Rapid and Selective Detection of Proteins by Dual Trapping Using Gold Nanoparticles Functionalized with Peptide Aptamers

A colorimetric platform for the fast, simple, and selective detection of proteins of medical interest is presented. Detection is based on the aggregation of two batches of peptide functionalized gold nanoparticles via the dual-trapping of the protein of interest. As proof of concept, we applied our platform to the detection of the oncoprotein Mdm2. The peptide aptamers used for the functionalization are based on the reported binding sequences of proteins p53 and p14 for the oncoprotein. Rapid aggregation, and a color change from red to purple, was observed upon addition of Mdm2 with concentrations as low as 20 nM. The selectivity of the system was demonstrated by the lack of response upon addition of bovine serum albumin (in large excess) or of a truncated version of Mdm2, which lacks one of the peptide binding sites. A linear response was observed between 30 and 50 nM of Mdm2. The platform reported here is flexible and can be adapted for the detection of other proteins when two binding peptide aptamers can be identified. Unlike current immunoassay methods, it is a one-step and rapid method with an easy readout signal and low production costs

Di-Arginine Additives for Dissociation of Gold Nanoparticle Aggregates: A Matrix-Insensitive Approach with Applications in Protease Detection

We report the reversible aggregation of gold nanoparticles (AuNPs) assemblies via a di-arginine peptide additive and thiolated PEGs (HS-PEGs). The AuNPs were first aggregated by attractive forces between the citrate-capped surface and the arginine side chains. We found that the HS-PEG thiol group has a higher affinity for the AuNP surface, thus leading to redispersion and colloidal stability. In turn, there was a robust and obvious color change due to on/off plasmonic coupling. The assemblies’ dissociation was directly related to the HS-PEG structural properties such as their size or charge. As an example, HS-PEGs with a molecular weight below 1 kDa could dissociate 100% of the assemblies and restore the exact optical properties of the initial AuNP suspension (prior to the assembly). Surprisingly, the dissociation capacity of HS-PEGs was not affected by the composition of the operating medium and could be performed in complex matrices such as plasma, saliva, bile, urine, cell lysates, or even seawater. The high affinity of thiols for the gold surface encompasses by far the one of endogenous molecules and is thus favored. Moreover, starting with AuNPs already aggregated ensured the absence of a background signal as the dissociation of the assemblies was far from spontaneous. Remarkably, it was possible to dry the AuNP assemblies and solubilize them back with HS-PEGs, improving the colorimetric signal generation. We used this system for protease sensing in biological fluids. Trypsin was chosen as the model enzyme, and highly positively charged peptides were conjugated to HS-PEG molecules as cleavage substrates. The increase of positive charge of the HS-PEG–peptide conjugate quenched the dissociation capacity of the HS-PEG molecules, which could only be restored by the proteolytic cleavage. Picomolar limit of detection was obtained as well as the detection in saliva or urine

Di-Arginine Additives for Dissociation of Gold Nanoparticle Aggregates: A Matrix-Insensitive Approach with Applications in Protease Detection

We report the reversible aggregation of gold nanoparticles (AuNPs) assemblies via a di-arginine peptide additive and thiolated PEGs (HS-PEGs). The AuNPs were first aggregated by attractive forces between the citrate-capped surface and the arginine side chains. We found that the HS-PEG thiol group has a higher affinity for the AuNP surface, thus leading to redispersion and colloidal stability. In turn, there was a robust and obvious color change due to on/off plasmonic coupling. The assemblies’ dissociation was directly related to the HS-PEG structural properties such as their size or charge. As an example, HS-PEGs with a molecular weight below 1 kDa could dissociate 100% of the assemblies and restore the exact optical properties of the initial AuNP suspension (prior to the assembly). Surprisingly, the dissociation capacity of HS-PEGs was not affected by the composition of the operating medium and could be performed in complex matrices such as plasma, saliva, bile, urine, cell lysates, or even seawater. The high affinity of thiols for the gold surface encompasses by far the one of endogenous molecules and is thus favored. Moreover, starting with AuNPs already aggregated ensured the absence of a background signal as the dissociation of the assemblies was far from spontaneous. Remarkably, it was possible to dry the AuNP assemblies and solubilize them back with HS-PEGs, improving the colorimetric signal generation. We used this system for protease sensing in biological fluids. Trypsin was chosen as the model enzyme, and highly positively charged peptides were conjugated to HS-PEG molecules as cleavage substrates. The increase of positive charge of the HS-PEG–peptide conjugate quenched the dissociation capacity of the HS-PEG molecules, which could only be restored by the proteolytic cleavage. Picomolar limit of detection was obtained as well as the detection in saliva or urine