µPED based electrode modified with nanocomposite for the detection of proline: An abiotic stress biomarker in plant
1Smart Agriculture Innovation Center, Kyungpook National University, Daegu, Republic of Korea.
2Major in Bio-industrial Machinery Engineering, Kyungpook National University, Daegu, Republic of Korea.
Plants when exposed to stressful conditions, accumulates array of metabolites. Among them proline plays significant and beneficial role as osmolyte, metal chelator and as anti-oxidative defense and regulator molecule. Thus the proline content is one of the potent bio-marker for the early detection of abiotic stress in plant. In this work a microfluidic paper-based electrochemical device (µPED) modified with copper oxide nanoparticles (CuO) decorated reduced graphene oxide (rGO) nanocomposite is utilized to develop disposable system for the detection of proline. Wax and screen printings were utilized to create microfluidic chambers and electrode pattern on paper device, respectively. Portable potentiostat was used for measuring the current in response from the addition of proline. The proposed sensor exhibits excellent electrochemical performance with broad linear responses over a proline concentration ranging from 0.1 mM to 7.5 mM (R2=0.99) and showed lower detection limit of 0.05 mM at 0.4 V vs. pseudo Ag/AgCl. Excellent electrochemical performance may be attributed to presence of electroactive rGO and catalytic activity towards proline is due to the coordination between proline and copper (II) oxide nanoparticles. These results demonstrate that the proposed paper-based electrochemical sensor can be utilized in disposable and portable way to detect proline content in the abiotic-stressed plants and has potential to be applied in on-site application.
abiotic stress, proline, paper-based electrochemical device, rGO, CuO
Recently, frequent occurrence of abiotic stress in plants (for instance, drought, salinity, flood, exposure to heavy metals and extreme temperature) due to rapid global climate changes bring frequent and severe extreme weather events makes plants vulnerable and disturb plant growth (Mittler, 2006). Proline (pyrrolidine-2-carboxylic acid) one of the primary amino acid plays very important role in maintaining homeostasis of plants when subjected to abiotic stress conditions. There are many reports indicates the positive role of proline accumulation and plant tolerant to various abiotic stress. For instances it acts as osmo-protectant, molecular chaperons and scavenges reactive oxygen species, metal chelator and also acts as potential nitrogen and carbon source (Ahmad and Wani, 2014; Gupta et al., 2015; Iqbal and Nazar, 2015).
Due to the strong co-relation between various stress condition and the accumulation of proline and to evaluate role of proline in various stress condition on different plant parts, there is high demand to develop simple, economical and portable detection system for quantification of proline is essential. Due to its importance, several analytical methods including gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC) with fluorescence and UV-based detection system, electrochemical methods and direct spectroscopic based methods based on reaction with ninhydrin and proline were developed (Edit et al., 2010; Lee et al., 2018; Liu et al., 2017). Chromatography based systems requires highly sophisticated instrument and are expensive and requires skilled labor for the operation. Ninhydrin based optical method is most commonly used method for proline detection but prone to interference from other related amino acids and requires high temperature for the reaction. Compared to the conventional analytical methods, paper-based microfluidic analytical device (µPADs) offers significant advantages including cost-effectiveness, ease of fabrication and use. These devices have gathered considerable attention for point-of care applications in food safety testing, environmental monitoring and clinical diagnostic (Adkins et al., 2017; Park et al., 2017; Wang et al., 2012; Yetisen et al., 2013). Recently, multilayer µPADs for colorimetric quantification of proline was developed by Choi et al (2020). Our group also developed smart-phone integrated semi-enclosed paper sensor for sensitive detection of proline water stressed A. Thaliana plants. These paper-based sensors demonstrated that they have better field applicability (Santhosh and Park, 2022).
Application of electrochemical methods for the detection of biomarkers in plants is gaining rapid attention due to their unique advantages of high accuracy, low detection limit, ease of portability, fast response and simple instrumentation. Few abiotic stress biomarker including phyto-hormones such as abscisic acid, salicyclic acid, indole acetic acid and ethylene were detected using electrochemical sensor (Li et al., 2021).
Amalgamation of electrochemical detection in paper based analytical devices is believed to be open more opportunities and combines exciting features of both methods. Additionally, it is demonstrated that effective utilization of carbon based nanomaterials such as carbon nanotubes and graphene could effectively enhances the analytical performance of these device. Moreover there is a growing demand for developing in-situ methods to monitor plant stress marker in simple, instrument-free, portable, non/minimally invasive and cost-effective way. In this regards paper as substrate offer great advantage due its ubiquitous availability, portability, low cost, ease to fabricate and can be easily integrated with electronic structure. Recently, paper-based analytical device for in-situ electrochemical detection of in diseased tomato leaves. Utilization of carbon nanotube/Nafion as electrode material significantly improves the response (Sun et al., 2014).
Inspired by the exciting integration of electrochemical methods on paper platform and potential advantages of these devices in real time monitoring of stress biomarker, in this work we have designed microfluidic paper based electrochemical device (µPEDs) for detection of proline. As a response layer we are utilizing rGO-CuO nanocomposite layer due to the fact that the proline is a known chelating agent and the formation of Copper (Cu (II)/Cu (I)) – proline complexes are spontaneous and thermodynamically favorable. Hence in this study, for the first time we are utilizing the favorable interaction of Cu (II/I) and proline for developing µPEDs electrodes for detection of proline. In the study µPEDs were modified with rGO decorated with CuO nanoparticles. The modified electrodes were electrocatalytically active toward the oxidation of proline and significantly reduce the oxidation potential. These results suggest that µPED based electrode could have the potential to become effective platform for monitoring abiotic stress marker in plants.
Materials and Methods
Chemicals and reagents
Proline, copper sulfate pentahydrate, phosphate buffer saline (PBS) tablet, potassium chloride (KCl), potassium ferric/ferrous cyanide Ag/AgCl conductive ink was purchased from Sigma-Aldrich (St. Louis, MO, USA). Whatman (no.1) chromatographic paper was purchased from GE health care. Graphene oxide (GO) was obtained from graphene supermarket. Carbon ink was purchased from Asahi chemical research laboratory (Gifu, Japan). All other reagents were of analytical grade and used without further purification. All solutions were prepared using deionized water (18.2 MΩ).
Design and fabrication of µPED based electrode
Figure 1 shows the design and dimension of 3D µPEDs. It consists of two rectangular wax patterned paper tab (auxiliary tab and sample tab) with sample zone of 1 cm diameter. Wax printing was carried out using Xerox wax printer (ColorQube 8570, Xerox, Norwalk, CT, USA). The counter and working electrodes were patterned on these paper tabs using custom-made screen-printing apparatus with silk screen frame of mesh size 100.
Fig. 1. Schematic representation of µPED design (A), picture of fabricated µPED (B) and assembled µPED with electrodes (C).
Synthesis of CuO decorated rGO
The GO was reduced by hydrazine method. In a typical reaction, 20 ml of reaction mixture containing 5 % NaOH, 10 mg of CuSO4 and 3 mg of GO was allowed to interact at 70 ℃ for half an hour after that 500 µl of 80 % hydrazine was added and the reaction mixture stirred continuously for another 45 min. the solution color changed to dark black color. The sample was centrifuged at 5,000 rpm. The pellet was collected and washed with several times. The obtained CuO-rGO was dried at 60 ºC for overnight. The scheme of synthesis of CuO decorated rGO is represented in figure 2.
Fig. 2. Schematic representation of synthesis of rGO decorated with CuO nanoparticles.
Measurement of electrochemical properties
All electrochemical work including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS and chrono-amperiometric (CA) experiments were carried out using PalmSens4 (Netherland) portable electrochemical interface. The figure 3 represents the experimental design setup. CV and CA were performed in PBS (10 mM, pH 7.0) with or without proline whereas EIS were conducted in PBS solution containing 25 mM of potassium a ferric /ferrous cyanide and 0.1 M KCl redox solution. The surface morphology of CuO-rGO and changes in the surface morphologies of the electrode during the electrode fabrication process were characterized by the field emission scanning electron microscope (Hitachi, SU8220), with EHT 3–5 kV.
Fig. 3. Experimental work set up for the electrochemical characterization of µPED electrode.
Results and discussion
Synthesis and characterization of CuO-rGO nano-conjugate
Figure 4 represent the UV-Vis spectra of GO, CuO and CuO-rGO. In case of GO, an absorption peak appeared at 232 nm, which was attributed to aromatic double bond and around 310 nm a peak appeared that was attributed to n → π* and π → π* transitions for C=O absorption. For the prepared rGO/CuO nanocomposite, absorbance maximum appeared at 300 nm, which was attributed to n–π* and π–π* transitions and these transitions occurred because of aromatic carbon–carbon bond and carbonyl group presence. The appearance of the absorption peak at 255 nm indicated the reduction of GO and replacement of OH and COOH group with CuO. It has been reported that red shift corresponds to the reduction of GO due to functionalization. The greater red shift indicated the complete reduction of GO.
Fig. 4. UV-Vis spectra of rGO (black trace), CuO nanoparticles (red trace), CuO-rGO nanocomposite (blue trace).
Morphological characterization of prepared CuO-rGO nano-conjugate were also studied using FESEM
The FESEM image of synthesized CuO-rGO is shown in figure 5(A) which clearly reveals the uniform sized CuO nanoparticles decorated on the surface of rGO nanosheets which appears as wrinkled sheets. CuO nanoparticles of size ranging from 50 nm to 60 nm were found to be evenly distributed on the surface of rGO. The surface morphologies of bare µPED electrode, rGO modified and CuO-rGO modified electrodes were represented in figure 5 (B), (C) and (D) respectively. Bare electrode displayed smooth surface as shown in figure 5(B), when they modified with rGO, they displayed a crumpled and wrinkled layer like morphology which is typical rGO nanosheets (figure 5C). After modification with CuO-rGO, the electrode surface displays that the rGO nanosheets with decorated CuO nanoparticles were fully covered on the electrode surface (figure 5D).These results indicate the successful immobilization of CuO-rGO nano composite on the µPED electrode surface.
Fig. 5. FESEM images of CuO-rGO nanocomposite (A), bare electrode (B), rGO modified electrodes (C) and CuO-rGO modified electrode surface (D).
Electrochemical characterization of the modified electrodes and electro-oxidation of proline
Electrochemical features of µPED electrode modified with rGO and CuO-rGO were studied by EIS and CV techniques. Figure 6(A) represents EIS spectra represented as Nyquist plot of the modified electrodes. Resistance to charge transfer (Rct) is usually represented as diameter of semi-circle of the Nyquist plot is high for bare µPED electrode (red trace), after modification with rGO, the diameter of the semi-circle is reduced indicating the conductive nature of rGO. After modification of µPED electrode with CuO-rGO the semi-circle of Nyquist plot was approximately straight line indicating the presence of CuO nanoparticle on rGO surface facilitated the electron transfer and is more electroactive compared to the bare and rGO modified µPED electrodes.
Fig. 6. EIS spectra represented as Nyquist plot of bare (red trace), CuO nanoparticles (purple trace) and CuO-rGO-modified µPED electrode (black trace) (A). The CV curves of GO (black trace), CuO-rGO –modified µPED (red trace) electrode in the presence of proline (blue trace) (B).
To investigate the catalytic activity of rGO and CuO-rGO modified electrodes, the CV studies were carried out. Figure 6(B) represents the cyclic voltammogram of the rGO, CuO-rGO modified electrodes in the presence of proline in PBS (pH 7.0). The rGO modified electrode CV doesn’t have any features in the potential scan range (black trace), after modification with CuO-rGO nanocomposite, an oxidation peak is observed at 0.1 V vs. pseudo Ag/AgCl. This peak is due to oxidation of exposed Cu to Cu (I) and Cu (II) species. In presence of proline another peak appears at 0.4 V vs pseudo Ag/AgCl is mainly due to the oxidation of proline to poly proline as indicated by equation.
2Proline + 2 CuO + 2 e− → H2O + poly − proline + Cu2O
CuO-rGO modified electrodes displays more current response demonstrating that these modified electrodes possesses outstanding electronic conductivity owing to the presence of uniformly decorates CuO on rGO, both of them possess excellent conductivity. While excellent electrocatalytic activity towards proline may be attributed to the co-ordination of proline with Cu, the unique pyrrolidine ring of the proline has inherent chelating activity. Once coordinated, proline is oxidized at the electrode surface to create ply-proline and hence the oxidation current increases with addition of proline.
CuO-rGO modified µPED electrodes for proline sensing
The CV of CuO-rGO modified µPED electrodes were shown to produce increased response at 0.4 V vs pseudo Ag/AgCl in the presence of proline. Chronoamperometry which measures current in the sensor over time with addition of analyte is conducted to generate current –time (IT) curve. Figure 7(A) shows the CA curves recorded at the CuO-rGO -modified µPED at various concentration of proline at potential of 0.4 V vs pseudo Ag/AgCl. The steady state current for each proline concentration is plotted against the concentration to extract a response curve, as shown in the figure 7(B) where the current recorded at 100 s is plotted against the corresponding proline concentration. At applied potential of 0.4 V, the CuO-rGO modified µPED electrodes exhibited long linear response range from 100 µM to 7.5 mM(R2 =0.99) and showed lower detection limit of 0.05 mM at 0.4 V vs. pseudo Ag/AgCl. The long linear range of the proposed sensors attributed to high electroactive surface area of the rGO and excellent electrocatalytic activity of the CuO nanoparticles.
Fig. 7. Amperometric responses of the CuO-rGO-modified µPED electrodes at different concentration of proline (A). Calibration curve generated from the response of modified electrode over proline concentration (B).
We have successfully utilized µPEDs modified with copper oxide nanoparticles (CuO) decorated reduced graphene oxide (rGO) nanocomposite to develop disposable system for the detection of proline. In-situ formed CuO nanoparticles appear to be uniformly decorated on rGO nanosheets act as efficient electro catalyst for the oxidation of proline. The excellent electrical conductivity and good catalytic activity of these nanocomposite electrode resulted in the good performance of the CuO-rGO modified µPED. The proposed sensor exhibited wide linear range., Extraction of proline from plant source is one of the critical issues for measuring proline content, traditionally sulphosailic acid based extraction method was utilized to extract proline from plant lysate. In the current paper-based assay we recommend to utilize the extracting reagent stored within the upper layer of µPED to extract proline from the plant. Plant parts been minimally damaged to ooze out liquid exudate which eventually moved to the lower detecting zone via upper layer. We also predict that the stable nanocomposite in the sensing layer is minimally affected by humidity and external temperature changes as it contains no biological sensing materials such as enzymes or antibodies, this sometime maybe unfavorable due to some potential interfering agents present in the complex sample matrix. However, with this modification the current paper-based disposable device with portable electrochemical reader has potential to be applicable for on-site measurement of proline content and monitor the stress in plants.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs Convergence Technologies Program for Educating Creative Global Leader, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Project No. 320001-4), Republic of Korea. We also acknowledge center for scientific instruments, Kyungpook National University, Daegu for providing FESEM and XPS instrumentation for the analysis.
Conflict of interest
All authors declare that they have no conflicts of interest.
Adkins JA, Boehle K, Friend C, Chamberlain B, Bisha B, Henry CS. 2017. Colorimetric and Electrochemical Bacteria Detection Using Printed Paper- and Transparency-Based Analytic Devices. Analytical Chemistry 89: 3613-3621.
Lee MR. Kim CS. Park T. Choi YS. Lee KH. 2018. Optimization of the ninhydrin reaction and development of a multiwell plate-based high-throughput proline detection assay. Analytical Biochemistry 556: 57-62.
Liu L. Zhang D. Jin Z. Zhang Z. Li S. Cang J. 2017. Development of an electrochemical approach for proline content detection in winter wheat. International Journal of Electrochemical Sciece 12: 3020-3029.
Sun LJ. Feng QM. Yan YF. Pan ZQ. Li XH. Song FM. Yang H. Xu JJ. Bao N. Gu HY. 2014. Paper-based electroanalytical devices for in situ determination of salicylic acid in living tomato leaves. Biosensors and Bioelectronics 60: 154-160.
Wang S. Ge L. Song X. Yu J. Ge S. Huang J. Zeng F. 2012. Paper-based chemiluminescence ELISA: Lab-on-paper based on chitosan modified paper device and wax-screen-printing. Biosensors and Bioelectronics 31: 212-218.