RESUMEN
The simultaneous detection of atherosclerotic cardiovascular disease (ACSVD) biomarkers was recently of great scientific interest. In this work, magnetic beads-based immunosensors for the simultaneous detection of low density lipoprotein (LDL) and malondialdehyde-modified low density lipoprotein (MDA-LDL) were presented. The approach proposed was based on the formation of two types of specific immunoconjugates consisting of monoclonal antibodies: anti-LDL or anti-MDA-LDL, together with redox active molecules: ferrocene and anthraquinone, respectively, coated on magnetic beads (MBs). The decrease in redox agent current in the concentration range: 0.001-1.0 ng/mL for LDL and 0.01-10.0 ng/mL for MDA-LDL, registered by square wave voltammetry (SWV), was observed upon the creation of complex between LDL or MDA-LDL and appropriate immunoconjugates. The detection limits of 0.2 ng/mL for LDL and 0.1 ng/mL for MDA-LDL were estimated. Moreover, the results of selectivity against the possible interferents were good, as human serum albumin (HSA) and high density lipoprotein (HDL), stability and recovery studies demonstrated the potential of platform proposed for early prognosis and diagnosis of ASCVD.
Asunto(s)
Aterosclerosis , Técnicas Biosensibles , Inmunoconjugados , Humanos , Lipoproteínas LDL , Metalocenos , Inmunoensayo , Anticuerpos Monoclonales , Antraquinonas , Fenómenos Magnéticos , MalondialdehídoRESUMEN
Low-density lipoprotein (LDL) is a cardiac biomarker identified in the pathology of cardiovascular disease (CVD). Typically, the level of LDL is calculated using the Friedewald relationship based on measured values of total cholesterol, high-density lipoproteins (HDL), and triglycerides. Unfortunately, this approach leads to some errors in calculation. Therefore, direct methods that can be used for fast and accurate detection of LDL are needed. The purpose of this study was to develop an electrochemical platform for the detection of LDL based on an antibody-ferrocene conjugate. An anti-apolipoprotein B-100 antibody labeled with ferrocene was covalently immobilized on the layer of 4-aminothiophenol (4-ATP) on the surface of gold electrodes. Upon interaction between LDL and the antibody-ferrocene conjugate, a decrease in the ferrocene redox signal registered by square wave voltammetry was observed, which depends linearly on the concentration from 0.01 ng/mL to 1.0 ng/mL. The obtained limit of detection was equal to 0.53 ng/mL. Moreover, the satisfied selectivity toward human serum albumin (HSA), HDL, and malondialdehyde-modified low-density lipoprotein (MDA-LDL) was observed. In addition, the acceptable recovery rates of LDL in human serum samples indicate the possible application of immunosensors presented in clinical diagnostics.
Asunto(s)
Técnicas Biosensibles , Inmunoconjugados , HDL-Colesterol , Humanos , Inmunoensayo , Lipoproteínas LDL , Metalocenos , TriglicéridosRESUMEN
An elevated level of low density lipoprotein (LDL) can lead to the cardiovascular system-related diseases, such as atherosclerosis and others. Therefore, fast, simple, and accurate methods for LDL detection are very desirable. In this work, the parameters characterizing the electrochemical immuno-and aptasensor for detection of LDL have been compared for the first time. An immunosensor has been designed, for which the anti-apolipoprotein B-100 antibody was covalently attached to 4-aminothiophenol (4-ATP) on the surface of the gold electrode. In the case of an aptasensor, the gold electrode was modified in a mixture of ssDNA aptamer specific for LDL modified with -SH group and 6-mercaptohexanol. Square-wave voltammetry has been used for detection of LDL in PBS containing redox active marker, [Fe(CN)6]3-/4-. Our results show the linear dependence of [Fe(CN)6]3-/4- redox signal changes on LDL concentration for both biosensors, in the range from 0.01 ng/mL to 1.0 ng/mL. The limit of detection was 0.31 and 0.25 ng/mL, for immuno- and aptasensor, respectively. Whereas slightly better selectivity toward human serum albumin (HSA), high density lipoprotein (HDL), and malondialdehyde modified low density lipoprotein (MDA-LDL) has been observed for aptasensor. Moreover, the other components of human blood serum samples did not influence aptasensor sensitivity.