Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres

Take an assay plate containing a mixture of magnetic polystyrene microspheres internally dyed with spectrally different fluorophores.

Each microsphere set is covalently coupled to distinct pathogenic bacteria-specific capture antibodies.

Add a heat-killed pathogenic bacterial mixture. Incubate.

Capture antibodies bind the target bacterial antigens, forming microsphere-bacteria complexes.

Use a magnetic separator to settle complexes. Discard unbound bacteria and wash with buffer.

Add buffer and biotinylated detection antibodies, which bind specifically to microsphere-bound bacteria.

Magnetically separate the complexes and wash with buffer.

Resuspend the complexes in buffer. Pipette reporter molecules comprising fluorophore-coupled streptavidin, which bind to the biotinylated detection antibodies.

Magnetically separate and wash the complexes. Resuspend in buffer.

During flow-based analysis, the first laser excites the microsphere's internal fluorophores, generating unique spectral signatures and identifying unique microsphere sets.

The second laser excites the fluorophore reporters bound to bacteria on microspheres, quantifying bacteria on various microsphere sets, enabling multiplexed quantitative bacterial detection.

For sample capturing, begin by adding fifty microliters of microspheres, or beads conjugated to capture antibodies into a 96-well plate. Then, add fifty microliters of the pathogenic bacteria followed by fifty microliters of PBS-TBN into background wells.

Cover the plate with a foil plate seal and incubate on a plate shaker at eight hundred rpm for an hour.

Thereafter, place the plate on the magnetic separator for 1 minute to separate the beads. Evacuate the remaining solution, while keeping the plate on the magnetic plate separator. Then, tap to dry on laboratory paper towels three to four times. Wash the wells thoroughly with PBS-TBN twice.

For sample detection, add fifty microliters of assay buffer followed by fifty microliters of biotinylated detection antibody at a concentration of four micrograms per milliliter into the wells.

Incubate the plate in the dark for an hour to allow efficient interaction between bacteria and detection antibodies. Subsequently, separate the beads using the magnetic separator and wash the beads with PBS-TBN, as demonstrated previously.

Resuspend the beads in fifty microliters of assay buffer and add fifty microliters of streptavidin-R-phycoerythrin or SAPE, a fluorescent reporter molecule. Cover the plate with a foil plate seal, and incubate on the plate shaker for 30 minutes.

Once the wells are washed, resuspend the beads in 100 microliters of PBS-TBN and load the plate on the flow-based analyzer.

Record the readings using appropriate software. The net median fluorescence intensity values for each organism can be evaluated through a standard curve to determine the bacterial load present in the sample.