Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Summary

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Abstract

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

Introduction

Efficient conjugation reactions are valuable tools for attaching bioactive molecules to surfaces for a variety of biotechnology applications. Recently, the very fast bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) has been used to label cell surfaces, subcellular structures, antibodies and nanoparticles.^1-7 Here, we use the [4+2] cycloaddition reaction in combination with antigen/antibody capture (GST/anti-GST) for reversible on-chip synthesis of multi-component structures for Surface Plasmon Resonance (SPR) interaction analysis and monitor the process in real-time (Figure 1

Protocol

1. Preparation of GST and Nanoparticle (NP) Conjugates

1. GST-TCO preparation:
   1. Add 8 μl of TCO-NHS solution (50 mM in DMSO) to 100 μl of GST (1 mg/ml in PBS) and shake mixture at RT for 1 hr.
   2. Remove excess reagent using a Zeba spin desalting column. The recovered filtrate containing the GST-TCO conjugate is stored at 4 °C prior to use.
2. GST-Tz preparation:
   1. Add 6 μl of Tz-NHS solution (25 mM in DMF) to 75 μl of GST (1 mg/ml in PBS) and shake mixture at RT for 1 hr.
   2. Dilute the reaction mixture with 25 μl of PBS and purify using a spin desalting column. The filtrat....

Results

Data and figures have been adapted from reference 8.

Efficient reversible immobilization of bioactive small molecules with control over orientation and density plays a key role in development of new biosensor applications. Using the fast bioorthogonal reaction between TCO and Tz, we describe a method for the stepwise assembly and regeneration of ligand surfaces with retention of biological activity. Figure 2 shows the real-time monitoring of Tz-BnNH₂ immobil.......

Discussion

The capture-cycloaddition method described here enables rapid, reversible immobilization of modified nanoparticles and small molecules for label-free chip-based interaction and kinetic studies. The immobilization protocol can be performed in minutes requiring <10 μM concentrations of small-molecule ligands. By modulating ligand concentration and contact time immobilization densities can be closely controlled. Our data show that on-chip bioorthogonal reactions preserve the ability of in situ functionaliz.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
Sensor Chip CM5	GE Healthcare	BR-1005-30
Amine coupling kit	GE Healthcare	BR-1000-50
GST capture kit	GE Healthcare	BR-1002-23
NAP-10 Columns	GE Healthcare	17-0854-01
GST, lyophilized in 1X PBS	Genscript	Z02039	1 mg/ml
rhFKBP12	R&D Systems	3777-FK
Surfactant P-20	GE Healthcare	BR-1000-54
Glycine 2.0	GE Healthcare	BR-1003-55
Zeba spin desalting column	Thermo	89882	7 K MWCO
Amicon Ultra 4	Fisher	UFC810096	100 K centrifugal filter
TCO-OH	Ref. 8		Synthesized in-house
TCO-NHS	Ref. 8		Synthesized in-house, *Commercially available from Click Chemistry Tools # 1016-25
Tz-BnNH2	Ref. 8		Synthesized in-house
Tz-NHS	Ref. 8	764701	Synthesized in-house, *Commercially available from Sigma Aldrich # 764701
NP-NH2 = CLIO-NH2	Ref. 8		Synthesized in-house
AP1497, AP1497-Tz	Ref. 8		Synthesized in-house
Equipment
SPR Biosensor	GE Healthcare	Biacore T100