Analytische Chemie
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Cancer is a leading cause of death worldwide, and its early detection and resultant treatment contributes significantly to patient recovery and survival. Detection is currently based on magnetic resonance imaging and computed tomography, methods that are expensive, while processing of the results is time consuming. There is a need for low-cost cancer-detection techniques that give conclusive results in the shortest time possible. Molecularly imprinted polymers (MIPs) targeting tumor markers on cancerous cells may provide a cheaper solution for cancer detection. Thin MIP layers immobilized on particle platforms are known to give faster response times and increased selectivity in comparison to bulk MIPs. It has been reported that a fluorescent monomer can be incorporated into the MIP layer, allowing for faster detection of the target group, thus significantly shortening the turn-around time for biopsies.
Changes in sialylation patterns of cell surface glycoproteins indicate malignancy. Here, we present the development of MIPs that target sialic acid-terminated glycoproteins (SA MIPs), prepared as a thin layer on a silica nanoparticle platform. A fluorescent monomer is incorporated into the MIP layer, and upon binding of the target group to the specific binding pockets in the MIP, the fluorescence signal is enhanced. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used for structural characterization. To validate the specificity, fluorescence changes of MIPs in the presence and absence of template are compared to their corresponding non-imprinted polymer particles (NIP). Initial binding experiments with tumor cells using fluorescence microscopy demonstrate that the presented technique shows promise as a cheaper alternative to current detection methods, while allowing for relatively shorter analysis of biopsy results.
Sialic acid (SA) is a cell surface glycan, which has a decisive role in many cell activities including differentiation, proliferation, and the immune response. The amount of SA has been found to correlate with cancer, with an upregulation on more aggressive cancers. Therefore, there is a great interest in developing methods for detection of SA on cancer cells. We are screening SA on cancer cell lines by using fluorescent molecularly imprinted polymers, SA-MIPs. Macrophages, which evolve from mono-cytes, are well known for their extraordinary ability to phagocytose foreign objects. This could lead to the hypothesis that the SA-MIPs can be recognized by macrophages as foreign object; thus leading to internalization and potentially degradation.
We have demonstrated that SA-MIPs can be detected after incubation with the RAW macrophage cells, with increasing fluorescence over time. The microscopy analysis shows that the RAW cells ingest the SA-MIP particles. This information is important when planning to use SA-MIPs in future in vivo applications.
Sialic acid (SA) is a cell surface glycan, which has a strong role in many cell activities including differentiation, proliferation, and the immune response. The amount of SA has been found to be correlated with cancer, with an upregulation on more aggressive cancers. Therefore, there is great interest in developing methods for detection of SA on cancer cells. We are screening SA on cancer cell lines by using fluorescent molecularly imprinted polymers, SA-MIPs.Macrophages, which evolve from mono-cytes, are well known for their extraordinary ability to phagocytose foreign objects. This could lead to the hypothesis that the SA-MIPs can be recognized by macrophages as foreign object; thus leading to internalization and potential degradation. We have discovered that SA-MIPs can be detected after incubation with the RAW macrophage cells, with increasing fluorescence over time. The microscopy analysis shows that the RAW cells ingest the SA-MIP particles. This information is important when planning to use SA-MIPs in future in vivo applications.
Cancer is modern medicine’s biggest challenge. It is now thought to be responsible for one in six deaths worldwide making early diagnosis, and treatment thereon, essential for better prognoses1. Protein phosphorylation is a post-translational modification of particular interest as a biomarker in the understanding of neurodegenerative diseases and a number of cancer pathways. There is therefore a need for robust, fast and low-cost techniques for the detection of these phosphorylations. Fluorescent molecularly imprinted polymers (MIPs) are a cheap and selective material for both the extraction and detection of a multitude of analytes. Often referred to as “plastic antibodies”, MIPs provide added robustness and chemical stability compared to their natural counterparts. Application of these fluorescent MIPs to a microfluidic lab-on-a-chip platform offers a fast, versatile method for the detection of biomarkers containing phosphorylated amino-acids such as phosphorylated tyrosine.
Here, we present a tuneable core-shell MIP system consisting of a polystyrene core, silica inner-shell and MIP outer-shell. The MIP outer-shell contains a urea-based fluorescent probe monomer co-polymerised into the polymer matrix that can detect phosphorylated-tyrosine based on a change in its optical properties. The phosphate group interacts with the fluorescent probe via hydrogen bonding interactions yielding a fluorescence enhancement in organic solvents. The fluorescent MIPs are to be applied to a microfluidic platform, for rapid extraction of the analyte from the aqueous sample phase and simple optical detection in the organic phase that contains the MIP microparticles.
Sialic acid (SA) is a cell surface glycan, which has been found to be upregulated on more aggressive cancers. Therefore, there is a great interest in developing methods for detection of SA on a great interest in developing methods for detection of SA o on cancer cells. We are screening SA on cell lines by fluorescent molecularly imprinted polymers, SA fluorescent molecularly imprinted polymers, SA fluorescent molecularly imprinted polymers, SA fluorescent molecularly imprinted polymers, SA fluorescent molecularly imprinted polymers, SA fluorescent molecularly imprinted polymers, SA -MIPs. Quantitative phase imaging (QPI) is a digital holographic technique. Various cellular parameters can be visualized and calculated from the particular hologram, including individual cell area, thickness, volume and population confluence and cell counts. The aim was to investigate the possible uptake of SA in macrophage cell lines in in vitro cell cultures and check if they affected the cell.
Glycosylation is a post-translational modification that is involved in the regulation of many biological processes. The glycosylation pattern in cancer cells differs from that in normal cells. One of the main alterations that has been observed in several cancers is the increase of sialic acids at the end of the glycan. The increase of sialic acids and other alterations affect development and progression of tumors and are found to play an important role in cancer invasiveness and metastasis. Molecularly imprinted polymers (MIPs) are synthetic recognition elements that show high selectivity and affinity for their targets. These polymers show promising applications in detection methods for cancer cells. In this study newly synthesized MIPs, labelled with a nitrobenzoxadiazole (NBD) fluorophore, are investigated for their specificity and utility in the detection of cancer cell-related sialic acids.
Cancer is the second leading cause of death globally according to the WHO 2018. Lung, prostate, colorectal, stomach and liver cancer are the most common types of cancer in men, while breast, colorectal, lung, cervix and thyroid cancer are the most common among women. Sialic acid (SA) plays an important role in a variety of biological processes in cells. Tumor cells express high levels of SA, which is often associated with poor prognosis due to increased invasive potential. In this study, we have performed a screening of SA-MIPs binding to different cancer cell lines. The overall aim is to use the SA-MIPs for detection of tumor cells, analyzed by flow cytometry and fluorescence microscopy. To confirm the levels of SA expression on the investigated cell lines, we started out by analyzing the binding of the lectins MAL I and SNA. The staining pattern of the two lectins binding to either α-2,3 or α-2,6 linkage SA, respectively, was compared with the staining pattern of the SA-MIPs. We show that the different cell types analyzed have a varying pattern of SA-MIP binding. The comparison with lectin binding will be further evaluated.
The reliable identification and quantification of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, noteworthy, to diagnose and treat diseases at an early developmental stage. Miniaturised sensing devices like microfluidic chips combined with “smart” detection chemistry, simple data assessment, processing and presentation are very attractive for benchtop use in clinical environments.
We developed novel synthetic probes targeting phosphorylated amino acids, based on core-shell microparticles consisting of a silica core coated with a molecularly imprinted polymer (MIP) shell. These “plastic antibodies” are extremely robust, resist denaturing solvents and elevated temperatures, can be reproducibly produced at low cost, and potentially overcome many of the practical problems in current bioanalytical detection strategies. The MIP layer contains a fluorescent probe monomer, binds selectively to phosphorylated tyrosine (pTyr) with a significant imprinting factor higher than 3.5 and responds with a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band toward the analyte.
In analogy to our previous work [4], the bead-based ratiometric detection scheme has also been successfully transferred to a microfluidic chip format to demonstrate its applicability to rapid assays. Such a miniaturised device could yield an automated pTyr measurement system in the future. The setup was built by coupling a modular microfluidic system [5] for amino acid functionalisation (Fmoc protection) and a multi-layer PDMS/Teflon/glass microfluidic chip [6] for buffering, extraction (micropillars co-flow extraction) and selective adsorption on the MIP core-shell particles.
A miniaturised optical assembly for low-light fluorescence measurements was also developed. Based on small opto-electronic parts and optical fibres, the emission from the MIP particles upon addition of pTyr concentrations from 0.5 – 200 μM could be monitored in real-time.
The reliable identification and quantification of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, noteworthy, to diagnose and treat diseases at an early developmental stage1. Miniaturised sensing devices like microfluidic chips combined with “smart” detection chemistry, simple data assessment, processing and presentation are very attractive for benchtop use in clinical environments.
We developed novel synthetic probes targeting phosphorylated amino acids, based on core-shell microparticles consisting of a silica core coated with a molecularly imprinted polymer (MIP) shell. These “plastic antibodies” are extremely robust, resist denaturing solvents and elevated temperatures, can be reproducibly produced at low cost, and potentially overcome many of the practical problems in current bioanalytical detection strategies. The MIP layer contains a fluorescent probe monomer, binds selectively to phosphorylated tyrosine (pY) with a significant imprinting factor higher than 3.5 and responds with a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band toward the analyte. In analogy to our previous work4, the bead-based ratiometric detection scheme has also been successfully transferred to a microfluidic chip format to demonstrate its applicability to rapid assays. Such a miniaturised device could yield an automated pY measurement system in the future. The setup was built by coupling a modular microfluidic system5 for amino acid functionalisation (Fmoc protection) and, as shown in Figure 1, a multi-layer PDMS/Teflon/glass microfluidic chip6 for buffering, extraction (micropillars co-flow extraction) and selective adsorption on the MIP core-shell particles.
A miniaturised optical assembly for low-light fluorescence measurements was also developed. Based on small opto-electronic parts and optical fibres, the emission from the MIP particles upon addition of pY concentrations from 0.5-200 μM could be monitored in real-time.
Cancer is a leading cause of death worldwide, and its early detection and resultant treatment contributes significantly to patient recovery and survival. Detection is currently based on magnetic resonance imaging and computed tomography, methods that are expensive, while processing of the results is time-consuming1. There is a need for low-cost cancer detection techniques that give conclusive results in the shortest time possible. When equipped with a reporter function, molecularly imprinted polymers (MIPs) targeting tumor markers on cancerous cells may provide a cheaper solution for imaging-based cancer detection. Thin MIP layers immobilized on particle platforms are ideal in this regard, because a fluorescence reporter can be integrated into the particle core and/or MIP shell and such core/shell nanoparticles show faster response times and increased selectivity in comparison to bulk MIPs.
Changes in sialylation patterns of cell surface glycans indicate malignancy2. Here, we present the development of MIPs that target sialic acid-terminated glycans (SA MIPs), prepared as a thin layer on a polystyrene core/silica shell nanoparticle platform. The MIP particles contain fluorescent emitters and can be applied in fluorescence imaging of malignant tumors. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) are used for structural characterization. Binding capacity of the MIPs to target glycans and competing sugars is also evaluated and compared to that of the corresponding non-imprinted polymer particles (NIP).