Fe₃O₄ Nanozyme-Labeled Lateral Flow Immunochromatography Strips for Rapid Detection of PVX and PVY

Abstract

Potato virus X (PVX) and potato virus Y (PVY) are major pathogens that threaten seed potato quality and yield. To improve the efficiency of field screening, we developed monovalent PVX, monovalent PVY, and bivalent PVX/PVY nanozyme strips using Fe₃O₄ nanozymes as labels in a double-antibody sandwich lateral flow immunochromatographic assay. Western blot analysis demonstrated that four monoclonal antibodies (PVX 2, PVX 6, PVY 2, and PVY 5) specifically recognized their corresponding viral coat proteins. Specificity testing showed that the nanozyme strips reacted only with the target viruses and did not cross-react with other common potato viruses, including Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), and Potato leafroll virus (PLRV). The PVX nanozyme strip detected PVX-positive extracts diluted up to 10³-fold, the PVY nanozyme strip up to 10⁴-fold, and the bivalent strip detected PVX/PVY co-infected samples diluted up to 10³-fold. In addition, detection results by strips from 12 samples of plantlets in vitro were fully consistent with RT-PCR. These nanozyme strips provide rapid, simple, specific, and sensitive methods that can be stored at ambient temperature, enabling field surveys, warehouse screening, and on-site testing and supporting early detection of potato virus diseases.

1. Introduction

The potato originated in the Andes Mountains of South America and is one of the most important food crops worldwide, playing a crucial role in food security [1]. However, the potato is threatened by a wide range of viruses, with more than 40 viruses reported to infect this crop [2]. Among them, potato virus Y (PVY) and potato virus X (PVX) are the most prevalent and damaging potato viruses [3]. Both occur in all potato-growing regions and induce potato degeneration and yield loss, and co-infections often cause particularly severe economic losses [4].

Commonly, detection methods for plant viruses include molecular methods, serological methods, and emerging isothermal amplification technologies. Reverse transcription-polymerase chain reaction (RT-PCR) offers high sensitivity but relies on specialized equipment and nucleic acid extraction procedures, resulting in longer workflows that are unsuited for on-site rapid testing [5]. Enzyme-linked immunosorbent assay (ELISA) is a widely used serological method suitable for batch screening, but it involves multiple steps and is time-consuming [6]. Isothermal methods such as recombinase polymerase amplification (RPA) do not require a PCR thermocycler, but reagent costs are relatively high, and ancillary equipment is typically needed [7]. By contrast, immunochromatography (lateral flow assays) is derived from ELISA and offers low cost, operational simplicity, and short turnaround time; it has been widely applied in medical diagnosis, environmental monitoring, and food safety [8].

Conventional immunochromatographic assays typically use colloidal gold as the label. However, its visual signal intensity and linear range are limited. In this study, nanozymes developed by Yan Xiyun et al. were used as alternative labels to prepare nanozyme strips [9]. Nanozymes possess enzyme-mimicking activities (e.g., peroxidase-like, oxidase-like, and superoxide dismutase-like) and can enable catalytic signal amplification in appropriate reaction systems. Compared to traditional biological enzymes (e.g., HRP, ALP), nanozymes offer superior structural stability, tolerance to a broader range of temperatures and pH, ease of scale-up for production, and facilitated surface functionalization (e.g., coupling with antibodies/aptamers). These properties make them suitable for integration with immunoassays, nucleic acid assays, and portable detection platforms for on-site rapid testing [10]. While lateral flow immunochromatographic systems utilizing traditional labels like colloidal gold and fluorescent microspheres are well-established, they face a trade-off between signal amplification capability and field adaptability. Colloidal gold relies primarily on the intrinsic color of the particles for visual signals, making it difficult to further enhance sensitivity without additional reading equipment [11]. Fluorescent microspheres can improve sensitivity but typically require excitation light sources and readers, thereby increasing equipment dependency and creating barriers for on-site deployment [12,13]. In contrast, nanozymes possess inherent potential for colorimetric visualization and good surface functionalization capability, allowing them to be used as labels in lateral flow systems [14]. Similar to traditional labels like colloidal gold, when the target is present, the immunocomplexes labeled with nanozymes accumulate at the test line, forming a visible band signal. This strategy enables on-site interpretation without extra instruments [15]. Furthermore, the intrinsic signal-amplification capability of nanozymes can potentially enhance sensitivity [16]. Additionally, nanomaterials offer potential advantages in terms of manufacturing consistency and tolerance to storage/transportation conditions, making them suitable for coupling with portable detection platforms for field testing [17]. Currently, nanozyme technology has been successfully applied in detecting human pathogens such as Ebola, Enterobacter sakazakii, and SARS-CoV-2 [18,19,20]. However, systematic research on applying nanozymes to lateral flow immunochromatography strips for plant and animal viruses remains relatively limited. Only a few studies have been reported to date, such as the application of nanozymes for detecting animal pathogens like avian leukosis virus [21]. Nevertheless, the majority of current rapid lateral flow immunochromatography strips for potato viruses employ colloidal gold as the label [22,23,24,25], and there is a lack of rapid diagnostic schemes utilizing nanozymes as labels for potato virus detection.

This study integrates nanozyme technology with lateral flow immunochromatography in potato virus detection. We developed PVX monovalent, PVY monovalent, and PVX/PVY bivalent nanozyme strips. Through systematic optimization, we comprehensively evaluated their specificity, sensitivity, and performance using real samples. This work developed a rapid, on-site detection method for PVX and PVY suitable for field application, providing technical support for early monitoring and control of potato viral diseases.

2. Results

2.1. Principle of Nanozyme Strip Detection

The detection method established in this study is based on the double-antibody sandwich principle (Figure 1). When the target virus is present in the sample, viral particles bind to the nanozyme-labeled antibodies on the conjugate pad, forming a “nanozyme-antibody-virus” complex. This complex migrates with the extraction buffer to the test line (T line) on the nitrocellulose (NC) membrane, where it is captured by the pre-immobilized capture antibodies. The nanozyme particles accumulate at the test line, forming a visually observable colored band. Unbound nanozyme-labeled control antibodies continue to migrate to the control line (C line), where they bind to the immobilized goat anti-chicken IgY and form a band, indicating the proper functioning of the nanozyme strip.

2.2. Specificity Testing of Monoclonal Antibodies

To assess the specificity of the monoclonal antibodies, total proteins were extracted from potato plantlets in vitro infected with PVX or PVY, as well as healthy controls, and analyzed by Western blot. The results, as shown in Figure 2, indicate that monoclonal antibodies PVX-2 and PVX-6 reacted specifically with the PVX-positive sample, producing a band at approximately 35 kDa corresponding to the PVX coat protein. Similarly, monoclonal antibodies PVY-2 and PVY-5 reacted specifically with the PVY-positive sample, producing a band at approximately 33 kDa corresponding to the PVY coat protein. None of the four monoclonal antibodies produced a specific band in the healthy control; PVX antibodies did not react with PVY-positive samples, and PVY antibodies did not react with PVX-positive samples. These findings confirm the high specificity of the antibodies, making them suitable for the subsequent preparation of nanozyme strips.

2.3. Development of the Nanozyme Strip System for PVX and PVY

To verify the effectiveness of the monovalent nanozyme strips in detecting their respective target viruses, PVX and PVY monovalent nanozyme strips were used to test corresponding virus-positive samples (PVX or PVY) and negative controls. The results indicated that both the PVX and PVY monovalent nanozyme strips produced positive reactions (both T and C lines visible) for their respective target viruses (Figure 3). In contrast, negative samples only showed the C line. This indicates the successful construction of the nanozyme strip system.

2.4. Specificity and Sensitivity of Nanozyme Strips for PVX and PVY

To evaluate the specificity of the nanozyme strips, samples infected with PVA, PVM, PVS, PVX, PVY, and PLRV, along with a negative control (healthy potato), were analyzed. The results (Figure 4A,C) demonstrated that both the PVX and PVY monovalent nanozyme strips produced positive signals exclusively for their respective target viruses and showed no cross-reactivity with other viruses or healthy samples, demonstrating excellent specificity. Furthermore, sensitivity tests were conducted. The results (Figure 4B,D) indicated that the PVX nanozyme strip could detect the virus at a dilution of up to 10³, while the PVY nanozyme strip could detect the virus at a dilution of up to 10⁴.

2.5. Development of a Bivalent PVX and PVY Nanozyme Strip

Based on the monovalent nanozyme strips, a PVX/PVY bivalency nanozyme strip was developed. It was tested with samples individually infected with PVX or PVY, co-infected with PVX/PVY, and negative samples (healthy potato). The results (Figure 5) showed that the bivalent nanozyme strip could simultaneously detect both PVX and PVY, accurately identifying samples with single infections as well as co-infections.

2.6. Sensitivity and Specificity of the Bivalent PVX and PVY Nanozyme Strip

The specificity test results for the bivalent nanozyme strip are shown in Figure 6A. The nanozyme strip only produced positive reactions with samples infected with PVX and/or PVY, while showing no cross-reactivity with samples positive for PVA, PVM, PVS, PLRV, and healthy samples, indicating its excellent specificity. The sensitivity test results, shown in Figure 6B, demonstrated that the bivalent nanozyme strip has a limit of detection at a 10³ dilution for samples co-infected with PVX and PVY, meeting the requirements for rapid field detection.

2.7. Evaluation of the PVX and PVY Nanozyme Strip Using Tissue-Cultured Plantlets

To further validate the applicability of the nanozyme strips for actual sample detection, 12 potato samples of plantlet in vitro, previously confirmed by RT-PCR, were selected for testing. The results showed that the PVX monovalent nanozyme strip detected 1 PVX-positive sample, the PVY monovalent nanozyme strip detected 6 PVY-positive samples, and the bivalent nanozyme strip detected 1 PVX-positive and 6 PVY-positive samples (Figure 7). All results were completely consistent with those results obtained by RT-PCR, with complete agreement within this limited panel (n = 12).

3. Discussion

As an asexually propagated crop, the potato suffers from progressive virus accumulation in tubers over generations, which is a major cause of seed potato degeneration [26]. Consequently, the early detection of viral diseases is essential for seed potato quality control. However, traditional detection methods often struggle to meet the demands of rapid on-site field screening. In this study, we integrated Fe₃O₄ nanozyme labeling with lateral flow immunochromatography to establish rapid detection assays for PVX and PVY in both monovalent and bivalent formats. These methods can complete detection within approximately 10 min, with results interpretable by the naked eye, making them suitable for on-site screening scenarios [27].

This study utilized Fe₃O₄ nanozyme as labeling probes for lateral flow immunochromatographic assays. The visual band signals generated by their accumulation at the T/C lines allow for rapid, instrument-free result interpretation [28]. The developed nanozyme strips showed no cross-reactivity with several common potato viruses in specificity tests, indicating their excellent specificity. Regarding sensitivity, under the sample preparation and result interpretation conditions adopted in this study, the detection sensitivity achieved is comparable to that of similar products [29]. The PVX monovalent nanozyme strip could detect the virus up to a 10³ dilution, while the PVY monovalent nanozyme strip could detect up to a 10⁴ dilution. The bivalent nanozyme strip could simultaneously differentiate between single infections of PVX or PVY and co-infections, detecting co-infected samples up to a 10³ dilution. This provides convenience for obtaining diagnostic information under field conditions involving mixed infections.

Compared with methods such as RT-PCR and ELISA, lateral flow immunochromatography strips eliminate the need for sophisticated instruments, feature simple operation, and are time-efficient. These advantages facilitate rapid screening during seed potato production, storage, transportation, and at the grassroots detection level. To more intuitively assess the practical utility of the proposed method, a comparison was conducted between the nanozyme strip developed in this study and commercial lateral flow immunochromatography strips, ELISA, and RT-PCR across key parameters, including cost, time, sensitivity, and portability [30]. Overall, the method presented here holds distinct advantages in terms of detection time and portability. Compared to traditional colloidal gold commercial lateral flow immunochromatography strips, nanozymes provide a platform that could further improve sensitivity if coupled with an added substrate-based amplification step; however, in this study, the strips were interpreted directly by the naked eye without such amplification. Compared to ELISA/RT-PCR, this method can significantly reduce the equipment and detection time for on-site screening, making it more suitable for rapid decision-making in seed potato field inspections (

Table 1. Comparison of representative PVX/PVY detection methods and key parameters.

Method	Time	Sensitivity	Equipment	On-Site Applicability	Cost	Accuracy	Ref.
RT-PCR	2–3 h	5/5	Required	No	High	High	[31,32,33]
ELISA	3–5 h	4/5	Required	No	High	High	[34,35,36]
Colloidal gold strip	10~20 min	3/5	Not required	Yes	Medium	Medium	[22,23,37]
Nanozyme strip	~10 min	3/5	Not required	Yes	Low	Medium	This work

).

In this study, the detection results for 12 plantlets in vitro samples were consistent with RT-PCR, demonstrating the potential of the nanozyme strips to provide results consistent with a reference molecular method. However, it must be noted that the purported advantages of nanomaterials in storage and transport stability typically require validation through accelerated stability or shelf-life studies. This study did not systematically evaluate strip performance under different temperature/time conditions, so related conclusions await further experimental support. Furthermore, field samples have more complex matrices (e.g., mucilage, polyphenols/polysaccharides from different cultivar leaves, soil, pesticide residues), which may cause non-specific adsorption or background signals. Validation with a larger-scale sample set is still needed. In addition, direct comparison of the limit of detection sensitivity with the colloidal gold strip across different studies is not straightforward due to differences in antibody affinity/pairing, sample matrices, strip materials, and limit of detection definitions. Meanwhile, the intra-batch and inter-batch consistency of the nanozyme strips (e.g., variations in NC membranes, conjugate pads, and antibody conjugation efficiency) should be systematically assessed using appropriate statistical metrics.

Future work will focus on the following aspects: (1) Optimizing the nanozyme conjugation and dispensing processes to further enhance sensitivity; (2) Conducting blind, controlled validation studies across multiple seasons and locations in main production areas, expanding sample size and performing consistency evaluation with RT-PCR/RT-qPCR; (3) Expanding the bivalent PVX/PVY detection platform to a multiplex format for simultaneous detecting of multiple potato viruses, and integrating smartphone imaging to achieve semi-quantitative analysis of band intensity, thereby meeting the needs for seed potato grading and field decision-making.

In conclusion, the rapid immunochromatographic detection method based on nanozymes established in this study represents a technological innovation for potato virus detection and meets the practical need for rapid field testing. With further optimization and refinement, this method is expected to become a valuable tool within the potato seed tuber quality detection system, offering support for plant quarantine and rapid plant disease diagnostics.

4. Materials and Methods

4.1. Materials and Reagents

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), 2-Morpholinoethanesulfonic acid (MES), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Co., LLC. (Shanghai, China). RIPA Lysis and Extraction Buffer was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). Skim milk powder was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Absorbent pads (CH37), glass fiber membranes (SB08), and PVC plates (SM31–40) were purchased from Shanghai Kinbio Tech. Co., Ltd. (Shanghai, China). Fe₃O₄ nanozymes (NMM0001) were purchased from Chongqing Chicano Biotechnology Co., Ltd. (Chongqing, China). Nitrocellulose membranes (CN140) were purchased from Sartorius (Göttingen, Germany). Goat anti-chicken IgY and chicken IgY were purchased from Nanjing Jingda Biotech Co., Ltd. (Nanjing, China). Paired monoclonal antibodies against PVX (PVX-2, PVX-6) and PVY (PVY-2, PVY-5) were prepared and stored in our laboratory.

4.2. Origin and Production of Monoclonal Antibodies

The anti-PVX monoclonal antibodies PVX-2 and PVX-6 and the anti-PVY monoclonal antibodies PVY-2 and PVY-5 were previously generated in our laboratory using recombinant coat protein (CP) as the immunogen. In this study, monoclonal antibodies were produced by the ascites method and purified by protein G affinity chromatography prior to Western blotting and nanozyme strip development. Antibody pairing (capture vs. detection) was determined by preliminary pairing tests to select the combination with the best visual signal-to-background performance on the nanozyme strips.

4.3. Sample Collection

Potato plantlets in vitro samples, previously confirmed as positive by RT-PCR, were maintained in the laboratory. These included samples singly infected with PVX, PVY, PVA, PVS, PVM, PLRV, and samples co-infected with PVX/PVY.

4.4. Specificity Detection of Monoclonal Antibodies

Total proteins were extracted from potato plantlets in vitro infected with PVX, PVY, and healthy controls using RIPA Lysis and Extraction Buffer. The extracts were mixed with loading buffer and heated in a boiling water bath for 10 min. Proteins were separated by 10% SDS-PAGE gel electrophoresis (constant voltage 120 V, 90 min) and then transferred onto a PVDF membrane (constant current 300 mA, 60 min). The membrane was subsequently blocked with 5% skim milk in TBST for 1 h. Primary antibodies (PVX-2, PVX-6, PVY-2, and PVY-5 monoclonal antibody ascites fluid, diluted 1:1000) were added separately and incubated at 4 °C overnight. After washing three times with TBST (10 min each), HRP-labeled goat anti-mouse IgG (diluted 1:5000) was added and incubated at room temperature for 1 h. Following another three TBST washes, the signals were developed and recorded using a chemiluminescence imaging system (ECL chemiluminescent substrate).

4.5. Preparation of Nanozyme Probe and Conjugate Pad

This method was adapted from the protocol by Duan Demin, with appropriate modifications [20]. Briefly, 5 mg of EDC and 5 mg of NHS were dissolved in 1 mL of MES buffer (50 mM, pH 6.0). Then, Fe₃O₄ nanozymes (5 mg) were added, and the mixture was activated at room temperature for 30 min. Following magnetic separation, the nanozymes were washed twice with MES buffer. Subsequently, 100 µg of detection antibody (PVX-6 or PVY-5) and 100 µg of control antibody (chicken IgY) were added. The mixture was vortexed and incubated at 4 °C overnight. Tris buffer (50 mM, pH 7.4) was added and reacted at room temperature for 30 min. After magnetic separation, the conjugates were washed once with PBS (20 mM, pH 7.2) supplemented with 5% BSA and further blocked at 4 °C for 4 h. Finally, the labeled probes were washed and resuspended in PBS containing 1% BSA and stored at 4 °C for later use.

The conjugate pad was cut into 7 mm × 300 mm using Programmable Sheet Cutter CTS300 (Shanghai Kinbio Tech. Co., Ltd.), immersed in processing solution (1% Triton X-100, 50 mM sodium borate, pH 8.0) for 30 min, and dried at 37 °C for 2 h. The prepared nanozyme conjugates were diluted with conjugate pad buffer (50 mM Tris, 10% trehalose, 5% BSA, 1% Triton X-100, 1% Tween-20, 0.05% Proclin-300, 1% PVP K30, pH 8.5) and uniformly sprayed onto the treated conjugate pad at a rate of 9.9 µL/cm using XYZ Platform Dispenser HM3035 (Shanghai Kinbio Tech. Co., Ltd.). The pad was then dried at 37 °C for 1 h.

4.6. Preparation of the Nanozyme Strips

The test strip consists of a sample pad, conjugate pad, nitrocellulose (NC) membrane, absorbent pad, and PVC backing card. The sample pad was cut into 11 mm × 300 mm using Programmable Sheet Cutter CTS300, immersed in processing solution (10 mM PBS, 1% Tween-20, 0.1 g/L PVP K30, pH 7.4) for 30 min, and dried at 40 °C for 2.5 h. The absorbent pad was cut into 22 mm × 300 mm using Programmable Sheet Cutter CTS300 and dried at 40 °C for 2.5 h. For NC membrane coating, antibodies were diluted to 1.0 mg/mL with coating buffer (10 mM PBS, 2% trehalose, pH 7.2). Using an XYZ Platform Dispenser, the antibodies were dispensed at a rate of 1 µL/cm: the test line (T line) of the PVX monovalent strip was coated with PVX-2; the T line of the PVY monovalent strip was coated with PVY-2; for the bivalent strip, the T1 line (coated with PVX-2) and the T2 line (coated with PVY-2) were established. The control line (C line) for all strips was coated with goat anti-chicken IgY. After coating, the membrane was dried at 37 °C for 1 h.

The sample pad, conjugate pad, NC membrane, and absorbent pad were sequentially overlapped and laminated onto the PVC backing card with 1–2 mm overlaps between adjacent components. Finally, the assembled sheet was cut into individual strips (4 mm in width) using a Programmable High-Speed Strip Cutter ZQ4500 (Shanghai Kinbio Tech. Co., Ltd.). The strips were assembled into plastic cassettes, sealed, and stored at room temperature.

4.7. Nanozyme Strips Test

Approximately 0.1 g of fresh potato leaf tissue was weighed and ground in extraction buffer (10 mM PBS, 1% skim milk powder, 1% Tween-20, pH 7.2) at a 1:10 (w/v) ratio. Then, 80 µL of the extraction solution was pipetted and added to the sample well of the test strip. After incubation at room temperature for 10 min, the color development on the T and C lines was observed visually. Result interpretation: A test was considered invalid if the C line did not develop. The result was positive if both the C line and T line(s) developed color. The result was negative if the C line developed color, but the T line(s) did not.

4.8. Evaluation of Nanozyme Strip Specificity and Sensitivity

Specificity analysis: Samples from potato plants infected with PVA, PVM, PVS, PVY, PVX, and PLRV, as well as healthy plants, were tested separately. Each sample was tested independently three times (n = 3). For each test, 80 µL of the sample was used. Results were read and recorded after a 10-minute reaction at room temperature.

Sensitivity analysis: Extracts from plants singly infected with PVX or PVY and co-infected with PVX/PVY were serially diluted (10¹ to 10⁵-fold). Each dilution was tested independently three times (n = 3). The limit of detection (LOD) was defined as the highest dilution factor at which a visible T line appeared at the 10-minute reading time point, with all three replicates testing positive.

Detection of actual samples: Twelve potato tissue-cultured seedling samples were selected and tested using the developed nanozyme strips. The results were compared with those obtained by RT-PCR, and the concordance rate was calculated.

5. Conclusions

In this study, we have developed Fe₃O₄ nanozyme strips for the rapid on-site detection of PVX and PVY. Preliminary tests have verified that the strips have high specificity, sufficient sensitivity (detection up to 10³–10⁴ dilutions), and complete agreement with RT-PCR within this limited panel (n = 12). This method shows potential as a practical tool for certified seed potato screening and field inspection, but requires further validation with larger sample sizes and under diverse field conditions.