Sensing Layer for Ni Detection in Water Created by Immobilization of Bioengineered Flagellar Nanotubes on Gold Surfaces

Abstract

The environmental monitoring of Ni is targeted at a threshold limit value of 0.34 μM, as set by the World Health Organization. This sensitivity target can usually only be met by time-consuming and expensive laboratory measurements. There is a need for inexpensive, field-applicable methods, even if they are only used for signaling the necessity of a more accurate laboratory investigation. In this work, bioengineered, protein-based sensing layers were developed for Ni detection in water. Two bacterial Nibinding flagellin variants were fabricated using genetic engineering, and their applicability as Ni-sensitive biochip coatings was tested. Nanotubes of mutant flagellins were built by in vitro polymerization. A large surface density of the nanotubes on the sensor surface was achieved by covalent immobilization chemistry based on a dithiobis(succimidyl propionate) cross-linking method. The formation and density of the sensing layer was monitored and verified by spectroscopic ellipsometry and atomic force microscopy. Cyclic voltammetry (CV) measurements revealed a Ni sensitivity below 1 μM. It was also shown that, even after two months of storage, the used sensors can be regenerated and reused by rinsing in a 10 mM solution of ethylenediaminetetraacetic acid at room temperature.

Flagellin-based electrochemical sensing layer for arsenic detection in water

“…Based on the results presented in this work, the advantageous properties of the previously published nickel sensing layer 1 , and in comparison with other protein-based sensors created for arsenic detection, we can conclude that mutant flagellins containing toxic metalion binder peptide motifs, which are well designed and therefore…”

“…In that work, various Ni-binding flagellin variants were constructed by mutations in the D3 domain or replacement with a histidine-rich polypeptide motif. We demonstrated that by immobilizing nanotubes built from mutant flagellins by in vitro polymerization on the surface of biochips, a Ni(II) ion concentration of 1 μM could be detected, which is close to the maximum allowable concentration (0.34 μM) 1 . These results prompted us to create novel flagellin-based sensor layers that, when incorporated into a biosensor, can be suitable for monitoring arsenic pollution in natural waters.…”

“…Since arsenic binding cysteines in both AfArsR (ArsR from A. ferrooxidans ) and EcArsR (ArsR from E. coli ) stem from the same protein monomer within a ten amino acid long polypeptide, however, they bear different cysteine arrangements (CXCX 2 C from E. coli and CCX 5 C from A. ferrooxidans ), we aimed to incorporate both motifs into a flagellin scaffold by replacing its D3 domain. The replacement was performed using the pKOT-based vector described earlier, which contains the gene of the D3 deficient flagellin protein 1 . The DNA segments coding for the arsenic-binding oligopeptides were synthesized and ligated into the above plasmid between XhoI and SacI restriction sites.…”

“…Cloning, expression and purification of As-binding flagellin variants. Complementary coding sequences of the different As-binding motifs (Table S2) were inserted into the gene of a D3-deleted variant of flagellin between the XhoI and SacI restriction sites of the pKOT-based plasmid as described earlier for the HG12 fusion variant 1 . Based on the result of in vivo filament formation experiments, expression of the fusion protein variants was carried out as described previously 33 , except that 25 mM dithiothreitol (DTT) (Promega, Madison, Wisconsin, US) was added to the purification buffer containing 20 mM tris(hydroxymethyl)aminomethane (Tris) (VWR International, Radnor, Pennsylvania, US), 150 mM NaCl (VWR) (pH 7.8) in order to avoid protein aggregation through intermolecular disulfide bond formation.…”

“…Cyclic voltammetry. CV measurements were performed as described earlier 1 . As 2 O 3 (Sigma Aldrich) was…”