Solid-Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles with a Reusable Template - "Plastic Antibodies"

Abstract

Molecularly Imprinted Polymers (MIPs) are generic alternatives to antibodies in sensors, diagnostics and separations. To displace biomolecules without radical changes in infrastructure in device manufacture, MIPs should share their characteristics (solubility, size, specificity and affinity, localized binding domain) whilst maintaining the advantages of MIPs (low-cost, short development time and high stability) hence the interest in MIP nanoparticles. Herein we report a reusable solid-phase template approach (fully compatible with automation) for the synthesis of MIP nanoparticles and their precise manufacture using a prototype automated UV photochemical reactor. Batches of nanoparticles (30-400 nm) with narrow size distributions imprinted with: melamine (d = 60 nm, K_d = 6.3 × 10^-8 m), vancomycin (d = 250 nm, K_d = 3.4 × 10^-9 m), a peptide (d = 350 nm, K_d = 4.8 × 10^-8 m) and proteins have been produced. Our instrument uses a column packed with glass beads, bearing the template. Process parameters are under computer control, requiring minimal manual intervention. For the first time we demonstrate the reliable re-use of molecular templates in the synthesis of MIPs (≥ 30 batches of nanoMIPs without loss of performance). NanoMIPs are produced template-free and the solid-phase acts both as template and affinity separation medium.

Keywords: Antibody Replacements; Automatic Reactor; Immobilized Template; Solid-Phase Synthesis; Template Re-use.

Figure 1

Schematic representation of the solid-phase synthesis of MIP nanoparticles. The monomer mixture is injected onto the column reactor with immobilized template and polymerization is initiated by UV-irradiation. The low-affinity particles, as well as unreacted monomers, are eluted at low temperature. The temperature is then increased and high-affinity particles are eluted from the column for collection.

Figure 2

Schematic diagram showing the mode of operation of the automated solid-phase MIP nanoparticle synthesizer. An engineering schematic is shown in the Supporting Information, Figure S1. Typical operational parameters using melamine as the immobilized target are: operation time: 2 h per cycle; yield of high affinity fraction: 6.6 ± 0.65 mg per cycle; column capacity: 23.5 g derivatized glass beads (solid phase).

Figure 3

Influence of the irradiation time on the yield and size of synthesized nanoparticles. Error bars represent ± 1 s.d. (n ≥ 7).

Figure 4

Influence of the size of the nanoparticles on their affinity (apparent dissociation constant) as determined by SPR. Dry size, measured by SEM and size in acetonitrile (in square brackets) measured by DLS. Inset: SEM of the 60 nm diameter MIP nanoparticles (the full field SEM can be seen in the Supporting Information, Figure S2). Error bars represent ± 1 s.d. (n = 3).