Electrochemical Sensor for Dopamine Detection Based on the CRISPR/Cas12 System ()
1. Introduction
Neurotransmitters are key messengers that perform a variety of functions in the nervous system. Dopamine (DA) is currently the most studied neurotransmitter due to its critical role in the human brain and nervous system, and this neurotransmitter is essential for neuronal development, attention, learning, and control of stress responses [1]. Its primary functions include the circulatory system, kidneys, hormones, central nervous system, and motivational actions [2]. Dopamine concentrations affect how the brain functions, feels, and behaves in both people and animals. A number of theories propose that diseases like schizophrenia, Parkinson’s disease, and attention deficit hyperactivity disorder are caused by anomalies in dopamine levels [3]. Damage to the neurons involved in dopamine synthesis is the root cause of many illnesses. A number of illnesses and neurological disorders, including hypertension, schizophrenia, attention deficit hyperactivity disorder, Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease, are frequently linked to abnormalities in the amounts of dopamine (DA) in biological fluids and tissues [4] [5]. Because of the low concentration levels of DA in biological samples, which are typically in the region of nM, the development of dependable, accurate, and affordable sensing methods for the detection of DA in biological samples is crucial in analytical and diagnostic applications [6] [7].
Dopamine has been detected using a variety of quantitative techniques up to this point, including spectrophotometry, high-performance liquid chromatography, and electrophoresis. These techniques might be costly and necessitate lengthy experiments [8] [9]. As a result, scientists have been working to develop more accurate and cost-effective techniques for detecting dopamine in biological fluids. Due to its affordability, ease of use, portability of the equipment, and the fact that dopamine is redox-active, electrochemical detection has been the method of choice up to this point [10]-[12]. Dopamine is less concentrated in physiological settings than other electrochemically active chemicals like uric acid and ascorbic acid, which presents a hurdle for the detection of dopamine utilizing electrochemical methods [13] [14]. Furthermore, biological samples may also contain catechol, norepinephrine, and adrenaline, among other compounds that are recognized as interfering substances due to their similar oxidation potential and competitive sensitivities, when it comes to the detection of dopamine in biological samples. For instance, it is typically discovered that ascorbic acid has hundreds to thousands of times the potency of DA [15]-[17]. Thus, it’s critical to overcome the selectivity problem in the creation of dopamine electrochemical sensors.
A workable method for the selective detection of dopamine is the creation of biosensors. Low detection limits and strong selectivity are two benefits of biosensors. As a result, biosensors can lessen the difficulty posed by the previously mentioned chemical interferents. A bioreceptor that has been subjected to a biorecognition process with the intended analyte is typically necessary for biosensors. Enzymes, antibodies, DNA, RNA, and whole cells can all function as these receptors. An aptamer is a particular DNA or RNA that has been chosen to bind to a particular target in an immunosensor, such as an antibody [18]. Short, manufactured ligands with a single strand that have a high affinity and specificity for their target molecules are known as nucleic acid aptamers. These features dictate how widely aptamers are used in the development of aptamer-based biosensors, commonly referred to as aptamer sensors [19]. Low cost, small size, easy synthesis, powerful recognition ability, and high binding affinity to targets are the characteristics of aptamers [20] [21]. Since these characteristics are superior to those of conventional antibodies, they can be used to improve the efficiency and specificity of electrochemical biosensors.
Because of their comparatively high reaction rate, specificity, and usage of comparatively basic and affordable equipment, electrochemical approaches have been developed. The electrochemical detection of dopamine and other neurotransmitters has been extensively studied [22]. Through, electrochemical techniques like cyclic voltammetry make it simple to identify dopamine in aqueous solutions. Aptamers have been studied by a number of research organizations as potential recognition components for dopamine sensors. 58 Nucleotide aptamers can bind to gold electrodes, and dopamine aptamers can electrostatically adsorb onto aminothiol-modified gold surfaces to create colorimetric dopamine sensors. The aptamer-dopamine binding process encourages the aggregation of the gold nanoparticles, which in turn causes the color change of the neurotransmitters. In order to complete the electrochemical sensor construction, cDNA and 6-mercapto-1-hexanol (6-MCH) were sequentially loaded onto the electrode. The sensor construction process was then applied to characterize and detect the sensor by applying cyclic voltammetry (CV) and by the amplitude of the anode and cathode currents of the electrodes. The CRISPR/Cas12a system detects dopamine using a specific group (ferrocene), which is labeled with ferrocene (Fc) into cDNA. The anodic and cathodic current amplitudes were utilized to calculate the dopamine concentration.
2. Experimental Design and Methodology
2.1. Materials and Equipment
The single-stranded DNA sulfhydrylated aptamer probe sequence was ACGTTTTATCTGCCCCAGTGTTCTC-(CH2)-6-SH, Apt single-stranded as Fc-GTCTCTGTGTGTGCGCCAGAGAGAACACTGGGGCAGATATGGGGCCAGCACAGAATGAGGCCC, and the nucleic acid sequence was purchased from Shanghai Sangong Bioengineering Technology Co. DA, MgCl2, KCl, NaOH, NaCl, and potassium ferricyanide was purchased from Shanghai Aladdin Biochemical Technology Co. Tris-HCl was purchased from Amresco. 0.3 µm, 0.05 µm Al2O3 was purchased from Shanghai Chenhua Instrument Co. The working rod gold electrode (Au), auxiliary platinum wire electrode (Pt), and reference electrode were Ag/AgCl. The electrochemical workstation (CHI660e) was used to perform cyclic voltammetry (CV) curves in 0.5 mm [Fe(CN)6]3−/4−. 0.5 mM [Fe(CN)6]3−/4− was configured with PBS solution. Ultrasonic oscillator (JYD-250) and water-bath constant temperature oscillator (SHA-C).
2.2. Preparation of Gold Electrodes
Bare gold electrodes were immersed in a tigerfish solution containing 30% H2O2 and 70% concentrated H2SO4 for 1 hour. The main objective was to completely remove the oxide layer and thus regenerate the surface of the gold electrode. After completing the treatment, the electrode was then thoroughly rinsed with a thorough rinse of deionized water to ensure that a flawless, oxide-free surface was obtained. Next, the surface of the gold electrodes was refined, starting with 0.3 µm and 0.05 µm aluminum oxide powder (Al2O3), with a precise polishing process lasting about 30 min to obtain a properly smooth surface. Subsequently, residual Al2O3 powder is removed by an ultrasonic cleaning process. This thorough cleaning procedure consisted of successive washes with deionized water, ethanol, and deionized water, each lasting 5 min, to ensure complete removal of all impurities. Upon completion of the cleaning procedure, the cleaned gold electrodes were subjected to another thorough washing with deionized water, followed by drying using nitrogen gas. Finally, the electrode was immersed in cDNA solution for 10 h to allow the cDNA to be completely modified on the electrode surface by Au-S bonding.
2.3. Preparation for DA Solution
10 × PBS was diluted 10-fold with deionized water to obtain 1 × PBS, which was used as a buffer. 0.038 g of DA was added to 100 mL of 1 × PBS to prepare a 20 μM DA solution, and the 20 μM DA solution was diluted with 1 × PBS in an appropriate ratio to obtain a lower concentration of DA solution.
2.4. Electrochemical Determination of DA
A bare gold electrode was used as the working electrode. Ag/AgCl and Pt electrodes were used as reference and auxiliary electrodes, respectively. The electrochemical behavior of different concentrations of DA on the modified electrode was analyzed using CV. The CV curves were recorded for 30 cycles using an electrochemical workstation (CHI660e) scanned in the voltage range of −0.2 - 0.4 V at a scan rate of 100 mV s.
3. Results and Discussion
3.1. DA electrochemical Biosensor Principle
Figure 1. DA electrochemical biosensor principle.
As shown in Figure 1, the cDNA was first loaded on the bare gold electrode, which was subsequently filled with 6-MCH, and the S-Au bond ensured the stability of the cDNA binding to the gold electrode, and finally the Fc-labeled dopamine Apt strand was bound to the cDNA, at which time the electrochemical signal was maintained at a higher level due to the presence of the Fc electrochemical signaling molecule on the electrode. When DA is contained in the solution to be measured, DA binds to Apt, resulting in a subsequent Fc-Apt movement away from the electrode, leading to a decrease in the electrochemical signal, thus realizing the detection of DA.
3.2. Electrochemical Characterization of DA Biosensors
The electrochemical behavior of the gold electrode was observed using electrochemical methods in potassium ferricyanide solution; the more loadings on the electrode, the lower the redox peak. We used CV to characterize the biosensor build change process. From Figure 2, it can be seen that the bare gold electrode has the largest redox peak (curve a), followed by a gradual decrease in the current of the redox peak when the cDNA signaling probe (curve b) and 6-MCH (curve c) are sequentially modified on the electrode, the electrode transfer of electrons is blocked. After continuing to load on Fc-Apt (curve d), the redox peak value continued to decrease, and when DA solution was added, Fc-Apt reacted with DA (curve e), Fc-Apt moved away from the electrode, and the redox peak value increased. This indicates that the electrochemical sensor was successfully constructed.
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Figure 2. a curve represents bare gold electrode, b curve represents cDNA loaded on gold electrode, c curve represents 6-MCH loaded on electrode, d curve represents loaded Fc-Apt, and e curve represents Fc-Apt after reaction with DA.
3.3. Optimization of Experimental Conditions
We carefully considered our options when choosing the experimental setup to ensure the electrochemical sensor operated at its peak efficiency. Included in this are the ideal DA concentration and reaction time. Since the concentration of the DA solution and the reaction time of the Apt chain with the DA solution are important, we set up various DA solution concentrations and calculate the time of the solution’s steady state as well as the ideal reaction concentration at steady state in order to determine the ideal reaction concentration and reaction time.
As shown in Figure 3(A), the current tends to stabilize when the reaction time reaches 10 min, and the current is stable at 15 min, so the optimal time for the solution to react to a steady state is 15 min. Configure different concentrations of DA solution 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, respectively, take the optimal reaction time of 15 min, and measure the electrochemical signals, as shown in Figure 3(B), the concentration of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, in that order, the 5 μM electrochemical signal response value is the largest, so we set the optimal response concentration as 5 μM.
Figure 3. Optimization of experimental conditions. (A) Currents for DA solution reaction times. (B) Currents for reactions with different DA solution concentrations.
3.4. DA Electrochemical Biosensor Platform Performance
The biosensor platform was used to measure different concentrations of DA solutions and analyze their electrochemical signal values to evaluate the sensitivity of the developed electrochemical biosensing platform to detect them. Figure 4(A), Figure 4(B) shows how different concentrations of DA solutions affect the responsiveness of electrochemical signals.
The initial DA concentration in this experiment was 5 μM/L. As the DA solution concentration continued to increase, the electrochemical signal was linearly related to the logarithm of the DA solution concentration (lgC, with C representing the DA solution concentration), and in this study, a linear regression equation was established, which showed that there was a linear relationship between the level of the electrochemical signal and the concentration of the DA solution, with Y = −1.45 × 10−9 LgC + 3.91 × 10−7, R2 = 0.993, and the limit of detection was determined to be 4.67 μmol/L according to the general equation LOD = 3σ/k, where k is the slope of the linear regression equation and σ is the standard deviation. Therefore, this biosensor is highly sensitive to the detection of the target target, which suggests that our constructed electrochemical biosensor can be used to detect DA.
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Figure 4. (A - B) Electrochemical signals of different concentrations of DA solutions after sensor treatment.
Figure 5. (A - B) Specificity of DA electrochemical biosensors.
3.5. Specificity of DA Electrochemical Biosensors
We chose various approaches for the measurement of sensor specificity in order to confirm, under ideal experimental conditions, that the biosensor is specific for the detection of DA. This study was divided into five groups: potassium chloride (KCL), dopamine solution (DA), uric acid solution (UA), 5% glucose solution (GLU), and blank control group (Blank). Figure 5 illustrates this: while the sensor responds to the electrochemical signals of the Glu, UA, and blank control group in some way, there is almost no difference, indicating that the sensor lacks specificity for any of these groups. In addition, the electrochemical signals of the DA group are much lower than those of other groups, which indicates that the experimental method has good selectivity for DA solution. This is enough to prove that the electrochemical sensor platform we developed has obvious specificity for the detection of DA solutions.
3.6. Determination of the Recovery of DA Solution
We simulated the samples in a serum environment, diluted GLU, UA, and KCL with PBS solution, and added various doses of DA to assess the recoveries in order to test the method’s applicability. Table 1 displays the method’s recoveries, which ranged from 83.93% to 96.26%. These results suggest that the electrochemical biosensor we created is useful for DA detection.
Table 1. DA sample recovery.
DA solution concentration (μM/L) |
Average value (μM/L) |
Recovery rate |
1 |
0.8393 |
83.93% |
5 |
4.4563 |
89.13% |
10 |
9.6256 |
96.26% |
4. Conclusion
As part of our ongoing effort to innovate scientific research, we have effectively created a novel approach to dopamine (DA) detection using the CRISPR/Cas12 gene editing technology. This method achieves quick and sensitive detection of DA by deftly combining gene editing and nucleic acid aptamer technologies, particularly by taking advantage of the high specificity of the CRISPR/Cas12a system. Our method maintains great specificity while drastically cutting detection time when compared to current detection techniques. With a low detection limit of 4.67 μmol/L, our assay shows significant promise for real-world uses. We intend to use this technique in upcoming research to identify human serum samples, which will strongly assist clinical diagnosis. We think that this method’s excellent sample recovery and high stability would greatly increase the effectiveness of DA detection, providing patients with neurodegenerative disorders like Parkinson’s disease with speedier and more precise treatment alternatives. Furthermore, our approach is very appropriate for usage in a clinical context due to its fast assay time and ease of operation. We believe this approach will offer a new tool for the early detection and treatment of Parkinson’s disease, enhancing patients’ quality of life with additional research and optimization.