Biotin-binding Sites Research Articles

A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein.

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.

Exploring the Complex Impact of Proteins on Dopamine Polymerization: Mechanisms and Strategies for Modulation.

Polydopamine (pDA) is a valuable material with wide-ranging potential applications. However, the complex and debated nature of dopamine polymerization complicates our understanding. Specifically, the impact of foreign substances, especially proteins, on pDA formation adds an additional layer of subtlety and complexity. This study delves into specific surface features of proteins that predominantly shape their impact on dopamine polymerization. Notably, the biotin-binding site emerges as a critical region responsible for the pronounced inhibitory effect of avidin and neutravidin on the dopamine polymerization process. The binding of biotin successfully mitigates these inhibitory effects. Moreover, several nucleases demonstrated a significant hindrance to pDA formation, with their impact substantially alleviated through the introduction of DNA. It is speculated that hydrogen bonding, electrostatic, cation-π, and/or hydrophobic interactions may underlie the binding between protein surfaces and diverse oligomeric intermediates formed by the oxidation products of dopamine. Additionally, we observed a noteworthy blocking effect on the dopamine polymerization reaction induced by erythropoietin (EPO), a glycoprotein hormone known for its role in stimulating red blood cell production and demonstrating neuroprotective effects. The inhibitory influence of EPO persisted even after deglycosylation. These findings not only advance our comprehension of the mechanisms underlying dopamine polymerization but also provide strategic insights for manipulating the reaction to tailor desired biomaterials.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.

A hand-held electrochemiluminescence biosensor for detection of carcinoembryonic antigen

After the development of portable glucose biosensor, challenges have remained to fabricate more portable devices for sensitive and reproducible detection of other biomarkers. Here, we fabricated a hand-held device for the quantification of carcinoembryonic antigen (CEA) or any other biomarkers based on electrochemiluminescence (ECL) using a bipolar electrode (BPE). The detection mechanism was based on a sandwich assay composed of a capture antibody and a secondary antibody conjugated with a robust ECL reporter. The ECL reporter was fabricated by conjugation of luminol on streptavidin-coated gold nanoparticle (Lum@SA-AuNP), leaving the biotin binding sites of the streptavidin intact for further conjugation with secondary antibody. This novel controlled functionalization strategy significantly enhanced the reproducibility and robustness of the biosensor. Moreover, an inventive parabolic reflector was implemented in the design, in order to maximize the lights to be captured by the photodiode (detector) and measured by a simple multimeter. Due to the synergetic signal amplification, the developed biosensor demonstrated a low sensitivity of 2.51 ng/ml with a linear detection range from 5 to 300 ng/ml with the ability to perform well in spiked-in samples. The designed sensing mechanism can definitely pave the way for further development of miniaturized devices in multiple formats.

A simple agglutination system for rapid antigen detection from large sample volumes with enhanced sensitivity

Lateral flow assays (LFAs) provide a simple and quick option for diagnosis and are widely adopted for point-of-care or at-home tests. However, their sensitivity is often limited. Most LFAs only allow 50 μL samples while various sample types such as saliva could be collected in much larger volumes. Adapting LFAs to accommodate larger sample volumes can improve assay sensitivity by increasing the number of target analytes available for detection. Here, a simple agglutination system comprising biotinylated antibody (Ab) and streptavidin (SA) is presented. The Ab and SA agglutinate into large aggregates due to multiple biotins per Ab and multiple biotin binding sites per SA. Dynamic light scattering (DLS) measurements showed that the agglutinated aggregate could reach a diameter of over 0.5 μm and over 1.5 μm using poly-SA. Through both experiments and Monte Carlo modeling, we found that high valency and equivalent concentrations of the two aggregating components were critical for successful agglutination. The simple agglutination system enables antigen capture from large sample volumes with biotinylated Ab and a swift transition into aggregates that can be collected via filtration. Combining the agglutination system with conventional immunoassays, an agglutination assay is proposed that enables antigen detection from large sample volumes using an in-house 3D-printed device. As a proof-of-concept, we developed an agglutination assay targeting SARS-CoV-2 nucleocapsid antigen for COVID-19 diagnosis from saliva. The assay showed a 10-fold sensitivity enhancement when increasing sample volume from 50 μL to 2 mL, with a final limit of detection (LoD) of 10 pg mL−1 (∼250 fM). The assay was further validated in negative saliva spiked with gamma-irradiated SARS-CoV-2 and showed an LoD of 250 genome copies per μL. The proposed agglutination assay can be easily developed from existing LFAs to facilitate the processing of large sample volumes for improved sensitivity.

Self-assembly of a dimeric avidin into unique higher-order oligomers.

The dimeric avidin family has been expanded in recent years to include many new members. All of them lack the intermonomeric Trp that plays a critical role in biotin-binding. Nevertheless, these new members of the avidins maintain the high affinity towards biotin. Additionally, all of the dimeric avidins share a very unique property: namely, the cylindrical oligomerization in the crystal structure. The newest member described here, agroavidin from the agrobacterium, Rhizobium sp. AAP43, shares their important structural features. However, the affinity of agroavidin towards biotin is lower than all other members of the avidin family, due to the presence of phenylalanine instead of a conserved tyrosine in the biotin-binding site. Mutating this phenylalanine into tyrosine regenerated the high affinity, which emphasizes the importance of this particular tyrosine residue. Another unique feature that distinguishes agroavidin from the other dimeric avidins is that it does not produce oligomers in its crystal structure. In order to understand the factors that promote oligomerization in dimeric avidins, we exchanged the C-terminal region of agroavidin with that of hoefavidin that produced octamers. This exchange resulted in a decamer rather than an octamer. This unusual outcome demonstrates the impact of the C-terminal region on the ability to produce oligomers. The decameric assembly of agroavidin expands the avidin-biotin toolbox even further and could well pave the path into new biotin-based technologies. Moreover, uncovering the factors that induce dimeric avidins into oligomeric assemblies may aid in better understanding the general molecular determinants that promote oligomerization.

Detection and analysis of phage M13KO7 using biosensor based on imaging ellipsometry

Antibody GP8 (anti-GP8) was used as a ligand to detect phage M13KO7 using a biosensor based on imaging ellipsometry (IE) without oriented ligand immobilization. When the phage M13KO7 in a sample interacted with the antibody in a microarray, a complex formed upon their affinity, and the layer covering the surface cell of the interaction became thicker. The variation in layer thickness was represented by gray-scale (or brightness) variation of the IE image. Direct and oriented immobilization of the antibody were analyzed to identify the best binding locations to capture the phage. In direct immobilization, the phage could be captured by anti-GP8 and not antibody GP3 (anti-GP3), which was attributed to different copy numbers of the coat proteins on the body of the phage specific to the two antibodies. This indicated that different binding locations could produce different detection efficiency. In oriented immobilization, the phage could be captured by the protein G‒Fc-anti-GP3 model, with a low detection efficiency. In contrast, the avidin‒biotin-based oriented immobilization could capture the phage with a high detection efficiency. Specifically, it could expose more Fab domains of anti-GP3 labeled with biotin; one avidin had four biotin-binding sites. To confirm detection and analyze binding location, the phage M13KO7 was imaged by transmission electron microscopy and atomic force microscopy. Our results suggest that binding location plays a significant role in the detection of big targets and contributes to enhancing biosensor detection sensitivity.

Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate

The rise of antibacterial-resistant bacteria is a major problem in the United States of America and around the world. Millions of patients are infected with antimicrobial resistant bacteria each year. Novel antibacterial agents are needed to combat the growing and present crisis. Acetyl-CoA carboxylase (ACC), the multi-subunit complex which catalyses the first committed step in fatty acid synthesis, is a validated target for antibacterial agents. However, there are at present, no commercially available antibiotics that target ACC. Ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) is a compound that has been shown to have antibacterial properties against Pseudomonas aeruginosa and Escherichia coli. SABA1 inhibits biotin carboxylase (BC), the enzyme that catalyses the first half reaction of ACC. SABA1 inhibits BC via an atypical mechanism. It binds in the biotin binding site in the presence of ADP. SABA1 represents a potentially new class of antibiotics that can be used to combat the antibacterial resistance crisis.

Probing Bioelectronic Connections Using Streptavidin Molecules with Modified Valency.

As molecular electronic components, proteins are distinguished by a remarkably long electronic decay length (∼10 nm) together with high contact resistance and extreme sensitivity to the chemical details of the contact. As a consequence, the conductance of even a large bioelectronic assembly is largely controlled by the conductance of the contacts. Streptavidin is a versatile linker protein that can tether together biotinylated electrodes and biotinylated proteins but with an ambiguity about the contact geometry that arises from its four possible binding sites for biotin. Here, we use engineered streptavidin tetramers, selected to contain a defined ratio of active monomers to "dead" monomers so as to define the biotin binding sites. We find a strong dependence of conductance on the separation of the biotin molecules, consistent with a short-range tunneling interaction within the streptavidin and in contrast to the long-range transport observed inside larger proteins. Hexaglutamate tails label the active monomers, and the additional negative charge enhances conductance significantly. This effect is quantitatively accounted for by an electronic resonance in the protein conductance.

Surface charge density measurement of a single protein molecule with a controlled orientation by AFM

The spatial distribution of functional groups causes a charge distribution that often has a close relationship with its biofunctions. To understand them of the protein molecules, measurements of the charge distribution under physiological conditions are desired. Atomic force microscopy (AFM) has been utilized to measure the surface charge density by measuring the electric double layer (EDL) force caused by the overlap of the EDLs on the surfaces of the AFM tip and the biomolecule. Here, we demonstrated the surface charge density measurement of a single streptavidin (SA) protein molecule by the three-dimensional force mapping method based on frequency modulation AFM (FM-AFM). The SA has a strong affinity to biotin because of the electrostatic interactions between the molecules. Therefore, the surface charge density measurements of the biotin-binding sites and other surface areas of the molecule have been anticipated. However, the surface charge density of the surfaces other than the biotin-binding side has never been measured. We demonstrate the surface charge density measurement of the top surface of the single SA molecule, which is perpendicular to the biotin-binding sides, with a controlled orientation using DNA origami as a template by FM-AFM in an electrolyte solution. The surface charge density of the top surface of the SA molecule was estimated by fitting the experimental force curves to the Derjaguin-Landau-Verwey-Overbeck theory. We found that the surface charge density of the top surface of the SA molecule is comparable to those reported earlier for the biotin-binding sides of the molecule. We expect that, by using the DNA origami technology, one can control the orientation of a biomolecule attached to the substrate and measure the surface charge density of the specific surface areas of the biomolecule to obtain information that will help us to understand the relationship between their structures and functions.

Insights into the structure of mature streptavidin C1 from Streptomyces cinnamonensis reveal the self-binding of the extension C-terminal peptide to biotin-binding sites.

The members of the avidin protein family are well known for their high affinity towards d-biotin and their structural stability. These properties make avidins a valuable tool for various biotechnological applications. In the present study, two avidin-like biotin-binding proteins (named streptavidin C1 and C2) from Streptomyces cinnamonensis were newly identified while exploring antifungal proteins against Fusarium oxysporum f. sp. cucumerinum. Streptavidin C1 reveals a low correlation (a sequence identity of approximately 64%) with all known streptavidins, whereas streptavidin C2 shares a sequence identity of approximately 94% with other streptavidins. Here, the crystal structures of streptavidin C1 in the mature form and in complex with biotin at 2.1 and 2.5 Å resolution, respectively, were assessed. The overall structures present similar tetrameric features with D 2 symmetry to other (strept)avidin structures. Interestingly, the long C-terminal region comprises a short α-helix (C-Lid; residues 169-179) andan extension C-terminal peptide (ECP; residues 180-191) which stretches into the biotin-binding sites of the same monomer. This ECP sequence (-180VTSANPPAS188-) is a newly defined biotin-binding site, which reduces the ability to bind to (strept)avidin family proteins. The novel streptavidin C1 could help in the development of an engineered tetrameric streptavidin with reduced biotin-binding capacity as well as other biomaterial tools.

Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks.

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin–biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin–biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin–biotin biotechnology in cellular uptake.

Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes.

The biotin–streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17 000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.

Recent advances in the engineering and application of streptavidin-like molecules.

Streptavidin (SA), and other related proteins, has been isolated from a wide range of organisms, including bacteria, fungi, frogs, fish, and birds. Although their original function is not well understood, they have found an important place in biotechnology based on their unique ability to bind biotin molecules with high affinity and specificity. The SA-biotin interaction is robust and easy to incorporate into different designs, and as such, it is used when reliable molecule interaction is needed under poorly controlled experimental conditions. There are continued efforts to engineer these proteins to modulate their size, valency, and affinity, since the optimum molecular properties vary depending on individual applications. This review will describe recent developments in streptavidin engineering to meet these requirements, including those that form novel oligomeric states, e.g., a monomer, have fewer functional biotin-binding sites, or bind biotin with reduced affinity. We also examine various reported applications of both natural or engineered SA constructs in cell biology, biochemistry, genetics, synthetic chemistry, cancer therapy, drug delivery, and nanotechnology to illustrate the breadth of modern science that is advanced by the endogenous and engineered SA-biotin interactions.

Orientation of Biotin-Binding Sites in Streptavidin Adsorbed onto the Surface of Polythiophene Films.

The orientation of biotin-binding sites of streptavidin adsorbed to thin films of three polythiophenes (PTs), namely, regioregular poly(3-hexylthiophene) (RP3HT), regiorandom poly(3-butylthiophene) (P3BT), and poly(3,3‴-didodecylquaterthiophene) (PQT12), has been investigated. Polymer films were examined prior to and after protein adsorption with atomic force microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA) applied to ToF-SIMS data revealed subtle changes in surface chemistry of polymer films and orientation of adsorbed streptavidin. PCA resolved the surface alignment of alkyl side chains and differentiated the ToF-SIMS data for PQT12, RP3HT, and P3BT, verifying an amorphous morphology for P3BT and a semicrystalline one for PQT12 and RP3HT. After the characterization of the polymeric films, streptavidin adsorption from solutions with different protein concentrations (up to 300 μg/mL) has been conducted. The PCA results distinguished between amino acids characteristic for external regions of streptavidin molecules adsorbed to different PTs suggest that streptavidin adsorbed to PQT12 exposes molecular regions rich in tryptophan and tyrosine, which are components of the biotin-binding sites. The latter results were confirmed using biotin-labeled horse radish peroxidase to estimate the exposed binding sites of streptavidin adsorbed onto the different PT films. The analysis of streptavidin structure suggests that interaction between polythiophene film and dipole moment of streptavidin subunit is responsible for orientation of biotin-binding sites.

Crystal structure of afifavidin reveals common features of molecular assemblage in the bacterial dimeric avidins.

The subfamily of bacterial dimeric avidins is being extended through the discovery of additional members originating from diverse sources. All of these newly discovered dimeric avidin forms exhibit high affinity towards biotin, despite their lack of critical Trp in the classical tetrameric forms. The common feature of forming cylinder-like multimers (hexamers and octamers) seems to be more than a random occurrence, which generally characterizes their apo forms in the crystalline state and also in some cases in solution. Afifavidin from the Gram-negative α-proteobacterium Afifella pfennigii is the fourth member of the subfamily of dimers, which, in the intact apo form, also congregates into octamers both in the solution and in the crystalline state, whereby the C-terminal extended segments stretch into the biotin-binding sites of adjacent non-canonical monomers. The intact apo afifavidin molecule self-assembles into toroid-shaped nanostructures that dissociate into the inherent dimers upon binding biotin. On removal of the C-terminal regions, the short-form of afifavidin forms dimers both in the solution and in the crystalline states. The high affinity of the dimeric forms of afifavidin towards biotin is maintained, due to the conserved disulfide bridge between L3,4 and L5,6 and the presence of Phe50 in L3,4 that compensate for the lack of the critical Trp in the tetrameric avidins. These cyclic multimeric-avidin assemblies may be exploited in the future to further diversify biotin-based nanotechnology or to serve as building blocks in the construction of bio-inspired materials. DATABASE: Structural data are available in the PDB databases under the accession numbers: 6HDV, 6HDS, 6HDT.

Fabrication of Oligomeric Avidin Scaffolds for Valency-Controlled Surface Display of Functional Ligands.

Multivalent surface display of biomolecules is crucial to study and utilize multivalent biological interactions. However, precise valency control of surface-displayed ligands remains extremely difficult. Now a series of new oligomeric avidin proteins were fabricated that allow facile control of surface multivalency of biotinylated ligands. Naturally dimeric rhizavidin (RA) was engineered to form a mixture of oligomeric avidin assemblies, and discrete RA oligomers from the dimer to octamer of RA, were homogeneously prepared. These oligomeric avidins are in polygonal forms with expected numbers of stable biotin binding sites. Upon immobilization on low-density biotin-coated gold surfaces, RA dimer, trimer, and tetramer scaffolds provided accurate mean residual valencies of 2, 3, and 4, respectively, for biotinylated proteins. Valency-controlled display of antibody binding protein G on these RA surfaces showed clear valency-dependent enhancement of antibody capturing stability.

Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10−16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10−6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

Tuning the chain length of new pyrene derivatives for site-selective photocleavage of avidin

Rational design of photoreagents with systematic modifications of their structures can provide valuable information for a better understanding of the protein photocleavage mechanism by these reagents. Variation of the length of the linker connecting the photoactive moiety with the protein anchoring-group allowed us to investigate the control of the protein photocleavage site. A series of new photochemical reagents (PMA-1A, PMA-2A and PMA-3A) with increasing chain lengths is examined in the current study. Using avidin as a model system, we examined the interaction of these probes by UV–Vis, fluorescence spectroscopic methods, photocleavage and computational docking studies. Hypochromism of the absorption spectrum was observed for the binding of these new photochemical reagents with estimated binding constants (Kb) of 6.2 × 105, 6.7 × 105 and 4.6 × 105 M−1, respectively. No significant changes of Stern-Volmer quenching constant (Ksv) with Co(NH3)6Cl3 has been noted and the data indicated that the probes bind near the surface of the protein with sufficient exposure to the solvent. Photoexcitation of the probe-avidin complex, in the presence of Co(NH3)6Cl3, resulted in protein fragmentation, and the cleavage yield decreased with the increase in the linker length, and paralleled with the observed Ksv values. Amino acid sequencing of the photofragments indicated that avidin is cleaved between Thr77 and Val78, as a major cleavage site for all the three photoreagents. This site is proximate to the biotin binding site on avidin, and molecular docking studies indicated that the H-bonding interactions between the polar end-group of the photoreagents and hydrophilic amino acids of avidin were important in positioning the reagent on the protein. The major cleavage site, at residues 77–78, was within 5 Å of the pyrenyl moiety of the probe, and hence, molecular tuning of the linker provided a simple approach to position the photoreagent along the potential photocleavage site.

Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo.

Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can be easily attached, also as a mixture of two or more molecules. This non-covalent bond is stable both in cell culture and after injection into mice in vivo. Thus, we have created a highly versatile system for many applications, from the delivery of biomolecules over the targeting of cells and tissue to in vivo imaging.