Ik-Bum Kim

Detection and identification of proteins using nanoparticle–fluorescent polymer ‘chemical nose’ sensors

A sensor array containing six non-covalent gold nanoparticle-fluorescent polymer conjugates has been created to detect, identify and quantify protein targets. The polymer fluorescence is quenched by gold nanoparticles; the presence of proteins disrupts the nanoparticle-polymer interaction, producing distinct fluorescence response patterns. These patterns are highly repeatable and are characteristic for individual proteins at nanomolar concentrations, and can be quantitatively differentiated by linear discriminant analysis (LDA). Based on a training matrix generated at protein concentrations of an identical ultraviolet absorbance at 280 nm (A280 = 0.005), LDA, combined with ultraviolet measurements, has been successfully used to identify 52 unknown protein samples (seven different proteins) with an accuracy of 94.2%. This work demonstrates the construction of novel nanomaterial-based protein detector arrays with potential applications in medical diagnostics.

Detection and differentiation of normal, cancerous, and metastatic cells using nanoparticle-polymer sensor arrays

Rapid and effective differentiation between normal and cancer cells is an important challenge for the diagnosis and treatment of tumors. Here, we describe an array-based system for identification of normal and cancer cells based on a ‘‘chemical nose/tongue’’ approach that exploits subtle changes in the physicochemical nature of different cell surfaces. Their differential interactions with functionalized nanoparticles are transduced through displacement of a multivalent polymer fluorophore that is quenched when bound to the particle and fluorescent after release. Using this sensing strategy we can rapidly (minutes/seconds) and effectively distinguish (i) different cell types; (ii) normal, cancerous and metastatic human breast cells; and (iii) isogenic normal, cancerous and metastatic murine epithelial cell lines.

Array-Based Sensing of Normal, Cancerous, and Metastatic Cells Using Conjugated Fluorescent Polymers

A family of conjugated fluorescent polymers was used to create an array for cell sensing. Fluorescent conjugated polymers with pendant charged residues provided multivalent interactions with cell membranes, allowing the detection of subtle differences between different cell types on the basis of cell surface features. Highly reproducible characteristic patterns were obtained from different cell types as well as from isogenic cell lines, enabling the identification of the cell type as well differentiating between normal, cancerous, and metastatic isogenic cell types with high accuracy.

Molecular Recognition Based on Low-Affinity Polyvalent Interactions: Selective Binding of a Carboxylated Polymer to Fibronectin Fibrils of Live Fibroblast Cells

To explore molecular recognition of biomolecules in the complex environment of the extracellular matrix, we utilized two fluorescent poly(p-phenyleneethynylene)s bearing either cationic alkylammonium or negatively charged carboyxlate side chains. While incubation of live NIH 3T3 fibroblast cells with the cationic polymer yielded perinuclear punctate staining reminiscent of endocytotic vesicles, the carboxylated polymer revealed a characteristic filamentous staining pattern. Histochemical and immunofluorescence studies demonstrated that the anionic PPE selectively binds to fibronectin fibrils of the extracellular matrix. An in vitro binding study revealed a dissociation constant of approximately 100 nM for the fibronectin-polymer complex. Both polymers showed bright two-photon excited emission as well as low toxicity, rendering them well-suited for live cell imaging studies. The studies demonstrate that selective molecular recognition of biomolecules in the complex environment of the extracellular matrix can be achieved by means of nonspecific low-affinity polyvalent interactions.

Fluorescence Self-Quenching of a Mannosylated Poly(p-phenyleneethynylene) Induced by Concanavalin A

We report that static quenching of a mannosylated conjugated polymer (sugar-PPE) by Concanavalin A is positively dependent upon sugar-PPE concentration, that is, the recorded Stern-Volmer constants increase with increasing sugar-PPE concentration. Comparison with data obtained from isothermal titration calorimetry (ITC) display the increased sensitivity of the quenching method when compared to ITC. The proposed mechanism suggests the interaction of two or more chains of PPE with one Con A molecule leading to a quenched sugar-PPE-Con A construct.

Nano-Conjugate Fluorescence Probe for the Discrimination of Phosphate and Pyrophosphate

We describe a pyrophosphate (PPi) probe that is based on a fluorescent dicarboxylate-substituted poly(para-phenyleneethynylene) (PPE) and 10 nm cobalt-iron spinel nanoparticles (NPs) in aqueous media. The spinel NPs efficiently quench the fluorescence of the PPE at a concentration of 20-30 pmol. Addition of phosphate anions to the PPE-NP construct displaces the quenched PPE to give rise to a fluorescent response; we found that PPi and phosphate (Pi) have significantly different binding affinities for the self-assembled materials. We can discern >40 nM PPi in the presence of 0.1 mM Pi at pH 7, which suggests that these assemblies may be useful in bio-analytical applications. This displacement assay was used to effectively determine the ability of pyrophosphatase to hydrolyze PPi to Pi.