17:15 〜 18:45
[AOS13-P13] Pseudoknot aptamer based cortisol biosensor for fish
キーワード：コルチゾール、アプタマー、バイオセンサー
Introduction
To increase fish culture efficiency, it is expected that stress of cultured fish can be measured in real time and then rearing environment can be controlled. Cortisol levels in plasma have been reported as an indicator of stress levels in fish. Cortisol is a steroid hormone secreted during a stress response. When fish are stressed, cortisol levels temporarily increase by an amount corresponding to the intensity of the stressor. Conventional cortisol detection methods include Liquid Chromatography-Mass Spectrometry (LC-MS) and Enzyme-Linked Immunosorbent Assay (ELISA). Although these two methods are very good methods for detecting hormonal substances, they both take time from the sampling of blood and other samples to the acquisition of detection results, resulting in indirect test results. In contrast, electrochemical biosensors are suitable for in-situ testing at fish culture site because they have the advantages of high sensitivity, detection with a small amount of sample, short detection time, easy miniaturization, and low cost. This study aimed to develop a biosensor capable of comprehensively diagnosing stress levels in fish by modifying aptamers for cortisol detection on gold electrode (Fig. 1).
Method
Design of aptamer for cortisol recognition
The sequence of the aptamer for cortisol detection used in this study is as follows. The number of bases is 71 bp, 5' end thiolate to enable aptamer modification via Au-S bond on the gold electrode. 3' end aminate to enable modification of the mediator via amide bond, ferrocenecarboxylic acid modification via an amide bond.
5'-SH-CTCTCGGGACGACGCCAGAAGTTTACGAGGATATGGTAACATAGTCGTCCCTTTT
TTTTTTTTGGTCTTCA-NH2-3'
Preparation of aptamer-modified electrode
2 micro M aptamer solution was allowed to stand at 95 degrees Celsius for 5 min, followed by 15 min at room temperature and 5 min on ice to form a loop structure. Then, 10 micro l was dropped onto a washed gold disk electrode and immobilized overnight, followed by washing with phosphate buffered saline (PBS) and air drying.
Preparation of ferrocene-labeled aptamer-modified electrode
By mixing equal amounts of 80 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride :WSC solution and 20 mM N-Hydroxysuccinimide :NHS solution and adding 20 mM ferrocenecarboxylic acid solution, WSC/NHS solution containing 2 mM ferrocenecarboxylic acid was prepared. The solution was stirred for 2 hours, and 10 micro l of the solution was dropped onto the prepared electrode and allowed to stand for 1.5 hours to modify the aptamer with ferrocene at the end.
Detection of cortisol
Electrochemical evaluation was performed using a three-electrode system with a working electrode, a platinum wire as a counter electrode, and a silver chloride electrode as a reference electrode, and cortisol solution was added in PBS at final concentrations of 1 nM to 10 micro M and evaluated by cyclic voltammetry (CV).
Results
CV results at different cortisol concentrations
The CV results before the addition of cortisol (Fig. 2 blue line) showed small ferrocene-derived oxidation and reduction peaks around +0.9 V and +0.56 V, respectively. Next, the CV results when cortisol was continuously added so that the final concentration ranged from 1 nM to 10 micro M are shown in Fig. 2 red line. The results show an increase in the oxidation peak around +0.9 V and the reduction peak around +0.56 V with increasing cortisol concentration. This is thought to be due to the binding of cortisol to the aptamer, which changed the steric structure of the aptamer, bringing the mediator closer to the electrode surface and increasing the electron transfer efficiency.
To increase fish culture efficiency, it is expected that stress of cultured fish can be measured in real time and then rearing environment can be controlled. Cortisol levels in plasma have been reported as an indicator of stress levels in fish. Cortisol is a steroid hormone secreted during a stress response. When fish are stressed, cortisol levels temporarily increase by an amount corresponding to the intensity of the stressor. Conventional cortisol detection methods include Liquid Chromatography-Mass Spectrometry (LC-MS) and Enzyme-Linked Immunosorbent Assay (ELISA). Although these two methods are very good methods for detecting hormonal substances, they both take time from the sampling of blood and other samples to the acquisition of detection results, resulting in indirect test results. In contrast, electrochemical biosensors are suitable for in-situ testing at fish culture site because they have the advantages of high sensitivity, detection with a small amount of sample, short detection time, easy miniaturization, and low cost. This study aimed to develop a biosensor capable of comprehensively diagnosing stress levels in fish by modifying aptamers for cortisol detection on gold electrode (Fig. 1).
Method
Design of aptamer for cortisol recognition
The sequence of the aptamer for cortisol detection used in this study is as follows. The number of bases is 71 bp, 5' end thiolate to enable aptamer modification via Au-S bond on the gold electrode. 3' end aminate to enable modification of the mediator via amide bond, ferrocenecarboxylic acid modification via an amide bond.
5'-SH-CTCTCGGGACGACGCCAGAAGTTTACGAGGATATGGTAACATAGTCGTCCCTTTT
TTTTTTTTGGTCTTCA-NH2-3'
Preparation of aptamer-modified electrode
2 micro M aptamer solution was allowed to stand at 95 degrees Celsius for 5 min, followed by 15 min at room temperature and 5 min on ice to form a loop structure. Then, 10 micro l was dropped onto a washed gold disk electrode and immobilized overnight, followed by washing with phosphate buffered saline (PBS) and air drying.
Preparation of ferrocene-labeled aptamer-modified electrode
By mixing equal amounts of 80 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride :WSC solution and 20 mM N-Hydroxysuccinimide :NHS solution and adding 20 mM ferrocenecarboxylic acid solution, WSC/NHS solution containing 2 mM ferrocenecarboxylic acid was prepared. The solution was stirred for 2 hours, and 10 micro l of the solution was dropped onto the prepared electrode and allowed to stand for 1.5 hours to modify the aptamer with ferrocene at the end.
Detection of cortisol
Electrochemical evaluation was performed using a three-electrode system with a working electrode, a platinum wire as a counter electrode, and a silver chloride electrode as a reference electrode, and cortisol solution was added in PBS at final concentrations of 1 nM to 10 micro M and evaluated by cyclic voltammetry (CV).
Results
CV results at different cortisol concentrations
The CV results before the addition of cortisol (Fig. 2 blue line) showed small ferrocene-derived oxidation and reduction peaks around +0.9 V and +0.56 V, respectively. Next, the CV results when cortisol was continuously added so that the final concentration ranged from 1 nM to 10 micro M are shown in Fig. 2 red line. The results show an increase in the oxidation peak around +0.9 V and the reduction peak around +0.56 V with increasing cortisol concentration. This is thought to be due to the binding of cortisol to the aptamer, which changed the steric structure of the aptamer, bringing the mediator closer to the electrode surface and increasing the electron transfer efficiency.