The bacteria Staphylococcus aureus is of major concern in modern healthcare. Once it spreads to a patient’s bloodstream the infection can lead to sepsis and death. Hospitalized patients are particularly vulnerable, especially those with weakened immune systems. Some strains of S. aureus have begun to demonstrate antibiotic resistance. For these reasons it is important to identify and contain infection early; rapid detection of bacteria in patient sera is key. Traditional culture methods are time-consuming and require transportation of samples to offsite clinical laboratories. Development of a rapid point-of-care diagnostic tool is desirable, and the magnetic nanoparticle is rapidly becoming the tool of choice.
First use biomagnetic separation to isolate the target molecule
Magnetic nanoparticles are fantastically customizable due to surface coatings and functional moieties, and are easily recoverable from solution. A functionalized nanoparticle will bind to its target after a short incubation time, and the conjugate can be isolated with the help of a magnetic separation rack. This technology allows for the rapid selection and concentration of a target molecule. Once the target molecules, in this case S. aureus bacteria, are isolated, further identification methods can be used. This is where a group of researchers working in Istanbul, Turkey became creative.
The group has engineered a S. aureus detection system using a mesoporous silica nanoparticle-oligonucleotide conjugate. The system’s limit of detection is 682 cells/mL, which indicates potential for use as a point-of-care diagnostic. The system detects Micrococcal nuclease (MNase), which is an enzyme specifically secreted by S.aureus. MNase degrades RNA or DNA, and is well known within the field to be a marker of S. aureus infection.
The system works as follows:
- A sample of blood (1 mL) is mixed with magnetic nanoparticles coated with silica and functionalized to bind to S. aureus.
- The nanoparticle-bacteria conjugate is isolated from solution by biomagnetic separation.
- The isolate is heated to release MNase out of the bacteria and into solution
- A non-magnetic mesoporous silica nanoparticle filled with fluorophore is added to the solution. The pores of this nanoparticle are sealed shut with oligonucleotides that are resistant to degradation by all nucleases except MNase. When the pores are capped the fluorophore is quenched and no fluorescence is visible.
- If MNase is present in solution the oligonucleotide caps are degraded, the nanoparticle pores open, and the flurofores are released. The free fluorphores are no longer quenched, and a fluorescence signal is measured.
Due to the resistance of the oligonucleotides to degradation by all nucleases except RNAse, fluorescence is only present when the isolated bacteria are S. aureus. Thus, the presence or absence of a fluorescent signal is all that is needed to evaluate the sample. The diagnostic is rapid and accurate and capable of identifying RNase even when the concentration of bacteria is low.
- Recent advances in protein purification using biomagnetic separation - Part 2
- Recent advances in protein purification using biomagnetic separation
- DNA isolation from bacteria using charge-reversible magnetic nanoparticles