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ABSTRACT The aggregation of plasmonic nanoparticles can lead to new and controllable properties useful for numerous applications. We recently showed the reversible aggregation of gold nanoparticles (AuNPs) via a small, cationic di‐arginine peptide; however, the mechanism underlying this aggregation is not yet comprehensively understood. Here, we seek insights into the intermolecular interactions of cationic peptide‐induced assembly of citrate‐capped AuNPs by empirically measuring how peptide identity impacts AuNP aggregation. We examined the nanoscale interactions between the peptides and the AuNPs via UV‐vis spectroscopy to determine the structure‐function relationship of peptide length and charge on AuNP aggregation. Careful tuning of the sequence of the di‐arginine peptide demonstrated that the mechanism of assembly is driven by a reduction in electrostatic repulsion. We show that acetylated N‐terminals and carboxylic acid C‐terminals decrease the effectiveness of the peptide in inducing AuNP aggregation. The increase in peptide size through the addition of glycine or proline units hinders aggregation and leads to less redshift. Arginine‐based peptides were also found to be more effective in assembling the AuNPs than cysteine‐based peptides of equivalent length. We also illustrate that aggregation is independent of peptide stereochemistry. Finally, we demonstrate the modulation of peptide‐AuNP behavior through changes to the pH, salt concentration, and temperature. Notably, histidine‐based and tyrosine‐based peptides could reversibly aggregate the AuNPs in response to the pH. 

We present a strategy for constructing activatable photoacoustic imaging (PAI) probes for in vivo enzyme activity measurements, based on a dissociation strategy previously applied to in vitro sensing. Unlike conventional nanoparticle aggregation strategies, dissociation minimizes false positives and functions effectively in complex biological environments. Overcoming the challenge of dissociating nanostructure aggregates, which arises from the strong van der Waals forces at short distances, we demonstrate the controlled assembly and dissociation of citrate-capped gold nanorods (AuNRs-citrate) using a diarginine peptide additive and a thiolated polyethylene glycol (HS-PEG-OMe), respectively. This assembly dissociation mechanism enables precise control of the optical and photoacoustic (PA) properties of AuNRs in both in vitro and in vivo settings. Building on these findings, we engineered an enzyme-sensitive PAI probe (AuNRs@RgpB) composed of AuNR assemblies and a PEG-peptide conjugate with a protease-specific cleavage sequence. The probe detects Arg-specific gingipain (RgpB), a protease expressed by Porphyromonas gingivalis associated with periodontal disease and Alzheimer’s disease. Proteolytic cleavage of the peptide sequence triggers AuNR dissociation, resulting in enhanced PA signal output. The probe was designed to be injected intrathecally for preclinical trials to image gingipains and investigate the value of gingipain inhibitors developed for Alzheimer’s disease. The probe’s performance was characterized in vitro using UV−vis spectroscopy and PAI, achieving detection limits of 5 and 20 nM, respectively. In vivo studies involved intracranial injection of AuNRs@ RgpB into RgpB-containing murine models, with PA monitoring over time. RgpB activity produced a four-fold PA signal increase within 2 h, while P. gingivalis-infected mice showed similar signal enhancement. Specificity was confirmed by negligible responses to Fusobacterium nucleatum, a non-RgpB-producing bacterium. Additionally, the system demonstrated utility in drug development by successfully monitoring the inhibition of RgpB activity using RgpB inhibitors (leupeptin and KYT-1) in vivo models. Beyond its immediate application to RgpB detection, this modular approach to plasmonic-based sensing holds significant potential for detecting other proteases, advancing both nanotechnology and protease-targeted diagnostics. 

Plasmonic nanoparticle-based biosensors often report a colorimetric signal through the aggregation or clustering of the nanoparticles (NPs), but these mechanisms typically struggle to function in complex biofluids. Here, we report a matrixinsensitive sensor array approach to detect bacteria, fungi, and viruses whose signal is based on the dissociation of the peptideaggregated NPs by thiolated polyethylene glycol (HS-PEG) polymers. We show that the HS-PEGs of differing sizes have varying capabilities to dissociate citrate-capped gold nanoparticle (AuNP) and silver nanoparticle (AgNP) assemblies. The dissociative abilities of the HS-PEGs were used in this sensor array to discriminate at the 90% confidence level the microorganisms Porphyromonas gingivalis, Fusobacterium nucleatum, and Candida albicans in water and saliva using linear discriminant analysis (LDA). We further demonstrate the versatility of the sensor array by detecting various subtypes of the viruses SARS-CoV-2 (beta, delta, and omicron) and influenza (H3N2) spiked in saliva samples using LDA. In the final demonstration, the sensor array design stratified healthy saliva samples from patient samples diagnosed with periodontitis as well as COVID-19. 

This review summarizes insights into colorant selection and signal mechanisms for the development of colorimetric sensing and POC sensors.