FluoBolt™ Noggin

Metal Enhanced Fluorescence (MEF) offers the possibility to increase the analytical sensitivity of systems based on fluorescence detection dramatically. MEF is based on the fact that excitation light interacts with the electrons of metal nano structures thus generating very high electromagnetic fields (Localized Surface Plasmons, LSPs) Therefore, such structures are also called “plasmonic structures” and the combination of (e.g. polymeric) support and structure is known as “plasmonic substrate”. These LSPs lead to an increase in emission output of fluorescent molecules (e.g. fluorescently labeled antibodies) when bound to surfaces with suitable nano metal structures that enhances the signal dramatically. FIANOSTICS has developed a new plasmonic enhanced immunoassay platform in cooperation with Sony DADC BioSciences (now STRATEC Consumables since July 1st 2016), that allows up to 300 fold gains of sensitivity. This platform is fully compatible to standard laboratory methodology using 96 well microtiter plate format and assays based on this technology can be run on any standard fluorescence microplate reader. Its unique features enable fluorescence immunoassays with highest sensitivity and without washing steps.

The quantification of serum biomarkers plays an important role in medical science. Very often their concentrations in human blood are very low, which creates a permanent need for raising the sensitivity of the assay methods used.

Metal-enhanced fluorescence (MEF) is a promising technology to deliver the required highly sensitive tests. It is well established, that MEF results from localized surface plasmons (LSPs) generated by the interaction of the excitation light with nanometer-sized noble metal structures, that dramatically increases the quantum yield of fluorescent molecules thus leading to highly sensitive detection systems (e.g. for fluorescent immunoassays).

In Fig. 1 a typical MEF measurement setup is sketched.

Fig. 1 Schematic representation of metal-enhanced fluorescence and measurement setup. The fluorescence of the near surface fluorophore is drastically enhanced in contrast to the fluorophore in the bulk solution.

Design and manufacturing reproducibility of the required metal structures is the key for a) optimizing the enhancement effect and b) reliability of the assay system they are used for.

This is why commercial application of this technology has failed so far.

STRATEC Consumables (former Sony DADC BioSciences) and FIANOSTICS successfully solved these problems by using highly reproducible nano-structuring technologies (patent pending), originally developed for Blu-Ray and DVD manufacturing. The manufactured MEF substrates were successfully used in the detection of fluorescence labelled antibodies and its application in fluorescence labelled immunoassays (MEF-FIAs) for biomarkers in human serum was demonstrated. This new MEF platform is compatible to any given assay format (e.g. microtiter plate, microfluidic chips, arrays or lateral flow devices).

Manufacturing method

The master structures were manufactured by laser lithography, copied into a Ni stamp by electroforming and replicated in a disc format by a vario-thermal injection compression process. For the polymer a medical grade of cycle olefin was used. The disc were selectively coated with Ag-spots by a DC sputtering process and protected by a surface adhesive film to avoid degradation of the Ag layer. Subsequently slide in microscope format were milled out and laser-welded onto 96-well microtiter plates.

Design of MEF substrate

The MEF substrate are polymer slides with a nanometer sized hexagonal arrays of well-like structures on the surface. The wells typically have diameters below 0.5μm, an aspect ratio of about 2 and a micron pitch. They are coated homogenously with several nanometer of Ag.

By adjusting the geometry of the wells-array and the thickness of the Ag-layer the MEF effect can be optimized.

Fig. 2 (a) SEM image of an uncoated MEF substrate; (b) AFM measurement of the MEF substrate coated with several nanometer of silver

Manufacturing method

The master structures were manufactured by laser lithography, copied into a Ni stamp by electroforming and replicated in a disc format by a vario-thermal injection compression process. For the polymer a medical grade of cycle olefin was used. The disc were selectively coated with Ag-spots by a DC sputtering process and protected by a surface adhesive film to avoid degradation of the Ag layer. Subsequently slide in microscope format were milled out and laser-welded onto 96-well microtiter plates.

Overview

Application

To demonstrate the functionality of the MEF substrate, Cy5 labeled antibody (goat anti-rabbit) were adsorbed to the MEF substrate and measured with a commercial fluorescence microplate reader. The measurements were done in bottom configuration, meaning that excitation and emission through the bottom of the plate. Depending on the fluorophore, antibody concentration in the sub-picomolar range can be clearly detected (see Fig. 3).

Fig. 3 Enhanced fluorescence signal of a Cy5 labeled antibody adsorbed a MEF substrate

MEF-FIA for NOGGIN

The developed MEF-platform was used to develop fluorescence immunoassays for Noggin and Asporin, two regulatory molecules which have a major impact on bone metabolism. Noggin acts as bone morphogenetic protein inhibitor and as such has an impact on bone/cartilage regeneration, limb development and fracture repair.

Summary and Outlook

The functionality of the MEF substrates could be successfully demonstrated in fluorescence labelled immunoassays for biomarkers in human serum. An enhancement of several orders of magnitudes could be achieved, allowing the quantification of biomolecules down to low picomolar and sub-picomolar concentrations. Preliminary tests have shown, that the MEF substrates can be also used for other fields like SERS (Surface Enhanced Raman Spectroscopy).

Additional material supplied with the kit

• 2 self-adhesive plastic films
• Protocol sheet
• Instruction manual for use
• Desiccant bag

Material and equipment required but not supplied

• Precision pipettes calibrated to deliver 10μl, 20μl, 50μl, 200μl, 500μl and disposable tips
• Distilled or deionized water
• Plate washer, multichannel pipette or manifold dispenser for washing
• Refrigerator with 4°C (2-8°C)
• Fluorescence microplate reader
• Graph paper or software for calculation of results

Reagents and sample preparation

All reagents of the kit are stable at 4°C (2-8°C) until expiry date stated on the label of each reagent.

Sample preparation

Collect venous blood samples by using standardized blood collection tubes for serum or plasma. We recommend performing plasma or serum separation by centrifugation as soon as possible, e.g. 10 min at 2000 x g, preferably at 4°C (2-8°C). The acquired plasma or serum samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C or lower. Do not freeze-thaw samples more than 4 times.

Lipemic or hemolysed samples may give erroneous results. Samples should be mixed well before assaying.

Reagent preparation

Add 250μl of distilled or deionized water to the lyophilized DS (Standards) and DC (Controls). Leave at room temperature (18-26°C) for 20min. Reconstituted DS and DC are stable at 25°C or lower until expiry date stated on the label. Reconstituted DS and DC can undergo 4 freeze thaw cycles.

Bring WP (Wash buffer) concentrate (20x) to room temperature. Make sure that the solution is clear and without any salt precipitates before further dilution. Dilute the WP to working strength by adding the appropriate amount of distilled or deionized water, e.g. 25ml of WP + 475ml water, prior to use in the assay. Undiluted WP is stable at 4°C (2-8°C) until expiry date on the label. Diluted WP is stable at 4°C (2-8°C) up to one month. Only use diluted WP in the assay.

All reagents and samples must be at room temperature (18-26°C) before use in the assay.

Mark position for standards, controls and samples on the protocol sheet. We recommend running samples and standards in duplicates.

Take the plasmonic enhanced microtiter plate out of the aluminum bag. Avoid touching the bottom of the plate with bare hands, because reading without washing is performed through the well bottom.

Seal all wells that will not be used in the following assay run with the accompanying adhesive film (cut to fit!).

In standard format, the kit is delivered with an AlexaFluor680 labeled detection antibody (DAA) because serum background fluorescence is minimal within this wavelength range. Therefore if your reader is equipped with monochromatic optics, please set Excitation/Emission to 679/702nm or if you are using an optical filter based reader, select a suitable filter pair (e.g. 670/720nm). On request the kit can also be delivered with FITC, Cy3 or Cy5 (Ex/Em = 495/518nm, 550/570nm or 650/670nm) labeled detection antibody.

1)      Add 25μl of the selected fluorescence labeled detection antibody (DAF or DA3 or DA5 or DAA) to all wells required. Swirl gently.

2)      Add 20μl of standard, control or sample to the wells according to the marked positions on the protocol sheet, swirl gently, cover tightly with the delivered adhesive film and incubate for 4 hours at 37°C or overnight at room temperature (18-26°C) in the dark.

3a)    If your reader allows bottom reading, read the plate without any further processing at the Ex/Em wavelength fitting to the delivered detection antibody (495/518 nm for DAF, 550/570nm for DA3, 650/670nm for DA5, 679/702nm for DAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0pM and the 250pM Noggin standard. Samples with signals exceeding the signal of the highest standard must be rerun with an appropriate dilution using sample diluent (DD).

3b)    If your reader has no bottom read option or if you want to store the plate for documentation purposes, discard or aspirate the content of the wells and wash 3x with diluted wash buffer. Use a minimum of 200μl wash buffer per well. After the final wash, remove remaining fluid by strongly tapping the plate against a paper towel.

Read the plate in top configuration without any further processing at the Ex/Em wavelength fitting to the chosen detection antibody (495/518nm for DAF, 550/570nm for DA3, 650/670nm for DA5, 679/702nm for DAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signals of the 0pM and the 250pM Noggin standard. Samples with signals exceeding the signal of the highest standard must be rerun with appropriate dilution using sample diluent (DD).

Hint: Quality of bottom reading (3a) may vary between microplate readers. For first time users we suggest to perform the washing step and follow protocol 3b.

4)      Store the plate with desiccant at 4°C (2-8°C) in the aluminum bag. Unused wells are stable until expiry date stated on the label. Fluorescence signals of standards, controls and samples remain detectable for at least two month, depending on signal intensity achieved.

Calculation of Results

Subtract the fluorescence intensity of the 0pM standard from all other standards, samples and controls. Construct a calibration curve from the fluorescence units (F.U.) of the standards using commercially available software or graph paper. Read sample and control concentrations from this standard curve. The assay was evaluated with 4PL algorithm. Different curve fitting methods need to be evaluated by the user.

The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit lot at production date.

Fluorescence intensity obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life.

However, this does not affect validity of results as long as the supplied kit controls read according to specifications (target ranges see labels).

Intra assay

4 samples of known concentrations were tested 3 times within 1 assay run

Inter assay

4 samples of known concentrations were tested in duplicates within 3 different assay runs

Spike/Recovery

The recovery of Noggin was evaluated by adding 2 known concentrations of human recombinant Noggin to 4 different human serum samples. Mean recovery was 113% (see table below).

Linearity

3 human serum samples were spiked with recombinant Noggin and diluted 1+1 and 1+2 with the sample diluent (DD) supplied with the kit. Mean linearity was 113% (see table below).

Analyte Specificity

This assay detects free, bioactive human Noggin, that is not bound to BMPs. Specificity was tested by titration of the serum based kit-calibrators with human recombinant BMP-2, -4 and -7.

As can be seen from the chart, addition of BMPs reduces the signal of the calibrator, which demonstrates that the assay only detects free, bioactive Noggin.

Species Specificity

This assay has been exclusively tested with human serum and plasma samples. No data are available for other species. However human Noggin shares 99%, 99%, 98%, 97% and 89% aa sequence identity with mouse, rat, bovine, equine and chicken Noggin. So using this assay for those species may be possible, but must be evaluated by the user.

Noggin is a secreted homodimeric glycoprotein that is an antagonist of bone morphogenetic proteins (BMPs). Human Noggin cDNA encodes a 232 amino acid (aa) precursor protein; cleavage of a 19 aa signal peptide generates the 213 aa mature protein which contains an N terminal acidic region, a central basic heparin binding segment and a C terminal cysteine knot structure. Secreted Noggin probably remains close to the cell surface due to its binding of heparin containing proteoglycans. Noggin is very highly conserved among vertebrates.

Noggin predominantly binds BMP–4 and BMP–2 and antagonizes their bioactivities by preventing binding to both type I and type II receptors. During embryogenesis Noggin is a crucial factor for regulation of various developmental processes like formation of neural tubes, cardiomyocyte growth and patterning as well as skeletal development where Noggin prevents chondrocyte hyperplasia, thus allowing proper formation of joints. Mutations within the cysteine–knot region of human Noggin are linked to multiple types of skeletal dysplasias that result in apical joint fusions. Noggin is expressed in defined areas of the adult central nervous system and peripheral tissues such as lung, skeletal muscle and skin.

In adults Noggin may be associated with dissemination of tumor cells to bone, ankylosing spondylitis or pulmonary arterial hypertension (PAH). Its value as biomarker remains to be established yet.

Research Applications:

Ankylosing Spondylitis (Morbus Bechterew)

Cell culture experiments have shown  that the imbalance of Noggin and BMP-2 levels may be the underlying pathophysiological cause for the imbalance between bone morphogenetic protein 2 and Noggin induces abnormal osteogenic differentiation of mesenchymal stem cells in Ankylosing Spondylitis.

Osteolytic Bone Metastases

It has been shown, that osteolytic carcinoma derived cell lines constitutively secret Noggin. This gives rise to research whether Noggin plays a crucial role in inhibiting bone repair by osteoblasts, thus facilitating the generation of osteolytic bone metastasis.

Pulmonary Arterial Hypertension

Aside from its role in bone metabolism Noggin has a profound impact on the proliferation of arterial smooth muscle (ASMC) cells. Researchers may study how a reduction of Noggin under hypoxia may lead to significant increase of ASMC proliferation resulting in a narrowing of the vessel and thus creation of hypertonia.

Nonalcoholic Fatty Liver Disease (NAFLD)

Nonalcoholic fatty liver disease (NAFLD) which may advance to cirrhosis and hepatocellular carcinoma is among the most common chronic liver diseases and affects globally approximately one fourth to one third of the general population worldwide. However, so far no biomarkers existed for non-invasive studies of this disease. A recent study using the high sensitivity FluoBolt™ Noggin assay could demonstrate, that the serum concentration of this biomarker is significantly reduced in subjects with NAFLD and approaches normal levels of healthy persons under vitamin E and spironolactone treatment. At present the FluoBolt™ Noggin assay is the only tool available for such investigations in subjects with NAFLD.