Method and system for microfluidic manipulation, amplification and analysis of fluids, for example, bacteria assays and antiglobulin testing
View Patent ↗A microfluidic system for isolation and amplification of DNA or RNA from aqueous solutions and detection of the DNA or RNA on a lateral flow detection strip, including a disposable microfluidic card for use in analysis of bacteria in platelets and an analysis of sexually transmitted diseases (STD) in urine. The card will include an embedded membrane that filters out cells and cellular debris. Any biological debris on the membrane will be lysed and the DNA or RNA amplified via PCR amplification protocol, including appropriate reagents and thermal cycling conditions. The amplified DNA or RNA are transferred to a lateral flow detection strip for a visual diagnostic read out. An alternate embodiment includes a microfluidic card for use in typing antiglobulin assays.
1. A method for microfluidic analysis of a fluid sample, comprising:
loading a microfluidic card with a fluid sample;
lysing the fluid sample to separate components of the fluid sample;
capturing the separated components on a solid substrate;
washing the separated components with wash buffers;
amplifying the washed components in an the amplification chamber; and
pumping the amplified components over a lateral flow strip for detection.
2. The method of claim 1 wherein the separated components are bacteria.
3. The method of claim 1 wherein the washing includes removing nucleic acid to prohibit interference with the amplifying the washed components.
4. The method of claim 2 wherein the bacteria are Escherichia. coli., Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp., Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae , β- Streptococcus, Serratia marcescens , and/or Bacillus cereus.
5. The method of claim 1 further including priming the separated components with DNA primers.
6. The method of claim 1 wherein the pumping includes visually detecting the presence of bacterial DNA on the lateral flow strip.
7. The method of claim 1 further including engaging the microfluidic card with a manifold of an instrument for purposes of pumping the fluid sample through the card.
8. A method of microfluidic analysis of a fluid sample on a microfluidic card, comprising:
collecting a fluid sample;
filtering the fluid sample through a membrane module wherein target cellular material is retained on membrane;
passing a wash buffer across the membrane such that target cellular material remains on the membrane;
passing induction solution across membrane;
passing a lysing solution across the membrane;
passing a wash buffer across the membrane to wash the lysing solution from the membrane;
passing a first NASBA solution across the membrane;
passing a wash buffer across the membrane to wash the first NASBA solution from the membrane;
passing a second NASBA solution across the membrane;
passing a wash buffer across the membrane to wash the second NASBA solution from the membrane;
passing a detection solution across the membrane;
amplifying a RNA signal by thermo-cycling the cellular material;
washing the detection probe solution from the membrane; and
exposing the washed detection probe solution to a lateral flow strip for visual detection of RNA.
9. The method of claim 8 wherein the membrane module is removed from a filtration apparatus and inserted into the microfluidic card.
10. The method of claim 8 wherein the induction solution is pipetted onto the card.
11. The method of claim 8 wherein the second NASBA solution includes enzymes.
12. The method of claim 8 wherein the microfluidic card in fluidly engaged with the manifold of a fluidic instrument for pumping the fluid throughout the card.
13. The method of claim 9 wherein the microfluidic card is removed from the fluid engagement of the manifold and detachably connected to a thermo-coupler for the amplification of the RNA signal.