BIOSENSORS; Biological and Technical Challenges Associated With In-Field Detection and Quantification

Biosensors :For detection of plant pathogens

Introduction

Plant pathogens are a major reason of reduced crop productivity and may lead to a shortage of food for both human and animal consumption. Although chemical control remains the main method to reduce foliar fungal disease incidence, frequent use can lead to loss of susceptibility in the fungal population. Furthermore, over-spraying can cause environmental contamination and poses a heavy financial burden on growers. To prevent or control disease epidemics, it is important for growers to be able to detect causal pathogen accurately, sensitively, and rapidly. To reach this goal, biosensors have been developed for  early and accurate pathogen detection. There is also great scope to translate innovative nanoparticle-based biosensor approaches developed initially for human disease diagnostics for early detection of plant disease-causing pathogens. 

Diagnostic Methods for Plant Pathogens

The earliest traditional method, still broadly used for disease and potentially pathogen diagnosis, is visual crop inspection, requiring an experienced grower or pathologist. As a consequence, recent efforts have focused on the development of earlier pathogen detection methods with greater sensitivity, accuracy, and identification speed. To date, these have comprised of three types of molecular assays, which are protein-based or nucleic acid technologies: Enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) assay and loop mediated isothermal amplification (LAMP) assay.

By far the most developed serology-based diagnostics method for fungal pathogens is ELISA. This enables pathogen detection via a colorimetric reaction, visualized by the naked eye or optical reader for quantification (Fang and Ramasamy, 2015). For improved specificity and sensitivity of detection, alternative approaches targeting nucleic acid sequences of target pathogens were developed. These often rely on PCR amplification of the target sequence, followed by amplicon detection and visualization. To improve portability of molecular diagnostic assays, the DNA amplification technique of LAMP was developed, which rapidly amplifies nucleic acids with high specificity and sensitivity under isothermal conditions (Notomi et al., 2000). LAMP-diagnostic assays were initially focused on detecting bacterial and viral pathogens (Obura et al., 2011; Bühlmann et al., 2013) and more recently on plant fungal pathogens (Tomlinson et al., 2013; Chen et al., 2016; Manjunatha et al., 2018).

Biosensor Technologies for Plant Pathogen Detection

Biosensors have appeared as advanced detection tools used in many research fields including environmental monitoring, detection of airborne pathogens, real-time detection of human blood components and pathogens and pesticide residues in foods and beverages (Liu et al., 2018).Biosensor are diagnostic devices, which typically integrates a biological sensing element and physicochemical transducer to generate an electronic signal when it contacts with a specific analyte of interest or pathogen in solution. Subsequently, a transducer converts a biomolecular interaction into a digital output (Elmer and White, 2018). The biological element that plays the role of a bioreceptor can be antibody, DNA, enzyme, tissue type, whole cell etc. These bioreceptors are responsible to provide recognition specificity to the biosensor through the selective nature of the biochemical interaction. Based on the transducer type, a biosensor may be classified as an electrochemical, optical, thermal, or piezoelectric biosensor (Sawant, 2017).

electrochemical biosensor was more sensitive than conventional PCR, able to diagnose infected plants before any symptoms of the disease appeared (Lau et al., 2017).

Electrochemical Biosensors

An electrochemical biosensor relies on two core components; a molecular recognition layer and an electrochemical transducer, which converts biological information that is derived from a binding event into an electrical signal that is subsequently shown on a readout device (Ronkainen et al., 2010). In other words, following the active interaction between the analyte and bio-recognition element, a signal generated on the electrode surface is transformed into an electrical signal for quantitative analysis. This class of biosensor is able to detect target pathogens under different conditions including in air, water, and on seeds within different platforms such as greenhouses, in-field and in postharvest storage vessels (Fang and Ramasamy, 2015). Among all the possible biological sensing components linked to a transducer, plant’s antibody and DNA are more advantageous and applied in point-of-care assays to detect plant pathogens.

FIGURE 1. Schematic representation of an (A) antibody-based and a (B) DNA/RNA-based biosensor for analyte detection.

TABLE 1. Examples of electrochemical biosensors developed for the detection of plant pathogens.

Optical Biosensors

Optical biosensors measure the interaction between a target analyte and ligand using a light source, an optical transmission medium, an immobilized biorecognition element, and a signal detection system. Ultimately, change in amplitude, phase, and frequency of the given light in response to physicochemical conversion (change) generated by the biorecognition process is measured (Ray et al., 2017). Among optical biosensors developed for plant pathogen detection, colorimetric biosensors, fluorescence-based assays-, and surface plasmon resonance-based biosensors are the most common.

TABLE 2. Examples of optical biosensors developed for the detection of plant pathogens.

Advantages of Biosensors over conventional methods

The accuracy for biosensor-based detection is largely dependent on the in-field sampling strategy. Detection limits for target pathogens may be greatly impacted by their biological concentrations on or within plant materials or other environmental samples tested. In conventional assays, sample preparation methods like centrifugation and precipitation have been used to solve these issues. However, these techniques are less conducive to in-field applications since they require multiple pieces of powered equipment and are time-consuming. Direct sequencing methods are even more sensitive to impurities in the input material and require high molecular weight pure DNA to produce reliable results. Although there are several commercial kits available for DNA extraction from pathogens, many fail to extract high quality DNA from environments that contain higher concentrations of acids such as humic acid in soil or phenolic acids in plant tissues. To overcome this challenge, modified nucleic acid (DNA or RNA) extraction techniques are required to remove background interference. However, these methods are laborious and time-consuming protocols that use liquid nitrogen or dry-ice for sample homogenizing .

As an alternative, the magnetic properties of metal nanoparticles themselves may be utilized, to separate and concentrate the bound target analytes. The further development of paramagnetic bead-based DNA extraction and purification methods will substantially improve the speed and quality of DNA extractions, while reducing the dependency on toxic reagents and powered centrifuge and heatblocks.Lab-made paramagnetic particles reagents or commercial kits, such as the CleanNA Clean Plant DNA Kit and MagBio HighPrep™ Plant DNA Plus Kit, should be further evaluated for their cost, time and labor-efficiency, and the quality and yield of the extracted DNA. This approach will be especially useful when analyte capture and separation are able to be performed as a twostep reaction in a single reaction tube with subsequent immediate loading onto the biosensor chip.Another main factor is affordability—massive reduction in cost per sample within nano-biosensor devices has already been achieved using bare screen-printed carbon electrodes  and streptavidin coated screen-printed carbon electrodes.

Need of Furthur Reasearch 

Despite the advantages of electrochemical and optical biosensing techniques over the conventional methods mentioned in this review, there is a need for further research on implementing this technology in plant pathogen quantification under in-field conditions. A validation of a portable sensor requires a specialized hardware, which can be expensive and difficult to operate for un-specialized such as farmers. Currently it is unknown what minimum inoculum levels are required to induce a disease in host plants. Establishing this threshold is essential to translate the biosensor quantified pathogen level to estimate the risk of disease..

Conclusion

Nano biosensing technologies and devices are fast replacing conventional and traditional diagnostic tools. With further optimization for application in a range of environments, the use and validation of these affordable, fast, and highly sensitive and specific tools for plant pathogen detection in the field will become widely adopted in the near future. It is highly likely that with further validation, these tools will also be used for modelling disease and will therefore become an essential part of a proactive and pre-emptive suit of IDM tools, for use by growers and agronomists ahead of epidemics.