Conductive tape. Purity >99.0% creatinine, uric acid (Shanghai yuan mubiotechnology co., Ltd).

Impedance analyzer (E4990A) is made by German technology (China) Co., Ltd. The electronic balance (FA2204B) is made by Shanghai Jingke Tianmei Scientific Instrument Co., Ltd. Xiangyi high speed centrifuge (TG16-WS) is manufactured by Shanghai Experimental Instrument Co., Ltd. Electric heating board (DB-1) is made by Jiangsu Jintan Jincheng Guosheng experimental instrument factory. Hand held digital Tesla meter (TD8620) is made by Changsha Tianheng measurement and Control Technology Co., Ltd. The (FX550) sound insulation box of ultrasonic cell grinder is made by Branson company. The pipette (50-1Bz) is made by Shanghai Kanu biological.

2.2. Fabrication of magnetic paper

The fabrication process of magnetic paper was as follows: firstly, the nitrocellulose paper was cut into three sizes (4 mm × 5 mm, 2 mm × 10 mm, 1.25 mm × 16 mm). Secondly, three concentrations (1, 2.5, 5 mg ml⁻¹) of Fe₃O₄@SiO₂–NH₂ dispersion solution were prepared by low-cost mixing method. Thirdly, the nitrocellulose paper was soaked in PBS dispersion solution of Fe₃O₄@SiO₂–NH₂ for 1 h until it was completely saturated, and finally it was heated and dried on a constant temperature at 60 ℃. Fe₃O₄@SiO₂–NH₂ were evenly doped on the surface of cellulose paper by soaking and drying repeatedly.

2.3. Antibody modification

EDC and NHS crosslinking agent were added into PRV antibody, and the mixture reaction was conducted at room temperature for 1 h to activate the carboxyl group on the surface of antibody. Then the magnetic test paper was immersed in the activated antibody solution and reacted for 2 h at room temperature, so that the carboxyl group on the surface of the activated antibody were covalently mixed with the amino group of the Fe₃O₄@SiO₂–NH₂. By this step, an immune magnetic bead modified with antibody on its surface has been accomplished. After that, the magnetic test paper modified with antibody was washed by PBS and then dried. Then the unconjugated carboxyl on the antibody's surface was sealed with 4 mg ml⁻¹ BSA for 30 min, and washed by PBS and dried. A magnetic paper biosensor with surface-functionalization of PRV antibody has been prepared.

2.4. Doping carbon nanotubes

Using MWCNTs to enhance the electrical conductivity of magnetic test paper: take 50 mg of MWCNTs with a diameter of 4–6 nm, length of 10–20 μm and purity of >98 wt% into a beater, add 10 ml of low-viscosity medium deionized water (which is good for dispersion), and then use ultrasonic cell crusher to set the magnetic test paper into a pulse mode ultrasonic treatment with a cycle of 4 s and a duty ratio of 50% for 5 min, and then immerse the magnetic test paper in the dispersed MWCNTs solution for 30 min, and then dry it at room temperature.

2.5. Sensing principles and device operation

Sensing principles: ① When the PRV antibody on the sensor specifically binds with PRV antigen, the immune complex will be produced, which leads to the increase of the volume of the immune magnetic beads distributed in the carbon nanotube conductive network, which makes the carbon nanotube network layer separate and the gap increase, resulting in the increase of the contact resistance between the carbon nanotubes on the whole paper biosensor. ② The original tissue structure and magnetization state of the immunomagnetic beads specifically combined with PRV antigen changed, which changed the magnetic characteristics of the magnetic paper biosensor. Magnetic permeability μ = 1 + Χ, Χ is magnetic susceptibility, which is related to magnetic intensity M and magnetic intensity H (M = X * H), and can change greatly with the change of material and magnetic state.

Device operation: ① The antigen was diluted to different concentrations (2.0, 1.5, 1.0, 0.5 mg ml⁻¹), and added 50 μl antigen to each test tube. ② Immersed the prepared paper-based biosensor in antigen solution and incubated at 37 °C for 1 h. ③ Took out the paper-based biosensor, and then washed and dried it, finally tested the impedance change and hysteresis loop of the paper-based biosensor.

2.6. Optimization experiment

To improve the sensitivity of the paper biosensor, we optimized the size of the magnetic paper and the doping concentration of Fe₃O₄@SiO₂–NH₂. When the magnetic test paper is bent under the force in the magnetic field, the tip offset and bending angle of the magnetic test paper are measured. The tip offset and bending angle increase with the increase of the magnetic field strength, and the higher the doping concentration of the magnetic microspheres, the greater the tip offset and bending angle. The optimal doping concentration of magnetic microspheres was determined. By analyzing and comparing the magnet response of three sizes (4 mm × 5 mm, 2 mm × 10 mm, 1.25 mm × 16 mm) of paper biosensors, the optimal size of paper biosensors was obtained. Testing the magnetic response of the magnetic paper: fix one end of magnetic paper with a clamp, and use the other end as the free end to induce the magnetic field, as shown in figure 1. The magnetic field intensity H (mT), the tip bending angle (°) and deflection (mm) at the free end were measured in the condition of different distances between the magnet and the magnetic paper in the horizontal direction X (mm) and vertical direction Y (mm). Comparing the magnetic response characteristics of the magnetic paper doped with different concentrations (1, 2.5, 5 mg ml⁻¹) of Fe₃O₄@SiO₂–NH₂ according to the changes of tip deflection and bending angle, the doping concentration of Fe₃O₄@SiO₂–NH₂ was optimized to improve the magnet response characteristic.

Figure 1. Refer to the following caption and surrounding text. — **Figure 1.** Schematic diagram of the testing device.
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In order to improve the sensitivity of the paper biosensor, the optimization of antibody modification concentration is an essential step. After diluting PRV polyclonal antibody with PBS buffer to four different concentrations (400, 200, 50, 25 μg ml⁻¹), two optimization experiments were carried out: ① The same concentration of PRV antigen was dripped onto the paper sensor modified by four different concentrations of antibody, and the impedance changes were measured. The optimal concentration of antibody modification was selected under different test frequencies. ② A series of gradient PRV antigens were dripped onto paper biosensor modified with four different concentrations of PRV. The impedance changes were measured. The optimal concentration of antibody was selected by comparing and analyzing the experimental data at the same test frequency.

2.7. Signal measurement

The paper biosensor was used to detect PRV antigen with a series of concentrations (10–1 mg ml⁻¹). The impedance change (ΔZ) was measured by E4990A impedance analyzer. In addition, the magnetic hysteresis loops of magnetic paper biosensor before and after the detection of antigen were tested in the magnetic field intensity range of ±1.5 T, and the test results were compared and analyzed at the same condition.

2.8. Specificity test

The solid creatinine powder, uric acid powder, PCV2 antigen, BSA and PRV antigen were all configured at the concentration of 1 mg ml⁻¹. The specificity of the paper biosensor was tested by detecting the five substances and comparing their ΔZ at the same condition.

3. Results and discussion

3.1. TEM morphology characterization

Figure 2 shows the TEM morphology and hysteresis loop diagram of the Fe₃O₄@SiO₂–NH₂. From figure 2(a), it can be seen that the shape of Fe₃O₄@SiO₂–NH₂ is uniform spherical shape with smooth surface and its dispersion is good. As shown in the figure 2(b), the magnetic saturation strength of Fe₃O₄@SiO₂–NH₂ is 80 emu g⁻¹. It has good superparamagnetism without remanence. Under the action of external magnetic field, it has good magnetic response and almost no hysteresis.

Figure 2. Refer to the following caption and surrounding text. — **Figure 2.** (a) TEM image of morphology; (b) Hysteresis loop diagram of Fe₃O₄@SiO₂–NH₂.
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3.2. Morphology characterization and element analysis

The process of the entire experiments was assessed by TEM. The paper surface was imaged and appeared smooth. It can be seen from figure 3(a) that the cellulose fibers of the paper appeared intricately overlapped to form a porous and closed cellulose fiber network. Figure 3(b) shows that the surface of the paper was doped with Fe₃O₄@SiO₂–NH₂, which were uniformly distributed on the surface of cellulose fibers. The macromolecular aggregates of PRV antibody can be seen from figure 3(c). The distribution of immune complex of PRV antibody and antigen on cellulose paper can be seen from figure 3(d).

Figure 3. Refer to the following caption and surrounding text. — **Figure 3.** SEM images of: (a) nitrocellulose paper; (b) Fe₃O₄@SiO₂–NH₂/paper; (c) antibody/Fe₃O₄@SiO₂–NH₂/MWCNTs paper; (d) PRV antigen/antibody/Fe₃O₄@SiO₂–NH₂/MWCNTs/paper.
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Figure 4 is the elemental analysis of the biosensor surface by energy dispersive spectrometer (EDS) measurement. According to figure 4(a), C and O are mainly on the nitrocellulose paper. Figure 4(b) shows that Fe is added after doping Fe₃O₄@SiO₂–NH_2. Figures 4(c) and (d) show that after the antibody is modified on the sensor, the content of C, N and O increase because the mainly components of the antibody are C, N and O. After the antibody combines with the antigen, C, N and O increase again, indicating that PRV antigen was bonded to the antibody.

Figure 4. Refer to the following caption and surrounding text. — **Figure 4.** EDS surface scanning electronic images of: (a) cellulose nitrate paper; (b) Fe₃O₄@SiO₂–NH₂/paper; (c) antibody/Fe₃O₄@SiO₂–NH₂/MWCNTs/paper; (d) PRV antigen/antibody/Fe₃O₄@SiO₂–NH₂/MWCNTs/paper.
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Figure 5 is chart of counting rate of four elements (C, N, O, Fe). It can be seen from figure 5 that after the nitrocellulose paper is developed into a paper biosensor, the amounts of C, N, O and Fe increase significantly. This is because the paper is doped with the Fe₃O₄@SiO₂–NH₂ which contain Fe, N and O and MWCNTs contain C. Whether the paper biosensor modified with antibody and combined with antigen or not, the count rate of Fe remained unchanged, because the component elements of antigen and antibody do not contain Fe. The amounts of C, N, and O in the paper biosensor modified with antibody was higher than that of the paper biosensor without modified with antibody, which indicated that the paper biosensor modified with antibody successfully. After the detection of PRV antigen, the number of C, N, and O were higher, which is because the specific binding of antigen and antibody.

Figure 5. Refer to the following caption and surrounding text. — **Figure 5.** The amounts change of (a) C; (b) N; (c) O; (d) Fe during the preparation of the paper biosensor.
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3.3. Optimization of the size of magnetic paper

The magnetic field intensity at the tip of three sizes of magnetic test paper are shown in figure 6. The part with good magnetic response is taken, the distance = (−6, −4, −2, 0, 2, 4, 6) mm, tip deflection = (4, 3, 2, 1) mm, bending angle = (15, 11, 7.6, 3.8)°. Comparing the figures 6(a)–(i), it can be seen that the larger the distance between magnet and magnetic test paper, the smaller the magnetic field intensity measured at the tip, no matter whether X changes in horizontal direction or Y changes in vertical direction. The comparison of figures 6(a)–(g) shows that when the doping concentration of Fe₃O₄@SiO₂–NH₂ is the same, the magnetic response of magnetic test paper with the size of 1.25 mm × 16 mm is better, and the magnetic field intensity of its tip is greater than that of 2 mm × 10 mm and 4 mm × 5 mm. And the longer the size of the magnetic test paper, the better the magnetic response. Because when the magnetic test paper is forced to bend in the magnetic field, the longer the size of the magnetic test paper, the greater the force on the unit area of the tip, the more prone to deformation. Thus, the magnetic test paper with the size of 1.25 mm × 16 mm with the most obvious deformation is selected to measure the magnet response property. But when MWCNTs with the same purity, state, diameter and length are doped into the paper biosensor, the paper of 4 mm × 5 mm with the same area is approximately square, showing stable conductivity, while the paper biosensor with long strip size is more prone to interference. Therefore, 4 mm × 5 mm is selected as the optimal size for the preparation of paper biosensor.

Figure 6. Refer to the following caption and surrounding text. — **Figure 6.** Magnetic field intensity at different distances between magnetic paper and magnet. The size of paper is 1.25 mm × 16 mm: (a) c = 5 mg ml⁻¹; (b) c = 2.5 mg ml⁻¹; (c) c = 1 mg ml⁻¹; the size of paper is 2 mm × 10 mm: (d) c = 5 mg ml⁻¹; (e) c = 2.5 mg ml⁻¹; (f) c = 1 mg ml⁻¹; the size of paper is 2 mm × 10 mm: (g) c = 5 mg ml⁻¹; (h) c = 2.5 mg ml⁻¹; (i) c = 1 mg ml⁻¹; c is the doping concentration of Fe₃O₄@SiO₂–NH₂.
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3.4. Optimization of the concentration of Fe₃O₄@SiO₂–NH₂

Figure 7(a) shows the change of tip deflection of the paper biosensor doped with Fe₃O₄@SiO₂–NH₂ with different concentrations (1, 2.5, 5 mg ml⁻¹). From figure 7(a), it can be seen that the higher the concentration, the larger the tip deflection. Figure 7(b) shows the change of bending angle caused by different concentrations (1, 2.5, 5 mg ml⁻¹). From figure 7(b), it can be seen that the higher the concentration, the larger the bending angle. So with the increase of doping concentration, the magnetic response characteristic of the biosensor is enhanced. But according to the experimental results, with the increase of the concentration, it finally reached to saturation. So the optimal doping concentration of Fe₃O₄@SiO₂–NH₂ is 5 mg ml⁻¹.

Figure 7. Refer to the following caption and surrounding text. — **Figure 7.** Magnetic response characteristics of the magnetic paper doped with different concentrations of Fe₃O₄@SiO₂–NH₂: (a) Tip deflection; (b) bending angle as functions of magnetic field.
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3.5. Optimization of concentration of antibody modification

Figure 8(a) shows the ΔZ of the paper biosensor modified with different concentrations (25, 50, 200, 400 μg ml⁻¹) of antibody to detect PRV antigen with the same concentration. From figure 8(a), we can see that the ΔZ is the largest when the modification concentration of antibody is 50 μg ml⁻¹, with the increase of antibody concentration ΔZ decreases. Therefore, when the concentration of antibody modification is 50 μg ml⁻¹, the sensitivity of the sensor is the highest. Meanwhile, from figure 8(a), it can be seen that ΔZ is the most obvious at the frequency of 20 Hz, so we choose 20 Hz as the test frequency in the next experiments. Figure 8(b) shows ΔZ of the paper biosensor modified with different concentrations of PRV antibody (25, 50, 200, 400 μg ml⁻¹) to detect PRV antigen with a series of gradient concentrations (0.5, 1, 1.5, 2 mg ml⁻¹). As can be seen from figure 8(b), ΔZ was the largest when the immobilization concentration of antibody was 50 μg ml⁻¹, corresponding to the detection of PRV antigen with different concentrations, that is, the sensitivity of detecting PRV antigen was the highest. Figures 8(a) and (b) proved that the optimal concentration of antibody modification is 50 μg ml⁻¹ simultaneously.

Figure 8. Refer to the following caption and surrounding text. — **Figure 8.** (a) ΔZ corresponding to different concentrations of PRV antibody immobilization at the same concentration of PRV detection; (b) ΔZ corresponding to different concentrations of PRV antibody immobilization at the different concentrations of PRV detection.
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3.6. PRV detection

Figure 9 shows the linear relationship between impedance change (ΔZ) and the logarithm of PRV antigen concentration. When PRV antigen combined with the antibody modified on the magnetic paper biosensor, producing immune complex, which leads to the size of immune magnetic bead bigger, which were distributed in the MWCNTs conductive network. It further makes the lamellar spacing of carbon nanotubes with porous structure increase, causing the contact resistance of MWCNTs in paper biosensors increase. It can be seen from figure 9, the higher the concentration of PRV antigen, the greater the ΔZ. The functional relationship between logarithm of antigen concentration and ΔZ was obtained by linear fitting: when f = 20 Hz, ΔZ = 5.5826 * logC + 6.3709, the linear correlation coefficient R² = 0.9113, the standard deviations = 9.715 73. Therefore, the linear range of the paper biosensor for PRV detection is 10–1 mg ml⁻¹, and the detection limit is 10 ng ml⁻¹.

Figure 9. Refer to the following caption and surrounding text. — **Figure 9.** Relationship between PRV concentration and ΔZ at test frequency f = 20 Hz.
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Figure 10 is the diagram of hysteresis loops of the paper biosensor before and after the detection of PRV antigen (2 mg ml⁻¹). As shown in figure 10, the magnetization intensity (M) of the paper biosensor after PRV detection is higher than that before PRV antigen detection under the same magnetic field intensity (H). According to the formula of M = cm * H, the magnetic susceptibility (cm) of the biosensor was increased. According to the relationship between relative permeability (μ) and magnetic susceptibility (cm): the μ of the paper biosensor is also increased after antigen detection. The principle is that Fe₃O₄@SiO₂–NH₂ modified with antibody combine with antigen specifically, which changes the microstructure and magnetization state of Fe₃O₄@SiO₂–NH₂, causing relative permeability (μ) and susceptibility (cm) of the paper biosensor increase.

Figure 10. Refer to the following caption and surrounding text. — **Figure 10.** Hysteresis loops before and after PRV antigen detection.
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Various detection methods for PRV are summarized in table 1. The paper biosensor is low in cost, simple to manufacture and easy to operate. After detecting different PRV concentrations, a wider linear range and detection limit were obtained. From table 1, we can see that the performance of the paper biosensor is comparable to or even better than other methods. Although the detection limit is not the lowest, the linear range is wider and the accuracy is higher, and the cost is the lowest. It is clear that the paper biosensor has great advantages when compared with other reported methods.

Table 1. Comparisons of performances between various methods for PRV detection.

Detection method	Linear range	Detection limit	Assay time	Ease of use	Reference
F-ICS	0.13–2.13 ng ml⁻¹	0.13 ng ml⁻¹	Several hours	Needs equipment	Shen et al (2018)
ELISA	10–1 mg ml⁻¹	10 ng ml⁻¹	Time-consuming	Needs skill	Covadonga et al (2020)
PCR		37.8 copies μl⁻¹	Several minutes	Low-accuracy	Tian et al (2020)
Paper biosensor	10–1 mg ml⁻¹	10 ng ml⁻¹	Several minutes	Minimum skill; smaller size	This work

3.7. Specificity measurement

To evaluate the specificity of the paper biosensor, different potential substances (BSA, uric acid, creatinine, PCV2 antigen) and PRV antigen with the same concentration (1 mg ml⁻¹) were detected. It can be clearly observed from figure 11 that the ΔZ caused by PRV antigen is almost five times greater than for other substances, indicating that the biosensor shows little response to these other substances. The results implied that the biosensor has high specificity for the detection of PRV.

Figure 11. Refer to the following caption and surrounding text. — **Figure 11.** Specificity measurement of the paper biosensor.
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4. Conclusions

In this study, a paper biosensor for rapid PRV detection was developed based on Fe₃O₄@SiO₂–NH₂ and MWCNTs. Fe₃O₄@SiO₂–NH₂ make the paper biosensor have magnetic response property, and MWCNTs enhance the electrical conductivity. The doping concentration of Fe₃O₄@SiO₂–NH₂ and immobilization concentration of PRV antibody and the sensor size were optimized. EDS and TEM analysis were used to characterize the morphology and elements of the paper biosensor. Based on the magnetic property and biocompatibility of Fe₃O₄@SiO₂–NH₂, and the excellent conductivity of MWCNTs, the PRV antigen concentration was reflected by ΔZ and magnetic characteristic parameter. After the PRV antigen was recognized and specifically combined to the antibody, the μ of the paper biosensor increases. The experimental results show that ΔZ of the biosensor is linear with the logarithm of PRV antigen concentration, in the range of 10–1 mg ml⁻¹. The detection limit is 10 ng ml⁻¹, which is lower than that of other literatures. The paper biosensor shows advantages of portability, low-cost, high specificity and sensitivity, providing an effective method for rapid detection of PRV.

Acknowledgments

The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 51975400, 61501316), Science and Technology Innovation Project of Colleges and Universities in Shanxi Province (2020L0076), National key Research and development program (2019YFB1310200), Patent Promotion and Implementation Funding Project in Shanxi (20200713), Science and Technology Innovation Project for Outstanding Talents of Shanxi Province (201805D211020), Beijing Natural Science Foundation (7202190), International Cooperation of Science and Technology Projects in Shanxi Province (201903D421063).

Data availability statement

The data that support the findings of this study are available upon reasonable request from the authors.

Author contributions

Xing Guo designed the study. Jianru Hou writed and changed the format of the article. Zhongyun Yuan and Hongmei Li performed the experiments. Shengbo Sang is the contact person for articles and research materials. All authors contributed towards data analysis, drafting and critically revising the paper, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

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