Screen printing is widely used to fabricate disposable and economical electrochemical sensors and has helped us to establish the route from 'lab-to-market' for a plethora of sensors. We overview ...recent developments in the field where screen printed electrochemical sensors are utilised. Starting with their fundamental understanding, through to highlighting new developments in bulk metal and mediator modified electrodes, as well as novel advantageous electrode designs, we demonstrate the wide and diverse range of applications that sensors based on this fabrication approach have achieved.
We present a simplified approach for the trace screening of toxic heavy metals utilizing bismuth oxide screen printed electrodes. The use of bismuth oxide instead of toxic mercury films facilitates ...the reliable sensing of lead(II), cadmium(II) and zinc(II). A linear range over 5 to 150 μg L−1 with detection limits of 2.5 and 5 μg L−1 are readily observed for cadmium and lead in 0.1 M HCl, respectively. Conducting a simultaneous multi‐elemental voltammetric detection of zinc, cadmium and lead in a higher pH medium (0.1 M sodium acetate solution) exhibited a linear range between 10 and 150 μg L−1 with detection limits of 5, 10 and 30 μg L−1 for cadmium, lead and zinc respectively. The sensor is greatly simplified over those recently reported such as bismuth nanoparticle modified electrodes and bismuth film coated screen printed electrodes. The scope of applications of this sensor with the inherent advances in electroanalysis coupled with the negliable toxicity of bismuth is extensive allowing high throughput electroanalysis.
Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and ...accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.
Non-membrane compartments or biomolecular condensates play an important role in the regulation of cellular processes including DNA repair. Here, an ability of XRCC1, a scaffold protein involved in ...DNA base excision repair (BER) and single-strand break repair, to form protein-rich microphases in the presence of DNA duplexes was discovered. We also showed that the gap-filling activity of BER-related DNA polymerase λ (Pol λ) is significantly increased by the presence of XRCC1. The stimulation of the Pol λ activity was observed only at micromolar XRCC1 concentrations, which were well above the nanomolar dissociation constant determined for the XRCC1–Pol λ complex and pointed to the presence of an auxiliary stimulatory factor in addition to protein–protein interactions. Indeed, according to dynamic light scattering measurements, the stimulation of the Pol λ activity by XRCC1 was coupled with microphase separation in a protein–DNA mixture. Fluorescence microscopy revealed colocalization of Pol λ, XRCC1, and gapped DNA within the microphases. Thus, stimulation of Pol λ activity is caused both by its interaction with XRCC1 and by specific conditions of microphase separation; this phenomenon is shown for the first time.
PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- ...and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.
Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to ...self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS–nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS’s affinity for a nucleic acid polymer. Some proteins—which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates—disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.
A bulk-modified screen-printed carbon electrode characterised for metal ion detection is presented. Bismuth oxide (Bi
2O
3) was mixed with graphite-carbon ink to obtain the modified electrode. The ...best composition was 2% Bi
2O
3 (wt%) in the graphite-carbon ink. The modified electrode with onboard screen-printed carbon counter and silver–silver chloride pseudo-reference electrodes exhibited good performance in the electrochemical measurement of lead (II) and cadmium (II). The electrode displayed excellent linear behaviour in the concentration range examined (20–300
μg
L
−1) with limits of detection of 8 and 16
μg
L
−1 for both lead (II) and cadmium (II), respectively. The analytical utility of the modified electrode was illustrated by the stripping chronopotentiometric determinations of lead (II) in soil extracts and wastewater samples.
Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and ...accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg2+ coordinated bonds with PAR polymers.
•Most proteins involved in BER self-associate via homodimerization.•Complexes of PARP1 with various BER proteins are heterotetramers.•The oligomerization state of XRCC1 heterocomplexes depends on the partner.•PARP1 forms huge conglomerates upon the autoPARylation stabilized by magnesium ions.
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