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The vision of a cheese sandwich is actually quite useful when trying to understand the sandwich hybridization technique. In this analogy the cheese represents the single strand (ss) target DNA or RNA. The bread represents the oligonucleotide probe. For each target there are two oligonucleotide probes. The probes are synthesized to be perfectly complementary to the ends of the target ssDNA or RNA. Therefore, during an incubation period each oligonucleotide probe will bind to its specific end of the target DNA: one at the 5’ end and one at the 3’ end.

The bacteria Staphylococcus aureusis of major concern in modern healthcare.Once it spreads to a patient’s bloodstreamthe infection can lead to sepsis and death. Hospitalized patients are particularly vulnerable, especially those with weakened immune systems. Some strains of S. aureushavebegun 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, andthe magnetic nanoparticleis 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 grouphas 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:

1. A sample of blood (1 mL) is mixed with magnetic nanoparticles coated with silica and functionalized to bind to S. aureus.
2. The nanoparticle-bacteria conjugate is isolated from solution by biomagnetic separation.
3. The isolate is heated to release MNase out of the bacteria and into solution
4. 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.
5. 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.