A Multi-Fluorescent DNA/Graphene Oxide Conjugate Sensor for Signature-Based Protein Discrimination

Abstract

Signature-based protein sensing has recently emerged as a promising prospective alternative to conventional lock-and-key methods. However, most of the current examples require the measurement of optical signals from spatially-separated materials for the generation of signatures. Herein, we present a new approach for the construction of multi-fluorescent sensing systems with high accessibility and tunability, which allows generating protein fluorescent signatures from a single microplate well. This approach is based on conjugates between nano-graphene oxide (nGO) and three single-stranded DNAs (ssDNAs) that exhibit different sequences and fluorophores. Initially, the three fluorophore-modified ssDNAs were quenched simultaneously by binding to nGO. Subsequent addition of analyte proteins caused a partial recovery in fluorescent intensity of the individual ssDNAs. Based on this scheme, we have succeeded in acquiring fluorescence signatures unique to (i) ten proteins that differ with respect to pI and molecular weight and (ii) biochemical marker proteins in the presence of interferent human serum. Pattern-recognition methods demonstrated high levels of discrimination for this system. The high discriminatory power and simple format of this sensor system should enable an easy and fast evaluation of proteins and protein mixtures.

Nano‐Graphene Oxide Based Multichannel Sensor Arrays towards Sensing of Protein Mixtures

“…Depending on overall charge, functionality and extension of aromatic moieties in biomolecule as well as dyes can control the tunable interaction. By utilizing this non‐covalent interactions, several research group have made sensing platform by attaching fluorescent labeled single‐stranded DNA for detection of proteins, bacteria and diseased cell lines . But the biggest challenge to build a multichannel optical sensor array to select and optimize a set of fluorophores which will have minimum FRET and efficient binding as well as detectable displacement by analytes.…”

“…By utilizing this non-covalent interactions, severalr esearchg roup have made sensing platform by attaching fluorescent labeled single-strandedD NA for detection of proteins,b acteria and diseased cell lines. [27,29,30] But the biggest challenge to build am ultichannelo ptical sensora rray to select and optimize as et of fluorophores which will have minimum FRET and efficient bindinga sw ell as detectable displacement by analytes.H ence to build am ultichannel array by using nGO as ar eceptor first we have screened several fluorophores (Table S17) and finally we have selected five fluorophores 7-hydroxy coumarin (7-HC), Fluorescein (Fl), Rhodamine 6G (Rh-6G), Rhodamine Bb ase (Rh-B) and Methylene Blue (MB)] which not only have the diversec harge and functionality but also the excitation and emission wavelengths have nominal overlap to minimize the FRET (Figure 2b,c). The selected fluorophores which are used in this study,h ave good quantum yield (Table S1), water solubility and high photo stability.…”

Composite‐Scattering Plasmonic Nanoprobes for Label‐Free, Quantitative Biomolecular Sensing

“…Figure A,B shows the results of hierarchical cluster analysis (HCA) for the shorter and longer wavelength resonance peaks of SNPG, respectively. The dendrograms demonstrate that the shorter wavelength peak for SNPG around 440 nm is extremely sensitive to the surrounding conditions and can be used to clearly distinguish between various dielectric media, notably water and 0.1 mmol L −1 BSA, which vary only by 0.001 in their RI.…”

“…Elucidating subtle RI-induced changes in these profiles (as shown in Figures S4 and S5 of the Supporting Information) is challenging by gross visual inspection alone but can be realized through application of multivariate analytics algorithms that harness the full spectral information. [58,59] Figure 4A,B shows the results of hierarchical cluster analysis (HCA) [60][61][62] for the shorter and longer wavelength resonance peaks of SNPG, respectively. The dendrograms demonstrate that the shorter wavelength peak for SNPG around 440 nm is extremely sensitive to the surrounding conditions and can be used to clearly distinguish between various dielectric media, notably water and 0.1 mmol L −1 BSA, which vary only by 0.001 in their RI.…”

Fingerprint-based Protein Identification in Cell Culture Medium Using Environment-sensitive Turn-on Fluorescent Polymer

“…Such probe arrays are constructed to exhibit the chemical diversity necessary to respond distinctively to a variety of analyte proteins, allowing the generation of target-specific response fingerprints. This strategy has been used to identify proteins dissolved in buffer solutions, (9)(10)(11)(12) serum, (13)(14)(15)(16) and urine. (17,18) Most of the previously reported fingerprint-based sensors, however, require multiple synthetic probes as sensor elements for the construction of an array with reliable discrimination ability, (7,8) which involves laborious synthetic efforts.…”