Core–Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum

We describe the application of a fluorescently labeled water-soluble core–shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. <i>p</i>-Aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles, which bound selectively to trypsin, were used for quantification. The MIP was prepared by a multistep solid-phase synthesis approach on glass beads functionalized with PAB, orientating all trypsin molecules in the same way. The core–MIP was first synthesized, using a thermoresponsive polymer based on <i>N</i>-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for postconjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core–MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core–shell MIP was endowed with a homogeneous population of high-affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into nondiluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications

Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins

We describe a potentially universal, simple and cheap method to prepare water-compatible molecularly imprinted polymer nanoparticles (MIP-NPs) as synthetic antibodies against proteins. The strategy is based on a solid phase synthesis approach where glass beads (GBs) are functionalized with a metal chelate, acting as a general affinity ligand to attract surface-bound histidines present on proteins. This configuration enables an oriented immobilization of the proteins, upon which thermoresponsive MIP-NPs are synthesized. The GBs play the role of both a reactor and a separation column since, after synthesis, the MIP-NPs are released from the support by a simple temperature change, resulting in protein-free polymers. The resulting MIP-NPs are endowed with improved binding site homogeneity, since the binding sites have the same orientation. Moreover, they are stable (no aggregation) in a buffer solution for prolonged storage time and exhibit apparent dissociation constants in the nanomolar range, with little or no cross-reactivity toward other proteins

Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging

War against cancer constantly requires new affinity tools to selectively detect, localize, and quantify biomarkers for diagnosis or prognosis. Herein, carbon nanodots (CDs), an emerging class of fluorescent nanomaterials, coupled with molecularly imprinted polymers (MIPs), are employed as a biocompatible optical imaging tool for probing cancer biomarkers. First, N-doped CDs were prepared by hydrothermal synthesis using starch as carbon source and l-tryptophan as nitrogen atom provider to achieve a high quantum yield of 25.1 ± 2%. The CDs have a typical size of ∼3.2 nm and produce an intense fluorescence at 450 nm upon excitation with UV light. A MIP shell for specific recognition of glucuronic acid (GlcA) was then synthesized around the CDs, using the emission of the CDs as an internal light source for photopolymerization. GlcA is a substructure (epitope) of hyaluronan, a biomarker for certain cancers. The biotargeting and bioimaging of hyaluronan on fixated human cervical cancer cells using CD core-MIP shell nanocomposites is demonstrated. Human keratinocytes were used as noncancerous reference cells and indeed, less staining was observed by the CD-MIP

Dual-Oriented Solid-Phase Molecular Imprinting: Toward Selective Artificial Receptors for Recognition of Nucleotides in Water

We describe the synthesis of water-soluble molecularly imprinted polymer nanoparticles (MIP-NPs) as a new artificial host receptor for the recognition of adenosine monophosphate (AMP), used herein, as a model nucleotide. MIP-NPs were prepared by solid-phase synthesis on glass beads (GB) using, for the first time, immobilized Fe­(III)-chelate as an affinity ligand to orientate the AMP via its phosphate group. A polymerizable thymine monomer which can induce complementary base-pairing with the adenine moiety of the nucleotide was synthesized and incorporated in the polymerization mixture to constrain the AMP in a dual-orientated configuration. The MIP-NPs were remarkably selective toward AMP as they did not bind other nucleotides, GMP, UMP, and CMP. This strategy of using the phosphate group of AMP as a hinge enables unhindered pairing of the nucleobase with its corresponding complementary base monomer and can be extended to the preparation of specific MIP receptors for other key nucleotides in aqueous conditions