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(searched for: doi:10.4236/ojab.2014.31001)
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Published: 9 January 2021
by MDPI
Abstract:
Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme’s secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications—particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning.
Suman Nayak, Paresh Kale,
International Journal of Environmental Analytical Chemistry pp 1-13; https://doi.org/10.1080/03067319.2020.1776864

Abstract:
In this study, individual and combinations of heavy metals were tested at different pH for their inhibition potential by horseradish peroxidase (HRP). At pH of 6.4, maximum velocities for a single substrate concentration of hydrogen peroxide was observed. The double reciprocal of Lineweaver-Burk plot gave the same values of Km and decreased Vmax represents the non-competitive type of inhibition by hexavalent chromium metal ions. The IC10 value of Cr6+ ion for free HRP was achieved at 0.55ppm, while inhibition for 1ppm of substrate concentration was 24.5%. For other tested heavy metals, no significant inhibition properties were observed except for Cd2+ and Mg2+ metal ions. However, when combined with Cr6+ ions, substantial changes were detected. Based on IC10 values, the sensitivity order for free HRP for individual metal ions was observed as Cd2+ > Cr6+ > Mg2+ > Cu2+ > Cr3+ > Mn2+ while the combination with Cr6+ gave the order as Cd2+ > Cu2+ > Cr3+ > Cr6+ > Mg2+ > Mn2+. The order of inhibition byquaternary combinations of metal ion were in the order (Cr6+ + Cd2+ + Cu2+ + Cr3+) > (Cr6+ + Cu2+ + Mg2+ + Cd 2+) ≥ (Cr6+ + Cd2+ + Mg2+ + Cr3+) ≥ (Cr6+ + Cu2+ + Mg2+ + Cr3+), while the quinary combination (Cr6+ + Cr3+ + Cu2+ + Mg2+ + Cd2+) was the most effective grouping. The studies shown excellent selectivity towards Cr6+ and suggest further use of HRP enzymes in the design and selection of biosensors for heavy metals and other pollutants.
Wan Mohd Ebtisyam Mustaqim Mohd Daniyal, , Nur Ain Asyiqin Anas, Nur Alia Sheh Omar, Nur Syahira Ramdzan, , Mohd Adzir Mahdi
Published: 17 December 2019
RSC Advances, Volume 9, pp 41729-41736; https://doi.org/10.1039/c9ra07368j

Abstract:
This work focus on sensitivity enhancement of surface plasmon resonance (SPR) optical sensor by modifying the gold thin film with nanocrystalline cellulose (NCC) based material for zinc ion (Zn2+) detection.
Eloy Povedano, Víctor Ruiz-Valdepeñas Montiel, , Verónica Serafín, María Pedrero, Ludmila Moranova, , Juan José Montoya, Paloma Yáñez-Sedeño, , et al.
Analytical and Bioanalytical Chemistry, Volume 412, pp 5031-5041; https://doi.org/10.1007/s00216-019-02219-w

Abstract:
This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin–horseradish peroxidase (Strep–HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (− 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA–RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP’s non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.
Published: 1 December 2016
Biosensors and Bioelectronics, Volume 86, pp 466-471; https://doi.org/10.1016/j.bios.2016.06.088

The publisher has not yet granted permission to display this abstract.
Joseph G. Ayenimo,
Published: 1 February 2016
Talanta, Volume 148, pp 502-510; https://doi.org/10.1016/j.talanta.2015.11.024

Abstract:
A sensitive and reliable inhibitive amperometric glucose biosensor is described for rapid trace metal determination. The biosensor utilises a conductive ultrathin (55 nm thick) polypyrrole (PPy) film for entrapment of glucose oxidase (GOx) to permit rapid inhibition of GOx activity in the ultrathin film upon exposure to trace metals, resulting in reduced glucose amperometric response. The biosensor demonstrates a relatively fast response time of 20 s and does not require incubation. Furthermore, a complete recovery of GOx activity in the ultrathin PPy-GOx biosensor is quickly achieved by washing in 2 mM EDTA for only 10 s. The minimum detectable concentrations achieved with the biosensor for Hg2+, Cu2+, Pb2+ and Cd2+ by inhibitive amperometric detection are 0.48, 1.5, 1.6 and 4.0 µM, respectively. Also, suitable linear concentration ranges were achieved from 0.48–3.3 µM for Hg2+, 1.5–10 µM for Cu2+, 1.6–7.7 µM for Pb2+ and 4–26 µM for Cd2+. The use of Dixon and Cornish-Bowden plots revealed that the suppressive effects observed with Hg2+ and Cu2+ were via non-competitive inhibition, while those of Pb2+ and Cd2+ were due to mixed and competitive inhibition. The stronger inhibition exhibited by the trace metals on GOx activity in the ultrathin PPy-GOx film was also confirmed by the low inhibition constant obtained from this analysis. The biosensor was successfully applied to the determination of trace metals in tap water samples.
Nawal Khedimallah, , Amel Sbartai,
IEEE Transactions on NanoBioscience, Volume 14, pp 694-699; https://doi.org/10.1109/tnb.2015.2461444

Abstract:
A new poly(vinylchloride) (PVC) membrane electrode based on tannin from the bark of Quercusmacrolepis (acorn) as the ionophore was prepared and modified onto the surface of a gold electrode. The electrochemical impedance spectroscopy (EIS) technique was used to study the sensitivity of the electrode that was modified with a thin layer of polymeric biomembrane, in order to detect heavy metals ions in solution. The device shows a good sensitivity for Zn 2+ , Ni 2+ ions and a little less for Cd 2+ . The electrode indicates a good linear response for the three metals over a wide concentration range from 1.0×10 -9 to 1.0×10 -4 M, with a detection limit of 1.0×10 -9 M.
Mahmoud S. Sharaf, Michael R. Van Den Heuvel, Don Stevens,
Free Radical Biology and Medicine, Volume 84, pp 142-153; https://doi.org/10.1016/j.freeradbiomed.2015.03.017

Abstract:
Zinc and calcium have highly interwoven functions that are essential for cellular homeostasis. Here we first present a novel real-time flow cytometric technique to measure mitochondrial redox state and show it is modulated by zinc and calcium, individually and combined. We then assess the interactions of zinc and calcium on mitochondrial H2O2 production, membrane potential (ΔΨm), morphological status, oxidative phosphorylation (OXPHOS), complex I activity, and structural integrity. Whereas zinc at low doses and both cations at high doses individually and combined promoted H2O2 production, the two cations individually did not alter mitochondrial redox state. However, when combined at low and high doses the two cations synergistically suppressed and promoted, respectively, mitochondrial shift to a more oxidized state. Surprisingly, the antioxidants vitamin E and N-acetylcysteine showed pro-oxidant activity at low doses, whereas at high antioxidant doses NAC inhibited OXPHOS and dyscoupled mitochondria. Individually, zinc was more potent than calcium in inhibiting OXPHOS, whereas calcium more potently dissipated the ΔΨm and altered mitochondrial volume and ultrastructure. The two cations synergistically inhibited OXPHOS but antagonistically dissipated ΔΨm and altered mitochondrial volume and morphology. Overall, our study highlights the importance of zinc and calcium in mitochondrial redox regulation and functional integrity. Importantly, we uncovered previously unrecognized bidirectional interactions of zinc and calcium that reveal distinctive foci for modulating mitochondrial function in normal and disease states because they are potentially protective or damaging depending on conditions.
, Lehutso R. Florence, Jonathan O. Okonkwo
Published: 23 December 2014
Sensors and Actuators B: Chemical, Volume 209, pp 898-905; https://doi.org/10.1016/j.snb.2014.12.059

The publisher has not yet granted permission to display this abstract.
Published: 27 June 2014
by MDPI
Molecules, Volume 19, pp 8995-9018; https://doi.org/10.3390/molecules19078995

Abstract:
The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many properties of enzymes such as performance in organic solvents, pH tolerance, heat stability or the functional stability. Increasing the structural rigidity of the protein and stabilization of multimeric enzymes which prevents dissociation-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their commercial value. We also present our view on the role that different immobilization methods play in the reduction of enzyme inhibition during biotechnological processes.
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