Aptasensor based on triplex switch for SERS detection of cytochrome c

Abstract

We report a sensitive approach for SERS detection of cytochrome c using target binding-induced conformational changes of signal transduction probe (STP). STP labeled with a SERS-active molecule, carboxy-X-rhodamine (ROX), is immobilized on the substrate where the formation of a rigid triplex switching structure with aptamers does not allow SERS amplification to take place. The target binding event leads to an enhancement in SERS intensity of ROX adsorbed on the gold surface. Meanwhile, we found that an appropriate STP surface density could shield the SERS signal produced by protein adsorption which would foul the sensing surface. In addition, cytochrome c aptamers used were not the original sequence but reorganized in the nonspecific binding site to adapt to our design. This method provides a low detection limit of 2 nM (10 fmol within 5 μL sample solution), and shows good selectivity toward cytochrome c compared to interfering proteins such as hemoglobin and immunoglobulin G. The general strategy of the method can also be extended to aptamer or DNA based sensors.

Surface-Enhanced Raman Scattering-Fluorescence Dual-Mode Nanosensors for Quantitative Detection of Cytochrome c in Living Cells

“…From the fluorescence standard curve (Figure D), the regression equation was: and the LOD was 52.136 nM. Compared with published methods (Table ), ,− our method not only had an advantage in the detection range and LOD in buffer to a certain extent but also could quantitatively detect the intracellular Cyt c.…”

Gold-Nanoparticle-Functionalized Cobalt–Nickel Phosphate 3D Nanoice Creams to Fabricate Stable and Sensitive Biosensors for the Cytochrome c Assay

“…Apoptosis, programmed cell death, is closely associated with degenerative disorders (excessive apoptosis), autoimmune disorders, and cancers (inadequate apoptosis). , The intrinsic apoptotic progress can be monitored via detecting the release of Cyt c from mitochondria. ,− Therefore, quantitative determination of Cyt c in cell lysates plays a key role for the diagnosis of early stage apoptotic cells. Many efficient methods containing fluorescence, surface-enhanced Raman scattering (SERS), mass spectrometry, electrochemistry, and chemiluminescence are developed for the determination of Cyt c. However, these proposed methods possess the disadvantages of low sensitivity, complex test steps, and expensive equipment. Electrochemiluminescence (ECL) has drawn great attention for Cyt c detection because of the merits of low background noise, high sensitivity, rapidness, and easy controllability. − Owing to its strong emission, low cost, and low oxidation potential, luminol has become the most widely applied ECL luminophor. − However, most of the ECL studies on luminol and its analogues have been limited to strong alkaline solution.…”

SERS Quantification and Characterization of Proteins and Other Biomolecules

“…Although it has not been extensively applied for proteins, the few examples available in the literature predict a huge potentiality also in the application to these targets. For example, the quantitative detection of cytochrome C was achieved with a triplex switch DNA structure bound to gold NP films (Figure A) . In this case, the thiolated oligonucleotide conjugated with a SERS-active molecule (carboxy-X-rhodamine, ROX) forms a rigid triplex structure on the gold surface with a thrombin-binding aptamer.…”

“…(A) Schematic illustration of the aptasensor based on a triplex switch for SERS detection of cytochrome c . Adapted with permission from ref . Copyright 2012 Royal Society of Chemistry.…”

“…For example, the quantitative detection of cytochrome C was achieved with a triplex switch DNA structure bound to gold NP films (Figure 7A). 88 In this case, the thiolated oligonucleotide conjugated with a SERS-active molecule (carboxy-X-rhodamine, ROX) forms a rigid triplex structure on the gold surface with a thrombin-binding aptamer.…”