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Talanta Aug 2024In this work we present the development of an electrochemiluminescence aptasensor based on electrografting molybdenum disulphide nanosheets functionalized with diazonium...
In this work we present the development of an electrochemiluminescence aptasensor based on electrografting molybdenum disulphide nanosheets functionalized with diazonium salt (MoS-N) upon screen-printed electrodes of graphene (SPEs GPH) for viral proteins detection. In brief, this aptasensor consists of SPEs GPH electrografted with MoS-N and modified with a thiolated aptamer, which can specifically recognize the target protein analyte. In this case, we have used SARS-CoV-2 spike protein as model protein. Electrochemiluminescence detection was performed by using the [Ru(bpy)]/TPRA (tripropylamine) system, which allows the specific detection of the SARS-CoV-2 spike protein easily and rapidly with a detection limit of 9.74 fg/mL and a linear range from 32.5 fg/mL to 50.0 pg/mL. Moreover, the applicability of the aptasensor has been confirmed by the detection of the protein directly in human saliva samples. Comparing our device with a traditional saliva antigen test, our aptasensor can detect the spike protein even when the saliva antigen test gives a negative result.
Topics: Graphite; Disulfides; Molybdenum; Aptamers, Nucleotide; Electrochemical Techniques; Biosensing Techniques; SARS-CoV-2; Humans; Luminescent Measurements; Spike Glycoprotein, Coronavirus; Limit of Detection; COVID-19; Electrodes; Saliva
PubMed: 38788383
DOI: 10.1016/j.talanta.2024.126293 -
Biosensors May 2024Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high...
Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms.
Topics: Biosensing Techniques; Nucleic Acid Amplification Techniques; Oligonucleotides; Streptococcus; Humans; DNA, Bacterial; Molecular Diagnostic Techniques
PubMed: 38785731
DOI: 10.3390/bios14050257 -
Biosensors May 2024Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease...
Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL, 0.77 ng∙μL, and 1.1 ng∙μL, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.
Topics: Fluorescence Resonance Energy Transfer; Aptamers, Nucleotide; Humans; Exosomes; Biosensing Techniques; Biomarkers; Nucleic Acid Amplification Techniques; Tetraspanin 30
PubMed: 38785707
DOI: 10.3390/bios14050233 -
Biosensors May 2024The development of rapid detection tools for viruses is vital for the prevention of pandemics and biothreats. Aptamers that target inactivated viruses are attractive for...
The development of rapid detection tools for viruses is vital for the prevention of pandemics and biothreats. Aptamers that target inactivated viruses are attractive for sensors due to their improved biosafety. Here, we evaluated a DNA aptamer (named as 6.9) that specifically binds to the inactivated SARS-CoV-2 virus with a low dissociation constant (K = 9.6 nM) for the first time. Based on aptamer 6.9, we developed a fiber-optic evanescent wave (FOEW) biosensor. Inactivated SARS-CoV-2 and the Cy5.5-tagged short complementary strand competitively bound with the aptamer immobilized on the surface of the sensor. The detection of the inactivated SARS-CoV-2 virus was realized within six minutes with a limit of detection (LOD, S/N = 3) of 740 fg/mL. We also developed an electrochemical impedance aptasensor which exhibited an LOD of 5.1 fg/mL and high specificity. We further demonstrated that the LODs of the FOEW and electrochemical impedance aptasensors were, respectively, more than 1000 and 100,000 times lower than those of commercial colloidal gold test strips. We foresee that the facile aptamer isolation process and sensor design can be easily extended for the detection of other inactivated viruses.
Topics: SARS-CoV-2; Aptamers, Nucleotide; Biosensing Techniques; Dielectric Spectroscopy; Limit of Detection; COVID-19; Humans; Fiber Optic Technology
PubMed: 38785705
DOI: 10.3390/bios14050231 -
Biosensors Apr 2024Graphene-based materials are actively being investigated as sensing elements for the detection of different analytes. Both graphene grown by chemical vapor deposition... (Comparative Study)
Comparative Study
Graphene-based materials are actively being investigated as sensing elements for the detection of different analytes. Both graphene grown by chemical vapor deposition (CVD) and graphene oxide (GO) produced by the modified Hummers' method are actively used in the development of biosensors. The production costs of CVD graphene- and GO-based sensors are similar; however, the question remains regarding the most efficient graphene-based material for the construction of point-of-care diagnostic devices. To this end, in this work, we compare CVD graphene aptasensors with the aptasensors based on reduced GO (rGO) for their capabilities in the detection of NT-proBNP, which serves as the gold standard biomarker for heart failure. Both types of aptasensors were developed using commercial gold interdigitated electrodes (IDEs) with either CVD graphene or GO formed on top as a channel of liquid-gated field-effect transistor (FET), yielding GFET and rGO-FET sensors, respectively. The functional properties of the two types of aptasensors were compared. Both demonstrate good dynamic range from 10 fg/mL to 100 pg/mL. The limit of detection for NT-proBNP in artificial saliva was 100 fg/mL and 1 pg/mL for rGO-FET- and GFET-based aptasensors, respectively. While CVD GFET demonstrates less variations in parameters, higher sensitivity was demonstrated by the rGO-FET due to its higher roughness and larger bandgap. The demonstrated low cost and scalability of technology for both types of graphene-based aptasensors may be applicable for the development of different graphene-based biosensors for rapid, stable, on-site, and highly sensitive detection of diverse biochemical markers.
Topics: Graphite; Natriuretic Peptide, Brain; Biosensing Techniques; Transistors, Electronic; Peptide Fragments; Humans; Limit of Detection; Gold; Aptamers, Nucleotide; Electrodes; Biomarkers
PubMed: 38785689
DOI: 10.3390/bios14050215 -
Biosensors Apr 2024The rapid and sensitive detection of pathogenic bacteria is becoming increasingly important for the timely prevention of contamination and the treatment of infections.... (Review)
Review
The rapid and sensitive detection of pathogenic bacteria is becoming increasingly important for the timely prevention of contamination and the treatment of infections. Biosensors based on nucleic acid aptamers, integrated with optical, electrochemical, and mass-sensitive analytical techniques, have garnered intense interest because of their versatility, cost-efficiency, and ability to exhibit high affinity and specificity in binding bacterial biomarkers, toxins, and whole cells. This review highlights the development of aptamers, their structural characterization, and the chemical modifications enabling optimized recognition properties and enhanced stability in complex biological matrices. Furthermore, recent examples of aptasensors for the detection of bacterial cells, biomarkers, and toxins are discussed. Finally, we explore the barriers to and discuss perspectives on the application of aptamer-based bacterial detection.
Topics: Biosensing Techniques; Aptamers, Nucleotide; Bacteria; Humans; Electrochemical Techniques; Biomarkers
PubMed: 38785684
DOI: 10.3390/bios14050210 -
The EMBO Journal Jul 2024During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises...
During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.
Topics: Animals; Ribosemonophosphates; Mice; Pentose Phosphate Pathway; eIF-2 Kinase; Macrophages; Aldose-Ketose Isomerases; RNA; Poly I-C; Nucleic Acids; Virus Replication; Phosphorylation
PubMed: 38778156
DOI: 10.1038/s44318-024-00100-w -
Development (Cambridge, England) May 2024In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal brain...
In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal brain infection, is associated with an increased risk of neuropsychiatric disorders in affected offspring. The cell types mediating the fetal brain response to maternal inflammation are largely unknown, hindering the development of novel treatment strategies. Here, we show that microglia, the resident phagocytes of the brain, highly express receptors for relevant pathogens and cytokines throughout embryonic development. Using a rodent maternal immune activation (MIA) model in which polyinosinic:polycytidylic acid is injected into pregnant mice, we demonstrate long-lasting transcriptional changes in fetal microglia that persist into postnatal life. We find that MIA induces widespread gene expression changes in neuronal and non-neuronal cells; importantly, these responses are abolished by selective genetic deletion of microglia, indicating that microglia are required for the transcriptional response of other cortical cell types to MIA. These findings demonstrate that microglia play a crucial durable role in the fetal response to maternal inflammation, and should be explored as potential therapeutic cell targets.
Topics: Animals; Microglia; Female; Pregnancy; Mice; Brain; Inflammation; Poly I-C; Fetus; Mice, Inbred C57BL; Gene Expression Regulation, Developmental; Neurons
PubMed: 38775708
DOI: 10.1242/dev.202252 -
Beilstein Journal of Organic Chemistry 2024Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine...
Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine ( = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.
PubMed: 38774272
DOI: 10.3762/bjoc.20.96 -
Scientific Reports May 2024Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed...
Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.
Topics: Toll-Like Receptor 9; Oligonucleotides, Antisense; Humans; CpG Islands; Animals; Mice; Nucleotides; Sugars; Oligodeoxyribonucleotides
PubMed: 38773176
DOI: 10.1038/s41598-024-61666-3