Physiologically responsive, mechanically adaptive polymer optical fibers for optogenetics.

The capability to deliver light to specific locations within the brain using optogenetic tools has opened up new possibilities in the field of neural interfacing. In this context, optical fibers are commonly inserted into the brain to activate or mute neurons using photosensitive proteins. While chronic optogenetic stimulation studies are just beginning to emerge, knowledge gathered in connection with electrophysiological implants suggests that the mechanical mismatch of conventional optical fibers and the cortical tissue may be a significant contributor to neuroinflammatory response. Here, we present the design and fabrication of physiologically responsive, mechanically adaptive optical fibers made of poly(vinyl alcohol) (PVA) that may mitigate this problem. Produced by a one-step wet-spinning process, the fibers display a tensile storage modulus E' of ∼7000 MPa in the dry state at 25°C and can thus readily be inserted into cortical tissue. Exposure to water causes a drastic reduction of E' to ∼35 MPa on account of modest swelling with the water. The optical properties at 470 and 590 were comparable with losses of 0.7±0.04 dB/cm at 470 nm and 0.6±0.1 dB/cm at 590 nm in the dry state and 1.1±0.1 dB/cm at 470 nm and 0.9±0.3 dB/cm at 590 nm in the wet state. The dry end of a partially switched fiber with a length of 10 cm was coupled with a light-emitting diode with an output of 10.1 mW to deliver light with a power density of >500 mW/cm2 from the wet end, which is more than sufficient to stimulate neurons in vivo. Thus, even without a low-refractive index cladding, the physiologically responsive, mechanically adaptive optical fibers presented here appear to be a very useful new tool for future optogenetic studies.

Lab-on-a-chip for multiplexed biosensing of residual antibiotics in milk.

A multiplexed immunoassay-based antibiotic sensing device integrated in a lab-on-a-chip format is described. The approach is multidisciplinary and involves the convergent development of a multi-antibiotic competitive immunoassay based on sensitive wavelength interrogated optical sensor (WIOS) technology and a polymer-based self-contained microfluidic cartridge. Immunoassay solutions are pressure-driven through external and concerted actuation of a single syringe pump and multiposition valve. Moreover, the use of a novel photosensitive material in a 'one step' fabrication process allowed the rapid fabrication of microfluidic components and interconnection port simultaneously. Pre-filled microfluidic cartridges were used as binary response rapid tests for the simultaneous detection of three antibiotic families - sulfonamides, fluoroquinolones and tetracyclines - in raw milk. For test interpretation, any signal lower than the threshold value obtained for the corresponding Maximum Residue Limit (MRL) concentration (100 microg L(-1)) was considered negative for a given antibiotic. The reliability of the multiplexed detection system was assessed by way of a validation test carried out on a series of six blind milk samples. A test accuracy of 95% was calculated from this experiment. The whole immunoassay procedure is fast (less than 10 minutes) and easy to handle (automated actuation).

Biosensors based on porous cellulose nanocrystal-poly(vinyl alcohol) scaffolds.

Cellulose nanocrystals (CNCs), which offer a high aspect ratio, large specific surface area, and large number of reactive surface groups, are well suited for the facile immobilization of high density biological probes. We here report functional high surface area scaffolds based on cellulose nanocrystals (CNCs) and poly(vinyl alcohol) (PVA) and demonstrate that this platform is useful for fluorescence-based sensing schemes. Porous CNC/PVA nanocomposite films with a thickness of 25-70 nm were deposited on glass substrates by dip-coating with an aqueous mixture of the CNCs and PVA, and the porous nanostructure was fixated by heat treatment. In a subsequent step, a portion of the scaffold's hydroxyl surface groups was reacted with 2-(acryloxy)ethyl (3-isocyanato-4-methylphenyl)carbamate to permit the immobilization of thiolated fluorescein-substituted lysine, which was used as a first sensing motif, via nucleophile-based thiol-ene Michael addition. The resulting sensor films exhibit a nearly instantaneous and pronounced change of their fluorescence emission intensity in response to changes in pH. The approach was further extended to the detection of protease activity by immobilizing a Förster-type resonance energy transfer chromophore pair via a labile peptide sequence to the scaffold. This sensing scheme is based on the degradation of the protein linker in the presence of appropriate enzymes, which separate the chromophores and causes a turn-on of the originally quenched fluorescence. Using a standard benchtop spectrometer to monitor the increase in fluorescence intensity, trypsin was detected at a concentration of 250 µg/mL, i.e., in a concentration that is typical for abnormal proteolytic activity in wound fluids.

Au-labeled antibodies to enhance the sensitivity of a refractometric immunoassay: detection of cocaine.

An integrated platform for a very sensitive detection of cocaine based on a refractometric biosensor is demonstrated. The system uses a waveguide grating biosensor functionalized with a cocaine multivalent antigen-carrier protein conjugate. The immunoassay scheme consists of the competitive binding of cocaine-specific antibodies to the immobilized conjugates. A 1000-fold enhancement of the sensor's sensitivity is achieved when using gold conjugated monoclonal antibodies instead of free antibodies. Together with the optimization of the assay conditions, the setup is designed for a quick identification of narcotics using automated sampling. The results show that the presence of cocaine in a liquid sample could be identified down to a concentration of 0.7 nM within one minute. This value can be reduced even further when longer binding time is allowed (0.2 nM after 15 min). Application of the system to detection of narcotics at airport security control points is discussed.

Integrated optical biosensor for in-line monitoring of cell cultures.

An analytical detection platform was developed to evaluate the induced toxicity in cell cultures exposed to foreign agents like growth factors or nanoparticles. Connecting a biosensing detection device to the cell culture flasks allows analyzing the composition of cell medium in real-time. The analysis relies on the quantification of inflammatory cytokines released by cells into the cell culture medium, by means of solid-phase immunoassays analyzed with the wavelength interrogated optical sensing (WIOS) instrument. A fluidic system for in situ measurements allows detecting cytokines in real-time, with a sensitivity of 1-100 ng/mL depending on the cytokine. In addition, integration of an in-line optical absorbance measurement unit, in combination with the standard AB cell proliferation assay, provides information on the cell viability in the culture. Fluidic connections between the cell culture flasks, the optical biosensor and the absorbance measurement unit simultaneously allow quantifying up to three cytokines (interleukin 8, interleukin 6 and the monocyte chemotactic protein), assessing cellular proliferation, and thus discriminating between naïve cells and cells exposed to foreign agents such as growth factors (tumor necrosis factor alpha) or nanoparticles. This analytical tool presents a high potential for assessing the cytotoxicity of nanoparticles and other chemicals in vitro.

Waveguide interrogated optical immunosensor (WIOS) for detection of sulfonamide antibiotics in milk.

An immunosensor was developed for the detection of sulfonamide antibiotics in milk. Detection relied on a competitive immunoassay format, using immunoreagents previously developed for the generic detection of sulfonamide antibiotics and evaluated by enzyme-linked immunosorbent assay. The immunoassay was implemented onto a microsystem platform, the wavelength interrogated optical sensing device, which uses the evanescent field to probe changes at the interface of a waveguiding layer, and thus allows sensitive detection of biomolecule adsorption. The immunoreagents were immobilized onto the surface of the waveguide chip, and a fluidic cell allowed flowing analyte and detection solutions above the surface. Sulfapyridine was used as reference sulfonamide and detected with the immunosensor in buffer and in milk with a limit of detection (IC(90)) of 0.2+/-0.1 microg L(-1) and 0.5+/-0.1 microg L(-1), respectively. The analysis time was below 30 min, including regeneration of the sensing surface, with minimum sample preparation required. The reproducibility of the detection was better than 10%. A blind assay allowed identifying milk samples that were contaminated with different sulfonamide antibiotics at or above the maximum residue limits established by the European Union for these compounds (100 microg L(-1)). Thus, the developed immunosensor presents great potential as a generic sensing device for the fast and early detection of food contaminants on the field by non-skilled users.

Three-dimensional microfluidic confinement for efficient sample delivery to biosensor surfaces. application to immunoassays on planar optical waveguides.

A microchip-based flow confinement method for rapid delivery of small sample volumes to sensor surfaces is described. For flow confinement, a sample flow is joined with a perpendicular makeup flow of water or sample medium. Under laminar flow conditions, the makeup flow confines the sample into a thin layer above the sensing area and increases its velocity. This can benefit mass transport limited processes such as DNA hybridization or heterogeneous immunoassays. For proof of concept, this method was applied to a high-affinity immunoassay with excess capture antibody. Rabbit IgG was immobilized onto a silicon nitride waveguide. Cy5-labeled anti-rabbit IgG was hydrodynamically pumped over the immobilized zone through an attached 3D-PDMS flow cell with 20-microm-deep microchannels. The degree of confinement was adjusted through the volume flow rate of the confining flow. Evanescent field-based fluorescence detection enabled monitoring of the binding event. Assays were allowed to reach equilibrium to enable sensorgram normalization for inter-run comparison. The corresponding assay completion times could be reduced from 55 min for static drop conditions to 13 min for 25:1 flow confinement (ratio of confining to sample flow). For typical analytical applications, where equilibrium formation is not required, the faster response should translate to very short analysis times. Concurrently with the faster binding, sample consumption was reduced by 96% compared to conventional whole-channel sample delivery. Diffusional loss of analyte into the confining layer was identified as the main limitation of flow confinement, particularly for long sensing pads.