Nanofilm-enhanced electrochemical DNA sensing: a breakthrough for yellow rust detection in wheat

This study showcases the development of a genosensor utilizing a nanoscale NiO thin film. The genosensor is constructed on a glass substrate coated with tin-doped indium oxide (ITO) and is designed for the specific detection of DNA sequences associated with Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat yellow rust. The detection process relies on the utilization of methylene blue (MB) as an electrochemical indicator, with NiO acting as the matrix and the electrochemical measurement system serving as the transducer. Various single-stranded DNA oligonucleotide sequences related to Pst pathogenesis are employed as probes to enable sensing. The electrochemical response of the nanoscale bioelectrode is characterized and studied using two distinct electrochemical techniques, cyclic voltammetry (CV) and differential pulse voltammetry (DPV), in conjunction with a potentiostat. The detection ranges spans from 40 pg μl−1 to 115 ng μl−1, demonstrating a linear correlation with exceptional precision. The absence of DNA-based biosensors for the detection of Puccinia striiformis f. sp. tritici (Pst) has prompted the need for a new method to address the limitations associated with previously reported technologies. Although surface plasmon resonance (SPR) immunoassays have been reported for Pst detection, the development of DNA-based biosensors specifically tailored for Pst detection remains unexplored. Introducing a novel method aims to overcome the challenges and shortcomings of existing techniques, providing a new approach to detect and combat the devastating effects of Pst on wheat crops. By leveraging the advantages of DNA-based biosensors, such as their sensitive and precise detection capabilities, this new method seeks to enhance the accuracy and efficiency of Pst detection, ultimately contributing to the development of effective strategies for disease management and crop protection. The developed nanoscale electrochemical DNA sensor offers outstanding sensitivity, extended shelf life, and reliable recovery, effectively minimizing the likelihood of obtaining erroneous results. A significant highlight of this study is the first-time utilization of conserved sequences associated with pathogenesis in selected Pst strains for the development of a nanoscale genosensor.


Introduction
Puccinia striiformis f. sp.tritici (Pst) is a devastating airborne wheat pathogen causing yellow rust/stripe rust disease throughout the world.The pathogen can mutate and multiply reducing wheat yield and adversely impacting the lives of farmers worldwide.The majority of winter wheat cultivars are either susceptible or resistant to this disease at a low level [1].In the early growing season, the disease causes stunted and weakened plants resulting in 70% losses and destruction of crops up to 100% in severe cases [2,3].This disease is generally confirmed by traditional methods including visually surveying symptoms of the disease and managed by pesticide sprays such as pyraclostrobin and propiconazole [4,5].The Yellow Rust symptoms appear very late and till that time the disease becomes epidemic, and for this reason, the timeline of pesticide sprays does not work anymore [1,6].
To date, conventional PCR (Polymerase chain reaction)-based strategies are employed in a variety of hostpathogen systems, yet the development and utilization of these techniques for plant diagnostics are lagging.Biosensors, highly sensitive and selective small analytical devices coupled with a biological recognition element to detect the presence and absence of an analyte, provide better options for overcoming these limitations.The researchers in their study developed a point-of-care (POC) photoelectrochemical (PEC) biosensing method for the detection of human papillomavirus-16 (HPV-16) using a portable electrochemical detection system.The biosensor incorporated CRISPR-Cas12a for the trans-cleavage of the G-quadruplex, which served as a biorecognition and amplification element.A hollow In2O3-In2S3-modified screen-printed electrode (In2O3-In2S3/SPE) was used as the photoactive material.
The biosensor exhibited high sensitivity, with a broad detection range of 5.0 to 5000 pM, and it could detect HPV-16 at a low concentration of 1.2 pM.The POC photoelectrochemical biosensor demonstrated its potential for rapid and accessible screening of HPV-16 concentration, particularly in remote areas and resource-limited settings where advanced instruments may not be available.Its innovative design and smartphone-based readout offer promising prospects for early detection of HPV-16 and improved disease management in healthcare settings [7].Mycotoxins, highly toxic secondary metabolites produced by fungi, are lurking in our food and posing serious threats to human health.These invisible killers have led to a growing concern for food safety worldwide.Detecting and monitoring mycotoxins in foodstuffs is of utmost importance to protect consumers from their harmful effects.Fortunately, cutting-edge technology has paved the way for innovative solutions in mycotoxin detection.Photoelectrochemical (PEC) biosensors, the superheroes of the analytical world offer a ray of hope, providing low-cost, rapid, and efficient methods for mycotoxin analysis.PEC biosensors harness the power of photoactive materials, acting as transducers that convert chemical information into detectable PEC signals.These materials play a vital role in the biosensing system, amplifying the accuracy and sensitivity of mycotoxin detection.By leveraging the unique properties of photoactive materials, PEC biosensors can swiftly and reliably identify the presence of mycotoxins in food samples [8].
Thus, biosensors can also be used for on-site detection of Pst at latent infection stages so that preventive measures can be taken early.Human and animal diseases have been detected with the help of biosensors.However, reports on plant pathogen detection using biosensors are still in their infancy.DNA (Deoxy ribonucleic acid)-based biosensors are gaining popularity because of their sensitive and precise detection of DNA target sequences.There is no report of DNA-based biosensors for Pst detection so far.
For the detection of pathogens several technologies, including microfluidic, quartz crystal microbalance (QCM), piezoelectric, Fourier-transform infrared spectroscopy (FTIR), acoustic sensors, microarray, etc. have been used [9][10][11][12]).These technologies have attracted the attention of the scientific community because of their accessibility, low-budget, high sensitivity, low detection limit, rapid, broad-spectrum range of detection, recovery, excellent stability, and portability [13].There are reportedly many varieties of fabrication materials, such as metallic nanoparticles [14], graphene, carbon nanotubes [15], metals, and their oxides, such as nickel, titanium, zinc, copper, tin, manganese oxide, etc.These materials have the characteristics of high affinity for biomolecules, excellent charge transfer, large surface-to-volume ratio, chemically stable, non-toxicity, and good electrochemical activity.For these reasons, they are used as fixed latent matrices [16,17].Therefore, they have been used in electrical biosensors for the immobilization of various biological elements, including microorganism DNA, Tissue, whole-cell, enzymes, antibodies, RNA (ribonucleic acid), microRNA, aptamers, antigens, and various other proteins [18][19][20].The materials under consideration exhibit several highly desirable characteristics for their application in biosensors for biomolecular interactions.In addition, these materials are also known for their chemical stability, ensuring the sensor's robustness and durability during prolonged usage.Their non-toxic nature further enhances their suitability for biological applications, ensuring minimal interference with the sample and maintaining sample integrity.Moreover, their good electrochemical activity enables efficient signal transduction and detection of biomolecular interactions.There is a study investigates the underlying mechanisms and principals involved in the gas sensing interface of 3D-SnS2 nanosheets.By leveraging the nanosheets' photoelectrochemical properties and the assistance of ultraviolet irradiation, the biosensor is designed to enable contactless detection of target analytes.The research delves into the detailed characterization of the 3D-SnS2 nanosheets, including their structural and optical properties, as well as their gas sensing performance.It also explores the sensing mechanism at the gas-solid interface and elucidates the influence of ultraviolet irradiation on the gas sensing process [21].There is a similar study titled 'Exploiting photoelectric activities and piezoelectric properties of NaNbO3 semiconductors for point-of-care immunoassay' delves into the exploration of utilizing the unique photoelectric activities and piezoelectric properties of NaNbO3 semiconductors for the advancement of point-of-care immunoassays.T It investigates the underlying mechanisms and principles governing these properties, shedding light on the material's structural composition, electronic behaviour, and their relationship to the observed photoelectric and piezoelectric responses.It explores how the photoelectric activities and piezoelectric properties of NaNbO3 semiconductors can be harnessed to improve the sensitivity, accuracy, and reliability of the immunoassay process [22].This integration of advanced functionalities offers the potential for more efficient and effective point-of-care diagnostics.
By exploiting the photoelectric activities and piezoelectric properties of these semiconductors, researchers are paving the way for more sophisticated, sensitive, and portable immunoassay devices that can significantly impact diagnostics and improve healthcare outcomes.
The Nano-NiO DNA biosensor, for example, is used for the detection of 'black fever', (Kala-azar) spread by sand-fly [23].For the detection of the Staphylococcus aureus nuc gene, a DNA-based biosensor was designed using a novel nanocomposite made by mixing chitosan, Co3O4 nanorod, and graphene [24].TiO 2 nanoparticles were made and used to develop a high-efficiency impedimetric genosensor to support the carbon ionic liquid electrode [8].For the detection of uric acid, an electrochemical biosensor, nanostructured CZTS capped with butylamine was fabricated on the ITO glass substrate [25].Liu et al [26] developed an electrochemical genosensor using a reducing graphene oxide gold nanoparticle-modified electrode for the detection of Mycobacterium tuberculosis.Due to its high electrical conductivity and high purity, NiO is widely employed as the manufacturing material for DNA biosensors for the detection of free cholesterol.Kaur et al [27] used the NiO/ITO electrode and found that NiO has an excellent ability to interact with biological materials, it is catalytically efficient, formations of stable nanostructures are easy, and have high isoelectric point (9.5).These unique properties of NiO made it a potential candidate for reagent-free biosensors.DNA probes were effectively anchored onto nanostructured nickel oxide (NiO) surfaces via electrostatic interactions facilitated by the low isoelectric point.This innovative approach has resulted in the creation of a DNA biosensor that is not only highly sensitive but also remarkably stable [28].A variety of technologies have been used for the synthesis of nanostructures including sol-gel, green synthesis, plasma processing, radio frequency, sputtering, laser deposition, aerosol condensation, thermal evaporation, etc [29][30][31].
For the first time, an electrochemical DNA biosensor was constructed in this study to detect the Pst pathogen utilizing consensus DNA sequences.The proposed biosensor eliminates the requirement of immunospecific analysis for Pst.This is due to the presence of a conserved consensus sequence that is intended to be active against all Pst strains.NiO nanocomposites are used as a matrix to connect -NH2-modified PDNA (Probe DNA) by physical adsorption, and MB is employed as an intercalating agent since its potential range is extremely near to the redox potential of biomolecules.MB has distinct redox reactions with single stranded (ss)-DNA and double stranded (ds)DNA due to its high discrimination affinity and is used to monitor native as well as hybridized states.Because of its attraction for free guanine bases, its reaction rises with PDNA contact, but decreases with the hybridization with TDNA (Target DNA), because it is inserted between the large number of double helices of dsDNA.Many researchers have used this interaction to develop electrochemical DNA biosensors [32,33].The results were obtained by performing electrochemical techniques such as cyclic voltammetry and differential pulse voltammetry and the resulting voltammogram was used to analyze basic information about redox reactions.In a groundbreaking study, Sengupta and Hussain [34] pioneered the use of graphene oxide-based electrochemical genosensors for the detection of SARS-CoV-2, highlighting the immense potential of biosensors in addressing global health challenges.Their research showcased the versatility of biosensor platforms in effectively tackling emerging infectious diseases, paving the way for rapid and accurate diagnostic tools.Additionally, Bommi et al [35] presented an innovative DNA biosensor with exceptional sensitivity and selectivity for the detection of cancer biomarkers.This study exemplified the broader applications of biosensors in precision medicine, enabling early detection of cancer and personalized treatment approaches.In the realm of food safety, Kuswandi et al [36] introduced a novel paper-based colorimetric biosensor for the rapid detection of food-borne pathogens.This low-cost and portable solution holds tremendous promise for ensuring the safety and quality control of food products, offering efficient and accessible detection methods.A comprehensive review conducted by Aggarwal et al [37] explored the recent advancements in plasmonic biosensors, shedding light on their underlying principles and highlighting their potential applications in biomedical settings.This review not only deepened our understanding of these innovative sensing platforms but also offered insights into their potential impact on various areas of healthcare.Furthermore, Ye et al [38] investigated the latest developments in wearable biosensors for healthcare monitoring.Their study emphasized the increasing significance of wearable technology in personalized healthcare and remote patient monitoring, showcasing the potential of biosensors in revolutionizing healthcare delivery.
Collectively, these remarkable studies exemplify the continuous progress in biosensor technology.They provide valuable insights and inspiration for future research, underscoring the immense potential of biosensors in diverse applications, including disease diagnostics, food safety, and personalized healthcare.

Materials
In this study, the pathogenesis-related oligonucleotide sequence of Pst was used as the probe.The probes are designed by Pst-specific genes (table 1).The sequence of Pst related to pathogenesis is downloaded from (http:// ncbi.nlm.nih.gov/)[39].Pst oligonucleotides were modified with an amine at the 5′ end.Nickel oxide thin film (100 nm) was deposited using high purity (99.9999%) nickel foil target by rf sputtering technique over ITOcoated glass substrates (Indium tin oxide, 2 cm × 1 cm) used as the matrix material.
Before deposition, the substrate was rigorously cleaned through a three-step process.The process included ultra-sonication of the substrate in trichloroethylene, acetone, and isopropyl alcohol for 10 min each followed by drying using nitrogen gas.
The liquefied solutions used in the study were prepared in deionized water to avoid contamination.The phosphate buffer saline (PBS) solution of pH 7.0 (50 mM) and Tris-EDTA buffer (10 mM Tris, pH 8.0, 1 mM EDTA) was prepared and further used in the study.

Nanostructured NiO thin film matrix synthesis
Sputtering was done applying basic vacuum pressure of approximately 6 × 10 −6 Torr.During the thin film deposition, a continuous supply of non-inert gaseous oxygen was introduced to maintain an oxygen atmosphere.The deposition pressure in the chamber was about 30 millitor.Alternating current was applied in the range of RF 40 W (voltage depends on the material and the objective size).Half of the substrate surface covering 1 cm × 1 cm was masked with aluminum foil to preserve its necessary electrical conductance in electrical measurements.Dejected NiO atoms deposited onto the unmasked area of the substrate were placed opposite to the sputtered target.

DNA bioelectrode fabrication
NiO/ITO electrode was used for sensing and different concentrations (1, 3, 5, 10, 50, and 115 ng μl −1 ) of probe DNA solution were prepared in TE buffer.The probe DNA solution (10 μl) was immobilized over the surface of the bare electrode followed by then keeping it in a humid chamber (incubator) for about 3 h at 25℃.The immobilization of the negatively charged DNA probe onto the positively charged nanostructured NiO matrix occurred via electrostatic interaction.The unbound probe DNA from (ss) DNA/NiO/ITO was removed by washing with TE buffer several times.For hybridization the (ss-DNA/NiO/ITO) electrode was incubated with varying concentrations of the target DNA at 25℃ for 3 h.Several piles of washings were done to remove the unbound probe DNA from the hybridized electrodes and electrochemical analysis was carried out.

Experimental characterizations of the bioelectrode
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) results are measured using an electrochemical instrument with high performance and research grade known as potentiostat/galvanostat [Gamry Inc. 600] with a three-electrode electrochemical cell.In the three-electrode system, the nickel oxide was used as a working electrode, platinum as an auxiliary electrode, and 5 mM [Fe(CN) 6 ] 3−/4− with Ag/AgCl as a capillary reference electrode (RE).The electrochemical response of the bioelectrode after hybridization for a given concentration of target DNA was carried out using a phosphate buffer solution containing MB (20 μM).The present study aimed at developing an electrochemical-based genosensor for the detection of wheat yellow rust pathogen.Pst-specific DNA sequences were attached to a NiO thin film constructed on an indium tin oxide (ITO) substrate.The applications of the NiO matrix in biosensors draw our attention in employing this matrix for Pst detection.Selected ss-DNA oligonucleotide sequences related to the pathogenesis of Pst have been employed as probes for sensing.These probes were immobilized onto NiO/ITO electrodes by physical adsorption and the electrochemical response of the bioelectrode was measured by performing cyclic voltammetry and differential pulse voltammetry studies in a MB mediated buffer using a potentiostat.A linear response with high sensitivity was obtained over a wide range of DNA concentrations.Eighteen NiO/ITO electrodes were manufactured for electrochemical study.DNA from yellow rust pathotype 78S84

Characterization of NiO/ITO electrodes:
Cyclic voltammetry studies were conducted to analyze the electrochemical performance of NiO/ITO electrodes using a potential range of −0.3 to 0.8 V. Ferro ferry cyanide mediated 50 mM PBS buffer solution was used as an external redox mediator to check the electrochemical response of the bare electrode, at a scan rate of 100 mV s −1 .The potential was scanned in both forward and reverse directions resulting in oxidation and reduction peaks.In the case of MB, no redox peak was observed (figure 1). Figure 2 shows that the value of peak oxidation current is too low when no external electrochemical indictor is present.In order to obtain a better biosensing response, it is very important to use an external redox mediator.Similarly, a pulse voltammetry study was carried out to analyze the electrochemical properties of NiO/ITO with and without MB.MB did not produce any redox peaks in the blank of the NiO/ITO electrode because it only emitted an electrochemical signal when intercalated with DNA.While using [Fe(CN) 6 ] 3/4− redox peaks were observed.DPV studies were found more sensitive than the CV and were mainly used for quantitative determinations while CV provides basic information, and the type of redox process present in the analysis.
Different concentrations of MB at 20 μM, 10 mM, and 1 mM were used in this study (figures 3 and 4).The electrochemical signal varied with different concentrations of methylene blue.The best results were obtained using 1 mM MB in 50 mM PBS (pH 7.0).Further, 1 mM MB concentration was used (figure 3). Figure 8 showed that when the concentration of MB was 20 μM, the peak oxidation current was too low.

Electrochemical characterization
In the present study, MB (methylene blue) was employed as an external redox mediator.At physiological pH, MB exhibits both oxidation and reduction reactions.Since MB carries a positive charge and both single-stranded DNA (ss-DNA) and double-stranded DNA (ds-DNA) possess a negative charge due to their sugar-phosphate backbone, electrostatic interactions occur among MB, ss-DNA, and ds-DNA.These interactions result in distinct electrochemical responses, as depicted in figure 5.When MB interacts with ss-DNA, an enhanced electrochemical response is observed.This can be attributed to the presence of free guanine bases in the ss-DNA sequence.MB readily binds to these free guanine bases, leading to an increased current response.Conversely, when MB reacts with ds-DNA, the electrochemical response diminishes.This is due to the formation of the DNA duplex, where the available guanine bases are paired with cytosine bases through hydrogen bonding.Consequently, no free guanine bases remain for MB to bind to.Instead, MB intercalates between the double helix structure of the ds-DNA, which results in a decrease in the observed current response.This behaviour is illustrated in figure 6.

Cyclic voltammetry studies for the detection of Pst
At first, the CV curve for ss-DNA/NiO/ITO and after that for varying concentrations of ds-DNA/NiO/ITO was obtained using 1 mM concentration of MB in a potential range of −0.3 V to 0.8 V at a scan rate of 100 mV s −1 .When ss-DNA as a probe was immobilized onto bare NiO/ITO electrode, a reduction in peak oxidation current was observed.The probe DNA was effectively bound to the electrode surface and blocked the conducting sites of the NiO/ITO electrode hindering the charge transportation.Further, reduction in current was observed when the complementary target DNA was immobilized and hybridization occurred amid the probe DNA and complementary target DNA.The decrease in current was due to the increase in the repulsive force between the anionic species present in the electrolyte and the negatively charged ds-DNA present on the electrode surface.
For the study of hybridization processes and electrochemical reactions, various concentrations of complementary target DNA ranging from 1 ng μl −1 to 115 ng/μl were used.The experiment was repeated with the same and different target DNA concentrations.In the repeat experiments also, a similar response was observed (figure 7).
A similar type of study was conducted by Tak et al [40] for the detection of meningitis by electrochemical DNA biosensor.In their study, they used Ni-ZnO thin film for the detection.The biosensor exhibits a detection range of 5 ng/ μl-200 ng μl −1 with a low detection limit of 5 ng/μl.

Differential pulse voltammetry (DPV) studies
The electrochemical response of the biosensor was also studied with DPV which is considered more sensitive as compared to CV because DPV current is measured immediately before each potential change thus providing a better current variation response.Electrochemical response for NiO/ITO, ss-DNA/NiO/ITO, and ds-DNA/ NiO/ITO was observed in MB (figure 8).The ss-DNA/NiO/ITO electrode was hybridized with varying  concentrations of target DNA (1, 3, 5, 10, 50, and 115 ng μl −1 ) and electrochemical response by DPV using potentiostat were recorded after every 30 s. Voltammogram for bare NiO/ITO electrode showed an appreciable MB peak at −0.18 V with an increase in current up to 0.042 mA.When ss-DNA/NiO/ITO was immobilized onto NiO/ITO electrode, DPV response showed reduced methylene blue peak current.As the concentration of target DNA increased (1-115 ng μl −1 ), a decrease in current was also observed.The saturation concentration of the ss-DNA / NiO / ITO bioelectrode was revealed at 115 ng/μl.No decrease in current was observed above the target DNA concentration.The calibration curve for the ss-DNA/NiO/ITO electrode with target DNA was established to assess the biosensor's sensitivity and detection capability.The response signals were measured and plotted against known concentrations of the target DNA.The resulting calibration curve provides a clear representation of the relationship between signal intensity and target DNA concentration.This curve serves as a crucial tool for quantifying unknown concentrations of target DNA in subsequent experiments, ensuring accurate and reliable detection within the biosensor system (figure 9).The results showed that single strands of probe DNA were hybridized into complementary target sequences.The experiment was repeated and similar electrochemical responses were observed.
The limit of detection for Pst using Ketopantoate reductase gene was found to be 0.4 ng μl −1 and 0.3 ng μl −1 respectively as below this concentration, no signals were observed.The sequence to design the probe was obtained from the sequencing of PCR amplified product of Pst pathotypes 78S84.The probe 2 sequences used for the biosensing were first checked for their uniqueness.BLAST alignment was done.The result showed that the sequence was 100% identical to Puccinia striiformis f. sp.tritici with 100% query cover.The genomic DNA of the Pst pathotype was isolated by the CTAB extraction method.The ds-DNA was given heat treatment to denature the double strands of the DNA followed by cooling at 75 °C for 3 min.The ss-DNA as a probe was immobilized onto the electrode and different concentrations of target DNA were used for the hybridization.

Electrochemical study for probe 1
DPV studies were performed on bare NiO/ITO, ss-DNA/NiO/ITO, and ds-DNA/NiO/ITO electrodes in PBS containing 1 mM methylene blue, and different electrochemical responses were recorded.For the ss-DNA/ NiO/ITO peak observed at −0.16 V, the current value is 0.07 mA.The ds-DNA/NiO/ITO bioelectrode was incubated with two different concentrations (1 ng μl −1 and 10 ng μl −1 ) of complementary target sequences.The electrochemical study was done by pulse voltammetry, and a decrease in current value was observed with increasing concentration of the target DNA (figure 10).

Electrochemical study for probe 2
Firstly, the probe was immobilized onto the bare NiO/ITO electrode.DPV studies were carried out on ss-DNA/ NiO/ITO and ds-DNA/NiO/ITO electrodes, and different electrochemical responses were obtained.The electrochemical reaction obtained aids in distinguishing the presence of the probe and ds-DNA on the electrode.For ss-DNA/NiO/ITO peak was observed at −0.16 V and the value of current was 0.075 mA.For ds-DNA/ NiO/ITO bioelectrode reduction in current value was observed when incubated with different target sequences (40 pg μl −1 , 1 ng μl −1 , and, 50 ng/μl).For the highest concentration of 50 ng μl −1 , the value of the current  detected was 0.012 mA.The lowest concentration of target DNA detected was 40 pg μl −1 (figure 11).Mohan et al [19] also performed a detection study for Leishmania donovani, causing Kala-azar.The ss-DNA probe was designed from 18 s rRNA gene sequences of the pathogen and hybridization was done with genomic DNA of L. donovani.They were able to produce a good linear response of DNA concentrations that ranges from 2 pg mL −1 -2 mg/ml showing the great potential of NiO in the development of sensitive and stable biosensors.

Reproducibility studies
To check the reproducibility, five NiO/ITO electrodes under similar processing conditions were prepared.10 μl of probe DNA solution (50 ng/μl) was used to immobilize all five electrodes.For hybridization, 50 ng μl −1 target DNA concentration was used.The repeatability of the bioelectrode was tested by a DPV response study, and it was found that even after one month of manufacture, the same biosensor gave the same response (within RSD 5%) indicating that it could be reused.

Conclusion
In this study, we successfully developed a cutting-edge DNA biosensor by harnessing the power of NiO/ITO bioelectrode technology for the accurate detection of Puccinia striiformis DNA sequences.Our biosensing platform employed a state-of-the-art nanostructured NiO-based thin film, a robust three-electrode system, and methylene blue as an intercalating agent.For analysis, we utilized the electrochemical techniques of cyclic voltammetry and differential pulse voltammetry.
Our developed biosensor exhibited exceptional performance, showcasing a rapid response time and demonstrating a broad-spectrum detection range spanning from 40 pg μl −1 to 115 ng/μl.It achieved remarkable precision with a relative standard deviation (RSD) of 5% and a remarkably low detection limit of 40 pg μl −1 .Moreover, the biosensor demonstrated remarkable reproducibility, enabling consistent and reliable results.
The integration of the nanostructured NiO film played a pivotal role in enhancing the biocompatibility, electrochemical activity, and shelf life of the electrode.This innovative approach effectively served as a tracer label for electrochemical detection, enabling highly selective and sensitive detection of the target Puccinia striiformis DNA.Furthermore, the biosensor exhibited reusability by interrupting the hybridization process onto the bioelectrode, enhancing its practicality and cost-effectiveness.Our developed biosensor holds immense potential for early detection of yellow rust, enabling the implementation of appropriate crop protection management strategies and facilitating swift recovery.Additionally, it can serve as a valuable screening tool in wheat breeding programs, assisting breeders and pathologists in selecting resistant germplasm or advanced lines that can combat Puccinia striiformis infection.As we look to the future, the latest research in biosensor technology, continues to drive advancements in the field.These ground-breaking studies underscore the expanding scope and transformative potential of biosensors in addressing global health challenges, precision medicine, and food safety.
In conclusion, our study has presented the development and characterization of a nanoscale genosensor utilizing a thin film of nanoscale NiO.This genosensor, designed for the specific detection of DNA sequences associated with Puccinia striiformis f. sp.tritici (Pst), represents a significant advancement in the field of biosensing.One of the key merits of our method lies in its ability to fill a critical gap in the detection of Pst.While previous methods, such as surface plasmon resonance (SPR) immunoassays, have been reported for Pst detection, the development of DNA-based biosensors tailored specifically for Pst detection remained unexplored.Our novel approach addresses this limitation, introducing a new method that overcomes the challenges and shortcomings of existing techniques.Moreover, our genosensor leverages the advantages of DNA-based biosensors, including their remarkable sensitivity and precision.This innovation enhances the accuracy and efficiency of Pst detection, making it a valuable tool for disease management and crop protection.The nanoscale electrochemical DNA sensor developed in this study offers outstanding sensitivity, extended shelf life, and reliable recovery, significantly reducing the likelihood of erroneous results.
A noteworthy highlight of our research is the pioneering use of conserved sequences associated with pathogenesis in selected Pst strains for the development of a nanoscale genosensor.This breakthrough not only contributes to the advancement of biosensing technology but also holds promise for improving disease management strategies and safeguarding wheat crops from the devastating effects of Pst.
In conclusion, our study showcases the successful development of an innovative DNA biosensor utilizing the NiO/ITO bioelectrode, enabling effective detection of Puccinia striiformis DNA sequences.This innovative approach showcases the potential of nanomaterials and electrochemical sensing in agriculture, paving the way for advanced disease detection and crop protection.Continued research and development in this field hold promise for the advancement of precision agriculture and sustainable food production.The future of biosensors holds tremendous promise in revolutionizing various domains, offering tailored solutions for real-time monitoring, early detection, and sustainable management.In the pursuit of advancing diagnostic capabilities, it is evident that there is a need for further research and development of novel rapid sensors capable of accurately detecting pathogens in samples of varying volumes.Currently available methods may not be optimized to handle different aliquot sizes, leading to potential limitations in sensitivity and performance.
To overcome these challenges, future research should prioritize the design and fabrication of multiplexed analytical sensors.These advanced sensors would integrate multiple assay techniques within a single device, offering a more comprehensive and synergistic approach to pathogen detection.By combining the strengths of different assay methods, such as molecular, immunological, and biochemical techniques, the sensor can achieve optimal sensitivity and accuracy.Moreover, the cost-effectiveness of such a sensor should be taken into account during the development process.Affordable and accessible diagnostic tools are crucial, especially in resourcelimited settings, where rapid and accurate detection of pathogens is vital for public health management.Secondly, an efficient and multiplexed sensor would reduce the need for multiple specialized devices or tests, streamlining the diagnostic process and saving valuable resources.
Overall, future research efforts focused on developing such a multiplexed analytical sensor have the potential to revolutionize pathogen detection, providing a significant leap forward in healthcare, agriculture and public health strategies.By creating a versatile and adaptable tool capable of accurate detection across different aliquot volumes, we can empower professionals with a more efficient means of diagnosing infections and responding to emerging infectious threats.

3. 1 .
Development of electrochemical DNA biosensor for detection of Pst on indium doped tin oxide (ITO) electrode fabricated with NiO thin film

Figure 3 .
Figure 3. Cyclic Voltammogram curve of ss-DNA/NiO/ITO with different concentration of target DNA using 1 mM and 10 mM concentrations of methylene blue.

Figure 4 .
Figure 4. Cyclic Voltammogram curve of ss-DNA/NiO/ITO with different concentration of target DNA using 20 μM of MB.

Figure 6 .
Figure 6.Schematic representation of bio sensing mechanism for ss-DNA and ds-DNA on NiO/ITO electrode with MB as redox indicator.

Figure 7 .
Figure 7. Cyclic Voltammogram with different concentration of target DNA using 1 mM of MB.

Figure 8 .
Figure 8. DPV with varying concentrations of target DNA using 1 mM concentration of MB.

Figure 9 .
Figure 9. (a) and (b) Calibration curve for the ss-DNA/NiO/ITO electrode with target DNA.

Figure 10 .
Figure 10.DPV curve with different concentration of target DNA using 1 mM of MB.

Figure 11 .
Figure 11.DPV curve with different concentration of target DNA using 1 mM of MB.

Table 1 .
The pathogenesis related oligonucleotide sequences of Pst, used in the study.