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83. Determination of petrophysical properties of sedimentary rocks by optical methodsDorota Korte, Dejan Kaukler, Mattia Fanetti, Humberto Cabrera, Efrain Daubronte, Mladen Franko, 2017, izvirni znanstveni članek Ključne besede: sedimentary rocks, beam deflection spectroscopy, grain size distribution, petrophysical
properties, chemical composition
Objavljeno v RUNG: 06.02.2017; Ogledov: 5339; Prenosov: 0
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84. Novel methods for detection of bioactive substances and their effects in organisms and in the environmentTatjana Radovanović Vukajlović, 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: 7224; Prenosov: 279
 Celotno besedilo (4,66 MB) |
85. Thermal and elastic characterization of glassy carbon thin films by photoacoustic measurementsDragan D. Markushev, J. Ordonez-Miranda, Mihajlo Rabasović, M. Chirtoc, D. M. Todorovic, S. E. Bialkowski, Dorota Korte, Mladen Franko, 2017, izvirni znanstveni članek Ključne besede: Thermal and elastic characterization, glassy
carbon, thin films, photoacoustic measurements
Objavljeno v RUNG: 23.01.2017; Ogledov: 5412; Prenosov: 0
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86. Determination of Trace Cr(VI) with Diphenylcarbazide by μFIA–Thermal Lens MicroscopyTatyana Gor’kova, Michail Proskurnina, Mingqiang Liu, Mladen Franko, 2016, izvirni znanstveni članek Opis: The optimum reaction parameters for the interaction of hexavalent chromium [Cr(VI)] with diphenylcarbazide in microfluidic
chips (μFIA) with thermal-lens microscopic detection were selected. The characteristic feature of the applied
flow scheme is the injection of the reagent into the stream containing the test metal, which enables in-field and
real-time monitoring of Cr(VI) simply by flowing the sample continuously through the microchip. The limit of detection
of Cr(VI) under the selected conditions (signal generating wavelength, 514.5 nm; excitation power, 100 mW; detection
position, 10 cm downstream from the mixing zone of the microchip; flow rate 10 μL min–1; injection volume,
1.4 μL) is 15 ng mL–1 (2.9 × 10–7 mol L–1). The linear range is 40 ng mL–1 – 10 μg mL–1 with a relative standard deviation
no higher than 10% in the concentration range 0.1–1 μg mL–1. The online monitoring by this scheme provides the
possibility of up to 360 analyses per hour.
Ključne besede: Photothermal lensing, thermal-lens microscopic detection, microfluidic applications, μFIA, hexavalent
chromium, diphenylcarbazide
Objavljeno v RUNG: 12.12.2016; Ogledov: 5504; Prenosov: 0
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87. One-pot synthesis of biocompatible silver nanoparticle composites from cellulose and keratin: characterization and antimicrobial activityChieu Ding Tran, Franja Prosenc, Mladen Franko, Gerald Benzi, 2016, izvirni znanstveni članek Opis: A novel, simple method was developed to synthesize biocompatible composites containing 50% cellulose (CEL) and 50% keratin (KER) and silver in the form of either ionic (Ag+) or Ag0 nanoparticle (Ag+NPs or Ag0NPs). In this method, butylmethylimmidazolium chloride ([BMIm+Cl-]), a simple ionic liquid, was used as the sole solvent and silver chloride was added to the [BMIm+Cl-] solution of [CEL+KER] during the dissolution process. The silver in the composites can be maintained as ionic silver (Ag+) or completely converted to metallic silver (Ag0) by reducing it with NaBH4. Results of spectroscopy (Fourier-transform infrared (FTIR), X-ray diffraction (XRD)) and imaging (scanning electron microscope (SEM)) measurements confirm that CEL and KER remain chemically intact and homogeneously distributed in the composites. Powder X-ray diffraction (XRD) and SEM results show that the silver in the [CEL+KER+Ag+] and [CEL+KER+Ag0] composites is homogeneously distributed throughout the composites in either Ag+ (in the form of Ag2O nanoparticles (NPs)) or Ag0NPs form with size of (9 ± 1) nm or (27 ± 2) nm, respectively. Both composites were found to exhibit excellent antibacterial activity against many bacteria including Escherichia coli, Staphylococus aureus, Pseudomonas aeruginosa, methicillin resistant Staphylococus aureus (MRSA), vancomycin resistant Enterococus faecalis (VRE). The antibacterial activity of both composites increases with the Ag+ or Ag0 content in the composites. More importantly, for the same bacteria and the same silver content, [CEL+KER+Ag0] composite exhibits relatively greater antimicrobial activity against bacteria compared to the corresponding [CEL+KER+Ag+] composite. Experimental results confirm that there was hardly any Ag0NPs release from the [CEL+KER+Ag0NPs] composite, and hence its antimicrobial activity and biocompatibility is due, not to any released Ag0NPs but rather entirely to the Ag0NPs embedded in the composite. Both Ag2ONPs or Ag0NPs were found to be toxic to human fibroblasts at higher concentration (>0.72 mmol), and that for the same silver content, [CEL+KER+Ag2ONPs] composite is relatively more toxic than [CEL+KER+Ag0NPs] composite. As expected, by lowering the Ag0NPs concentration to 0.48 mmol or less, the [CEL+KER+Ag0NPs] composite can be made biocompatible while still retaining its antimicrobial activity against bacteria such are E. coli, S. aureus, P. aeruginosa, MRSA, VRE. These results together with our previous finding that [CEL+KER] composites can be used for controlled delivery of drugs such as ciprofloxacin clearly indicate that the [CEL+KER+Ag0NPs] composite possess all required properties for successfully used as high performance dressing to treat chronic ulcerous infected wounds.
Ključne besede: Celulose, Keratin, Cmposites, Biocompatible, Ag nanoparticles, one-pot synthesis, Antibacterial, Antiviral
Objavljeno v RUNG: 28.11.2016; Ogledov: 6167; Prenosov: 0
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89. Applications of Thermal Lens Microscopy in Microfluidic SystemsMladen Franko, 2016, objavljeni povzetek znanstvenega prispevka na konferenci (vabljeno predavanje) Opis: Detection in microfluidic systems requires highly sensitive analytical methods, because of the very short optical interaction length, which is usually in the range of 100 μm or shorter. Furthermore, the amounts of analytes in detection volumes are extremely small (femto- or attomoles). Thermal lens spectrometry and particularly thermal lens microscopy (TLM) appear as techniques of choice for detection in microfluidic and lab-on-a-chip systems, since they enable measurements of absorbance’s or absorbance changes as low as 10-7. In addition to ultra-high sensitivity, TLM offers high spatial resolution (≈1 μm) and sufficient temporal resolution (ms range), which is required for studies of processes in microfluidic systems.
Recent development of TLM theory and instrumentation lead to experimental confirmations of the effects of microfluidic flows on the TLM signal, which affects the sensitivity. On the other hand, these observations have enabled optimization of TLM instruments [1]. As a result of these advancements, applications of TLM were extended from simple laminar flows [2], to highly complex systems such as Tylor-type flows, where TLM detection provided data for description of diffusion processes in n-octane/methanol binary liquid systems [3]. The major streamline of TLM applications was however focused on the development of vanguard analytical systems [4], which are needed in various fields of chemical analysis, including food safety and quality control, environmental monitoring as well as biomedical research and diagnostics. Such systems are used as sample screening systems (sample filters or selectors) when the information is needed quickly to make immediate decisions in relation to the analytical problem. They provide simplicity (e.g. little or no sample pre-treatment), low cost, rapid and reliable response, and frequently give just binary responses. However, their major weakness is low metrological quality of results. Therefore, uncertainties of up to 5–15% are usually accepted as a toll for rapidity and simplicity, which are essential even though in contradiction with conventional analytical concepts. With the objective of developing new vanguard analytical systems, a relevant goal is to exploit the advantages offered by microfluidic lab-on-a-chip systems on one hand, and TLM detection on the other. In such combinations, the FIA approach simplifies sample handling (e.g. volume measurements) and transport to the detector, while microfluidic lab-on-a-chip technology can facilitate and speed up processes including colorimetric reactions, antigen–antibody or enzyme–substrate interactions in bioanalytical systems, and even extraction and preconcentration steps by introducing continuous flow processing and micro unit operations in chemical analysis [2]. High sensitivity of TLM in such systems offers low limits of detection, which also contribute to low uncertainties that are typically below 10%. An important advantage of microfluidic systems lies in the fact that small dimensions of such systems, which consist of capillaries and micro reactors with dimensions about 10 to 100 μm, significantly reduce the molecular diffusion time, which is inversely proportional to the second power of distance. For example, the time required for completion of an ELISA immunoassay for NGAL a biomarker of acute kidney failure was reduced from four hours to only 30 mins. [5, 6] when transferring the assay into a microfluidic system, while maintaining or even improving the sensitivity. Even more evident improvement in sample throughput (reduction of analysis time from 10 hours to 30 minutes) was achieved for determination of antibodies for human papilloma virus (anti L1 HPV 16) in blood plasma, after immobilizing adequate pseudovirions as antigens on magnetic nanobeads [6]. Other health-related applications include detection of toxins, such as microcystin, or carcinogenic substances such as Cr(VI), which offers improved limits of detection as compared to spectrophotometry as well as sample throughput, which can reach 20 samples/min. [7].
[1] M. Liu and M. Franko, Crit. Rev. Anal. Chem. 44, 328-353 (2014).
[2] T. Kitamori, M. Tokeshi, A. Hibara, and K. Sato, Anal. Chem. 76, 52A-60A (2004).
[3] M. Lubej, U. Novak, M. Liu, M. Martelanc, M. Franko and I. Plazl, Lab Chip (2015) DOI:10.1039/c4lc01460j.
[4] M. Valcárcel and B. Lendl. Trends Anal. Chem. 23, 527-534 (2004).
[5] T. Radovanović, M. Liu, P. Likar, M. Klemenc and M. Franko, Int. J. Thermophys. (2014) DOI:10.1007/s10765-014-1699-9.
[6] T. Radovanović, Dissertation, University of Nova Gorica (2016).
[7] M. Franko, M. Liu, A. Boškin, A. Delneri, and M.A. Proskurnin, Anal. Sci. 32, 23-30 (2016).
Ključne besede: Spektrometrija TLM, mikrofluidni sistemi, biomarkerji, alergeni, pesticidi, acetilholinesteraza
Objavljeno v RUNG: 07.11.2016; Ogledov: 6650; Prenosov: 45
Celotno besedilo (42,25 MB) |
90. Optimized frequency dependent photothermal beam deflection spectroscopyMladen Franko, Dorota Korte, Humberto Cabrera, 2016, izvirni znanstveni članek Opis: In the paper the optimization of the experimental setup for photothermal beam deflection spectrometry is performed by analyzing the influence of its geometrical parameters (detector and sample position, probe beam radius and its waist position etc) on the detected signal. Furthermore,
the effects of fluid’s thermo-optical properties, for optimized geometrical configuration, on the
measurement sensitivity and uncertainty determination of sample thermal properties is also studied.
The examined sample is a recently developed CuFeInTe3 material. Results show, that it is a complex problem to choose the proper geometrical configuration as well as
sensing fluid to enhance the sensitivity of the method. A signal enhancement is observed at low modulation frequencies by placing the sample in acetonitrile (ACN), while at high modulation frequencies the sensitivity is higher for measurements made in air. For both, detection in air and
acetonitrile the determination of CuFeInTe3 thermal properties is performed. The determined values of thermal diffusivity and thermal conductivity are (0.048 ± 0.002) × 10−4 m2 s−1 and 4.6 ± 0.2 W m−1 K−1 and (0.056 ± 0.005) × 10−4 m2 s−1 and 4.8 ± 0.4 W m−1 K−1 for ACN and air,
respectively. It is seen, that the determined values agree well within the range of their measurement uncertainties for both cases, although the measurement uncertainty is two times lower for the measurements in ACN providing more accurate results. The analysis is performed by the use of
recently developed theoretical description based on the complex geometrical optics. It is also shown, how the presented work fits into the current status of photothermal beam deflection spectroscopy.
Ključne besede: Optitermi, na spektrometrija, toplotna difuzivnost, toplotna prevodnost, polprevodniki, vpliv topila
Objavljeno v RUNG: 07.11.2016; Ogledov: 6003; Prenosov: 0
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