Evaluation of an Enzyme-Linked Magnetic Electrochemical Assay for Hepatitis a Virus Detection in Drinking and Vegetable Processing Water

Abstract

Globally, waterborne viral infections significantly threaten public health. While current European Union regulations stipulate that drinking water must be devoid of harmful pathogens, they do not specifically address the presence of enteric viruses in water used for irrigation or food production. Traditional virus detection methods rely on molecular biology assays, requiring specialized personnel and laboratory facilities. Here, we describe an electrochemical sandwich enzyme-linked immunomagnetic assay (ELIME) for the detection of the hepatitis A virus (HAV) in water matrices. This method employed screen-printed electrodes as the sensing platform and utilized commercially available pre-activated magnetic beads to provide a robust foundation for the immunological reaction. The ELIME assay demonstrated exceptional analytical performance in only 185 min achieving a detection limit of 0.5 genomic copies per milliliter (g.c./mL) and exhibiting good reproducibility with a relative standard deviation (RSD) of 7% in HAV-spiked drinking and processing water samples. Compared with the real-time RT-qPCR method described in ISO 15216-1, the ELIME assay demonstrated higher sensitivity, although the overall linearity of the method was moderate. These analytical attributes highlight the potential of the ELIME assay as a rapid and viable alternative for HAV detection in water used for agriculture and food processing.

1. Introduction

The hepatitis A virus (HAV), a non-enveloped, single-stranded positive-sense RNA virus within the Picornaviridae family, is the causative agent of acute viral hepatitis and is responsible for numerous cases and outbreaks globally [1,2]. HAV is primarily spread via the fecal–oral route, either through direct contact with infected individuals or through the ingestion of contaminated food (such as bivalve shellfish and soft fruits) or water [3,4]. The prevalence of foodborne and waterborne viral diseases represents a substantial public health challenge worldwide. A comprehensive review of HAV prevalence across various water matrices—including untreated and treated wastewater, surface water, groundwater, and drinking water—reported an overall prevalence of 16.7%, with the highest prevalence in untreated wastewater (31.4%) and the lowest in drinking water (0.3%) [5].

Currently, European Union legislation on water intended for human consumption (EU Dir. 2020/2184) depends on fecal indicator bacteria (FIB) for microbiological safety, which is more straightforward to measure compared with the full range of pathogens that pose health risks [6]. However, FIB does not adequately indicate the presence of pathogenic viruses. Similarly, regulations on microbial contamination in food products and hygiene in primary production (Reg. EC 2073/2005, 853/2004, 852/2004, and subsequent amendments) mandate the use of water deemed “suitable for human consumption” or “clean water”, yet lack specific microbiological criteria for enteric viruses in waters used for food production or irrigation [7,8,9]. Public health management strategies for hepatitis A emphasize pre-exposure vaccination for high-risk groups; education on good personal hygiene, particularly hand hygiene; and the provision of adequate sanitation and housing. Countries with high income levels and robust sanitary conditions typically exhibit low infection rates. Understanding the environmental sources of HAV is critical for controlling the disease. Studies have identified HAV in foods, treated and untreated wastewater, and various aquatic environments [10,11,12].

The gold standard for the quantitative detection of HAV in food and bottled water is reverse transcription-quantitative real-time PCR (RT-qPCR), supported by an internationally validated method (ISO 15216-1:2017) [13,14]. However, while real-time RT-qPCR is sensitive and effective, this technique has some significant limitations [15]. In particular, to ensure high sensitivity of detection, molecular methods require the use of matrix-specific concentration procedures, both to optimize viral recovery from samples and to remove PCR inhibitors that may negatively affect the efficiency of polymerization. This often implies the need for multi-step sample preparation procedures, which may significantly affect both sample turnaround and laboratory capacity. Further to this, to minimize the risk of sample cross-contamination and molecular reagents contamination and ensure quality of results, appropriate working environments are required for molecular methods, therefore limiting their use to laboratory environments, with few applications in the field or production lines. More recently, progress in the available molecular solutions has reduced some of these limitations [16]. The introduction and progressive diffusion of digital and digital droplet PCR, which show higher tolerance to inhibitors deriving from complex matrices [17], have reduced the need for laborious inhibitors’ removal treatment and, thanks to their independence from calibration curves for target quantification, have improved the precision and accuracy of viral quantification. The use of other nucleic acid amplification technologies such as loop-mediated isothermal amplification (LAMP), which performs DNA synthesis in isothermal conditions (e.g., also in a simple water or heat block), has improved both the turnaround time and the portability of molecular methods in non-laboratory settings, with a potential for on-site use [18]. Further improvements to the molecular detection of viruses in food and water matrices are expected with the progressive implementation of viability assays [19], exploitation of CRISP technology [20], and combination of molecular assays with artificial intelligence tools [21]. On the other hand, immunoassays, based on the enzyme-linked immunosorbent assay (ELISA), have been known for decades for their easiness, high throughput, and ability to detect specific antigens through targeted antibodies [20,21,22]. ELISA tests are generally more cost-effective than PCR, making them suitable for high-throughput screening, and allowing for the rapid testing of large sample numbers though sensitivity issues precluded extensive use beyond clinical diagnostics [15,23]. Screen-printed electrodes (SPE) have been linked to recent improvements in sensitivity. SPEs are used as a signal transducer for the detection of the enzymatic product of the enzyme conjugated with the antigen or antibody, depending on the format used to highlight the immunological chain formed, as well as a support for the construction of the immunological chain. The advantages of the association of electrochemical detection in ELISA over conventional optical detection are reported in a 2007 review [22]. The development of electrochemical immunosensors has significantly advanced thanks to the practical and creative fusion of various signal-amplified components with enzyme catalysis.

Furthermore, the introduction of magnetic supports—such as secondary antibody-modified microparticles and nanoparticles—has demonstrated in more recent times how immunoassays can be made more sensitive and repeatable even when dealing with complicated matrices [23]. Additionally, the magnetic particles demonstrate their exceptional benefits in the pre-concentration and isolation of targets from intricate biological materials. To increase the sensitivity of electrochemical immunosensors and support their use in clinical, food, and environmental fields, enzyme catalysis can be seamlessly combined with these amplification methods [24]. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are two of the most often employed enzyme reporters among the many enzymes used as markers in immunoassays. However, several inherent issues, including a non-specific staining response, activity inhibition by Cu+ ions, high background from the electrochemical reduction in the H₂O₂ substrate, and the inability to use voltammetric methods (except for amperometry) [25] for electrochemical detection, may affect the application of HRP. As a reporter enzyme for signal amplification, however, AP has garnered a lot of interest because of its superior qualities, including high catalytic activity, a large turnover number, and a broad range of electrochemical techniques for the detection, such as differential pulse voltammetry (one of the most sensitive electrochemical techniques) [26,27].

Compared with traditional ELISA or electrochemical immunosensors, the ELIME (enzyme-linked immunosorbent magnetic electrochemical) method offers a larger surface-to-volume ratio due to magnetic particles and improved sensitivity of electrochemical detection over spectrophotometric analysis [28,29,30].

This study highlights the advantages of ELIME over real-time PCR and ELISA, demonstrating the sensitivity and reliability of the electrochemical sandwich ELIME technique, which employs commercially preactivated magnetic beads for HAV detection in processing water samples [31]. Our findings reveal significant benefits of this method compared with the real-time RT-qPCR protocol outlined in ISO 15216-1 [32], presenting promising results in analytical performance comparisons.

2. Materials and Methods

2.1. Reagents

Unless otherwise specified, chemical reagents were obtained from Merck^® (Bari, Italy). Goat Anti-Mouse IgG magnetic beads were purchased from New England BioLabs^® (Hitchin, UK) and stored at +4 °C. Anti-Mouse IgG was covalently coupled to a ~0.5 µm diameter and 1 μm nanoporous paramagnetic microparticle; 1 mL suspension in PBS Buffer (pH 7.4), 0.05% Tween 20, 0.1% BSA, and 0.05% NaN3 contains 3.65 × 10¹⁰ particles. The primary monoclonal hepatitis A virus antibody (MAbI) clone M5112922 used in this study was provided by Fitzgerald^® (Crossville, TN, USA), with 1000 μg/mL in 10 mM PBS, pH 7.2, with 0.1% NaN₃, whereas the primary monoclonal hepatitis A virus antibody labelled with alkaline phosphatase (MAbI-AP) clone 7E7 was supplied by Pantec^® (Milano, Italy) in 10 mM PBS, pH 7.2, with 0.1% NaN₃. Both Abs were stored at −20 °C.

The viral standard for the hepatitis A virus was purchased from Mediagnost^® (Reutlingen, Germany). Information provided by the supplier, concerning the amount of HAV virus in the sample, indicated that the suspension contained 4.8 × 10⁶ genome copies (g.c.)/mL (measured by real-time RT-qPCR according to ISO 15216-1:2017) [14] and 100 International Unit (UI)/mL. The hepatitis A virus strain HM175/18f (American Type Culture Collection, ATCC VR-1402), used for spiking drinking water samples, was provided by the Istituto Superiore di Sanità (ISS) at a concentration of 7.5 × 10⁸ g.c./mL (measured by real-time RT-qPCR). For virus production, HAV HM-175/18f was replicated in Frp3 cells (non-human primate cell line derived from fetal kidney) cultured in minimum essential medium with Earle’s salts (MEM) supplemented with 1% glutamine, 2% non-essential amino acids, and 2% FBS. The virus was incubated in 5% CO₂ at 37 °C with daily checks of modifications of the cell monolayer. The cell culture medium and supplements were provided by EuroClone (Milan, Italy). The viral suspension was prepared by three freeze and thaw lysis cycles of infected monolayers, clarified using low-speed centrifugation (800× g) to remove residual cell debris, then divided in aliquots and kept at −80 °C for long-term storage and at −20 °C during experiments.

The buffers used for the experiments were the following: solution A (washing magnetic beads solution), 50 mM sodium phosphate buffer (PB), pH 7.4, containing 100 mM KCl and 0.05% (v/v) of Tween 20; solution B (store magnetic beads solution), 50 mM PB buffer, pH 7.4, containing 0.1% BSA and 0.02% NaN₃; solution C (extraction solution), 100 mM Tris, 50 mM Gly, and 0.1% of beef extract, pH 9.5 (TGBE buffer).

2.2. Materials

2.2.1. Screen-Printed Electrodes (SPEs)

Screen-printed electrodes were produced by a screen printer DEK 245 (DEK, Weymouth, UK) on polyvinyl chloride (PVC) strips with eight printed sensors each. The inks were from Acheson (Mezzago, Milan, Italy). The SPE included three electrodes: graphite for working and counter electrodes and silver/silver chloride (Ag/AgCl) for the reference electrode. The diameter of the working electrode was 0.3 cm, with an apparent geometric area of 0.07 cm² [33,34].

2.2.2. Filters

Zeta Plus™ 1MDS discs (CUNO, 3M Company, CUNO, 3M Company, Meriden, CT, USA), 47 mm, 0.45 μm pore size, were used in this study. Filters were characterized by an electropositive, charge-modified, cellulose medium double open-ended (flat nitrile gasket) configuration. The filters used electrokinetic adsorption to capture and concentrate waterborne viruses.

2.2.3. Instrumentations

Electrochemical measurements were performed at room temperature, using a computer-controlled system, AUTOLAB model GPSTAT-12 with GPES software (Ecochemie, Utrecht, The Netherlands). Differential pulse voltammetry (DPV) measurements were carried out in the potential range from 0 to 600 mV with a pulse width of 50 ms, a pulse amplitude of 70 mV, and a scan speed of 100 mV/s.

2.3. Methods

2.3.1. ELIME Assay

In this work, an enzyme-linked immuno-magnetic electrochemical assay (ELIME), with a sandwich format, was developed. Goat Anti-Mouse IgG magnetic beads (10 μL) were first washed twice in 50 mM PBS, pH 7.4, by using an external magnet to allow easy separation. They were coated, then, with primary monoclonal hepatitis A virus antibody (MAbI) in 50 mM carbonate buffer, pH 9.6, shaking for 45 min at room temperature, separated with the magnet, and washed again twice with solution A. The coated beads were resuspended in 3% BSA in 50 mM carbonate buffer, pH 9.6, shaking for 45 min at room temperature, washed again twice with solution A, and resuspended in solution B. The beads were stored at +4 °C overnight, separated, and washed twice with solution A. HAV in 50 mM PBS, pH 7.4, containing 0.1% BSA was added. After shaking for 45 min at room temperature, the beads were washed twice with solution A. An alkaline phosphatase (AP) labeled anti-HAV monoclonal antibody (MAb-AP) in 50 mM PBS, pH 7.4, was used to complete the immunological chain constituted by HAV and MAbI, immobilized directly on the beads, to reveal the sandwich complex format.

The beads were shaken for 45 min at room temperature, washed twice with solution A, and resuspended in 50 mM PBS, pH 7.4. After the immunochemical steps, the beads were localized onto the surface of SPEs with the aid of a magnet. The enzyme-substrate 1-naphthyl-phosphate (1-NPP) in 0.97 diethanolamine (DEA) buffer, pH 9.8, containing 1 mM MgCl₂ and 100 mM KCl was added to measure the electroactive product (Figure 1).

2.3.2. Spiking of Drinking Water and Water for Vegetable Processing

Drinking water was collected from the public water system of Rome, Italy. Vegetable processing water was collected from an industrial setting performing the processing of ready-to-eat leafy vegetables and included water taken after its use for vegetable washing. One hundred mL of drinking water and vegetable processing water were spiked with different concentrations of HAV (strain HM175/18f) at concentrations ranging between 0 (level 0) and ~10.000 g.c./mL (level 9). The exact inoculum value (and the expected value for quantification by real-time RT-qPCR and ELIME assay) was confirmed through analysis of the highest concentration of the HAV suspension used for spiking.

2.3.3. Sample Concentration for HAV Detection

The concentration method for HAV was obtained by a modification of an organic solvent-based method taken from the literature [8,9] and the reference ISO method for HAV detection in food and water samples (ISO 15216-1:2017) [14,35]. Samples were filtered on the wrinkled side of the Zeta Plus™ 1MDS disc using an EZ-Stream^® vacuum laboratory pump (Merck Millipore, Rome, Italy). Viruses adsorbed on the filter were eluted and recovered using 40 mL of solution C, spinning at 35–40 rpm for 15 min. After adjusting the pH to 7.0 ± 0.2 with HCl 5 N, the suspension was divided into two aliquots of equal volume, for ELIME assay detection and real-time RT-qPCR analysis. The aliquot intended for real-time RT-qPCR was concentrated according to ISO 15216-1:2017 [14] and centrifuged at 4000× g for 15 min using Amicon Ultra centrifugal filter devices ultracel 100 KDa (Merck Millipore, Rome, Italy), and the viral concentrate was eluted with 0.5 mL of sterile PBS (Figure 2).

In order to underline the reproducibility of the analysis for HAV concentrations near the LOD, 10 aliquots of drinking water sample were spiked with 0.7 g.c./mL, and the extraction method was applied.

2.3.4. RNA Extraction and Real-Time RT-qPCR Protocol

Nucleic acid extraction was performed using the NucliSens extraction kit and the MiniMag semi-automatic platform (bioMerieux, Paris, France) according to the manufacturer’s instructions. Eluted RNA (100 μL) was stored at −80 °C until molecular analysis. Real-time RT-qPCR was conducted using the amplification conditions, primers, probes, and reagents reported in the annexes of the ISO 15216 method [14,31,35,36]. Briefly, for the assay the RNA UltraSense One-Step qRT-PCR System kit (Life Technologies, Carlsbard, CA, USA) was used. A 25 μL reaction mixture was prepared with 5 μL of RNA, reverse primer (900 nM), forward primer (500 nM), and probe (250 nM). For each sample, duplicate reactions were carried out on a 7700 Sequence Detection System (Applied Biosystems, Waltham, MA, USA). The following amplification conditions were applied: 55 °C for 60 min for reverse transcription and 95 °C for 5 min for RT inactivation followed by 95 °C for 15 s, 60 °C for 60 s, and 65 °C for 60 s (45 cycles). For standard curve construction, tenfold dilutions of a quantified plasmid containing the target sequence were used (range 10¹–10⁵ copies/μL); quantifications were considered acceptable if standard curves displayed a slope between −3.1 and −3.6 and an R² ≥ 0.98 [35].

2.3.5. Calibration Curve for Immunosensor

The dose–response curve of the ELIME was constructed by fitting experimental data with a nonlinear four-parameter logistic calibration plot, as shown in the equation [33,37]

$$y=y_0+{\displaystyle \frac{a}{1+{\left(\frac{x}{x_0}\right)}^b}}$$

(1)

in which a and y₀ are the asymptotic maximum and minimum values, x₀ is the IC₅₀, and b is the slope. Each value of current I (μA) was normalized using the formula [33]

$$\mathrm{I}\%={\displaystyle \frac{{\mathrm{I}}_{\mathrm{x}}-{\overline{\mathrm{I}}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}{{\overline{\mathrm{I}}}_{\mathrm{m}\mathrm{a}\mathrm{x}}-{\overline{\mathrm{I}}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}}\times 100\%$$

(2)

with ${\overline{\mathrm{I}}}_{\mathrm{m}\mathrm{i}\mathrm{n}}$ being the average value of the current at minimum concentration of HAV and ${\overline{\mathrm{I}}}_{\mathrm{m}\mathrm{a}\mathrm{x}}$ being the average value of the current at the maximum concentration of HAV.

The LOD, Equation (3), and the limit of quantification (LOQ), Equation (4), were calculated as follows [33,37]:

Y_LOD = SB + 3 SD

(3)

Y_LOQ = SB + 10 SD

(4)

where SB is the signal measured in the absence of HAV and SD is the standard deviation of the blank (10 replications). The obtained values were introduced in the logistic equation, and the relative concentrations were extrapolated.

3. Results and Discussion

3.1. ELIME Optimization

In this paper, a rapid system for HAV detection, based on ELIME assay, was evaluated. The system was based on the use of Goat Anti-Mouse IgG magnetic beads as a solid support for the immunochemical chain, using an array of eight screen-printed electrodes (SPEs) as sensing platforms. An alkaline phosphatase-labelled anti-HAV monoclonal antibody (MAb-AP) was used as an electroactive reporter and to complete the immunological chain formed by HAV and primary monoclonal antibody against HAV (MAbI), immobilized directly on magnetic beads to reveal the sandwich complex. A general schematization of the ELIME functioning is depicted in Figure 3.

The magnetic beads were kept on SPEs with the support of a magnet, and the enzyme-substrate 1-naphthyl-phosphate (1-NPP) was added to measure the electroactive product. For this goal, the analytical parameters were optimized. To establish the best conditions for the sandwich ELIME, different dilutions of MAbI were tested using a fixed concentration of HAV (10⁻⁸ UI/mL), 3% dry milk (DM) as a blocking agent, and MAb-AP diluted 1:10,000 (v/v) in PBS with incubation times of 45 min (parameters from previous studies) [21] (Figure 4). The maximum electrochemical signals were observed at a concentration of 10 μg/mL MAbI, whereas no further current increase was observed at higher concentrations.

Different blocking agents were evaluated to reduce non-specific adsorption onto the surface of coated beads. Three percent bovine serum albumin (BSA), 3% dry milk (DM), and 1% polyvinyl alcohol (PVA) were tested by adding MAb-AP diluted 1:5000, 1:10,000, and 1:25,000 (v/v) in PBS with no other bioreagent. The experimental results (Figure 5) revealed that 3% BSA was the most effective blocking agent for MAb-AP diluted 1:25,000 (v/v) in PBS from the stock solution.

A layer-by-layer electrochemical (LbL-EC) characterization of the primary modification step of the SPE surface was conducted at this stage. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed as complementary techniques for this characterization. Visual inspection reveals a progressive increase in peak-to-peak separation with decreasing electron transport efficiency as the bare screen-printed electrode (SPE) is modified and with each subsequent modification step. This observation is corroborated not only by the CV results but also by the EIS experiments, which show a progressively increasing charge transfer resistance (Rct) (Figure 6).

3.2. Analytical Performances in Buffer and Real Sample Solution

After optimizing the antibody concentration and the blocking agent, a calibration curve was constructed using the HAV strain HM175 (Figure 7). This ELIME assay achieved a detection limit (LOD) of 0.5 g.c./mL and an LOQ of 0.7 g.c./mL for the quantitative determination of HAV.

To investigate the applicability of the proposed methods for practical analysis, the immunosensor was used to evaluate the detection of different concentrations of HAV spiked in drinking water samples and in washing water used for ready-to-eat vegetable processing. The results were compared with those obtained by the standard real-time RT-qPCR analysis (ISO 15216-1:2017) [14], which is typically used to estimate the presence of HAV in food and water samples. To compare the results of the two methods (ELIME and RT-qPCR), a correlation was established on the HM175/18f strain supplied by ISS between the units used in ELIME (IU/mL) and those used in RT-qPCR (g.c./mL), revealing that 1 UI/mL = 2 × 10⁶ g.c./mL with R² = 0.997.

In experimentally contaminated water samples, the ELIME assay detected HAV at the lowest spiking level 1 in both types of water samples (

Table 1. Assessment of the ELIME assay on drinking water. The results were compared with RT-qPCR on the same samples.

Spiking Level	HAV Expected Value (g.c./mL) †	HAV Measured Value
		ELIME Assay & (g.c./mL)	RT-qPCR & (g.c./mL)
0	-	-	-
1	0.8 ± 0.1	0.9 ± 0.1	-
2	4.1 ± 0.6	1.2± 0.2	0.56 * ± 0.3
3	8 ± 1	1.5 ± 0.2	0.56 * ± 0.2
4	41 ± 6	2.6 ± 0.4	2.70 ± 0.8
5	82 ± 12	3.2 ± 0.5	4.22 ± 0.7
6	411 ± 60	4.4 ± 0.6	35.44 ± 3
7	822 ± 121	104 ± 15	119 ± 9
8	4108 ± 603	531 ± 78	952 ± 65
9	8216 ± 1206	1089 ± 159	2684 ± 154

† Based on quantification of HAV stock used for spiking of samples. * Value below the analytical LOQ of the real-time RT-qPCR. ^& Replicate analysis n = 3 for ELIME and n = 2 for qRT-PCR for each sample.

and

Table 2. Assessment of the ELIME assay on vegetables rinsing water. The results were compared with RT-qPCR on the same samples.

Spiking Level	HAV Expected Value (g.c./mL) †	HAV Measured Value
		ELIME Assay & (g.c./mL)	RT-qPCR & (g.c./mL)
0	-	-	-
1	0.9 ± 0.3	1.5 ± 0.5	-
2	5 ± 2	1.7 ± 0.7	0.19 * ± 0.16
3	9 ± 3	1.67 ± 0.6	0.40 * ± 0.09
4	469 ± 17	1.98 ± 0.07	0.53 * ± 0.36
5	91 ± 33	2.18 ± 0.8	0.96 * ± 0.73
6	456 ± 167	4 ± 1	4.04 ± 2.01
7	912 ± 333	31 ± 11	6.46 ± 0.30
8	4559 ± 1667	136 ± 50	86.85 ± 5.21
9	9118 ± 3334	229 ± 84	93.02 ± 3.53

† Based on quantification of HAV stock used for spiking of samples. * Value below the analytical LOQ of the real-time RT-qPCR. ^& n = 3 for ELIME and n = 2 for qRT-PCR for each sample.

), demonstrating a greater sensitivity than the ISO 15216-1 method [14] and validating a detection limit of 0.5 g.c./mL (roughly equivalent to 0.5 viruses/mL, given the correspondence between a genome and a viral particle). The linearity of the results was modest, particularly in vegetable washing water, where organic and disinfectant residues reduced the efficiency of both the ELIME and PCR analysis. However, the underestimation of the viral concentration in the spiked samples did not negatively affect the overall ELIME sensitivity.

To test the reproducibility of the proposed assay and the extraction method, 0.7 g.c./mL was added to 10 aliquots of drinking water, and the extraction procedure was applied. The results showed a satisfactory reproducibility with RSD% equal to 14%.

3.3. Comparison of the Performance and Cost–Benefit Analysis

The ELIME sensor’s performance herein was compared with earlier HAV sensors reported in the literature and detailed in

Table 3. Comparison of ELIME sensor’s performance with earlier HAV sensors, reported in the literature.

Transductor	LOD	Sample	Reference
MNPs–pDA ELIME	10−11 IU/mL	Tap water	[21]
MIP-SPE	4.6 × 10−4 ng/mL	Human plasma	[38]
DNA-SPE	6.94 fg/µL	PBS	[39]
ELIME	0.5 g.c./mL	Drinking and vegetable rinsing water	This work

. Indeed, examples of this technology are already present in the literature; for example, Micheli and her coworkers implemented an ELIME for detecting the hepatitis A virus (HAV), utilizing polydopamine-modified magnetic nanobeads. This ELIME system demonstrated a detection limit of 1·10⁻¹¹ IU/mL and a working range of 10⁻¹⁰ to 5·10⁻⁷ IU mL⁻¹. In addition, a promising low cross-reactivity with Coxsackie B4 was found with an assay average relative standard deviation (RSD) of less than 5% for same-day tests and 7% for different-day tests. Another interesting biosensor for HAV detection is the one proposed by Antipchik et al. (2022). Their sensor employs a synthetic recognition element created through molecular imprinting technology, forming a molecularly imprinted polymer (MIP) that recognizes the HAV envelope protein E2 (E2-MIP). This MIP sensor, when tested in human plasma, showed a promising detection limit of 4.6 × 10⁻⁴ ng/mL for HCV-MPs. However, analytical performance characterization was limited to the sensitivity in real samples, and important parameters such as cross-reactivity, storage stability, and reusability were not investigated. We compared the performances of our device also with the DNA-based biosensors proposed by Manzano and his coworkers (2018). This device demonstrated a LOD of 0.65 pM with comparable outcomes to nRT-PCR (LOD limit of 6.4 fg/µL). The analytical performance of the proposed ELIME method is of paramount importance, particularly considering its straightforward assembly and an anticipated cost of approximately five times lower than that of the alternatives. Additionally, it is important to mention that the characteristics of the ELIME assay described in this study tackle some critical issues related to the microbiological control of water sources used for food production purposes as it allows monitoring on production lines, reduces costs and, therefore, supports a more extensive application of controls. Therefore, this ELIME should be considered a crucial advancement tool for HAV detection in complex real samples.

4. Conclusions

Currently, spectrophotometric ELISA and qPCR are the primary methods for diagnosing HAV infections in humans and monitoring the virus in food, respectively. Notably, serological testing for HAV, commonly used to investigate abnormal liver function, may face challenges in accurately diagnosing acute infection, particularly when IgG testing is inconclusive or low-level anti-HAV IgM results are observed.

In this study, we present a robust analytical method as an alternative to real-time RT-qPCR for detecting HAV in water samples. The developed ELIME assay effectively identified HAV in both drinking water and vegetable rinsing water, demonstrating high comparability to conventional reference methods. These findings highlight its potential application in agricultural contexts, such as the monitoring of irrigation water, and in food processing environments for real-time checks in washing systems, where viruses detached from treated vegetables may remain suspended and pose a risk of cross-contamination to subsequent batches. Furthermore, due to its straightforward design, this method can be adapted for the detection of a wide range of environmentally significant pathogens by simply substituting the analyte-specific bioreceptor.