Optical biosensing of nitric oxide using the metalloprotein cytochrome c′

Abstract

The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO). The metalloprotein was encapsulated in a porous silicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements. Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre. Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period. The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO. However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%). An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate working range of 10-400 mumol l-1.

Aptamer entrapment in microfluidic channel using one‐step sol‐gel process, in view of the integration of a new selective extraction phase for lab‐on‐a‐chip

“…Indeed, it was shown that enzymes retain their activity toward their target and antibodies are able to bind their corresponding antigen . These interesting properties triggered the development of biosensors for medical or environmental diagnosis using metalloproteins , enzymes , or antibodies entrapped in silica gels.…”

Solid state nitric oxide sensor prepared by sol–gel entrapment of iron-diethyldithiocarbamate in a siloxane matrix

“…The physical entrapment of molecules using the solgel process [10,11] has been used for the development of NO electrochemical and optical sensors. Sensors entrapped in gels offer numerous advantages when compared with liquid based systems: they are easier to manipulate, allow species detection and concentrations measurements with less contamination of the sample, can be used for continuous sensing and are normally more stable.…”

Electrochemical Control of Protein Monolayers at Indium Tin Oxide Surfaces for the Reagentless Optical Biosensing of Nitric Oxide

“…We are interested in developing optical biosensors and in this article report the use of cytochrome c , as the biological sensing molecule, deposited onto ITO electrodes for the optical detection of NO. Previously we have encapsulated cytochrome c , and another heme protein, cytochrome c ‘, within silica sol−gel matrixes for the measurement of NO. Such biosensing measurements relied on the use of chemical reductants and oxidants to regenerate the heme center for multiple use.…”