Employment of Iron-Binding Protein from Haemophilus influenzae in Functional Nanopipettes for Iron Monitoring

Abstract

Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus inf luenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 μM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

Perturb–Scan–Pull: A Novel Method Facilitating Conformational Transitions in Proteins

“…However, phosphate ion is not essential for iron binding as confirmed by site-directed mutagenesis studies whereby crystal structures complex to iron without the synergistic anion. , It has been suggested that phosphate might be regulating FBP function by stabilizing the closed conformation . However, a small-angle X-ray scattering (SAXS) study indicated that the holo form tends to adopt an open conformation even in the presence of phosphate ion . In fact, all mutated FBP proteins display open conformation while they still have iron or phosphate ion attached to them. ,,, …”

“…89 However, a small-angle X-ray scattering (SAXS) study indicated that the holo form tends to adopt an open conformation even in the presence of phosphate ion. 95 In fact, all mutated FBP proteins display open conformation while they still have iron or phosphate ion attached to them. 88,93,94,96 Site-directed mutagenesis of iron-coordinating residues as well as phosphate-coordinating residues indicate that, unlike transferrin, only one tyrosine is sufficient for iron binding and transfer process of bacterial FBP.…”

Nano‐sensing and nano‐therapy targeting central players in iron homeostasis

“…The free iron can also be detected through a different approach via biological recognition. A ferrous iron‐binding binding protein (FBP) obtained from Haemophilus influenzae was synthesized and immobilized onto the inner surface of a nanopipette system for ferrous detection (Bulbul et al, 2018). With two Ag/AgCl electrodes in‐ and outside the nanopipette followed by current recording, the existence and concentration of ferrous iron can be monitored in real‐time (Figure 5c).…”

“…Copyright 2013 American Chemical Society); (b) Iron binding properties of ferritin were utilized for ferric iron and apoferritin detection (Reprinted with permission from Han et al (2016). Copyright 2016 Elsevier); (c) Ferrous iron was measured by electrodes in nanopipette modified with iron binding proteins (Reprinted with permission from Bulbul et al (2018). Copyright 2018 American Chemical Society); (d) Fluorescence can be restored through ferric ion‐catalyzed destruction of the quenching encapsulation of fluorescent NPs (Reprinted with permission from W. Zhang et al (2019).…”

Silica-Based Nanopipettes for Rapid Living Single-Cell Transfection

“…Nanopipettes also have many other applications, such as small-molecule detection, − single-molecule/particle detection, − scanning ion conductance microscopy for imaging, , scanning tunneling microscopy, and surface-enhanced Raman scattering; therefore, their controlled fabrication is a highly attractive requirement. The geometrical parameters of nanopipettes directly affect the experiments and results. ,, Thus, nanopipettes having a range of tip sizes and geometries that can be reproducibly and precisely fabricated are urgently needed. , However, nanopipette fabrication has the disadvantages of poor controllability, high cost, and difficult operation.…”