Label-free Optical Biosensor Research Articles

Label-Free Detection of Peptide Nucleic Acid−DNA Hybridization Using Localized Surface Plasmon Resonance Based Optical Biosensor

The development of label-free optical biosensors for DNA and other biomolecules has the potential to impact life sciences as well as screening in medical and environmental applications. In this report, we developed a localized surface plasmon resonance (LSPR) based label-free optical biosensor based on a gold-capped nanoparticle layer substrate immobilized with peptide nucleic acids (PNAs). PNA probe was designed to recognize the target DNA related to tumor necrosis factor. The nanoparticle layer was formed on a gold-deposited glass substrate by the surface modified silica nanoparticles using silane-coupling reagent. The optical properties of gold-capped nanoparticle layer substrate were characterized through monitoring the changes in the absorbance strength, as the thickness of the biomolecular layer increased with hybridization. The detection of PNA-DNA hybridization with target oligonucleotides and PCR-amplified real samples were performed with a limit of detection value of 0.677 pM target DNA. Selective discrimination against a single-base mismatch was also achieved. Our LSPR-based biosensor with the gold-capped nanoparticle layer substrate is applicable to the design of biosensors for monitoring of the interaction of other biomolecules, such as proteins, whole cells, or receptors with a massively parallel detection capability in a highly miniaturized package.

Characteristics of Dynamic Mass Redistribution of Epidermal Growth Factor Receptor Signaling in Living Cells Measured with Label-Free Optical Biosensors

This paper reported the identification of a novel optical signature for epidermal growth factor (EGF) receptor signaling in human epidermoid carcinoma A431 cells mediated by EGF. The optical signature was based on dynamic mass redistribution (DMR) in living cells triggered by EGFR activation, as monitored in real time with resonant waveguide grating biosensors. Analysis of the modulation of the EGF-induced DMR signals by a variety of known modulators provided links of various targets to distinct steps in the cellular responses. Results showed that the dynamic mass redistribution in quiescent A431 cells mediated by EGF required EGFR tyrosine kinase activity, actin polymerization, and dynamin and mainly proceeded through MEK. The DMR signals obtained serve as integrated signatures for interaction networks in the EGFR signaling.

Engineering a signal transduction mechanism for protein-based biosensors.

Hybridization-induced conformational changes have been successfully used in biosensors for the transduction of DNA-binding events into readily observable optical or electronic signals. Similar signal transduction has not, however, proven of equal utility in protein-based biosensors. The discrepancy arises because, unlike ssDNA, most proteins do not undergo significant conformational changes upon ligand binding. Here, we describe a solution to this problem. We show that an arbitrarily selected, normally well folded protein can be rationally engineered such that it undergoes ligand-induced folding. The engineered protein responds rapidly (milliseconds) and selectively to its target, and it couples recognition with the largest possible conformational change: folding. These traits suggest that ligand-induced folding could serve as an ideal signal-transduction mechanism. Consistent with this claim, we demonstrate a label-free optical biosensor based on the effect that is sufficiently selective to detect its target even in complex, contaminant-ridden samples such as blood serum.

Localized surface plasmon resonance based optical biosensor using surface modified nanoparticle layer for label-free monitoring of antigen–antibody reaction

In recent years, label-free biosensors not requiring external modifications have been receiving intense attention. A label-free optical biosensor, which retains many of the desirable features of conventional surface plasmon resonance (SPR) reflectometry, namely, the ability to monitor the kinetics of biomolecular interactions in real-time without a label has been developed with several important advantages: the biosensor device is easy to fabricate, and simple to implement, requiring only an UV–Vis spectrophotometer or flatbed scanner. Importantly, the label-free optical biosensor can be easily multiplexed to enable high-throughput monitoring of biomolecular interactions in an arraybased format. In this research,the development of a localized surface plasmon resonance (LSPR)-based label-free optical biosensor using a surface modified nanoparticle layer is aimed. This optical detection method promises to offer a massively parallel detection capability in a highly miniaturized package. The two-dimensional nanoparticle layer was formed by the surface modified silica nanoparticles. The optical properties and surface analysis of nanoparticle layer substrate were characterized through transmissionmeasurements and atomic force microscopy (AFM). Simultaneously, the nanoparticle layer substrate was applied to the optical LSPR-based biosensor for label-free monitoring of the antigen–antibody reaction. The anti-fibrinogen antibody wasimmobilized onto the nanoparticle layer substrate surface. Different concentrations of fibrinogen were introduced to the anti-fibrinogen antibody immobilized nanoparticle layer substrate surface, and the change in the absorption spectrum, caused by the antigen–antibody reaction, was observed. By using this anti-fibrinogen antibody immobilized nanoparticle layer substrate; the detection limit of this optical LSPR-based biosensor was 10 ng/ml.

Fabrication of Photonic Crystal Biosensors using Micro-molding of Nanoporous Glass

ABSTRACTWe demonstrate a micro-molding method for submicron patterning of a low-index sol-gel nanoporous glass for the purpose of fabricating large-area (∼3”×”) label-free photonic crystal optical biosensors. SEM images show that the sol-gel exhibited minimal shrinkage and good substrate adhesion and depict precise and uniform pattern transfer over the fabricated area within the limits of measurement resolution. A unique characterization approach accurately and quickly revealed porous glass patterned over a large area with geometrical and material properties uniform within 1%. We suggest that this robust method is an excellent approach for photonic crystal sensor fabrication, and may also find applications in integrated optics and electronics.

Monitoring an antibody affinity chromatography with a label-free optical biosensor technique

In the production of polyclonal antibody, a purification step is necessary which is often done by affinity chromatography. We present a biosensor system based on reflectometric interference spectroscopy (RIfS) to monitor the quantity and quality in terms of affinity and kinetic constants of the antibody during this procedure. Biosensors are rapid compared with ELISA, which is done in practice and can work fully automated. They provide additional information about the active antibody to protein concentration ratio and the affinity of the antibody. We show how to determine these values very accurate. In addition, we describe a new rapid method to monitor the affinity chromatography in process. This gives the possibility to select antibody fractions with best properties in respect to the application.

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.

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.

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.

Label-free detection of biomolecular interaction by optical sensors.

Since the first label-free optical biosensor was commercialized in 1990 a rising number of publications have demonstrated the benefits of direct biomolecular interaction analysis (BIA) for biology and biochemistry. This article first gives an overview of the historical development of different transducer principles used for the detection of BIA. Subsequently, the four major parts of a biosensor system: transducer, sample handling, surface/immobilization chemistry and test formats/data evaluation will be discussed, with a main focus on the test formats and data evaluation. The intention of this review is to present an introduction to the field and to point out the difficulties most frequently encountered.