The development of a cholesterol biosensor using a liquid crystal/aqueous interface in a SDS-included β-cyclodextrin aqueous solution
Graphical abstract
Introduction
Cholesterol analysis has been subjected to considerable scrutiny owing to its importance in clinical diagnosis. Excess cholesterol content in the blood serum results in the development of plaque in the blood vessels, which in turn leads to cardiovascular diseases. Several assays and methods, including chemical, colorimetric, fluorimetric, and polarographic assays, as well as thin layer chromatography, high-performance liquid chromatography (HPLC), gas chromatography, and enzyme-based biosensors have been used to detect cholesterol [1], [2], [3], [4]. Among these, enzyme-based biosensors rely on use of cholesterol-selective enzymes, such as cholesterol esterase, cholesterol oxidase, and per oxidase, which are expensive and prone to denaturation. Therefore, alternative, simple, and cost-effective methods for cholesterol detection need to be identified.
Cyclodextrins (CDs) are a series of cyclic oligosaccharides containing 6, 7, or 8 glucose units connected by an α-1, 4 linkage. CDs with 6, 7, and 8 glucose units are called α, β, and γ- CD, respectively. The outer surface of CD is hydrophilic because of the presence of hydroxyl groups; conversely, the inner surface of the molecular cavity is hydrophobic. This structure provides the CDs with a unique ability to form host-guest inclusion complexes with a wide range of suitably sized guest molecules [5]. The guest molecule is held within the CD cavity in these complexes. The complexation largely depends on the dimension of CD and the particular steric arrangement of the functional groups in the guest molecule [5]. The CDs usually favor the unionized guest molecules displaying a higher level of hydrophobicity, compared to the ionized molecules [5]. The host-guest interaction between β-CD and cholesterol has been widely used in the selective extraction of cholesterol from bio-environments, such as food, cell membranes, blood serum, and cultured cell [6], [7], [8], [9].
Nanoparticle-functionalized β-CD have been used in the selective detection and separation of cholesterol [10], [11]. Previous studies have suggested that the β-CD can be used as an alternative to enzyme-based biosensors for cholesterol detection. However, the interaction between β-CD and cholesterol does not alter the electrical conductance, pH, or optical appearance. Therefore, this interaction cannot be used to detect cholesterol using electrochemical or potentiometric methods. The fluorescence-based detection of cholesterol has been reported using fluorescence dye-included β-CD hybrid systems. For example, Zhang et al. designed gold nanoparticles functionalized with a fluorescent dye-included β-CD hybrid system. Cholesterol was detected via fluorescence recovery from the released dye, which occurred as a result of the replacement of quenched dyes in the β-CD with cholesterol [10]. Li et al. reported the preparation of superparamagnetic nanoparticles conjugated with β-CD, and their selective binding to cholesterol [11]. Mondal et al. designed a β-CD/graphene hybrid system and incorporated a fluorescence dye, rhodamine 6G, for the optical detection of cholesterol; here, the β-CD allowed for selective cholesterol detection, while the graphene transduced the signal emitted by the replacement of rhodamine 6G by cholesterol in the hybrid into an optical signal [12]. This system showed a superior optical response compared to the previously reported nanoparticles/β-CD hybrid systems [10], [11]. However, the difficulties in synthesis of nanoparticles and graphene, as well as the use of fluorescent materials, result in the increase in complexity and cost of these methods.
A diverse range of dynamic and equilibrium processes can occur when molecular self-assembly and specific bimolecular recognition events take place at the interfaces formed between nematic liquid crystals (LCs) and immiscible aqueous phases. The orientation of LCs is extraordinarily sensitive to the change in the adjacent (contact) interface. The optical amplification of LCs make them a unique optical probe for sensing chemical reactions, such as enzymatic reactions [15], [16], ligand-receptor binding [13], [14], [15], [16], [17] and peptide-lipid [18] interactions at the LC/aqueous interface. In this study, a transmission electron microscopy (TEM) grid on an octadecyltrichlorosilane (OTS)-coated glass filled with 4-cyano-4′-pentylbiphenyl (5CB) (a nematic liquid crystal at room temperature) was used to detect cholesterol via host (β-CD)/guest (cholesterol) interactions. Sodium dodecyl sulphate (SDS)-included β-CD (β-CDSDS) was initially introduced to the 5CB/aqueous interface. The SDS present in the TEM grid cell containing β-CDSDS was replaced with cholesterol upon injection of a cholesterol solution. The planar-to-homeotropic (P–H) change in orientation of 5CB due to adsorption of the excluded SDS at the interface was observed through a polarized optical microscope (POM) under crossed polarizers. This cholesterol detection system was observed to be simple, cost-effective, and highly stable. Therefore, it could be applied as a new alternative enzyme-based cholesterol biosensor.
Section snippets
Materials
Microscopic glass slides (Duran group, Wertheim, Germany) were cleaned using a piranha solution in order to remove organic contaminants (Caution: Piranha solution is extremely corrosive, necessitating proper care). These were subsequently washed with distilled water, dried under nitrogen, and coated with OTS (Sigma–Aldrich, St. Louis, MO) which aligns the 5CB mesogens perpendicular to the glass surface. Copper TEM specimen grids (G75, grid hole width 285 μm, pitch 340 μm, bar width 55 μm,
Preparation of SDS-included β-CD
In order to determine the optimum concentration of SDS for complete inclusion within β-CD (without any free SDS in water), the pH of the pure SDS and β-CDSDS aqueous solutions was measured at different concentrations. The optimum concentration must be accurately determined as the orientation of the TEM grid cell must be altered by exclusion of SDS via cholesterol inclusion (not by free SDS). Fig. 1 shows the pH change in pure SDS and β-CDSDS aqueous solutions as a function of CSDS. The β-CDSDS
Conclusion
A cholesterol biosensor was fabricated using a TEM grid cell containing a SDS-included β-CD aqueous solution (TEMβ-CD/SDS grid cell). The maximum concentration of SDS for complete inclusion of SDS within β-CDSDS without any free SDS in the solution was determined at 340 μM; this was confirmed by pH measurement and HPLC analysis. The TEMβ-CD/SDS grid cell detected cholesterol through changes in the optical appearance using a POM (at a detection limit of (3 μM)) without considerable interference
Acknowledgment
This work was supported by the National Research Foundation of Korea (NRF-2011-0020264 and NRF-2014R1A2A1A11050451)
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