Novel Aptasensor Platform Based on Ratiometric Surface-Enhanced Raman Spectroscopy

Abstract

A novel aptasensor platform has been developed for quantitative detection of adenosine triphosphate (ATP) based on a ratiometric surface-enhanced Raman scattering (SERS) strategy. The thiolated 3'-Rox-labeled complementary DNA (cDNA) is first immobilized on the gold nanoparticle (AuNP) surface and then hybridizes with the 3'-Cy5-labeled ATP-binding aptamer probe (Cy5-aptamer) to form a rigid double-stranded DNA (dsDNA), in which the Cy5 and Rox Raman labels are used to produce the ratiometric Raman signals. In the presence of ATP, the Cy5-aptamer is triggered the switching of aptamer to form the aptamer-ATP complex, leading to the dissociation of dsDNA, and the cDNA is then formed a hairpin structure. As a result, the Rox labels are close to the AuNP surface while the Cy5 labels are away from. Therefore, the intensity of SERS signal from Rox labels increases while that from Cy5 labels decreases. The results show that the ratio between the Raman intensities of Rox labels and Cy5 labels is well linear with the ATP concentrations in the range from 0.1 to 100 nM, and the limit of detection reaches 20 pM, which is much lower than that of other methods for ATP detection and is also lower than that of SERS aptasensor for ATP detection. The proposed strategy provides a new reliable platform for the construction of SERS biosensing methods and has great potential to be a general method for other aptamer systems.

On-demand quantitative SERS bioassays facilitated by surface-tethered ratiometric probes

“… 18 – 20 Although a few ratiometric SERS detection methods were also proposed recently, most of them involved the aggregation of metal colloids to form the hot spots. 21 – 23 Such aggregation based detection methods have to face difficulties to get a reproducible SERS response for quantitative assessment because of many uncontrollable factors, including the difference in aggregation degree, the inherent heterogeneity in particle structure, the random adsorption and orientation of the SERS reporter molecules on the metal surfaces, and so forth. Additionally, the thermodynamic instability of the aggregated colloidal NPs adds further complications by requiring the test results to be read within a specified time interval to guarantee their validity.…”

A Mass-Ratiometry-Based CD45 Barcoding Method for Mass Cytometry Detection

“…As previous literature has indicated, the ratiometry method is an effective strategy to overcome the instability of detection intensity. 25 Furthermore, using mass-ratiometry barcoding, we expect to extend the two-state signal combination, which is based on a signal’s presence or absence, into multistate signals.…”

“…26,27 Nevertheless, its wide application is hampered by data variations of different samples and limited barcoding dimensions. 16 As the ratiometric method provides an effective approach to overcome the instability of single signals, 25 this study provides a new barcoding strategy based on a CD45 mass-ratiometry The occupancies of specific cell clusters (CD3 -, CD3 + , CD3 + CD4 + , CD3 + CD8 + , CD3 + CD4 -CD8 -, CD3 -CD20 + , CD16 + CD56 + , and CD14 -CD16 + ) among entire lymphocytes in each barcoded sample and nonbarcoded sample were determined from gating statistics. The plots, in which each dot stands for a human PBMC sample, compare the cell cluster occupancies acquired in barcoded samples with nonbarcoded samples.…”

Thrombin Assessment on Nanostructured Label-Free Aptamer-Based Sensors: A Mapping Investigation via Surface-Enhanced Raman Spectroscopy

“…The investigation presented here is based on the quantitative detection of a label-free biomarker, differently from a number of reports mainly focused on this method of analysis, also involving the design of a specific detection and measurement protocol [ 25 – 27 ]. Clearly, the latter one can easily support the recognition of the target, but it implies additional manipulations of the sample before evaluation as labelling biomolecules is a time-consuming procedure and can imply the loss of biological activity [ 24 ].…”