In today’s point-of-care testing (POCT), there is an ever-increasing demand for novel and more efficient devices for early diagnosis, especially in cardiovascular diseases (CVD). Early detection of CVD markers, such as Troponin present in the bloodstream, is a key factor for reducing CVD mortality rates.
Thiol-ene coupling (TEC) and Light Assisted Molecular Immobilization (LAMI) are photonic techniques leading to immobilization of bioreceptors, such as, antibodies which recognize cardiac markers. These techniques present advantages compared to traditional immobilization techniques since, e.g., there are no thermal or chemical steps and they work in water media. TEC reaction takes place at close-to-visible wavelengths (λ=365nm) which induces the formation of thiol radicals which bind to alkene functional group on the surface through a thioether bond. LAMI secures molecular immobilizations in a spatially oriented, localized and covalent coupling of biomolecules onto thiol reactive surfaces down to submicrometer spatial resolution. LAMI is possible due to a conserved structural motif in proteins: the spatial proximity between aromatic residues and disulfide bridges. When aromatic residues are excited with UV light (275- 295nm), disulphide bridges are disrupted and free thiol groups are formed that can bind covalently to a surface decorated with thiol groups.
We have achieved successful immobilization of anti-troponin and anti-myoglobin antibodies with both photonic immobilization techniques. The microarrays of immobilized monoclonal antibodies have successfully detected the CVD biomarkers troponin I and myoglobin, as confirmed by fluorescence imaging. A sandwich immunoassay was carried out, Troponin I and Myoglobin were detected down to 10 ng/mL and 1 ng/mL, respectively.
Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.
The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases. EGFR activation upon binding of ligands (such as EGF and TGF-α) results in cell signaling cascades that promote cell proliferation, survival and apoptosis inhibition. As reported for many solid tumors, EGFR overactivation is associated with tumor development and progression, resistance to cancer therapies and poor prognosis. Therefore, inhibition of EGFR function is a rational cancer therapy approach. We have shown previously that 280 nm UV illumination of two cancer cell lines overexpressing EGFR could prevent phosphorylation of EGFR and of its downstream signalling molecules despite the presence of EGF. Our earlier studies demonstrated that UV illumination of aromatic residues in proteins leads to the disruption of nearby disulphide bridges. Since human EGFR is rich in disulphide bridges and aromatic residues, it is likely that structural changes can be induced upon UV excitation of its pool of aromatic residues (Trp, Tyr and Phe). Such changes may impair the correct binding of ligands to EGFR which will halt the process of tumor growth. In this paper we report structural changes induced by UV light on the extracellular domain of human EGFR. Steady state fluorescence spectroscopy and binding immunoassays were carried out. Our goal is to gain insight at the protein structure level that explains the way the new photonic cancer therapy works. This technology can be applicable to the treatment of various forms of cancer, alone or in combination with other therapies to improve treatment outcome.
Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high
signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have
developed an image processing methodology that may remove/reduce blur significantly for any type of microscopy. A
total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1
second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object
in each frame. We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore
be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size as
the individual image. We have also reconstructed the image using a 3 fold larger grid with a pixel size of 10 nm. The
resulting images reveal details at the diffraction limit. In principle we can only compensate for inter-image drift – thus
the drift that takes place during the acquisition time for the individual image is not corrected. We believe that our results
are of general applicability in microscopy and other types of imaging. A prerequisite for our method is the presence of a
trackable object in the image such as a cell nucleus.
We have developed a photonic technology that allows for precise immobilisation of proteins to sensor
surfaces. The technology secures spatially controlled molecular immobilisation since the coupling of each
molecule to a support surface can be limited to the focal point of the UV laser beam, with dimensions as
small as a few micrometers. The ultimate size of the immobilized spots is dependent on the focal area of
the UV beam. The technology involves light induced formation of free, reactive thiol groups in molecules
containing aromatic residues nearby disulphide bridges. It is not only limited to immobilizing molecules
according to conventional patterns like microarrays, as any bitmap motif can virtually be used a template
for patterning. We now show that molecules (proteins) can be immobilized on a surface with any arbitrary
pattern according to diffraction patterns of light. The pattern of photo-immobilized proteins reproduces the
diffraction pattern of light expected with the optical setup. Immobilising biomolecules according to
diffraction patterns of light will allow achievement of smaller patterns with higher resolution. The
flexibility of this new technology leads to any patterns of photo-imprinted molecules, with micrometer
resolution, thus being of relevance for present and future applications in nanotechnologies.
A combination of bioinformatics, biophysical, advanced laser studies and cell biology lead to the realization that laser-pulsed
UV light stops cancer growth and induces apoptosis. We have previously shown that laser-pulsed UV (LP-UV)
illumination of two different skin-derived cancer cell lines both over expressing the EGF receptor, lead to arrest of the
EGFR signaling pathway. We have investigated the available sequence and experimental 3D structures available in the
Protein Data Bank. The EGF receptor contains a Furin like cystein rich extracellular domain. The cystein content is
highly unusual, 25 disulphide bridges supports the 621 amino acid extracellular protein domain scaffold (1mb6.pdb).
In two cases a tryptophan is neighboring a cystein in the primary sequence, which in itself is a rare observation.
Aromatic residues is observed to be spatially close to all observed 25 disulphide bridges. The EGF receptor is often
overexpressed in cancers and other proliferative skin disorders, it might be possible to significantly reduce the
proliferative potential of these cells making them good targets for laser-pulsed UV-light treatment. The discovery that
UV light can be used to open disulphide bridges in proteins upon illumination of nearby aromatic amino acids was the
first step that lead to the hypothesis that UV light could modulate the structure and therefore the function of these key
receptor proteins. The observation that membrane receptors (EGFR) contained exactly the motifs that are sensitive to
UV light lead to the prediction that UV light could modify these receptors permanently and stop cancer proliferation.
We hereby show that the EGFR family of receptors has the necessary structural motifs that make this family of
proteins highly sensitive to UV light.
We present a new photonic technology and demonstrate that it allows for precise immobilisation of biomolecules to
sensor surfaces. The technology secures spatially controlled molecular immobilisation since immobilisation of each
molecule to a support surface can be limited to the focal point of the ultraviolet (UV) beam, as small as a few
micrometers. We can immobilise molecules according to any pattern, from classical microarrays to diffraction patterns
creating unique watermarking safety patterns. Given that suitable protein markers exists for all relevant diseases it is
entirely feasible to test for a range of disease indicators (antigens and other markers) in a single test. Few micrometer
spotsize allows for a virtually unlimited number of protein spots in a multipotent microarray. This new technology
produces radically new photonics based microarray sensing technology and watermarking and has clear potential for
biomedical, bioelectronic, surface chemistry, security markers production, nanotechnology and therapeutical
applications. We also show an in depth analyses of the immobilized patterns and of the microarrays with our software
Photonic induced immobilization of biosensor molecules is a novel technology that results in spatially oriented and
spatially localized covalent coupling of a large variety of biomolecules onto thiol reactive surfaces, e.g. thiolated glass,
quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of
disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids resulting in the formation of reactive
molecules that will form covalent bonds with thiol reactive surfaces. This new technology has the potential of replacing
present micro dispensing arraying technologies, where the size of the individual sensor spots are limited by the size of the
dispensed droplets. Using light-induced immobilization the spatial resolution is defined by the area of the sensor surface
that is illuminated by UV light and not by the physical size of the dispensed droplets of sensor molecules. This new
technology allows for dense packing of different biomolecules on a surface, allowing the creation of multi-potent
functionalized materials, such as biosensors with micrometer sized individual sensor spots. Thus, we have developed the
necessary technology for preparing large protein arrays of enzymes and fragments of antibodies, with micrometer
resolution, without the need for liquid micro dispensing.
We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either gold or thiolated quartz or silicium. The reaction mechanism behind the reported new technology involves light induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. The protein molecules in general retain their function. The size of the immobilization spot is determined by the dimension of the UV beam. In principle, the spot size may be as small as 1 micrometer or less. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated Image Processing Software has been developed for making quality assessment of the protein arrays. A multitude of important application areas such as drug carriers and drug delivery, bioelectronics, carbon nanotubes, nanoparticles as well as protein glue are discussed.
Systematic investigations of luminescence lifetimes of organic phenylene nanofibers are presented as a function of intrinsic parameters such as morphology or bleaching factor as well as extrinsic parameters such as substrate material, coating or excitation intensity. By varying either one of these parameters, the decay times of the electronic excitation can be varied. This should have a strong influence on the efficiency of nanolasing, which is observed by increasing the excitation intensity of a femtosecond pump laser. Lasing action starts at pump fluences as low as a few μJ/cm2 per pulse. In ensemble measurements, the number of lasing modes depends strongly on the density of contributing nanofibers. In spatially resolved measurements, the nonlinear optical response of individual nanofibers is investigated. This enables us to make a correlation between the morphological features of the nanofibers, as deduced from atomic-force microscopy, and their lasing properties.