Enzyme-induced Aggregation of Plasmonic Nanoparticles for the Development of Label-free and Ultrasensitive Detection of Bacterial DNA

Sensitive detection of pathogens is critical to ensure the safety of food supplies and to prevent bacterial disease infection and outbreak at the first onset. While conventional techniques such as cell culture, ELISA, PCR, etc. have been used as the predominant detection workhorses, they are however limited by either time-consuming procedure, complicated sample pre-treatment, expensive analysis and operation, or inability to be implemented at point-of-care testing. Here, we present our recently developed assay exploiting enzyme-induced aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. In the experiments, AuNPs are first functionalized with specific, single-stranded RNA probes so that they exhibit high stability in solution even under high electrolytic condition thus exhibiting red color. When bacterial DNA is present in a sample, a DNA-RNA heteroduplex will be formed and subsequently prone to the RNase H cleavage on the RNA probe, allowing the DNA to liberate and hybridize with another RNA strand. This continuously happens until all of the RNA strands are cleaved, leaving the nanoparticles ‘unprotected’. The addition of NaCl will cause the ‘unprotected’ nanoparticles to aggregate, initiating a colour change from red to blue. The reaction is performed in a multi-well plate format, and the distinct colour signal can be discriminated by naked eye or simple optical spectroscopy. As a result, bacterial DNA as low as pM could be unambiguously detected, suggesting that the enzyme-induced aggregation of AuNPs assay is very easy to perform and sensitive, it will significantly benefit to development of fast and ultrasensitive methods that can be used for disease detection and diagnosis.

The Use of a Pressure-Indicating Sensor Film to Provide Feedback upon Hydrogel-Forming Microneedle Array Self-Application In Vivo

PURPOSE:
To evaluate the combination of a pressure-indicating sensor film with hydrogel-forming microneedle arrays, as a method of feedback to confirm MN insertion in vivo.
METHODS:
Pilot in vitro insertion studies were conducted using a Texture Analyser to insert MN arrays, coupled with a pressure-indicating sensor film, at varying forces into excised neonatal porcine skin. In vivo studies involved twenty human volunteers, who self-applied two hydrogel-forming MN arrays, one with a pressure-indicating sensor film incorporated and one without. Optical coherence tomography was employed to measure the resulting penetration depth and colorimetric analysis to investigate the associated colour change of the pressure-indicating sensor film.
RESULTS:
Microneedle insertion was achieved in vitro at three different forces, demonstrating the colour change of the pressure-indicating sensor film upon application of increasing pressure. When self-applied in vivo, there was no significant difference in the microneedle penetration depth resulting from each type of array, with a mean depth of 237 μm recorded. When the pressure-indicating sensor film was present, a colour change occurred upon each application, providing evidence of insertion.
CONCLUSIONS:
For the first time, this study shows how the incorporation of a simple, low-cost pressure-indicating sensor film can indicate microneedle insertion in vitro and in vivo, providing visual feedback to assure the user of correct application. Such a strategy may enhance usability of a microneedle device and, hence, assist in the future translation of the technology to widespread clinical use.