DEOXYRIBOZYMES IN DETECTION OF PATHOGENIC BACTERIA

Aim. The purpose of the review was to analyze the use of DNAzyme biosensors for the detection of pathogens. In the recent years, deoxyribozymes (DNAzymes) have a significant impact as biosensors in diverse fields, from detection of metal ions in the environment to theranostic applications and detection of microorganisms. Although routinely used sophisticated instrumental methods are available to detect pathogenic bacterial contamination, they involve time-consuming, complicated sample pre-treatment and expensive instruments. As an alternative, pathogen-specific DNAzymes have demonstrated a series of advantages: a non-destructive rapid analysis technique with in situ and real-time detection of bacteria with high sensitivity and selectivity. A wide range of pathogen-specific DNAzymes has been developed using colorimetric and fluorescence-based detections for pathogenic bacterial contamination in various samples. The current review summarizes the in vitro selection of pathogen-specific DNAzymes, various strategies utilized in the sensor designs, and their potential use in theranostic applications.

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Secondary Structure Libraries for Artificial Evolution Experiments

Molecules ◽

10.3390/molecules26061671 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1671

Author(s):

Ráchel Sgallová ◽

Edward A. Curtis

Keyword(s):

Secondary Structure ◽

Sequence Space ◽

In Vitro Selection ◽

Random Sequence ◽

Random Mutagenesis ◽

Nucleotide Composition ◽

Artificial Evolution ◽

Wide Range ◽

Range Of Functions

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.

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Aminoluciferin 4-hydroxyphenyl amide enables bioluminescence detection of endogenous tyrosinase

Organic & Biomolecular Chemistry ◽

10.1039/c8ob01777h ◽

2018 ◽

Vol 16 (47) ◽

pp. 9197-9203 ◽

Cited By ~ 2

Author(s):

Chunchao Tang ◽

Lei Jin ◽

Yuxing Lin ◽

Jing Su ◽

Yingai Sun ◽

...

Keyword(s):

High Sensitivity ◽

Living Cells ◽

Tyrosinase Activity ◽

Cell Permeability ◽

Sensitivity And Selectivity ◽

Good Cell

We report a new BL probe,TyrBP-3, which not only exhibits high sensitivity and selectivity for imaging tyrosinase in vitro, and good cell-permeability for detecting tyrosinase in living cells, but can also visualize the level of tyrosinase activity in tumors of living animals.

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Label-Free Split Aptamer Sensor for Femtomolar Detection of Dopamine by Means of Flexible Organic Electrochemical Transistors

Materials ◽

10.3390/ma13112577 ◽

2020 ◽

Vol 13 (11) ◽

pp. 2577 ◽

Cited By ~ 5

Author(s):

Yuanying Liang ◽

Ting Guo ◽

Lei Zhou ◽

Andreas Offenhäusser ◽

Dirk Mayer

Keyword(s):

Detection Limit ◽

Electrochemical Sensors ◽

Limit Of Detection ◽

High Sensitivity ◽

Label Free ◽

Dopamine Detection ◽

Electrochemical Transistor ◽

Sensitivity And Selectivity

The detection of chemical messenger molecules, such as neurotransmitters in nervous systems, demands high sensitivity to measure small variations, selectivity to eliminate interferences from analogues, and compliant devices to be minimally invasive to soft tissue. Here, an organic electrochemical transistor (OECT) embedded in a flexible polyimide substrate is utilized as transducer to realize a highly sensitive dopamine aptasensor. A split aptamer is tethered to a gold gate electrode and the analyte binding can be detected optionally either via an amperometric or a potentiometric transducer principle. The amperometric sensor can detect dopamine with a limit of detection of 1 μM, while the novel flexible OECT-based biosensor exhibits an ultralow detection limit down to the concentration of 0.5 fM, which is lower than all previously reported electrochemical sensors for dopamine detection. The low detection limit can be attributed to the intrinsic amplification properties of OECTs. Furthermore, a significant response to dopamine inputs among interfering analogues hallmarks the selective detection capabilities of this sensor. The high sensitivity and selectivity, as well as the flexible properties of the OECT-based aptasensor, are promising features for their integration in neuronal probes for the in vitro or in vivo detection of neurochemical signals.

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Cancer-Specific Biomarker hNQO1-Activatable Fluorescent Probe for Imaging Cancer Cells In Vitro and In Vivo

Cancers ◽

10.3390/cancers10120470 ◽

2018 ◽

Vol 10 (12) ◽

pp. 470 ◽

Cited By ~ 8

Author(s):

Surendra Punganuru ◽

Hanumantha Madala ◽

Viswanath Arutla ◽

Kalkunte Srivenugopal

Keyword(s):

Cancer Cells ◽

Fluorescent Probe ◽

Stokes Shift ◽

High Sensitivity ◽

Cell Permeability ◽

Quinone Oxidoreductase ◽

Brain Tumor Cells ◽

Sensitivity And Selectivity

Human NAD(P)H quinone oxidoreductase-1 (hNQO1) is an important cancer-related biomarker, which shows significant overexpression in malignant cells. Developing an effective method for detecting NQO1 activity with high sensitivity and selectivity in tumors holds a great potential for cancer diagnosis, treatment, and management. In the present study, we report a new dicyanoisophorone (DCP) based fluorescent probe (NQ-DCP) capable of monitoring hNQO1 activity in vitro and in vivo in both ratiometric and turn-on model. NQ-DCP was prepared by conjugating dicyanoisophorone fluoroprobe with hNQO1 activatable quinone propionic acid (QPA), which remain non-fluorescent until activation by tumor-specific hNQO1. NQ-DCP featured a large Stokes shift (145 nm), excellent biocompatibility, cell permeability, and selectivity towards hNQO1 allowed to differentiate cancer cells from healthy cells. We have successfully employed NQ-DCP to monitor non-invasive endogenous hNQO1 activity in brain tumor cells in vitro and in xenografted tumors developed in nude mice.

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Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro

Australian Journal of Experimental Agriculture ◽

10.1071/ea02199 ◽

2004 ◽

Vol 44 (1) ◽

pp. 27 ◽

Cited By ~ 16

Author(s):

M. Bajji ◽

P. Bertin ◽

S. Lutts ◽

J-M. Kinet

Keyword(s):

Durum Wheat ◽

Somaclonal Variation ◽

Drought Resistance ◽

In Vitro Selection ◽

Culture Media ◽

Field Studies ◽

Resistance Mechanisms ◽

Wide Range ◽

Triticum Durum Desf

Somaclonal variation associated with in vitro selection has been used as a source of variability to improve drought resistance of 3 durum wheat (Triticum durum Desf.) cultivars (Selbera, Sebou, and Kyperounda). In a previous study, R0 plants with improved drought resistance-related characters were regenerated after selection on culture media containing polyethylene glycol (PEG). This improvement was transmitted to the R1 progeny. The present study analysed the behaviour of the selected tissue culture-derived lines in subsequent R2, R3 and R4�generations. Differences in electrolyte leakage, chlorophyll fluorescence (Fv/Fm), stomatal conductance and days to heading were found between the parental cultivars and most of their in vitro-derived lines. The changes may differ from one cultivar to another. Many promising somaclonal lines still presented improvement for at least 3 of the 4�parameters measured comparatively to initial cultivars. Somaclonal variation thus appears to induce a wide range of modifications among individual components of drought-resistance mechanisms. These improved traits could be valuable if shown to be inherited and to give enhanced agronomic performances in future field studies.

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Brewing and Probiotic Potential Activity of Wild Yeasts Hanseniaspora Uvarum PIT001, Pichia Kluyveri LAR001 and Candida Intermedia ORQ001

10.21203/rs.3.rs-772136/v1 ◽

2021 ◽

Author(s):

Renan Eugênio Araujo Piraine ◽

Gustavo M Retzlaf ◽

Vitória S. Gonçalves ◽

Rodrigo C Cunha ◽

Fabio Pereira Leivas Leite

Keyword(s):

Pathogenic Bacteria ◽

Inhibitory Effect ◽

Starter Cultures ◽

Mixed Fermentation ◽

Candida Intermedia ◽

Probiotic Potential ◽

Hanseniaspora Uvarum ◽

Wide Range ◽

Pichia Kluyveri

Abstract Non-conventional yeasts can be isolated from a wide range of environmental sources, often found in beverage industry in mixed fermentations, in which the microorganisms’ inoculum usually is not fully known. It is important to know starter cultures, since in addition to favoring reproducibility, other properties can be discovered. Thus, the objective of this work was to identify and characterize yeasts isolated from environment, evaluating their probiotic potential and possible use in brewery. Isolates were obtained from flowers, fruits, leaves and mixed-fermentation beers, being identified by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and presence of organic acids. To explore probiotic potential, in vitro tests were performed of antimicrobial activity and co-aggregation with food pathogens, auto-aggregation, and survival in simulated gastrointestinal tract conditions. In our study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates. P. kluyveri was the only one that tolerated pH 2.5. Lactic acid was not inhibitory, while acetic acid and incubation at 37 °C had a partially inhibitory effect on yeasts growth. All yeasts tolerated α-acids from hops and NaCl up to 1%. It is suggested that isolates are able to adhere to intestinal cells and influence positively the organism in combating pathogens, as they showed auto-aggregation rates above 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, however were more sensitive to pancreatic conditions. We conclude that isolated non-conventional yeasts showed probiotic potential and promising application in beer fermentation.

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Nanomaterials-Based Electrochemical Sensors for In Vitro and In Vivo Analyses of Neurotransmitters

Applied Sciences ◽

10.3390/app8091504 ◽

2018 ◽

Vol 8 (9) ◽

pp. 1504 ◽

Cited By ~ 17

Author(s):

Sharmila Durairaj ◽

Boopathi Sidhureddy ◽

Joseph Cirone ◽

Aicheng Chen

Keyword(s):

Electrochemical Sensors ◽

Point Of Care ◽

Electrochemical Techniques ◽

High Sensitivity ◽

Electrochemical Analysis ◽

Detection Techniques ◽

Efficient Detection ◽

Sensitivity And Selectivity

Neurotransmitters are molecules that transfer chemical signals between neurons to convey messages for any action conducted by the nervous system. All neurotransmitters are medically important; the detection and analysis of these molecules play vital roles in the diagnosis and treatment of diseases. Among analytical strategies, electrochemical techniques have been identified as simple, inexpensive, and less time-consuming processes. Electrochemical analysis is based on the redox behaviors of neurotransmitters, as well as their metabolites. A variety of electrochemical techniques are available for the detection of biomolecules. However, the development of a sensing platform with high sensitivity and selectivity is challenging, and it has been found to be a bottleneck step in the analysis of neurotransmitters. Nanomaterials-based sensor platforms are fascinating for researchers because of their ability to perform the electrochemical analysis of neurotransmitters due to their improved detection efficacy, and they have been widely reported on for their sensitive detection of epinephrine, dopamine, serotonin, glutamate, acetylcholine, nitric oxide, and purines. The advancement of electroanalytical technologies and the innovation of functional nanomaterials have been assisting greatly in in vivo and in vitro analyses of neurotransmitters, especially for point-of-care clinical applications. In this review, firstly, we focus on the most commonly employed electrochemical analysis techniques, in conjunction with their working principles and abilities for the detection of neurotransmitters. Subsequently, we concentrate on the fabrication and development of nanomaterials-based electrochemical sensors and their advantages over other detection techniques. Finally, we address the challenges and the future outlook in the development of electrochemical sensors for the efficient detection of neurotransmitters.

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Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes

Epidemiology and Infection ◽

10.1017/s0950268813002677 ◽

2013 ◽

Vol 142 (8) ◽

pp. 1671-1677 ◽

Cited By ~ 13

Author(s):

M. KARANI ◽

I. SOTIRIADOU ◽

J. PLUTZER ◽

P. KARANIS

Keyword(s):

Lamp Assay ◽

High Sensitivity ◽

Leishmania Species ◽

18S Rrna Gene ◽

Isothermal Amplification ◽

Rrna Gene ◽

Bench Scale ◽

Loop Mediated Isothermal Amplification ◽

Wide Range

SUMMARYWe developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

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Microwave Based Pretreatment for Efficient Extraction of Biologically Active Compounds from Fucus vesiculosus

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.850.190 ◽

2020 ◽

Vol 850 ◽

pp. 190-195

Author(s):

Sarmīte Janceva ◽

Aleksandr Arshanica ◽

Līga Lauberte ◽

Anna Andersone ◽

Lilija Jashina ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Reference Sample ◽

Microwave Treatment ◽

Biologically Active ◽

Target Compound ◽

Treatment Efficiency ◽

Pea Seedlings ◽

Wide Range ◽

Efficient Extraction ◽

Pre Treatment

Fucus V e s i culosus (F. Vesiculosus ) by its composition is a unique raw for obtaining of variety substances with a wide range of consumer properties. The composition of the polyphenolic fraction of F.Vesiculosus is characterized by a predominant content of phloroglucinol polymers - phlorotannins, which are the target compound of this study. Microwave assisted pre-treatments of algae were carried out in order to intensify the extraction of target compound. The pre-treatment efficiency was evaluated by determining the yield of extractives, polyphenols and phlorotannins. The chemical composition of the polyphenols fraction of algae was studied. The obtained results show that microwave treatment of F.Vesiculosus allows to increase 1.6 times the yield of target compound with lower energy consumption compared to the reference sample. The obtaining extracts obtained have a high antibacterial effect on gram-positive and gram-negative bacteria, and also stimulate the growth of pea seedlings and can be recommended for use in agriculture as a growth stimulator in preparing seeds for sowing and also as an antibacterial agent, protecting as seeds and seedlings from pathogenic bacteria.

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Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

Toxins ◽

10.3390/toxins13010018 ◽

2020 ◽

Vol 13 (1) ◽

pp. 18

Author(s):

Janne Leivo ◽

Markus Vehniäinen ◽

Urpo Lamminmäki

Keyword(s):

In Vitro Selection ◽

Specific Binding ◽

Binding Properties ◽

Phage Displays ◽

Synthetic Antibody ◽

Wide Range ◽

Antibody Libraries ◽

Rapid Generation ◽

Selection Of

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

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