Novel methods for detection of bioactive substances and their effects in organisms and in the environmentTatjana Radovanović
, doktorska disertacija
Opis: Since the concentration of bioactive substances and infectious agents in organisms and in the environment are low highly sensitive techniques such as: chromatography technology coupled with mass spectrometry (GC/MS, LC/MS and LC–MS/MS) and transmission electron microscopy (TEM) are needed for their detection. These techniques are highly sensitive, but time consuming, requiring use of expensive apparatus and large quantities of reagents and organic solvents which are harmful for the environment. Because there is a growing need for analysis of a large number of environmental samples it is necessary to develop new, so called vanguard methods that enable rapid and reliable screening of large numbers of samples in the shortest possible time. Analysis with such “screening” methods are often less accurate or even semi-quantitative, but nevertheless allow reliable identification of nonproblematic samples and in practice they limit the use of demanding classical analytical methods to only a few percent of all the samples.
Therefore, general objectives of the thesis were development of novel methods for sensitive, fast and cost effective detection of pharmaceuticals, viruses and viral particles in waters and biological fluids and for detection of their effects in organisms.
Novel methods were based on the combination of TLS (Thermal Lens Spectrometry), microfluidics and immunological methods such as ELISA. TLS as highly sensitive technique (allowing detection of absorbances of less than 10-6) coupled with microfluidic technology allows detection of very low analyte concentration, shorter time for analysis, higher sample throughput and low consumption of reagents. In such combination microfluidic technology can simplify or speed up antigen-antibody or enzyme-substrate interactions in bioanalytical systems. Decisive advantage of microfluidic systems lies in the fact that small dimensions of such systems, composed of capillaries and micro-reactors with dimensions from about 10 to 100 µm, significantly reduce diffusion time, which is inversely proportional to second power of distance.
However, highly sensitive detection techniques are needed in microfludic systems, because the amounts of analytes in detection volumes are generally small and optical interaction lengths are two to three orders of magnitude shorter than in conventional spectrometric techniques.
By combining microscopic TLS (TLM) with microfluidic technique it is possible to reach very low limits of detection and at the same time shorten ELISA analysis time from 20 h to 20 minutes as was described before in the literature for detection of BNP (brain natriuretic peptide). TLM furthermore allows measurements of extremely small volumes (sub-microliter) as well as fast signal response (milliseconds).
In this Dissertation specific goals were the development of new methods for detection of selected bioactive substances and infectious agents:
-iodinated contrast agents
-NGAL (neutrophil gelatinase associated lipocalin) as a new biomarker of contrast induced nephropathy (CIN)
-antibodies against human papilloma viruses (HPV) viruses and HPV-16 pseudovirions.
For the development of new method for detection of iodinated contrast agents chemical degradation of iodinated contrast agents was investigated as well, as a potential method for their removal from waste water.
For the determination of NGAL, a commercially available ELISA kit was used as the basis for method development. In the initial experiments the final product of the reaction of substrate with enzyme HRP (horse radish peroxidase) was transferred from microtiter plate into a microfluidic system, which served just for the sample transport to TLM detector on microchip. With comparable speed analysis we achieved LOD of 1.4 pg/mL which is 7 times lower in comparison to commercial ELISA test (LOD=10 pg/mL). For further development of the method for detection of NGAL with µFIA-TLM magnetic nanobeads were used as a solid support for primary antibodies of ELISA assay. By applying appropriate magnetic field the antibodies were kept in microfluidic system, which also enabled binding of NGAL, secondary antibodies and reaction of substrate with HRP. Developed method for NGAL detection with LOD of 2.3 pg/mL compares favorably with LOD for commercial ELISA tests (10 pg/mL) in standard microtiter plates and significantly reduces the analysis time. TLM in combination with microchip for NGAL detection reduces the duration of individual incubation steps (from one hour to 5 minutes) and at the same time shortens total analysis time from four hours for commercial ELISA test to 35 minutes allowing higher sample throughput. Analysis of real blood samples was also performed and it has shown good agreement between NGAL concentrations measured by magnetic nanobeads based µFIA-TLM with the concentrations measured by a commercial ELISA test. Such short analysis time of analysis and possible further optimizations are opening new possibilities for application of µFIA-TLM in medical diagnostics and clinical research. By using appropriate antibodies the method for developed NGAL detection could be easily adopted for detection of different pharmaceuticals or pollutants in environmental samples.
We have also developed a magnetic nanobeads based ELISA assay for detection of anti-HPV-16 L1 antibodies in the sera of HPV-16 infected women. To ensure the selectivity, HPV-16 pseudovirions were used as an antigen for anti-HPV-16 L1 antibodies, which were detected with secondary HRP labeled antibodies. Initially the ELISA assay for antibodies against HPV pseudovirions was performed on a microtiter plate and an LOD of 3.8 ng/mL was achieved by measurement on a microtiter plate reader. When performing a µFIA-TLM measurement of the final ELISA solution the LOD was reduced to 0.9 ng/mL. Similar to the method for NGAL detection based on magnetic nanobeads, these were used as solid support for HPV pseudovirions and after carrying out all the incubation steps of the ELISA test in microfluidic chip the final product of the reaction of substrate with HRP was detected on TLM. With magnetic nanobeads based ELISA assay with µFIA-TLM for measurement of antibodies against PsVs of HPV-16 virus an LOD of 0.6 ng/mL was achieved, which is six times lower in comparison to classic ELISA on microtiter plate. Furthermore, the analysis time was reduced from ten hours to 30 minutes. The novel method was successfully validated by analysis of real sera samples from women who were previously diagnosed for infection with HPV-16 virus.
For determination of iodinated MRI contrast agents we developed a new method based on the measurement of concentration of released iodide which allows indirectly semi-quantitative detection of concentration of iodinated contrast agents. For iodide release from parent molecule of contrast agent we applied a chemical reaction with Cu2+ ions in the presence of H2O2. Released iodide was first oxidized into iodine and then extracted into chloroform. Contrast agents degradation reaction showed 70 % of efficiency for removal of iomeprol, taking into account the 60 % overall efficiency of iodide oxidation and extraction. The extract was injected into microfluidic chip and iodine concentration was determined with TLM. Chloroform as organic solvent with low thermal conductivity and high temperature coefficient of refractive index is a good choice for TLM measurement due to high TLS enhancement factor, which theoretically provides 40 times higher sensitivity of TLM measurements as compared to water and a four time improvement in sensitivity for each milliwatts of excitation power, when compared to spectrophotometry. The developed µFIA-TLM method for indirect determination of contrast agents based on detection of iodine provides around 60 times lower LOD, with low reagent and sample consumption in comparison to spectrophotometry. The LOD of 18 ng/mL for iomeprol achieved with TLM is 16 times lower in comparison to LOD of 294 ng/mL for iomeprol determination with HPLC. In comparison to LOD of 133 ng/mL for iomeprol achieved with detection of released iodide by ion chromatography, µFIA-TLM enables around 7 times lower LOD.
HPLC and HPLC/MS analysis showed that the parent compounds is completely removed after 120 min. of chemical degradation and that different degradation products are formed by cleavage of one or two iodine atoms. By this we have shown that the applied chemical degradation is efficient for removal of iomeprol and could be applied for treatment of waste waters after further optimization and reduction of reaction time.
New analytical methods developed within this work provide limits of detection for the selected compounds which are significantly lower (up to 60 times) in comparison to conventional analytical techniques based on transmission mode measurements. At the same time the new methods allows shorter time of analysis and higher sample throughput for the purpose of fast screening methods. Magnetic nanobeads based µFIA-TLM ELISA assays developed within this work offer several advantages in comparison to commercial ELISA tests on microtiter plates such as: higher surface for antibody binding, lower reagent consumption, and shorter analysis time.
Although the TLS technique didn’t reach appropriate stage of development and applicability for routine chemical analysis, improved methods for detection of NGAL and antibodies against HPV viruses could be applied for clinical studies and development of commercial tests for detection of viruses or other bioactive substances, which are needed for diagnostic purposes in hospitals.
Ključne besede: ELISA, NGAL, PsVs, kontrastna sredstva, TLM
Objavljeno v RUNG: 02.02.2017; Ogledov: 5208; Prenosov: 272
Celotno besedilo (4,66 MB)