In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we report a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). Through boronate formation, the carbohydrate moiety within the constant domain, Fc, of the antibody can be specifically and covalently linked to a boronic acid-functionalized MNP (BA@MNP) without hindering the antigen binding domain, Fab. The performance was evaluated by immunoaffinity extraction of multiple serum antigens. Compared with the random immobilization of antibody on a MNP, the antibody self-oriented immunoprobe provides long-term stability (>2 months) and 5-fold extraction efficiency. It also provides 5-fold improved sensitivity at a low nM range (0.4 nM), presumably through enhanced antibody@MNP activity. In addition, false-positive detections arising from nonspecific binding can be completely minimized by effective surface protection using concentration-dependent dextran blocking. Compared with conventional antibody site-specific immobilization through protein G, this new BA-mediated covalent antibody immobilization provides interference-free extraction resulting from noncovalent immobilization of antibody by protein G. The new immunoassay was applied in comparative profiling of serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP) in human serum. Our triple immunoassay revealed a distinct pattern among normal patients, patients with cancer, and patients with cardiovascular disease. Using the previously reported quantization capability of the MALDI MS readout, we expect that this site-specific immunonanoprobe-based immunoassay can be highly active, rapid, and accurate in nanodiagnosis.
The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle-based biosensors.
A straightforward method for fabricating a stable and covalent carbohydrate microarray based on boronate formation between the hydroxyl groups of carbohydrate and boronic acid (BA) on the glass surface was used to identify carbohydrate-protein interactions.
We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.
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