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Due to the absence of labels and fast analyses, optical biosensors promise major advances in biomedical diagnostics, security, environmental, and food safety applications. However, the sensitivity of the most advanced plasmonic biosensor implementations has a fundamental limitation caused by losses in the system and/or geometry of biochips. Here, we report a “scissor effect” in topologically dark metamaterials which is capable of providing ultrahigh-amplitude sensitivity to biosensing events, thus solving the bottleneck sensitivity limitation problem. We explain how the “scissor effect” can be realized via the proper design of topologically dark metamaterials and describe strategies for their fabrication. To validate the applicability of this effect in biosensing, we demonstrate the detection of folic acid (vitamin important for human health) in a wide 3-log linear dynamic range with a limit of detection of 0.22 nM, which is orders of magnitude better than those previously reported for all optical counterparts. Our work provides possibilities for designing and realizing plasmonic, semiconductor, and dielectric metamaterials with ultrasensitivity to binding events.

When combined with immunotherapy, image-guided targeted delivery of chemotherapeutic agents is a promising direction for combination cancer theranostics, but this approach has so far produced only limited success due to a lack of molecular targets on the cell surface and low therapeutic index of conventional chemotherapy drugs. Here, we demonstrate a synergistic strategy of combination immuno/chemotherapy in conditions of dual regioselective targeting, implying vectoring of two distinct binding sites of a single oncomarker (here, HER2) with theranostic compounds having a different mechanism of action. We use: (i) PLGA nanoformulation, loaded with an imaging diagnostic fluorescent dye (Nile Red) and a chemotherapeutic drug (doxorubicin), and functionalized with affibody ZHER2:342(8 kDa); (ii) bifunctional genetically engineered DARP-LoPE (42 kDa) immunotoxin comprising of a low-immunogenic modification of therapeutic Pseudomonasexotoxin A (LoPE) and a scaffold targeting protein, DARPin9.29 (14 kDa). According to the proposed strategy, the first chemotherapeutic nanoagent is targeted by the affibody to subdomain III and IV of HER2 with 60-fold specificity compared with nontargeted particles, while the second immunotoxin is effectively targeted by DARPin molecule to subdomain I of HER2. We demonstrate that this dual targeting strategy can enhance anticancer therapy of HER2-positive cells with a very strong synergy, which made possible 1000-fold decrease of effective drug concentration in vitroand a significant enhancement of HER2 cancer therapy compared to monotherapy in vivo. Moreover, this therapeutic combination prevented the appearance of secondary tumor nodes. Thus, the suggested synergistic strategy utilizing dual targeting of the same oncomarker could give rise to efficient methods for aggressive tumors treatment.

Nanoparticle surface engineering can change its chemical identity to enable surface coupling with functional biomolecules. However, common surface coupling methods such as physical adsorption or chemical conjugation often suffer from the low coupling yield, poorly controllable orientation of biomolecules, and steric hindrance during target binding. These issues limit the application scope of nanostructures for theranostics and personalized medicine. To address these shortfalls, we developed a rapid and versatile method of nanoparticle biomodification. The method is based on a SiO2-binding peptide that binds to the nanoparticle surface and a protein adaptor system, Barnase*Barstar protein pair, serving as a “molecular glue” between the peptide and the attached biomolecule. The biomodification procedure shortens to several minutes, preserves the orientation and functions of biomolecules, and enables control over the number and ratio of attached molecules. The capabilities of the proposed biomodification platform were demonstrated by coupling different types of nanoparticles with DARPin9.29 and 4D5scFv—molecules that recognize the human epidermal growth factor receptor 2 (HER2/neu) oncomarker—and by subsequent highly selective immunotargeting of the modified nanoparticles to different HER2/neu-overexpressing cancer cells in one-step or two-step (by pretargeting with HER2/neu-recognizing molecule) modes. The method preserved the biological activity of the DARPin9.29 molecules attached to a nanoparticle, whereas the state-of-the-art carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysulfosuccinimide method of conjugation led to a complete loss of the functional activity of the DARPin9.29 nanoparticle–protein complex. Moreover, the method allowed surface design of nanoparticles that selectively interacted with antigens in complex biological fluids, such as whole blood. The demonstrated capabilities show this method to be a promising alternative to commonly used chemical conjugation techniques in nanobiotechnology, theranostics, and clinical applications.

The progress in the design of hybrid photoluminescent assemblies based on upconversion nanoparticles (UCNPs) meant for biomedical applications is analyzed. The key approaches to the synthesis of bright UCNPs and their surface modification, including encapsulation into hydrophilic shells and providing with therapeutic and targeting agents, are described. The range of potential applications of UCNPs in analytical biochemistry, biomedical diagnostics and therapy of cancer and infectious diseases is considered.The bibliography includes 145 references.

•Silver nanowires possess intrinsic peroxidase-like activity with TMB, OPD, and DAB.•AgNW with OPD can serve as a robust hydrogen peroxide sensor and pH-sensor.•Competitive ELISA with AgNW for chloramphenicol (CAP) detection was realized.•Lateral flow test for CAP detection was realized based on the LSPR property of AgNWs.•AgNWs are extremely stable without light protections and preservatives for > 2 years.

To date, a number of biomolecule-mediated nanoparticle self-assembly systems have been developed that are amenable to controllable disassembly under relatively gentle conditions. However, for some applications such as design of self-assembled multifunctional theragnostic agents, high stability of the assembled structures can be of primary importance. Here, we report extraordinarily high durability of protein-assisted nanoparticle self-assembly systems yielding bifunctional colloidal superstructures resistant to extreme denaturing conditions intolerable for most proteins (e.g., high concentrations of chaotropic agents, high temperature). Among the tested systems (barnase–barstar (BBS), streptavidin–biotin, antibody–antigen, and protein A–immunoglobulin), the BBS is notable due to the combination of its high resistance to severe chemical perturbation and unique advantages offered by genetic engineering of this entirely protein-based system. Comparison of the self-assembly systems shows that whereas in all cases the preassembled structures proved essentially resistant to extreme conditions, the ability of the complementary biomolecular pairs to mediate assembly of the initial biomolecule–particle conjugates differs substantially in these conditions.

Chimeric immunotoxins that combine antigen recognition domains of antibodies and cytotoxic RNases have attracted much attention in recent years as potential targeted agents for cancer immunotherapy. In an attempt to obtain a structurally minimized immunofusion for folding/stability studies, we constructed the chimeric protein VL–barnase. The chimera comprises a small cytotoxic enzyme barnase, ribonuclease from Bacillus amyloliquefaciens, fused to the C‐terminus of the light chain variable domain (VL) of the anti‐human ferritin monoclonal antibody F11. While the individual VL domain was expressed in Escherichia coli as insoluble protein packed into inclusion bodies, its fusion to barnase resulted in a significant (∼70%) fraction of soluble protein, with only a minor insoluble fraction (∼30%) packed into inclusion bodies. The in vivo solubilizing effect of barnase was also observed in vitro and suggests a chaperone‐like role that barnase exerted with regard to the N‐terminal VL domain. Cytoplasmic VL–barnase was analyzed for structural and functional properties. The dimeric state of the chimeric protein was demonstrated by size‐exclusion chromatography, thus indicating that fusion to barnase did not abrogate the intrinsic dimerization propensity of the VL domain. Ferritin‐binding affinity and specificity in terms of constants of association with isoferritins were identical for the isolated VL domain and its barnase fusion, and RNase activity remained unchanged after the fusion. Intrinsic fluorescence spectra showed a fully compact tertiary structure of the fusion protein. However, significantly altered pH stability of the fusion protein versus individual VL and barnase was shown by the pH‐induced changes in both intrinsic fluorescence and binding of ANS. Together, the results indicate that VL–barnase retained the antigen‐binding affinity, specificity and RNase activity pertinent to the two individual constituents, and that their fusion into a single‐chain chimeric protein resulted in an altered tertiary fold and pH stability.

Recombinant barnase was proposed as a label in the enzyme‐linked immunosorbent assay (ELISA). Barnase‐conjugated pig transferrin was prepared by the periodate oxidation procedure. Solid‐phase bound barnase activity was determined from the change in RNA‐ethidium bromide complex fluorescence upon RNA hydrolysis. The sensitivity of transferrin‐barnase conjugate determination in ELISA was no less than 5 ng per well. The conjugate was applied in competition ELISA for free transferrin determination.

A tandem of recombinant mouse/human immunoglobulin (Ig) genes was constructed and inserted into the plasmid pGEM1 under the control of T7 phage RNA polymerase promoter. Sp2/0 lymphoid cell line and Chinese Hamster Ovary (CHO) cells were used as the targets for gene transfection. Both cell lines contained in their genomes a T7 RNA polymerase gene modified with a nuclear-located signal derived from SV40 large T-antigen. Cell lines transfected with the gene tandem effectively synthesized mRNA (up to 9 × 103bp) that hybridized with ε-and ϰ-gene probes. Separate transcripts corresponding to mRNAs of individual heavy and light chains were not detected in either transfected cell line. It follows from these data that transcription in the transfected cells is controlled mainly by the T7 phage polymerase promoter. Both lymphoid and nonlymphoid cell lines transfected with the gene tandem synthesized the ε-heavy (70 kDa) and ϰ-light (25 kDa) Ig polypeptide chains. Production of chimeric antibodies by the myeloma Sp2/0 cells was higher than that by the CHO cells. Individual clones synthesized up to 150 ng/mL chimeric IgE. However, only lymphoid Sp2/0 cells were capable of efficient secretion of the recombinant antibodies. The mechanism of translation of mRNA synthesized in eukaryotic cells by T7 phage RNA polymerase is discussed.

The pET(scF11) plasmid was constructed comprising the gene of a single‐chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable VHdomain, (Gly4Ser)3linker peptide, and light chain variable VLdomain. The correctly processed scF11 antibody was expressed in Escherichia colias an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion‐exchange chromatography. ScF11 demonstrated only a ∼8‐fold decrease in the affinity (Ka=5.1×108M−1in RIA and 1.8×108M−1in ELISA) vs. the parent IgG2a/κ monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant‐induced fluorescence ‘dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8‐anilino‐1‐naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single‐chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding.

Safe application of nanoparticles in medicine requires full understanding of their pharmacokinetics including catabolism in the organism. However, information about nanoparticle degradation is still scanty due to difficulty of long-term measurements by invasive techniques. Here, we describe a magnetic spectral approach for in vivomonitoring of magnetic particle (MP) degradation. The method noninvasiveness has allowed performing of a broad comprehensive study of the 1-year fate of 17 types of iron oxide particles. We show a long-lasting influence of five parameters on the MP degradation half-life: dose, hydrodynamic size, ζ-potential, surface coating, and internal architecture. We observed a slowdown in MP biotransformation with an increase of the injected dose and faster degradation of the particles of a small hydrodynamic size. A comparison of six types of 100 nm particles coated by different hydrophilic polymer shells has shown that the slowest (t1/2= 38 ± 6 days) and the fastest (t1/2= 15 ± 4 days) degradations were achieved with a polyethylene glycol and polyglucuronic acid coatings, respectively. The most significant influence on the MP degradation was due to the internal architecture of the particles as the coverage of magnetic cores with a solid 39 nm polystyrene layer slowed down the half-life of the core–shell MPs from 48 days to more than 1 year. The revealed deeper insights into the particle degradation in vivomay facilitate rational design of nano- and microparticles with predictable long-term fate in vivo.

A recombinant tandem of ‘chimeric’ mouse/human immunoglobulin (Ig) genes was constructed and inserted into plasmid pGEM 1 under the control of the T7 bacteriophage RNA polymerase promoter. Lymphoid (Sp2/0) and non‐lymphoid (CHO) cell lines used for transfection contained in their genomes a semisynthetic gene of T7 RNA polymerase and steadily expressed this enzyme. It was shown for the first time that a stable polycystronic transcription of the Ig gene tandem occurs under the control of a single T7 phage promoter, both in lymphoid and non‐lymphoid cells. Synthesis of κ‐light and ε‐heavy Ig chains and functionally active antibodies was observed in the above‐mentioned transfected cell lines.

The progress in the design of hybrid photoluminescent assemblies based on upconversion nanoparticles (UCNPs) meant for biomedical applications is analyzed. The key approaches to the synthesis of bright UCNPs and their surface modification, including encapsulation into hydrophilic shells and providing with therapeutic and targeting agents, are described. The range of potential applications of UCNPs in analytical biochemistry, biomedical diagnostics and therapy of cancer and infectious diseases is considered.