Nanobiosensors and nanoformulations in agriculture: new advances and challenges for sustainable agriculture

Agriculture can greatly benefit from practical applications of nanotechnology. For example, the development of more efficient and less polluting agrochemicals (nanoformulations) or the design of devices that helps to detect biotic and abiotic stresses in plants before they affect plant production (nanobiosensors). Advancements in these fields will assist in mitigating some of the serious problems and challenges that agriculture faces in the coming years. These include reducing the negative environmental impact that agricultural activities have on the environment, dealing with issues related to climate change (such as altered precipitation patterns, temperature changes, and the emergence of new plant pathogens), and overcoming the plateau in crop yields [1]. The number of nanotechnological applications being tested in agriculture is continually increasing and expanding to various areas, including soil and water management, pathogen detection, and plant production amongst others [1–4] (Figure 1). However, in this mini-review, we will focus on the two most important applications: nanobiosensors and nanoformulations (NFs).

Biosensors incorporating nanoparticles (NPs) are defined as nanobiosensors. The properties at the nano-scale of NPs result on a more organised and efficient material (enhanced optical and mechanical properties, and high chemical customisation among others) in comparison with conventional biosensors. Nanobiosensors can play a crucial role in agriculture by detecting and monitoring various parameters related to plant health and environmental conditions [5,6]. These small devices can detect the presence of pathogens, pests, and diseases at an early stage, allowing for timely intervention and minimising crop losses [7]. Nanobiosensors can also provide real-time analytical data, including the detection of nutrient levels, metabolites, pesticides, soil moisture, and temperature, aiding in precision farming practices and optimising resource usage [5,8].

NFs refer to the development of agrochemicals, such as pesticides and fertilisers, in nano-sized formulations. By reducing the particle size, these formulations offer several advantages. They enhance the efficiency of the active ingredients, allowing for lower doses while maintaining effectiveness [9], which reduces environmental pollution and minimises the potential negative impacts on beneficial organisms. NFs also improve the targeted delivery of agrochemicals, ensuring that they reach the intended plant tissues or pests more effectively [10], and decrease damage to non-targeted sites of plants, controlling the detrimental effects of chemicals on the environment.

Overall, nanotechnology offers promising solutions to address agricultural challenges and enhance productivity while minimising environmental impact. Continued research and development in this field has the potential to revolutionise farming practices and contribute to sustainable and efficient agriculture. However, the use of nanotechnology in agriculture requires educating farmers to ensure appropriate use of this technology, which is still a worldwide challenge for governments.

One of the major challenges in crop production is monitoring their health status. Acting quickly against incipient diseases or compensating for nutritional or water deficiencies can result in significant savings, not only economically but also environmentally, especially if such interventions are localised only where and when they are needed. Smart farming aims to address this need by employing advanced technologies such as drones, remote operations, real-time information, etc. Within these technologies, the development of nanobiosensors plays a crucial role. These devices can monitor markers for specific stresses (drought, disease, etc.) and detect them in advance, even at the level of individual plants.

Nanobiosensors are found either in the field or in the laboratory for analysis. The combination of nanoscience, computer, biology, and electronics is allowing researchers to develop nanobiosensors with powerful sensing abilities. Ray et al. [11] listed the ideal characteristics of nanobiosensors. Briefly, (i) nanobiosensors should be cheap, biocompatible, non-toxic, and portable; (ii) they should be stable when stored at regular conditions; (iii) the interaction with the analytes to be determined should be extremely specific, so that minimum reactivity is required; (iv) received signals from nanobiosensors should be precise, accurate, and reproducible.

A nanobiosensor needs to be able to convert the chemical signals from plants into digital information that can be measured and monitored by external electronic devices. Tipically, a nanobiosensor has three components: (i) bioreceptor; (ii) transducer; and (iii) detector (Figure 2). The bioreceptors are generally biological materials (antibodies, enzymes, microorganisms, nucleic acids, and etc.) that interact and accept the chemical signal from the analyte to be detected, and then transfer it to the transducer. Next, the transducer works as an interface which measures the signal from the bioreceptor and transforms it into a quantifiable electrical signal in the detector. Finally, the detector processes the signals to amplify and analyse them (Figure 2). A list of representative nanobiosensors used for crop detection problems, their targets, and their detection mechanism is presented in Table 1.

There are several ways to achieve the conversion from the presence of a specific chemical compound to an electrical or luminous signal correlated with the amount of compound to be detected. For example, there are nanobiosensors with a receptor for the analyte and two chromophores (molecules that emit fluorescence) of different colours. When the receptor is empty, the chromophores are separated, and only one of them can emit its characteristic fluorescence. However, when the analyte binds to the receptor, the chromophores come into contact, and the emitted fluorescence is different (based on a phenomenon called Förster Resonance Energy Transfer — FRET (see Table 1). In this way, the presence or absence of the analyte can be linked to the type of emitted fluorescence, and its concentration can even be measured based on the intensity of that fluorescence. This type of nanobiosensor can provide in vivo information of molecule concentrations, protein activity (enzymes), and ion dynamics [23].

Variations in the content of reactive oxygen species (H₂O₂), sugars (glucose, sucrose), ions (Ca²⁺, H⁺), and plant hormones (ethylene, abscisic acid, jasmonic acid, etc.) are often related to the health status of the plant, and there are nanobiosensors designed to measure their concentrations [23]. Nanobiosensors also play valuable roles in plant breeding and culturing, with the purpose of improving crop productivity and quality. Many nanomaterials are used to deliver nutrients and pesticides to particular target points in plants [5,24]. Detecting and controlling the use of pesticides, fertilisers, as well other chemical compounds, provide convenient information for precision farming [25], where detection of pesticides residues has several advantages such as low detection, sensitivity, high selectivity, and fast responses. Several types of nanobiosensors (enzyme, optical, and electrochemical sensors) have been developed to detect pesticide mixtures [12,26], organophosphorus pesticides [13] or even particular pesticides such as methyl parathion or chlorpyrifos [14]. In addition, an accurate and rapid estimation of nutrient concentrations in soil would increase the productivity, reduce cost to farmers, and reduce leaching of unutilised fertilisers. Thus, detection of urea, urease activity, and urease inhibition are possible by the development of a nanobiosensor based on gold nanopaticles (Au NPs) [15].

Designs based on carbon nanotubes, quantum dots (QDs), and plant viruses take advantage of the inherent properties of nanomaterials to convert chemical signals into optical signals [23]. Non-invasive electronic systems have also been developed to be placed on leaf surfaces using nano-scale carbon structures, which can be highly useful for measuring volatile substances [16]. The use of nanobiosensors for pest detection has provided rapid and accurate results compared with the time-consuming conventional methods (manually observing), allowing for plants to be quickly protected. Detailed reviews on NP-based biosensors used for plant pathogen detection, their detection mechanisms, and performance have been published elsewhere [5,7,8,10,25,26].

Inorganic nanomaterials such as Ag, Au and metal oxide NPs are commonly used as nanobiosensors. For example, Khaledian et al. [17] used Au NPs functionalised with single-stranded oligonucleotides to detect the genomic DNA of the devastating causal agent of potato bacterial wilt, Ralstonia solanacearum. Gold NPs have also been used in the detection of the plant bacterial pathogen Pantoea stewartii [18]. The detection method was developed using horseradish peroxidase labelled antibodies in an electrochemical enzyme linked immunoassay. Silver nanorods have been employed for the identification of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman spectroscopy [19]. Siddiquee et al. [20] developed a DNA biosensor using zinc oxide NPs/chitosan nanocomposites to identify the presence of Trichoderma harzianum, a soil-borne fungus that has been successfully used as a biological control agent against various plant pathogens.

Carbon dots (CDs) are the new alternative to the conventional semiconductors QDs. Their low toxicity and high biocompatibility are ideal characteristics for using them as nanobiosensors in the detection of microorganism. For example, Safarnejad et al. described a QD-based complex nanobiosensor against the Citrus Tristeza virus, which is responsible for one of the most important diseases of citrus in the world. To achieve this goal, a fluorescence resonance energy transfer approach was used [21]. Additionally, surface-functionalised QDs are used as controlled release NFs for delivering the natural antibiotic kasugamycin in plant disease management [22].

Our agriculture is intricately linked to the application of agrochemicals to maintain yields, whether to improve the nutritional status of crops or to combat pests and diseases. However, the efficiency of both fertilisers and pesticides remains relatively low, with a significant amount of the product being lost due to factors such as volatilisation, leaching, immobilisation, decomposition, etc. When chemical fertilisers are applied in bulk, plants cannot take them all at once, resulting in a cycle of re-application that increases costs [27]. In fact, studies have revealed that half of the applied nitrogen fertilisers are lost into water and air [28]. Besides the financial cost, this poses an environmental problem, as all the active substance that does not reach its target (the plant or the pathogen) ends up in the surrounding ecosystem (soil, water, other living organisms, etc.). Furthermore, the pesticide contamination of surface and ground water has resulted in loss of freshwater content available for human consumption [29]. This is where nanotechnology can be useful, making a significant contribution by increasing the efficiency and safety of treatments through NFs (Figure 3). As with every new technology that has advantages, there are also drawbacks using nanotechnology in agriculture. Some of the biggest concerns associated with its use go from high reactivity and variability, environmental impact, to safety concerns for farmers and consumers. However, using nanomaterials as platforms for the application of active substances (whether fertilisers or pesticides) offers several powerful advantages [30]:

• Protection against degrading agents (pH, light, temperature, microorganisms, etc.): encapsulating the product in a nanocapsule provides a protective cover that prevents the deterioration of the active substance, allowing for a reduction in the effective applied dose. This also opens the possibility of using naturally occurring agrochemicals, which easily decompose in the environment and are less persistent, whose sensitivity to degradation has hindered their use until now [31,32].

• Controlled release: nanocapsules and NPs can be designed and prepared to release the active substance suddenly in response to a specific stimulus (e.g. a pH change when reaching an insect's stomach or the action of an enzyme) or to release it continuously over time [33,34] (Figure 3).

• Control of solubility in both aqueous and lipid media, facilitating penetration and movement in different tissues [35].

• Attachment of biomolecules for specific functions: nucleic acids (DNA, RNA), antibodies, proteins, lipids, etc., can be attached to the surface of nanomaterials with the ability to recognise other substances. In this way, nanomaterials can be directed and concentrated in specific areas of organisms, tissues or cells, where they can later release their chemical cargo [36,37].

NFs are new-age materials with endless nano-scale based delivery systems. Some representative NFs designs, which vary in size from 10 nm to more than 100 nm, are based on: liposomes, niosomes, phytosomes, microgels, multiple emulsions, nanoemulsions, microclusters, solid lipid NPs, nanostructured lipid carriers, biopolymeric NPs, gold NPs, self-assembled micelle, silica NPs, graphene NPs, and mesoporous NPs [10,38]. To improve the properties of active components, NFs are synthesised through encapsulation, i.e. entrapment of the active molecule inside the nanocarriers, or complexation, i.e. covalent attachment of active molecules to nanocarriers. The interaction, uptake, and translocation of NFs by plants is highly dependent of several factors such as plant physiology, surface charge of NFs itself, shape, size, and physico-chemical properties [39]. Thus, diverse crops belonging to different species have shown diverse patterns of accumulation and absorption of different types of NFs [40,41]. Table 2 summarises some examples of NFs for crop protection and production.

These NFs can be used to improve existing strategies and products traditionally used in agriculture or to create new approaches and solutions [54] (Figure 3). In the former case, the aim is often to achieve better application through controlled and localised release of existing and marketed active substances. This type of strategy is particularly useful for fertilisers, as it prevents losses due to leaching and decomposition, allowing nutrients to be available to the plant when and where they are needed [55]. Nanofertilisers or smart fertilisers are NFs (polymers) mainly based on NPs that provide nutrients to plants or enhance the effect of conventional fertilisers when they are applied in small amounts. The size and high volume to surface area make them more efficient than the conventional ones. In addition, they move faster than the conventional fertilisers. Based on the exogenous cargo, there are three categories of nano fertilisers: (i) macronutrient nanofertilisers, (ii) micronutrient nano- fertilisers, and (iii) nanoparticulate nano fertilisers [10]. There are different types of NPs employed for the generation of nanofertilisers with different compositions, benefits, and active compounds; these have been addressed elsewhere [8,10,28,56].

As alternative solutions to the conventional agrochemicals, some NPs have the ability to stimulate the natural defences of plants, possess inherent microbiological activity or incorporate new substances and products with highly specific action or natural origin [30,57]. NFs offer an excellent alternative for safe and highly efficient delivery of biomolecules such as microRNA, CRISPR–Cas, and RNA interference (RNAi) for plant protection [36]. For example, the technology of RNAi is a perfect candidate to be coupled with NFs that protect the nucleic acid and allow its release at specific times and locations to silence specific phytopathogenic genes and prevent infections [30,52]. Siddiqui et al. [58] suggested that silicon dioxide NPs (SiO₂ NPs) may improve the natural defence mechanisms of Cucurbita pepo plants in response to salt stress by improving the transpiration rate, water use efficiency, total chlorophyll, and carbonic anhydrase activity.

NPs also protect plants from abiotic stress including drought, temperature fluctuations, salinity, alkalinity, and mineral and metal toxicity [10]. Thus, NPs, especially Au counterparts, can mimic the activity of antioxidant enzymes working as nano-enzymes in mitigating the oxidative stress [59]. Furthermore, cerium NPs (CeO NPs) could increase the tolerance against abiotic stress in plants by regulating essential pathways. Thus, CeO NPs are well-suitable to mitigate the effects of excessive ROS production by acting as ROS scavengers when transitioning easily between the Ce³⁺ and Ce⁴⁺ oxidation states [60,61]. Wu et al. [62] demonstrated that CeO NPs could increase Arabidopsis plants tolerance to salinity stress by directly acting on the cytosolic Na⁺/K⁺ ratio (improving K⁺ retention and reducing Na⁺ efflux), resulting in improving plant leaf photosynthetic performance and biomass. Additionally, phytohormones play important roles in responses against abiotic stress [33]. Silica NPs loaded with abscisic acid resulted in prolonged release of the exogenous cargo in Arabidopsis thaliana plants, improving the drought resistance in seedlings [63]. In addition to the enzymatic defence system against abiotic stress provided by the NPs indicated so far, titanium dioxide NPs (TiO₂ NPs) protect plants by using an alternative gene regulation mechanism. For example, TiO₂ NPs were able to trigger the expression of essential non-coding RNA to protect plants in response to heavy metal stress [64].

While the published results in the field of nanotechnology in agriculture are increasingly promising, and there are already numerous patents and commercial products available [54], it is difficult to adapt such products into practical applications in the field. First, research work in this area tends to be highly complex and requires multidisciplinary teams with expertise in chemistry, physics, biology, and agronomy, among others, along with the use of sophisticated equipment (e.g. particle accelerators — synchrotrons). Second, the behaviour of nanomaterials can depend significantly on factors such as plant species and physiology so results obtained from one crop (especially regarding NFs) may not be directly applicable to others [39]. Third, agriculture often operates on tight profit margins, so the cost of using nanomaterials for large-scale field applications must be relatively low, otherwise, farmers may not be able to adopt them [39,54]. Additionally, it is crucial to consider the potential environmental impact and food safety of these products, which has resulted in a growing trend towards using natural nanomaterials (biopolymers) that are non-toxic and easily degradable [39,54].

Finally, one of the major challenges is the application of many of these nanodevices to crops, as it often incurs additional costs for farmers. A nanoagrochemical should ideally be applicable in the same way as conventional agrochemicals (using machinery, irrigation, etc.), depending on its mode of action and function. However, it would be highly advantageous if they could be incorporated as seed coatings, which would significantly reduce costs. Nanobiosensors can be more problematic in this regard, as specific methods would need to be designed for their release onto crops; some of them may be useless or may be too costly in many situations, such as non-invasive electronic systems on leaves, unless they are used for perennial crops (e.g. fruit trees).

To address many of these challenges, it is imperative to develop research focused on field applications [39,54]. Many of the studies conducted so far lack real-field trials and are limited to extrapolating results obtained in the laboratory or under controlled conditions (greenhouses or growth chambers). Field trials in agronomy are equivalent to clinical trials in medicine, and even if a product performs well in the laboratory, it does not guarantee effectiveness under real conditions, or there may be challenges in its manufacturing and/or distribution. Therefore, conducting experiments with different crops in experimental plots, comparing them to conventional products to assess efficiency, and following the usual practices of farmers is the only way to validate whether a nanotechnology application will have a significant impact on agriculture.

• The study of NFs–plant interaction is an important point to consider in the understanding of the physiological and biochemical responses in plants.

• Nanobiosensors and NFs have the potential to revolutionise farming practices and contribute to sustainable and efficient agriculture.

• Continued research and development in nanotechnology for agriculture is needed to address challenges such as climate change, crop yield plateaus, and negative environmental impacts.

• There is a requirement to execute large trials based on the lack of knowledge in some critical areas such as toxicity and bioaccumulation of the NFs and physico-chemical interaction of NFs with soil.

• Governments should develop open awareness programs to educate farmers and consumers about nanotechnology for its successful implementation in agriculture. This way, people, and specially farmers, would not be as reluctant to purchase products developed using the technology of novel NFs-based agrochemicals.

The authors declare that there are no competing interests associated with the manuscript.

CDs

carbon dots

NFs

nanoformulations

NP

nanoparticle

QDs

quantum dots

RNAi

RNA interference