Stable Hemoglobin-Based Biosensor based on Coordination-Assisted Microfluidic Technology for Hydrogen Peroxide Determination

Hemoglobin (Hb), a special redox protein, has received widespread attention because of its good selectivity to hydrogen peroxide, thus, has been employed in numerous sensing technologies. However, because Hb is easily inactivated after being separated from the biological environment, tremendous efforts have been devoted to enhance its stability and application potential. In recent years, a large number of bioconjugation strategies have been developed to immobilize Hb, such as using (bio-) polymers, metal organic framework materials to encapsulate hemoglobin, sol-gel methods, porous materials to adsorb Hb, etc. Nevertheless, the factors affecting Hb activity are not only related to the strength of the interaction force between the immobilized carrier and hemoglobin, but are also affected by the immobilized microenvironment. Generally, the bio-friendly microenvironment for preparing an enzyme-based biosensor should: (i) maintain high intrinsic activity; (ii) limit exposure time to environmental stress; and (iii) not hinder the conformational dynamic mobility of the enzyme. While most traditional methods can improve the stability of Hb and enhance catalytic activity, only a few can maintain the conformational dynamic mobility of immobilized Hb.<br/>In addition, hemoglobin is generally “inactive” and exhibits a closed form in aqueous media due to its active sites covered by hydrophobic amino acid chains. And the electroactive centers of Hb are deeply buried in its hydrophobic cavity, delaying electron transfer and subsequently limiting the practical application of hemoglobin sensors. HIL is composed entirely of anions and cations and takes on liquid form at or near room temperature, also showing good solubility, absorption, and stability. Interestingly, HIL can act as a “two-handed weapon.” On one hand, the imidazole cation of HIL coordinates with the ferrous ion of Hb, realizing the effective immobilization of Hb. The combination of HIL and hemoglobin driven by hydrophobic and electrostatic interaction, which helps the expansion of the temporal polypeptide of Hb and ensures the dynamic migration of its conformation. On the other hand, the hydrophobic surface of HIL helps to “open” Hb, thereby exposing its active center – a process called “interfacial activation” and enhancing its biocatalytic activity. However, because of the strong trapping power of HIL, Hb can easily aggregate in HIL, which greatly decreases its biocatalytic activity.<br/>The mussel foot protein is secreted by the shellfish, the concentrated protein solution and the metal were stored respectively. The two types of secretion capsules of ions are mixed in the microfluidic channel-like duct network and form protein-metal bonds in the newly formed bypass. Metal coordination provides strong adhesion and enhances the mechanical stability of silk protein. Herein, we used a microfluidic channel as a microreactor to immobilize Hb in HIL and construct an active layer of HIL@Hb, which can effectively prevent the aggregation of Hb. More wrinkles formed on the surface of the electrode surface synthesized using the microfluidic channel, subsequently enhancing the effective sensing area for more target molecules. Moreover, to obtain a sensitive biosensor, the effective collection and rapid transfer of electrons between Hb and the electrode is also highly important. Thus, ultra-thin MXene-Ti₃C₂ nanosheets were introduced to modify the electrode surface. The rough 2D structure of MXene-Ti₃C₂ is conducive to adhere HIL@Hb on the electrode surface; while the excellent conductivity of MXene-Ti₃C₂ can effectively reduce contact resistance and support fast electron transfer. Interestingly, the good selective sensing of MXene-Ti₃C₂ to H₂O₂ can improve the sensitivity of the sensor by synergistic catalysis. Overall, this work proposes an effective strategy to construct a stable and sensitive enzyme sensing platform, which is promising for enhancing the sensor’s stability and sensitivity.