An activatable chemiluminescence probe based on phenoxy-dioxetane scaffold for biothiol imaging in living systems

Quantification of biothiols in living systems is essential to understand their biological applications. Here, we developed two activatable chemiluminescence probes (SHCL and NCCL) and investigated their utility in the bioimaging of intracellular biothiols by directly tethering 2,4-dinitrobenzenesulfonyl to the hydroxyl group of phenoxy-dioxetane. The design of these two probes differed in substituents of phenol-dioxetane, i.e., SHCL contained the ortho chlorine, whereas NCCL had the para hydroxymethyl. Upon glutathione (GSH) cleavage, both probes emitted significantly “turn-on” chemiluminescent signals. However, the chemiluminescence intensity based on NCCL declined with increasing GSH level above 5 mM, while SHCL exhibited much higher chemiluminescent intensity and a wider concentration range (0.5 μM-50 mM), which was much more suitable for sensing endogenous biothiols.
We further demonstrated that chlorine substitution in SHCL played an important role in bioimaging owing to the halogen effect, providing a lower pKa value and significant enhancement of the chemiluminescent emission. SHCL imaged the biothiols effectively in tumor cells and tumor-bearing mice. Additionally, this novel chemiluminescence probe can be easily used to evaluate the in vitro activity of acetylcholinesterase. Overall, we anticipate that SHCL may provide a facile and intuitive tool for studying the role of biothiols in diseases.

Nitrogen doped graphene quantum dots based long-persistent chemiluminescence system for ascorbic acid imaging.

  • High photo-intensity and sluggish flight attenuation are important to highly sensitive chemluminescence imaging. Herein, we present a copper ion catalyzed long-persistent chemiluminescent imaging system of nitrogen-doped graphene quantum dots (NGQDs) for ascorbic acid detection in fruit. NGQDs as luminescent probe are fabricated, emitting out chemluminescence with the direct oxidation by H2O2.
  • In addition, Cu2+ ion enlarges over two order magnitudes of NGQDs CL intensity (214 times) due to its catalyzed Fenton-like reaction for H2O2 decomposition, and displaying unique specificity against other metal ions. As a result, the twinkling luminescence of NGQDs is boosted and changes to hold persistent with small decay in the presence of copper ion exhibiting potential for CL imaging.
  • As an imaging model, a visual sensor based on Cu2+/NGQDs/H2O2 is developed for AA quantitative monitoring with a limit of detection (LOD) of 0.5μM (S/N=3) and applied in real AA detection in fruit. The CL imaging method demonstrated with high stability and proper sensitivity would provide a convenient and visual tool for AA determination, displaying promising candidates for imaging sensing.

Imaging systems for westerns: chemiluminescence vs. infrared detection.

Western blot detection methods have traditionally used X-ray films to capture chemiluminescence. The increasing costs for film, reagents, and maintenance have driven researchers away from darkrooms to more sensitive and technologically advanced digital imaging systems. Cooled charge coupled devices (CCD) cameras capture both chemiluminescence and fluorescence images, with limitations for each detection method. Chemiluminescence detection is highly sensitive and relies on an enzymatic reaction that produces light, which can be detected by a CCD camera that records photons and displays an image based on the amount of light generated. However, the enzymatic reaction is dynamic and changes over time making it necessary to optimize reaction times and imaging.
Fluorescent detection with a CCD camera offers a solution to this problem since the signal generated by the proteins on the membrane is measured in a static state. Despite this advantage, many researchers continue to use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum by imaging fluorphores in the infrared spectrum.
Infrared imaging is equally sensitive to chemiluminescence and more sensitive to visible fluorescence due in part to reduced autofluorescence in the longer infrared wavelength. Furthermore, infrared detection is static, which allows a wider linear detection range than chemiluminescence without a loss of signal.
A distinct advantage of infrared imaging is the ability to simultaneously detect proteins on the same blot, which minimizes the need for stripping and reprobing leading to an increase in detection efficiency. Here, we describe the methodology for chemiluminescent (UVP BioChemi) and infrared (LI-COR Odyssey) imaging, and briefly discuss their advantages and disadvantages.

Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown.
The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 x 10(-18) mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images.

New advanced oxidation progress with chemiluminescence behavior based on NaClO triggered by WS 2 nanosheets

As one integral part of coping strategies for addressing water pollution, advanced oxidation progresses (AOPs) get enormous attentions in recent years. However, the complex synthesis and high cost of H2O2 and K2S2O8 hampered their developments. Herein, a novel AOP with the chemiluminescence (CL) property based on economic NaClO and WS2 nanosheets was proposed to achieve efficient decomposition of organic pollutants.
In this AOP, WS2 nanosheets exhibited a dual-function feature of the catalyst and energy acceptor. It demonstrated that the reaction order of WS2 nanosheets was equal to 0.8271 and enormous singlet oxygen (1O2),·ClO and hydroxyl radical (·OH) were generated in rhodamine B (RhB) degradation process. Interestingly, a strong CL emission was observed and reflected the relative concentration of 1O2 and·OH for adjusting the oxidizing capability in WS2 nanosheets-NaClO system.
Through a series of degradation tests, RhB, methylene blue (MB), p-nitrophenol and phenol were decomposed and the degradation efficiency of over 90% was achieved. Therefore, this study not only builds a chemiluminescent AOPs to eliminate organic pollutants, but also broadens the applications of WS2 nanomaterials and CL in environmental field.

PolyTek HRP Anti-Rabbit Polymerized Imaging System

PIP080 ScyTek Laboratories 1 kit(s) 156 EUR

PolyTek Anti-Mouse (DAB) Polymerized HRP Imaging System

PIR080 ScyTek Laboratories 70 Slides 156 EUR

Accuris SmartDoc 2.0 Imaging Enclosure

E5001-SD Benchmark Scientific 1 PC 417.35 EUR

SuperBrite? ELISA HRP Chemiluminescence Substrate Kit

K4005-500 Biovision 180 EUR

MycoLight™ Live Bacteria Fluorescence Imaging Kit

22409 AAT Bioquest 100 Tests 306 EUR

ProtoBlock System

NAT1004 National Diagnostics 1KIT 102 EUR

GloBrite chemiluminescence reagent kit for western blotting 200 mL

GLB1 Detroit R&D 200 mL 145 EUR

GloBrite chemiluminescence reagent kit for western blotting 500 mL

GLB2 Detroit R&D 500 mL 234 EUR

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K002M T-Pro Biotechnology 250ml*2/Kit 204 EUR

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K002S T-Pro Biotechnology 100ml*2/Kit 152 EUR

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K004M T-Pro Biotechnology 250ml*2/Kit 239 EUR

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K004S T-Pro Biotechnology 100ml*2/Kit 170 EUR

High-Sensitive ECL Chemiluminescence Detection Kit (Ready-to-Use)

E412-01 Vazyme 2 × 50 ml 150 EUR

High-Sensitive ECL Chemiluminescence Detection Kit (Ready-to-Use)

E412-02 Vazyme 250 ml 328 EUR

Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit

15290 AAT Bioquest 100 Tests 480 EUR

ROS Brite™ 700 *Optimized for in Vivo Imaging*

16004 AAT Bioquest 1 mg 219 EUR

data acquisition system

D230 Consort ea 1704 EUR

photo documentation system

VLBPCX4-20M Consort ea 6352 EUR

photo documentation system

VLBPCX4-20MX Consort ea 6774 EUR

photo documentation system

VLDP-HOOD Consort ea 5084 EUR

A microfluidic DNA library preparation platform for next-generation sequencing

A microfluidic DNA library preparation platform for next-generation sequencing

Next-generation sequencing (NGS) is emerging as a strong device for elucidating genetic info for a large range of functions. Unfortunately, the surging recognition of NGS has not but been accompanied by an enchancment in automated strategies for preparing formatted sequencinglibraries.

To tackle this challenge, we have now developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for evaluation by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) pattern handling with peripheral modules to create a fully-integrated, sample-in library-out platform.

A microfluidic DNA library preparation platform for next-generation sequencing
A microfluidic DNA library preparation platform for next-generation sequencing

In this report, we use our automatedsystem to arrange NGSlibraries from samples of human and bacterial genomic DNA. E. coli libraries ready on-device from 5 ng of whole DNA yielded glorious sequence protection over the complete bacterial genome, with>>99% alignment to the reference genome, even genome protection, and good high quality scores.

Furthermore, we produced a de novo meeting on a beforehand unsequenced multi-drug resistant Klebsiella pneumoniae pressure BAA-2146 (KpnNDM). The new methodology described right here is quick, strong, scalable, and automated. Our machine for librarypreparation will help within the integration of NGS know-how into all kinds of laboratories, including small analysis laboratories and scientific laboratories.

We have developed an automated high quality management (QC) platform for next-generation sequencing (NGSlibrary characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent supply unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a ready pattern droplet was actuated into place between the bottom electrode and the inlet of the separation capillary to finish the circuit for an electrokinetic injection.

Using a DNA ladder as an inner normal, the CE module with a compact LIF detector was able to detecting dsDNA within the vary of 5-100 pg/μL, appropriate for the quantity of DNA required by the Illumina Genome Analyzer sequencing platform.

This DMF-CE platform consumes tenfold much less pattern quantity than the present Agilent BioAnalyzer QC method, preserving treasured pattern whereas offering obligatory sensitivity and accuracy for optimum sequencing efficiency.

The means of this microfluidic system to validate NGSlibrarypreparation was demonstrated by inspecting the consequences of limited-cycle PCR amplification on the scale distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the scale distribution of the library towards undesirable bigger fragments.

Automated digital microfluidic pattern preparation for next-generation DNA sequencing

Next-generation sequencing (NGS) know-how is a promising device for figuring out and characterizing unknown pathogens, however its usefulness in time-critical biodefense and public well being functions is presently restricted by the dearth of quick, environment friendly, and dependable automated DNA pattern preparation strategies.

To tackle this limitation, we’re growing a digital microfluidic (DMF) platform to operate as a fluid distribution hub, enabling the mixing of a number of subsystem modules into an automatedNGSlibrary pattern preparationsystem.

A novel capillary interface allows extremely repeatable switch of liquid between the DMF machine and the exterior fluidic modules, permitting each continuous-flow and droplet-based pattern manipulations to be carried out in a single built-in system. Here, we spotlight the utility of the DMF hub platform and capillary interface for automating two key operations within the NGS pattern preparation workflow.

Using an in-line contactless conductivity detector at the side of the capillary interface, we display closed-loop automated fraction assortment of goal analytes from a continuous-flow pattern stream into droplets on the DMF machine. Buffer trade and pattern cleanup, probably the most repeated steps in NGSlibrarypreparation, are additionally demonstrated on the DMF platform utilizing a magnetic bead assay and reaching a mean DNA restoration effectivity of 80%±4.8%.