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Mediated amperometric immunosensing using single walled carbon nanotube forests.

A prototype amperometric immunosensor was evaluated based on the adsorption of antibodies onto perpendicularly oriented assemblies of single wall carbon nanotubes called SWNT forests. The forests were self-assembled from oxidatively shortened SWNTs onto Nafion/iron oxide coated pyrolytic graphite electrodes. The nanotube forests were characterized using atomic force microscopy and resonance Raman spectroscopy. Anti-biotin antibody strongly adsorbed to the SWNT forests. In the presence of a soluble mediator, the detection limit for horseradish peroxidase (HRP) labeled biotin was 2.5 pmol ml(-1) (2.5 nM). Unlabelled biotin was detected in a competitive approach with a detection limit of 16 nmol ml(-1) (16 microM) and a relative standard deviation of 12%. The immunosensor showed low non-specific adsorption of biotin-HRP (approx. 0.1%) when blocked with bovine serum albumin. This immunosensing approach using high surface area, patternable, conductive SWNT assemblies may eventually prove useful for nano-biosensing arrays.

Adsorption↗

Nanomaterial-based electrochemical biosensors.

The unique properties of nanoscale materials offer excellent prospects for interfacing biological recognition events with electronic signal transduction and for designing a new generation of bioelectronic devices exhibiting novel functions. In this Highlight I address recent research that has led to powerful nanomaterial-based electrical biosensing devices and examine future prospects and challenges. New nanoparticle-based signal amplification and coding strategies for bioaffinity assays are discussed, along with carbon-nanotube molecular wires for achieving efficient electrical communication with redox enzyme and nanowire-based label-free DNA sensors.

Biosensing Techniques↗

Biomolecule detection via target mediated nanoparticle aggregation and dielectrophoretic impedance measurement.

A new biosensing system is described that is based on the aggregation of nanoparticles by a target biological molecule and dielectrophoretic impedance measurement of these aggregates. The aggregation process was verified within a microchannel via fluorescence microscopy, demonstrating that this process can be used in a real time sensor application. Positive dielectrophoresis is employed to capture the nanoparticle aggregates at the edge of thin film electrodes, where their presence is detected either by optical imaging via fluorescence microscopy or by measuring the change in electrical impedance between adjacent electrodes. The electrical detection mechanism demonstrates the potential for this method as a micro total analysis system (microTAS).

Avidin↗

Chitosan-mediated in situ biomolecule assembly in completely packaged microfluidic devices.

We report facile in situ biomolecule assembly at readily addressable sites in microfluidic channels after complete fabrication and packaging of the microfluidic device. Aminopolysaccharide chitosan's pH responsive and chemically reactive properties allow electric signal-guided biomolecule assembly onto conductive inorganic surfaces from the aqueous environment, preserving the activity of the biomolecules. A transparent and nonpermanently packaged device allows consistently leak-free sealing, simple in situ and ex situ examination of the assembly procedures, fluidic input/outputs for transport of aqueous solutions, and electrical ports to guide the assembly onto the patterned gold electrode sites within the channel. Both in situ fluorescence and ex situ profilometer results confirm chitosan-mediated in situ biomolecule assembly, demonstrating a simple approach to direct the assembly of biological components into a completely fabricated device. We believe that this strategy holds significant potential as a simple and generic biomolecule assembly approach for future applications in complex biomolecular or biosensing analyses as well as in sophisticated microfluidic networks as anticipated for future lab-on-a-chip devices.

Biosensing Techniques↗

Silica microspheres for biomolecular detection applications.

Microsphere-based biosensors have been attracting the attention of the photonics community due to their high sensitivity, selectivity and implementation. Microspheres, with their high quality-factor (Q-factor) morphology dependent resonances, are very sensitive to refractive index and size changes. The perturbation of the microsphere morphology dependent resonances can be used for the detection of biomolecules. Adsorption of different biomolecules on the surface of microspheres causes a change of effective size and refractive index leading to the shift of resonance wavelengths. A biosensor, based on this phenomenon, can detect a single molecule sensitively depending on the configuration that needs to be designed and optimised. Silica with a refractive index of 1.5, which is very close to that of bimolecular agents, is a suitable photonic material to use for biosensing applications. The transverse electric and transverse magnetic elastic scattering spectra at 90 degrees and 0 degrees are calculated at 1.55 microm with the associated shifts after adding a layer on it. 90 degrees scattering is used to monitor the scattered signal, whereas 0 degrees scattering is used to monitor the transmission signal.

Biopolymers↗

Biological applications of localised surface plasmonic phenomenae.

Researchers and industrialists have taken advantage of the unusual optical, magnetic, electronic, catalytic, and mechanical properties of nanomaterials. Nanoparticles and nanoscale materials have proven to be useful for biological uses. Nanoscale materials hold a particular interest to those in the biological sciences because they are on the same size scale as biological macromolecules, proteins and nucleic acids. The interactions between biomolecules and nanomaterials have formed the basis for a number of applications including detection, biosensing, cellular and in situ hybridisation labelling, cell tagging and sorting, point-of-care diagnostics, kinetic and binding studies, imaging enhancers, and even as potential therapeutic agents. Noble metal nanoparticles are especially interesting because of their unusual optical properties which arise from their ability to support surface plasmons. In this review the authors focus on biological applications and technologies that utilise two types of related plasmonic phenomonae: localised surface plasmon resonance (LSPR) spectroscopy and surface-enhanced Raman spectroscopy (SERS). The background necessary to understand the application of LSPR and SERS to biological problems is presented and illustrative examples of resonant Rayleigh scattering, refractive index sensing, and SERS-based detection and labelling are discussed.

Biology↗

Biosensors from conjugated polyelectrolyte complexes.

A charge neutral complex (CNC) was formed in aqueous solution by combining an orange light emitting anionic conjugated polyelectrolyte and a saturated cationic polyelectrolyte at a 1:1 ratio (per repeat unit). Photoluminescence (PL) from the CNC can be quenched by both the negatively charged dinitrophenol (DNP) derivative, (DNP-BS(-)), and positively charged methyl viologen (MV(2+)). Use of the CNC minimizes nonspecific interactions (which modify the PL) between conjugated polyelectrolytes and biopolymers. Quenching of the PL from the CNC by the DNP derivative and specific unquenching on addition of anti-DNP antibody (anti-DNP IgG) were observed. Thus, biosensing of the anti-DNP IgG was demonstrated.

Animals↗

Label-free THz sensing of genetic sequences: towards 'THz biochips'.

THz-wave-based approaches for the label-free characterization of genetic material are described. Time-resolved THz spectroscopic analysis of genetic sequences (polynucleotides) demonstrate a distinct complex refractive index in the THz frequency range as a function of the binding state of the analysed DNA sequences. By monitoring THz signals, one can thus infer the binding state of oligo- and polynucleotides, enabling the label-free determination of the genetic composition of target polynucleotides by sensing their binding to known probe molecules. Here we review integrated THz sensing array developments exhibiting high sensitivity and single-base mutation detection capabilities. Recent achievements using functionalized biosensing arrays of high-Q resonators are illustrated.

Biosensing Techniques↗

Solidly mounted ZnO shear mode film bulk acoustic resonators for sensing applications in liquids.

Solidly mounted film bulk acoustic resonators (FBAR) operating at 850 MHz in the shear vibration mode have been fabricated. C-axis inclined zinc oxide (ZnO) thin films realized by modified reactive magnetron sputtering were used: Coupling factors k2 of 1.7% and Q-factors of 312 were determined in air. Q-factors of 192 were measured in water, making these devices attractive for sensing applications in liquids, e.g., biosensing.

Biosensing Techniques↗

Regulation of protein activity with small-molecule-controlled inteins.

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.

Allosteric Site↗

Development of organic dye-doped silica nanoparticles for bioanalysis and biosensors.

The combination of two silica precursors, tetraethylorthosilicate and phenyltriethoxysilane, were utilized to synthesize organic dye-doped silica nanoparticles. The hydrophobic nature of phenyltriethoxysilane keeps the organic dye in the silica matrix, whereas the hydrophilic tetraethylorthosilicate-formed silica allows the resulting nanoparticles to be dispersed in aqueous solutions. Characterization of the nanoparticles showed that they could be synthesized in the nanometer range with high photostability and minimal dye leakage. The silica matrix of the nanoparticles allows different routes of surface biomolecular modification for biosensor and bioanalysis applications. We have shown different applications of the nanoparticles in bioanalysis and in biosensing. Biotin interaction of avidin-coated nanoparticles can be used for the determination of biotinylated bovine serum albumin, and the immobilization of glutamate dehydrogenase on the nanoparticle surfaces enables the nanoparticles to be used as biosensors for glutamate determination.

Biosensing Techniques↗

Silver growth on micropatterned DNA chips: effect of growth conditions and morphology on I-V behavior.

An approach to the design of DNA-based electronics is presented, in which standard microfabrication processes are integrated with lithographic patterning of single-stranded oligonucleotides followed by hybridization to gold-labeled, complementary oligonucleotides and subsequent silver enhancement for signal amplification. The resulting bioinorganic devices demonstrate micron-sized geometric features, very little non-specific silver growth, a distinct silver morphology in the patterned region, and a 10(9)-fold increase in conductivity across an electrode gap when compared with control devices. This approach may prove useful for the fabrication of high fidelity, high-density arrays for DNA-based biosensing applications.

Biosensing Techniques↗

Nanopatterns with biological functions.

Both curiosity and a desire for efficiency have advanced our ability to manipulate materials with great precision on the micrometer and, more recently, on the nanometer scale. Certainly, the semiconductor and integrated circuit industry has put the pressure on scientist and engineers to develop better and faster nanofabrication techniques. Furthermore, our curiosity as to how life works, and how it can be improved from a medical perspective, stands to gain a great deal from advances in nanotechnology. Novel nanofabrication techniques are opening up the possibilities for mimicking the inherently nano-world of the cell, i.e., the nanotopographies of the extracellular matrix (ECM) and the nanochemistry presented on both the cell membrane and the ECM. In addition, biosensing applications that rely on fabrication of high-density, precision arrays, e.g., DNA or gene chips and protein arrays, will gain significantly in efficiency and, thus, in usefulness once it becomes possible to fabricate heterogeneous nanoarrays. Clearly, continued advances in nanotechnology are desired and required for advances in biotechnology. In this review, we describe the leading techniques for generating nanopatterns with biological function including parallel techniques such as extreme ultraviolet interference lithography (EUV-IL), soft-lithographic techniques (e.g., replica molding (RM) and microcontact printing (muCP)), nanoimprint lithography (NIL), nanosphere lithography (NSL) (e.g., colloid lithography or colloidal block-copolymer micelle lithography) and the nanostencil technique, in addition to direct-writing techniques including e-beam lithography (EBL), focused ion-beam lithography (FIBL) and dip-pen nanolithography (DPN). Details on how the patterns are generated, how biological function is imparted to the nanopatterns, and examples of how these surfaces can and are being used for biological applications will be presented. This review further illustrates the rapid pace by which advances are being made in the field of nanobiotechnology, owing to an increasing number of research endeavors, for an ever increasing number of applications.

Animals↗

Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on the Mach-Zehnder configuration.

A high-sensitivity surface plasmon resonance (SPR) biosensor based on the Mach-Zehnder interferometer design is presented. The novel feature of the new design is the use of a Wollaston prism through which the phase quantities of the p and s polarizations are interrogated simultaneously. Since SPR affects only the p polarization, the signal due to the s polarization can be used as the reference. Consequently, the differential phase between the two polarizations allows us to eliminate all common-path phase noise while keeping the phase change caused by the SPR effect. Experimental results obtained from glycerin-water mixtures indicate that the sensitivity limit of our scheme is 5.5 x 10(-8) refractive-index units per 0.01 degrees phase change. To our knowledge, this is a significant improvement over previously obtained results when gold was used as the sensor surface. Such an improvement in the sensitivity limit should allow SPR biosensors to become a possible replacement for conventional biosensing techniques based on fluorescence. Monitoring of the bovine serum albumin (BSA) binding reaction with BSA antibodies is also demonstrated.

Antigen-Antibody Complex↗

D-type fiber biosensor based on surface-plasmon resonance technology and heterodyne interferometry.

A D-type fiber biosensor based on surface-plasmon resonance (SPR) technology and heterodyne interferometry is presented. The sensing device is a single-mode optical fiber in which half the core is polished away and a thin-film layer of gold is deposited. We measure the phase-difference variations instead of the light intensity as in traditional SPR techniques. Therefore the accuracy and resolution of our method are very high. Its sensitivity can reach 2 x 10(-6) refractive-index units. The sensor has some merits, e.g., tunable high sensitivity, small size, lower cost, smaller sample volume, and suitability for in vivo testing. This novel method of a D-type fiber biosensor based on SPR technology and heterodyne interferometry is valuable for chemical, biological, and biochemical sensing, and the novel method of D-type fiber biosensing is a feasible means of study.

Alcohols↗

Common-path phase-shift interferometry surface plasmon resonance imaging system.

Surface plasmon resonance (SPR) and common-path phase-shift interferometry (PSI) techniques are integrated in a biosensing imaging system for measuring the two-dimensional spatial phase variation caused by biomolecular interactions on a sensing chip without the need for additional labeling. The common-path PSI technique has the advantage of long-term stability, even when it is subjected to external disturbances. Hence the system meets the requirements of the real-time kinetic studies involved in biomolecular interaction analysis. The proposed SPR-PSI imaging system demonstrates a detection limit of a 2 x 10(-7) refractive-index change, a long-term phase stability of 2.5 x 10(-4) pi rms for 4 h, and a spatial phase resolution of 10(-3) pi with a lateral resolution of 100 microm.

Biosensing Techniques↗

Highly sensitive detection of biomolecules with the magneto-optic surface-plasmon-resonance sensor.

The characteristics of a novel magneto-optic surface-plasmon-resonance (MOSPR) sensor and its use for the detection of biomolecules are presented. This physical transduction principle is based on the combination of the magneto-optic activity of magnetic materials and a surface-plasmon resonance of metallic layers. Such a combination can produce a sharp enhancement of the magneto-optic effects that strongly depends on the optical properties of the surrounding medium, allowing its use for biosensing applications. Experimental characterizations of the MOSPR sensor have shown an increase in the limit of detection by a factor of 3 in changes of refractive index and in the adsorption of biomolecules compared with standard sensors. Optimization of the metallic layers and the experimental setup could result in an improvement of the limit of detection by as much as 1 order of magnitude.

Biopolymers↗

Electrochemical biosensors for environmental monitoring.

Highly sensitive electrochemical biosensors offer precision, sensitivity, rapidity, and ease of operation for on-site environmental analysis. An electrochemical biosensor is an analytical device in which a specific biological recognition element (bioreceptor) is integrated within or intimately associated with an electrode (transducer) that converts the recognition event to a measurable electrical signal for the purpose of detecting a target compound (analyte) in solution. The signal generated allows both qualitative and quantitative measurements of an analyte in real time. In most cases, a miniaturized electrochemical cell contains a low volume of analyte, which is vital when dealing with hazardous materials and makes such devices ideal for environmental monitoring. This approach not only provides the means for on-site analysis but also removes the time delay and sample alteration that can occur during transport to a centralized laboratory. We first address the basic principles of electroanalytical measurement and the merger of electrochemistry and biology into a biosensing system, and then we discuss various environmental monitoring strategies involving this technology.

Biosensing Techniques↗