Strain promoted azide alkyne cycloaddition, an efficient surface functionalization strategy for microRNA magnetic separation

In an emergency context, the detection of microRNA (miRNA), which are potential biomarkers of cardiovascular and muscular pathologies is hampered by the time required for the various steps involved: treatment of the patient sample, reverse transcription (RT), and finally polymerase chain reaction (PCR). In this context, magnetically assisted extraction of miRNA could shorten the sample treatment duration and enable a potential pre-concentration to gain in sensitivity. To this goal, magnetic nanoparticles (MNP) functionalized with DNA were developed. Maghemite nanoparticles were coated with a layer of silica, doubly functionalized with PEG chains to guarantee colloidal stability and limit non-specific protein adsorption, and amine functions to allow post-functionalization with DNA which have complementary sequences of the targeted miRNA. Two post-functionalization strategies were tested, one based on the reaction between a thiolated DNA and a maleimide group at the surface of the MNP, the other between an azide-functionalized DNA and a dibenzocyclooctyne group on the surface of the MNP. The strain-promoted azide alkyne cycloaddition (SPAAC click chemistry) proved to be the most efficient functionalization strategy. Optimization of the complementary DNA grafting protocol, carried out by varying the ratios between the quantities of dibenzocyclooctyne groups and DNA and between the quantities of DNA and MNP, revealed on the one hand that the quantity of DNA grafted per nanoparticle could be finely controlled, and on the other hand that the grafting on the MNP’s surface of an excessive quantity of dibenzocyclooctyne groups led to a reduction in grafting yields. The optimized process was used for grafting the complementary DNA of 6 different miRNA, including 4 biomarkers of muscle injury (miR-1, 133a, 133b, and 206). It led to the grafting of approximately 4 complementary DNA per MNP, whatever the nucleotide sequence considered. Capture of the targeted miRNA by magnetic separation was then studied in buffered model media and in complex biological media. This study showed that MNP allowed magnetically-assisted extraction of target miRNA with good hybridization yields, even in biological media for which the capture capacity was preserved at around 75 %, with rapid kinetics and satisfactory selectivity.