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Laccase-Based SPR Biosensor for Highly Sensitive Dopamine Detection

In a recent article published in Scientific Reports, researchers introduced a novel surface plasmon resonance (SPR) biosensor for direct detection of dopamine using laccase as the recognition element. Enzymes are highlighted as effective recognition elements for sensor applications, with laccase specifically chosen for its industrial and biotechnological applications.

The challenge of maintaining biomolecule activity after immobilization on the sensor surface is addressed, emphasizing the need for efficient biosensor design.

Laccase-Based SPR Biosensor for Highly Sensitive Dopamine Detection
Study: Laccase-Based SPR Biosensor for Highly Sensitive Dopamine Detection. Image Credit: Artur Wnorowski/Shutterstock.com

Background

Enzyme-based SPR techniques have been widely explored for their specificity and sensitivity in detecting analytes. Laccase, a multicopper-oxidase enzyme, is utilized in this study due to its ability to interact with dopamine, a phenolic substrate.

The immobilization of laccase on the SPR- carboxymethyldextran (CMD) chip using an amine coupling procedure is crucial for enhancing specificity towards dopamine. The reversible and non-covalent interaction between dopamine and laccase allows for easy regeneration of the biosensor surface, ensuring stability and reproducibility in dopamine detection.

The Current Study

The laccase immobilization process on the CMD chip was conducted using the SPRSR7500DC instrument from XanTec bioanalytics GmbH, Germany. The protocol involved the activation of surface carboxyl groups using N-ethyl-N′-(3-diethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) esters.

A 10-minute pulse of 250 μl NHS/EDC injection was administered to activate the surface, followed by the injection of laccase (0.2 mg/ml) in 10 mM acetate buffer (pH 4.5) to immobilize the enzyme on the activated chip.

The deactivation of remaining esters was carried out using 1 M ethanolamine (pH 8.5) to remove any electrostatically bound enzyme. This deactivation step was crucial for ensuring the removal of any non-specifically bound enzyme molecules. Subsequently, all operations were rinsed with a phosphate buffer solution to maintain the integrity of the immobilized laccase on the CMD chip.

The SPR measurements were performed using the SPRSR7500DC instrument with an automatic flow injection system. The instrument allowed for real-time monitoring of the interactions between dopamine and the immobilized laccase on the CMD chip. The SPR signal changes induced by the binding of dopamine to the laccase active site were recorded and analyzed to determine the sensitivity and specificity of the biosensor.

To ensure precise and reproducible detection of dopamine, the experimental steps for biomolecular interactions were meticulously optimized. Parameters such as flow rates, buffer compositions, and reaction times were fine-tuned to enhance the sensitivity and specificity of the biosensor.

The optimization process aimed to maximize the signal-to-noise ratio and minimize non-specific interactions, thereby improving the accuracy and reliability of dopamine detection using the laccase-based SPR biosensor.

The reversible and non-covalent interaction between dopamine and laccase allowed for the easy regeneration of the biosensor surface. Mild running buffer solutions were used to regenerate the surface without causing damage, enabling multiple analyses to be conducted with the same biosensor chip. The regeneration process was essential for maintaining the stability and reproducibility of dopamine detection over multiple measurements.

Results and Discussion

The laccase-based SPR biosensor demonstrated remarkable sensitivity and specificity in detecting dopamine, with a linear range from 0.1 to 18.9 μg/ml and a lower detection limit of 0.1 ng/ml. These results surpass those of other enzyme-based biosensors for dopamine detection, highlighting the effectiveness of laccase as a recognition element.

The high sensitivity of the biosensor can be attributed to the specific interaction between dopamine and the laccase active site, leading to detectable changes in the SPR signal.

Comparative analysis with existing enzyme-based biosensors for dopamine detection revealed the superiority of the laccase-based SPR biosensor in terms of sensitivity and linear range. The biosensor's ability to detect dopamine at low concentrations with high precision positions it as a valuable tool for therapeutic monitoring of dopamine levels.

The simplicity and cost-effectiveness of the biosensor design further enhance its potential for clinical applications, particularly in the diagnosis of central nervous system disorders associated with dopamine imbalance.

The reversible and non-covalent interaction between dopamine and laccase allowed for the easy regeneration of the biosensor surface. This feature enables multiple analyses to be conducted using the same biosensor chip, enhancing the cost-effectiveness and efficiency of dopamine detection. The mild running buffer used for regeneration prevents surface damage, ensuring the stability and reproducibility of the biosensor over multiple measurements.

Conclusion

The study successfully develops a laccase-based SPR biosensor for direct detection of dopamine, showcasing its high sensitivity and specificity. The biosensor's ability to maintain enzyme activity post-immobilization, regenerate the surface, and optimize biomolecular interactions highlights its potential for clinical applications.

This cost-effective and efficient biosensor design offers a valuable tool for precise and reproducible dopamine detection in various biomedical and healthcare settings.

Journal Reference

Jabbari S, Dabirmanesh B, et al. (2024). The potential of a novel enzyme-based surface plasmon resonance biosensor for direct detection of dopamine. Scientific Reports 14(1):14303. doi: 10.1038/s41598-024-64796-whttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC11192927/

Dr. Noopur Jain

Written by

Dr. Noopur Jain

Dr. Noopur Jain is an accomplished Scientific Writer based in the city of New Delhi, India. With a Ph.D. in Materials Science, she brings a depth of knowledge and experience in electron microscopy, catalysis, and soft materials. Her scientific publishing record is a testament to her dedication and expertise in the field. Additionally, she has hands-on experience in the field of chemical formulations, microscopy technique development and statistical analysis.    

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