Dendronized Gold Nanoparticles as Carriers for gp160 (HIV-1) Peptides: Biophysical Insight into Complex Formation
Langmuir, 2021
The unavailability of effective and safe human immunodeficiency virus (HIV) vaccines incites seve... more The unavailability of effective and safe human immunodeficiency virus (HIV) vaccines incites several approaches for development of the efficient antigen/adjuvant vaccination composite. In this study, three different dendronized gold nanoparticles (AuNPs 13-15) were investigated for a complexation ability with gp160 synthetic peptides derived from an HIV envelope. It has been shown that HIV peptides interacted with nanoparticles as evident from the changes in their secondary structures, restricted the mobility of the attached fluorescence dye, and enhanced peptide helicity confirmed by the fluorescence polarization and circular dichroism results. Transmission electron microscopy visualized complexes as cloud-like structures with attached nanoparticles. AuNP 13-15 nanoparticles bind negatively charged peptides depending on the number of functional groups; the fastest saturation and peptide retardation were observed for the most dendronized nanoparticle as indicated from dynamic light scattering, laser Doppler velocimetry, and agarose gel electrophoresis experiments. Dendronized gold nanoparticles can be considered one of the potential HIV peptide-based vaccination platforms.
Novel electrochemical aptasensor toward cytochrome c (Cyt c) has been developed on the base of gl... more Novel electrochemical aptasensor toward cytochrome c (Cyt c) has been developed on the base of glassy carbon electrode (GCE) modified with electropolymerized neutral red (Poly-NR) and decacarboxylated pillar[5]arene (P[5]A-COOH) bearing terminal neutral red (NR) and aminated aptamer specific to Cyt c. Addition of Cyt c resulted in decrease of the cathodic peak current of NR on cyclic voltammogram due to suppression of the electron exchange between reduced and oxidized NR forms in the surface layer. The implementation of Cyt c in the surface layer was confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Depending on the content of the surface layer and assembling protocol, the limits of detection (LODs) varied from 0.02 to 1.0 nM and linear range of concentrations was within three orders of magnitude. Interfering influence of some proteins and polyethylene glycol was characterized. The aptasensors developed can find application in detection of Cyt c as apoptosis agent in blood serum. This has been partially validated in model blood serum mimicking the ionic composition of the plasma.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
We analyzed the possibility of the detection of cytochrome c (cyt c) being physically adsorbed on... more We analyzed the possibility of the detection of cytochrome c (cyt c) being physically adsorbed on lipid films or covalently bounded to 11-mercapto-1-undecanoic acid (MUA) chemisorbed on the gold layer using quartz crystal microbalance with dissipation monitoring (QCM-D). The negatively charged lipid film composed of a mixture of zwitterionic DMPC and negatively charged DMPG phospholipids at a molar ratio of 1:1 allowed the formation of a stable cyt c layer. Addition of DNA aptamers specific to cyt c, however, resulted in removal of cyt c from the surface. The interaction of cyt c with the lipid film and its removal by DNA aptamers were accompanied by changes in viscoelastic properties evaluated using the Kelvin–Voigt model. Cyt c covalently bound to MUA also provided a stable protein layer already at its relatively low concentrations (0.5 μM). A decrease in the resonant frequency following the addition of gold nanowires (AuNWs) modified by DNA aptamers was observed. The interaction ...
Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for t... more Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination of food. In this work, an aptasensor based on a thickness shear mode acoustic method (TSM) with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow’s milk. The frequency variation and dissipation data demonstrated the correct immobilization of the components. The analysis of viscoelastic properties suggests that DNA aptamers bind to the surface in a non-dense manner, which favors the binding with bacteria. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the sensor’s antifouling properties, which is based on 3-dithiothreitol propanoic acid (DTTCOOH) antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-m...
The 2nd International Electronic Conference on Biosensors, Feb 14, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Quartz crystal microbalance (QCM) biosensor for detection plasmin (PLA) in milk using β-casein as... more Quartz crystal microbalance (QCM) biosensor for detection plasmin (PLA) in milk using β-casein as a substrate immobilized at piezocrystal surface was reported. The cleavage of β-casein induced by PLA within the concentration range 0.1-40 nM resulted in an increase of series resonant frequency, f s , and a decrease of motional resistance, R m , due to removal of short peptide fragments from the crystal surface and decrease of viscosity contribution, respectively. The sensor was verified in both raw and spiked milk samples of various origins (cow, goat, sheep). The sensor response has also been studied for trypsin and α-chymotrypsin activities. Sensor technology achieved a limit of PLA detection (LOD) 167.16 ± 39.36 pM at pH 7.4. Similar LOD, 121.98 ± 18.30 pM, was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) test performed in-parallel with the biosensor measurements. Atomic force microscopy (AFM) confirmed cleavage effect of PLA on the surface covered by β-casein.
Calmodulin (CaM) interactions with bilayer lipid membranes (BLM) were studied by measuring modulu... more Calmodulin (CaM) interactions with bilayer lipid membranes (BLM) were studied by measuring modulus of elasticity in direction perpendicular to the membrane plane (E perpendicular) and intramembrane potential delta psi. Upon interaction of CaM with egg phosphatidylcholine and asolectin BLM the parameter E perpendicular grew slightly (not more than by 10% as compared to the respective vale for nonmodified BLM), suggesting a weak effect on the ordering of the hydrophobic moiety of the lipid bilayer. In the presence of mesocaine (Mes), a calmodulin inhibitor, CaM affected the incorporation of Mes into the membrane. It can be concluded that CaM affects the ordering of the polar (superficial) membrane region.
The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties ... more The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties of liposomes and planar bilayer lipid membranes (BLM) were studied. The method of fluorescence spectroscopy and the electrostriction method based on measurement of higher current harmonics were used. At low concentrations (10(-5)-5 x 10(-5) mol/l), 9AA increased fluorescence intensity, while in liposomes from soybean phosphatidylcholine fluorescence quenching occurred at higher probe concentration. Fluorescence quenching occurred over the entire concentration range tested (10(-5)-10(-4) mol/l) in liposomes made from a mixture of egg phosphatidylcholine and cardiolipin. In contrast to 9AA, AT, thanks to its hydrophobic chain, penetrates deeper into the hydrophobic membrane moiety; thus, immobilization of the molecule and an increase in fluorescence intensity was always observed. Probes adsorbed to membranes, leaving their electric capacitance effectively unchanged. Adsorption of charged d...
Plasmin protease plays an important role in many processes in living systems, including milk. Mon... more Plasmin protease plays an important role in many processes in living systems, including milk. Monitoring plasmin activity is important for control of the nutritional quality of milk and other dairy products. We designed a biosensor to detect the proteolytic activity of plasmin, using multiharmonic quartz crystal microbalance with dissipation (QCM-D). The β-casein immobilized on the hydrophobic surface of 1-dodecanethiol on the AT-cut quartz crystal was used to monitor plasmin activity. We demonstrated detection of plasmin in a concentration range of 0.1–20 nM, with the limit of detection about 0.13 ± 0.01 nM. The analysis of viscoelastic properties of the β-casein layer showed rapid changes of shear elasticity modulus, μ, and coefficient of viscosity, η, at plasmin sub-nanomolar concentrations, followed by modest changes at nanomolar concentrations, indicating multilayer architecture β-casein. A comparative analysis of viscoelastic properties of β-casein layers following plasmin and...
Spectrally-and time-resolved microscopy study of the interaction of microplastics with Chlorella ... more Spectrally-and time-resolved microscopy study of the interaction of microplastics with Chlorella sp. algae
Milk is a significant foodstuff around the world, being produced and consumed in large quantities... more Milk is a significant foodstuff around the world, being produced and consumed in large quantities. The safe consumption of milk requires that the liquid has an acceptably low level of microbial contamination and has not been subjected to spoiling. Bacterial safety limits in milk vary by country but are typically in the thousands per mL of sample. To rapidly determine if samples contain an unsafe level of bacteria, an aptamer-based sensor specific to Escherichia coli bacteria was developed. The sensor is based on an ultra-high frequency electromagnetic piezoelectric acoustic sensor device (EMPAS), with the aptamer being covalently bound to the sensor surface by the anti-fouling linker, MEG-Cl. The sensor is capable of the selective measurement of E. coli in PBS and in cow’s milk samples down to limits of detection of 35 and 8 CFU/mL, respectively, which is well below the safe limits for commercial milk products. This sensing system shows great promise for the milk industry for the pu...
Mycotoxins are secondary metabolites of fungi that contaminate agriculture products. Their releas... more Mycotoxins are secondary metabolites of fungi that contaminate agriculture products. Their release in the environment can cause severe damage to human health. Aptasensors are compact analytical devices that are intended for the fast and reliable detection of various species able to specifically interact with aptamers attached to the transducer surface. In this review, assembly of electrochemical and surface plasmon resonance (SPR) aptasensors are considered with emphasis on the mechanism of signal generation. Moreover, the properties of mycotoxins and the aptamers selected for their recognition are briefly considered. The analytical performance of biosensors developed within last three years makes it possible to determine mycotoxin residues in water and agriculture/food products on the levels below their maximal admissible concentrations. Requirements for the development of sample treatment and future trends in aptasensors are also discussed.
An impedimetric aptasensor for ochratoxin A (OTA) detection has been developed on the base of a g... more An impedimetric aptasensor for ochratoxin A (OTA) detection has been developed on the base of a gold electrode covered with a new modifier consisting of electropolymerized Neutral Red and a mixture of Au nanoparticles suspended in the dendrimeric polymer Botlorn H30 ®. Thiolated aptamer specific to OTA was covalently attached to Au nanoparticles via Au-S bonding. The interaction of the aptamer with OTA induced the conformational switch of the aptamer from linear to guanine quadruplex form followed by consolidation of the surface layer and an increase of the charge transfer resistance. The aptasensor makes it possible to detect from 0.1 to 100 nM of OTA (limit of detection: 0.02 nM) in the presence of at least 50 fold excess of ochratoxin B. The applicability of the aptasensor for real sample assay was confirmed by testing spiked beer samples. The recovery of 2 nM OTA was found to be 70% for light beer and 78% for dark beer.
A highly sensitive electrochemical DNA sensor for detection of the chemotherapeutic drug idarubic... more A highly sensitive electrochemical DNA sensor for detection of the chemotherapeutic drug idarubicin mediated by Methylene blue (MB) has been developed. DNA from fish sperm has been immobilized at the electropolymerized layers of Azure B. The incorporation of MB into the DNA layers substantially increased the sensor sensitivity. The concentration range for idarubicin determination by cyclic voltammetry was from 1 fM to 0.1 nM, with a limit of detection (LOD) of 0.3 fM. Electrochemical impedance spectroscopy (EIS) in the presence of a redox probe ([Fe(CN)6]3−/4−) allowed for the widening of a linear range of idarubicin detection from 1 fM to 100 nM, retaining LOD 0.3 fM. The DNA sensor has been tested in various real and artificial biological fluids with good recovery ranging between 90–110%. The sensor has been successfully used for impedimetric idarubicin detection in medical preparation Zavedos®. The developed DNA biosensor could be useful for the control of the level of idarubicin...
Proceedings of The 1st International Electronic Conference on Biosensors, 2020
Proteases play an important role in various biological as well as dairy industrial processes. In ... more Proteases play an important role in various biological as well as dairy industrial processes. In this work, we focused on chymotrypsin, which is an important protease in human digestion. Chymotrypsin also cleaves milk proteins, which makes it useful in the study of activity of milk proteases. In this work, we focused on detection of chymotrypsin using optical and gravimetric techniques, their peculiarities, and the limit of detection (LOD) (0.15 ± 0.005 nM and 0.64 ± 0.31 nM for optical and gravimetric methods, respectively). While the optical detection is easier to implement, the gravimetric method is more robust for sample preparation. Both methods could represent a novel assay for detection of chymotrypsin and other milk proteases.
Proceedings of The 1st International Electronic Conference on Biosensors, 2020
The determination of protease activity is very important for disease diagnosis, drug development,... more The determination of protease activity is very important for disease diagnosis, drug development, and quality and safety assurance for dairy products. Therefore, the development of low-cost methods for assessing protease activity is critical. Here, we demonstrate that an acoustic wave-based biosensor operated in the thickness-shear mode (TSM) enables the low-cost detection of protease activity in real time. The TSM sensor was based on a protein substrate (PS) β-casein immobilized on a piezoelectric quartz crystal electrode. The β-casein layer was immobilized onto a gold surface by a carboxylate terminated self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA). The carboxylic acid terminal was activated by the reaction of a mixture of watersoluble N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) on the electrode surfaces. We demonstrated that β-casein can form a stable assembly on a piezoelectric quartz crystal electrode. After an enzymatic reaction with trypsin, it cleaved the surface-bound β-casein substrate, which increased the frequency of the crystal in a sigmoidal manner. Trypsin was detected in the range of concentrations from 1 to 50 nM. The limit of detection was 0.2 nM. Initial reaction rates measured at different enzyme concentrations have been used to construct a calibration curve. Considering the results obtained, we believe that the TSM biosensor is a useful tool for protease analysis.
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