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Conjugation of Oligonucleotides to Carboxylated Fluorescent Microspheres

Introduction


This tech note briefly describes the conjugation of oligonucleotides to Cytodiagnostics polymer microspheres with a carboxylated surface through a one-step carbodiimide conjugation reaction.

 

In a one-step carbodiimide conjugation reaction all components, i.e. carboxyl microspheres (polystyrene beads), EDC and the ligand to be conjugated are mixed together simultaneously.


The advantage of a one-step reaction over the two-step conjugation procedure, besides being simpler, is that it may provide higher conjugation efficiency in the presence of excess EDC during the reaction process.


One potential complication is that EDC may induce inter-molecular crosslinking instead of specifically conjugating the ligand to the microsphere surface. However, this side-reaction only occurs when the ligand to be conjugated has both accessible primary amines and carboxyl groups that compete with microsphere surface carboxyl groups. Thus the optimal ratio between microspheres and molecules to be conjugated may be different from two-step conjugation procedure.


A one-step conjugation procedure is ideal for low molecular weight molecules and ligands such as amine-modified oligonucleotides. See below for a recommended starting protocol.

Zepto Ultra Carboxyl Fluorescent Microspheres
Figure 1. Fluorescent microscopy image of a mixture of Cytodiagnostics 450nm, 525nm and 665nm emitting Zepto™ Ultra beads. Beads were excited using a single UV laser (405nm) and the image acquired using a color camera.

Conjugation Procedure


The following protocol is for conjugating amine-modified oligo strands to our Zepto™ Carboxylated Fluorescent Microspheres. A typical conjugation reaction utilizes 10 million microspheres. The following reagents are required for the conjugation reaction not including polystyrene beads with a carboxyl surface groups:

  • Activation buffer: 50 mM MES buffer (2-(N-morpholino)ethanesulfonic acid), pH 5.5
  • Washing buffer: 0.1X Phosphate-buffered saline with 0.05% (w/v) Tween 20 (0.1X PBST)
  • Storage buffer: 1% bovine serum albumin in 0.1X PBST, 0.09% sodium azide
  • EDC (Sigma Cat.# E1769): 1 mg/ml in activation buffer (Note: always prepare fresh)
  • Amine-modified oligonucleotide: 10 µM in ddH2O or buffer (Note: buffer cannot contain any primary amines, e.g. Tris)
  1. Vortex the bottle/vial containing the beads thoroughly before use to ensure homogeneous suspension of the beads.
  2. Immediately aliquot 10 million beads (10 µL of stock solution as provided) into a 1.5 ml microcentrifuge vial.
  3. Add 1 ml of EDC prepared as described above.
  4. Add 20 µL of oligonucleotide stock solution (10 µM).
  5. Mix well and incubate for 4 hours at room temperature with constant mixing.
  6. Sonicate the mixture in a sonicator bath for 10 seconds if microspheres agglomerate or adhere to tube surface.
  7. Add TWEEN 20 to the final concentration of 0.05% (w/v).
  8. Pellet the beads by centrifuging in a microcentrifuge at 500 x g for 5 minutes.
  9. Discard the supernatant and add 500 µL of washing buffer.
  10. Pellet the beads by centrifuging in a microcentrifuge at 500 x g for 5 minutes.
  11. Add 500 µL of storage buffer and mix well.
  12. The beads are now conjugated to your oligonucleotide and ready for your use in your assay. Alternatively, store at 4°C protected from light until use.

* The centrifugation speed for pelleting microspheres is a range between 100 to 10,000 g. A lower/more gentle centrifugation speed with longer time is recommended to form a good microsphere pellet, especially after conjugation. Prolonged centrifugation at high speed may aggregate microspheres irreversibly.