# Reversible chemical modifications of graphene oxide for enhanced viral capture and release in water

Pilar Ferré-Pujol, Seiji Obata, Jésus Raya, Alberto Bianco, Hiroyuki

Katayama, Takashi Kato, Yuta Nishina

► To cite this version:

Pilar Ferré-Pujol, Seiji Obata, Jésus Raya, Alberto Bianco, Hiroyuki Katayama, et al. Reversible chemical modifications of graphene oxide for enhanced viral capture and release in water. Carbon, 2025, 234, pp.120015. 10.1016/j.carbon.2025.120015. hal-05337665

HAL Id: hal-05337665

https://hal.science/hal-05337665v1

Submitted on 29 Oct 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Reversible chemical modifications of graphene oxide for enhanced viral capture and release in water

Pilar Ferré-Pujol<sup>a,1 ID</sup>, Seiji Obata<sup>a</sup>, Jésus Raya<sup>b</sup>, Alberto Bianco<sup>a,c ID</sup>, Hiroyuki Katayama<sup>d,e ID</sup>, Takashi Kato<sup>e,f</sup>, Yuta Nishina<sup>a,* ID</sup>

## ARTICLE INFO

### Keywords:

- Carbon nanomaterials

- Functionalization

- Adsorption

- Desorption

- Pathogens

## ABSTRACT

Detecting low concentrations of viruses in sewage water is crucial for monitoring the spread of emerging viral diseases. However, current detection methods, which involve concentrating viruses using traditional materials such as gauze or cotton, have limitations in effectively accomplishing this task. This study demonstrates that graphene oxide (GO), a two-dimensional carbon material, possesses strong viral adsorption capabilities. However, it lacks efficiency for effective viral release. Therefore, we designed a series of new GO-based materials, which exhibited a viral adsorption similar to pristine GO, while significantly enhancing their release performance by attaching alkyl chains and hydrophilic functional groups. Among the synthesized materials, 1,8-aminooctanol grafted to GO (GO-NH₂C₈OH) has emerged as the most promising candidate, achieving a viral release rate higher than 50 %. This superior performance can be attributed to the synergistic effect of the alkyl chain and the terminal OH group, which enhances both its affinity for viruses and water dispersibility. Furthermore, we have successfully applied GO-NH₂C₈OH in a new protocol for concentrating viruses from sewage wastewater. This approach has demonstrated a 200-fold increase in virus concentration, allowing PCR detection of this type of pathogens present in wastewater below the detection limit by direct analysis, underscoring its significant potential for virus surveillance.

## 1. Introduction

Throughout history, viral infections have been a persistent and serious health issue for humanity. The emergence of new viruses emphasizes the critical need for effective monitoring and fast response strategies to avoid uncontrolled virus spread. A defining feature of numerous viral infections is that while some patients may experience severe symptoms, many others may have mild or no symptoms [1]. Consequently, identifying all individuals who have been infected poses real challenges, underscoring the need for novel systems to detect viral spread before an epidemic takes hold. Although the human PCR test is “the practical” tool to confirm viral infections [2], conducting mass testing using this method remains virtually unfeasible. Furthermore,

many asymptomatic patients are reluctant to undergo PCR testing, inadvertently contributing to viral transmission [3]. Another limitation of PCR testing for virulent viruses is the requirement for laboratories with biosafety measures at a minimum Level 2 [4,5], making those working in close contact with the virus to use proper personal protective equipment to prevent infection [6,7]. Consequently, attention has turned to wastewater-based epidemiological surveillance as an indicator of community infection status, leveraging the extensive sewerage network present in populated areas. In many cases, viruses can infect and replicate in the cells of the small intestine [8], henceforth, the feces of all infected individuals, whether symptomatic or not, will contain the virus [9,10]. Therefore, analyzing viral contents present in wastewater can help to identify emerging clusters of affected population and

<footnote><sup>a</sup> Research Institute for Interdisciplinary Science, Okayama University, Tsushimanaka, Kita-ku, Okayama, 700-8530, Japan</footnote>

<footnote><sup>b</sup> Institut de Chimie, UMR 7177 CNRS, Université de Strasbourg, Strasbourg, 67000, France</footnote>

<footnote><sup>c</sup> CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR3572, University of Strasbourg, ISIS, Strasbourg, 67000, France</footnote>

<footnote><sup>d</sup> Department of Urban Engineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8656, Japan</footnote>

<footnote><sup>e</sup> Research Center for Water Environment Technology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8656, Japan</footnote>

<footnote><sup>f</sup> Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8656, Japan</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: nisina-y@cc.okayama-u.ac.jp (Y. Nishina).</footnote>

<footnote n="1">¹ Current address: Field Science Center, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchū-shi, Tokyo 183-8509, Japan.</footnote>
determine those regions where the infection has not yet occurred [11, 12].

Currently, passive sampling stands as the standard method for wastewater-based epidemiological surveillance. This technique involves submerging cotton buds or gauze pads in sewage wastewater for at least 24 h to capture viruses [13]. Despite its effectiveness in virus capture, the release of viruses from the cotton/gauze fibers is minimal (~7 %) [14], rendering it unsuitable for detecting highly diluted viruses. Consequently, the current wastewater-based epidemiological surveillance protocols fall short in epidemic prediction.

Techniques such as ultracentrifugation [15] and centrifugal filtration [16] have been developed to concentrate viruses present at low levels in water samples. However, these methods are resource-intensive, time-consuming, and applicable only to small water volumes (up to 200 mL). Moreover, their concentration capabilities are restricted to a laboratory setting, with a range of up to 30-fold [16], and for wastewater viral concentration, the efficiency reaches only 30–50 % [9,17]. Thus, novel materials are needed for efficiently sensing diluted viruses in wastewater.

Nanocarbons have gained attention as potential virus adsorbents due to their high specific surface area and controllable surface properties. For instance, single-walled and multi-walled carbon nanotubes have exhibited impressive virus adsorption rates of 99.9 % or higher [18,19]. However, these materials are unsuitable for wastewater-based epidemiological surveillance, as they necessitate specialized devices with vertically oriented nanotubes and only apply to small clinical samples (~50 µL) [20]. Additionally, previous studies have not addressed the desorption process to concentrate the viruses [18–20]. Therefore, our study focused on identifying a material that could be mass-produced and readily applied to large wastewater volumes. Our investigation was centered on two-dimensional materials that uniformly present a high surface area, regardless of the material size. Graphene oxide (GO) is a material derived from oxidation and exfoliation of graphite [21], and its synthesis on a >100 g scale is achievable even at the laboratory level [22]. Furthermore, chemical modification can tailor and enhance its properties [22]. Although GO is promising in bacteria and virus adsorption [23–25], its exact mechanism of action remains unclear. In general, the adsorption capacity of viral particles depends on surface charge, size, stability, and conformation [26,27]. Therefore, the capture and release performance of viruses can be fine-tuned by optimizing the type, quantity, and size of functional groups on the surface of GO. In this study, we aimed to achieve superior performance to conventional virus-concentrating materials for diluted virus detection toward precise wastewater surveillance by utilizing modified GO materials (Fig. 1).

## 2. Experimental section

### 2.1. Chemicals and equipment

Graphite was obtained from Bay Carbon Inc. (Michigan, USA). 1-Butylamine, 1-octylamine, 1-octanol, 1,8-octanediol, 1,8-diaminoocytane, and 8-amino-1-octanol were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Hydrogen peroxide ($H_2O_2$) was acquired from Kanto Chemical Co. Inc. (Tokyo, Japan); potassium permanganate ($KMnO_4$), sulfuric acid ($H_2SO_4$, 96 %), and hipolypepton were sourced from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Yeast extract and magnesium sulfate heptahydrate ($MgSO_4 \times 7H_2O$) were acquired from Nacalai-Tesque (Kyoto, Japan). Agar was obtained from Funakoshi (Tokyo, Japan), and Gibco™ beef extract powder was procured from Thermo Fisher Scientific (Michigan, USA). Solmix (a mixture of ethanol: 85.5 %, n-propyl alcohol: 9.6 %, methanol: 4.9 %, water: less than 0.2 %) was purchased from Japan Alcohol Trading Co., Ltd. (Tokyo, Japan). All chemicals were used as received. Unless otherwise indicated, reverse osmosis water (RO water) was utilized for all the experiments.

The microorganisms employed in this study, *Escherichia coli* (NBRC: 106373) and Qβ phage (NBRC: 20012), were obtained from the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC, Tokyo, Japan). For the activation, propagation, and culture of microorganisms, medium 702 consisting of 1 % w/v hipolypepton, 0.2 % w/v yeast extract, and 0.01 % w/v $MgSO_4 \times 7H_2O$ at a pH of 7.0 was used. For phage quantification via plaque assay, medium 702 was supplemented with 1 % w/v agar. All media and buffers used in this study were autoclaved for 20 min at 121 °C before use and were stored at room temperature (RT) until usage, except for medium 702 supplemented with agar, which was maintained at 60 °C.

The GO conjugates were characterized using various techniques: Fourier Transform Infrared (FT-IR) spectroscopy (Shimadzu, IR Tracer 100), UV-Vis spectrophotometry (JASCO, V670), X-ray Diffraction (XRD) analysis (Malvern Panalytical, Aeris), Thermogravimetric Analysis (TGA) (Shimadzu, DTG-60AH), X-ray Photoelectron Spectroscopy (XPS) (JEOL, JPS-9030), Nuclear Magnetic Resonance (NMR) (Bruker™, AVANCE III 750 MHz), and zeta potential analysis (Otsuka Electronics, ELSZ-2000 N).

### 2.2. Preparation of GO

GO was prepared using a modified version of Hummers' method [21, 22]. In summary, graphite (3.0 g) was stirred in 95 % $H_2SO_4$ (75 mL). While maintaining the solution temperature below 10 °C with an ice bath, $KMnO_4$ (9.0 g) was gradually added. The solution was continuously stirred at 35 °C for 2 h. The resulting mixture was diluted with water (75 mL) under vigorous stirring and kept cool to prevent the

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Protocols for wastewater-based epidemiological surveillance using GO-derived materials.</figcaption></figure>
temperature from exceeding 50 °C. A 30 % aqueous solution of H₂O₂ (7.5 mL) was then added to the mixture. Finally, the resulting graphite oxide suspension was purified through repeated centrifugation before being exfoliated in water to obtain a 1 % w/v stock solution of GO (10 mg/mL), which was stored at 4 °C until use.

## 2.3. Functionalization of GO

To chemically modify the GO surface, various alkyl chain-containing compounds were utilized: 1-butylamine, 1-octylamine, 1-octanol, 1,8-octanediol, 1,8-diaminooctane, and 8-amino-1-octanol. Controls included water and solmix.

The protocol for GO functionalization at a 0.5:1 (w/w) compound: GO ratio was as follows. First, 10 mg of each compound was dissolved in 8 mL of solmix, and 2 mL of GO (10 mg/mL water dispersion) was added. The mixture was vortexed vigorously to ensure homogeneity and left at RT for 1 h. Subsequently, the mixture was centrifuged at 9800 rpm for 40 min, and the supernatant was removed. This step was repeated 3 times with solmix and 4 times with water to purify the functionalized GOs. Finally, the concentrations of functionalized GOs were adjusted to 0.1 % (w/v), and the materials were stored at 4 °C until they were used for the virus adsorption/desorption experiments.

## 2.4. Structural analyses of functionalized GO

The incorporation of functional molecules onto GO was verified using Fourier Transform Infrared (FT-IR) spectroscopy, covering a spectral range of 4000-700 cm⁻¹. The FT-IR spectra of both pristine and functionalized GO were obtained by placing freeze-dried samples on the ATR prism. Datasets were collected from 45 replicates to reduce background noise.

TGA analysis was performed to further characterize the functionalized GO materials. Samples of at least 1 mg were placed in alumina pans and heated from RT to 600 °C with a ramp of 10 °C min⁻¹ under a nitrogen atmosphere using a flow rate of 300 mL/min. When heating, the temperature was kept for 60 min at 80 °C to ensure complete removal of any water present in the samples. XRD analysis for the functionalized materials was conducted via a standard Cu-Kα source having λ = 1.5406 nm, with 40 kV and 15 mA. XPS analysis of the materials was performed with a basic chamber pressure of <1 × 10⁻⁶ Pa with an anode using MgKα radiation (hv = 1253.6 eV) with 12 kV and 25 mA.

The water dispersibility of all successfully functionalized GO materials was evaluated through UV-Vis absorbance measurements of supernatants after 30 min of settling. As all materials contained GO, with its UV-Vis spectra showing a peak at 230 nm [28], the absorbance at this wavelength was assessed by point read method.

## 2.5. NMR characterization

Solid-state NMR experiments were performed at RT on an AVANCE 750 MHz wide-bore spectrometer (Bruker™) operating at a frequency of 188.5 MHz for ¹³C. All the samples were spun at 30 kHz in a double resonance MAS (Magic Angle Spinning) probe designed for 2.5 mm o.d. zirconia rotors closed with vespal caps. A detailed description of the MAS NMR experiments is reported in Supporting Information.

## 2.6. Virus adsorption and release experiments

For the virus adsorption and release experiments, stock solutions containing approximately 10⁵ PFU/mL of Qβ phage were prepared by diluting freshly produced stocks of approximately 10⁹ PFU/mL Qβ phage in 100 mM phosphate buffer (PB, pH 7.2). These solutions were stored at 4 °C until use. Initially, 9 mL of the phage stock solution (approximately 10⁵ PFU/mL) were mixed with 1 mL of GO (0, 0.1, 0.05, 0.01, 0.005, or 0.001 % w/v) and co-incubated at RT for 30 min in a 15 mL centrifuge tube. Subsequently, the samples were centrifuged at

10,000 rpm for 30 min, and 1 mL of the supernatant was subjected to serial dilutions (10⁰-10⁵) in PB. This was followed by plaque assay in culture dishes, which were then incubated at 37 °C for 24 h. The phage adsorption ratio was determined using the log10 reduction method by quantifying clear areas (plaques) [29].

For the analysis of phage release, the pellet that remained after adsorption and centrifugation was resuspended in 10 mL of either 10 % or 3 % beef extract solution. These beef extract solutions are commonly used to facilitate virus release without deactivating the virus [30,31]. After thorough homogenization by vortexing, the sample was incubated for 30 min at RT and then subjected to centrifugation (10,000 rpm, 30 min). Finally, the ratio of released phage in the supernatant was calculated using plaque assay, employing the following equations:

$$V_A = \frac{V_i}{100} \times V_c \qquad \text{eq. 1}$$

$$V_r = \frac{V_s}{V_A} \times 100 \qquad \text{eq. 2}$$

where V_A is the amount of virus adsorbed by GO, V_i indicates the initial amount of virus used in the experiment, V_c indicates the percentage of virus captured after adsorption, V_r represents the percentage of virus released after the desorption protocol, and V_s refers to the virus detected in the supernatant after the release procedure.

Among the functionalized GO samples synthesized, only those materials that could be successfully employed following our established protocol for phage adsorption and release, specifically those that demonstrated successful precipitation through centrifugation (GO/sol-mix, GO-NH₂C₄, GO-NH₂C₈, and GO-NH₂C₈OH), were subjected to the testing procedure as elucidated earlier. In these experiments, the concentration of functionalized GO was standardized at 0.01 % w/v, as this concentration represented the minimal quantity necessary to achieve a 99.9 % phage adsorption rate on pristine GO.

## 2.7. Structural analysis of GO and GO-NH₂C₈OH after virus adsorption and release by beef extract

In order to gain deeper insights into the mechanism by which the Qβ phage adsorbs onto our materials (GO or GO-NH₂C₈OH), a concentrated and purified quantity of Qβ phage (approximately 10¹⁰ PFU/mL) was introduced using Vivaspin® in PB buffer, followed by a 30-min incubation at RT. After centrifugation, the residual supernatant was discarded, and the pellet was resuspended in 10 mL of a 70 % (v/v) aqueous ethyl alcohol solution for a minimum of 30 min, with the objective of inactivating any remaining phages. Subsequently, the samples underwent repeated washing with water to eliminate any non-adhered phages. This material was then subjected to freeze-drying to facilitate subsequent structural analysis using FT-IR. Additionally, a parallel analysis was conducted on the pellet subsequent to the introduction of beef extract solution for virus release. Briefly, after the virus release protocol, the supernatant containing beef extract was decanted, and the remaining pellet was resuspended in a 70 % (v/v) aqueous ethyl alcohol solution for 30 min to ensure phage inactivation. After serial washes with water, the sample underwent freeze-drying for FT-IR analysis (see Supporting Information).

## 2.8. Detection of viruses from sewage wastewater

Sewage wastewater was obtained from Koto sewage treatment center in Okayama (Japan) and stored in 2 L plastic containers for transport. The samples were maintained at 4 °C until they were utilized for the assessment of SARS-CoV-2 presence. Initial removal of suspended particulate matter was achieved through filtration using qualitative filter paper of grade number 2 (Advantec Toyo Kaisha Ltd., Tokyo, Japan). Subsequently, 1 mL of the filtered sewage wastewater underwent testing for the presence of the SARS-CoV-2 nucleocapsid gene regions N1 and
N2 [32] using the Ampdirect™ 2019-nCoV Detection Kit and the medical-grade automatic PCR system, AutoAmp™ (Shimadzu Corp., Kyoto, Japan), in order to confirm negative results. Subsequently, 200 mL of the filtered wastewater were combined with 2 mL of 0.1 % w/v **GO-NH₂C₈OH** and mixed at RT for 30 min on a horizontal shaker. The resulting suspension was partitioned into four 50 mL centrifuge tubes and centrifuged at 10,000 rpm for 30 min to precipitate the virus captured by **GO-NH₂C₈OH**. The pellet obtained was transferred to a 15 mL centrifuge tube, followed by the addition of 1 mL of 10 % w/v beef extract. The mixture was vigorously vortexed to attain a homogeneous dark suspension, which was then left at RT for 30 min while placed on a horizontal shaker. Following a final centrifugation step (10,000 rpm, 30 min), 1 mL of the supernatant was subjected to evaluation using the AutoAmp™ PCR system to detect SARS-CoV-2.

## 2.9. Statistical analysis

For the virus adsorption and release experiments, data derived from a minimum of 4 replicated experimental trials were subjected to one-way or two-way ANOVA utilizing IBM SPSS® Statistics Software, Version 28.0 for Windows. All data are presented as mean ± SD or percentage ± SEM. Statistical significance was considered at $P < 0.05$, and in the event of a significant effect, values were compared using Tukey's multiple comparisons test.

# 3. Results and discussion

## 3.1. Virus adsorption and release performance of pristine GO

Although previous studies have reported that virus adsorption using GO is possible [24,25], a comprehensive investigation of the detailed structure, morphology, and optimal dosage of GO employed has not been achieved. In our present study, we utilized fully exfoliated single-layer GO, characterized by a specific surface area exceeding 1000 m²/g in aqueous media [33]. The results of the assessment of pristine GO virus capture capability are presented in Table 1. Notably, a concentration of 0.01 % w/v (equivalent to 0.1 mg/mL) of GO resulted in the removal of Qβ phage (an RNA bacteriophage virus) from the solution to the order of approximately 3 log10 (99.9 % reduction), determined by plaque assay. Remarkably, even at a lower GO concentration of 0.001 % w/v, substantial phage reduction occurred, yielding a decrease to the order of approximately 2 log10 (99 % reduction) from the solution.

We concurrently conducted a PCR analysis on the supernatant post-centrifugation to ascertain whether the interaction between GO and Qβ phage resulted in effective capture and subsequent removal of the virions from the solution. As a result, we confirmed a substantial reduction in the presence of this viral RNA from the supernatant, thereby establishing the efficacy of GO in removing the phage from the solution via physical and chemical interactions [24,34]. This finding corroborates the role of GO in actively capturing the phage rather than just

<table><caption>**Table 1**
Capacity of different concentrations of pristine GO to reduce the amount of Qβ phage in water.</caption><thead><tr><th>Concentration of GO (w/v)</th><th>Phage concentration [(PFU/mL) ± SD]</th><th>Adsorption (% ± SEM)</th></tr></thead><tbody><tr><td>(Initial phage)</td><td>3.99 × 10<sup>5</sup> ± 2.12 × 10<sup>5</sup></td><td>–</td></tr><tr><td>0.1 %</td><td>2.09 × 10<sup>2</sup> ± 1.13 × 10<sup>2</sup></td><td>99.96 ± 0.004<sup>a</sup></td></tr><tr><td>0.05 %</td><td>3.99 × 10<sup>2</sup> ± 4.63 × 10<sup>2</sup></td><td>99.92 ± 0.02<sup>a</sup></td></tr><tr><td>0.01 %</td><td>6.20 × 10<sup>2</sup> ± 4.12 × 10<sup>2</sup></td><td>99.88 ± 0.01<sup>a</sup></td></tr><tr><td>0.005 %</td><td>2.04 × 10<sup>3</sup> ± 1.06 × 10<sup>3</sup></td><td>99.27 ± 0.20<sup>b</sup></td></tr><tr><td>0.001 %</td><td>2.24 × 10<sup>3</sup> ± 1.30 × 10<sup>3</sup></td><td>99.28 ± 0.14<sup>b</sup></td></tr></tbody></table>

inactivating its infectivity.

Subsequently, we explored the possibility of releasing the adsorbed phages from the GO surface. For this purpose, we investigated a widely recognized organic flocculation method employing a beef extract solution (3 % w/v or 10 % w/v) to facilitate the release of adsorbed phages [30,31]. The organic flocculation method is a water treatment technique that employs organic compounds, typically polymers or nanomaterials, to aggregate and remove suspended particles and impurities from water. However, the release efficiencies achieved with GO were low (Table S1). These outcomes motivated us to explore the effect of surface modification of GO through chemical functionalization on the interaction with the captured virus. We aimed to enhance phage release while upholding the high adsorption performance of GO.

## 3.2. GO functionalization for the enhancement of viral release

It is known that the hydrophobicity of a surface plays a pivotal role in influencing virus affinity; surfaces with mild hydrophobicity promote virus adsorption [35], whereas elevated hydrophobicity inhibits the interaction with the virus [36]. Based on this premise, we systematically examined the impact of the alkyl chain length of alkyl amines, namely 1-butylamine (C₄NH₂) and 1-octylamine (C₈NH₂). GO can be functionalized with amines via the epoxide ring opening reaction or ionic interaction by forming a charge stabilized complex between the carboxyl/hydroxyl groups on GO and amine (Scheme S1 in Supplementary Information) [37,38]. In this research, we focused on the reversible interaction of amines with GO via ionic interactions to facilitate viral release (Fig. 2A). Following the functionalization, 1-octylamine-functionalized GO (GO-NH₂C₈) displayed a subtle hydrophobicity, which was not observed in 1-butylamine functionalization (GO-NH₂C₄) (Fig. S1), prompting the selection of the C₈ alkyl chain to investigate the collective influence of functional groups at its termini (e.g., NH₂ or OH). Next, hydrophilic terminal groups such as OH and NH₂ were explored by utilizing 1,8-diaminooctane (NH₂C₈NH₂) and 8-amino-1-octanol (NH₂C₈OH), in an endeavor to assess the combined impact of hydrophobic alkyl chains and hydrophilic functional moieties. For comparison, industry-denatured alcohol (solmix), 1-octanol (C₈OH), and 1, 8-octanediol (HOC₈OH) were also investigated to ascertain the role of the amino group for GO functionalization.

Structural characterization of the functionalized GO samples was conducted to verify the successful grafting of alkyl chains onto the GO sheets. FT-IR spectrum of pristine GO exhibited the characteristic peaks at 3000–3700, 1732, 1633, and 1162 cm⁻¹, corresponding to O–H stretching, and C=O (carbonyl/carboxyl), C=C, and C=O bending modes, respectively (Fig. 2B) [39]. FT-IR analysis of functionalized GO samples revealed the presence of alkyl chains around 2900 cm⁻¹ [40], accompanied by a decreased C=O stretching at 1732 cm⁻¹ due to the deprotonation by the amines leading to a shift of the peak below 1700 cm⁻¹ supporting the formation of a charged complex (Scheme S1) [41]. The 1225 cm⁻¹ peak associated with the epoxy groups in GO did not significantly change after the reaction with C₄NH₂, C₈NH₂, NH₂C₈NH₂, and NH₂C₈OH, indicating that the epoxide ring opening is very low due to the mild reaction conditions compared with our previous research [37,42,43]. Conversely, the reaction of GO with solmix, C₈OH, and HOC₈OH (forming GO/solmix, GO/C₈OH, and GO/HOC₈OH, respectively) did not show a similar functionalization, as the nucleophilicity of OH towards epoxide ring-opening reactions is insufficient, and the deprotonation of the acidic functional groups on GO also cannot occur.

The successful and uniform integration of amines onto GO was further confirmed through X-ray diffraction (XRD) analysis. The interlayer distance of GO sheets remained unchanged when the short alkyl chain amine, C₄NH₂, was introduced (0.86 nm, Fig. 2Ci and ii). In contrast, GO-NH₂C₈, GO-NH₂C₈NH₂, and GO-NH₂C₈OH showed larger sheet distances, corresponding to 0.95, 0.94, and 0.96 nm, respectively (Fig. 2Ciii-v). Thermogravimetric analysis (TGA) is also an indicator of functionalization. For pristine GO, a significant weight loss occurred up

<footnote>a, b Different superscript letters indicate significant differences amongst the values in the same column ($P < 0.01$). Data for phage concentrations was obtained from 5 to 10 independent replicates, whereas data for viral reduction was obtained from 5 independent replicates.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Material design and structural analysis of the functionalized GO. (A) Concept of GO functionalization. (B) FT-IR analysis of (i) pristine GO, (ii) GO/solmix, (iii) GO-NH₂C₄, (iv) GO-NH₂C₈, (v) GO/C₈OH, (vi) GO/HOC₈OH, (vii) GO-NH₂C₈NH₂, and (viii) GO-NH₂C₈OH. (C) XRD analysis of (i) pristine GO, (ii) GO-NH₂C₄, (iii) GO-NH₂C₈, (iv) GO-NH₂C₈NH₂, and (v) GO-NH₂C₈OH. (D) TGA weight loss at 300 °C of (i) pristine GO, (ii) GO/solmix, (iii) GO-NH₂C₄, (iv) GO-NH₂C₈, (v) GO-NH₂C₈NH₂, and (vi) GO-NH₂C₈OH.</figcaption></figure>

to 300 °C (Fig. S2), attributed to the degradation of the labile oxygen groups on its surface [44]. GO/solmix showed a weight loss similar to GO (33 %) because the alcohols do not react with GO [45]. In contrast, the functionalized materials GO-NH₂C₄, GO-NH₂C₈, GO-NH₂C₈NH₂, and GO-NH₂C₈OH exhibited a slightly lower loss of 29.7 %, 27.3 %, 24.9 %, and 28.7 %, respectively (Fig. 2D). Even though the reaction conditions used for the functionalization were mild, some labile oxygen functionalities were removed from the surface of GO during the reaction, making the interpretation of the TGA data difficult [46]. To estimate the degree of functionalization more accurately, elemental analysis was performed. Calculating from the nitrogen content of GO and functionalized GO samples, the amounts of amines on GO for GO-NH₂C₄, GO-NH₂C₈, GO-NH₂C₈NH₂, and GO-NH₂C₈OH were 8.5 %, 15.2 %, 15.5 %, and 22.2 %, respectively (Table S2). XPS analysis further corroborated the successful functionalization of GO with amines; 8–22 wt% of amines were grafted on GO (Fig. S3, Table S2).

Finally, to assess the applicability of the functionalized GO samples, their dispersibility in water was evaluated by UV-Vis measurements.

GO, GO/solmix, GO-NH₂C₄, and GO-NH₂C₈OH demonstrated high dispersibility in water, whereas GO-NH₂C₈ and GO-NH₂C₈NH₂ displayed significantly lower dispersibility (Fig. S4A). The longer alkyl chains in GO-NH₂C₈ rendered the conjugate more hydrophobic, making it partially immiscible with water. The terminal amino groups in GO-NH₂C₈NH₂ did not help to enhance the dispersibility, likely due to the zwitterionic character of the conjugate where the terminal amino groups of the alkyl chain can interact with the negatively charged surface of GO, causing aggregation. In addition, even after 60 min of centrifugation, complete precipitation was not achieved, rendering GO-NH₂C₈NH₂ unsuitable for viral adsorption/release experiments (Fig. S4B).

### 3.3. Qβ phage adsorption and release performance of functionalized GOs

Following the characterization of the different functionalized GO conjugates, we assessed their viral adsorption and release capabilities. While no significant differences were observed in the adsorption rates for all the samples (>98 % phage reduction, Table S3), notable
differences were observed in phage release when 10 % beef extract treatment was used, depending on the functional molecules (Fig. 3). Similar to pristine GO, as described above (see section 2.1), GO/solmix also exhibited a robust interaction with Qβ phages on its surface, leading to the lowest release rates (i.e., 3.8 % and 6.5 %, respectively). The functionalization of GO with the short alkyl chain (GO-NH₂C₄) exhibited a slightly improved release rate (i.e., 15.4 %), while the presence of a longer chain (e.g., GO-NH₂C₈) resulted in an improved release performance up to 36.2 %. Remarkably, among the samples, a longer chain with the terminal hydrophilic group (GO-NH₂C₈OH) showed the best release performance, corresponding to 55.8 %.

Next, the minimum dosage of GO-NH₂C₈OH required for efficient Qβ phage adsorption and subsequent release was investigated; even at a minimal concentration of 0.001 % w/v, GO-NH₂C₈OH was able to adsorb over 97 % of the virus. When different conditions of phage release were evaluated, no significant differences were observed between the use of 3 % or 10 % w/v beef extract solutions (Table 2). This observation suggests that the concentration of beef extract may not have a significant effect on the release process, possibly indicating a robust and consistent performance of GO-NH₂C₈OH across all the concentrations studied.

### 3.4. Mechanisms of virus adsorption and release

In non-enveloped viruses, the functional groups on the protein capsid significantly influence the viral surface charge [47]. The Qβ phage has an isoelectric point of 4.3, similar to conventional viral particles that manifest a slightly negative charge when immersed in water [48]. Upon exposure to a neutral buffer, the charge of the Qβ phage tends to approach its isoelectric point (+0.39 mV) [49]. GO has a negative charge character in water (−39.84 mV) and in neutral buffer (−30.75 mV) [50]; therefore, GO can capture the Qβ phage effectively via electrostatic interactions. Similarly, the functionalized material, GO-NH₂C₈OH, showed a substantial negative charge in an aqueous environment (−36.89 mV), although a significant shift towards a less negative charge in a neutral buffer (−22.91 mV) was measured likely due to the effect of the attachment of the 8-amino-1-octanol to the surface of GO. The negative charges inherent to both GO and GO-NH₂C₈OH would facilitate the adsorption of the Qβ phage. Intriguingly, in FT-IR spectra, a new band emerged around 1230 cm⁻¹ on pristine GO after phage adsorption, indicating the attachment of viral particles (see spectrum (iv) in Fig. S5). Upon treatment of GO with beef extract, a shift of the C=O peak from 1732 cm⁻¹ to 1650-1600 cm⁻¹ region was again observed, suggesting the interaction between the carboxyl groups on GO and the positively charged functions present in the beef extract solution, such as amino acids and peptides (Fig. S5). Further corroborative evidence for the interaction between GO and beef extract compounds is seen by the zeta-potential shift to less negative values (from −39.84 mV to −15.82 mV and −6.43 mV) upon treatment with 3 % and 10 % beef extract solutions, respectively.

The FT-IR and zeta potential analyses indicate that the amino acid moieties in Qβ phage and beef extract can form strong complexes stabilized by electrostatic interactions with GO. Therefore, beef extract solution per se is unable to release the adsorbed phages when pristine GO is used. In contrast, functionalizing GO with NH₂C₈OH occludes reactive sites, tuning the strong interactions between GO and viral particles and facilitating their release by the beef extract. To confirm the virus adsorption/release mechanisms for GO-NH₂C₈OH, we further conducted FT-IR analysis after virus release treatments. The weakening of the CH stretching bands at 2900 cm⁻¹ in GO-NH₂C₈OH indicates a partial removal of NH₂C₈OH (Fig. S5), most likely due to the components of beef extract, including amino acids and peptides, being exchanged with the amine molecules on GO. Further evidence of the attachment of the components of the beef extract to the material is confirmed by the zeta-potential shift to less negative values (from −22.91 mV to −15.54 mV and −6.31 mV) when GO-NH₂C₈OH is treated with 3 % and 10 % beef extract solutions for virus retrieval.

Considering the functional groups onto the surface and the edges of GO, there are at least three interaction modes between GO and NH₂C₈OH: (i) covalent bonding through epoxy ring opening (ii) electrostatic (e.g., positive/negative charge), interaction between the acidic functional groups and the amino function of NH₂C₈OH, and (iii) dynamic covalent bonding onto GO. To understand the bonding mode of NH₂C₈OH, high-resolution X-ray photoelectron spectroscopy (XPS) analysis at the N 1s region was performed (Fig. 4). The deconvolution of the N 1s peak allowed to calculate a ratio of 1:1.5 between the free amines (at 399 eV) and the protonated amines/ammonium groups (at 401 eV), suggesting that ionic interactions of NH₂C₈OH and possible protonation of the amino groups are present. The NH₂C₈OH functionalized by ionic interaction could be replaced by components present in beef extract, facilitating the virus release.

To confirm the covalent bonding through epoxy ring-opening or a simple complexation stabilized by the acidic nature of the carboxyl and hydroxyl groups prone to easily deprotonate in the presence of amino functions, we performed solid-state NMR. The ¹³C spectrum of GO shows the presence of the typical peaks in the aromatic region around 130 ppm and the sp³ carbons corresponding to the C-OH and C-O-C (i.e., epoxide) groups at ~70 and ~60 ppm [43], respectively (Fig. S6A). Compared to pristine GO, the ¹³C spectrum of GO-NH₂C₈OH clearly evidences the presence of new methylene groups of the aliphatic chain of NH₂C₈OH at 27 ppm together with the CH₂ carbon linked to the nitrogen atom at 40 ppm (Fig. S6B). A similar cross-correlation peak at ~40 ppm, attributed to the methylene group in alpha to a nitrogen atom, was evidenced in a previous work, where GO was functionalized with a triethylene glycol chain via the epoxide ring opening reaction [43]. Comparing the intensities of the epoxide peaks around 60 ppm before and after the addition of NH₂C₈OH, we can see no difference confirming that the opening reaction is occurring to a non-detectable extent, as it likely requires higher amounts of the nucleophile and longer reaction times, thus supporting mainly the formation of a charge stabilized complex. To further evidence the presence of the functional groups, we did an advanced solid-state NMR 2D ¹H-¹³C correlation experiment (e.g., Frequency-Switched Lee-Goldburg HETeronuclear CORrelation, FSLG-HETCOR) at high magnetic field and spinning rate. The cross-polarized ¹H-¹³C spectra allowed to identify the short (Fig. 5A) and long (Fig. 5B) range spin contacts between the GO surface and the linked NH₂C₈OH. In the first spectrum, we can only see the aliphatic carbon in the region between 20 and 75 ppm, corresponding to 4 intense cross-peaks that can be associated with the spatial proximity and close contact between protons and carbons in CH₂, C-NH, C-OH and C-O-C groups (Fig. 5A); in contrast, the long-range correlation spectrum (Fig. 5B) allows to observe the cross peaks in the aromatic region between 120 and 135 ppm. These peaks rise from the closed spatial contact between the protons of the hydroxyl and methylene groups and the C=C aromatic bonds onto GO. The methylene protons at 0.7-1.0 ppm

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Rates of Qβ phage release by GO samples after resuspension in 10 % (w/v) beef extract solution. a-d Different letters indicate significant differences amongst the values (P < 0.01). Data was obtained from at least 4 independent replicates.</figcaption></figure>
<table><caption>**Table 2**
Comparison of various concentrations of GO-NH₂C₈OH on Qβ phage adsorption and its release efficiency.</caption><thead><tr><th>GO-NH₂C₈OH concentration (w/v)</th><th>Beef extract concentration</th><th>Initial phage concentration [(PFU/mL) ± SD]</th><th>Adsorption (% ± SEM)</th><th>Released virus (% ± SEM)</th></tr></thead><tbody><tr><td>0 (Initial virus)</td><td></td><td>1.35 × 10<sup>5</sup> ± 0.48 × 10<sup>5</sup></td><td>–</td><td>–</td></tr><tr><td>0.01 %</td><td>3 %</td><td></td><td>99.60 ± 0.10<sup>a</sup></td><td>46.41 ± 5.50</td></tr><tr><td>0.005 %</td><td></td><td></td><td>98.98 ± 0.64<sup>a</sup></td><td>43.56 ± 6.98</td></tr><tr><td>0.001 %</td><td></td><td></td><td>97.51 ± 0.58<sup>b</sup></td><td>44.15 ± 9.44</td></tr><tr><td>0.01 %</td><td>10 %</td><td></td><td>99.67 ± 0.10<sup>a</sup></td><td>59.02 ± 11.72</td></tr><tr><td>0.005 %</td><td></td><td></td><td>99.23 ± 0.14<sup>a</sup></td><td>49.71 ± 7.62</td></tr><tr><td>0.001 %</td><td></td><td></td><td>97.40 ± 1.20<sup>b</sup></td><td>32.36 ± 2.48</td></tr></tbody></table>

a,b Different superscript letters indicate significant differences amongst the values in the same column (P < 0.01). Data was obtained from at least 4 independent replicates.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** | High-resolution XPS N 1s spectrum of GO-NH₂C₈OH. The deconvolution evidences the presence of free amines (399 eV) and protonated amines/ammonium groups (401 eV).</figcaption></figure>

correlate with C=C carbons at 123.2 ppm, indicating the aliphatic chain is likely in contact with the surface of GO. The protons at 7.0–7.4 ppm, likely corresponding to the hydroxyl and the amino (free or protonated) groups of NH₂C₈OH, cross-correlate with carbons at 127.7 and 130.7 ppm of the sp² domains (Fig. 5B). Although the 2D NMR can be considered a reliable technique to prove the formation of covalent bonds in chemically functionalized materials, in this case, NH₂C₈OH is primarily non-covalently bound to the surface of GO via the formation of ionic interactions with the carboxylic functions and the acidic hydroxyl groups, as also shown by the high-resolution N 1s XPS analysis (Fig. 4). The two cross-peaks at 37.5 and 39.9 ppm likely correspond to the CH₂ attached to NH group generated by the limited epoxide ring opening, and the ammonium group following the deprotonation of the carboxyl and acidic hydroxyl groups at the surface of GO, respectively (Fig. 5B).

Based on these results, we would like to propose the mechanisms that underlie the differences in virus complexation and release between pristine GO and functionalized GO. In the case of GO, the carboxyl and hydroxyl groups can form ionic interactions with the virus. However, the treatment with beef extract is not capable of competing with the strong forces between the negative charges on the surface of GO and the positive charges of the capsid proteins, disabling the release of the virus (Fig. 6A). We also cannot exclude a certain contribution of the hydrophobic domain of GO on the complexation with the virus. On the contrary, the surface of functionalized GO is masked by the functional groups introduced during the opening of the epoxides and the deprotonation and amine neutralization of the GO acidic groups, limiting close contact of the viral particles with the charged surface of GO. Predominantly, weak hydrophobic and ionic interactions occur between the functionalized GO and the virus. Consequently, beef extract

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** | NMR analysis of GO-NH₂C₈OH. ¹H–¹³C short (A) (134 µs contact-time) and long (B) range (4 ms equivalent contact time) correlation spectra.</figcaption></figure>

treatment facilitates an efficient virus release (Fig. 6B).

### 3.5. Detection of SARS-CoV-2 in sewage wastewater using GO-NH₂C₈OH

Sewage wastewater samples taken from Okayama city in Japan were analyzed using an automated PCR system, targeting the detection of low concentration of viruses. Initial processing involved filtration of the collected wastewater using grade number 2 filter paper for preventing particulate matter contamination [51]. Since our experiments had demonstrated that GO was unable to efficiently release viruses (see section 3.3 and 3.4), we performed a preliminary PCR analysis of the filtered wastewater for the presence of the virus (SARS-CoV-2), to
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Schematic illustration of the interactions between GO materials and a viral particle. (A) GO and (B) GO-NH₂C₈OH. Thick yellow arrows indicate strong interactions, while thin arrows indicate weak interactions. For simplicity, only ionic interactions between GO and NH₂C₈OH are shown. A more detailed chemical structure of GO-NH₂C₈OH is shown in Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

confirm a negative result in the initial wastewater, which indicated a viral content below the detection limit of this technique. After that, GO-NH₂C₈OH (0.001 % w/v) was introduced into the filtered wastewater (200 mL). Following a 30-min incubation, centrifugation was conducted, and the supernatant was removed. The resultant pellet was resuspended in 1 mL of 10% beef extract solution to facilitate the virus release, and it was subsequently subjected to PCR analysis. The outcomes revealed the positive presence of the N1 gene region of SARS-CoV-2 in 9 out of 12 samples (75%), while the N2 region was undetectable in all samples, suggesting virus denaturation (Table S3). It is important to note that viruses in wastewater are often non-viable due to environmental stress, and not all genetic regions may remain intact. This explains why some gene regions, such as the N2 region in this study, were not detected despite the successful concentration and release of the virus. Our method enables a 200-fold concentration enrichment (from 200 mL to 1 mL). The primary goal of our study is to demonstrate the feasibility of using GO-NH₂C₈OH in conjunction with a fully automated medical-grade PCR system for wastewater-based epidemiological surveillance. This approach enables simple, rapid, and reliable detection without complex laboratory procedures. In this study, we highlight the utility of our method for enabling large-scale, real-world applications of wastewater surveillance, while providing a simple way to assess epidemic progression in a Biosafety Level 1 facility, unlike direct human PCR testing for active viruses.

## 4. Conclusion

In this study, we investigated the reversible surface functionalization of GO with amine-containing molecules. This modification demonstrated highly efficient capture and release properties towards viral particles in aqueous solutions. All the materials exhibited exceptional virus capture efficiencies of over 99%. Notably, the GO conjugate modified with 8-amino-1-octanol displayed the most promising release performance, enabling a viral recovery of over 50%. 8-Amino-1-octanol reacts with GO by ionic and covalent bond formation. In this research, we clarified the significance of reversible ionic interactions for capturing and releasing viruses. Leveraging this functional GO material, we have devised a simple method to concentrate viruses in sewage wastewater,

allowing detection of low levels of viruses in a Biosafety Level 1 facility. This innovative approach holds great promise for future applications in wastewater-based epidemiological surveillance and represents a significant step toward enhancing public health care. Looking ahead, developing materials with selective adsorption properties could involve functionalizing the surface of GO with recognition elements, such as antibodies or aptamers. This approach holds significant potential for capturing specific viruses. Additionally, advancements in mass production [52] and the exploration of diverse applications [53] are expected to further unlock the potential of 2D carbon materials.

## CRediT authorship contribution statement

**Pilar Ferré-Pujol:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Seiji Obata:** Writing – review & editing, Methodology, Data curation. **Jésus Raya:** Methodology, Formal analysis. **Alberto Bianco:** Writing – review & editing, Funding acquisition, Formal analysis, Data curation. **Hiroyuki Katayama:** Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. **Takashi Kato:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **Yuta Nishina:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

## Data availability statement

All data needed to draw the conclusions of the paper are present in the paper and/or the Supplementary Materials. Additional datasets that support the findings of this study are available from the corresponding authors upon reasonable request.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgments

This research was supported by CREST (JPMJCR24S6, JPMJCR20H3), and KAKENHI (JP19H05715 and JP22H04548), and the Centre National de la Recherche Scientifique (CNRS) through the International Research Project MULTIDIM between I2CT Unit and Okayama University.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbon.2025.120015.

## References

[1] C.R. Bailie, et al., Symptoms and laboratory manifestations of mild COVID-19 in a repatriated cruise ship cohort, Epidemiol. Infect. 149 (2021) e44, https://doi.org/10.1017/S0950268821000315.

[2] C.J. Burrell, C.R. Howard, F.A. Murphy, Laboratory diagnosis of virus diseases, in: Fenner and White's Medical Virology, 2017, pp. 135–154, https://doi.org/10.1016/B978-0-12-375156-0.00010-2.

[3] F.J. Pérez-Reche, K.J. Forbes, N.J.C. Strachan, Importance of untested infectious individuals for interventions to suppress COVID-19, Sci. Rep. 11 (2021) 20728, https://doi.org/10.1038/s41598-021-00056-5.

[4] D.T. Mourya, G. Sapkal, P.D. Yadav, S.K.M. Belani, A. Shete, N. Gupta, Laboratory biosafety measures of SARS-CoV-2 at containment level 2 with particular reference to its more infective variants, Indian J. Med. Res. 151 (2020) 172–176, https://doi.org/10.4101/ijmr.LJM7_763_20.

[5] S.A. Hussain Gardezi, A. Ikram, Application of biosafety principles in laboratory analysis of clinical samples from patients with COVID-19, J. Pakistan Med. Assoc. 70 (2020) S48-S51, https://doi.org/10.5455/JPMA.10.

[6] A. Waris, U.K. Atta, M. Ali, A. Asmat, A. Baset, COVID-19 outbreak: current scenario of Pakistan, New Microbes New Infect 35 (2020) 100681, https://doi.org/10.1016/j.jmni.2020.100681.

[7] Z. Wu, J.M. McGoogan, JAMA 323 (2020) 1239–1242, https://doi.org/10.1001/jama.2020.2648.

[8] R. Zang, et al., TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes, Sci. Immunol. 5 (2020) eabc3582, https://doi.org/10.1126/sciimmun.albc3582.

[9] S. Sharif, et al., Detection of SARS-CoV-2 in wastewater using the existing environmental surveillance network: a potential supplementary system for monitoring COVID-19 transmission, PLoS One 16 (2021) e0249568, https://doi.org/10.1371/journal.pone.0249568.

[10] N. Wiktorczyk-Kapischke, et al., SARS-CoV-2 in the environment-Non-droplet spreading routes, Sci. Total Environ. 770 (2021) 145260, https://doi.org/10.1016/j.scitotenv.2021.145260.

[11] R.S. López-Pénalver, et al., Predictive potential of SARS-CoV-2 RNA concentration in wastewater to assess the dynamics of COVID-19 clinical outcomes and infections, Sci. Total Environ. 886 (2023) 163935, https://doi.org/10.1016/j.scitotenv.2023.163935.

[12] D.A. Larsen, K.R. Wigginton, Tracking COVID-19 with wastewater, Nat. Biotechnol. 38 (2020) 1151–1153, https://doi.org/10.1038/s41587-020-0690-1.

[13] E.F. Valenzuela, H.C. Menezes, Z.L. Cardeal, Passive and grab sampling methods to assess pesticide residues in water. A review, Environ. Chem. Lett. 18 (2020) 1019–1048, https://doi.org/10.1007/s10311-020-00998-8.

[14] B. Fattal, E. Katzenelson, Evaluation of gauze pad method to recover viruses from water, Water Res. 10 (1976) 1135–1140, https://doi.org/10.1016/0043-1354(76)90047-6.

[15] C. Prata, A. Ribeiro, À. Cunha, N.C. Gomes, A. Almeida, Ultracentrifugation as a direct method to concentrate viruses in environmental waters: virus-like particle enumeration as a new approach to determine the efficiency of recovery, J. Environ. Monit. 14 (2012) 64–70, https://doi.org/10.1039/c1em10603a.

[16] N. Bonilla, J.J. Barr, Phage on Tap: a quick and efficient protocol for the preparation of bacteriophage laboratory stocks, Methods Mol. Biol. 1838 (2018) 37–46, https://doi.org/10.1007/978-1-4939-8682-8_4.

[17] J. Trottier, et al., Post-lockdown detection of SARS-CoV-2 RNA in the wastewater of Montpellier, France, One Health 10 (2020) 100157, https://doi.org/10.1016/j. onehlt.2020.100157.

[18] A.S. Brady-Estévez, S. Kang, M. Elimelech, A single-walled-carbon-nanotube filter for removal of viral and bacterial pathogens, Small 4 (2008) 481–484, https://doi.org/10.1002/smll.200700863.

[19] A.S. Brady-Estévez, M.H. Schnoor, C.D. Vecitis, N.B. Saleh, M. Elimelech, Multi-walled carbon nanotube filter: improving viral removal at low pressure, Langmuir 26 (2010) 14975–14982, https://doi.org/10.1021/la102783v.

[20] Y.T. Yeh, et al., A rapid and label-free platform for virus capture and identification from clinical samples, Proc. Natl. Acad. Sci. U.S.A. 117 (2020) 895–901, https://doi.org/10.1073/pnas.1910113117.

[21] W.S. Hummers Jr., R.E. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80 (1958) 1339, https://doi.org/10.1021/ja01539a017, 1339.

[22] N. Morimoto, T. Kubo, Y. Nishina, Tailoring the oxygen content of graphite and reduced graphene oxide for specific applications, Sci. Rep. 6 (2016) 21715, https://doi.org/10.1038/srep21715.

[23] S. Ye, et al., Antiviral activity of graphene oxide: how sharp edged structure and charge matter, ACS Appl. Mater. Interfaces 7 (2015) 21571–21579, https://doi.org/10.1021/acsmami.5b06876.

[24] S. Gungordu Er, T.A. Tabish, M. Edisinghe, R.K. Matharu, Antiviral properties of porous graphene, graphene oxide and graphene foam ultrafine fibers against Phi6 bacteriophage, Front. Med. 9 (2022) 1032899, https://doi.org/10.3389/fmed.2022.1032899.

[25] R.K. Matharu, H. Porwal, B. Chen, L. Ciric, M. Edisinghe, Viral filtration using carbon-based materials, Med. Devices Sens. 3 (2020) e10107, https://doi.org/10.1002/mds3.10107.

[26] C. Dika, M.H. Ly-Chatain, G. Francius, J.F.L. Duval, C. Gantzer, Non-DLVO adhesion of F-specific RNA bacteriophages to abiotic surfaces: importance of surface roughness, hydrophobic and electrostatic interactions, Colloids Surf. A 435 (2013) 178–187, https://doi.org/10.1016/j.colsurfa.2013.02.045.

[27] C. Dika, J.F. Duval, G. Francius, A. Perrin, C. Gantzer, Isoelectric point is an inadequate descriptor of MS2, Phi X 174 and PRD1 phages adhesion on abiotic surfaces, J. Colloid Interface Sci. 446 (2015) 327–334, https://doi.org/10.1016/j.jcis.2014.08.055.

[28] S. Guo, S. Garaj, A. Bianco, C. Ménard-Moyon, Controlling covalent chemistry on graphene oxide, Nat. Rev. Phys. 4 (2022) 247–262, https://doi.org/10.1038/s42254-022-00422-w.

[29] S. Rizvi, P.T. Mora, Bacteriophage plaque-count assay and confluent lysis on plates without bottom agar layer, Nature 200 (1963) 1324–1325, https://doi.org/10.1038/2001324a0.

[30] E.F. Landry, J.M. Vaughn, M.Z. Thomas, T.J. Vicale, Efficiency of beef extract for the recovery of poliovirus from wastewater effluents, Appl. Environ. Microbiol. 36 (1978) 544–548, https://doi.org/10.1128/aem.36.4.544–548.1978.

[31] P.A. Shields, S.R. Farrah, Concentration of viruses in beef extract by flocculation with ammonium sulfate, Appl. Environ. Microbiol. 51 (1986) 211–213, https://doi.org/10.1128/aem.51.1.211-213.1986.

[32] X. Lu, et al., US CDC Real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2, Emerg. Infect. Dis. 26 (2020) 1654–1665, https://doi.org/10.3201/eid2608.201246.

[33] G. Reina, N.D.Q. Chau, Y. Nishina, A. Bianco, Graphene oxide size and oxidation degree govern its supramolecular interactions with siRNA, Nanoscale 10 (2018) 5965–5974, https://doi.org/10.1039/c8nr00333e.

[34] M. Fukuda, et al., Lethal interactions of SARS-CoV-2 with graphene oxide: implications for COVID-19 treatment, ACS Appl. Nano Mater. 4 (2021) 11881–11887, https://doi.org/10.1021/acsanm.1c02446.

[35] M. Liu, U. Chida, S. Matsuyama, T. Kato, H. Katayama, Surface modification of cotton gauze for increased adsorption of enveloped and nonenveloped viruses from water, ACS EST Water 4 (2024) 368–376, https://doi.org/10.1021/acsestwater.3c00358.

[36] C.P. Gerba, Applied and theoretical aspects of virus adsorption to surfaces, Adv. Appl. Microbiol. 30 (1984) 133–168, https://doi.org/10.1016/s0065-2164(08)70054-6.

[37] R. Khan, R. Nakagawa, B. Campeon, Y. Nishina, A simple and robust functionalization of graphene for advanced energy devices, ACS Appl. Mater. Interfaces 12 (2020) 12736–12742, https://doi.org/10.1021/acsmami.9b21082.

[38] O.C. Compton, D.A. Dikin, K.W. Putz, L.C. Brinson, S.T. Nguyen, Electrically conductive “alkylated” graphene paper via chemical reduction of amine-functionalized graphene oxide paper, Adv. Mater. 22 (2010) 892–896, https://doi.org/10.1002/adma.200902069.

[39] R.M. Silverstein, G.C. Bassler, Spectrometric identification of organic compounds, J. Chem. Educ. 39 (1962) 546, https://doi.org/10.1021/ed039p546.

[40] Y. Ikeda, et al., Proton conductivity of graphene oxide hybrids with covalently functionalized alkylamines, Chem. Lett. 42 (2013) 1412–1414, https://doi.org/10.1246/cl.130606.

[41] S. Wu, T. Shi, L. Zhang, Preparation and properties of amine-functionalized reduced graphene oxide/waterborne polyurethane nanocomposites, High Perform. Polym. 28 (2015) 453–465, https://doi.org/10.1177/0954008315587124.

[42] R. Shibahara, K. Kamiya, Y. Nishina, Grafting chelating groups on 2D carbon for selective heavy metal adsorption, Nanoscale Adv. 3 (2021) 5823–5829, https://doi.org/10.1039/d1na00435b.

[43] I.A. Vacchi, C. Spinato, J. Raya, A. Bianco, C. Ménard-Moyon, Chemical reactivity of graphene oxide towards amines elucidated by solid-state NMR, Nanoscale 8 (2016) 13714–13721, https://doi.org/10.1039/C6NR03846H.

[44] S. Stankovich, et al., Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558–1565, https://doi.org/10.1016/j.carbon.2007.02.034.

[45] A. Iakunkov, et al., Swelling of graphene oxide membranes in alcohols: effects of molecule size and air ageing, J. Mater. Chem. A 7 (2019) 11331–11337, https://doi.org/10.1039/C9TA01902B.

[46] I.A. Vacchi, S. Guo, J. Raya, A. Bianco, C. Ménard-Moyon, Strategies for the controlled covalent double functionalization of graphene oxide, Chem. Eur. J. 26 (2020) 6591–6598, https://doi.org/10.1002/chem.201905785.

[47] G. Bastin, C. Gantzer, G. Sautrey, New method to quantify hydrophobicity of non-enveloped virions in aqueous media by capillary zone electrophoresis, Virology 568 (2022) 23–30, https://doi.org/10.1016/j.viroi.2022.01.004.

[48] A. Armanious, et al., Viruses at solid-water interfaces: a systematic assessment of interactions driving adsorption, Environ. Sci. Technol. 50 (2016) 732–743, https://doi.org/10.1021/acs.est.5b04644.

[49] J. Heffron, B.K. Mayer, Virus isoelectric point estimation: theories and methods, Appl. Environ. Microbiol. 87 (2021) 1–17, https://doi.org/10.1128/AEM.02319-20.
[50] F. Baskoro, et al., Graphene oxide-cation interaction: inter-layer spacing and zeta potential changes in response to various salt solutions, J. Membr. Sci. 554 (2018) 253–263, https://doi.org/10.1016/j.memsci.2018.03.006.

[51] F.M. Wellings, A.L. Lewis, C.W. Mountain, Demonstration of solids-associated virus in wastewater and sludge, Appl. Environ. Microbiol. 31 (1976) 354–358, https://doi.org/10.1128/aem.31.3.354-358.1976.

[52] Y. Nishina, Mass production of graphene oxide beyond the laboratory: bridging the gap between academic research and industry, ACS Nano 18 (2024) 33264–33275, https://doi.org/10.1021/acsnano.4c13297.

[53] G.S. Lee, J.G. Kim, J.T. Kim, C.W. Lee, S.J. Cha, G.B. Choi, J.W. Lim, S.P. Sasikala, S.O. Kim, 2D materials beyond post-AI era: smart fibers, soft robotics and single atom catalysts, Adv. Mater. 36 (2024) 2307689.