Magnetic detection of small entities

#The present invention relates to a novel detection or quantifying method for biological entities or molecules such as, but not limited to, DNA, microorganisms and pathogens, proteins and antibodies, that by themselves are target molecules or from which target molecules are extracted. The methods comprise the steps of i) forming target molecule-dependent volume-amplified entities, ii) allowing magnetic nanoparticles to bind to said volume-amplified entities, and iii) measuring changes in dynamic magnetic response of the magnetic nanoparticles caused by the increase in hydrodynamic volume of said magnetic nanoparticles or measuring the magnetic field due to the magnetic nanoparticles as they bind to a sensor surface functionalized with a secondary capturing probe.

#The present invention further relates to biosensors and kits to be used in such a method.

#There is a need for novel simple assay formats for sensitive DNA sequence analyses as well as for detection of RNA and proteins, suitable for e.g. a clinical setting, bed-side applications, military purposes, forensic analyses, food and pharmaceutical product analyses, as well as analyses of microorganisms in the environment.

#The following detailed description will be more fully understood in view of the drawing in which:

#FIG. 1 shows some examples of probing technologies where reacted probes create nucleic acids circles upon specific recognition of target molecules. I) Proximity probes recognize one or more target molecules (in this case the antibodies have recognised and bound to a protein target) and together with accompanying oligonucleotides enable the formation of a nucleic acids circle. A ligase enzyme can then join the ends of the probes and form a closed nucleic acids circle. II) A padlock probe recognizes a specific DNA or RNA target molecule. Correct hybridization between the padlock probe and the DNA or RNA target molecule enables joining of the padlock probe ends, which forms a nucleic acids circle which may be joined by the addition of a ligase enzyme. III) A selector probe recognizes a specific DNA or RNA target molecule. Correct hybridization between the selector probe and the DNA or RNA target molecule enables joining of the DNA or RNA target molecule ends, which forms a nucleic acids circle which may be joined by the addition of a ligase enzyme. The circles produced in I, II and III, may then be amplified by Rolling Circle Amplification (RCA).

#FIG. 2 demonstrates volume-amplification by RCA or hybridization of passive particles functionalized with secondary capturing probes. I) Preformed nucleic acids circles (created as shown in FIG. 1 or by another suitable strategy), are amplified with RCA, and the RCA products are detected with magnetic nanoparticles functionalized with primary capturing probes or according to any of the procedures illustrated in FIG. 3. II) Preformed nucleic acids circles (created as shown in FIG. 1: I-III or by another strategy) are captured with magnetic nanoparticles functionalized with primary capturing probes and amplified with RCA. The RCA products are thereby linked to the magnetic nanoparticles. The RCA products can be detected with additional magnetic nanoparticles functionalized with primary capturing probes or according to any of the procedures illustrated in FIG. 3. III) DNA or RNA target molecules are captured by magnetic nanoparticles functionalized with selector probes. Correct hybridization between the selector probe and the DNA or RNA target molecule enables joining of the DNA or RNA target molecule ends, which forms a nucleic acids circle, which is amplified with RCA. The RCA products are therefore linked to the magnetic nanoparticles. The RCA products can be detected with additional magnetic nanoparticles functionalized with primary capturing probes or according to any of the procedures illustrated in FIG. 3. IV) DNA or RNA target molecules are captured by magnetic nanoparticles functionalized with primary capturing probes and padlock probes are added. Correct hybridization between the padlock probe and the DNA or RNA target molecule enables joining of the padlock probe ends, which forms a nucleic acids circle, which is amplified with RCA. The RCA products are then linked by hybridization to the magnetic nanoparticles. The RCA products can be detected with additional magnetic nanoparticles functionalized with primary capturing probes or according to any of the procedures illustrated in FIG. 3. V) DNA or RNA target molecules are captured by magnetic nanoparticles functionalized with primary capturing probes. Hybridization of passive particles functionalized with secondary capturing probes amplifies the hydrodynamic volume of the magnetic nanoparticles (sandwich type assay).

#As mentioned above, the present invention relates to a method for quantifying and/or detecting a target molecule, comprising the steps of:

#1) forming a target molecule-dependent volume-amplified entity;

#2) allowing a magnetic nanoparticle, functionalized with a primary capturing molecule, to bind to said volume-amplified entity via said primary capturing molecule;