Methods for isolating target analytes from urine samples using ATPS and magnetic beads
The present disclosure relates to methods, compositions, and kits for concentrating and purifying at least one target analyte from a clinical biological sample. In some embodiments, the methods involve one or more aqueous two-phase system (ATPS) compositions and at least one solid phase medium. Some embodiments provide a kit comprising one or more ATPS compositions, a binding buffer; and a solid phase medium. Other embodiments provide methods of treating cancers or infectious diseases in a patient in need thereof.
1. A method for concentrating and purifying at least one target analyte from a clinical biological sample, wherein the clinical biological sample is a urine sample, comprising the steps of
(a) combining the clinical biological sample with a first aqueous two-phase system (ATPS) composition comprising a polymer and a salt component dissolved in an aqueous solution to form a target-rich phase solution and a target-poor phase solution, such that the target analyte is concentrated in the target-rich phase solution, wherein the polymer comprises 5-60% PEG with a molecular weight of at least 400, and the salt component comprises 0.1-6% K 2 HPO 4 and 0.1-18% KH 2 PO 4 ;
(b) collecting the target-rich phase solution;
(c) adding the target-rich phase solution to a second ATPS composition comprising a second polymer and a second salt component dissolved in an aqueous solution to form a second target-rich phase solution and a second target-poor phase solution, such that the target analyte is concentrated in the second target-rich phase solution, and collecting the second target-rich phase solution, wherein the second polymer comprises 2-25% PEG with a molecular weight of 200 or less, and the second salt component comprises 0.1-50% K 2 HPO 4 and 4-40% KH 2 PO 4 ;
(d) optionally mixing the target-rich phase solution from step (b) or the second target-rich phase solution from step (c) with a binding buffer to form a mixed solution;
(e) contacting the the second target-rich phase solution from step (c) or the mixed solution from step (d) with a plurality of magnetic beads configured to selectively bind the target analyte such that the plurality of magnetic beads binds to the target analyte; and
(f) eluting and collecting the target analyte from the plurality of magnetic beads with an eluting solution, resulting in a final solution containing the concentrated and purified target analyte.
2. The method of claim 1 , further comprising the step of washing the plurality of magnetic beads with one or more solvents to remove impurities after step (e) and before step (f).
3. The method of claim 2 , wherein the solvent is the binding buffer.
4. The method of claim 1 , further comprising the step of treating the clinical biological sample with a lysing composition before step (a).
5. The method of claim 1 , wherein the binding buffer comprises a chaotropic agent comprising an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.
6. The method of claim 1 , wherein the target analyte is a nucleic acid, and the nucleic acid is gDNA, cDNA, plasmid DNA, mitochondrial DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, cell-free fetal DNA, cell-free microbial DNA, micro RNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA) or combinations thereof.
7. The method of claim 1 , wherein step (e) comprises the following steps:
(i) contacting a portion of the mixed solution with the plurality of magnetic beads such that the target analyte binds to the plurality of magnetic beads to form a beads-analyte complex;
(ii) spinning the beads-analyte complex and discarding the supernatant; and
(iii) optionally repeating steps (i) and (ii).
8. The method of claim 7 , wherein
the target analyte is a nucleic acid less than a target size;
the plurality of magnetic beads binds to the target analyte and to other nucleic acids;
the eluting solution is a fractionation buffer that, when contacted with the beads during the elution step (f), causes the target analyte to be released while not releasing the other nucleic acids, resulting in a final solution containing the concentrated and purified target analyte(s); wherein the fractionation buffer comprises a polymer, a chaotropic agent, or any combination thereof.
9. The method of claim 1 , further comprising the step of analyzing the final solution from step (f) using ELISA, nucleic acid sequencing, or a PCR-based assay.
10. The method of claim 1 , wherein the target analyte is a biomarker indicating the presence or risk of a medical condition or disease in a patient, wherein the medical condition or disease is an infectious disease, cancer, or a genetic disease.
11. The method of claim 1 , wherein the urine sample has a volume >10 mL, further comprising the following step before step (a): dividing the urine sample into at least two aliquots of a sample solution;
wherein each aliquot is separately combined with the first ATPS in steps (a) and optionally the collected target-rich phase solution from step (b) is separately combined with the second ATPS in step (c) to form each aliquot's second target-rich phase solution; wherein each aliquot's target-rich phase solution from step (b) or each aliquot's second target-rich phase solution from step (c) is combined together to form a final target rich phase for step (d).