SENSOR FOR PREDNISOLONE DETECTION IN SPORTS DOPING

ABSTRACT Introduction: Prednisolone causes pro-inflammatory impulses to be inhibited and anti-inflammatory signals to be promoted. As a result, it alters how the body's immune system reacts to certain diseases. The World Anti-Doping Agency, however, has banned SNP and other glucocorticosteroids. An electrochemical sensor can be developed using a gold nanocomposite, polypyrrole nanoparticles and synthesized carbon nanotubes (Au-PPy NPs@CNTs). Objective: Develop an electrochemical sensor to detect prednisolone. Method: Au-PPy NPs@CNTs nanocomposite was chemically synthesized with a modified glassy carbon electrode (GCE) surface. Results: According to SEM data, the nanocomposite was composed of amorphous Au NPs, and PPy NPs deposited in tubes strongly entangled in a CNTs network. The wide linear range and low detection limit of the Au-PPy NPs@CNTs/GCE as prednisolone sensors were attributed to the combined catalytic performance of the Au and PPy NPs@CNTs nanostructures. Conclusion: The results of prednisolone detection in each specimen using the amperometric method indicated good accuracy. The accuracy and precision of Au-PPy NPs@CNTs/GCE for prednisolone detection were explored in blood samples from 5 young athletes aged 20-24 years who used prednisolone tablets (RSD less than 4.25%). In addition to monitoring prednisolone concentrations in athletes’ serum, Au-PPy NPs@CNTs/GCE can be used as a reliable prednisolone sensor. Level of evidence II; Therapeutic studies - investigating treatment outcomes.


INTRODUCTION
Prednisolone (PNS) is a steroid that is used to treat a wide range of ailments, including arthritis, hormone imbalances, ulcerative colitis, psoriasis, allergic disorders, and kidney and blood cell issues 1 .In order for receptor-glucocorticoid developments to migrate into cell nucleus, somewhere they advantages of this method by the Binding area and link to glucocorticoid reaction basics, PNS molecules diffuse through cell membranes and link to glucocorticoid receptors. 2It causes pro-inflammatory impulses to be inhibited and anti-inflammatory signals to be promoted.As a result, PNS alters how the body's immune system reacts to certain diseases.Prednisolone's most frequent adverse effects include excessive perspiration, weight gain, indigestion, and insomnia.
The World Anti-Doping Agency, however, has prohibited PNS and other glucocorticosteroids. PNS can be administered orally or intravenously to muscles to lessen the discomfort and inflammation that are frequently brought on by vigorous exercise.Athletes claim that glucocorticosteroids assist them endure the discomfort of intense exercise and enable them to recuperate more quickly for the following event. 3NS use for a brief period of time can significantly boost performance in submaximal exercise.As a result, it might make it impossible for there to be fair competition.As a result, a lot of research has been done using techniques like liquid chromatography, 4 mass spectrometry, 5 gas chromatography, 6 ultraviolet detection, 7 fluorogenic DNA Probes, 8 Raman spectroscopy, 9 and electrochemical methods [10][11][12] to determine PNS in clinical specimens and body fluid.Between these techniques, electrochemical methods have been established as a straightforward, inexpensive, and accurate solution for the food and medical industries, as well as for environmental monitoring.By modifying electrodes with a variety of nanostructures, bio-recognition components, and compositions, electrochemical sensors' durability, selectivity, and sensitivity can be improved.In order to detect PNS as just doping agents in sport, this study absorbed on the development of electrochemical sensors are based on an electrode modified with Au-PPyNPs@CNTs.

METHOD Exprimental details
The precursor was ultrasonically added to 1 ml of L-cysteine hydrochloride monohydrate and 1 ml of cysteamine aqueous solution for the production of Au NPs. 13 1 ml of 10 mM NaBH 4 aqueous solution was gradually added to the solution after 20 minutes of ultrasonication.The resulting mixture was then allowed to react for 120 minutes.Six grams of polyvinyl alcohol were added to the solution and stirred magnetically for 30 minutes in order to create the Au-PPy NPs. 14 Au NPs and PPy NPs have a constant mass ratio of 1:1.The polymerization reaction was then started by adding 3 g of FeCl 3 to the product while magnetic stirring it at 6°C.After 10 hours, the liquid phase was filtered to remove the sediments, and the sediments were then washed with deionized water.The generated Au-PPy NPs were disseminated in ethanol using ultrasonography.
CNTs were refluxed for an entire night at 90 °C in 3 M HNO 3 , which led to minor oxidation and functionalization of the side walls of the CNTs. 15The treated CNTs were then filtered, washed with ultrasonic energy, and dried.After that, the CNTs were dissolved using ultrasonic technology in ethanol and introduced to the suspension of Au-PPy NPs.The resulting mixture was then poured onto GCE and allowed to dry to create the Au-PPy NPs@CNTs nanocomposites.The mass ratio of CNTs to Au-PPy NPs was set at 3:1.
Five young athletes, aged 20 to 24, whose blood serum was provided, used PNS pills, which each contain 10 mg of PNS.After 3 days of taking the tablets, plasma serum samples were given, and they were centrifuged at 2000 rpm for 10 min.The supernatants that were produced were harvested and used to make 0.1M PBS that was used as actual samples.Au-PPy NPs@CNTs were used in produced genuine samples from blood samples at -0.95V to use the amperometric to detect the PNS concentration in serum samples.PNS levels in blood serum samples were also examined using enzyme-linked immunosorbent test kit (ELISA).Scanning electron microscopy (SEM) was used to describe the morphologies of changed electrodes.All the procedures and the investigation in human subjects were conducted based on the Malaysian Guideline for Independent Ethics Committee Registration and Inspection, and National Statement on Ethical Conduct in Human Research with approval H21REA1103/HREC.

RESULTS AND DISCUSSIONS
The SEM images of the CNTs and Au-PPyNPs@CNTs materials are displayed in Figure 1. Figure 1a's SEM picture of the CNTs film shows a network of densely tangled tubes with sizes between 50-100 nm that are dispersed across the GCE during in the drop casting.Figure 1b showed that the Au-PPy NPs@CNTs nanocomposite was made up of Au-PPy NPs that had been deposited on the f-CNT network.The Au-PPy NPs are attributed to the adsorption of CNTs by chemical complex interaction as a result of the abundant functional groups of CNTs.As can be seen, pure Au NPs are covered by the PPy layer because the surfaces of Au-PPy NPs is uniform. 16The electrocatalytic activity may be improved by the formation of additional active sites on Au-PPyNPs@CNTs.Additionally, the polymer matrix makes it easier for CNTs to effectively transmit load from a polymer matrix. 17µm The DPV curves for GCE,CNTs/GCE, Au@CNTs/GCE, and Au-PPy NPs@CNTs with 0.1M PBS at a scanning speed of 10mV/s in the absence and presence of 5µM PNS are shown in Figure 2 for potential ranges 2. DPV curves ofGCE,CNTs/GCE,Au@CNTs/GCE andAu-PPy NPs@CNTs in 0.1M PBS at 10mV/s scan rate into 5µM PNS.  of -0.1 to 0.6V.Prior to the addition of 5µM PNS, no redox peak could be seen on the DPV graphs of GCE,CNTs/GCE, Au@CNTs/GCE and Au--PPy NPs@CNTs.Anodic peak at 0.25V after adding 5µM PNS to the electrochemical cell was attributed to the decrease of PNS. 11The keto group at position 3 of prednisolone, which provide a carbonyl group coupled with the double bond and produce matching hydroxyl into 2H + , 2e reaction, are the sites suitable for reduction. 10,11A Au-PPy NPs@ CNTs exhibits the larger electrocatalytic current when the DPV curves of the electrodes are compared, showing a sensitive reaction because of the synergic catalytic action of the nanocomposite Au-PPy NPs@ CNTs.Additionally, the CNTs/GCEand Au@CNTs/GCE comparisons demonstrate how Au nanostructures enhance catalytic activity, and the mixture of Au NPs and CNTs has produced a new category of hybrid nanostructures with effective ionic diffusion, that also led to an increase in charge transport due to the formation of globular diffusion regions from around electrode materials. 18dditional research was done to assess the stability of the electrodes' response to the additional PNS.First and 40th DPV curve for the GCE,CNTs/GCE, Au@CNTs/GCE, and Au-PPy NPs@CNTs within 0.1M PBS containing 5µM PNS are shown in Figure 3 at scan rates of 10mV/s and potential ranges of -0.1 to 0.5V, respectively.After 40 further scans, it can be seen that the maximum peak density of electrodes at -0.95V decreases.The peak current change for GCE, CNTs/GCE, Au@CNTs/GCE, and Au-PPy NPs@CNTs has been observed to be 24 percent, 13 percent, 14 percent, and 3 percent, respectively, after 40 continuous scans.This shows the advanced stability of the electrochemical reactions of Au-PPy NPs@CNTs due to the mixture of -ATP molecule with the very stable nanostructure of CNTs and AuNPs.
Additionally, self-assembled of -ATP molecule was generated on the CNTs and Au nanostructures by thiol groups during the electropolymerization, which pointed to the material's high stability. 19,20For the following electrochemical investigations on PNS, Au-PPy NPs@ CNTs were employed as a extremely sensitive and improved electrode.
Au-PPy NPs@CNTs were successively injected with 20M PNS solution with 0.1M PBS, and Figure 4 shows the amperometric reaction and subsequent calibration curve.With each PNS injection into the electrochemical cell, I can see a linear improvement in the amperometric current.The suggested sensor's sensitivity, detection limit, and linear-range are determined to be 0.1984 µA/µM, 0.005 µM, and 0-200 µM, respectively.The wide-linear range and reduced limit of detection of Au-PPy NPs@CNTs as a PNS sensor, which are due to the nanocomposite's synergistic catalytic action, were demonstrated when these sensing properties are compared to those of other PNS sensors.Due to their strong conductivity and substantial specific surface area, CNTs and Au nanostructures improve sensitivity. 21In order to produce stable host-guest complexes, the functional monomer ρ-ATP interacts strongly with the nanocomposite. 22,23 ing the amperometry technique in 0.1M PBS, the sensitivity of Au-PPy NPs@CNTs as just a PNS sensor was further investigated in the presence of numerous metabolic compounds in bodily fluids and medications as interfering agents.After adding 1µM PNS and 20µM interfering agents successively, the resultant amperometric current is shown in Table 1.The amperometric reaction of Au-PPy NPs@CNTs to the addition of PNS was demonstrably higher than that of interference agents, and trying to interfere agents exhibit negligible electrocatalytic signals, according to the results.As a result, employing Au-PPy NPs@CNTs to determine PNS is not hindered by the interference agents in Table 1.The effective recognition locations inside the molecularly imprinted polymeric membrane for target molecule are related to the sensitivities of Au-PPy NPs@CNTs. 24Additionally, the polymerization of polymer electrolytes onto Au@CNTs nanocomposites, that supports biocompatibility and electroactivity, improves the conductance of molecularly imprinted sensors. 25

CONCLUSIONS
In order to identify PNS as a sport doping agent, Au-PPy NPs@CNTs were electrochemically studied in this work.In order to create the sensors, the Au@CNTs composites was electropolimerized with ATP and TBAP on the Au@CNTs/GCE surface.According to the results of SEM and XRD investigations, the Au NPs were efficiently electropolimerized on Au@ CNTs/GCE and were uniformly deposited on CNT porous networks in

Figure 4 .
Figure 4. Amperometric and calibration plots of Au-PPy NPs@CNTs to consecutive injection of 20µM PNS solutions into 0.1M PBS.