Redox-Type Label-Free ATP Image Sensor for Highly Sensitive in Vitro Imaging of Extracellular ATP

2021 ◽  
Author(s):  
Hideo Doi ◽  
Tomoko Horio ◽  
Young-Joon Choi ◽  
Kazuhiro Takahashi ◽  
Toshihiko Noda ◽  
...  
Keyword(s):  
Image Sensor ◽  
Extracellular Atp ◽  
Label Free ◽  
Highly Sensitive

scholarly journals CMOS-Based Redox-Type Label-Free ATP Image Sensor for In Vitro Sensitive Imaging of Extracellular ATP

Sensors ◽  
2021 ◽  
Vol 22 (1) ◽  
pp. 75
Author(s):  
Hideo Doi ◽  
Tomoko Horio ◽  
Yong-Joon Choi ◽  
Kazuhiro Takahashi ◽  
Toshihiko Noda ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Spatiotemporal Analysis ◽  
Image Sensor ◽  
Extracellular Atp ◽  
Nerve Tissue ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Electron Mediator ◽  
Extracellular Signaling

Adenosine 5′-triphosphate (ATP) plays a crucial role as an extracellular signaling molecule in the central nervous system and is closely related to various nerve diseases. Therefore, label-free imaging of extracellular ATP dynamics and spatiotemporal analysis is crucial for understanding brain function. To decrease the limit of detection (LOD) of imaging extracellular ATP, we fabricated a redox-type label-free ATP image sensor by immobilizing glycerol-kinase (GK), L-α-glycerophosphate oxidase (LGOx), and horseradish peroxidase (HRP) enzymes in a polymer film on a gold electrode-modified potentiometric sensor array with a 37.3 µm-pitch. Hydrogen peroxide (H2O2) is generated through the enzymatic reactions from GK to LGOx in the presence of ATP and glycerol, and ATP can be detected as changes in its concentration using an electron mediator. Using this approach, the LOD for ATP was 2.8 µM with a sensitivity of 77 ± 3.8 mV/dec., under 10 mM working buffers at physiological pH, such as in in vitro experiments, and the LOD was great superior 100 times than that of the hydrogen ion detection-based image sensor. This redox-type ATP image sensor may be successfully applied for in vitro sensitive imaging of extracellular ATP dynamics in brain nerve tissue or cells.

scholarly journals Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

2018 ◽  
Author(s):  
Mohammed Mahamdeh ◽  
Steve Simmert ◽  
Anna Luchniak ◽  
Erik Schäeffer ◽  
Jonathon Howard
Keyword(s):  
High Speed ◽  
Signal To Noise Ratio ◽  
Dark Field ◽  
Fluorescent Labeling ◽  
Wide Field ◽  
Differential Interference Contrast ◽  
Label Free ◽  
High Speed Imaging ◽  
Highly Sensitive

SummaryWhen studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

scholarly journals Computational Design of a Molecularly Imprinted Polymer for the Biomonitoring of the Organophosphorous Metabolite Chlorferron

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 192
Author(s):  
Bakhtiyar Qader ◽  
Issam Hussain ◽  
Mark Baron ◽  
Rebeca Jiménez-Pérez ◽  
Guzmán Gil-Ramírez ◽  
...  
Keyword(s):  
Biological Samples ◽  
Limit Of Detection ◽  
Computational Design ◽  
Signal Change ◽  
Highly Sensitive ◽  
Limit Of Quantitation ◽  
Molecularly Imprinting Polymers ◽  
Mip Sensor ◽  
Parasitic Mites

Coumaphos is an organophosphorus compound used as insecticide and frequently used by beekeepers for the management of parasitic mites. The most important metabolite, chlorferron (CFN), has been identified in biological samples and foodstuff. The need to quickly identify the presence of typical metabolites, as an indication of interaction with coumaphos has driven the need to produce a highly sensitive electrochemical method for chlorferron analysis, based on molecularly imprinting polymers (MIP) technology. It showed irreversible behaviour with mixed diffusion/adsorption-controlled reactions at the electrode surface. A monoelectronic mechanism of reaction for oxidation has also been suggested. The linear range observed was from 0.158 to 75 µM. Median precision in terms of %RSD around 3% was also observed. For DPV, the limit of detection (LOD) and the limit of quantitation (LOQ) for the CFN-MIP were 0.158 µM and 0.48 µM, respectively. The obtained median % recovery was around 98%. The results were also validated to reference values obtained using GC-MS. Urine and human synthetic plasma spiked with CFN were used to demonstrate the usability of the method in biological samples, showing the potential for biomonitoring. The developed imprinted sensor showed maximum signal change less than 16.8% when related metabolites or pesticide were added to the mix, suggesting high selectivity of the MIP sensor toward CFN molecules. The results from in vitro metabolism of CMP analysed also demonstrates the potential for detection and quantification of CFN in environmental samples. The newly developed CFN-MIP sensor offers similar LoDs than chromatographic methods with shorter analysis time.

scholarly journals Optical Monitoring of the Production Quality of Si-Nanoribbon Chips Intended for the Detection of ASD-Associated Oligonucleotides

Micromachines ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 147
Author(s):  
Kristina A. Malsagova ◽  
Tatyana O. Pleshakova ◽  
Vladimir P. Popov ◽  
Igor N. Kupriyanov ◽  
Rafael A. Galiullin ◽  
...  
Keyword(s):  
Production Quality ◽  
Autism Spectrum ◽  
Covalent Immobilization ◽  
Biological Macromolecules ◽  
Label Free ◽  
Raman Scattering Spectroscopy ◽  
Dna Oligonucleotides ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Gas-phase etching and optical lithography were employed for the fabrication of a silicon nanoribbon chip (Si-NR chip). The quality of the so-fabricated silicon nanoribbons (Si-NRs) was monitored by optical Raman scattering spectroscopy. It was demonstrated that the structures of the Si-NRs were virtually defect-free, meaning they could be used for highly sensitive detection of biological macromolecules. The Si-NR chips were then used for the highly sensitive nanoelectronics detection of DNA oligonucleotides (oDNAs), which represent synthetic analogs of 106a-5p microRNA (miR-106a-5p), associated with the development of autism spectrum disorders in children. The specificity of the analysis was attained by the sensitization of the Si-NR chip sur-face by covalent immobilization of oDNA probes, whose nucleotide sequence was complementary to the known sequence of miR-106a-5p. The use of the Si-NR chip was demonstrated to al-low for the rapid label-free real-time detection of oDNA at ultra-low (~10−17 M) concentrations.

scholarly journals Nanoimprinted multifunctional nanoprobes for a homogeneous immunoassay in a top-down fabrication approach

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hubert Brueckl ◽  
Astrit Shoshi ◽  
Stefan Schrittwieser ◽  
Barbara Schmid ◽  
Pia Schneeweiss ◽  
...  
Keyword(s):  
Thin Film ◽  
Nanoimprint Lithography ◽  
Thin Film Deposition ◽  
Film Deposition ◽  
In Vitro Diagnostics ◽  
Top Down ◽  
Highly Sensitive ◽  
Optical And Magnetic Properties ◽  
Lift Off

AbstractMultifunctional nanoparticles are discussed as versatile probes for homogeneous immunoassays for in-vitro diagnostics. Top-down fabrication allows to combine and tailor magnetic and plasmonic anisotropic properties. The combination of nanoimprint lithography, thin film deposition, and lift-off processing provides a top-down fabrication platform, which is both flexible and reliable. Here, we discuss the material compositions and geometrical designs of monodisperse multicomponent nanoparticles and their consequences on optical and magnetic properties. The rotational hydrodynamics of nanoparticles is measured and considered under the influence of magnetic shape anisotropy in the framework of the Stoner-Wohlfarth theory. The plasmon-optical properties are explained by discrete-dipole finite-element simulations. Rotational dynamical measurements of imprinted nanoprobes for two test proteins demonstrate the applicability as highly sensitive biomolecular nanoprobes.

scholarly journals Anemoside B4 ameliorates TNBS-induced colitis through S100A9/MAPK/NF-κB signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yong Zhang ◽  
Zhengxia Zha ◽  
Wenhua Shen ◽  
Dan Li ◽  
Naixin Kang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Tca Cycle ◽  
Mapk Signaling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Trinitrobenzene Sulfonic Acid ◽  
Therapeutic Effects ◽  
Triterpenoid Saponin ◽  
Label Free

Abstract Background Despite the increased morbidity of ulcerative colitis (UC) in the developing countries, available treatments remain unsatisfactory. Therefore, it is urgent to discover more effective therapeutic strategies. Pulsatilla chinensis was widely used for the treatment of inflamed intestinal diseases including UC for thousands of years in China. Anemoside B4, the most abundant triterpenoid saponin isolated from P. chinensis, exerts anti-inflammatory and antioxidant effects and may be the most active compounds, which is responsible for the therapeutic effects. However, the mechanism how anemoside B4 executes its biological functions is still elusive. Methods Here, we used the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model to evaluate the therapeutic effect of anemoside B4. Blood samples of colitis rats were collected for hematology analysis. The inflammation-associated factors were investigated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis was determined with EdU cell proliferation assay and TUNEL assay. The proteins regulated by anemoside B4 were identified by label-free quantitative proteomics. The significantly down-regulated proteins were verified by Western blotting analysis. mRNA expression was analyzed by quantitative real-time RT-PCR. Results The results showed that anemoside B4 ameliorated TNBS-induced colitis symptoms, including tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine production, apoptosis and slowed proliferation in colon. Quantitative proteomic analyses discovered that 56 proteins were significantly altered by anemoside B4 in the TNBS-induced rats. These proteins mainly clustered in tricarboxylic acid (TCA) cycle and respiratory electron transport chain. Among the altered proteins, S100A9 is one of the most significantly down-regulated proteins and associated with NF-κB and MAPK signaling pathways in the pathogenesis of UC. Further experiments revealed that anemoside B4 suppressed the expression of S100A9 and its downstream genes including TLR4 and NF-κB in colon. In vitro, anemoside B4 could inhibit the NF-κB signaling pathway induced by recombinant S100A9 protein in human intestinal epithelial Caco-2 cells. Moreover, anemoside B4 inhibits neutrophils recruitment and activation in colon induced by TNBS. Conclusions Our results demonstrate that anemoside B4 prevents TNBS-induced colitis by inhibiting the NF-κB signaling pathway through deactivating S100A9, suggesting that anemoside B4 is a promising therapeutic candidate for colitis.

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