44 results on '"Farka Z"'
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2. The pros and cons of nucleic acid-amplified immunoassays-a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification.
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Dekaliuk M, Farka Z, and Hildebrandt N
- Subjects
- Immunoassay methods, Humans, Male, Fluorescence Resonance Energy Transfer methods, Prostate-Specific Antigen blood, Prostate-Specific Antigen analysis, Nucleic Acid Amplification Techniques methods, Limit of Detection
- Abstract
Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs)., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).) more...
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- 2024
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3. Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen.
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Zikmundová E, Sklenárová D, Kočí E, Zatloukalová T, Bačová T, Makhneva E, Holub D, Macháčová E, Kopřivová H, Vytisková K, Pořízka P, Novotný K, Skládal P, Farka Z, and Kaiser J
- Subjects
- Humans, Immunoassay methods, Spectrum Analysis methods, Yttrium chemistry, Yttrium radiation effects, Male, Microspheres, Prostate-Specific Antigen analysis, Prostate-Specific Antigen immunology, Prostate-Specific Antigen blood, Lasers, Limit of Detection
- Abstract
Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determination of cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0 pg·mL
-1 , representing two orders of magnitude improvement compared with equivalent microtiter plate-based assay (LOD of 460 pg·mL-1 ). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.) more...- Published
- 2024
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4. Luminescent photon-upconversion nanoparticles with advanced functionalization for smart sensing and imaging.
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Skládal P and Farka Z
- Subjects
- Humans, Fluorescent Dyes chemistry, Animals, Neoplasms diagnostic imaging, Neoplasms diagnosis, Optical Imaging, Nanoparticles chemistry, Photons
- Abstract
Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.) more...
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- 2024
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5. Hyaluronic Acid Nanoparticles with Parameters Required for In Vivo Applications: From Synthesis to Parametrization.
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Matějková N, Korecká L, Šálek P, Kočková O, Pavlova E, Kašparová J, Obořilová R, Farka Z, Frolich K, Adam M, Carrillo A, Šinkorová Z, and Bílková Z
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- Humans, Biocompatible Materials chemistry, Biocompatible Materials chemical synthesis, Animals, Particle Size, Hyaluronan Receptors metabolism, Hyaluronic Acid chemistry, Nanoparticles chemistry
- Abstract
Hyaluronic acid is an excellent biocompatible material for in vivo applications. Its ability to bind CD44, a cell receptor involved in numerous biological processes, predetermines HA-based nanomaterials as unique carrier for therapeutic and theranostic applications. Although numerous methods for the synthesis of hyaluronic acid nanoparticles (HANPs) are available today, their low reproducibility and wide size distribution hinder the precise assessment of the effect on the organism. A robust and reproducible approach for producing HANPs that meet strict criteria for in vivo applications ( e.g. , to lung parenchyma) remains challenging. We designed and evaluated four protocols for the preparation of HANPs with those required parameters. The HA molecule was cross-linked by novel combinations of carbodiimide, and four different amine-containing compounds resulted in monodisperse HANPs with a low polydispersity index. By a complex postsynthetic characterization, we confirmed that the prepared HANPs meet the criteria for inhaled therapeutic delivery and other in vivo applications. more...
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- 2024
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6. Changes in the fatty acid profiles and health indexes of bovine colostrum during the first days of lactation and their impact on human health.
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Farková V, Křížová L, Dadáková K, Farka Z, Mascrez S, Eggermont D, Purcaro G, and Kašparovský T
- Abstract
Our objective was to analyze the changes in fatty acid (FA) profiles of bovine colostrum and immature milk during the first four days of lactation and assess their potential impact on human health. Colostrum and immature milk samples were collected from Czech Fleckvieh cows during their first to third lactation and the FA profiles were analyzed using multidimensional gas chromatography with a vacuum ultraviolet detector (GC×GC-VUV). The colostrum of primiparous cows contained lower levels of medium-chain and saturated fatty acids, and higher levels of mono- and unsaturated fatty acids compared to that of multiparous cows. The atherogenic and thrombogenicity indexes, as well as the hypocholesterolemic-to-hypercholesterolemic fatty acid ratio, were more favourable in primiparous cows. This makes colostrum fat an attractive product for human nutrition. To obtain the maximum health benefits, we recommend collecting and processing the colostrum of primiparous cows and immature milk at the end of the milk transition separately., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.) more...
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- 2024
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7. Single-molecule microfluidic assay for prostate-specific antigen based on magnetic beads and upconversion nanoparticles.
- Author
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Sklenárová D, Hlaváček A, Křivánková J, Brandmeier JC, Weisová J, Řiháček M, Gorris HH, Skládal P, and Farka Z
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- Humans, Microfluidic Analytical Techniques instrumentation, Male, Nanoparticles chemistry, Immunoassay instrumentation, Immunoassay methods, Limit of Detection, Prostatic Neoplasms diagnosis, Prostatic Neoplasms blood, Prostate-Specific Antigen analysis, Prostate-Specific Antigen blood
- Abstract
Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL
-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%). more...- Published
- 2024
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8. Upconversion Nanoparticle-Based Dot-Blot Immunoassay for Quantitative Biomarker Detection.
- Author
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Máčala J, Makhneva E, Hlaváček A, Kopecký M, Gorris HH, Skládal P, and Farka Z
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- Humans, Immunoassay methods, Quantum Dots chemistry, Serum Albumin, Human analysis, Serum Albumin, Human urine, Male, Nanoparticles chemistry, Limit of Detection, Prostate-Specific Antigen blood, Prostate-Specific Antigen analysis, Biomarkers blood, Biomarkers urine, Biomarkers analysis
- Abstract
Dot-blot immunoassays are widely used for the user-friendly detection of clinical biomarkers. However, the majority of dot-blot assays have only limited sensitivity and are only used for qualitative or semiquantitative analysis. To overcome this limitation, we have employed labels based on photon-upconversion nanoparticles (UCNPs) that exhibit anti-Stokes luminescence and can be detected without optical background interference. First, the dot-blot immunoassay on a nitrocellulose membrane was optimized for the quantitative analysis of human serum albumin (HSA), resulting in a limit of detection (LOD) of 0.19 ng/mL and a signal-to-background ratio ( S / B ) of 722. Commercial quantum dots were used as a reference label, reaching the LOD of 4.32 ng/mL and the S / B of 3, clearly indicating the advantages of UCNPs. In addition, the potential of UCNP-based dot-blot for real sample analysis was confirmed by analyzing spiked urine samples, reaching the LOD of 0.24 ng/mL and recovery rates from 79 to 123%. Furthermore, we demonstrated the versatility and robustness of the assay by adapting it to the detection of two other clinically relevant biomarkers, prostate-specific antigen (PSA) and cardiac troponin (cTn), reaching the LODs in spiked serum of 9.4 pg/mL and 0.62 ng/mL for PSA and cTn, respectively. Finally, clinical samples of patients examined for prostate cancer were analyzed, achieving a strong correlation with the reference electrochemiluminescence immunoassay (recovery rates from 89 to 117%). The achieved results demonstrate that UCNPs are highly sensitive labels that enable the development of dot-blot immunoassays for quantitative analysis of low-abundance biomarkers. more...
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- 2024
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9. Comparison of single and double pulse laser-induced breakdown spectroscopy for the detection of biomolecules tagged with photon-upconversion nanoparticles.
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Farka Z, Vytisková K, Makhneva E, Zikmundová E, Holub D, Buday J, Prochazka D, Novotný K, Skládal P, Pořízka P, and Kaiser J
- Subjects
- Humans, Spectrum Analysis methods, Immunoassay methods, Lasers, Metals, Nanoparticles chemistry
- Abstract
Background: Laser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity., Results: This paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL
-1 to 0.29 ng mL-1 . In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples., Significance and Novelty: In this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jozef Kaiser reports financial support was provided by Czech Science Foundation. Karolína Vytisková reports financial support was provided by Brno University of Technology. Ekaterina Makhneva reports financial support was provided by Ministry of Education, Youth and Sports of the Czech Republic., (Copyright © 2024 Elsevier B.V. All rights reserved.) more...- Published
- 2024
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10. Influence of surface modification and size of lanthanide-doped upconverting nanoparticles on wheat seedlings.
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Hosseinifard M, Jurga N, Brandmeier JC, Farka Z, Hlaváček A, Gorris HH, Grzyb T, and Ekner-Grzyb A
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- Triticum, Seedlings, Polyethylene Glycols, Lanthanoid Series Elements toxicity, Nanoparticles toxicity, Nanostructures
- Abstract
In recent years, nanotechnology has found widespread applications in environmental monitoring, medical applications, plant fertilisers, cosmetics and others. Therefore, it is important to study nanomaterials' influence and subsequent risks to the environment and organisms (from production to disposal). Therefore, in the present study, the toxic effects of two surface modifications (poly (ethylene glycol)-neridronate, PEG-Ner and poly (acrylic acid), PAA) in comparison to unmodified, 26 nm- and 52 nm-sized core@shell lanthanide-doped upconverting nanoparticles (UCNPs, NaYF
4 :Yb3+ ,Er3+ @NaYF4 ) were analysed. Wheat seedlings (Triticum aestivum L.) were chosen as a model organism since this species is one of the most widely cultivated crops. The influence of UCNPs (at concentrations of 0, 10, 50, and 100 μg/mL) on germination percentage, germination rate and growth was studied based on morphological parameters such as root number, root and hypocotyl length, and root and hypocotyl mass. In addition, an assay based on Evans blue staining was conducted to analyse damaged cell membranes and cell death. The type, size and concentration of UCNPs influenced the growth but not the germination of wheat. 52-nm-sized ligand-free UCNPs and the 26-nm-sized UCNPs/PAA decreased plant growth. Moreover, the ligand-free 26-nm-sized UCNPs interacted with the root cell membranes of seedlings. No significant changes were observable regarding viability (tetrazolium chloride reduction assay), oxidative stress and electrolyte leakage from root cells in plants incubated with ligand-free 26-nm-sized UCNPs. Overall, we have shown that the ligand-free UCNPs (of both sizes) had the strongest toxic effect; PAA-modified UCNPs were toxic only at smaller sizes and PEG-Ner-modified UCNPs had no toxic impact. Therefore, PEG-Ner was identified as the safest surface compound among the UCNPs investigated in the study, which may neutralise the harmful effects of nanoparticles on plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.) more...- Published
- 2024
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11. Nanoparticle-Based Bioaffinity Assays: From the Research Laboratory to the Market.
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Farka Z, Brandmeier JC, Mickert MJ, Pastucha M, Lacina K, Skládal P, Soukka T, and Gorris HH
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- Immunoassay, Sensitivity and Specificity, Nanoparticles
- Abstract
Advances in the development of new biorecognition elements, nanoparticle-based labels as well as instrumentation have inspired the design of new bioaffinity assays. This review critically discusses the potential of nanoparticles to replace current enzymatic or molecular labels in immunoassays and other bioaffinity assays. Successful implementations of nanoparticles in commercial assays and the need for rapid tests incorporating nanoparticles in different roles such as capture support, signal generation elements, and signal amplification systems are highlighted. The limited number of nanoparticles applied in current commercial assays can be explained by challenges associated with the analysis of real samples (e.g., blood, urine, or nasal swabs) that are difficult to resolve, particularly if the same performance can be achieved more easily by conventional labels. Lateral flow assays that are based on the visual detection of the red-colored line formed by colloidal gold are a notable exception, exemplified by SARS-CoV-2 rapid antigen tests that have moved from initial laboratory testing to widespread market adaption in less than two years., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.) more...
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- 2024
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12. Bright photon upconversion in LiYbF 4 :Tm 3+ @LiYF 4 nanoparticles and their application for singlet oxygen generation and in immunoassay for SARS-CoV-2 nucleoprotein.
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Drozdowski A, Jurga N, Przybylska D, Brandmeier JC, Farka Z, Gorris HH, and Grzyb T
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- Humans, Singlet Oxygen, SARS-CoV-2, Immunoassay, COVID-19 diagnosis, Nanoparticles
- Abstract
Photon upconversion is an intensively investigated phenomenon in the materials sciences due to its unique applications, mainly in biomedicine for disease prevention and treatment. This study reports the synthesis and properties of tetragonal LiYbF
4 :Tm3+ @LiYF4 core@shell nanoparticles (NPs) and their applications. The NPs had sizes ranging from 18.5 to 23.7 nm. As a result of the energy transfer between Yb3+ and Tm3+ ions, the synthesized NPs show intense emission in the ultraviolet (UV) range up to 347 nm under 975 nm excitation. The bright emission in the UV range allows for singlet oxygen generation in the presence of hematoporphyrin on the surface of NPs. Our studies show that irradiation with a 975 nm laser of the functionalized NPs allows for the production of amounts of singlet oxygen easily detectable by Singlet Oxygen Sensor Green. The high emission intensity of NPs at 800 nm allowed the application of the synthesized NPs in an upconversion-linked immunosorbent assay (ULISA) for highly sensitive detection of the nucleoprotein from SARS-CoV-2, the causative agent of Covid-19. This article proves that LiYbF4 :Tm3+ @LiYF4 core@shell nanoparticles can be perfect alternatives for the most commonly studied upconverting NPs based on the NaYF4 host compound and are good candidates for biomedical applications., Competing Interests: Declaration of Competing Interest The authors do not declare any competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.) more...- Published
- 2023
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13. Staphylococcus aureus Prophage-Encoded Protein Causes Abortive Infection and Provides Population Immunity against Kayviruses.
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Kuntová L, Mašlaňová I, Obořilová R, Šimečková H, Finstrlová A, Bárdy P, Šiborová M, Troianovska L, Botka T, Gintar P, Šedo O, Farka Z, Doškař J, and Pantůček R
- Subjects
- Humans, Staphylococcus aureus genetics, Lysogeny, Staphylococcus, Staphylococcus Phages genetics, Membrane Proteins genetics, Prophages genetics, Staphylococcal Infections microbiology
- Abstract
Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus , mediated by the membrane-anchored protein designated Pdp
Sau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau , and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy. more...- Published
- 2023
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14. Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay.
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Brandmeier JC, Jurga N, Grzyb T, Hlaváček A, Obořilová R, Skládal P, Farka Z, and Gorris HH
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- Humans, SARS-CoV-2, Enzyme-Linked Immunosorbent Assay, Nucleocapsid Proteins, Antibodies, Viral, Sensitivity and Specificity, Immunosorbents, COVID-19 diagnosis
- Abstract
The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout. more...
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- 2023
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15. Concentration and diffusion of the redox probe as key parameters for label-free impedimetric immunosensing.
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Lacina K, Věžník J, Sopoušek J, Farka Z, Lacinová V, and Skládal P
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- Dielectric Spectroscopy methods, Electrodes, Oxidation-Reduction, Electrochemical Techniques, Gold chemistry, Biosensing Techniques methods, Nanopores
- Abstract
Nanoporous surfaces are promising for label-free electrochemical biosensing. We formed nanopores directly on the electrode surface by means of assembling a dense layer of nonconductive nanoparticles. In our model affinity biosensor, covalent attachment of albumin protein on top of 40 nm polystyrene nanoparticles represented a capture of an analyte, resulting in blockage of the nanopores. Different bulk concentrations of the ferro/ferricyanide redox pair were probed by Faradaic electrochemical impedance spectroscopy and fast chronoamperometry. The character of the redox probe permeation towards the electrode surface differed in dependence on its concentration. These data were compared with the theoretical behavior of the free diffusion according to the Cottrell equation. Both the bulk concentration of the redox probe and the timescale of the experiment affected the performance of the electrochemical detection, demonstrating the importance of controlling these parameters in immunosensing applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.) more...
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- 2023
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16. Influence of Label and Solid Support on the Performance of Heterogeneous Immunoassays.
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Makhneva E, Sklenárová D, Brandmeier JC, Hlaváček A, Gorris HH, Skládal P, and Farka Z
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- Male, Humans, Immunoassay methods, Limit of Detection, Streptavidin, Magnetics, Immunosorbents, Nanoparticles
- Abstract
Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labels─horseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)─UCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection. more...
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- 2022
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17. PMVEMA-coated upconverting nanoparticles for upconversion-linked immunoassay of cardiac troponin.
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Shapoval O, Brandmeier JC, Nahorniak M, Oleksa V, Makhneva E, Gorris HH, Farka Z, and Horák D
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- Immunoassay methods, Limit of Detection, Luminescence, Nanoparticles chemistry, Troponin I analysis
- Abstract
Surface engineering of upconverting nanoparticles (UCNPs) is crucial for their bioanalytical applications. Here, an antibody specific to cardiac troponin I (cTnI), an important biomarker for acute myocardial infection, was covalently immobilized on the surface of UCNPs to prepare a label for the detection of cTnI biomarker in an upconversion-linked immunoassay (ULISA). Core-shell UCNPs (NaYF
4 :Yb,Tm@NaYF4 ) were first coated with poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) and then conjugated to antibodies. The morphology (size and uniformity), hydrodynamic diameter, chemical composition, and amount of coating on the of UCNPs, as well as their upconversion luminescence, colloidal stability, and leaching of Y3+ ions into the surrounding media, were determined. The developed ULISA allowed reaching a limit of detection (LOD) of 0.13 ng/ml and 0.25 ng/ml of cTnI in plasma and serum, respectively, which represents 12- and 2-fold improvement to conventional enzyme-linked immunosorbent based on the same immunoreagents., (Copyright © 2022 Elsevier B.V. All rights reserved.) more...- Published
- 2022
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18. Colloidal lithography as a novel approach for the development of Ni-nanocavity insulin sensor.
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Šišoláková I, Petruš O, Shepa J, Farka Z, Oriňak A, and Oriňaková R
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- Electrochemical Techniques methods, Electrodes, Insulin, Biosensing Techniques methods, Nanostructures
- Abstract
In this study, a highly sensitive, fast, and selective enzyme-free electrochemical sensor based on the deposition of Ni cavities on conductive glass was proposed for insulin detection. Considering the growing prevalence of diabetes mellitus, an electrochemical sensor for the determination of insulin was proposed for the effective diagnosis of the disease. Colloidal lithography enabled deposition of nanostructured layer (substrate) with homogeneous distribution of Ni cavities on the electrode surface with a large active surface area. The morphology and structure of conductive indium tin oxide glass modified with Ni cavities (Ni-c-ITO) were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The diameter of the resulting cavities was approximately 500 nm, while their depth was calculated at 190 ± 4 nm and 188 ± 18 nm using AFM and SEM, respectively. The insulin assay performance was evaluated by cyclic voltammetry. Ni-c-ITO exhibited excellent analytical characteristics, including high sensitivity (1.032 µA µmol
-1 dm3 ), a low detection limit (156 µmol dm-3 ), and a wide dynamic range (500 nmol dm-3 to 10 µmol dm-3 ). Finally, the determination of insulin in buffer with interferents and in real blood serum samples revealed high specificity and demonstrated the practical potential of the method., (© 2022. The Author(s).) more...- Published
- 2022
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19. Up and down the spectrum: upconversion nanocrystal and semiconductor material fused into a single nanocomposite.
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Gorris HH and Farka Z
- Abstract
A nanocomposite consisting of a cubic EuSe semiconductor material grown on a hexagonal upconversion nanoparticle has overcome the crystal lattice mismatch that typically prevents the epitaxial growth of such heterogeneous nanocrystals. Eu
3+ at the interface layer shows its characteristic red emission band both under UV excitation light due to energy transfer from the semiconductor and under NIR excitation light due to energy transfer after photon-upconversion. Data storage and security applications are suggested for this new nanocomposite., (© 2022. The Author(s).) more...- Published
- 2022
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20. Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging.
- Author
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Hlaváček A, Farka Z, Mickert MJ, Kostiv U, Brandmeier JC, Horák D, Skládal P, Foret F, and Gorris HH
- Subjects
- Biomarkers, Tumor, Humans, Immunosorbents, Male, Polyethylene Glycols chemistry, Silicon Dioxide chemistry, Streptavidin, Nanoparticles chemistry, Neoplasms diagnosis
- Abstract
The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.) more...
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- 2022
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21. Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin.
- Author
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Brandmeier JC, Raiko K, Farka Z, Peltomaa R, Mickert MJ, Hlaváček A, Skládal P, Soukka T, and Gorris HH
- Subjects
- Humans, Immunoassay, Particle Size, Photons, Troponin, Nanoparticles
- Abstract
Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL
-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals., (© 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.) more...- Published
- 2021
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22. Atomic force microscopy and surface plasmon resonance for real-time single-cell monitoring of bacteriophage-mediated lysis of bacteria.
- Author
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Obořilová R, Šimečková H, Pastucha M, Klimovič Š, Víšová I, Přibyl J, Vaisocherová-Lísalová H, Pantůček R, Skládal P, Mašlaňová I, and Farka Z
- Subjects
- Animals, Humans, Microscopy, Atomic Force, Staphylococcus aureus, Surface Plasmon Resonance, Bacteriophages, Staphylococcal Infections
- Abstract
The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy. more...
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- 2021
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23. Laser-induced breakdown spectroscopy as a readout method for immunocytochemistry with upconversion nanoparticles.
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Pořízka P, Vytisková K, Obořilová R, Pastucha M, Gábriš I, Brandmeier JC, Modlitbová P, Gorris HH, Novotný K, Skládal P, Kaiser J, and Farka Z
- Subjects
- Antibodies, Immobilized immunology, Biomarkers, Tumor immunology, Cell Line, Tumor, Feasibility Studies, Fluorides chemistry, Fluorides radiation effects, Humans, Immunohistochemistry methods, Light, Nanoparticles radiation effects, Receptor, ErbB-2 immunology, Spectrum Analysis methods, Thulium chemistry, Thulium radiation effects, Yttrium chemistry, Yttrium radiation effects, Biomarkers, Tumor analysis, Nanoparticles chemistry, Receptor, ErbB-2 analysis
- Abstract
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are widely used to identify cancerous cells within tissues and cell cultures. Even though the optical microscopy evaluation is considered the gold standard, the limited range of useful labels and narrow multiplexing capabilities create an imminent need for alternative readout techniques. Laser-induced breakdown spectroscopy (LIBS) enables large-scale multi-elemental analysis of the surface of biological samples, e.g., thin section or cell pellet. It is, therefore, a potential alternative for IHC and ICC readout of various labels or tags (Tag-LIBS approach). Here, we introduce Tag-LIBS as a method for the specific determination of HER2 biomarker. The cell pellets were labeled with streptavidin-conjugated upconversion nanoparticles (UCNP) through a primary anti-HER2 antibody and a biotinylated secondary antibody. The LIBS scanning enabled detecting the characteristic elemental signature of yttrium as a principal constituent of UCNP, thus indirectly providing a reliable way to differentiate between HER2-positive BT-474 cells and HER2-negative MDA-MB-231 cells. The comparison of results with upconversion optical microscopy and luminescence intensity scanning confirmed that LIBS is a promising alternative for the IHC and ICC readout. more...
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- 2021
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24. Detection of organic dyes by surface-enhanced Raman spectroscopy using plasmonic NiAg nanocavity films.
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Petruš O, Macko J, Oriňaková R, Oriňak A, Múdra E, Kupková M, Farka Z, Pastucha M, and Socha V
- Abstract
This work presents the NiAg nanocavity film for the detection of organic dyes by surface-enhanced Raman spectroscopy (SERS). Nanocavity films were prepared by colloidal lithography using 518-nm polystyrene spheres combined with the electrochemical deposition of Ni supporting layer and Ag nanoparticles homogeneous SERS-active layer. The theoretical study was modelled by finite-difference time-domain (FDTD) simulation of electromagnetic field enhancement near the nanostructured surface and experimentally proven by SERS measurement of selected organic dyes (rhodamine 6G, crystal violet, methylene blue, and malachite green oxalate) in micromolar concentration. Furthermore, the concentration dependence was investigated to prove the suitability of NiAg nanocavity films to detect ultra-low concentrations of samples. The detection limit was 1.3 × 10
-12 , 1.5 × 10-10 , 1.4 × 10-10 , 7.5 × 10-11 mol·dm-3 , and the standard deviation was 20.1%, 13.8%, 16.7%, and 19.3% for R6G, CV, MB, and MGO, respectively. The analytical enhancement factor was 3.4 × 105 using R6G as a probe molecule. The principal component analysis (PCA) was performed to extract the differences in complex spectra of the dyes where the first and second PCs carry 42.43% and 31.39% of the sample variation, respectively. The achieved results demonstrated the suitability of AgNi nanocavity films for the SERS-based detection of organic dyes, with a potential in other sensing applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...- Published
- 2021
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25. Competitive upconversion-linked immunoassay using peptide mimetics for the detection of the mycotoxin zearalenone.
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Peltomaa R, Farka Z, Mickert MJ, Brandmeier JC, Pastucha M, Hlaváček A, Martínez-Orts M, Canales Á, Skládal P, Benito-Peña E, Moreno-Bondi MC, and Gorris HH
- Subjects
- Chromatography, Liquid, Food Contamination analysis, Immunoassay, Peptides, Tandem Mass Spectrometry, Zea mays, Biosensing Techniques, Mycotoxins analysis, Zearalenone analysis
- Abstract
Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF
4 :Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL-1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and β-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...- Published
- 2020
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26. Versatile Bioconjugation Strategies of PEG-Modified Upconversion Nanoparticles for Bioanalytical Applications.
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Kostiv U, Farka Z, Mickert MJ, Gorris HH, Velychkivska N, Pop-Georgievski O, Pastucha M, Odstrčilíková E, Skládal P, and Horák D
- Subjects
- Polyethylene Glycols, Nanoparticles
- Abstract
Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL). more...
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- 2020
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27. Functionalizable Antifouling Coatings as Tunable Platforms for the Stress-Driven Manipulation of Living Cell Machinery.
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Víšová I, Smolková B, Uzhytchak M, Vrabcová M, Chafai DE, Houska M, Pastucha M, Skládal P, Farka Z, Dejneka A, and Vaisocherová-Lísalová H
- Subjects
- Acrylamides chemistry, Cell Line, Tumor, Extracellular Matrix metabolism, Humans, Oligopeptides chemistry, Proto-Oncogene Proteins c-yes metabolism, Stress, Mechanical, Biofouling prevention & control, Coated Materials, Biocompatible chemistry, Mechanotransduction, Cellular
- Abstract
Cells are continuously sensing their microenvironment and subsequently respond to different physicochemical cues by the activation or inhibition of different signaling pathways. To study a very complex cellular response, it is necessary to diminish background environmental influences and highlight the particular event. However, surface-driven nonspecific interactions of the abundant biomolecules from the environment influence the targeted cell response significantly. Yes-associated protein (YAP) translocation may serve as a marker of human hepatocellular carcinoma (Huh7) cell responses to the extracellular matrix and surface-mediated stresses. Here, we propose a platform of tunable functionable antifouling poly(carboxybetain) (pCB)-based brushes to achieve a molecularly clean background for studying arginine, glycine, and aspartic acid (RGD)-induced YAP-connected mechanotransduction. Using two different sets of RGD-functionalized zwitterionic antifouling coatings with varying compositions of the antifouling layer, a clear correlation of YAP distribution with RGD functionalization concentrations was observed. On the other hand, commonly used surface passivation by the oligo(ethylene glycol)-based self-assembled monolayer (SAM) shows no potential to induce dependency of the YAP distribution on RGD concentrations. The results indicate that the antifouling background is a crucial component of surface-based cellular response studies, and pCB-based zwitterionic antifouling brush architectures may serve as a potential next-generation easily functionable surface platform for the monitoring and quantification of cellular processes. more...
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- 2020
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28. Advances in Optical Single-Molecule Detection: En Route to Supersensitive Bioaffinity Assays.
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Farka Z, Mickert MJ, Pastucha M, Mikušová Z, Skládal P, and Gorris HH
- Subjects
- Binding Sites, Biomarkers analysis, Enzyme-Linked Immunosorbent Assay, Fluorescent Dyes chemistry, Limit of Detection, Nanostructures chemistry, Nucleic Acids analysis, Polymerase Chain Reaction methods, Proteins analysis, Signal-To-Noise Ratio, Viruses isolation & purification, Single Molecule Imaging methods
- Abstract
The ability to detect low concentrations of analytes and in particular low-abundance biomarkers is of fundamental importance, e.g., for early-stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single-molecule bioaffinity assays. While many review articles have highlighted the potentials of single-molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real-world applications as one should expect. This Review provides a theoretical background on single-molecule-or better digital-assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single-molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.) more...
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- 2020
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29. Surface design of photon-upconversion nanoparticles for high-contrast immunocytochemistry.
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Farka Z, Mickert MJ, Mikušová Z, Hlaváček A, Bouchalová P, Xu W, Bouchal P, Skládal P, and Gorris HH
- Subjects
- Biomarkers, Tumor metabolism, Cell Line, Tumor, Diphosphonates chemistry, Humans, Luminescence, Nanoconjugates chemistry, Polyethylene Glycols chemistry, Signal-To-Noise Ratio, Streptavidin chemistry, Immunohistochemistry methods, Nanoparticles chemistry, Photons
- Abstract
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are routinely employed for the microscopic identification and diagnosis of cancerous cells in histological tissues and cell cultures. The maximally attainable contrast of conventional histological staining techniques, however, is low. While the anti-Stokes emission of photon-upconversion nanoparticles (UCNP) can efficiently eliminate optical background interference, excluding non-specific interactions of the label with the histological sample is equally important for specific immunolabeling. To address both requirements, we have designed and characterized several UCNP-based nanoconjugates as labels for the highly specific detection of the cancer biomarker HER2 on various breast cancer cell lines. An optimized streptavidin-PEG-neridronate-UCNP conjugate provided an unsurpassed signal-to-background ratio of 319, which was 50-fold better than conventional fluorescent labeling under the same experimental conditions. In combination, the absence of optical interference and non-specific binding lays the foundation for computer-based data evaluation in digital pathology. more...
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- 2020
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30. Functional Plasma Polymerized Surfaces for Biosensing.
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Makhneva E, Barillas L, Farka Z, Pastucha M, Skládal P, Weltmann KD, and Fricke K
- Subjects
- Atmospheric Pressure, Oxygen analysis, Polymerization, Surface Properties, Biosensing Techniques, Oxygen chemistry, Polymers chemistry, Water chemistry
- Abstract
The capabilities of biosensors for fast, economic, and user-friendly analysis of complex samples has led to the exploitation of analytical devices for detection, quantification, and monitoring of specific chemical species for various applications. For a sufficiently high surface reactivity toward the adopted bioreceptors, a thin functional layer is required to enable coupling of the target biomolecules and to provide good stability in the presence of a sample matrix. In this work, the generation of water-stable oxygen-rich plasma polymerized (pp) films deposited by atmospheric-pressure jet plasma for reliable immobilization of biomolecules is presented. Three types of pp films were developed and characterized. All of the obtained pp films were successfully used as a matrix layer in the SPR immunosensors, which provided excellent level of sensitivity, stability, and regenerability. The achieved results show that atmospheric pressure plasma-induced polymerization is a powerful alternative method for the preparation of matrix layers for a wide range of applications in the biological field. more...
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- 2020
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31. Maleic anhydride and acetylene plasma copolymer surfaces for SPR immunosensing.
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Makhneva E, Farka Z, Pastucha M, Obrusník A, Horáčková V, Skládal P, and Zajíčková L
- Subjects
- Biosensing Techniques, Limit of Detection, Microscopy, Atomic Force, Sulfhydryl Compounds chemistry, Surface Properties, Acetylene chemistry, Maleic Anhydrides chemistry, Plasma Gases, Polymers chemistry, Surface Plasmon Resonance methods
- Abstract
We report on the successful application of carboxyl-rich plasma polymerized (PP) films as a matrix layer for bioreceptor immobilization in surface plasmon resonance (SPR) immunosensing. Composition and chemical properties of the carboxyl-rich PP films deposited from a mixture of maleic anhydride and acetylene were investigated. Changes in the films stored in air, water, and buffer were studied and the involved chemical changes were described. Performance in SPR immunosensing was evaluated on interactions of human serum albumin (HSA) with a specific monoclonal antibody. The comparison with the mixed self-assembled monolayer of mercaptoundecanoic acid and mercaptohexanol (MUA/MCH) and one of the most widely used surfaces for SPR, the 2D and 3D carboxymethylated dextran (CMD), was presented to show the efficacy of plasma polymerized matrix layers for biosensing. The PP film-based SPR immunosensor provided a similar detection limit of HSA (100 ng/mL) as MUA/MCH- (100 ng/mL) and 3D CMD (50 ng/mL)-based sensors. However, the response levels were about twice higher in case of the PP film-based immunosensor than in case of MUA/MCH-based alternative. The PP film surfaces had similar binding capacity towards antibody as the 3D CMD layers. The response of PP film-based sensor towards HSA was comparable to 3D CMD-based sensor up to 2.5 μg/mL. For the higher concentrations (> 10 μg/mL), the response of PP film-based immunosensor was lower due to inaccessibility of active sites of the immobilized antibody inside the flat PP film surface. We have demonstrated that due to its high stability and cost-effective straightforward preparation, the carboxyl-rich PP films represent an efficient alternative to self-assembled monolayers (SAM) and dextran-based layers in label-free immunosensing. Graphical abstract. more...
- Published
- 2019
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32. Laser-induced breakdown spectroscopy as a novel readout method for nanoparticle-based immunoassays.
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Modlitbová P, Farka Z, Pastucha M, Pořízka P, Novotný K, Skládal P, and Kaiser J
- Subjects
- Biomarkers blood, Gold chemistry, Humans, Particle Size, Silver chemistry, Surface Properties, Immunoassay methods, Lasers, Metal Nanoparticles chemistry, Serum Albumin, Human analysis
- Abstract
Laser-induced breakdown spectroscopy (LIBS) was examined as a novel method for readout of microtiter plate immunoassays involving nanoparticles (NP). The so-called Tag-LIBS technique is a sensitive method for the detection of specific biomarkers. It was applied to the determination of NP labels using nanosecond ablation sampling. The NP labels were examined from the bottom of a standard 96-well microtiter plate. Thanks to the flexibility of LIBS instrumentation, both the plasma emission collection and the focusing optics arrangements can be collinearly arranged. The experiments showed that silver NPs and gold NPs can be readily quantified on the bottom of the microtiter plate. Utilizing this technique, a sandwich immunoassay for human serum albumin using streptavidin-coated AgNP labels was developed. The assay has a 10 ng·mL
-1 detection limit which is comparable to the sensitivity of fluorometric readout. The main advantage of this LIBS technique is its wide scope in which it enables a detection of almost any type of NP labels, irrespective to any fluorescence or catalytic properties. Owing to the immediate signal response, the relatively simple instrumentation also enables assay automation. The LIBS capability of multi-elemental analyses makes it a promising and fast alternative to other readout techniques, in particular with respect to multiplexed detection of biomarkers. Graphical abstract Laser-induced breakdown spectroscopy (LIBS) is used as a novel readout method of nanoparticle-based immunoassays in microtiter plates. After formation of sandwich immunocomplex, the analyte concentration is quantified as the signal of Ag nanoparticle labels determined by LIBS. more...- Published
- 2019
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33. Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay.
- Author
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Mickert MJ, Farka Z, Kostiv U, Hlaváček A, Horák D, Skládal P, and Gorris HH
- Subjects
- Dermoscopy methods, Diphosphonates, Humans, Male, Nanoparticles chemistry, Photons, Polyethylene Glycols, Streptavidin, Immunoassay methods, Immunosorbent Techniques, Prostate-Specific Antigen blood, Single Molecule Imaging methods
- Abstract
Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL
-1 , 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment. more...- Published
- 2019
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34. Magnetic nanoparticles for smart electrochemical immunoassays: a review on recent developments.
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Pastucha M, Farka Z, Lacina K, Mikušová Z, and Skládal P
- Subjects
- Animals, Humans, Electrochemistry methods, Immunoassay methods, Magnets chemistry, Nanoparticles chemistry
- Abstract
This review (with 129 refs) summarizes the progress in electrochemical immunoassays combined with magnetic particles that was made in the past 5 years. The specifity of antibodies linked to electrochemical transduction (by amperometry, voltammetry, impedimetry or electrochemiluminescence) gains further attractive features by introducing magnetic nanoparticles (MNPs). This enables fairly easy preconcentration of analytes, minimizes matrix effects, and introduces an appropriate label. Following an introduction into the fundamentals of electrochemical immunoassays and on nanomaterials for respective uses, a large chapter addresses method for magnetic capture and preconcentration of analytes. A next chapter discusses commonly used labels such as dots, enzymes, metal and metal oxide nanoparticles and combined clusters. The large field of hybrid nanomaterials for use in such immunoassays is discussed next, with a focus on MNPs composites with various kinds of graphene variants, polydopamine, noble metal nanoparticles or nanotubes. Typical applications address clinical markers (mainly blood and urine parameters), diagnosis of cancer (markers and cells), detection of pathogens (with subsections on viruses and bacteria), and environmental and food contaminants as toxic agents and pesticides. A concluding section summarizes the present status, current challenges, and highlights future trends. Graphical abstract Magnetic nanoparticles (MNP) with antibodies (Ab) capture and preconcentrate analyte from sample (a) and afterwards become magnetically (b) or immunospecifically (c) bound at an electrode. Signal either increases due to the presence of alabel (b) or decreases as the redox probe is blocked (c). more...
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- 2019
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35. Click-conjugated photon-upconversion nanoparticles in an immunoassay for honeybee pathogen Melissococcus plutonius.
- Author
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Poláchová V, Pastucha M, Mikušová Z, Mickert MJ, Hlaváček A, Gorris HH, Skládal P, and Farka Z
- Subjects
- Animals, Antibodies, Bacterial immunology, Bees growth & development, Enterococcaceae immunology, Larva immunology, Larva metabolism, Limit of Detection, Photons, Silicon Dioxide chemistry, Bees microbiology, Enterococcaceae isolation & purification, Immunoassay methods, Nanoparticles chemistry
- Abstract
European foulbrood (EFB) is an infectious disease affecting honeybee larvae caused by the bacterium Melissococcus plutonius. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for antibody-based bacteria detection, however, its sensitivity is not high enough to reveal early-stage EFB infection. Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanomaterials that emit light of shorter wavelength under near-infrared (NIR) excitation and thus avoid optical background interference. After conjugation with specific biorecognition molecules, UCNPs can be used as ultrasensitive labels in immunoassays. Here, we introduce a method for conjugation of UCNPs with streptavidin based on copper-free click chemistry, which involves surface modification of UCNPs with alkyne-modified bovine serum albumin (BSA) that prevents the non-specific binding and provides reactive groups for conjugation with streptavidin-azide. To develop a sandwich upconversion-linked immunosorbent assay (ULISA) for M. plutonius detection, we have prepared a rabbit polyclonal anti-Melissococcus antibody. The specific capture of the bacteria was followed by binding of biotinylated antibody and UCNP-BSA-streptavidin conjugate for a highly sensitive upconversion readout. The assay yielded an LOD of 340 CFU mL-1 with a wide working range up to 109 CFU mL-1, which is 400 times better than the LOD of the conventional ELISA. The practical applicability of the ULISA was successfully demonstrated by detecting M. plutonius in spiked real samples of bees, larvae and bottom hive debris. These results show a great potential of the assay for early diagnosis of EFB, which can prevent uncontrolled spreading of the infection and losses of honeybee colonies. more...
- Published
- 2019
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36. Time-Dependent Growth of Silica Shells on CdTe Quantum Dots.
- Author
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Modlitbová P, Klepárník K, Farka Z, Pořízka P, Skládal P, Novotný K, and Kaiser J
- Abstract
The purpose of this study is to investigate the time dependent growth of silica shells on CdTe quantum dots to get their optimum thicknesses for practical applications. The core/shell structured silica-coated CdTe quantum dots (CdTe/SiO₂ QDs) were synthesized by the Ströber process, which used CdTe QDs co-stabilized by mercaptopropionic acid. The coating procedure used silane primer (3-mercaptopropyltrimethoxysilane) in order to make the quantum dots (QDs) surface vitreophilic. The total size of QDs was dependent on both the time of silica shell growth in the presence of sodium silicate, and on the presence of ethanol during this growth. The size of particles was monitored during the first 72 h using two principally different methods: Dynamic Light Scattering (DLS), and Scanning Electron Microscopy (SEM). The data obtained by both methods were compared and reasons for differences discussed. Without ethanol precipitation, the silica shell thickness grew slowly and increased the nanoparticle total size from approximately 23 nm up to almost 30 nm (DLS data), and up to almost 60 nm (SEM data) in three days. During the same time period but in the presence of ethanol, the size of CdTe/SiO₂ QDs increased more significantly: up to 115 nm (DLS data) and up to 83 nm (SEM data). The variances occurring between silica shell thicknesses caused by different methods of silica growth, as well as by different evaluation methods, were discussed. more...
- Published
- 2018
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37. Short-term assessment of cadmium toxicity and uptake from different types of Cd-based Quantum Dots in the model plant Allium cepa L.
- Author
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Modlitbová P, Pořízka P, Novotný K, Drbohlavová J, Chamradová I, Farka Z, Zlámalová-Gargošová H, Romih T, and Kaiser J
- Subjects
- Adsorption, Microscopy, Fluorescence, Onions chemistry, Plant Roots chemistry, Plant Roots drug effects, Cadmium Compounds analysis, Cadmium Compounds toxicity, Onions drug effects, Quantum Dots analysis, Quantum Dots toxicity
- Abstract
We report on the toxicity and bioaccumulation of three different types of Cd-based quantum dots (QDs), dispersed in aqueous medium, for a model plant Allium cepa L. It is believed that encapsulation of nanoparticles should reduce their toxicity and increase their stability in different environments; in this work we studied how QD encapsulation affects their phytotoxicity. Core, core/shell, and core/shell/shell QDs (CdTe, CdTe/ZnS, and CdTe/CdS/ZnS QDs capped by 2-mercaptopropionic acid) were tested and CdCl
2 was used as a positive control. After 24-h and 72-h exposure, total Cd content (MCd ) and bioaccumulation factors (BAFs) were determined in all parts of A. cepa plants (roots, bulb, shoot), and the total length of the root system was monitored as a toxicity end-point. Measurements of total Cd content versus free Cd2+ content (with Differential Pulse Voltammetry, DPV) in exposure media showed differences in chemical stability of the three QD types. Correspondingly, selected QDs showed different toxicity for A. cepa and different Cd bioaccumulation patterns. CdTe QDs were the most toxic; their effect was similar to CdCl2 due to the release of free Cd2+ , which was confirmed by the DPV measurements. Plants exposed to CdTe QDs also bioaccumulated the most Cd among all QD exposure groups. CdTe/ZnS QDs showed no toxicity and very low bioaccumulation of Cd in A. cepa; the main source of measured Cd in the plants were QDs adsorbed on their roots, which was confirmed by fluorescence microscopy. On the contrary, CdTe/CdS/ZnS QD toxicity and bioaccumulation patterns were similar to those of CdTe QDs and pointed to unstable CdS/ZnS shells., (Copyright © 2018 Elsevier Inc. All rights reserved.) more...- Published
- 2018
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38. Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich Nanozyme-Linked Immunosorbent Assay.
- Author
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Farka Z, Čunderlová V, Horáčková V, Pastucha M, Mikušová Z, Hlaváček A, and Skládal P
- Subjects
- Animals, Antibodies, Bacterial immunology, Catalysis, Humans, Limit of Detection, Milk microbiology, Salmonella typhimurium immunology, Salmonella typhimurium isolation & purification, Serum Albumin, Human immunology, Serum Albumin, Human urine, Biosensing Techniques methods, Ferrocyanides chemistry, Immunosorbent Techniques, Nanoparticles chemistry
- Abstract
Enzyme immunoassays are widely used for detection of analytes within various samples. However, enzymes as labels suffer several disadvantages such as high production cost and limited stability. Catalytic nanoparticles (nanozymes) can be used as an alternative label in immunoassays overcoming the inherent disadvantages of enzymes. Prussian blue nanoparticles (PBNPs) are nanozymes composed of the Fe
4 [Fe(CN)6 ]3 -based coordination polymer. They reveal peroxidase-like activity and are capable of catalyzing the oxidation of colorless 3,3',5,5'-tetramethylbenzidine in the presence of H2 O2 to form intensely blue product. Here, we introduce the method for conjugation of PBNPs with antibodies and their application in nanozyme-linked immunosorbent assay (NLISA). Sandwich NLISA for detection of human serum albumin in urine was developed with limit of detection (LOD) of 1.2 ng·mL-1 and working range up to 1 μg·mL-1 . Furthermore, the microbial contamination of Salmonella Typhimurium in powdered milk was detected with LOD of 6 × 103 colony-forming units (cfu)·mL-1 and working range up to 106 cfu·mL-1 . In both cases, a critical comparison with the same immunoassay but using native peroxidase as label was realized. The achieved results confirmed the suitability of PBNPs for universal and robust replacement of enzyme labels. more...- Published
- 2018
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39. Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers.
- Author
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Farka Z, Mickert MJ, Hlaváček A, Skládal P, and Gorris HH
- Subjects
- Biomarkers analysis, Immunosorbents immunology, Luminescence, Nanoparticles chemistry, Immunoassay methods, Immunosorbents chemistry, Limit of Detection, Prostate-Specific Antigen analysis
- Abstract
The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL
-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays. more...- Published
- 2017
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40. Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges.
- Author
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Farka Z, Juřík T, Kovář D, Trnková L, and Skládal P
- Subjects
- Animals, Humans, Biosensing Techniques, Immunoassay, Nanoparticles chemistry
- Abstract
We review the progress achieved during the recent five years in immunochemical biosensors (immunosensors) combined with nanoparticles for enhanced sensitivity. The initial part introduces antibodies as classic recognition elements. The optical sensing part describes fluorescent, luminescent, and surface plasmon resonance systems. Amperometry, voltammetry, and impedance spectroscopy represent electrochemical transducer methods; electrochemiluminescence with photoelectric conversion constitutes a widely utilized combined method. The transducing options function together with suitable nanoparticles: metallic and metal oxides, including magnetic ones, carbon-based nanotubes, graphene variants, luminescent carbon dots, nanocrystals as quantum dots, and photon up-converting particles. These sources merged together provide extreme variability of existing nanoimmunosensing options. Finally, applications in clinical analysis (markers, tumor cells, and pharmaceuticals) and in the detection of pathogenic microorganisms, toxic agents, and pesticides in the environmental field and food products are summarized. more...
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- 2017
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41. Enzymatic Precipitation Enhanced Surface Plasmon Resonance Immunosensor for the Detection of Salmonella in Powdered Milk.
- Author
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Farka Z, Juřík T, Pastucha M, and Skládal P
- Subjects
- Animals, Microscopy, Atomic Force, Powders chemistry, Food Contamination analysis, Immunoassay, Milk microbiology, Salmonella typhimurium isolation & purification, Surface Plasmon Resonance
- Abstract
Contamination of food by pathogenic bacteria has always been a serious threat for human health. The amount of food exports and imports has been increasing in recent years which requires precise food quality control with short analysis time and simplified sample treatment. Surface plasmon resonance (SPR) immunosensor enhanced by biocatalyzed precipitation was developed for the analysis of Salmonella in dairy products. The specific capture antibody was immobilized on the SPR chip which allowed a direct label-free detection of Salmonella Typhimurium with the limit of detection (LOD) of 10
4 CFU·mL-1 and the analysis time of 10 min. Alternatively, the secondary detection antibody was conjugated with horseradish peroxidase to provide a signal enhancement by the biocatalyzed conversion of 4-chloro-1-naphthol to insoluble benzo-4-chlorocyclohexadienone. The formation of precipitate was studied in detail by atomic force microscopy (AFM). The sensitivity was increased 40 times in case of the precipitation-enhanced detection compared to the label-free approach. The optimized method provided LOD of 100 CFU·mL-1 with linear range up to 106 CFU·mL-1 . The total time of analysis including bacteria binding and enhancement step was below 60 min. The capability to analyze real samples with complex matrices was demonstrated on the detection of Salmonella in powdered milk. The developed sensor represents simple and robust approach for routine monitoring of food contamination. more...- Published
- 2016
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42. Competitive Upconversion-Linked Immunosorbent Assay for the Sensitive Detection of Diclofenac.
- Author
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Hlaváček A, Farka Z, Hübner M, Horňáková V, Němeček D, Niessner R, Skládal P, Knopp D, and Gorris HH
- Subjects
- Drinking Water chemistry, Nanoparticles chemistry, Particle Size, Silicon Dioxide chemistry, Surface Properties, Diclofenac analysis, Immunoassay methods, Immunosorbents chemistry, Water Pollutants, Chemical analysis
- Abstract
Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-infrared excitation and thus avoid optical background interference. We have exploited this unique photophysical feature to establish a sensitive competitive immunoassay for the detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter) exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody. We investigated the structure and monodispersity of the nanoconjugates in detail. Using a highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of 0.05 ng DCF per mL. This performance came close to a conventional enzyme-linked immunosorbent assay (ELISA) without the need for an enzyme-mediated signal amplification step. The ULISA was further employed for analyzing drinking and surface water samples. The results were consistent with a conventional ELISA as well as liquid chromatography-mass spectrometry (LC-MS). more...
- Published
- 2016
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43. Rapid detection of microorganisms based on active and passive modes of QCM.
- Author
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Farka Z, Kovář D, and Skládal P
- Subjects
- Antibodies metabolism, Calibration, Electric Impedance, Immobilized Proteins metabolism, Immunoassay, Microscopy, Atomic Force, Surface Plasmon Resonance, Escherichia coli isolation & purification, Quartz Crystal Microbalance Techniques methods
- Abstract
Label-free immunosensors are well suited for detection of microorganisms because of their fast response and reasonable sensitivity comparable to infection doses of common pathogens. Active (lever oscillator and frequency counter) and passive (impedance analyzer) modes of quartz crystal microbalance (QCM) were used and compared for rapid detection of three strains of E. coli. Different approaches for antibody immobilization were compared, the immobilization of reduced antibody using Sulfo-SMCC was most effective achieving the limit of detection (LOD) 8 × 104 CFU·mL-1 in 10 min. For the passive mode, software evaluating impedance characteristics in real-time was developed and used. Almost the same results were achieved using both active and passive modes confirming that the sensor properties are not limited by the frequency evaluation method but mainly by affinity of the antibody. Furthermore, reference measurements were done using surface plasmon resonance. Effect of condition of cells on signal was observed showing that cells ruptured by ultrasonication provided slightly higher signal changes than intact microbes. more...
- Published
- 2014
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44. Detection of aerosolized biological agents using the piezoelectric immunosensor.
- Author
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Kovář D, Farka Z, and Skládal P
- Subjects
- Air Microbiology, Antibodies immunology, Quartz Crystal Microbalance Techniques, Aerosols analysis, Biosensing Techniques methods, Escherichia coli isolation & purification
- Abstract
Airborne microorganisms are a major cause of respiratory diseases. Detection of pathogenic bacteria in the form of bioaerosols is required not only in peacetime but also in the threat of biological attacks. The label-free and direct detection of aerosolized biological agents is presented here. A desktop bioaerosol chamber for safe work with aerosolized microbial cells was constructed, and its functionality was tested. The model organisms (Escherichia coli) were disseminated using an aerosol generator in the chamber filled with either common laboratory indoor air or sterile air. The particles from the generated aerosol were collected using the cyclone SASS 2300, suspended in buffer and then analyzed using the piezoelectric immunosensor modified with specific capture antibodies. The frequency shifts indicated presence of the model biological agent with limit of detection of 1.45 × 10(4) CFU·L(-1) of air. The total time from sample collection to detection was 16 min. The system was fully automated and controlled remotely through a local network. more...
- Published
- 2014
- Full Text
- View/download PDF
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