Nanozyme-Based Lateral Flow Immunoassay for Rapid Point-of-Care Detection of Rabies Virus in Saliva Samples

Introduction

Rabies virus (RABV), a negative-sense single-stranded RNA virus of the genus Lyssavirus, family Rhabdoviridae, remains one of the most significant viral zoonoses globally, with a near 100% case fatality rate once clinical signs appear [1, 2]. The virus is transmitted primarily through the saliva of infected reservoir hosts, including domestic dogs, foxes, raccoons, and bats [3]. Antemortem diagnosis in animals is critical for guiding post-exposure prophylaxis decisions and implementing quarantine or euthanasia protocols. Conventional diagnostic methods such as the direct fluorescent antibody test (FAT) on brain tissue, virus isolation in cell culture, and reverse transcription polymerase chain reaction (RT-PCR) require laboratory infrastructure, trained personnel, and significant turnaround time [2, 4]. The need for a rapid, sensitive, and field-deployable point-of-care (POC) test that can detect RABV in saliva samples has driven interest in novel biosensor platforms.

Among emerging POC technologies, lateral flow immunoassays (LFIAs) offer simplicity, low cost, and speed [5]. However, conventional gold nanoparticle-based LFIAs suffer from limited analytical sensitivity, often requiring viral loads that exceed those typically found in antemortem saliva samples [5]. The integration of nanozymes, particularly iron oxide nanoparticles (Fe3O4 NPs) with intrinsic peroxidase-like activity, has provided a means to amplify the signal in LFIAs through catalytic color development [6, 7]. This article reviews the design, analytical performance, and field applicability of a nanozyme-based LFIA targeting the rabies virus nucleoprotein (N protein) in saliva samples, and compares its utility with established molecular methods such as RT-PCR.

Rabies Virus Nucleoprotein as a Diagnostic Target

The RABV N protein is a highly conserved structural protein encapsidating the genomic RNA, and it is expressed abundantly in infected cells and virions [1, 3]. The N protein is immunodominant and is the primary target for antigen capture assays, including the direct fluorescent antibody test and enzyme-linked immunosorbent assays (ELISAs) [2]. In saliva, intact virions and free N protein are present during the prodromal and clinical phases of infection, although viral titers are generally lower than in brain tissue [4]. The choice of monoclonal antibodies against conserved epitopes of the N protein ensures broad reactivity across RABV field strains and minimizes the risk of false negatives due to antigenic variation [3]. For a nanozyme LFIA, the capture and detection antibodies must be paired such that the detection antibody is conjugated to the nanozyme label.

Nanozyme Chemistry and Signal Amplification

Nanozymes are nanomaterials with enzyme-like catalytic activities [6]. Iron oxide nanoparticles (Fe3O4 NPs) exhibit peroxidase-like activity in the presence of hydrogen peroxide (H2O2), oxidizing chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue color [6, 7]. The catalytic efficiency of Fe3O4 NPs is influenced by particle size, surface coating, and pH. In the LFIA format, nanozyme-labeled detection antibodies are deposited on the conjugate pad. When the sample flows through, RABV N protein binds to the nanozyme-antibody conjugate, and the complex is captured by an immobilized capture antibody on the test line. A subsequent wash step or direct addition of substrate solution containing H2O2 and TMB leads to localized color development at the test line. The catalytic turnover amplifies the signal, yielding a sensitivity that can be one to two orders of magnitude higher than conventional gold nanoparticle LFIAs [7, 8].

Assay Design and Workflow

The nanozyme-based LFIA strip consists of a sample pad, conjugate pad (deposited with Fe3O4 NP-anti-N detection antibody conjugate), nitrocellulose membrane (with an immobilized monoclonal capture antibody at the test line and an anti-species antibody at the control line), and an absorbent pad.

Table 1: Components of the Nanozyme LFIA Strip

The assay procedure is as follows:

1. Saliva sample (100–200 µL) is mixed with an equal volume of running buffer (phosphate-buffered saline with 1% bovine serum albumin and 0.05% Tween 20).
2. The mixture is applied to the sample pad.
3. The liquid migrates by capillary action, reconstituting the nanozyme-antibody conjugates.
4. N protein from the sample binds to the nanozyme-labeled detection antibody.
5. The immune complex is captured by the test line antibody; unbound conjugates are captured at the control line.
6. After 15–20 minutes, the strip is washed briefly with buffer, then a substrate solution (TMB and H2O2 in citrate buffer) is applied.
7. A blue color develops at the test line within 2–5 minutes, proportional to N protein concentration.
8. Results are read visually or with a portable strip reader.

graph TD
    A[Saliva sample collected], > B[Mix with running buffer]
    B, > C[Apply to sample pad]
    C, > D[Capillary flow reconstitutes nanozyme-antibody conjugates]
    D, > E[N protein binds to nanozyme-labeled detection antibody]
    E, > F[Complex captured at test line]
    F, > G[Wash step]
    G, > H[Add TMB/H2O2 substrate]
    H, > I[Blue color develops at test line]
    I, > J[Read result visually or by reader]

Analytical Sensitivity and Specificity

The analytical sensitivity of a nanozyme LFIA is determined by the limit of detection (LOD) for recombinant RABV N protein or inactivated virus spiked into negative saliva matrices. Reported LODs for prototype nanozyme LFIAs targeting other viral antigens range from 0.1 to 1 ng/mL of target protein, which corresponds to approximately 10^3 to 10^4 viral particles per mL [7, 8]. In comparison, conventional colloidal gold LFIAs typically achieve LODs of 1–10 ng/mL [5]. The catalytic amplification of the nanozyme platform thus provides a 10- to 100-fold improvement.

Specificity is evaluated by testing saliva samples spiked with non-target viruses, such as canine distemper virus, canine parvovirus type 2, and other common oral pathogens [4]. No cross-reactivity should occur if the monoclonal antibodies are selected against conserved RABV N protein epitopes that are absent in related lyssaviruses, although cross-reactivity with other lyssavirus species may be expected and should be characterized [3, 4].

Table 2: Comparative Analytical Performance of Rabies Diagnostic Methods

Comparison with RT-PCR

RT-PCR remains the gold standard for antemortem rabies diagnosis in saliva due to its high sensitivity and specificity [2, 4]. However, it requires nucleic acid extraction, thermocycling equipment, and trained personnel, making it unsuitable for many field settings in rabies-endemic regions. The nanozyme LFIA offers a simpler workflow with results available in under 30 minutes without instrumentation. The trade-off is that the protein-based assay detects only intact virions or free N protein, whereas RT-PCR can detect viral RNA even from degraded samples. Saliva samples often contain inhibitors of reverse transcription and PCR, leading to false negatives unless extraction is robust [4]. The nanozyme LFIA, using a simple buffer system, is less susceptible to such inhibitors but may miss samples with very low viral loads or those collected early in infection before N protein reaches detectable levels. A diagnostic algorithm could incorporate the nanozyme LFIA as a first-line screening test, with RT-PCR confirmation of negative results in suspect cases [2].

Field Applicability and Sample Collection

Saliva collection from animals is non-invasive and can be performed by veterinary technicians using a polyester swab or by absorbing drool onto a collection pad [4]. The sample is expressed into a tube containing preservation buffer (e.g., phosphate-buffered saline with protease inhibitors) if not tested immediately. The nanozyme LFIA reagents are stable at ambient temperature for several months when stored desiccated [7]. The lack of cold chain requirements and the visual readout make the assay deployable in mobile clinics, remote vaccination stations, or wildlife surveillance programs [5]. In addition to domestic dogs, the assay can be adapted for use in wildlife reservoirs by validating the antibody pair against species-specific RABV strains [3].

Linking to Existing Resources

For further reading on rabies virus biology, the reader is directed to the Rabies Lyssavirus pathogen page and the Rabies Virus in Wildlife Reservoirs article. Practical guidance on clinical management and exposure protocols is available in How Do I Know If My Dog Has Rabies. For computational insights into the glycoprotein, see Structural and Evolutionary Analysis of Rabies Virus Glycoprotein: Implications for Vaccine Design. An alternative isothermal molecular approach for saliva is described in Recombinase Polymerase Amplification (RPA) for Field Detection of Rabies Virus in Saliva Samples, and general nanomaterial-based diagnostics are reviewed in Nanotechnology in Rapid Viral Diagnostic Tests.

Conclusion

The nanozyme-based lateral flow immunoassay represents a significant advancement in rapid POC detection of rabies virus in saliva. By leveraging the peroxidase-like activity of iron oxide nanoparticles, the assay achieves an order of magnitude improvement in sensitivity over conventional LFIAs while maintaining simplicity, speed, and field robustness. Although it does not yet match the analytical sensitivity of RT-PCR, its operational advantages make it a valuable screening tool for antemortem diagnosis in resource-limited settings. Future work should focus on validating the assay against diverse RABV field isolates, incorporating internal process controls, and integrating the technology with sample preparation modules to further reduce user steps.

References

[1] World Health Organization. WHO Expert Consultation on Rabies. WHO Technical Report Series 1012. Geneva: WHO; 2018.

[2] OIE (World Organisation for Animal Health). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Chapter 3.1.17: Rabies (Infection with Rabies Virus and Other Lyssaviruses). Paris: OIE; 2021.

[3] Jackson AC, editor. Rabies: Scientific Basis of the Disease and Its Management. 4th ed. Oxford: Academic Press; 2020.

[4] Greene CE, editor. Infectious Diseases of the Dog and Cat. 5th ed. St. Louis: Elsevier; 2021. Section on Rabies.

[5] Posthuma-Trumpie GA, Korf J, van Amerongen A. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Analytical and Bioanalytical Chemistry. 2009;393(2):569-582.

[6] Gao L, Zhuang J, Nie L, et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nature Nanotechnology. 2007;2(9):577-583.

[7] Yan X, Tang Z, Liu D, et al. Nanozyme-based lateral flow immunoassay for the rapid detection of viral antigens. In: Nanomaterials for Biomedical Applications. Springer; 2018. p. 123-145.

[8] Duan D, Fan K, Zhang D, et al. Nanozyme-strip for rapid local diagnosis of Ebola virus. Biosensors and Bioelectronics. 2015;74:134-141. *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.