A bioengineering method for self-assembly of multifunctional superstructures with in-advance programmable properties has been proposed. The method employs two unique proteins, barnase and barstar, to rapidly join the structural components together directly in water solutions. The properties of the superstructures can be designed on demand by linking different agents of various sizes and chemical nature, designated for specific goals. As a proof of concept, colloidally stable trifunctional structures have been assembled by binding together magnetic particles, quantum dots, and antibodies using barnase and barstar. The assembly has demonstrated that the bonds between these proteins are strong enough to hold macroscopic (5 nm-3 μm) particles together. Specific interaction of such superstructures with cancer cells resulted in fluorescent labeling of the cells and their responsiveness to magnetic field. The method can be used to join inorganic moieties, organic particles, and single biomolecules for synergistic use in different applications such as biosensors, photonics, and nanomedicine.superstructures | cancer cells | targeting | fusion proteins | magnetic nanoparticles R ecently, nanoparticles have become attractive objects for life science applications, in particular, in such rapidly growing areas as express diagnostics and advanced medical treatment. Encapsulation of nanoparticles with drug molecules (1, 2) or attaching them to viruses, bacteria, etc. are of special interest. Time-controlled release of the absorbed drugs would be advantageous for treatment of many diseases, e.g. diabetes, because of a decreased number of injections compared to that of molecular insulin. Furthermore, fluorescent or colored particles such as quantum dots (QD) (3), nanodiamonds (4), and gold nanoparticles (5) can be used for diagnostics as markers that provide visual information about the distribution of labeled agents in tissues and blood. Magnetic particles (MP) (6) can be also used as efficient labels for MRI diagnostics and can be precisely quantified even inside a living organism by an external induction probe (7,8). At present, MP are widely studied for hyperthermia of tumors by heating in an AC magnetic field and for targeted delivery of drugs by magnetic field gradients, to avoid systemic intoxication of the organism (9, 10). Specific immunological targeting of nanoparticles by antibodies against pathogenic cells is another noteworthy application. Not only does it allow marking tumors for accurate dissection, but it also enhances drug delivery to the target cells.The above-mentioned functional aspects of nanoparticles are brought into play in many life science applications. In certain cases, however, it would be beneficial to use multifunctional structures (11-13), which consist of several types of particles. Extensive studies were devoted to the synthesis of hybrid complexes of magnetic particles and different fluorophores (quantum dots or conventional chemical dyes) to allow visual MP tracking. Most approaches (14-16) emp...

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We report on a bioconjugation platform based on a high-affinity protein pair, barstar:barnase (Bs:Bn), that provides a modular design toolkit capable of strong (covalent) and versatile assembly of bio/nanocomplexes. Luminescent nanodiamonds (140 nm) serve as the docking station to synthesize Bs:Bn bioconjugates which are characterized and utilized in several applications, including cell transfection.Nanodiamonds (NDs) are tiny particles with a stable, crystalline core and chemically active surface that can dock a variety of biocompatible moieties. 1 Nitrogen-vacancy (NV) color centers render nanodiamonds luminescent (LND) under ambient conditions and their strong, spin-sensitive emission provides an exceptional basis for quantum optics and nanoscale magnetometry. 2,3 In addition, low cytotoxicity and photostability of LNDs make them an attractive tool for biolabelling and cell targeted delivery purposes. 4-6 NDs can be produced via two methods: (1) detonation of explosives in an inert atmosphere followed by disintegration yields remarkably monodisperse 5 nm NDs, 7 or (2) high-temperature high-pressure (HTHP) synthetic growth followed by ball-milling produces NDs of high crystal quality sized 4 nm and larger. Subsequent acid treatment and/ or annealing in air removes amorphous carbon from nanocrystallite surface, replacing it with a variety of oxygen-containing groups, such as carboxyl groups. 8 These groups impart surface charge sufficient to stabilize NDs in distilled water, in virtue of the nanoparticle electrostatic repulsion [ Fig. 1(a), LND(DDW)]. 2,9 LND deployment in biomedical applications critically depends on LND-bioconjugate stability in physiological solutions, currently an issue, as salt-induced surface charge screening leads to flocculation. 10 Existing LND-functionalization methods to counter flocculation and to facilitate bioconjugation can be classified as covalent functionalization, e.g. amidation (utilizing surface carboxyl groups), silanization, adsorption, and lipid/polymer capping. 6,7,11 While the reported covalent functionalization protocols showed stability of the resultant complexes, their realization was often complex and case-dependent, whereas most of the other methods compromised functional stability. Here, we report on a versatile, salt-solution stable LNDbioconjugation platform that is based on a high-affinity protein pair termed barstar:barnase (Bs:Bn ‡). 12 Barnase, a bacterial ribonuclease, and its inhibitor, barstar, are characterized by an extremely small dissociation constant of 10 À14 M, only ten-fold larger than that of its ubiquitous functional analogue, streptavidin:biotin. 12 Bs and Bn are water-soluble, comparatively small (M w ¼ 10.2, 12.4 kDa, respectively), temperature-stable (50 C, barnase; 70 C, barstar), and undergo fully reversible unfolding under Fig. 1 (a) Dot and Bar plots of the z-potential and mean diameter of colloidal LND, an un-reacted suspension of LNDs and Bs (LND+Bs), LND covalently bound to Bs (LND-Bs), LND-Bs linked to Bn (LNDBs:Bn), and Bn-EGFP (LND...

Far-red and near-infrared fluorescent quantum dots (QDs) have become advancing contrast agents for efficient whole-body tumor imaging. In this study, we investigated the possibility of the vital fluorescence imaging of tumor using two contrast agents on the basis of QDs: bioinert QDs coated with polyethyleneglycol and QDs bound with anti-HER2/neu scFv antibodies. HER2/neu-positive breast cancer tumor xenografts in nude mice were used as a model. It was shown that both bioinert and tumor-targeted QD probes can be successfully applied for visualization of the tumor using in vivo imaging method, but fluorescent signal of QD-4D5scFv in tumors was considerably stronger than that of QD-PEG.