Methods and related aspects for multiplexed analyte detection using sequential magnetic particle elution
Provided herein are magnetofluidic cartridges of use in a wide variety of sample analysis applications, including nucleic acid amplification assays. The magnetofluidic cartridges include sample inlet wells and sample analysis wells for performing controlled serial elution techniques that enables execution of extraction/purification and splitting of analytes for multiplex detection via magnetic actuation only. Related magnetofluidic devices, kits, and methods are also provided.
1 . A method of detecting multiple biomolecules in a sample comprising a first biomolecule species and a second biomolecule species using a magnetofluidic cartridge comprising a sample inlet well, a first sample analysis well, and a second sample analysis well, wherein the first sample analysis well fluidly communicates with the sample inlet well and the second sample analysis well fluidly communicates with the first sample analysis well, the method comprising:
contacting, in the sample inlet, a plurality of magnetic particles and an aliquot of the sample such that the first biomolecule species and the second biomolecule bind to the plurality of magnetic particles and produce a first bound biomolecule species and a second bound biomolecule species;
moving the first bound biomolecule and the second bound biomolecule from the sample inlet well to the first sample analysis well using at least one magnet;
producing a first unbound biomolecule species in the first sample analysis well by eluting a portion of the first bound biomolecule species from the plurality of magnetic particles;
moving the remainder of the first bound biomolecule species and the second bound biomolecule species from the first sample analysis well to the second sample analysis using the at least one magnet;
producing a second unbound biomolecule species in the second sample analysis well by eluting a portion of the second bound biomolecule species from the plurality of magnetic particles;
detecting, in the first sample analysis well, the first unbound first biomolecule species or a derivative thereof or both; and
detecting, in the second sample analysis, the second unbound biomolecule species or a derivative thereof or both.
2 . The method of claim 1 , further comprising washing the first bound biomolecule species and the second bound biomolecule species prior to the eluting the portion of the first bound biomolecule species from the plurality of magnetic particles or the eluting the portion of the second bound biomolecule species from the plurality of magnetic particles.
3 . The method of claim 1 wherein the magnetofluidic cartridge further comprises:
a top layer;
a bottom layer spaced apart from the top layer in a substantial parallel orientation with respect to the top layer, which bottom layer including a bottom layer surface and defining a plurality of wells protruding from the bottom layer surface, wherein the plurality of wells comprises the sample inlet well, the first sample analysis well and the second sample analysis well;
a spacer layer operably connected to the top layer and the bottom layer;
a channel defined by the top layer, the bottom layer, and the spacer layer, which channel fluidly communicates with the plurality of wells;
at least one port disposed through the top layer and proximal to the sample inlet well, which at least one port fluidly communicates with the channel;
a sealing mechanism operably connected to the top layer, which sealing mechanism is configured to seal the port in a closed position;
the plurality of magnetic particles disposed in the sample inlet well; and
a different processing reagent disposed in each sample analysis well of the plurality of sample analysis wells.
4 . The method of claim 1 wherein the eluting the portion of the first bound biomolecule species from the plurality of magnetic particles and the eluting the portion of the second bound biomolecule species from the plurality of magnetic particles comprise tuning one or more elution conditions.
5 . The method of claim 1 wherein the one or more elution conditions comprise one or more defined parameters selected from the group consisting of:
a temperature, a duration, a buffer composition, a reaction mixture composition, and a pH level.
6 . The method of claim 1 , wherein the first biomolecule species comprises a first locus of a genomic DNA or a first variant of a genetic locus and the second biomolecule species comprises a second locus of the genomic DNA or a second variant of the genetic locus, wherein the second locus is different from the first locus and the first variant is different from the second variant.