Label-free Optical Method Research Articles

Detection of biological macromolecules on a biochip dedicated to UV specific absorption

This work describes an ultraviolet biosensing technique based on specific molecular absorption detected with a previously developed spectrally selective aluminum gallium nitride (AlGaN) based detector. Light absorption signal of DNA and proteins, respectively at 260 nm and 280 nm, is used to image biochips. To allow detection of protein or DNA monolayers at the surface of a biochip, we develop contrast-enhancing multilayer substrates. We analyze them through models and experiments and validate the possibility of measuring absorptions of the order of 10 −3. These multilayer structures display a high reflectivity, and maximize the interaction of the electric field with the biological element at the chip surface. Optimization of the experimental absorption, which includes effects such as roughness of the biochip, spectral and angular resolution of the optics, illumination, etc., is carried out with an inorganic ultraviolet absorber (titanium dioxide) deposit. We obtained an induced absorption contrast enhanced by a factor of 4.0, conferring enough sensitivity to detect monolayers of DNA or proteins. Experimental results on an Escherichia coli histidine-tagged methionyl-tRNA synthetase protein before and after complexation with an anti-polyHis specific antibody validate our biosensing technique. This label-free optical method may be helpful in controlling biochip coatings, and subsequent biological coupling at the surface of a biochip.

Interferon Gamma: Activity and ELISA Detection Comparisons

Interferon-gamma (IFN-γ) is a pleiotropic cytokine secreted by Natural Killer (NK) cells and many T cells. It is the sole representative of Type II IFN. IFN-γ has pleiotropic activities including: inducing an antiviral state in cells, upregulating cell surface markers such as Class I and II MHC, and regulating the activity of macrophages, NK cells, neutrophils, B-cells, and T-cells. As such, IFN-γ is one of the most widely studied proteins [Scroder et al (2004) J. Leuk. Biol. 75:163; Young and Bream (2007) Curr. Top. Microbiol. Immunol. 316:97].

A label-free optical detection method for biosensors and microfluidics

A label-free detection method is developed for biosensors and for microfluidic applications. Multiple optical modes, including a conventional surface plasmon resonance (SPR) mode and many bulk modes, are created in a microfluidic channel with metallic coatings on top and bottom. The bulk modes are bulk sensitive and detect analytes in the channel with 20 times higher sensitivity than the SPR, which is particularly suitable for microfluidic devices. The method can be operated either in the SPR mode for detection of molecular binding on a surface or in one of the bulk optical modes for monitoring analytes in the channel.

Label-free optical imaging of mitochondria in live cells

The far-field optical imaging of mitochondria of live cells without the use of any label is demonstrated. It uses a highly sensitive photothermal method and has a resolution comparable to confocal fluorescence setups. The morphological states of mitochondria were followed under different physiological treatments, and the role of cytochrome c was ruled out as the main origin of the photothermal signals. This label free optical method provides a high contrast imaging of live mitochondria and should find many applications in biosciences.

New direct optical biosensors for multi-analyte detection

A new label-free optical biosensing method has been developed, which can be called Spectral Correlation Method (SCM). The method is based on measurements of a correlation signal of two interferometers in an original optical scheme. The first interferometer is a scanned Fabry–Perot interferometer, in which the distance between two mirrors is periodically changed by a peizoelectric driver. The second interferometer is formed by a biochip, which is a simple glass plate with thickness of several tens or hundreds microns with different recognition spots or wells, or flow channels. In our experiments, a microscope cover glass was successfully used as a biochip to detect reactions of binding or detachment of different biological agents in real time. Two- and 96-channel SCM devices have been designed to register bio- and chemical-components in real time. The sensogram drifts for these devices expressed in terms of layer thickness were 20 and 40 pm/h, respectively.

Label-free parallel screening of combinatorial triazine libraries using reflectometric interference spectroscopy.

The parallel reflectometric interference spectroscopy is presented as a label-free optical detection method. A new setup was adapted to accommodate sample carriers in a 96-well microplate. It allows for the first time simultaneous plate imaging by a CCD camera for the parallel detection of specific biomolecular interaction in the microplate wells at heterogeneous phase using direct optical monitoring. The detection of binding events with time resolution enables a highly parallel functional biomolecular interaction analysis (BIA). The combination of this new screening setup with combinatorial solid-phase synthesis is performed in the wells of glass-bottom microplates to accomplish the synthesis and the screening platform within one device. As a model system for a solid-phase substance library, synthesis of a triazine library and the subsequent BIA with four different antibodies were carried out. The presented setup enables a time resolution of 18 s with a total screening time of less than 35 min including baseline adjustment, BIA, and regeneration of the screening device for 96 samples in parallel. The binding studies reveal a fast classification of the different monoclonal and polyclonal antibodies and enable the detection of triazines with high binding affinity. The presented prototype is the first parallelized optical label-free detection system for biomolecular interaction analysis that is suitable for a high-throughput screening based on the 96-well microplate format.

A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface.

This paper presents a new label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of colloidal gold on glass, as a function of biomolecular binding to the surface of the immobilized colloids. Using this approach, we demonstrate proof of principle of a label-free optical biosensor to quantify biomolecular interactions in real time on a surface in a commercially available UV-visible spectrophotometer and of a colorimetric end-point assay using an optical scanner. The spectrophotometric sensor shows concentration-dependent binding and a detection limit of 16 nM for streptavidin. The sensor is easy to fabricate, is reproducible in its performance, has minimal technological requirements, namely, the availability of an UV-visible spectrophotometer or an optical scanner, and will enable high-throughput screening of biomolecular interactions in real time in an array-based format.

The effect of surface probe density on DNA hybridization.

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.