Polyphenol Laccase Biosensor Based On Composite Carbon Nanofibers

Electrochemical biosensor is a sort of multidisciplinary advanced technology, which offers a kind of facile, high-efficient, fast, low-cost and accurate detection method. It has been widely developed in the days of decades since it was created. However, poor stability and short service life are still the main problem limiting practical application of electrochemical biosensor. It is an efficient method to improve sensitivity, stability and service life of biosensor by introducing nanomaterials in the modification layers of enzyme electrodes. Carbon nanofiber, as a kind of high-performance electrode material, has been widely applied in the fields of energy, environment, etc, which was seldom applied in electrochemical biosensor. In this work, electrospun carbon nanofibers were combined with laccase to construct novel electrochemical biosensor used in the detection of catechol and hydroquinone existing in water environment. A series of synthesis methods were employed to prepare functionalized composite carbon nanofibers which were utilized as modified materials for sensor electrodes. These composite nanofibers could enhance the direct electron transfer and futher improve the response sensitivity and stability of sensors, broaden linear detection range. The main research contents and conclusions are shown as below:Carbon nanofibers were prepared by combining electrospinning with high temperature carbonization technique. And a polyphenol biosensor was fabricated by blending the obtained CNFs with laccase and Nafion. Raman spectrum, Fourier Transform Infrared Spectroscopy(FTIR) and scanning electron microscope(SEM) were respectively employed to investigate the structures and morphologies of the CNFs and the mixtures. Cyclic voltammetry and chronoamperometry were employed to study the electrocatalysis of the biosensor to catechol. The results indicated that the sensitivity of the biosensor was 41μA/mM, the detection limit was 0.63 μM, the linear range was 1—1310 μM and the response time was within 2 seconds.This novel biosensor also demonstrated its promising application in detecting catechol in real water samples. This study expanded the application research of electrospun carbon nanofiber in enzyme-based biosensor.To improve the response sensitivity of sensor, reduce the detection limit, some methods like carbonization, hydrothermal reduction, and electrostatic self-assembly were used to realize surface functionization of carbon nanofiber, obtaining composite carbon nanofiber with higher sensibilization. Meanwhile, linear sweep voltammetry and square wave voltammetry with higher detection sensitivity were employed to detect catechol and hydroquinone in water. The as-prepared composite carbon nanofibers were NiCu alloy nanoparticles loaded composite carbon nanofiber(NiCuCNFs), ZnO nanoparticles loaded composite carbon nanofiber(ZnO/CNFs) and grapheme loaded composite carbon nanofiber(G/CNFs). FTIR, SEM, Raman spectrum, X-ray diffraction(XRD), and transmission electron microscopy(TEM) were respectively employed to investigate the structures and morphologies of the composite CNFs. And novel biosensing platforms were constructed by blending the obtained composite CNFs with laccase and Nafion. The functionalized alloy nanoparticles, metal oxide nanoparticles and graphene can enlarge the electroactive specific area, enhance the direct electron transfer, and perform concerted catalysis, leading to higher response sensitivity of sensor. The results indicated that quasi reversible electrochemical redox reaction occurred on the electrode surface. According to Faraday’s law, the calculated surface coverage values for the electroactive laccase were higher than the theoretically calculated value reported in a previous study. These three laccased based biosensors all possessed high sensitivity, low detection limit and wide linear range, and all were applied in the trace detection of polyphenol pollutants.The biosensors with wide linear detection range have attracted increasingly attention in online monitor of environmental pollutants. In this study, a composite of PDA-Lac-NiCNFs was synthesized by one-pot Lac-catalyzed oxidation of dopamine(DA) in an aqueous suspension containing laccase(Lac), nickel nanoparticle loaded carbon nanofibers(NiCNFs) and DA. Subsequently, a magnetic glass carbon electrode(MGCE) was employed to separate and immobilize the composite to prepare novel biosensor for detection of catechol. The site-specific immobilization of laccase was realized by this method, resulting in enhanced electron transfer rate and response sensitivity. Moreover, the electroactive specific area of NiCNFs was enlarged by Ni nanoparticles, which also offered large number of enzyme immobilization sites. The calculated surface coverage for the electroactive laccase was 8.31×10-10 mol/cm2, and the apparent Michaelis–Menten constant of laccase was calculated to be 0.31 mM. And the novel biosensor displayed outstanding electrocatalysis toward catechol with high sensitivity, wide linear range and low detection limit. Furthermore, the biosensor also showed good repeatability, reproducibility and stability and the biosensor was successfully applied in detecting catechol in lake water. The study offered technical support and theoretical foundation for exploiting phenolic biosensors with wide detection linear range.