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In this review article, we discuss state-of-the-art methods for immobilizing functional receptors in microfluidic devices. Strategies used to immobilize receptors in such devices are essential for the development of specific, sensitive (bio)chemical assays that can be used for a wide range of applications. In the first section, we review the principles and the chemistry of immobilization techniques that are the most commonly used in microfluidics. We afterward describe immobilization methods on static surfaces from microchannel surfaces to electrode surfaces with a particular attention to opportunities offered by hydrogel surfaces. Finally, we discuss immobilization methods on mobile surfaces with an emphasis on both magnetic and non-magnetic microbeads, and finally, we highlight recent developments of new types of mobile supports.

Immuno-PCR (IPCR) combines the versatile ELISA antigen detection with ultrasensitive PCR signal amplification, thereby enabling the highly sensitive detection of a broad range of targets with a typically very large dynamic detection range. The quantification of the antigen is usually achieved by real-time PCR, which provides a correlation between the target concentration and amplified DNA marker. We here report on the implementation of digital droplet PCR as a means for direct quantification of DNA copies to enable the highly sensitive detection of protein biomarkers. To this end, two alternative approaches, based on either magnetic microbead-based IPCR or a microplate-release IPCR were tested. The latter format worked well and revealed an extraordinary high robustness and sensitivity. While rtIPCR already fulfills typical immunoassay acceptance criteria, ddIPCR enables improved accuracy and precision of the assay because signal response and analyte concentrations are directly correlated. The utility of the novel ddIPCR technology is demonstrated at the example of two cytokines, interleukin 2 and interleukin 6 (IL2, IL6, respectively), with an overall average CV% of 5.0 (IL2) and 7.4 (IL6).

[EN] We demonstrate a redox-sensitive nanofluidic diode whose ion rectification is modulated by the oxidation and reduction of chemical moieties incorporated on its surface. To achieve this goal, we have first synthesized the chemical compounds 1-(4-aminobutyl)-3-carbamoylpyridin-1-ium (Nic-BuNH2) and 3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium (Nic-DNP). Then, the surface of track-etched single asymmetric nanopores is decorated with the redox-sensitive Nic-BuNH2 and Nic-DNP molecules using carbodiimide coupling chemistry and Zincke reaction, respectively. The success of the modification reactions is monitored through the changes in the current¿voltage (I¿V) curves prior to and after pore functionalization. Upon exposing the modified pore to solutions of hydrogen peroxide (oxidizing agent) and sodium dithionite (reducing agent) the surface charge is reversibly modulated from positive to neutral, leading to measurable changes in the electronic readout of ion current passing through the nanopore. On oxidation, the quaternary nicotinamide units impart positive charge to the pore surface, resulting in the ion current rectification (anion-selective pore). On the contrary, the complementary reduced dihydronicotinamide moieties resulted in the loss of surface charge and ohmic behaviour (non-selective pore). The experimental results are further theoretically described by using Poisson-Nernst-Planck, M.A., S.N. and W.E. acknowledge the funding from the Hessen State Ministry of Higher Education, Research and the Arts, Germany, under the LOEWE project iNAPO. P. R. and S. M. acknowledge financial support by the Generalitat Valenciana (Program of Excellence Prometeo/GV/0069), the Spanish Ministry of Economic Affairs and Competitiveness (MAT2015-65011-P), and FEDER. I.A. and C.M.N. acknowledge financial support through the Helmholtz programme BioInterfaces in Technology and Medicine. The authors are also thankful to Prof. C. Trautmann, Department of Materials Research from GSI, for support with irradiation experiments.

This review provides an overview of the developments in semisynthetic DNA-protein conjugates. Both covalent and noncovalent bioconjugation strategies are used to combine natural proteins and synthetic nucleic acid building blocks to produce nanoscale functional devices. Examples include the formation of supramolecular assemblies from proteins and chemically modified DNA molecules that can serve as diagnostic tools and allow fundamental research in nanosciences. The approach allows one to fabricate model systems for the investigation of catalytic cascade reactions or light-harvesting devices. Semisynthetic DNA-protein conjugates can be used in the biofunctionalization of planar surfaces for micro- and nanoarray technologies. Moreover, examples are given in which inorganic nanoparticles (NPs) are decorated with DNA and proteins to enable applications in sensing, materials science, and catalysis.

The oxidation of 10 polycyclic aromatic hydrocarbons (PAH) by cytochrome P450(BSbeta) using three different electron acceptors is reported. Three PAH were found to be substrates for the oxidation by P450(BSbeta), namely anthracene, 9-methyl-anthracene and azulene. The respective oxidation products were identified by reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry. In addition, 10 drug-like compounds were investigated for their effects on the catalytic activity of P450(BSbeta) by carrying out inhibition studies. The stability of P450(BSbeta) against hydrogen peroxide, cumene, and ter-butyl hydroperoxide was determined. Overall, the results of this study suggested that the P450(BSbeta) enzyme represents a powerful catalyst in terms of the catalytic activity and operational stability.

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.

Nucleic acid amplification techniques are used for signal generation in antibody-based immunoassays, thereby dramatically enhancing the sensitivity of conventional immunoassays. Methodological aspects, as well as applications of this novel approach, are summarized in this review, with an emphasis on immuno-polymerase chain reaction (IPCR). IPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100-10,000-fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. The evolution of IPCR included the development of efficient reagents, the design of assay formats and the maintenance of functionality, even within complex biological matrices. Eventually, IPCR crossed the border from being a research method to a routine laboratory technique, enabling a broad range of applications in immunological research and clinical diagnostics.

Self-organization of colloidal metal nanoparticles into micro- and nanostructured assemblies is currently of tremendous interest promising to find new size- and structure-dependent physical properties. Owing to its unique recognition capabilities and physicochemical stability, DNA can be used as a molecular linker for gold nanoparticles and is a promising construction material for their precise spatial positioning. Due to the enormous specificity of nucleic acid hybridization, the site-specific immobilization of DNA-functionalized gold colloids (1–40 nm) to solid supports, previously functionalized with a complementary DNA array, allows the fabrication of novel nanostructured surface architectures. Scanning force microscopy (SFM), used to characterize the intermediate steps of the DNA-directed immobilization (DDI) on a gold substrate, provides initial insight into the specificity and efficiency of this technique.

Thioesters can be O-borylated using optically active chloro-2,5-diphenylborolane to produce chiral boron enolates which show complete reagent control in aldol additions to chiral α-amino aldehydes.

The simultaneous identification and quantification of several different proteins is crucial for the understanding of cellular processes at the protein level, and for detecting changes in protein expression signatures due to disease, drugs or any biological perturbation. High-throughput, high sensitivity, nanotechnology-based analytical tools, with low requirements on the sample amount and hands-on processing time, are prerequisites for further developments in this field.We use atomic force microscopy nanografting (AFM-NG) to fabricate innovative nano-arrays for immuno-assay, with complete and efficient control on protein/antibody immobilization. By combining NG and DNA-directed immobilization (DDI), different protein-ssDNA conjugates are driven simultaneously on top of a nano-array of complementary ssDNA strands, previously prepared on a gold film surface functionalized with a protein repellent reference carpet. The integrity and biochemical functionality of the immobilized proteins were demonstrated as a proof of principle by AFM topography measurements and immunological assays. Different proteins were detected within a complex matrix of standardized human serum, demonstrating the specificity of the biomolecular recognition, and the absence of any nonspecific binding and cross-reactivity. Protein folding was tested by studying protein-ligand interactions.In a parallel approach we promote protein immobilization in controlled orientation, by means of nitrilotriacetic acid (NTA)-terminated thiols. NTA binds specifically to a polyhistidine tag of the protein (through Ni chelation), which can be genetically engineered in recombinant proteins in a known position. This immobilization is reversible and ensures surface re-usability. Technically, the know-how for both DNA and NTA nanografting based protein immobilization can be combined to exploit the extreme selectivity and parallelizability of the former with the versatility of the latter. Specifically, by using a ssDNA-NTA conjugate, the added to our protein nano-arrays the opportunity to immobilize in an oriented manner through the histidine tag an enormous variety of proteins.