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Superbugs, or bacteria that are resistant to currently available antibiotic treatments, are of growing concern to human health worldwide. Many of these superbugs are present in hospitals, and are  frequently colonizing surgical sites and causing life-threatening infections and sepsis. The presence of these bacteria in blood is commonly detected by traditional culture methods that require one to two days. There is a need for a faster method to identify the presence of the bacteria and to take measures to prevent the spread of infection  as rapidly as possible. One proposed technology is to use immunomagnetic separation with conjugated fluorescent probes to selectively bind bacteria and quickly visualize their presence in whole blood samples. Recently, fluorescent magnetic multifunctional carbon dots have been coupled with superbug-specific antibodies for the identification of the drug-resistant superbugs Staphylococcus aureus (MRSA) and Salmonella enterica serotype typhimurium definitive phage type 104 (DT 104) present in whole blood samples.

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Red and blue fluorescent carbon dots

The carbon dots used in this system are quantum dots, which are nanocrystals of approximately 5 nm in size. When nanocrystals are this small they demonstrate fascinating optical properties. The emission fluorescence of a quantum dot is dependent on its size, so quantum dots of the same composition but different size will emit different wavelengths of light. The emission spectrum of a quantum dot is very narrow, but the excitation spectrum is broad. This means that UV or white light can be used to excite multiple quantum dots of different sizes and they will fluoresce at different colors. There are two carbon dots used in this study—red (630 nm emission) and blue (462 nm emission), and they are both excited by UV light (380 nm).

General structure of the multifunctional nanoparticles

The carbon-dot antibody functionalized superparamagnetic nanoparticles were formed during a multistep reaction. First, magnetic nanoparticles were synthesized with a surface coating of carboxylic acid functional groups. Blue or red carbon dots were then attached to the magnetic nanoparticles by amide coupling. Finally, the antibodies were coupled to the carbon dots by another amide coupling reaction involving PEG. The anti-salmonella antibody was attached to the blue carbon dot magnetic nanoparticles, and the anti-MRSA antibody was attached to the red carbon dot magnetic nanoparticles.

Capturing and identifying superbugs from infected blood

The red anti-MRSA multifunctional nanoparticles were incubated with MRSA-infected blood for 30 minutes, and demonstrated close to 100% capture efficiency with magnetic separation. The capture of MRSA was confirmed by RT-qPCR. Additionally, successful capture could be fluorescently imaged for quick readout. The anti-MRSA nanoparticles were specific to the MRSA bacteria and did not capture the salmonella. This specificity was also true of the anti-salmonella nanoparticles.

This multifunctional magnetic nanoparticle is versatile and could potentially be used to capture a variety of target bacteria from blood. The carbon dot color could be changed, or the antibody bound to the surface of the particle could be modified. Therefore, this rapid identification system has the potential to identify many different kinds of superbugs in blood, and may be optimized for various imaging systems.

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Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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