Immunosensing with Near-Infrared Plasmonic Optical Fiber Gratings.

Surface Plasmon resonance (SPR) optical fiber biosensors constitute a miniaturized counterpart to the bulky prism configuration and offer remote operation in very small volumes of analyte. They are a cost-effective and relatively straightforward technique to yield in situ (or even possibly in vivo) molecular detection. They are usually obtained from a gold-coated fiber segment for which the core-guided light is brought into contact with the surrounding medium, either by etching (or side-polishing) or by using grating coupling. Recently, SPR generation was achieved in gold-coated tilted fiber Bragg gratings (TFBGs). These sensors probe the surrounding medium with near-infrared narrowband resonances, which enhances both the penetration depth of the evanescent field in the external medium and the wavelength resolution of the interrogation. They constitute the unique configuration able to probe all the fiber cladding modes individually, with high Q-factors. We use these unique spectral features in our work to sense proteins and extra-cellular membrane receptors that are both overexpressed in cancerous tissues. Impressive limit of detection (LOD) and sensitivity are reported, which paves the way for the further use of such immunosensors for cancer diagnosis.

Cancer biomarker sensing using packaged plasmonic optical fiber gratings: Towards in vivo diagnosis.

This work presents the development of an innovative plasmonic optical fiber (OF) immunosensor for the detection of cytokeratin 17 (CK17), a biomarker of interest for lung cancer diagnosis. The development of this sensing platform is such that it can be assessed in non-liquid environments, demonstrating that a surface plasmon resonance (SPR) can be excited in this case. For this purpose, detections have been first carried out on CK17 encapsulated in gel matrix in the aim of mimicking tissue samples. Gold-coated OF immunosensors were embedded in a specifically designed packaging providing enough stiffness to penetrate into soft matters. Resulting reflected spectra have revealed, for the first time, the presence of a stable SPR signal recorded in soft matters. Experiments conducted to detect CK17 trapped in a porous polyacrylamide gel matrix have highlighted the specific and selective biosensor response towards the target protein. Finally, the packaged OF immunosensor has been validated by a preliminary test on human lung biopsy, which has confirmed the ex-vivo CK17 detection. Consequently, this work represents an important milestone towards the detection of biomarkers in tissues, which is still a clinical challenge for minimally-invasive in vivo medical diagnosis.

Small biomolecule immunosensing with plasmonic optical fiber grating sensor.

This study reports on the development of a surface plasmon resonance (SPR) optical fiber biosensor based on tilted fiber Bragg grating technology for direct detection of small biomarkers of interest for lung cancer diagnosis. Since SPR principle relies on the refractive index modifications to sensitively detect mass changes at the gold coated surface, we have proposed here a comparative study in relation to the target size. Two cytokeratin 7 (CK7) samples with a molecular weight ranging from 78 kDa to 2.6 kDa, respectively CK7 full protein and CK7 peptide, have been used for label-free monitoring. This work has first consisted in the elaboration and the characterization of a robust and reproducible bioreceptor, based on antibody/antigen cross-linking. Immobilized antibodies were then utilized as binding agents to investigate the sensitivity of the biosensor towards the two CK7 antigens. Results have highlighted a very good sensitivity of the biosensor response for both samples diluted in phosphate buffer with a higher limit of detection for the larger CK7 full protein. The most groundbreaking nature of this study relies on the detection of small biomolecule CK7 peptides in buffer and in the presence of complex media such as serum, achieving a limit of detection of 0.4 nM.

Fiber-Optic SPR Immunosensors Tailored To Target Epithelial Cells through Membrane Receptors.

We report, for the first time, the use of a surface plasmon resonance (SPR) fiber-optic immunosensor for selective cellular detection through membrane protein targeting. The sensor architecture lies on gold-coated tilted fiber Bragg gratings (Au-coated TFBGs) photoimprinted in the fiber core via a laser technique. TFBGs operate in the near-infrared wavelength range at ∼1550 nm, yielding optical and SPR sensing characteristics that are advantageous for the analyses of cellular bindings and technical compatibility with relatively low-cost telecommunication-grade measurement devices. In this work, we take consider their numerous assets to figure out their ability to selectively detect intact epithelial cells as analytes in cell suspensions in the range of 2-5 × 10(6) cells mL(-1). For this, the probe was first thermally annealed to ensure a strong adhesion of the metallic coating to the fiber surface. Its surface was then functionalized with specific monoclonal antibodies via alkanethiol self-assembled monolayers (SAMs) against extracellular domain of epidermal growth factor receptors (EGFRs) and characterized by peak force tapping atomic force microscopy. A differential diagnosis has been demonstrated between two model systems. The developed immunosensors were able to monitor, in real time, the specific attachment of single intact cells in concentrations from 3 × 10(6) cells mL(-1). Such results confirm that the developed probe fits the lab-on-fiber technology and has the potential to be used as a disposable device for in situ and real-time clinical diagnosis.

EPR spectroelectrochemical investigation of guanine radical formation and environment effects.

Guanine radical detection was carried out by a new convenient and efficient method coupling electron paramagnetic resonance spectroscopy and indirect electrooxidation of guanine in different biological environments, from the free nucleotide to several types of DNA substrates. Compared to the widely used photoirradiation method, this method appeared more selective in the choice of the electrochemical mediator. Carried out in presence of a ruthenium mediator and PBN as spin trap, this method revealed two types of EPR spectra depending of the environment of the guanine radical. Both EPR spectra show the trapping of the neutral guanine radical G(-H)(•) obtained after fast deprotonation of the radical cation G(•+). However, they differ by the atom where the trapped radical is centered. This difference highlights the structural dependency of the environment on the nature of the radical formed. This work gave the evidence of an innovative method to detect in situ the guanine radical.

Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy.

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedance spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulated macrophages. This production was not observed in the case of Leishmania-infected macrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC)(2) as NO radical spin trap were carried out.

Electrochemical impedance spectroscopy to study physiological changes affecting the red blood cell after invasion by malaria parasites.

The malaria parasite, Plasmodium falciparum, invades human erythrocytes and induces dramatic changes in the host cell. The idea of this work was to use RBC modified electrode to perform electrochemical impedance spectroscopy (EIS) with the aim of monitoring physiological changes affecting the erythrocyte after invasion by the malaria parasite. Impedance cell-based devices are potentially useful to give insight into cellular behavior and to detect morphological changes. The modelling of impedance plots (Nyquist diagram) in equivalent circuit taking into account the presence of the cellular layer, allowed us pointing out specific events associated with the development of the parasite such as (i) strong changes in the host cell cytoplasm illustrated by changes in the film capacity, (ii) perturbation of the ionic composition of the host cell illustrated by changes in the film resistance, (iii) releasing of reducer (lactic acid or heme) and an enhanced oxygen consumption characterized by changes in the charge transfer resistance and in the Warburg coefficient characteristic of the redox species diffusion. These results show that the RBC-based device may help to analyze strategic events in the malaria parasite development constituting a new tool in antimalarial research.

Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species.

BACKGROUND: Parasite concentration methods facilitate molecular, biochemical and immunological research on the erythrocytic stages of Plasmodium. In this paper, an adaptation of magnetic MACS(R) columns for the purification of human Plasmodium species is presented. This method was useful for the concentration/purification of either schizonts or gametocytes. RESULTS AND CONCLUSIONS: The magnetic removal of non-parasitized red blood cells (in vivo and in vitro) using magnetic columns (MACS) was evaluated. This easy-to-use technique enriched schizonts and gametocytes from Plasmodium falciparum in vitro cultures with a very high degree of purity. In addition, all haemozoin-containing stages (schizonts and/or gametocytes) from the peripheral blood of infected patients could be concentrated using this method. This method is particularly useful for the concentration of non-falciparum species, which do not grow in culture and are otherwise difficult to obtain in large amounts.