The estimation ofcholesterol on a periodical basis is essential for living beings because the abnormal levels of cholesterol are related to heart diseases. In addition, cholesterol and its fatty acid esters are essential components of nerve cells and are precursors of many biomaterials such as bile acid and steroid hormones.
Here I`m reporting the results of mine studies relating to cholesterol biosensor basedon fisical immobilization of cholesterol oxidase (ChOx) onto polystyrene (PS) nanoparticles decorated with Congo red (CR).
Here, Our aims was develop a biosensor device from cholesterol oxidase immobilized onto Congo red decorated onto polymer nanoparticles.
NpPS/PEG was characterized by means of dynamic light scattering (DLS) and zeta (ζ) potential. The adsorption of Congo red (CR)onto PS/PEG particles was evidenced by adsorption isotherms, decrease of ζ potential values and increase in the mean diameter. Cholesterol oxidase (ChOx), the main enzyme in the oxidation of cholesterol, adsorbed onto PS/PEG and PS/PEG/CR particles, as evidenced by the increase in the particles mean size and spectrophotometry. The enzymatic activity of free and immobilized ChOx was determined as afunction of time by means of a coupled reaction with horseradish peroxidase.
Figure 1: Particle size (zeta-average diameter, D) and size distribution were determined using the ZetaPlus-ZetaPotential . Scanning electron microscopy (SEM) analyses were performed to determine the morphology and mean diameter of dried particles. Here, both histograms indicate bimodal size distribution withpredominant diameter values of 120 nm and 480 nm. The mean diameter values calculated from DLS and SEM data were (247 ± 4) nm and (240 ± 2) nm, respectively.
Figure 2: shows the adsorption isotherm obtained for Congo red (CR) onto PS/PEG particles at (23 ( 1) oC and pH 7. The adsorbed amount of CR (QCR) onto polymer particles increased with CR concentration up to the onset for theadsorption plateau, at 7.3 (mol L-1 CR, and Q value of 4.73 ( 0.02 (mol g-1.
Figures 3a and 3b show that as QCR values increased, the ζ-potential values became less negative and the mean particle diameter (D) increased, respectively. Desorption experiments indicated changes of less than 5% in the QCR values, evidencing high affinity between CR molecules and PS/PEG particles. Considering thatthe adsorption takes place at pH 7and that the pKb of CR is 4.6 ± 0.2 , the amount of CR protonated amine group is small, but might be enough to bind to the sulfate groups on the particle surface and to increase the ζ-potential values from - (70 ± 4) mV up to - (46 ± 2) mV. Moreover, H bonding between the CR uncharged amine groups and PEG oligomers on the particle surface might favor theadsorption.
In order to evaluate possible changes of ChOx secondary structure in the presence of CR, circular dichroism (CD) spectra were recorded for ChOx in the absence and in the presence of CR, as shown in Figure 4. As expected, CR presented no CD signal in the range between 190 nm and 700 nm. CD spectra determined for pure ChOx presented typical α-helix features with minima at 208 nmand 220 nm. Changes in the structure of CR due to binding to ChOx could not be detected by CD. Thus, the interaction between CR and ChOx was monitored by changes in the electronic spectra of CR in the visible range by increasing addition of ChOx.
Figures 5b and 5c show the histograms corresponding to PS/PEG/CR at the plateau value and PS/PEG/CR/ChOx particles at the highest QChOx...