Biomolecule-nanoparticle hybrids as functional units for nanobiotechnology

Biomolecule-metal or semiconductor nanoparticle (NP) hybrid systems combine the recognition and catalytic properties of biomolecules with the unique electronic and optical properties of NPs. This enables the application of the hybrid systems in developing new electronic and optical biosensors, to synthesize nanowires and nanocircuits, and to fabricate new devices. Metal NPs are employed as nano-connectors that activate redox enzymes, and they act as electrical or optical labels for biorecognition events. Similarly, semiconductor NPs act as optical probes for biorecognition processes. Double-stranded DNA or protein chains that are modified with metallic nanoclusters act as templates for the synthesis of metallic nanowires. The nanowires are used as building blocks to assemble nano-devices such as a transistor or a nanotransporter.     

DNA-embedded Au/Ag core-shell nanoparticles

Here, we synthesized highly stable DNA-embedded Au/Ag core-shell nanoparticles (NPs) by a straightforward silver-staining of DNA-modified Au nanoparticles (AuNPs); unlike conventional DNA-surface modified NPs that present particle stability issues, DNA-embedded core-shell NPs offer an extraordinary stability with nanoscale controllability of silver shell thickness; these DNA-embedded core-shell NPs show excellent biorecognition properties and Ag shell-thickness-based optical properties, distinctively different from those of a mixture of AuNPs and AgNPs or Ag/Au alloy nanoparticles.     

Semiconductor quantum dots for bioanalysis

Semiconductor nanoparticles, or quantum dots (QDs), have unique photophysical properties, such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching. These properties enable the use of QDs as optical labels for the multiplexed analysis of immunocomplexes or DNA hybridization processes. Semiconductor QDs are also used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, or the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of QD-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. Examples are the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations. Semiconductor nanoparticles are also used as labels for the electrochemical detection of DNA or proteins: Semiconductor NPs functionalized with nucleic acids or proteins bind to biorecognition complexes, and the subsequent dissolution of the NPs allows the voltammetric detection of the related ions, and the tracing of the recognition events.     

Real-time PCR in a plastic chip based on solid state FRET

We show a novel solid state optical detection platform, integrated in a plastic biochip, based on colloidal nanocrystal FRET donors. The approach exploits a "smart" polymeric layer with both optical and biorecognition properties that allows real-time monitoring of biomolecular interactions and quantitative analyses of real-time PCR. The proposed strategy, demonstrated here for DNA detection, may open interesting perspectives for a wide range of applications, such as for proteomic studies.     

Molecularly templated materials in chemical sensing

Biologically-based recognition elements (e.g., antibodies, aptamers, enzymes, etc.) are used as the recognition element within a wide variety of assays and sensor systems. There are, however, compelling reasons for researchers to develop inexpensive, robust, and reusable alternatives for these expensive and unstable biorecognition elements. This review summarizes recent research efforts on the development of molecularly templated (sometimes called molecularly imprinted) organic and inorganic polymers as possible replacements for expensive/labile biorecognition elements. The review begins with a briefing on biosensing and the pertinent issues and limitations. The focus then swings toward molecularly templating within organic and inorganic (xerogels) polymers to create materials with analyte binding characteristics akin to a biorecognition element. The review then describes several recent developments wherein analyte recognition and an analyte-dependent transduction methodology are simultaneously incorporated directly within the templated materials. The review ends by outlining the current state-of-the-art and the remaining issues and impediments.     

Single-step DNA immobilization on antifouling self-assembled monolayers covalently bound to silicon (111)

Hydrosilylation of alkenes with epoxide-terminated tri(ethylene oxide) moieties on Si-H surfaces yields homogeneous monolayers for the efficient coupling of biomolecules. The wetting properties of the epoxide-functionalized surface allow for the spotting of solutions of biomolecules, making the surface amenable to microarraying. Immobilization of thiolated DNA was achieved in a single step to fabricate biorecognition interfaces showing the hybridization of complementary DNA at low concentrations and negligible binding of noncomplementary DNA.     

Glyconanocavities: Cyclodextrins and Beyond

Cyclodextrins (CDs) represent a unique example of complementarity between nanotechnology and biotechnology. Their molecular nanocavity character and the possibility of selective functionalization offer an excellent opportunity for chemical elaboration of unique nanostructures in a three-dimensional network. Several approaches from our laboratories, aimed at endowing CDs with biorecognition properties by incorporation of saccharide ligands, are discussed. Applications range from site-specific drug delivery systems to more fundamental studies on carbohydrate–protein interactions. Results on the de novo synthesis of a new family of glyconanocavities constructed from α,α-trehalose building blocks, namely cyclotrehalans (CTs), and on their complexing properties are also presented.     

Characterisation and determination of stability and functionality of biofunctionalised colloidal gold nanoparticles

Colloidal gold nanoparticles were conjugated with oligonucleotides to create biorecognition nanomodules. The efficiency of conjugation was determined by fluorescence using a FITC-labelled thiolated model probe and by enzyme-linked nanoparticle assay (ELINA) using a digoxigenin-labelled thiolated model probe. The thermal stability of the conjugation was determined by displacement and fluorescence measurement of the FITC probe. Functionality for hybridisation was determined by enzyme-linked oligonucleotide assay (ELONA). It was found that the equilibrium oligonucleotide surface coverage reached 37% of the total nanoparticle area. These results could be verified by ELINA. Under hybridisation conditions that allowed the detection of 4-point mutations on a target 19-mer sequence (1 h at 65 °C), it was found that the biofunctionalised nanomodules lost between 10 and 30% of the conjugated biorecognition molecules.     

Peptides on GaAs surfaces: comparison between features generated by microcontact printing and dip-pen nanolithography

Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) were employed to understand the size, composition, and conformation of lithographic patterns composed of peptide molecules. GaAs surfaces were patterned by microcontact printing (microCP) and dip-pen nanolithography (DPN) using a peptide sequence composed of 15 amino acids. The detailed surface evaluation showed that the patterns have similar chemical compositions but differ in the bonding among the molecules anchored on the GaAs substrate. Both types of patterns were crystalline-like in nature. The features created by DPN exhibited interchain hydrogen bonding, while the ones generated by microCP displayed non-hydrogen bonding. The differences in the lithographic structures can be utilized in future biorecognition experiments that take advantage of the electronic properties of the GaAs substrate and the tunable behavior of the covalently anchored biomolecules on the surface.     

Nano-chemistry and scanning probe nanolithographies

The development of nanometer-scale lithographies is the focus of an intense research activity because progress on nanotechnology depends on the capability to fabricate, position and interconnect nanometer-scale structures. The unique imaging and manipulation properties of atomic force microscopes have prompted the emergence of several scanning probe-based nanolithographies. In this tutorial review we present the most promising probe-based nanolithographies that are based on the spatial confinement of a chemical reaction within a nanometer-size region of the sample surface. The potential of local chemical nanolithography in nanometer-scale science and technology is illustrated by describing a range of applications such as the fabrication of conjugated molecular wires, optical microlenses, complex quantum devices or tailored chemical surfaces for controlling biorecognition processes.     

Biorecognizable polymers: Structure-properties relationship

The biocompatibility of polymers depends on their chemical composition, as well as physical and physico-chemical structure. However, biocompatibility is not an intrinsic polymer property since it relates to a particular localization of the polymer in the living organism. Any foreign macromolecule may be recognized by the living organism as “non-self”. The degree of recognition can be altered by modification of the structure of macromolecules or biomaterial surfaces. Modification of biomacromolecules (e.g., enzymes¹) or biomaterial surfaces with hydrophilic polymers, e.g., poly(ethylene oxide²) or other semitelechelic hydrophilic macromolecules³ has been shown to minimize biorecognition. On the other hand, to achieve specific recognition by cell receptors and/or antigens, complementary structures have to be incorporated as a part of the polymer structure^{4, 5}. The biorecognition of fluorescently labeled N-(2-hydroxypropyl)-methacrylamide copolymers containing side-chains terminated in acylated galactosamine by Hep G2 cells on the cellular and subcellular levels can be visualized by confocal fluorescence microscopy. The recognition of modified water soluble macromolecules strongly depends on their solution properties⁶. Incorporation of stimuli sensitive moieties into copolymer side-chains permits to control recognition by external stimuli, e.g., irradiation^{7, 8}, resulting in the change of the copolymer conformation. On the other hand, the biorecognition of unsoluble (crosslinked) copolymers depends mainly on their surface structure. The rationale for the structure modification of synthetic copolymers to regulate biorecognition will be reviewed. The principles will be described for the interaction of synthetic polymers with enzymes⁹, cell surface receptors and/or antigens^{10, 11}, lectins of the gastrointestinal tract^{12, 13}, and immobilized lectins¹⁴.     

Optimized biorecognition of cytochrome c 551 and azurin immobilized on thiol-terminated monolayers assembled on Au(111) substrates

Molecular recognition between two redox partners, azurin and cytochrome c 551, is studied at the single-molecule level by means of atomic force spectroscopy, after optimizing azurin adsorption on gold via sulfhydryl-terminated alkanethiol spacers. Our experiments provide evidence of specific interaction between the two partners, thereby demonstrating that azurin preserves biorecognition capability when assembled on gold via these spacers. Additionally, the measured single-molecule kinetic reaction rate results are consistent with a likely transient nature of the complex. Interestingly, the immobilization strategy adopted here, which was previously demonstrated to favor electrical coupling between azurin (AZ) and the metal electrode, is also found to facilitate AZ interaction with the redox partner, if compared to the case of AZ directly adsorbed on bare gold. Our findings confirm the key role of a well-designed immobilization strategy, capable of optimizing both biorecognition capabilities and electrical coupling with the conductive substrate at the single-molecule level, as a starting point for advanced applications of redox proteins for ultrasensitive biosensing.     

Sol–Gel‐Derived Biohybrid Materials Incorporating Long‐Chain DNA Aptamers

Sol–gel‐derived bio/inorganic hybrid materials have been examined for diverse applications, including biosensing, affinity chromatography and drug discovery. However, such materials have mostly been restricted to the interaction between entrapped biorecognition elements and small molecules, owing to the requirement for nanometer‐scale mesopores in the matrix to retain entrapped biorecognition elements. Herein, we report on a new class of macroporous bio/inorganic hybrids, engineered through a high‐throughput materials screening approach, that entrap micron‐sized concatemeric DNA aptamers. We demonstrate that the entrapment of these long‐chain DNA aptamers allows their retention within the macropores of the silica material, so that aptamers can interact with high molecular weight targets such as proteins. Our approach overcomes the major limitation of previous sol–gel‐derived biohybrid materials by enabling molecular recognition for targets beyond small molecules.     

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals

Reactions catalyzed by artificial allosteric enzymes, chimeric proteins with fused biorecognition and catalytic units, were used to mimic multi-input Boolean logic systems. The catalytic parts of the systems were represented by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). Two biorecognition units, calmodulin or artificial peptide-clamp, were integrated into PQQ-GDH and locked it in the OFF or ON state respectively. The ligand-peptide binding cooperatively with Ca²⁺ cations to a calmodulin bioreceptor resulted in the enzyme activation, while another ligand-peptide bound to a clamp-receptor inhibited the enzyme. The enzyme activation and inhibition originated from peptide-induced allosteric transitions in the receptor units that propagated to the catalytic domain. While most of enzymes used to mimic Boolean logic gates operate with two inputs (substrate and co-substrate), the used chimeric enzymes were controlled by four inputs (glucose – substrate, dichlorophenolindophenol – electron acceptor/co-substrate, Ca²⁺ cations and a peptide – activating/inhibiting signals). The biocatalytic reactions controlled by four input signals were considered as logic networks composed of several concatenated logic gates. The developed approach allows potentially programming complex logic networks operating with various biomolecular inputs representing potential utility for different biomedical applications.     

Biorecognition-modulated ion fluxes through functionalized gold nanotubules as a novel label-free biosensing approach

A novel biosensing principle is presented, based on the potentiometric monitoring of an indicator ion such as Ca2+, whose zero-current flux through chemically modified nanochannels is altered by biorecognition events.     

Development of a stable dual functional coating with low non-specific protein adsorption and high sensitivity for new superparamagnetic nanospheres

To overcome major challenges of non-specific protein adsorption on nanoparticles for nanosensing and nanodiagnosis, an efficient method for robust chemical modification was developed to achieve excellent specific biorecognition and long-term stability in complex biomedia. This method is demonstrated by a highly specific and sensitive immunoassay (IA), using superparamagnetic nanospheres (NSs) with high magnetite content. The non-specific protein adsorption on the NSs was suppressed dramatically when modified with dual functional poly(carboxybetaine methacrylate) (polyCBMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and chemically grafted with antibodies of the β subunit of human chorionic gonadotrop (anti-β-hCG). The response to hCG of IA NSs with polyCBMA coatings was highly consistent in either phosphate-buffered saline (PBS) or 50% fetal bovine serum (FBS), which is far less variable than the response of the IA NSs without polyCBMA coatings. After all, a very robust platform for IA NSs with excellent specific biorecognition was obtained. It is expected that this method for nanoparticle modification could be widely used in ultrasensitive nanosensing and nanodiagnosis in the future.     

Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials

This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.     

Hydrogels: From soft contact lenses and implants to self‐assembled nanomaterials

Hydrogels were the first biomaterials designed for clinical use. Their discovery and applications as soft contact lenses and implants are presented. This early hydrogel research served as a foundation for the expansion of biomedical polymers research into new directions: design of stimuli sensitive hydrogels that abruptly change their properties upon application of an external stimulus (pH, temperature, solvent, electrical field, biorecognition) and hydrogels as carriers for the delivery of drugs, peptides, and proteins. Finally, pathways to self‐assembly of block and graft copolymers into hydrogels of precise 3D structures are introduced. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5929–5946, 2009     

Graphene as signal amplifier for preparation of ultrasensitive electrochemical biosensors

Early diagnosis of diseases with minimal cost and time-consumption has become achievable due to recent advances in the development of biosensors. These devices use biorecognition elements for the selective interaction with an analyte and the signal read-out is obtained via different types of transducers. The operational characteristics of biosensors have been reported as improving substantially when a diverse range of nanomaterials is employed. This review presents the construction of electrochemical biosensors based on graphene, atomically thin 2D carbon crystals, a nanomaterial currently the subject of intensive studies. Here, the most attractive directions for graphene applications in biosensor preparation are discussed, including novel detection and amplification schemes exploiting graphene’s unique electrochemical, physical and chemical properties. There is probably a very bright future for graphene-based biosensors, but much further work is required to fulfill the high expectations.